Sincèrement Patrick

Aux membres de mon jury À Fabienne Vennet, merci de m'avoir accueillie dans votre laboratoire au tout début de ma thèse, un grand merci pour vos conseils et l'intérêt porté à mon travail.

À Benjamin Glenn-Choustermann, merci de l'aide, des conseils et des corrections apportées pour la rédaction de notre article.

À Pierre Asfar, merci pour nos échanges et vos conseils lors de mon comité de suivi de thèse ainsi que pour toutes les corrections et les recommandations pour l'écriture de notre article.

À Julie Helms, merci de l'intérêt porté à mon travail et d'avoir accepté d'évaluer mes travaux de thèse.

A mes encadrants

Je remercie Bruno de m'avoir fait confiance et d'avoir fait confiance à Antoine pour l'encadrement au jour le jour de ma thèse.

Je pense que ma première encadrante a été Chaojie, qui m'a fait, autorisez-moi l'expression, une passation de pouvoir pendant mon master 2 et qui malgré quelques encombres au démarrage m'a donné envie de faire cette thèse.

Et bien sûr, je remercie infiniment Antoine, sans qui je ne serais jamais arrivée à ce stade.

Merci de m'avoir encadrée pendant ces trois ans, sans faille, sans compter les heures passées malgré votre emploi du temps plus que chargé. Je ne pense pas en faire trop (quoi que vont en dire tous mes collègues, j'entends déjà les blagues) en disant que vous avez été un chef parfait. Pas présent au laboratoire, mais pourtant toujours là pour moi, disponible pour n'importe quel problème, que, vous avez raison, nous pouvons toujours résoudre en étant méthodique et rigoureux ! Votre optimisme et votre passion pour nos recherches m'ont énormément motivée tout au long de ma thèse. Je pense sincèrement que, grâce à vous, ces trois années se sont extrêmement bien déroulées. Je sais que vous essayez toujours de vous améliorer et je suis sûre que le prochain doctorant que vous encadrerez sera aussi épanoui que je l'ai été. Je ne suis pas encore au point, mais je vais essayer dans ma future vie professionnelle de manager à votre façon.

A mes collègues

Merci Huguette de m'avoir appris tout ce que je sais désormais en cytométrie, de m'avoir aidée à mettre en place tous mes protocoles, d'avoir cherché avec moi les solutions à mes problèmes, d'avoir participé à l'élaboration et à la réflexion scientifique de tout mon projet.

Outre le travail, merci pour tous ces repas, ces cafés, ces bières. J'en viendrais presque à te remercier pour tes engueulades, mais non, je vais m'arrêter là.

Je ne peux pas séparer Huguette de ses deux acolytes, alors je remercie beaucoup Cécile, à qui aucun problème ne résiste au laboratoire. Merci pour ta bonne humeur et pour m'avoir défendue dans n'importe quelle situation, avec un peu d'aveuglement, on peut l'avouer maintenant. Merci à Simon pour ta gentillesse et pour l'aide que tu apportes à tout le monde.

Et surtout merci pour tes blagues incessantes sur les vosgiens (et sur tout le reste).

Un énorme merci également à deux collègues devenus deux amis, sans qui ma vie au labo n'aurait pas été si agréable. Céline, je suis très heureuse d'avoir passé ces trois ans avec toi, sans avoir besoin de nous parler pour nous comprendre, alors merci pour tous ces regards complices. Alex, je ne sais pas comment j'ai fait pour te supporter mais j'espère que j'aurai encore à le faire, allez, pars avec moi, on va continuer à travailler ensemble ! Merci également à Véronique, pour t'être toujours occupée de mes animaux et de l'animalerie.

Je remercie également tous les collègues, Jeremy, Zhor, Nathalie, Allyson, Simon, pour l'aide technique et les conseils que vous m'avez apportés, et pour toutes les discussions autour des repas.

Aux secrétaires, merci de m'avoir toujours aidée pour la partie administrative, merci Audrey pour ton aide, ta générosité et ton soutien.

Je remercie tous les doctorants, Camille qui a laissé un grand vide en partant, Yun et Ilona pour avoir partagé mon bureau et je souhaite le meilleur à Célia et Mathieu pour la fin de leur thèse.

Je tiens également à remercier les membres de mon comité de thèse, Jean-Pol Frippiat et Mervyn Singer, pour leur disponibilité et leurs précieux conseils

Tables

 Table 1:
Résumé de la thèse

Situation du sujet L'histoire naturelle d'une infection grave est un continuum qui débute par le sepsis et qui évolue en choc septique. Le choc septique se définit par un état infectieux associé à une hypotension, marqueur d'une dysfonction d'organe, persistante malgré l'expansion volémique et requérant l'administration de drogues vasoactives. Son évolution conduit, en l'absence de prise en charge, à un dysfonctionnement multi-organes potentiellement létal.

Le sepsis et le choc septique sont l'une des principales causes d'admission en unité de soins intensifs et sont reconnus par l'Organisation Mondiale de la Santé comme un problème de santé publique majeur [1]. En 2017, les données mondiales, provenant principalement des pays à revenu élevé, estimaient à 19,4 millions le nombre de cas de sepsis grave et de chocs septiques, entraînant 5,3 millions de décès par an [2]. Ces statistiques étaient sans doute sousestimées de par le manque d'informations provenant des pays à faible revenu. En effet, en 2020, une étude incluant un plus grand nombre de données provenant de pays à faible revenu et à revenu intermédiaire a estimé à 48,9 millions le nombre de cas de sepsis graves et de chocs septiques avec en conséquence un nombre de décès deux fois plus élevé que décrit précédemment [3].

Malgré des progrès dans sa prise en charge, le choc septique est toujours associé à une mortalité de 40% [4]. Aucune thérapeutique spécifique testée au cours de ces quatre dernières décennies n'a montré, pour l'heure, une quelconque efficacité sur la mortalité. Les essais cliniques de phase III pour le traitement du choc septique tels que la protéine C activé, les anticorps anti-TNF, anti-LPS ou l'antithrombine III, ont tous échoué à améliorer la survie malgré des études positives de phase II et des études pré-cliniques hautement favorables [5][6][7]. L'amélioration des processus de prise en charge, bien qu'ayant largement diminué la mortalité du choc septique, montre aujourd'hui ses limites. Il y a donc un besoin autant sur le plan individuel qu'en terme de santé publique de développer de nouvelles stratégies thérapeutiques [8].

Le choc septique est associé à une activation physiologique du système nerveux sympathique (SNS). L'activation du SNS, en libérant de la noradrénaline et de l'adrénaline, est une réponse essentielle à l'infection à la phase initiale de tout processus septique. Ces effets bénéfiques sont une augmentation de l'inotropisme, une tachycardie et une vasoconstriction, qui contrecarrent les effets toxiniques et/ou inflammatoires initiaux du processus infectieux [9]. Néanmoins, la persistance de cette activation dans le temps à un haut niveau d'intensité peut aussi être délétère et entraîner un syndrome de dysautonomie sympathique. Cette dernière est marquée par une perte de la variabilité cardiovasculaire, une désensibilisation des adrénorécepteurs et une immunosuppression adaptative à l'origine d'une surmortalité [10][11][12][13][14].

L'immunosuppression induite par le choc septique est caractérisée par une déplétion et une dysfonction des cellules immunitaires conduisant à l'incapacité du patient à répondre à une seconde infection [15]. Cette dysfonction touche les cellules de l'immunité innée, caractérisée par une diminution de la production cytokinique, de la cytotoxicité et de la capacité d'orchestrer la réponse adaptative. Ces cellules de l'immunité adaptative sont également impactées. Les lymphocytes présentent une diminution de leurs capacités prolifératives, des secrétions de cytokines et un dérèglement de leurs fonctions.

Les lymphocytes T CD4 sont plus particulièrement touchés par l'immunosuppression septique. Le nombre de lymphocytes T CD4 circulants diminue drastiquement lors d'un choc septique. L'analogie avec le VIH peut être pertinente car dans cette pathologie chronique, source d'un déficit T CD4, les infections opportunistes font le lit du pronostic défavorable de cette maladie. Contrairement aux autres sous populations lymphocytaires, plusieurs études décrivent une augmentation du pourcentage de lymphocytes T régulateurs (Tregs) circulants [16][17][18][19]. Cette augmentation est observée immédiatement après le début du sepsis et persiste chez les patients non survivants [16,20]. L'augmentation des proportions de Tregs participe à la diminution des capacités prolifératives des lymphocytes, induit la production de cytokines anti-inflammatoires et réprime les fonctions de cellules de l'immunité innée jouant un rôle majeur dans l'immunosuppression.

L'adrénaline et la noradrénaline, deux inopresseurs majeurs ayant principalement des effets adrénergiques α 1 et β 1 adrénergiques (β 1 -AR), ont été récemment confirmés comme étant des facteurs cruciaux dans la genèse de l'immunosuppression [21]. Lors d'un choc septique, la noradrénaline favorise la sécrétion de cytokines anti-inflammatoires et est associée à des effets inhibiteurs de la prolifération des lymphocytes T et de la production de cytokines [22][23][24].

Ainsi, la modulation du SNS pourrait être une approche thérapeutique intéressante dans la prise en charge du choc septique. Pour moduler l'interaction entre le SNS et la réponse inflammatoire, l'utilisation d'un traitement β 1 -bloquant semble la plus justifiée.

Les premières études chez l'homme ont principalement porté sur l'effet bénéfique de la réduction de la fréquence cardiaque par le blocage β 1 -AR sur la fonction cardiovasculaire lors d'un choc septique [25]. Cependant, les effets cardiovasculaires protecteurs des β 1bloquants semblent plutôt associés aux effets anti-inflammatoires de ces molécules qu'à la réduction de la fréquence cardiaque. En effet, l'adjonction de doses non-chronotropes d'un β 1 -AR bloquant dans un modèle expérimental de choc septique est associée à une cardioprotection in vivo et à une meilleure vasoréactivité ex vivo [26]. Ces effets sont probablement en partie médiés par une régulation de l'inflammation.

Parallèlement, il a été démontré que les cellules de l'immunité innée et adaptative exprimaient des β-AR [27][28][29][30][31]. Des études expérimentales ont exploré l'effet inflammatoire du blocage β 1 -AR et ont retrouvé une pondération globale des cytokines plasmatiques pro-et anti-inflammatoires ainsi qu'une diminution de l'inflammation tissulaire chez des animaux septiques β 1 -AR bloqués [32]. Cependant, l'effet de ce blocage sur la réponse immunitaire adaptative, et plus particulièrement sur les dysfonctions lymphocytaires T CD4 survenant au cours du choc septique n'a jamais été étudié.

Objectifs du travail

L'objectif général de cette thèse est d'explorer l'impact de la modulation β 1 -AR sur l'hémodynamique et le système inflammatoire et plus particulièrement sur les lymphocytes T CD4 dans un modèle murin de sévère de sepsis induit par ligature et ponction du caecum (LPC).

Méthodologie générale

En utilisant un modèle de sepsis polymicrobien sévère induit par LPC les effets hémodynamiques et inflammatoires du blocage β 1 -AR ont été étudiés :

-Chez des souris sauvages (WT), auxquelles un β 1 -AR bloquant (esmolol, 18 mg/kg/h) est administré ou non trois heures après l'opération via une mini-pompe osmotique sous cutanée.

-Chez des souris knock-out (KO) pour le β 1 -AR.

Six groupes ont été définis :

1) WT sham,

2) WT sham + esmolol, 3) WT LPC, 4) WT LPC + esmolol, 5) KO Sham, 6) KO LPC.

Dix-huit heures après l'induction du choc septique, quatre séries d'expériences ont été réalisées :

1) Caractérisation hémodynamique et inflammatoire (n=74) :

-La fonction cardiaque in vivo a été évaluée par échocardiographie afin de mesurer la fréquence (FC), le débit cardiaque (DC) et la puissance myocardique de chaque souris,

-La pression artérielle moyenne (PAM) in vivo a été mesurée par cathétérisation de la carotide,

-L'infiltration des leucocytes dans le myocarde a été évaluée par immunohistochimie (marquage CD45+),

-Le dosage plasmatique de 46 médiateurs pro-et anti-inflammatoires a été réalisé par Luminex®,

-Les différentes populations lymphocytaires de la rate (T CD3+, T CD8+, T CD4+, Th1, Th2, Th17, Treg) ont été immunophénotypées par cytométrie en flux.

2) Un test de prolifération des lymphocytes T CD4 sur 72 heures en présence d'un agent mitogène a été réalisé et analysé par cytométrie en flux (n=44).

3) La prolifération et a mortalité des lymphocytes T CD4 en présence de Tregs pré-activés par des catécholamines (dobutamine, β 1 -AR agoniste ; phénylephrine, α 1 -AR agoniste ; noradrénaline, α 1 -et β 1 -AR agoniste) provenant de splenocytes de souris non septiques a été mesurée in vitro en temps réel pendant cinq jours (Incucyte®) (n=4).

4) Une étude de survie sur cinq jours a été réalisée dans les groupes de souris suivant : WT LPC, WT LPC + esmolol administré pendant 24 heures et KO LPC (n=60).

Résultats obtenus

Cette étude confirme que l'induction d'une septicémie par LPC reproduisait les caractéristiques clinico-biologiques usuelles de cette pathologie :

1) Cliniquement : une hypotension artérielle, une diminution du DC et une diminution de la puissance cardiaque ;

2) Biologiquement : une augmentation de l'ensemble des médiateurs pro et antiinflammatoires, une diminution des proportions de lymphocytes T CD4 corrélée à une augmentation des proportions de Tregs dans la rate ainsi qu'une diminution des capacités prolifératives des lymphocytes T CD4.

Dans ce travail, il était retrouvé que le blocage β 1 -AR lors du choc septique, qu'il soit pharmacologique ou génétique, avait pour conséquence une préservation de la proportion de lymphocytes T CD4 et de la proportion des Tregs, restant à un niveau comparable à celui des souris sham. De plus, ce blocage β 1 -AR permettait le maintien des capacités prolifératives des lymphocytes T CD4.

Nous avons également démontré le rôle direct de la simulation des β 1 -ARs sur les fonctions immunorégulatrices des Tregs. En effet, les capacités prolifératives des lymphocytes T CD4 tendent à diminuer en présence de Treg pré-stimulé à la dobutamine, un β 1 -AR agoniste.

Ces résultats se retrouvaient au travers d'une diminution des cytokines plasmatiques pro-et anti-inflammatoires. De plus, malgré une FC diminuée chez les souris β 1 -AR bloquées, la PAM, le DC ainsi que la puissance myocardique étaient maintenus.

L'étude de survie sur cinq jours a également montré un taux de survie de 60% chez les souris β 1 -AR KO en choc septique. Chez les souris WT, le taux de survie était de 36% chez les souris septiques, et de 37% lorsqu'un β 1 -AR bloquant leur était administré pendant les 24 premières heures.

Discussion

Caractéristiques du modèle

Pour étudier le rôle immunitaire et cardiovasculaire des β 1 -ARs dans le sepsis, nous avons délibérément choisi un modèle murin de LPC non réanimé, afin d'éviter toute interférence entre l'administration de catécholamines exogènes et le système sympathique.

De ce fait le terme choc septique ne peut pas être utilisé. Ainsi, les animaux ont tous présenté un sévère sepsis typique caractérisé par une hypotension et un bas débit cardiaque associé à une augmentation des concentrations plasmatiques de médiateurs pro-et antiinflammatoires. Ce modèle reproduit au niveau splénique de nombreuses altérations immunitaires observées au niveau périphérique chez les patients en choc septique, caractéristique de l'immunosuppression septique : une diminution des proportions des lymphocytes T CD4 corrélée à une augmentation des proportions de Tregs dans la rate a été retrouvée. De plus, les capacités de proliférations des lymphocytes T CD4 étaient altérées.

La stimulation β 1 -AR induit un dysfonctionnement des cellules T CD4

Les cellules de l'immunité innée comme adaptative expriment les récepteurs adrénergiques. Cependant leur caractérisation, particulièrement pour les β-AR, est moins bien définie. Plusieurs études ont démontré la présence de β-AR sur les différentes souspopulations lymphocytaires, à l'exception des lymphocytes Th2. Cependant, jusqu'à récemment, seule la présence de β 2 -AR et non β 1 -AR sur les lymphocytes a été étudiée. De ce fait, le β 1 -AR n'est pas considéré comme un acteur de la réponse inflammatoire. Toutefois, une étude publiée en 2020 apporte une preuve de l'expression de β 1 -AR à la surface des lymphocytes T CD4 dans la rate.

Dans cette étude, l'impact des β 1 -AR bloquants sur le dysfonctionnement des cellules T CD4 chez des souris cirrhotiques est examiné. Ainsi, il a été démontré qu'un traitement à la dobutamine, β 1 -AR agoniste, est capable d'activer les Tregs, suggérant la présence de β 1 -AR à leur surface [33]. De la même manière, notre étude démontre que la prolifération in vitro des lymphocytes T CD4 tend à réduire en présence de Tregs préalablement traités à la dobutamine, révelant l'impact direct de la stimulation β 1 -AR sur la fonction inhibitrice des Tregs.

Le blocage β 1 -AR réduit l'immunosuppression septique Dans de nombreuses maladies telles que l'insuffisance cardiaque aiguë, les brûlures, l'insuffisance hépatique chronique ou le choc septique, le blocage β 1 -AR est associé à des effets protecteurs hémodynamiques et inflammatoires [26,34]. De façon similaire, dans cette étude, nous avons trouvé une pondération globale des médiateurs pro-inflammatoires et antiinflammatoires chez les animaux en état de choc septique traités avec un β 1 -AR bloquant.

Cependant, les mécanismes sous-jacents ne sont toujours pas élucidés. Il a été observé chez des souris infectées par Listeria monocytogenes qu'un stress aigu, substitut de la stimulation adrénergique sympathique, inhibe la réponse des lymphocytes T et augmente la production de cytokines. Dans ce modèle, le blocage pharmacologique β 1 -mais pas β 2 -AR a permis de diminuer cet état pro-inflammatoire et de raccourcir le temps de clairance bactérienne [35].

Dans notre étude, le blocage β [36,37]. L'induction d'une septicémie chez la souris est le plus souvent associée à une diminution marquée du métabolisme énergétique avec pour conséquence une diminution de la FC [37]. À l'inverse, chez l'homme, le métabolisme est le plus souvent augmenté, entraînant une tachycardie adaptative précoce.

Perspectives

Les β 1 -ARs, mais également les β 2 -ARs sont exprimés par les cellules immunitaires et notamment par les lymphocytes [30]. La stimulation β 2 -AR semble avoir des effets antiinflammatoires et favoriser la différenciation des lymphocytes Th2 [38]. Pour soutenir cette hypothèse, il a été démontré chez des patients en choc septique que certains polymorphismes β 2 -ARs étaient associés à une surmortalité [39]. Dans la présente étude, l'effet du blocage β 1 -AR sur les lymphocytes T CD4 a été analysé. Une suite logique serait de s'intéresser à l'impact de la modulation β 2 -AR sur l'immunosuppression septique, grâce à un blocage pharmacologique (ICI 118,551) et à un modèle de souris β 2 -AR KO que nous possédons au laboratoire.

L'étude du rôle des β 3 -ARs, exprimés par les cellules endothéliales, durant le choc septique pourrait également être prometteur. En effet, il est démontré que l'administration de nébivolol, un agoniste des β 3 -ARs, favorise la vasorelaxation [40]. De plus, une étude préliminaire a révélé dans la graisse brune chez le rat que l'esmolol pourrait être un antagoniste des β 3 -ARs [41]. Il sera donc intéressant de regarder si les β 1 -ARs bloquants ont également des effets sur les β 3 -ARs, exprimés par les cellules endothéliales. Plusieurs études expérimentales et cliniques ont montré que l'administration d'un β 1 -AR bloquant diminuait l'hyporéactivité vasculaire aux catécholamines, alors que ce récepteur n'est pas exprimé dans les vaisseaux [25,32]. L'éventuel effet antagoniste d'un β 1 -AR bloquant sur les β 3 -ARs pourrait expliquer l'amélioration de la réactivité vasculaire aux catécholamines lors d'un choc septique expérimental.

Conclusion

Durant le processus septique, la pondération de la dysautonomie adrénergique par l'administration d'un β 1 -AR bloquant est associée à une amélioration de la fonction immunitaire adaptative. Ces résultats sont dans notre étude, pour la première fois, aussi retrouvés chez les souris génétiquement invalidées pour le récepteur β 1 -AR. Ainsi, au lieu de considérer le blocage de β 1 -AR comme un simple traitement hémodynamique, ces résultats ouvrent la voie à une approche immuno-régulatrice rétablissant l'homéostasie lymphocytaire T CD4 en cas de septicémie.

Background Definition of septic shock

The definition of sepsis has evolved in the past few years to improve the diagnosis and the management provided to patients. Septic shock was first defined by the American College of Chest Physicians (ACCP) and Society of Critical Care Medicine (SCCM) consensus conference in 1991 [42]. This definition included a continuous worsening process starting with systemic inflammation response syndrome (SIRS) (Table 1). SIRS with presumed or proven infection is defined as sepsis. Finally, septic shock is referred to severe sepsis with signs of persistent hypoperfusion despite adequate fluid resuscitation. According to the reference: [42] Term Criteria SIRS Two of the four following criteria: Temperature >38°C or <36°C Heart rate >90 min -1 Hyperventilation evidenced by respiratory rate >20 min -1 or arterial CO 2 lower than 32 mmHg White blood cell count >12000 cells.μl -1 or <4000 cells. μl -1

Sepsis

SIRS criteria with presumed or proven infection

Severe sepsis

Sepsis with organ dysfunction

Septic shock

Sepsis with hypotension despite adequate fluid resuscitation However, this definition has many limitations. Some patients may not have symptoms meeting two SIRS criteria despite having an infection and develop organ failure. Kaukonen et al., in a retrospective study from 2000 to 2013 with more than 1 million of patients with infection and organ failure, confirmed that the SIRS criteria rule missed one patient in eight with severe sepsis [43].

An improvement in our understanding of sepsis pathophysiology, such as the early activation of both pro-and anti-inflammatory responses, led the Sepsis Definition Task Force members to update sepsis definitions between January 2014 and January 2015 [44]. The updated definition includes both pathophysiological and clinical heterogeneities of septic shock. Thus, sepsis is defined as organ dysfunction induced by an infection. Organ dysfunction can be summarised with the Sequential Organ Failure Assessment (SOFA) score of 2 points or more. This score aims to describe the degree of organ dysfunction or failure over time in patients by assessing the respiratory, cardiovascular, coagulation, renal, hepatic, and central nervous systems. A SOFA score of 2 or more associated with a suspected or documented infection defines sepsis.

Patients with septic shock are treated with a vasopressor aiming to maintain mean arterial pressure above 65 mmHg and have a serum lactate level greater than 2 mmol.l -1 despite adequate volume resuscitation (Table 2). The terminology of severe sepsis has been removed. According to the reference: [44]

Sepsis

Septic shock Definition

Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection Septic shock is a subset of sepsis in which underlying circulatory and cellular/metabolic abnormalities are profound enough to substantially increase mortality Clinical criteria Suspected or documented infection and an acute increase of ≥ 2 SOFA points (a proxy for organ dysfunction) Sepsis and vasopressor therapy needed to elevate MAP ≥65 mmHg and lactate >2 mmol.l -1 (18 mg.dl -1 ) despite adequate fluid resuscitation

In order to facilitate the identification of sepsis at the bedside, patients with a suspected infection who are likely to have a prolonged intensive care unit (ICU) stay or to die during hospital stay can be promptly identified at the bedside if they have at least two of the quick SOFA (qSOFA) clinical criteria. The latter is a surrogate of SOFA score. The qSOFA score calculation just needs the respiratory, the consciousness and the systolic blood pressure (Table 3). However, Raith et al. demonstrates that, among adults with suspected infection admitted to an ICU, an increase in the SOFA score of 2 or more points had greater prognostic accuracy for in-hospital mortality than qSOFA, suggesting that qSOFA may have limited use for predicting mortality in an ICU setting [45]. According to the reference: [44]

qSOFA (quick SOFA) criteria

Respiratory rate ≥22 min -1 Altered mentation Systolic blood pressure ≤100 mmHg

Epidemiology of septic shock

Incidence

The description of septic shock epidemiology is crucial as 31,5 million cases of sepsis and 19,4 million cases of severe sepsis were estimated each year in high-income-countries.

Fleischmann et al. in a meta-analysis, including 27 studies from seven countries, showed that the incidence was 288 hospital-treated cases of sepsis and 148 hospital-treated cases of severe sepsis per 100,000 person-years between 1979 to 2015. In the last decade, the incidence was 437 cases of sepsis and 270 cases of severe sepsis per 100,000 person-years [2].

Gram positive strain was Staphylococcus aureus. Microbiological patterns varied around the globe; for example, in south Asia Gram-positive infections are much less frequent (21.4%) than in other regions. The prevalence of bacterial species also varies according to continents.

Pseudomonas spp. was most common in South America (20.4%) and eastern Europe (21.1%) and infection with fungal organisms was found in 14.5% and 14.8 % of cases, respectively, in western and eastern Europe, but only 5.1% in North America [1]. Similarly, Vincent et al. showed that 70% of infected patients have positive microbial samples, 62% of them were Gram negative organisms, 47% were Gram + , and 19% were fungi [62].

Site of infection

The primary origin of sepsis remains virtually unchanged over time. Thus, in 1997Thus, in -1998, in decreasing order of frequency, the infected sites were from respiratory, digestive tract, and urinary; bacteraemia represented more than 80% of the infectious aetiologies of patients with septic shock [51]. Thereafter, a retrospective cohort including 32 ICUs in the Paris area including 31,251 patients in septic shock found that the most common site of infection was still pulmonary (50.9%) in the period from 1998 to 2010 [63]. This was confirmed in many further studies [4,60,64].

Risk factors

Risk factors of death in septic shock patients are well identified. Regardless of the time period studied, severity scores are reliable prognostic markers. Thus, the SOFA score is systematically found as a prognostic marker. Age and sex are also strongly associated with mortality, as well as the presence of at least one comorbidity [4,65].

Historically, Vincent et al. showed that Simplified Acute Physiology Score (SAPS) II score was independently associated with mortality and a higher risk of infection [62].

However, the clinical pattern at admission is continuously evolving over time. Thus, new scores are being implemented, such as SAPS III or APACHE III scores. The latter improve risk prediction of poor outcome compared to APACHE II or SAPS II [66,67]. Moreover, specific subgroups deserve an independent analysis and have been studied. Galbois et al., investigated septic shock patients with cirrhosis. Thus, from 1998 to 2010, the ICU mortality of cirrhotic patients with septic shock markedly decreased, from 84.7% to 68.5% for patients receiving mechanical ventilation and from 91.2% to 78.4% for those with renal replacement therapy [63].

Diabetes mellitus is also a common comorbidity in septic shock patients. Diabetes is associated with chronic organ dysfunction. One epidemiologic study from 1979 to 2003 including 2,070,459 septic patients with diabetes are less likely to develop an acute respiratory failure and more likely to have an acute kidney injury compared to non-diabetic patients.

Interestingly, there was no difference in mortality between septic patients with and without diabetes [68].

Mortality

Sepsis accounts for more than five million deaths around the world each year, and it is accepted that sepsis is the leading cause of death in ICUs around the world [62,69] 19.7% of deaths worldwide. The estimated number of sepsis-related deaths was two-fold higher than previously described [2]. This increase could be accountable to the inclusion of more data from low-income and middle-income countries for which the incidence and mortality were considerably higher and from which data were previously lacking [3].

Several studies have found a decrease in mortality related to sepsis in the three last decades [46,47]. From 1990 to 2017, Rudd et al. estimated that sepsis mortality was reduced by 52.8% worldwide [3]. In a retrospective study from Australia and New Zealand performed between 2000 to 2012, it was found that absolute septic mortality decreased from 35.0% to 18.4%, representing an annual decrease rate of 1.3% [70]. Furthermore, Stevenson el al., in a comparative meta-analysis, observed a reduction in mortality from 46.9% from 1991-1995 to 29% from 2006-2009, representing an annual mortality decrease of 3.0% [71]. Since there is no new efficient therapy for sepsis introduced in the last for decades, it can be assumed that mortality decrease in high income countries could be attributed to the early detection and to the prompt management of septic patients [72].

Clinical management of patients

The Surviving Sepsis Campaign consortium established guidelines for the management of sepsis and septic shock. They were first published in 2004 [73] and revised in 2008 [74], 2012 [75], and 2016 [8]. This consensus defined the best practice for managing septic and septic shock patients. The first principle was that the success in treating septic shock is timedependent. Management of sepsis and septic shock patients can be summarised in three points which must be handled simultaneously: infection source control, hemodynamic stabilisation, and the management of organ failure.

The early diagnosis of the infection source is essential based on a clinical examination, bacteriological samples, imaging such as echography of chest X-ray. As soon as possible after the diagnosis, broad-spectrum antibiotic therapy should be administered. Kumar et al. have shown that a delay in the initiation of effective antibiotic therapy is associated with increased mortality [76]. Similarly, prescription of an inappropriate antibiotic regimen is also associated with increased mortality [77]. The last part of the SSC guidelines is about supportive care, which is also crucial. For instance, treatments of respiratory, kidney, or coagulation failure are most often required during the septic shock management. For more detail, readers should refer to the guidelines [8].

Cardiovascular physiopathology of septic shock

Cardiac physiopathology

Myocardial dysfunction

Left ventricular dysfunction

The failure of the cardiovascular system due to sepsis involves both peripheral vascular and myocardial dysfunction, with as both cause and consequence an an increase in circulating catecholamine and cytokine levels [78,79]. Sepsis-induced myocardial dysfunction occurs in approximately 50% of septic patients, and both diastolic and systolic dysfunction are observed [80]. A study by Parker et al. published in 1984 was the first to demonstrate that myocardial dysfunction is of major importance in septic shock patients [START_REF] Parker | Profound but reversible myocardial depression in patients with septic shock[END_REF]. In this work, using the cineangiography technique and pulmonary artery catheterisation, left ventricular (LV) ejection dysfunction (<40%) was initially found in 10 of 13 surviving patients and was completely resolved 7 to 10 days later [START_REF] Parker | Profound but reversible myocardial depression in patients with septic shock[END_REF]. Then, Ellrodt et al. and Ognibene et al. showed that the LV stroke work index was depressed in 94% of septic patients [START_REF] Ellrodt | Left ventricular performance in septic shock: reversible segmental and global abnormalities[END_REF][START_REF] Ognibene | Depressed left ventricular performance. Response to volume infusion in patients with sepsis and septic shock[END_REF].

Using cardiac ultrasound measurements, Jardin et al. also found a decrease in the LV ejection fraction (LVEF) and preload dependence, despite fluid resuscitation, in non-survivors [80]. Recent studies also confirmed these findings [START_REF] Orde | Outcome prediction in sepsis: speckle tracking echocardiography based assessment of myocardial function[END_REF][START_REF] Sanfilippo | Left ventricular systolic function evaluated by strain echocardiography and relationship with mortality in patients with severe sepsis or septic shock: a systematic review and meta-analysis[END_REF]. Interestingly, Sanfilippo et al., in a meta-analysis, found that a decrease in myocardial strain was a good predictor of adverse outcome in severe sepsis or septic shock, while such association was not found for LVEF [START_REF] Sanfilippo | Left ventricular systolic function evaluated by strain echocardiography and relationship with mortality in patients with severe sepsis or septic shock: a systematic review and meta-analysis[END_REF].

Diastolic dysfunction, while less characterised and much more difficult to diagnosed, is also frequently reported in septic shock patients. Barraud et al. reported an increase in enddiastolic pressure resulting from a reduction in LV compliance in anaesthetised endotoxaemic rabbits [START_REF] Barraud | Levosimendan restores both systolic and diastolic cardiac performance in lipopolysaccharide-treated rabbits: comparison with dobutamine and milrinone[END_REF]. Landersberg et al., in a cohort of 262 ICU patients with severe sepsis and septic shock, found that 9.1% of patients had systolic dysfunction, 14.1% had combined systolic and diastolic dysfunction, and 38% had isolated diastolic dysfunction [START_REF] Landesberg | Diastolic dysfunction and mortality in severe sepsis and septic shock[END_REF]. This study demonstrated a six-fold higher mortality in septic patients with diastolic dysfunction. Further studies also confirmed the strength of this association [START_REF] Sanfilippo | Diastolic dysfunction and mortality in septic patients: a systematic review and meta-analysis[END_REF][START_REF] Sanfilippo | Tissue Doppler assessment of diastolic function and relationship with mortality in critically ill septic patients: a systematic review and meta-analysis[END_REF].

Right ventricular dysfunction

Right ventricular (RV) dysfunction in septic patients was discovered early but received far less attention and investigation than LV dysfunction [START_REF] Kimchi | Right ventricular performance in septic shock: a combined radionuclide and hemodynamic study[END_REF][START_REF] Dhainaut | Right ventricular dysfunction in patients with septic shock[END_REF]. RV dysfunction is reported in 30-60% of septic patients and is most often associated with LV dysfunction [START_REF] Lakshmanadoss | Right Ventricle Failure in Sepsis: A Case Report[END_REF]. Thus, Vallabhajosyula et al. reported that, in a cohort of 388 patients admitted to the ICU for severe sepsis or septic shock, RV dysfunction was diagnosed in 55%. Forty-seven percent of them had isolated RV dysfunction and 53% combined bi-ventricular dysfunction [START_REF] Vallabhajosyula | Prognostic impact of isolated right ventricular dysfunction in sepsis and septic shock: an 8-year historical cohort study[END_REF]. During septic shock, RV dysfunction can be characterised by either dilation or a decreased ejection fraction, or both [START_REF] Dhainaut | Right ventricular dysfunction in patients with septic shock[END_REF]94]. The latter is multifactorial and can be due to an increase in RV afterload, or sepsis-related myocardial fibre depression. In acute respiratory distress syndrome with septic shock, RV failure is most often worsened by hypoxaemia, hypercapnia, and the high positive end-expiratory pressure level, which increases the RV afterload [START_REF] Dessap | Acute cor pulmonale during protective ventilation for acute respiratory distress syndrome: prevalence, predictors, and clinical impact[END_REF].

Molecular mechanism of cardiac dysfunction

Pathogen-associated molecular patterns and Toll-like receptors Intramyocardial inflammation, involving many pathways of the inflammatory response, is a major cause of myocardial dysfunction [START_REF] Walley | Sepsis-induced myocardial dysfunction[END_REF]. The Toll-like receptors (TLRs) expressed on cardiomyocytes are activated by pathogen-associated molecular patterns (PAMPs) originating from the pathogen [START_REF] Boyd | Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response[END_REF] and by endogenous damage-associated molecular patterns (DAMPs) [START_REF] Zhou | Knockout of Toll-like receptor 4 improves survival and cardiac function in a murine model of severe sepsis[END_REF]. TLR stimulation leads to nuclear factor-κB (NF-κB) activation and the subsequent release of pro-inflammatory cytokines [START_REF] Akira | Pathogen recognition and innate immunity[END_REF]. Indeed, the administration of endotoxin (molecule released by the lysis of Gram negative bacteria) in healthy volunteers is responsible for a reduction in both LVEF and LV performance, leading to the typical hemodynamic pattern of sepsis-related myocardial dysfunction [100]. TLR-4, by itself, plays a pivotal role in sepsis-induced myocyte dysfunction. Taverner et al. demonstrated that TLR-4 was required to induce myocyte and mitochondrial dysfunction in mice with endotoxaemia. and Zhou et al. reported that septic TLR-4 knock-out (KO) mice were protected against sepsisinduced cardiac dysfunction due to decreased myocardial neutrophils infiltration, attenuated cardiomyocytes apoptosis, and decreased pro-inflammatory cytokines release in heart tissue [START_REF] Zhou | Knockout of Toll-like receptor 4 improves survival and cardiac function in a murine model of severe sepsis[END_REF]101].

Cytokines

The inflammatory cytokines released during septic shock play a pivotal role in cardiac dysfunction. Kumar et al. showed that tumour necrosis factor-α (TNF-α) and interleukin-1β Nitric oxide is considered as to be beneficial and detrimental to cardiac function. NO can be produced by three forms of NOS: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) [113,114]. The activity of eNOS is dependent on the cytosolic calcium concentration and results in low levels of NO, which is involved in several physiological processes. [133][134][135][136]. This increase in catecholamines is responsible for inadequate tachycardia and an increase in oxygen requirements, both of which are associated with a higher mortality rate [137]. Several experimental studies have demonstrated in septic rats that these high catecholamine levels were associated with a desensitisation process at the cardiomyocyte level, characterised by the downregulation of β-adrenergic receptors (β-AR) [138,139]. This desensitisation process is described in another section.

In addition to the decrease in myocardial adrenoceptor density, signal transduction is also disrupted by an increase in the expression of inhibitory G-proteins, while stimulatory G proteins (G s ) are reduced (Figure 1) [140][141][142].

Vascular hyporeactivity is defined by the inability to increase blood pressure despite the administration of vasopressor agents in septic shock patients. Thus, the constriction of vascular smooth muscle cells is not able to maintain transparietal pressure and thus arterial blood pressure. Experimentally, vascular reactivity can be assessed in myography chambers by exposing segments of isolated vessels to vasopressor or vasodilator agents [146,147].

Dose-response studies with phenylephrine, a pure alpha agonist, allow for the detection of vessel contractility, while dose-response studies with acetylcholine allow for the detection of endothelium-dependent relaxation capacity and those with sodium nitroprusside (SNP) allow for the detection of the endothelium-independent relaxation capacity of the vessel. In clinical practice, dose-response studies with phenylephrine can also be performed; in this specific case, vascular hyporesponsiveness can be defined as a lower increase in arterial pressure for the same dose of phenylephrine or NE administered to patients compared to healthy volunteers [148].

There is considerable clinical evidence of vascular hyporeactivity during septic shock.

Immune response in septic shock

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection characterised by deep circulatory, cellular, and metabolic abnormalities.

Historically, an excessive pro-inflammatory response was thought to lead to an early mortality in the first days of sepsis, and the compensatory anti-inflammatory response was thought to induce mortality later by immune suppression and long-term organ failure (Figure 2, Theory 1) [166,167].

However, new insights gathered from septic patients have identified that both proinflammatory and anti-inflammatory responses occur early and simultaneously during sepsis [168]. The first part is a dominant hyper-inflammatory response characterised by hemodynamic instability, fever, and hyper-metabolism, mediated by the release of proinflammatory cytokines from innate immune cells. In the first few days, the early death is mainly due to a hyper-inflammatory cytokine storm [15]. In the second theory, it is postulated that the primary infection persists and that mortality is associated with a late dysregulated immune-inflammatory state. Alterations in the adaptive immune response could occur simultaneously with the persistent activation of inflammation and innate immunity (Figure 2, Theory 2) [169].

In the third theory, which is the most accepted one, the pro-inflammatory state predominates in the first days and then decreases. Failure of both innate and adaptive immune responses occurs in the second state, and death is due to the immune inability to cope with a second infection (Figure 2

Recovery

Early deaths due to overwhelming inflamma5on This first stage can be the cause of an early death (top red line, day 3), typically due to a global hyperinflammatory 'cytokine storm' response. The intensity of this response, induced by innate immunity cells, depends on the cause of the infection, patient comorbidities, and genetic factors. In most patients, recovery can take five days. In this theory, the late death of septic patients results from a persistent activation of the innate immune response, associated with adaptive responses causing intractable inflammation and organ injuries.

Theory 3: The first stage is similar to that of the second theory, thereafter, patients enter a profound immunosuppressive state characterised by inability of the immune system to cope a new infection. Most of patients will die from a nosocomial infection.

According to the references: [15,167].

Activation of the immune innate system

The innate immune system is mainly based on physical and chemical barriers such as the skin, vascular endothelium, mucosal surfaces, and ciliated respiratory epitheliums. Cells of innate immunity include antigen presentation dendritic cells (DCs), macrophages and granulocytes and also, natural killer (NK), and γδ T lymphocytes.

After the onset of an infection, the pathogen is recognised by the host innate immune system, mediated by the interaction between PAMPs and pathogen recognition receptors (PRRs). PRRs are proteins expressed in the cytoplasm and on the membrane of innate immune cells. Their role is to identify two classes of molecules: PAMPs and DAMPs. PAMPs are conserved motifs expressed by pathogens, not found on eukaryotic cells, including bacterial and viral nucleic acids, peptidoglycan, LPS, fungal β-glucan, α-mannan, and the bacterial protein flagellin. DAMPs are intracellular proteins or mediators that are released from dying cells, also known as 'alarmins', including high-mobility group box 1 protein (HMGB1), IL-1α, heat shock protein (HSP), and uric acid [171].

PRRs are located on macrophages and DC, but also on various non-immune cells such as epithelial cells [172]. Four classes of PRRs have been identified: TLRs and C-type lectin receptors, which are transmembrane proteins, and retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) and NOD-like receptors (NLRs), which are cytoplasmic proteins [173]. The recognition of PAMPs and DAMPs by PRRs induces the transcription of genes involved in the inflammatory response.

During sepsis, high levels of PAMPs and DAMPs lead to overstimulation of immune cells, resulting in a 'cytokine storm' state.

Toll-like receptors

TLRs are PRRs that have been most studied in the context of sepsis. TLRs are transmembrane proteins whose ectodomains contain leucine-rich domain repeats that mediate the recognition of PAMPs; the intracellular TIR (toll-interleukin 1 receptor) domain is required to downstream the signal transduction, leading to inflammatory mediator transcription [174]. Thirteen TLRs have been identified: 12 receptors in mice and 10 in humans.

To describe the signalling pathway, we will take the classic example of TLR4 (Figure 3). TLR4 responds to LPS, a component of the outer membrane of Gram negative bacteria. In the blood, LPS combines with LPS binding protein (LBP). The LPS/LBP complex is recognised by the cluster of differentiation CD14 [175]. TLR4 is associated with MD-2 protein, which binds to the extracellular domain of the receptor. All of these are essential to activate TLR4 downstream signalling [176]. The cytoplasmic portion of TLR4 activates adaptors such as myeloid differentiation factor 88 (MyD88) and TIR-associated protein (TIRAP) to the TIR-domain of the receptor, leading to IκBα/β ubiquitination and degradation by the proteasome complexes. The consequence is the release of NF-κB, which is translocated of into the nucleus. The latter initiates the transcription of pro-inflammatory cytokines such as IL1-β and TNF-α [START_REF] Akira | Pathogen recognition and innate immunity[END_REF]. [179][180][181]. Targeted therapies such as antibodies and anti-TNF-α, anti-IL-1, or anti IL-1 receptor antagonist (IL-1Ra) have been tested, and have provided interesting preclinical results in terms of reducing plasma cytokine levels in mouse models [182]. However, these treatments consistently failed to reduce overall sepsis mortality in the clinic [7,183,184].

Osuchowki et al., using a caecal ligation and puncture (CLP) mouse model of septic shock, observed an elevation at 24 hours of both pro-and anti-inflammatory cytokines. The proinflammatory cytokines peak was resolved within 48 hours, while the anti-inflammatory peak persisted for several days. Mortality was associated with the degree of both pro-and antiinflammatory cytokine levels [185].

The most investigated anti-inflammatory interleukins are IL-1Ra and IL-10. IL-1Ra, a specific inhibitor of IL-1, is secreted by immune and epithelial cells and blocks the action of IL-1α or IL-1β inflammatory signal [186]. IL-1Ra treatment in mice with polymicrobial peritonitis or Gram negative pneumonia reduces the acute hemodynamic instability and decreases mortality [183,187]. IL-10, a pleiotropic cytokine produced by both T cells and macrophages, is the main central anti-inflammatory cytokine [188]. High levels of IL-10 have been found 15 days after the onset of sepsis and may contribute to or amplify sepsis-induced immunosuppression [189]. In a two-hit model of sepsis, Muenzer et al. showed that blocking IL-10 can reverse sepsis immunosuppression. In this study, four days after CLP, mice were challenged with Pseudomonas aeruginosa bacteria and treated with an anti-IL-10 antibodies.

Mice treated with the anti-IL-10 antibody showed a recovery of interferon-γ (IFN-γ) production, an increase in pro-inflammatory cytokines, and a decrease in bacterial growth [190].

Nitric oxide

Please refer to chapter 'Cardiovascular physiopathology of septic shock', page XX.

Coagulation

Coagulation abnormalities may occur in 50 to 70% of patients with septic shock [191].

In septic patients, various mechanisms at different sites act simultaneously, resulting in a procoagulant state that can lead to disseminated intravascular coagulation (DIC). The. Latter is associated with organ dysfunction and/or hemorrhage [192]. Pro-inflammatory cytokines are the main mediators that disrupt the coagulation system [193]. The interaction between inflammatory cytokines and tissue factor trigger the pro-coagulant state. Tissue factor, the main starter of thrombin generation, is expressed on endothelial cells and mononuclear phagocytes such as monocytes and macrophages [194]. Its activation leads to the production of thrombin. The combination of activated platelets to thrombin induces blood clot formation [195]. In addition, downregulation of fibrinolysis by endothelial cells promotes intravascular fibrin deposition [196]. The pathogenic pathways involved in coagulation alteration during septic shock are described in Figure 4.

During sepsis, both damaged endothelial cells and activated circulating mononuclear cells produce proinflammatory cytokines that mediate the activation of coagulation system. Tissue factor expression by the endothelium and activated mononuclear cells initiates the activation of coagulation. In addition, impairment of anticoagulant mechanisms and inhibition of fibrinolysis by the endothelium promote intravascular fibrin

deposition. The latter results in a procoagulant state in septic shock patients. According to the reference: [196] Immunoparalysis

Innate immune cells

Polynuclear neutrophils

Neutrophils are the first line of defence in protecting the host against infection. Under usual conditions, neutrophils have a short half-life (7-12 hours in vivo) while during septic shock an extended lifespan is observed [197][198][199]. This increase is beneficial on the one hand, giving more time for neutrophils to resolve sepsis, while on the other hand it is detrimental, by increasing neutrophil-dependent inflammation and tissue damage [START_REF] Lakshmanadoss | Right Ventricle Failure in Sepsis: A Case Report[END_REF]. Delayed neutrophil apoptosis is also associated with decreased ROS secretion, potentially due to impaired TLR

The immune system develops a state of leukocytosis for the first 2-4 days after the onset of septicaemia, characterised by an increase in circulating neutrophils and monocytes and an increased in levels of both circulating pro-and anti-inflammatory cytokines. Thereafter lymphopenia takes hold with a marked decrease in the number of adaptive immune cells such as B cells, CD4 and CD8 T cells, as well as innate immune cells including NK cells and DCs. For most patients, one

month is required a full recovery. However, septic shock patients suffer from a long-lasting state of immunoparalysis despite a normal number of leukocytes. The state of immunoparalysis is characterised by a reduced ability in the control of new infections and byfunctional alterations in both innate and adaptive immune cells. According to the reference: [236]. Memory CD8 T cells show an impairment in both Ag-dependent and independent functions with a reduction in Ag-sensitivity, proliferative capacity, and in the ability to produce cytokines [248]. Defective naïve and memory CD8 T cells contribute to the increased susceptibility of septic shock survivors to new infections [247,248].

Time

Sepsis onset

Leukocytosis

Monocytes & neutrophils

Systemic cytokines

Immunoparalysis

Several studies also demonstrate, in CLP models, increased expression of inhibitory receptors such as programmed cell death 1 (PD-1) and 2B4, a reduced ability to produce effector cytokines (IFN-γ and TNF-α), and a reduced ability to clear the infection, indicating the exhaustion of CD8 T cells [249][250][251]. The latter plays a major role in lingering viral infection but is less pronounced than CD4 T cell exhaustion in sepsis-induced immunosuppression.

CD4 T lymphocytes CD4 T cell loss and recovery

The number of CD4 T cells is considerably reduced after the onset of sepsis [252][253][254].

Thus, T cell apoptosis plays a crucial role in reducing the number of circulating T cells [255,256]. Lymphocyte apoptosis during sepsis results from both the extrinsic (death receptor- They found an inability of stimulated splenocytes to produce IFN-γ, TNF-α, IL-6, and IL-10 and an increase in the expression of some inhibitory receptors. These results provide evidence of the functional unresponsiveness of T cells, which appears to be a state of cell exhaustion [253,266,267]. This point is strengthened by several studies showing a decrease in the mRNA transcription levels of the T-bet, GATA3, and RORγt transcription factors that regulate differentiation into the Th1, Th2, and Th17 CD4 T cells subsets, respectively [254,268,269].

The inability to proliferate and the over-expression of inhibitory receptors are surrogates of functional alterations. PD-1 is known to induce an inhibitory signal in T cells characterised by cell cycle arrest, the inability to proliferate, and reduced cytokine synthesis [270]. In septic shock, several studies have shown an increase in the expression of PD-1 in CD4 T cells, which is associated with increased mortality and the occurrence of secondary nosocomial infections [271,272]. Moreover, a role for the receptor TRAIL (targeting TNFrelated apoptosis-inducing ligand) in immune suppression during septic shock has been identified. Targeting the TRAIL/TRAIL receptor pathway could be a potential target for therapeutic intervention for restoring T cell functions [273,274]. Thus, septic shock is responsible of global functional defects regulating in CD4 T cell responses, which are summarised Table 5.

Changes in CD4 T cells subsets: increased regulatory T cell proportions

One of the characteristics of CD4 T cells is their ability to differentiate into subtypes.

The latter have specific functions best fitted to fight against specific threats [275]. As described in the previous sections, sepsis disrupts both numbers and function of CD4 T cells, including the Th1, Th2, Th17, and Treg subsets.

A number of studies have described an increased percentage of circulating Treg cells in septic shock patients [16][17][18][19]. This increase was observed immediately after the onset of sepsis but persisted only in septic shock patients who subsequently died [16,20]. The increased number of circulating Treg cells could be the result of preferential loss of other subsets (i.e., Th1, Th2, and Th17) [276,277]. This elevated proportion of the Treg subset might

Category

Effects References

Loss and recovery CD4 T cell number is dramatically reduced [252-254]

Apoptosis results from both the intrinsic and extrinsic pathways [257][258][259] One month is required to recover the preseptic number of T cells [252,253,260] There is an incomplete recovery for some epitope specificities

[254]

Functional defects

Global anergy:

• Reduced ability to produce cytokines [170,253,266,267] • Reduced ability to proliferate [254,261,269] • Increased expression of inhibitory receptors

[271-274]

Changes in subset representation

There is a decreased representation of Th1, Th2, and Th17 subsets [276,277] There is a decreased transcript levels of T-bet, GATA3, and RORγt [254,268,269] There is an increased Treg cell representation leading to decreased in T cell proliferation and innate cells [16-19, 278, 279] Experimental therapies to alleviate sepsis-induced immunoparalysis Therapies designed to alleviate sepsis-induced immunoparalysis have been focused on reducing immune cell death, on increasing the number of surviving immune cells, and on restoring their functions. At first sight, studies investigating anti-apoptotic molecules, i.e. Bcl-2 or caspase inhibitors, to block lymphocyte apoptosis showed a survival benefit in septic mouse models [283][284][285][286]. However, the therapeutic potential has not been widely adopted Dysregulation of the sympathetic nervous system following septic shock

Sympathetic nervous system

The sympathetic nervous system (SNS) is one of the three components of the autonomic nervous system (ANS), the other two being the parasympathetic and enteric systems. The main function of the SNS is to maintain both basal and stress-related homeostasis [295]. The sympathetic system is located in the locus coeruleus and comprises four anatomical levels (cervical, thoracic, lumbar, and pelvic), with two neurons connected in series at each level by a para-spinal ganglion fibre that innervates all parts of the body.

The SNS is activated by different inputs from sensory afferent nerves (via the hypothalamus), or by central nervous stimuli (cortical areas, limbic system). Conceptually, activation of the sympathetic nervous system releases NE by the second post-ganglionic sympathetic neuron in specific organs. Stimulation by acetylcholine released from splanchnic pre-ganglionic fibres on the adrenal gland turns on the production and release by the medulla of NE and epinephrine in the bloodstream [295]. NE and epinephrine bind to AR, a G-protein coupled receptor. There are two main classes of AR: α and β. The distribution, signalling pathways, effects of activation and inhibition of these AR on hemodynamics and inflammation are detailed in Table 6. When homeostasis is disrupted or threatened by internal or external challenges, the SNS is activated, resulting in an increased plasma level of catecholamines, which play a crucial role in maintaining the homeostasis. In a shock state, activation of the SNS increases HR, cardiac inotropism, and vascular contractility. These effects aim to preserve and/or restore organ perfusion. In addition, SNS activation has much broader effects, including bronchodilation, relaxation of smooth muscle and the intestine sphincter, gluconeogenesis, glycolysis and lipolysis, and anti-inflammatory regulation [298]. This schematic representation of SNS activation is closely associated with other response systems, such as the parasympathetic and hypothalamic-pituitary-adrenal systems.

β-AR and their signalling pathways Interaction between the sympathetic system and immune system

Many organs, such as the heart and the gastrointestinal tract, receive both sympathetic and parasympathetic innervation. However, in both primary (bone marrow and thymus) and secondary (spleen and lymph nodes) lymphoid organs, sympathetic innervation is predominant [307,308]. A recent study demonstrated, with immunofluorescent staining of neurofilaments, the innervation of splenic sub-compartments, including splenic nodules, marginal zones, and red pulp [309]. The findings supported the location of sympathetic nerves

Adrenergic regulation of innate immune cells

Recent findings provide evidence that the SNS regulates the innate immune response, which is summarised in CD4 T cells [22,38,327]. In addition, independently of IL-10 receptor activation and transforming growth factor-β (TFG-β) release, activation of β 2 -AR on DCs leads to an increase in Treg cells [22].

Adrenergic regulation of adaptive immune cells

Numerous studies demonstrated that almost all lymphocyte subtypes express β-AR and that adrenergic agents can suppress T cell activity and proliferation [23,38,[328][329][330].

Treatment with NE is able to suppress cytokine production by T cells, illustrating its immunosuppressive capability [23]. Furthermore, several studies have demonstrated in a cancer context that adrenergic signalling impaired the antitumor CD8 T cell response, which was reversed by a β-AR blocker administration [331][332][333].

To date, mostly the β 2 -AR subtype has been described on T cells: naive T cells, Th1, found that noradrenaline treatment enhanced the suppressive activity of Treg cells [330,335,341].

Until recently, β 1 -ARs were far less reported on [335,338,342]. A recent study investigating the impact of β-AR blockers on CD4 T cell dysfunction in cirrhotic mice reported that β 1 -AR were overexpressed by spleen CD4 T cells. In addition, chronic β-AR blocker treatment was accompanied by the redistribution of T cell subsets, leading to the normalisation of Th2 and Treg levels in the spleen [33].

Modulation of the sympathetic system by β 1 -AR blocker

Which molecules could be used to modulate the sympathetic nervous system during septic shock?

The peripheral therapeutic target regarding SNS overactivation seems naturally to be β-AR. β-AR blockers are therefore the molecules indicated in this context. studies showed that its inhibition and its genetic polymorphisms were associated with a poor prognosis in septic shock [39,343]. Conversely, in an experimental setting, several studies have demonstrated that β 2 -AR stimulation has beneficial effects in terms of reducing organ failure [344]. However, these results were never translated to the clinic [345].

Esmolol, a cardioselective β 1 -AR blocker with a half-life of two minutes and a half-life elimination of nine minutes, could be used. The main advantages of this molecule are its very short duration of action, its short half-life, and its intravenous administration, which make its use possible in the management of septic shock. Other β 1 -AR blockers could be used, such as landiolol, which has a higher β 1 -cardioselectivity and an even shorter elimination half-life (three to four minutes).

A central approach could also be added to the abovementioned peripheral approach to the modulation of the SNS. Central modulation of the SNS is also an approach to regulating adrenergic overstimulation. Similarly to the peripheral approach, this is associated with a recovery of the contractile, cardiac, and vascular response to catecholamines through the reexpression of AR on cardiac and vascular smooth muscle cells [346]. Thus, the stimulation of α 2 -ARs in the locus coeruleus drives a decrease in the peripheral sympathetic adrenergic response [295]. Dexmedetomidine or clonidine, used for light sedation and alcohol withdrawal, respectively, are the two best-known and most tested α 2 -AR agonist molecules.

Although there is yet no clinical evidence, numerous preclinical studies have demonstrated that, during experimental septic shock, central modulation of the sympathetic nervous system was associated with improved vasoreactivity, microcirculation, and a reduction in tissue and plasma inflammation [347][348][349]. The combination of the peripheral approach with esmolol and the central approach with dexmedetomidine has also been experimentally evaluated, demonstrating the feasibility of this combination. This strategy needs to be confirmed in patients in a randomized trial [350]. Indeed, it should be remembered that it is counterintuitive in septic shock to reduce the main factors of arterial oxygen transport, which are HR and inotropism. It is therefore appropriate, as a first step, to accumulate experimental and clinical data assessing the tolerance to and the clinical effects of such an approach.

Pharmacology of esmolol

Esmolol is an ultra-short acting cardioselective b 1 -blocker. The structure of esmolol

(methyl 3-[4-(2-hydroxy-3-[isoprolylamino] propoxy) phenyl] propinonate) is shown in Figure 9.
Esmolol is a well-tolerated therapy in patients with tachycardia and critical cardiovascular conditions that require a β 1 -AR blocker with fast-acting and above all fast-reversible properties. Esmolol is an ultra-short-acting β 1 -AR blocker with a half-life of distribution of 2 The initial search strategy identified 218 studies, of which 199 were fully reviewed.

One hundred and twenty were clinical studies and 79 were experimental studies. Editorials and case reports were excluded as were studies in which using β 1 -AR blocker treatment in sepsis was not the main topic. Twelve clinical studies and 24 experimental studies were included as described in the PRISMA flow chart (Figure 10).

Experimental support for β-AR blocker use in septic shock

Experimental studies in small and large animals assessed two aspects of β-AR blockade in septic shock: cardiovascular and inflammatory effects. The models used were mainly polymicrobial by CLP or endotoxaemia induced by the injection of LPS, leading to various sepsis-like phenotypes, ranging from mild to severe sepsis (Table 7). Most of the studies administered a β 1 -AR blocker, with as the main goal a 20% reduction in HR. Overall, the 20% HR reduction target was always achieved. Regardless of the animal model and the administration route of the β 1 -AR blocker, a cardioprotective effect was found.

Ackland et al. showed that administration of metoprolol or atenolol, both cardioselective β 1 -AR blockers, in a moderately severe septic shock rodent model resulted in a decrease of HR, an increased of systolic ejection volume and a maintained cardiac output. In contrast, intracerebral administration of a β 1 -AR blocker did not improve survival in rats with septic shock, suggesting a peripheral rather than a central β 1 -AR mechanism [34].

All studies performed in rodents showed either a maintenance or an increase in cardiac output despite the decrease in HR. One possible explanation could be an increase in diastole duration in these chronodependant animals secondary to the HR reduction. As a result, left ventricular filling was increased, allowing a similar cardiac output despite the reduction in HR.

External myocardial work was reduced, which led to better overall myocardial efficiency at a lower energy cost [36].

This result was surprisingly associated with an improvement in vascular function.

Theoretically, β 1 -ARs are not expressed on vessels, so β 1 -AR blockers should have no direct effect on vasoreactivity. However, an improvement in vasoreactivity was found during septic shock, in both animal models and humans after β 1 -AR blockade [32,374]. In the pig, a large animal model, two studies demonstrated that the administration of esmolol resulted in an increase in systemic vascular resistance in vivo [366,368]. Similarly, in a model of severe septic shock in rats, Wei et al. found that, ex vivo, the administration of esmolol induced a restoration of vascular contractility capacity to phenylephrine in dose response studies. In the same work, low concentrations of esmolol, without a direct hemodynamic effect, led to a similar improvement in vasoreactivity [26]. An improved prognosis in animals with septic shock receiving a β 1 -AR blocker was also found in all survival studies conducted in small animals [32,34,[357][358][359]361].

Another crucial point is effects on inflammation induced by the the use of β 1 -AR blockers in septic shock. In the early phase of septic shock, the 'cytokine storm' is consistently observed, characterised by a significant increase in the level of pro-inflammatory cytokines, and anti-inflammatory cytokines, which promotes organ failure and participates in the sepsisinduced immunosuppressed state [15].

The administration of β 1 -AR blockers in animal models of septic shock induces an overall decrease in inflammation, at both the systemic and tissue levels. Hagiwara et al.

showed that early administration of landiolol in a rodent LPS model significantly decreased cytokine production, including IL-6 and TNF-α, allowing a reversal of cardiac inflammation and contractile dysfunction [356]. Regardless of the model and the time of assessment, administration of a β 1 -AR blocker is most often associated with an overall pro-and antiinflammatory systemic and tissue weighting (Table 7). Mori et al. similarly showed that intraperitoneal TNF-α production was decreased by the administration of esmolol in CLP rodent model of septic shock. They also demonstrated a decrease in functional and structural alterations of the intestinal barrier in treated rats [357]. Other studies showed similar results on the kidney, liver, vessels, and lung [26,[361][362][363].

Clinical indication of the utility of β 1 -AR blockers in septic shock

The clinical data in Tables 8,9 are limited to the period from 1990 to September 2020 [375]. It was also found that the reduction in HR did not induce a decrease in cardiac output [376,377]. In one of the two publications, milrinone (a phosphodiesterase type 3 inhibitor, adrenoreceptor-independent positive inotropic) was co-administered to counteract the negative inotropic effects of the β 1 -AR blocker [376].

Only one major clinical study was published in 2013 by Morelli et al. [25]. This monocentric, prospective, randomised, open-label, esmolol vs. placebo study included a population of 154 patients in septic shock, who remained with a tachycardi above 95 beats per minute 24 hours after admission. The primary objective was to determine the efficiency of esmolol administration in reducing HR between 80-94/minutes and to assess the associated safety. The primary outcome was an effective reduction of HR in the esmolol group within the target range. Cardiac output was broadly maintained (although decreased in the first 24 hours) despite the reduction in HR. The explanation for this was an increase in systolic ejection volume secondary to the prolongation of the diastolic period in the esmolol group.

Levosimendan (an inotropic calcium sensitiser, independent of adrenoreceptors) was also administered in a similar manner in both groups (esmolol: 49.4%, placebo: 40.3%, p=0.39).

The required dose of NE was lower in the esmolol group than in the placebo group and vascular resistance was increased in the esmolol group compared to the placebo group.

Moreover, mortality was reduced from 80% in the control group to 31% in the esmolol group compared to 80% mortality in the control group. Considering the dramatic decrease in mortality, these results were the subject of an intense debate [385][386][387][388][389][390][391].

Four points should be highlighted concerning the effects of esmolol treatment:

-The effectiveness of HR reduction; -The excellent tolerance to esmolol, bearing in mind that 50% of the population included had received an inotropic agent (levosimendan); -The vasoactive effects, as the reduction in NE doses in the esmolol group is unexplained;

-The increase in survival rate, which should be moderated by the unexplained high mortality rate in the control group.

Based on the results of Morelli et al., further studies aimed to confirm the tolerance and physiological effects of this treatment. However, in each further study, the guidelines for septic shock management were systematically not followed. For instance, arterial pressure was most often beyond the recommended values [381][382][383][384].

Finally, given the methodological weaknesses of these clinical studies, it is premature to recommend the use of esmolol during septic shock, mainly because the targeted population, in whom this treatment could be beneficial, is not clearly defined. Indeed, a feasibility study, including patients with usual baseline characteristics, showed that only 10/186 patients were eligible to receive esmolol [392].

Objective β 1 -AR blockade is one therapeutic approach currently under investigation, aimed at downregulating the excessive adrenergic stimulation induced by septic shock. Early human studies have mainly shown the hemodynamic benefits of lowering HR through β 1 -AR blockade, on the improvement of cardiac efficiency, with one also finding survival benefit [25,376,377]. Some experimental studies have investigated the inflammatory effects of β 1 -AR blockade in septic shock and demonstrated a global decrease in both pro-and anti-inflammatory plasma cytokines [26,361,363]. However, little is known about the effects of β 1 -AR blockade on the early adaptive immune response, specifically on CD4 T cells expressing β-AR [393,394].

Accordingly, the present study aimed to address this question in a murine model of high-grade sepsis. Then heterozygote mice were intercrossed to generate β 1 +/+ and β 1 -/-mice.

Methodology

After obtaining β 1 -/-mice in the laboratory, heterozygotes were generated by backcross with C57BL/6J mice. Mice were genotyped and two strains, β 1 -/-and these littermates β 1 +/+ were used in this study.

Genotyping of mouse ear DNA via PCR

DNA extraction

Mice were tattooed for identification and a piece of ear was cut between 14-21 days of age. Sample was stored at -80°C. DNA was extracted using KAPA Mouse Genotyping Kit (KAPA BIOSYSTEMS, Sigma Aldrich, Saint-Louis, MO) which contains a thermostable protease, designed for rapid sample lysis. A mix was prepared in 0,2 ml tube containing the piece of ear. Tubes were placed in thermocycler MyiQ TM (BioRad, Hercules, CA) 10 minutes at 75°C followed by 5 minutes at 95°C. DNA samples were stored at -20°C.

Polymerase Chain Reaction (PCR)

Principle PCR uses the DNA polymerase that drives the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. When upstream and downstream primers are annealed to a longer template DNA, DNA polymerase adds nucleotides to their 3' end to extend primers. Under the action of DNA polymerase, each primer is elongated in the direction 5' to 3' of a sequence complementary to the template strand. For an exponential amplification of the target gene sequence, the denaturation, the annealing and the elongation cycles are repeated.

Procedure

Extracted DNA was amplified using KAPA Mouse Genotyping Kit (KAPA BIOSYSTEMS, Sigma Aldrich) containing DNA polymerase, dNTPs, MgCl 2 and stabilizers, 1μl of DNA and 1 μl of primers (forward and reverse primers) were added to the solution. The primers used are described in Table 10. DNA amplification was realised with a real-time PCR Detection System MyiQ TM (BioRad). A cycle of amplification was composed of 3-step runs after pre-heating DNA for 10 minutes at 94°C.

1) Denaturation: the DNA was heated at 94°C for 30 seconds to render it single-stranded;

2) Annealing: 62°C for 15 seconds for both primers to bind to the appropriate complementary strand;

3) Extension: 72°C for 30 seconds for DNA polymerase extending the primer by polymerase activity.

This cycle was repeated 30 times followed by a step at 72°C for 10 min.

An agarose gel was prepared (1,5% to separate 200-400 kDa DNA fragments) by dissolving the agarose powder in Tris-Borate-EDTA buffer (TBE 1%). Ethidium bromide (10 μl, Sigma Aldrich) was added to the agarose solution before it had gelled to visualise DNA under UV light. Amplified DNA samples were loaded in the wells. Agarose gel electrophoresis was done horizontally in a submarine mode whereby the slab gel was completely submerged in TBE buffer during electrophoresis (100 volts, 45 minutes). The gel was viewed in UV light with a Gel Doc TM XR+ (BioRad), the DNA stained with ethidium bromide appeared as glowing bands (Figure 11).

Experimental design

First, the dose of esmolol (a selective b 1 -AR blocker) responsible for a 20% reduction HR was determined in b 1 +/+ mice (n=25). Then, b 1 +/+ mice were randomly assigned to four groups: sham, sham + esmolol, CLP or CLP + esmolol. Esmolol (Esmocard®, Amomed, Meudon, France) was infused at 18 mg/kg/h (8 μl/h volume) through the subcutaneous mini-osmotic pump which was activated 3 hours post-insertion. b 1 -/-mice were randomly assigned to two groups: sham or CLP. Eighteen hours after surgery, echocardiography was performed, plasma 11. Intra-arterial blood pressure measurement

Principle

The invasive intra-arterial blood pressure measuring systems consist of a column of fluid directly connecting the arterial system to a pressure transducer (hydraulic coupling). The pressure waveform of the arterial pulse is transmitted via the column of fluid, to a pressure transducer where it is converted into an electrical signal. This electrical signal is then processed, amplified and converted into a visual display by a microprocessor.

Procedure

Anesthesia was maintained by 1.5% Isoflurane. Temperature was maintained by a plate heated at 37°C. After shaving the fur and disinfecting the skin, an incision was performed in the middle of the anterior cervical region, followed by dissection of the muscles and isolating the right carotid artery. The distal end of the carotid artery was ligated with a suture and the proximal end was clamped with an artery clamp. A hole was cut with a micro-scissor, then a catheter (PE-50, Braintree Scientific®, Braintree, MA) was inserted towards the proximal end. The catheter was connected to the pressure transducer (P23XL, BD Bioscience, San Jose, CA) and filled with heparinized saline (50 UI/ml). The catheter was fixed with a suture and the clamp was removed. Blood pressure was monitored with Ponemah P3Plus software for about 5-10 minutes until it became stable and mean arterial pressure (MAP) was reported.

Sample Collection

Blood was collected via the catheter into the carotid in EDTA tubes (BD vacutainer ® , BD Bioscience) and centrifuged 10 minutes at 1750 rpm for plasma extraction. Heart tissues were removed and rinsed in PSB for immunohistochemistry and spleen was removed and rinsed in PBS for immune cells analysis.

Quantification of cytokines

In 5-6 mice from each group, the plasma levels of 46 inflammatory mediators were measured in duplicate using the Magnetic Luminex® Performance Assay -Mouse XL Discovery Premixed kit (Bio-Techne, Minneapolis, MN) according to the manufacturer's instructions.

Results were expressed as pg/ml

Immunohistochemistry

Hearts were sectioned in a transverse slice and embedded in optimal cutting temperature compound (CellPath, Newtown, UK), frozen in liquid nitrogen and stored until use at -80°C. Hearts were cut by Leica CM1950 cryostat (LeicaBiosystem, Nussloch, Germany) into 7 μm thick sections at -20°C. The sections were rinsed with PBS and fixed in 4% paraformaldehyde (PFA, Sigma Aldrich). Heart section were stained in Autostainer Link 48

Dako (Agilent, Santa Clara, CA) with rabbit polyclonal primary antibody to CD45 (1/150, ab10558, Abcam, Cambridge, UK). Staining was visualised using the appropriate secondary antibody (goat anti-rabbit HRP polymer, ab214880, Abcam) and 3,3ʹ-diaminobenzidine with peroxidase substrate (Dako Real DAB Chromogen, Agilent). The sections were counterstained with hematoxylin. For each heart section the CD45 positives cells were count using a 40x objective lens in 10 fields of the microscope, in blind, by three different experimenters.

Splenocytes isolation

Spleens were harvested and immediately placed in cold PBS (Sigma Aldrich) and gently grounded between frosted slides to produce a single-cell suspension. Suspensions were filtered through 70-μm nylon filters (Biologix, Shandong, China) and centrifuged at 300g for 10 minutes at 4°C. Red blood cells were lysed using the BD PharmLyse TM (BD Bioscience) solution. Then, splenocytes were washed twice in PBS before being resuspended in RPMI 1640 medium (Gibco TM , ThermoFisher Scientific, San Diego, CA) supplemented with 10% heatinactivated fetal bovine serum (FBS, Sigma Aldrich). The preparation was left overnight at 4°C. conjunction with a protein transport inhibitor containing Brefeldin A (BD GolgiPlug, BD Bioscience) resulting in the accumulation of cytokines in the golgi complex.

Cell surface staining

Cells were washed with PBS and Fc receptors were blocked with antimouse CD16/CD32-purified antibodies (eBioscience TM , Thermo Fisher Scientific) for 15 min. The plasma concentrations of 31 pro-inflammatory and 15 anti-inflammatory mediators were measured. Briefly, all inflammatory mediators were increased in b 1 +/+ CLP mice compared to sham controls (Table 13). Compared to b 1 +/+ CLP mice, esmolol-treated CLP mice showed an overall decrease in plasma concentrations of 58% (18/31) and 60% (9/15) for pro-and anti-inflammatory mediators, respectively (Figure 28). For instance, compared to b 1 +/+ CLP mice, administration of a b 1 -AR blocker reduced plasma TNF-a by 66% vs. CLP (p=0.02), and IL-10 by 82% vs. CLP (p=0.01). In b 1 -/-mice, sepsis induced an overall increase in both pro-and anti-inflammatory mediators compared to sham animals (Table 13).

Sex does not affect the b 1 -AR blockade effect

Sensitivity analyses were performed on results obtained for major hemodynamic parameters (Figure 30) and immune effects (Figure 31) according to sex. No difference was found between male and female mice.

Discussion

In the present study, we confirm that experimental high-grade sepsis induces three hallmark features of sepsis-induced immunosuppression: a significant decrease in splenic CD4

T cells, an increase in splenic Treg cells, and a decrease in CD4 T cell proliferation capacity [15].

A notable finding was the marked increase in Treg cells in the spleen of septic mice, which could be counteracted by pharmacologically or genetically blocking β 1 -AR signalling. This observed decrease in Treg cells in treated animals was associated with a simultaneous increase in CD4 T cells and a global decrease in circulating pro-and anti-inflammatory mediators, indicating the involvement of β 1 -AR in the septic-immune phenotype. These results also translated into an in vivo global improvement in cardiac efficiency.

Model characteristics

To investigate the immune and cardiovascular roles of β 1 -AR in sepsis, we deliberately chose a non-resuscitated murine model of CLP to avoid any interference between exogenous catecholamine administration and the immune system, therefore the term septic shock cannot be used [397]. This model depicts all hemodynamic and survival characteristics of a typical high-grade sepsis, with an increase in both plasma pro-and anti-inflammatory mediators and a decrease in CD4 T cell proportion [37,396,398,399]. Analysis of CD4+ subtypes revealed that early after onset of sepsis, the percentages of Th1, Th2 and Th17 cells were unchanged. Many studies usually report a switch from Th1 to Th2 phenotypes, and a decrease in Th17 cell percentages [262,264,400,401]. However, other studies found that sepsis could also disrupt in a time-dependent manner both the representation and the function of CD4 T cell subsets, including Th1, Th2, Th17 [170,225,254,268]. Unlike other lymphocyte populations, Treg cells are increased during trauma in both animals and humans, as well as during sepsis and septic shock (as seen in our study) [170,225]. This increase in Treg cells contributes to lymphocyte paralysis and, more broadly, enhances the post-septic immunosuppressive process [236].

β-AR stimulation contributes to CD4 T cell dysfunction

During sepsis and septic shock, overstimulation of the sympathetic system is associated to the massive release of plasma β and ⍺ adrenergic agonists such as norepinephrine and epinephrine [342]. Norepinephrine is known to enhanced IL-10 concentration, to decrease the release of pro-inflammatory cytokines and to contribute to sepsis-induced immunoparalysis [24]. If in both innate and adaptive immune cells, adrenergic receptors are widely expressed, although they are not well characterised, particularly for the β subtype [295]. Numerous studies demonstrated the presence of β-AR on almost all lymphocyte subtypes [38,330,334]. However, until recently, only β 2 -AR but not β 1 -AR subtypes were found on T cells [335,342]. As a consequence, β 1 -AR stimulation was not considered as a major actor of inflammation. Indeed, in an early study, a short β 1 -AR stimulation in healthy volunteers challenged with endotoxins did not influence the inflammatory pattern [402]. It is only very recently that β 1 -AR were found to be widely expressed on splenic CD4 T cells. In a study investigating the impact of β-AR blockers on CD4

T cell dysfunction in cirrhotic mice, dobutamine, a β 1 -AR agonist, was able to re-activate inhibited Treg cells, suggesting the presence of β 1 -AR on their surface [33]. In accordance, we too found that in vitro CD4 T cell proliferation tended to be reduced by Treg cells pre-treated with dobutamine.

What is the best balance between stimulation and inhibition of β-ARs?

Cardioselective β 1 -AR blockers seem to be the natural target to treat SNS overactivation. However, this strategy may appear counterintuitive. Indeed, the recommended treatment for fluid-unresponsive sepsis-related hypotension remains the prescription of adrenergic agents. To date, norepinephrine, an α1-AR and β 1 -AR agonist, is the main inopressor used for the treatment of both cardiac and vascular failures. By stimulating the α1-AR widely expressed on VSMCs, norepinephrine agent is consistently confirmed as a very potent and reliable vasopressor. The latter also increases the cardiac work by increasing MAP without any concomitant increase in heart rate [403].

The adrenergic system is also a powerful modulator of cell immunity. Norepinephrine displays numerous adverse effects on immune, metabolic and coagulation functions [21,24,404]. Overall, in-vitro effects of catecholamines can be summed-up as a roughly inhibition of Another option, which we have investigated in the present study, is the use of βblockers. In this work, we found that some of the adverse immunologic effects of norepinephrine could be attenuated by blocking, at least partially, β1-AR. This strategy, in experimental models, prevents organ dysfunctions due to the sympathetic overstimulation.

β-AR blockade reversed sepsis-induced dysautonomia

In numerous conditions, such as acute heart failure, burn injuries, chronic hepatic failure, the peri-operative period and septic shock, β 1 -AR blockade has been consistently associated with protective effects on inflammation and hemodynamics [26,414,415]. As others, we too found a global decrease in pro-and anti-inflammatory mediators in animals with high-grade sepsis treated by β 1 -AR blockade [26,34,36]. Underlying mechanisms are still not elucidated. In Listeria monocytogenes infected mice acute cold or restraint stress (surrogates of sympathetic adrenergic stimulation) inhibited the host T cell response and increased cytokine production [416]. In this model, pharmacological blockade of the β 1 -AR but not β 2 -AR could downregulate this pro-inflammatory state and shorten time to bacterial clearance [35].

In β 1 -AR blocked animals with high-grade sepsis, we found the decrease in Treg cells in the spleen was associated with a concomitant increase in CD4 T cells, as reported previously [417]. In addition, we found that proliferative capacity of splenic CD4 T cells in septic animals was maintained when the β 1 -AR was blocked either pharmacologically or genetically. This suggests that β 1 -AR activation contributes to sepsis-induced immunosuppression, which could be reversed by β 1 -blockade. By comparison, volunteers challenged with lipopolysaccharide, and then being infused with norepinephrine for 5 hours showed a shift to an antiinflammatory state (decreased TNF-⍺/IL-10 ratio) which could be counterbalanced by β-AR blockade (increased TNF-⍺/IL-10 ratio) [24]. β 1 -AR blockade in our septic animals reduced the inflammatory burden and, taken as a whole, helped to shift from an immunosuppressive state to a state of immune homeostasis.

Beside this newly observed immune rebalancing in experimental sepsis, it is of utmost importance to reiterate that β 1 -AR blockade also has major cardiovascular effects, mostly driven by its negative chronotropic effects. In agreement with most previously published experimental or clinical studies, the septic mice with β-AR blockade did not show an alteration in cardiac power, despite a decrease in HR, suggesting a better cardiac efficiency [25,26,32,36]. In fact, in the present study, it was found a similar cardiac power, a surrogate of external cardiac work, in non-treated and treated groups [418]. Considering that HR, a major determinant of oxygen consumption, was reduced in treated animals, our result suggests a better cardiac efficiency in esmolol treated animals.

While the association between an increased Treg cell percentage during sepsis and an immunosuppressive state is well proven, effects on survival remain inconclusive [419,420].

The benefits of β 1 -AR blockade on immune and cardiovascular function should improve the survival rate. However, in the present study, and without any treatment (antibiotics, fluid or vasopressors), no improvement was seen in survival. In a similar CLP surgery model, Ackland et al. also found that metoprolol infusion, commenced 6 hours after the onset of fecal peritonitis, did not confer any benefit in improving survival whereas pretreatment was effective [34]. A dose effect may be relevant as the β 1 -AR blocker was infused only during the first 24 hours after CLP surgery. In a single-center study prolonged administration of esmolol in septic shock patients, titrated to maintain heart rate between 80-94 bpm, was associated with increased survival [25].

Study limitations

In our model, CLP animals were not resuscitated, preventing us from mentioning a model of septic shock, and no antibiotic was administered in contrast to the usual management of sepsis. Models of resuscitated septic mice have already been described using miniaturised equipment to reproduce the ICU environment, i.e. catheterisation of the femoral artery for for arterial pressure measurements, catheterisation of the external jugular vein for fluid resuscitation, and drug administration and tracheotomy for mechanical ventilation [421].

In our model, we aimed to limit the consequence of highly aggressive ICU procedures and thus to limit cofounding factors. In addition, administration of adrenergic agonists, recommended for sepsis management, would have interfered with our goals. Nevertheless, our results will require further confirmation in resuscitated models.

Our animal model also presents conceptual limits. Indeed, in mice for which the HR is greater than 400 bpm at rest, sepsis induction is most often associated with a marked decrease in metabolism with, as a surrogate, a decrease in HR [37]. In this setting, HR in wild type septic mice was lower compared to previous studies [422]. Thus, the use of β 1 -AR blockade could appear counterintuitive. Moreover, the reduced HR in this model could have been the consequence of the mouse strain. Wild type littermates β 1 +/+ were used as the closest comparators to β 1 -/-mice. The complex genetic background comes from three different strains, which might explain the unexpected reduction in HR observed in the untreated groups. Moreover, sepsis-induced hypothermia is also known to reduce HR in murine models of sepsis [37].

In this model, the percentages of spleen Th1, Th2, and Th17 lymphocytes were unmodified while several studies found a shift from Th1 to Th2 in sepsis. One explanation could be the timing of the assessment. Indeed, in mice, the immune cell pattern depends on several factors, for instance, the mouse strain chosen, the method for inducing sepsis, the delay between sepsis induction, and the measurement or the method used to collect immune cells could have impacted the immune cell pattern [423][424][425]. It seems that the shift from Th1 to Th2 in mice occurs later compared to our time of assessment which was at 18 hours postsurgery. Most studies investigating the Th1/Th2 balance have used a 48-hour sepsis model [426]. Moreover, to date, no study has assessed the immunologic pattern of the β 1 -/-mouse strain.

Future researches, should firstly assess the tolerance pattern of this treatment in septic patients, secondly should define the best endpoints (HR reduction, inflammation modulation), thirdly, should precise which population is the most likely to benefit from this approach.

Finally, experimental studies demonstrated the inflammatory weighting and the reduced sepsis-induced immunosuppression following β 1 -AR blockers administration in sepsis models.

This effect has never been investigated in human septic shock and might not require high doses of β-AR blockers which could be extremely interesting in hemodynamically unstable patients [4,25]. Studies aiming to define more precisely the safety pattern in septic shock patients are ongoing (NCT02120404, NCT02369900).

Perspectives

Impact of β 2 -AR modulation on inflammatory pattern in experimental sepsis β 1 -AR but also β 2 -AR are widely expressed on both innate and adaptive immune cells.

Specifically, it has been reported that naïve CD4 T cells, Th1, Th17, and Treg cells, but not Th2, express β 2 -AR [30,334]. β 2 -AR stimulation promotes a shift from a Th1 to Th2 inflammatory pattern and could activate Treg suppressive functions [330,338]. Reports have also found that β 2 -AR stimulation enhances the macrophage M2 anti-inflammatory phenotype and favours IL-10 production by CD4 T cells [38]. The beneficial effects of β 2 -AR modulation remain unclear and very little is known about β 2 -AR signalling in CD4 T cells during sepsis. In a murine model of endotoxemia, β 2 -AR blockade reduced survival and, in septic pigs, amplified sepsis-induced hepatic injuries [38,427,428]. In humans, it has been reported that, during sepsis, β 2 -AR polymorphism is associated with increased mortality [39]. In the present study, we only investigated the impact of β 1 -AR blockade on the alteration of CD4 T cell patterns in the spleen. Therefore, it will be crucial to investigate, first in a murine model of sepsis, the impact of β 2 -AR blockade on hemodynamic and CD4 T cells, and secondly the impact of non-selective β-AR blockade in this setting.

Methodology

To achieve this goal, similarly to the model used in the present study, the hemodynamic and inflammatory effects of β 2 -AR blockade will be studied using a CLP polymicrobial model of sepsis. Two strains of mice will be used: β 2 -AR (C57BL/6J, CD-1, FVB/N) knockout mice (β 2 -/-) and wild type littermates (b 2 +/+ ), to which a β 2 -AR blocker (CI 118,551) or non-selective β 1 /β 2 -AR blocker (propranolol) will be administered 3 hours after the surgery via a subcutaneous mini-osmotic pump.

Hemodynamic assessment:

-Echocardiography and mean arterial pressure measurements will be performed.

-Cardiac pressure-volume loop will be analyzed using a conductance catheter.

Immune function assessment:

-The immunophenotyping of lymphocyte subpopulations will be investigated by flow cytometry.

β-AR receptors on lymphocytes will be quantified by quantitative PCR and western blot.

-Cytokines will be measured in b-AR stimulated lymphocyte culture medium,

-Lymphocyte function will be assessed.

Impact of β-AR modulation on vascular leakage in experimental sepsis

During sepsis, dysautonomia is also responsible for the desensitisation of Several studies have demonstrated that β 1 -AR blockers can also have effects on β 3 -AR, depending on the molecule used. Thus, nebivolol, used in heart failure, is a β 3 -AR agonist promoting vasorelaxation, a beneficial effect in heart failure [40]. Conversely, one preliminary study found that esmolol might be a β 3 -AR antagonist in brown fat in rats [41].

To date, β 1 -AR blockers have not been found act directly on vascular function since β 1 -AR are poorly expressed in vessels. Nevertheless, in the clinical study conducted by Morelli et al., septic shock patients in the esmolol group received less norepinephrine than patients in the placebo group [25]. Moreover, administration of esmolol in CLP rats treated with norepinephrine did not change MAP and decreased the hyporesponsiveness to vasopressor treatment and the sepsis-induced endothelial dysfunction [32].

Given the short time between the administration of the treatment and the observation of the effect, these results probably cannot be explained only by an inflammatory pathway.

Other mechanisms, acting earlier, are probably involved in the improvement of vascular function during esmolol-treated sepsis. Therefore, it will be interesting to assess if β1-AR blockers, also have effects on β 3 -AR expressed on endothelial cells. The β 3 -AR antagonistic effect of esmolol could explain the improvement in vascular responsiveness to catecholamines during experimental sepsis.

Methodology

In vitro study of the antagonistic effects of esmolol on β 3 -AR

Endothelial cell culture

Two cell types will be studied: primary aortic endothelial cells from β 1 +/+ and β 1 -/-mice and human aortic endothelial cells (HAEC).

-1/ The β 1 -AR, β 2 -AR and β 3 -AR receptors will be quantified by quantitative PCR and western blot in control and LPS conditions.

-Cells will be fixed and labelled by immunochemistry in order to visualize the β-AR and cells differentiation markers of endothelial cells types by confocal microscopy (Dil-ac-LDL, vWB, CD31, eNOS, iNOS).

-2/ These cells will be cultured in the presence or not of β 3 -AR agonist (CL316243) and β 3 -AR antagonists (SR59230A) in control and LPS conditions.

-3/ These cells will be cultured in the presence or not of β 1 -AR antagonists (esmolol)+ β 2 -AR antagonist (ICI118551) in control and LPS conditions.

-4/ Effect of β 3 -AR agonist (CL316243) and β 3 -AR antagonists (SR59230A) in control and LPS will be tested on cells culture 3/ to rule out the involvement of these two receptors.

For 2/ 3/ and 4/ NO, eNOS, cGMP and cAMP will be measured.

Endothelial permeability assay

Endothelial cells will be challenged with LPS and the effect of β-AR blockers on the endotoxin-mediated barrier disruption will be evaluated in the presence or not of β 1 -AR (esmolol) and β 3 -AR (CL316243) antagonists.

-The barrier function will be evaluated by measuring the leakage of the Evans blue dye through an endothelial monolayer.

-The quantity of NO will be measured.

-Adherents junctions markers (vascular endothelial (VE)-cadherin) and tight junctions markers (occludins and claudins) will be measured.

In vivo study of antagonistic effects of esmolol on β 3 -AR

High-grade sepsis will be induced in mice by CLP. Animals will be randomly divided into five groups and assessed 18 hours after the onset of sepsis: sham, CLP, CLP + esmolol, CLP + CL316243, and CLP + esmolol + CL316243.

Parameter evaluated:

-The arterial pressure will be measured by carotid catheterization.

-Left ventricular function will be analyzed by echocardiography.

-Vasoreactivity to vasoconstrictors and vasodilators of aortic and mesenteric rings will be analysis by wire myography.

-The lung wet-to-dry ratio will be assessed to measure the total amount of lung water.

-The blood vessel permeability will be measured the leak of Evans blue dye in tissues.

-The cytokines profile will be analyzed in plasma.

In summary, the precise contribution of each subtype of β-AR on the dysfunction of different organs and systems occurring in the context of sepsis remains to be studied. A better understanding of β-AR mechanisms and their interactions on the cardiac, vascular, and immune processes could actually be one key feature for new therapeutic strategies for sepsis.

394. [START_REF] Sanders | The role of adrenoceptor-mediated signals in the modulation of lymphocyte function[END_REF] Durant le choc septique, cette dysautonomie survient de façon précoce, et son intensité, reflet de la suractivation sympathique adrénergique, est associée à la gravité clinique et à la mortalité [4,5]. Pour le clinicien, ce paradigme est contreintuitif, car il suggère que l'excès de catécholamines est délétère alors que la pierre angulaire du traitement du choc septique tel qu'il est recommandé, outre l'expansion volémique et le contrôle infectieux, repose sur l'administration exogène de catécholamines (noradrénaline, adrénaline, dobutamine).

Ainsi, l'hypothèse de l'administration d'un traitement visant à diminuer le stress adrénergique périphérique pourrait se justifier par la diminution précoce des effets délétères de la suractivation sympathique et éventuellement améliorer le pronostic des patients en choc septique. Ce concept de rééquilibration adrénergique ou de « décatécholaminisation », en particulier évalué avec des β-bloquants cardiosélectifs, repose sur des bases scientifiques de plus en plus robustes [6,7].

Dans ce travail, nous ferons la synthèse des travaux scientifiques, expérimentaux et cliniques ayant évalué l'efficacité d'un traitement par β-bloquants pour améliorer la prise en charge du choc septique. Une recherche suivant les recommandations PRISMA a été réalisée via Pubmed en utilisant, à partir du constructeur de requêtes bibliographiques médicales, les termes Mesh suivants « choc septique » ou « sepsie », traduction française de « sepsis » et « propanolamines » sur la période de 1990 à 2018 incluse (Fig. 1). Toutes les langues ont été prises en compte. La sélection des travaux s'est concentrée sur les termes suivants lors de la lecture de l'abstract : hémodynamique, survie et inflammation.

Présentation du système nerveux sympathique et concept dysautonomique

Système nerveux sympathique

Le système sympathique, situé au niveau du locus caeruleus dans le tronc cérébral, comprend anatomiquement quatre niveaux (cervical, thoracique, lombaire et pelvien) avec deux neurones en série à chaque niveau, connectés par un relais ganglionnaire paravertébral. De façon schématique, l'activation du système nerveux sympathique va, par l'intermédiaire du deuxième neurone postganglionnaire sympathique, libérer de la noradrénaline sur les organes cibles. La libération de l'adrénaline dans la circulation sanguine via la médullosurrénale est secondaire à la stimulation de la glande par l'acétylcholine libérée des fibres préganglionnaires splanchniques [8].

La réponse à ces deux hormones est médiée par des protéines G couplées à des adrénorécepteurs. Ces derniers comprennent deux classes : α et β. La répartition, les voies de signalisation et les effets de l'activation et de l'inhibition de ces adrénorécepteurs sur l'hémodynamique et l'inflammation sont détaillés dans le tableau 1.

Les effets cliniques hémodynamiques de cette activation les plus connus se résument à une tachycardie, une augmen-tation de l'inotropisme cardiaque ainsi qu'une vasoconstriction permettant de maintenir la stabilité cardiovasculaire. Les effets sont en réalité beaucoup plus larges et incluent aussi une bronchodilatation, une relaxation des muscles lisses intestinaux et des sphincters, une augmentation de la néoglucogenèse, de la glycolyse, de la lipolyse et une régulation Tableau 1 Classification, localisation, signalisation, agonistes, antagonistes, effets hémodynamiques et inflammatoires des principaux adrénorécepteurs (d'après Scanzano et Cosentino,de Montmollin et al. et Tank et Lee Wong [9,10,11]) anti-inflammatoire [11]. La représentation schématisée de ce système de réponse suraiguë à l'agression ne doit pas faire perdre de vue qu'il est relié et dépendant d'autres systèmes de réponse à l'agression, comme par exemple le système parasympathique (beaucoup moins bien connu) ou encore le système hypothalamo-hypophyso-corticosurrénalien.

Adréno
Dysautonomie sympathique (Fig. 2)

La dysautonomie est un terme générique qui englobe l'ensemble des effets considérés comme délétères liés à la persistance dans le temps d'un haut niveau d'activation du système nerveux sympathique [6]. Les marqueurs biolo-giques de cette suractivation sont les concentrations d'adrénaline et de noradrénaline de 5 à 100 fois plus élevées par rapport à un système non suractivé [4]. Ces hautes concentrations sont associées à des altérations des fonctions cardiaque et vasculaire, de la coagulation et de l'inflammation.

Participation dysautonomique à la cardiomyopathie septique

La noradrénaline mais surtout l'adrénaline vont, par la stimulation β 1 -adrénergique, augmenter la consommation en adénosine triphosphate (ATP) de la cellule cardiomyocytaire afin d'augmenter l'inotropisme et le chronotropisme cardiaque. Sur le plan fonctionnel, l'excès de stimulation dans le temps va altérer la réponse β 1 -adrénergique d'une part, par une internalisation des β 1 -adrénorécepteurs et, d'autre part, par une altération de la fonction énergétique mitochondriale diminuant ainsi globalement l'effet des catécholamines [12][13][14]. Sur le plan morphologique, l'excès de stimulation adrénergique est associé à des lésions histologiques (apoptose cardiomyocytaire, oedème interstitiel), à des nécroses de bandes de contraction et à une infiltration macrophagique [15,16]. Néanmoins, cette atteinte est bien plus fonctionnelle que morphologique, étant donné la constante réversibilité de la cardiomyopathie septique en rapport avec l'élimination des facteurs dépresseurs myocardiques comme l'interleukine-1 (IL-1), le tumor necrosis factor-α (TNF-α) et le monoxyde d'azote (NO) [17].

Participation dysautonomique à la vasoplégie

L'altération de la vasoréactivé durant l'état de choc septique comprend deux versants :

• une diminution de la réponse contractile vasculaire à l'augmentation de la stimulation catécholaminergique vasopressive ;

• une altération de la relaxation vasculaire à l'augmentation des doses d'acétylcholine.

Les mécanismes sont aujourd'hui assez bien caractérisés avec la place centrale du monoxyde d'azote [18]. Similairement au myocarde, l'hypovasoréactivité contractile est en partie médiée par des mécanismes de désensibilisation des α-adrénorécepteurs situés sur les cellules musculaires lisses vasculaires [19].

Participation dysautonomique à l'immunoparalysie (Fig. 3) La dysautonomie est aussi responsable d'une altération de la réponse immunitaire. En effet, les cellules de l'immunité présentent à leur surface des adrénorécepteurs principalement de type β 2 [8]. Schématiquement, la stimulation sympathique Fig. 2 Activation du système nerveux sympathique au cours du choc septique. Lors d'une infection, les lipopolysaccharides (LPS), un composant majeur de la membrane bactérienne, activent les toll-like receptors 4 (TLR4) présents sur les cellules de l'immunité innée. L'activation des récepteurs TLR4 induit la translocation de NFκB dans le noyau, permettant la transcription de cytokines pro-inflammatoires telles que IL-1 ou TNF-α. Ces cytokines activent le système nerveux sympathique au niveau central, dans le locus coeruleus et au niveau périphérique via les ganglions sympathiques, induisant une libération massive de catécholamines. Ces catécholamines activent les récepteurs adrénergiques conduisant, dans des conditions physiologiques, à une augmentation de l'inotropisme, de la vasoréactivité et une diminution de l'inflammation. Lorsque cette activation persiste dans le temps, elle devient pathologique et implique une diminution de l'inotropisme, de la vasoréactivité et une immunosuppression. Ce phénomène, se produisant lors d'un choc septique, est appelé dysautonomie. adrénergique qui initialement durant le processus septique a un rôle bénéfique de régulation anti-inflammatoire entraîne, lorsqu'elle se poursuit dans le temps à un haut niveau d'activité, une immunoparalysie pouvant favoriser la survenue d'une deuxième infection [20]. Les β 2 -adrénorécepteurs situés sur les cellules présentatrices d'antigènes (cellules dendritiques, monocytes…) et les lymphocytes T helper 1 (Th1) vont, lorsqu'ils sont activés, entraîner un déplacement de la réponse inflammatoire Th1 vers une réponse préférentielle Th2. Les monocytes et les lymphocytes Th2 ainsi stimulés vont libérer des cytokines telles que l'IL-4, l'IL-6, l'IL-10 et l'IL-13 qui inhibent la réponse Th1 ; prises ensemble, la réponse immunitaire semble s'orienter vers une réponse humorale de type anti-inflammatoire. Sa persistance dans le temps est une des explications de l'immunoparalysie secondaire du choc septique [21].

Participation dysautonomique à la mortalité L'impact de la dysautonomie sympathique sur la mortalité des patients en choc septique a pu être évalué à partir de l'étude de la variabilité cardiovasculaire. De façon simplifiée, la variabilité cardiovasculaire explore dans les domaines temporels et fréquentiels (ou spectraux) la fréquence cardiaque (FC), les pressions artérielles systoliques, diastoliques et moyennes. Ces variables sont intimement liées au système nerveux sympathique. Durant le choc septique, la perte de la variabilité cardiovasculaire et un haut niveau de catécholamines endogènes ou exogènes sont constamment associés à un pronostic défavorable, tandis que le maintien de la variabilité est associé à un sevrage plus précoce des catécholamines [4,5,22,23].

Ainsi, le système nerveux sympathique est un système central de la réponse précoce à l'agression septique. Néanmoins, s'il est indispensable à la phase initiale, de très nombreux experts considèrent qu'au regard de la littérature sa persistance dans le temps est délétère et, donc, que sa modulation pourrait être bénéfique [3,24]. Regroupée sous différents termes comme la « décatécholaminisation », la modulation du système nerveux sympathique, qu'elle soit centrale ou périphérique, est une voie thérapeutique envisageable, mais qui, comme nous allons le voir par la suite, reste encore au stade de la preuve de concept [7].

Quelles molécules seraient utilisables pour moduler le système nerveux sympathique dans le choc septique ?

La cible thérapeutique sur le système nerveux sympathique semble naturellement être les récepteurs β-adrénergiques. Les β-bloquants sont donc, a priori, les molécules indiquées dans ce cadre. Néanmoins, les β-bloquants non cardiosélectifs, c'est-à-dire avec une activité à la fois β 1 et β 2 bloquante, sont probablement moins adaptés que les β 1 -bloquants cardiosélectifs. En effet, l'inhibition β 2 est contre-intuitive étant donné que l'activation β 2 -adrénergique semble centrale dans la balance inflammatoire [8]. Les travaux expérimentaux autant que cliniques montrent que son inhibition ou l'existence d'un certain polymorphisme du gène codant pour cet adrénorécepteur semble associée à une moins bonne évolution et à un moins bon pronostic lors du choc septique [25,26]. À l'inverse, sur le plan expérimental, sa stimulation semble présenter des effets plutôt bénéfiques en terme de réduction des défaillances d'organes [27].

L'esmolol (methyl 3-[4-(2-hydroxy-3-[isoprolylamino] propoxy) phenyl] propionate) est un β 1 -bloquant cardiosélectif dont la demi-vie de distribution est de deux minutes et la demi-vie d'élimination de neuf minutes. Cette molécule est hydrolysée dans le sang par des estérases et métabolisée dans le foie en formant ses principaux métabolites, que sont le méthanol et l'acide propionique. Si la demi-vie d'élimination de ces métabolites est beaucoup plus longue, seul l'esmolol a des effets β 1 -bloquants. Ainsi, aucun effet mesurable n'est retrouvé à 30 minutes de l'arrêt du traitement par esmolol [28]. Les principaux avantages de ce type de molécules sont : son très court délai d'action, sa courte demi-vie et son Fig. 3 Effet des catécholamines sur la balance lymphocytaire Th1/ Th2. Les catécholamines activent les récepteurs adrénergiques présents sur les cellules présentatrices d'antigènes. La stimulation des récepteurs β2-adrénergiques entraîne la libération de cytokines anti-inflammatoires induisant la différenciation en lymphocytes Th2 et réprimant la différenciation de lymphocytes Th1. Inversement, il semblerait que la stimulation des récepteurs β1-adrénergiques entraîne la libération de cytokines pro-inflammatoires induisant la différenciation de lymphocytes Th1 et réprimant la différenciation de lymphocytes Th2, cela restant à mettre en évidence plus précisément [21].

administration par voie intraveineuse qui rendent son utilisation éventuellement possible lors de la prise en charge d'un état de choc septique. D'autres molécules de ce type existent comme le landiolol, possédant une forte cardiosélectivité et dont la demi-vie d'élimination est encore plus courte (trois à quatre minutes).

Cette approche périphérique de la modulation du système nerveux sympathique pourrait être complétée aussi par une approche centrale. Théoriquement, il se pourrait que la modulation centrale du système nerveux sympathique permette de pondérer l'hyperactivation adrénergique. Cette pondération serait associée à une restauration de la réponse contractile, cardiaque et vasculaire aux catécholamines via une réexpression des adrénorécepteurs sur les cellules musculaires cardiaques et vasculaires [29]. Ainsi, la stimulation des α 2 -adrénorécepteurs présents au sein du locus coeruleus entraîne une diminution centrale de la réponse adrénergique sympathique [8]. La dexmédétomidine ou la clonidine sont les deux molécules α 2 -agonistes les plus connues et les plus testées pour cette approche. Sans qu'il existe encore de preuves cliniques, de nombreux travaux ont démontré que durant le choc septique expérimental, la modulation centrale du système nerveux sympathique était associée à une amélioration de la vasoréactivité, de la microcirculation et une diminution de l'inflammation tissulaire et plasmatique [30][31][32]. L'association d'une approche périphérique par esmolol et centrale par dexmédétomidine a aussi été évaluée expérimentalement, démontrant que l'approche combinée est envisageable [33].

Malgré ces concepts intéressants, il faut tout de même rappeler qu'il est contre-intuitif lors du choc septique de réduire les principaux facteurs du transport artériel en oxygène, que sont la FC et l'inotropisme. Il convient donc, dans un premier temps, de synthétiser les données expérimentales et cliniques, évaluant la tolérance et les effets cliniques de l'administration d'un traitement β 1 -bloquant chez le patient en choc septique, en se concentrant plus spécifiquement sur la diminution du débit cardiaque, la vasoréactivité, la mortalité et l'inflammation.

Preuves expérimentales de l'intérêt des β-bloquants dans le choc septique

Le diagramme de la recherche systématique des articles expérimentaux est détaillé sur la figure 1.

Les études expérimentales chez le petit et le gros animal ont évalué deux aspects de l'effet des β-bloquants dans des modèles de choc septique : les effets sur la fonction cardiovasculaire et les effets inflammatoires. Les modèles utilisés sont principalement polymicrobiens par ligature et ponction du caecum (CLP) ou endotoxinique par injection de lipopolysaccharides (LPS) et conduisent à des sepsis de sévérité variable, allant du sepsis peu grave au choc septique sévère (Tableau 2). La majorité des travaux ont administré un β 1 -bloquant, avec pour objectif une diminution de 20 % de la FC.

Globalement, l'objectif de diminution de la FC de 20 % était toujours atteint. Quels que soient le modèle animal et la modalité d'administration du β 1 -bloquant, un effet cardioprotecteur était retrouvé. Par exemple, Ackland et al. ont montré que l'administration de métoprolol ou d'aténolol, des β 1 -bloquants cardioséléctifs, chez des rats en choc septique moyennement sévère, avait pour conséquence une augmentation du volume d'éjection systolique combiné au maintien du débit cardiaque, tout en réduisant la FC. En revanche, l'administration de β 1 -bloquant en intracérébral, contrairement à l'administration en intrapéritonéal dans cette étude, n'améliore pas la survie des rats en choc septique, suggérant ainsi un mécanisme β 1 -adrénergique périphérique plutôt que central [37].

L'ensemble des études réalisées chez les rongeurs montrent soit un maintien, soit une augmentation, du débit cardiaque malgré la diminution de la FC. Une explication possible serait, chez ces animaux chronodépendants, un allongement de la diastole secondaire à la réduction de la FC. Du fait de cet allongement, le remplissage ventriculaire gauche est augmenté permettant un maintien du débit cardiaque. Le travail externe myocardique est diminué, ce qui permet une meilleure efficience myocardique globale pour un moindre coût énergétique [34].

Ce résultat s'associe, de façon étonnante, à une amélioration de la fonction vasculaire. Théoriquement, les β 1 -adrénorécepteurs n'étant pas présents sur les vaisseaux, les β 1 -bloquants n'ont aucun effet direct sur la vasoréactivité. Cependant, une amélioration de la vasoréactivité est retrouvée lors du choc septique, aussi bien dans les modèles animaux que chez l'homme [43,54]. Dans un modèle de gros animal tel que le cochon, deux études ont démontré que l'administration d'esmolol permettait in vivo une augmentation des résistances vasculaires systémiques [48,50]. De même, dans un modèle de choc septique sévère chez le rat, Wei et al. ont retrouvé que l'administration d'esmolol permet, ex vivo, de restaurer la contractilité vasculaire à l'augmentation des doses de phényléphrine. Dans ce même travail, des concentrations faibles d'esmolol, sans effet hémodynamique direct, conduisaient à une amélioration de la vasoréactivité [45].

Ces améliorations hémodynamiques sont associées à une diminution de la lactatémie qui pourrait être le reflet d'une demande en oxygène moins importante par les tissus et donc un meilleur métabolisme oxydatif [33]. Une amélioration du pronostic chez les animaux en choc septique recevant un β 1 -bloquant est aussi retrouvée dans toutes les études de survie réalisées chez le petit animal. Le second versant le plus étudié est l'impact des β 1 -bloquants sur l'inflammation. Lors de la phase aiguë du choc septique, on observe un « orage cytokinique » caractérisé par une hausse importante des taux de cytokines proinflammatoires telles que l'IL-6, l'IL-1 β ou le TNF-α, et anti-inflammatoires telle que l'IL-10, favorisant les défaillances d'organes et conduisant dans un second temps, lorsque l'état de choc persiste, à une profonde immunodépression [20].

L'administration de β 1 -bloquants chez les animaux en choc septique induit une diminution globale de l'inflammation, aussi bien systémique que tissulaire. Hagiwara et al. ont montré que l'administration de landiolol chez des rats, après injection de LPS, permettait de diminuer significativement le pic des cytokines caractéristique de la phase initiale du choc septique, incluant l'IL-6 et le TNF-α, molécules favorisant, entre autres, l'inflammation cardiaque et la dysfonction contractile [36]. Quels que soient le modèle et le moment de l'évaluation, l'administration d'un β 1 -bloquant est associée à une pondération globale pro-et anti-inflammatoire systémique et tissulaire (Tableau 2). Mori et al. ont de même montré que la production de TNF-α intrapéritonéale est diminuée par l'administration d'esmolol chez des rats après CLP. Ils démontrent également une diminution des altérations fonctionnelles et structurelles de la barrière intestinale chez les rats traités [38]. D'autres travaux retrouvent des résultats similaires sur le rein, le foie, les vaisseaux et le poumon [42,[44][45][46].

Preuves cliniques de l'intérêt des β-bloquants dans le choc septique

Le diagramme de la recherche systémique des articles cliniques est détaillé sur la figure 1.

Les données cliniques dans les tableaux 3 et 4 se limitent à la période s'étendant de 1990 à juillet 2018. Auparavant, la prise en charge du choc septique était beaucoup plus hétérogène, et les travaux sur le sujet ne permettent pas de conclure.

On retiendra que l'ensemble des travaux cliniques des tableaux 3 et 4 ont cherché à évaluer l'impact en termes Ils concluaient à la bonne tolérance de ce traitement [55]. Ensuite, les travaux ont essentiellement été monocentriques, observationnels, sur de petites populations légèrement plus sévères de choc septique, et ont retrouvé que la réduction de la FC n'induisait pas de diminution du débit cardiaque avec des résistances vasculaires qui restaient similaires [56,57]. Dans une des deux publications, la milrinone (inhibiteur des phosphodiestérase de type 3, inotrope positif indépendant des adrénorécepteurs) était coadministrée pour contrecarrer les effets inotropes négatifs du β-bloquant administré [56].

Une seule étude clinique d'envergure a été publiée en 2013 par Morelli et al. [61]. Ce travail monocentrique, prospectif, randomisé, esmolol versus placebo en ouvert incluait une population de 154 patients en choc septique 24 heures après l'admission qui restait tachycarde à plus de 95 battements par minute. L'objectif principal portait sur l'efficacité de l'administration de l'esmolol à diminuer la FC entre 80-94/min et sur l'évaluation de la tolérance qui y était associée. Le résultat principal est une réduction efficace de la FC dans la cible visée dans le groupe esmolol. Le débit cardiaque est globalement non diminué malgré la réduction de la FC, en partie grâce à l'augmentation du volume d'éjection systolique secondaire à l'allongement de la période diastolique dans le groupe esmolol. Du lévosimendan (un inotrope calcium sensibilisateur, indépendant des adrénorécepteurs) était administré de façon similaire dans les deux groupes (esmolol : 49,4 %, placebo : 40,3 %, p = 0,39). La posologie de noradrénaline requise était inférieure dans le groupe esmolol par rapport au groupe placebo. Les résistances vasculaires étaient augmentées par rapport au groupe placebo. La mortalité était réduite de 31 % dans le groupe esmolol avec 80 % de mortalité dans le groupe témoin. Ces résultats ont suscité d'intenses débats dans la littérature [66][67][68][69][70][71][72]. On retiendra surtout quatre points :

• l'efficacité sur la réduction de la FC est certaine ;

• la tolérance semble excellente, mais la prise en charge est en dehors des pratiques usuelles, car près de 50 % de la population reçoit du lévosimendan ;

• la réduction de la dose de noradrénaline dans le groupe esmolol est inexpliquée ;

• la diminution de la mortalité est à pondérer à la mortalité du groupe témoin qui est inhabituellement très élevée et en dehors de ce qui était attendu en 2013.

Fondés sur les résultats de Morelli et al., les travaux qui ont suivi ont cherché à confirmer la tolérance et les effets physiologiques de ce traitement. Néanmoins, les populations incluses ne correspondent jamais aux recommandations de prise en charge du choc septique : soit les patients ont des objectifs de pression artérielle trop élevés, soit les patients ont une prise en charge non usuelle ou un niveau de sévérité qui est trop faible [62][63][64][65]. Finalement, étant donné les faiblesses méthodologiques des travaux présentés, il est prématuré de recommander l'utilisation d'esmolol durant le choc septique, essentiellement en raison de l'absence d'identification d'une population à qui l'administrer. En effet, une étude de faisabilité, incluant des patients d'une population comparable à celle que nous avons dans nos services, démontre que seuls 10/186 étaient éligibles à l'administration d'esmolol [73].

Les β-bloquants ont-ils une place dans la prise en charge du choc septique ?

Si les études expérimentales suggèrent que l'administration précoce intraveineuse d'un β 1 -bloquant est bénéfique en terme hémodynamique et pour un certain nombre d'études sur la survie, les travaux cliniques sont néanmoins soit peu concluants, soit méthodologiquement fragiles. Ces différences de résultat peuvent s'expliquer par le modèle et les conditions expérimentales appliqués. Ainsi, les études expérimentales sont réalisées essentiellement chez le petit animal, les souris ou les rats, dont les FC au repos sont supérieures à 450/min. Le débit cardiaque est donc en cas de choc septique étroitement relié à la FC et à la durée de la diastole et donc au remplissage ventriculaire en fin de diastole. Ainsi, dans un travail chez la souris en choc septique par ligature et ponction caecale, la survie était améliorée lorsque le ventricule gauche se dilatait permettant ainsi un meilleur remplissage ventriculaire [74]. Les modèles de choc septique utilisant les petits animaux sont d'ailleurs loin d'être tous tachycardes, et plus particulièrement chez la souris, où l'induction d'un choc septique provoque une régulation négative du métabolisme conduisant à une diminution de la FC [75]. Au contraire, chez l'homme, la survenue d'un choc septique induit le plus souvent une tachycardie dès la phase initiale. De même, le moment d'administration du β-bloquant est toujours défini lors d'une étude expérimentale, le plus souvent précoce alors qu'il est par définition hautement variable chez l'homme et le plus souvent tardif [61,76]. Finalement, si le modèle du petit animal est intéressant sur le plan conceptuel, la transposition des méthodes appliquées, chez le gros animal et plus encore chez l'homme, ne produit pas des résultats équivalents.

En pratique, les résultats de la seule étude randomisée sur le sujet restent encore à confirmer de façon multicentrique. Plus précisément, la tolérance clinique reste sujette à caution au regard de la mortalité de la population en choc septique du groupe non traité (80,5 %) incluse dans l'étude princeps, différente de celle rencontrée habituellement (42 %) [61,77].

Par ailleurs, des études tendent à démontrer que les critères d'application de ce traitement, c'est-à-dire le patient cliniquement stable à 24 heures mais présentant une tachycardie persistante supérieure à 95/min, ne sont que rarement rencontrés [73]. De plus, il n'existe pas d'indicateur simple permettant de différencier une tachycardie adaptative (une hypovolémie par exemple) d'une tachycardie dysautonomique, c'est-à-dire qui est le reflet d'un hyperadrénergisme central inadapté [78,79].

Au regard de la littérature, il n'existe pas de preuves cliniques suffisantes pour recommander l'administration d'un traitement β 1 -bloquant chez le patient en choc septique, stabilisé mais toujours tachycarde avec pour objectif la réduction de la FC en vue de l'amélioration de son pronostic vital. Les études qui permettront de mieux définir le profil de tolérance des patients en choc septique pouvant éventuellement bénéficier d'un traitement bradycardisant par β 1 -bloquant sont actuellement en cours (NCT02120404, NCT02369900).

Si les données précliniques révèlent des effets intéressants sur le plan de la rééquilibration inflammatoire, une meilleure compréhension des mécanismes sous-jacents à cette régulation est un prérequis nécessaire à une meilleure définition des patients qui éventuellement pourraient bénéficier, par la suite, de ce traitement.

Conclusion

La dysautonomie, surstimulation du système sympathique, est une complication précoce du choc septique associée à une altération des fonctions cardiaque et vasculaire et une immunosuppression. Son association à la mortalité semble avérée. Sa modulation est un enjeu qui pourrait se faire via l'inhibition des adrénorécepteurs β 1 par un traitement β 1bloquant. Néanmoins, si les preuves expérimentales sont en faveur de cette approche, l'accumulation des preuves cliniques n'est pas encore suffisante.

Remerciements : Ce travail est soutenu par la Fondation pour la recherche médicale (numéro de subvention FRM ECO20170637495 à MD). Sepsis is the consequence of a highly complex and dysregulated inflammatory host response to infection leading to life-threatening organ dysfunction 4 . After initial activation, the immune systemboth innate and adaptiveis downregulated, leading to immunosuppression that may be long-lasting and predispose the patient to secondary infection. A disproportionate and prolonged activation of the sympathetic nervous system (dysautonomia) also occurs, characterized by loss of cardiovascular variability, tachycardia, and excessive release of catecholamines such as epinephrine and norepinephrine 5 . These hormones, mainly possessing α 1 and β 1 adrenergic effects, are crucial factors underlying immunosuppression 6 . Sepsis-induced immunosuppression is characterized by immune cell depletion; dysregulated cytokine production and compromised T helper cell (CD4 T cells) function. There is an associated increase in regulatory T cells (Treg cells) leading to a decreased ability to respond to subsequent infectious insults 7 . In this context, norepinephrine favors an anti-inflammatory cytokine imbalance, associated with inhibitory effects on T cell growth and cytokine production 8-10 .

Beta-1 adrenoreceptor (β 1 -AR) blockade is a therapeutic approach aimed at downregulating the excessive adrenergic stimulation induced by sepsis. Initial single-center studies in human

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

6 septic shock have mainly reported hemodynamic benefits with improvements in cardiac efficiency, with one also finding survival benefit 11-13 . Multicenter trial will begin soon (EudraCT: 2017-002138-22). Some experimental studies have investigated the inflammatory effects of β 1 -AR blockade in sepsis and demonstrated a global decrease in both pro-and anti-inflammatory plasma cytokines 14-16 . However, little is known about the effects of β 1 -AR blockade on the early adaptive immune response, specifically on CD4 T cells 17, 18 . The present study aimed to address this question in a murine model of high-grade sepsis.

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

Methods

Sepsis model

1 -AR (C57BL/6J, DBA/2, 129Sv) knockout mice ( and subsequently maintained through the procedure with 2% isoflurane. A laparotomy was performed, 75% of the caecum is being ligated and then punctured with an 18-gauge needle (high-grade sepsis) 19 . The caecum was re-inserted into the abdomen and the incision closed by suture. Sham-operated mice underwent the same procedure except for the caecal ligation and puncture. An incision was performed between the scapulae in all mice and a mini-osmotic pump (2001D, Alzet, Cupertino, CA) implanted subcutaneously in animals to be treated with the selective 1 -AR blocker, esmolol (Esmocard®, Amomed, Meudon, France).

After 20 minutes, mice received subcutaneous analgesia with buprenorphine 0.2 mg/kg (Buprécare®, Axience, Pantin, France) in physiological saline solution (0.9% NaCl; 5ml/100g; Baxter, Guyancourt, France).

Experimental design

8

(Figure S1). 1 -/-mice were randomly assigned to two groups: sham or CLP. Eighteen hours after surgery, echocardiography was performed, plasma collected and spleens immunophenotyped (n=74). Splenic CD4 T cell proliferation assays were also performed (n=44). In 1 +/+ non-septic mice, Treg cells and CD4 T cells were isolated and co-cultured (n=4). A 5-day survival study was performed in 3 groups of mice: 1 +/+ CLP (n=20), 1 +/+ CLP + esmolol (n=20), and 1 -/-CLP mice (n=20). The number and reasons for failed measurements and excluded mice are provided in Table S1.

Splenocyte isolation

Spleens were harvested, immediately placed in cold PBS (Sigma Aldrich, Saint-Louis, MO), gently ground between frosted slides, then dispersed through 70 μm nylon filters (Biologix,

Co-culture of CD4 T cells with catecholamine-stimulated Treg cells

Treg cells were stimulated with 1 μM dobutamine (a 1 -AR agonist), norepinephrine (a mixed 1 -and 1 -AR agonist), phenylephrine ( 1 -AR agonist); (all from Sigma-Aldrich) or PBS for 30 minutes, then washed twice to remove the catecholamines. Pre-treated Treg cells were co-cultured with CD4 T cells at a 1:4 ratio in complete RPMI 1640 medium (supplemented with 10% FBS, 2 mM L-glutamine, 20 U/ml penicillin, 20 μg/ml streptomycin, 1% sodium pyruvate, 1% HEPES, 50 μM 2-mercaptoethanol and 30 U/ml IL-2) in 96-well plates coated with 50 μL of 0.01% poly-L-ornithine solution (Sigma Aldrich). Cells were activated with anti-CD3/CD28 beads (bead-to-cell ratio 1:1; T cell activation/expansion kit, MiltenyiBiotec) and imaged every 2 hours for 5 days (37 °C, 5% CO2) using the 20× objective of the IncuCyte® S3 Live-Cell Analysis System (Sartorius, Goettingen, Germany). Proliferation was measured by cell counting in the phase contrast mode. Dead cells were identified using the green fluorescent Cytotox Green dye (Sartorius; final concentration, 250 nM). Analysis was performed using IncuCyte™ Basic Software (Figure S3).

Quantification of inflammatory mediators

In 5-6 mice from each group, the plasma levels of 46 inflammatory mediators were measured in duplicate using the Magnetic Luminex® Performance Assay -Mouse XL Discovery Premixed kit (Bio-Techne, Minneapolis, MN) according to the manufacturer's instructions. Results were expressed as pg/ml.

Survival study

Survival was studied in 1 +/+ CLP mice, 1 +/+ CLP + esmolol (18 mg/kg/h) mice, and 1 -/-CLP mice. Census of animals was undertaken twice a day for 5 days. Administration of esmolol (Esmocard®, Amomed) was started 3 hours post-CLP and continued for a total infusion period of 24 hours. The one animal that died before the first count was considered a technical failure and excluded from the study.

Statistical analysis

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

Results are expressed as mean and standard deviation (mean ± SD). Results in 1 -/-mice were compared using a Mann-Whitney test while the Kruskal-Wallis test was used to compare 1 +/+ groups. When the Kruskal-Wallis test was significant, post hoc comparisons were performed between sham, CLP, and CLP + esmolol groups using Dunn's multiple comparisons and a Bonferroni correction.

Variations were calculated as follows: 100-((mean group 1/mean group 2)x100).

To analyze the effect of catecholamines on Treg cells co-cultured with CD4 T cells, we firstly assessed the interaction between mouse at each time point. Considering the mouse has a random effect, the p-value of the test for the treatment effect was calculated. Only the last p-value (obtained at 132 hours) is shown.

For measurement of plasma inflammatory mediators, groups were compared using the Mann-Whitney test. A Benjamini-Hochberg adjustment was performed for multiple comparisons.

Kaplan-Meier curves were drawn. Significance was assessed using the log-rank test.

P values <0.05 were considered statistically significant. Statistical analysis was performed using R, version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria), using Graph-Pad Software 6.0, (San Diego, CA) and using NCSS Statistical Software 9 (East Kaysville, UT).

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

13

Results

-AR contributes to post-septic immunosuppressive phenotype.

Male and female mice were used in equal numbers for all experiments (sex ratio=1.02).

Eighteen hours after surgery, the spleens of 1 +/+ and 1 -/-mice were harvested and immunophenotyped to study T lymphocyte subpopulations. ) and Th17 (IL-17+)) in these experimental groups (Figure S5). These findings were not caused by a difference in cell viability among groups (Figure S6).

-AR activation induces a decrease in CD4 T cell proliferation during sepsis.

After culturing splenocytes for 72 hours with PHA stimulation, the proliferative response of CD4 T cells was measured to assess sepsis-induced immune suppression (Figure 2, panel A).

In 1 +/+ mice, sepsis reduced the proliferative capacity of CD4 T cells (-15% vs. sham, p=0.04).

However, administration of a 1 -AR blocker limited this effect (-8% vs. sham, p=0.38). In Hemodynamic parameters were assessed by echocardiography (Table 1). In 1 +/+ septic mice, administration of a 1 -AR blocker induced a decrease in HR (-24% vs. sham, p<0.001; -14% vs. CLP, p=0.07). In 1 -/-mice, sepsis did not affect HR (+9% vs. sham, p=0.24).

Compared to

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

15 CLP, p=0.99). In 1 -/-mice, sepsis induced a decrease in MAP (-19% vs. sham, p=0.04), SV (-47% vs. sham, p<0.001), CI (-37% vs. sham, p=0.002) and CPI (-50% vs. sham, p<0.001).

1 -AR modulates the concentration of both pro-and anti-inflammatory circulatory mediators.

The plasma levels of 31 pro-inflammatory and 15 anti-inflammatory mediators were measured. Briefly, all inflammatory mediators were increased in 1 +/+ CLP mice compared to sham controls (Table S2). Compared to 1 +/+ CLP mice, esmolol-treated CLP mice showed an overall decrease in plasma concentrations of 58% (18/31) and 60% (9/15) for pro-and antiinflammatory mediators, respectively (Figure 4). For instance, compared to 1 +/+ CLP mice, administration of a 1 -AR blocker reduced plasma TNF-by 66% vs. CLP (p=0.02), and IL-10 by 82% vs. CLP (p=0.01). In 1 -/-mice, sepsis induced an overall increase in both pro-and anti-inflammatory mediators compared to sham animals (Table S2).

1 -AR modulates survival during sepsis.

The survival rate was documented in 1 +/+ CLP, 1 +/+ CLP + esmolol and 1 -/-CLP mice (Figure 5). Survival at 110 hours decreased from 60% in 1 -/-CLP mice, to 37% in 1 +/+ CLP + esmolol mice and 35% in 1 +/+ CLP mice (log rank p=0.13).

Sex does not affect the 1 -AR effect.

Sensitivity analyses were performed on results obtained for major hemodynamic parameters (Figure S9) and immune effects (Figure S10) according to sex. No differences were found between male and female mice.

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

16

Discussion

In the present study, we confirm that experimental high-grade sepsis induces three hallmark features of sepsis-induced immunosuppression: a significant decrease in splenic CD4 T cells, an increase in splenic Treg cells, and a decrease in CD4 T cell proliferation capacity 7 . A notable finding was the marked increase in Treg cells in the spleen of septic mice, which could be counteracted by pharmacologically or genetically blocking β 1 -AR signaling. This observed decrease in Treg cells in treated animals was associated with a simultaneous increase in CD4 T cells and a global decrease in circulating pro-and anti-inflammatory mediators, indicating the involvement of β 1 -AR in the septic-immune phenotype. These results also translated into an in vivo global improvement in cardiac efficiency. Figure 6 shows a graphical summary of the effect of β 1 -AR blockade on cardiac and immune function during sepsis induced-dysautonomia.

Model characteristics

To investigate the immune and cardiovascular roles of β 1 -AR in sepsis, we deliberately chose a non-resuscitated murine model of CLP to avoid any interference between exogenous catecholamine administration and the immune system 20 . This model depicts all hemodynamic and survival characteristics of a typical high-grade sepsis, with an increase in both plasma pro-and anti-inflammatory mediators and a decrease in CD4 T cell proportion 19,[21][22][23] . Analysis of CD4 T subtypes revealed that early after onset of sepsis, the percentages of Th1, Th2 and Th17 cells were unchanged. Many studies usually report a switch from Th1 to Th2 phenotypes, and a decrease in Th17 cell percentages 24-27 . However, other studies found that sepsis could also disrupt in a time-dependent manner both the representation and the function of CD4 T cell subsets, including Th1, Th2, Th17 28-31 . Unlike other

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

lymphocyte populations, Treg cells are increased during trauma in both animals and humans, as well as during sepsis and septic shock (as seen in our study) 29, 30 . This increase in Treg cells contributes to lymphocyte paralysis and, more broadly, enhances the post-septic immunosuppressive process 32 .

ß-adrenergic stimulation contributes to CD4 T cell dysfunction

In both innate and adaptive immune cells, adrenergic receptors are widely expressed though not well characterized, particularly for the β subtype 33 . Numerous studies have demonstrated the presence of β-AR on almost all lymphocyte subtypes 34-36 . However, until recently, only β 2 -AR but not β 1 -AR subtypes were found on T cells 37, 38 . As a consequence, β 1 -AR stimulation was not considered to have a major role in inflammation 39 . β 1 -AR were found to be widely expressed on splenic CD4 T cells only recently. In cirrhotic mice, the β 1 -AR agonist dobutamine, could re-activate inhibited Treg cells, suggesting the presence of β 1 -AR on their surface 40 . In accordance, we too found that in vitro CD4 T cell proliferation tended to be reduced by Treg cells pre-treated with dobutamine.

ß-adrenoreceptor blockade reversed sepsis-induced dysautonomia

In numerous conditions, such as acute heart failure, burn injuries, chronic hepatic failure, the peri-operative period and septic shock, β 1 -AR blockade has been consistently associated with protective effects on inflammation and hemodynamics 15, 41, 42 . As others, we too found a global decrease in pro-and anti-inflammatory mediators in animals with high-grade sepsis treated by β 1 -AR blockade 15, 43, 44 . Underlying mechanisms are still not elucidated. In Listeria monocytogenes infected mice acute cold or restraint stress (surrogates of sympathetic adrenergic stimulation) inhibited the host T cell response and increased cytokine production 18 45 . In this model, pharmacological blockade of the β 1 -AR but not β 2 -AR could downregulate this pro-inflammatory state and shorten time to bacterial clearance 46 .

In β 1 -AR blocked animals with high-grade sepsis, we found the decrease in Treg cells in the spleen was associated with a concomitant increase in CD4 T cells, as reported previously 47 .

In addition, we found that proliferative capacity of splenic CD4 T cells in septic animals was maintained when the β 1 -AR was blocked either pharmacologically or genetically. This suggests that β 1 -AR activation contributes to sepsis-induced immunosuppression, which could be reversed by β 1 -blockade. By comparison, volunteers challenged with lipopolysaccharide, and then being infused with norepinephrine for 5 hours showed a shift to an anti-inflammatory state (decreased TNF-⍺/IL-10 ratio) which could be counterbalanced by β-AR blockade (increased TNF-⍺/IL-10 ratio) 10 . β 1 -AR blockade in our septic animals reduced the inflammatory burden and, taken as a whole, helped to shift from an immunosuppressive state to a state of immune homeostasis.

Beside this immune rebalancing in experimental high-grade sepsis, it is important to reiterate that β 1 -AR blockade also has major cardiovascular effects. In agreement with most other experimental or clinical studies, septic mice receiving β-AR blockade showed no alteration in cardiac power, despite a decrease in HR, suggesting improved cardiac efficiency 13,15,43,48 .

While the association between an increased Treg cell percentage during sepsis and an immunosuppressive state is well proven, effects on survival remain inconclusive 49, 50 19 vasopressors), no improvement was seen in survival. In a similar CLP surgery model, Ackland et al. also found that metoprolol infusion, commenced 6 hours after the onset of fecal peritonitis, did not confer any benefit in improving survival whereas pretreatment was effective 44 . A dose effect may be relevant as the β 1 -AR blocker was infused only during the first 24 hours after CLP surgery. In a single-center study prolonged administration of esmolol in septic shock patients, titrated to maintain heart rate between 80-94 bpm, was associated with increased survival 13 .

Perspectives β 1 -AR and β 2 -AR are widely expressed on both innate and adaptive immune cells.

Specifically, it has been reported that naïve CD4 T cell subtypes Th1, Th17 and Treg cells, but not Th2, express β 2 -AR 34, 51 . β 2 -AR stimulation promotes a shift from Th1 to Th2 antiinflammatory patterns and could activate Treg suppressive functions 35, 52 . β 2 -AR stimulation may enhance the macrophage M2 anti-inflammatory phenotype, favoring IL-10 production by CD4 T cells 36 . Beneficial effects of β 2 -AR modulation remain unclear and little is known about β 2 -AR signaling in CD4 T cells during sepsis. In a murine endotoxemia model, β 2 -AR blockade reduced survival while, in septic pigs, it amplified sepsis-induced hepatic injury 36, 53, 54 . In humans, it has been reported that β 2 -AR polymorphism is associated with increased mortality in septic shock 55 . In this study, we only investigated the impact of β 1 -AR blockade on alteration of CD4 T cell patterns. The impact of selective β 2 -AR modulation as well as nonspecific β-AR blockade on CD4 T cells during sepsis merits investigation.

In summary, in the murine model tested, the β 1 -AR contributes to sepsis-induced immunosuppression through modulation of regulatory T cell inhibitory functions.

Figures and

Supplemental Figures and

Supplemental Figures and

Abstract

Rationale: Sepsis typically induces dysautonomia, an over-activation of the sympathetic nervous system. This can lead to impaired cardiovascular function and immunosuppression, both of which are associated with poor outcome. Beta1-adrenoreceptor blockade downregulates dysautonomia. Although cardiovascular benefits of β1-adrenoreceptor blockade have been described in sepsis, little is known about its impact on the adaptive immune response, specifically CD4 T cells.

Objective: To investigate the effects of β1-adrenoreceptor modulation on CD4 T cell function in a murine model of sepsis. Methods and results: High-grade sepsis was induced by caecal ligation and puncture (CLP) in wild-type mice (b1+/+) with or without esmolol (a selective β1-adrenoreceptor blocker), or in β1adrenoreceptor knockout mice (b1-/-). At 18 hours, b1+/+ mice exhibited characteristics of highgrade sepsis and three surrogate markers of immunosuppression, namely decreased splenic CD4 T cells, reduced CD4 T cell proliferation and increased Treg cell proportions. Pharmacologic and genetic β1-adrenoreceptor blockade reversed the impact of sepsis on CD4 and Treg proportions and maintained CD4 T cell proliferative capacity. β1-adrenoreceptor blocked mice also exhibited a global decrease in both pro-and anti-inflammatory mediators and improved in-vivo cardiovascular efficiency with a maintained cardiac power index despite the expected decrease in heart rate. Conclusion: β1-adrenoreceptor activation enhances Treg inhibitory function and thus contributes to sepsis-induced immunosuppression. This can be attenuated by β1-adrenoreceptor blockade, suggesting a potential immunoregulatory role for this therapy in the treatment of sepsis.

Figure 1 :

 1 Figure 1: Influence of sepsis in adrenergic signalling. ...............................................

Figure 2 :

 2 Figure 2: Theories of the host immune response in sepsis. ......................................

Figure 3 :

 3 Figure 3: TLR4 signalling pathway. ...........................................................................

Figure 4 :

 4 Figure 4: Pathogenic pathways involved in the activation of coagulation in sepsis. .

Figure 5 :

 5 Figure 5: Effects of septic shock on immune cell subsets. ........................................

Figure 6 :

 6 Figure 6: Intracellular mechanisms behind the effects of β-AR stimulation on cytokine

Figure 7 :

 7 Figure 7: Several adrenergic mechanisms contribute to sepsis-induced

Figure 8 :

 8 Figure 8: Sympathetic nervous system activation during septic shock. ....................

Figure 9 :

 9 Figure 9: Structural formula of esmolol. ....................................................................

Figure 10 :

 10 Figure 10: Flow diagram of researched studies. .......................................................

Figure 11 :

 11 Figure 11: Genotyping of mouse DNA by PCR. ........................................................

Figure 12 :

 12 Figure 12: Anatomical locations for caecal ligation of mice. ......................................

Figure 13 :

 13 Figure 13: Representative measurement of Velocity Time Integral by

Figure 14 :

 14 Figure 14: Gating strategy for the identification of T cells subsets by flow cytometry.

Figure 15 :

 15 Figure 15: Gating strategy for the identification of Th1, Th2 and Th17 subsets by flow

Figure 16 :

 16 Figure 16: Gating strategy for the identification of monocytes by flow cytometry. ....

Figure 17 :

 17 Figure 17: Isolation of CD4 T cells and Treg cells. ..................................................

Figure 18 :

 18 Figure 18: Co-culture of CD4 T cells and Treg cells pre-treated with catecholamines.

Figure 19 :

 19 Figure 19: Effect of esmolol on heart rate in septic mice. .......................................

Figure 20 :

 20 Figure 20: b 1 -AR blockade in sham treated mice does not change lymphocyte

Figure 21 :

 21 Figure 21: b 1 -AR does not change monocytes proportions in sepsis. .....................

Figure 22 :

 22 Figure 22: b 1 -AR contributes to the post-septic immunosuppressive phenotype. ...

Figure 23 :

 23 Figure 23: b 1 -AR does not change the CD4 T, Th1, Th2, Th17 subpopulation

Figure 24 :

 24 Figure 24: b 1 -AR does not change lymphocyte viability in sepsis. ..........................

Figure 25 :

 25 Figure 25: b 1 -AR activation decreases CD4 T cell proliferation during sepsis. .......

Figure 26 :

 26 Figure 26:Treg cell stimulation by catecholamines increases their

Figure 27 :

 27 Figure 27: Leukocytes infiltration is not modified by the sepsis in the heart. ..........

Figure 28 :

 28 Figure 28: b 1 -AR modulates the concentration of both pro-and anti-inflammatory

Figure 29 :

 29 Figure 29: b 1 -AR blockade modulates survival during SS. ......................................

Figure 30 :

 30 Figure 30: sex does not affect the hemodynamic effects of b 1 -AR. .........................

Figure 31 :

 31 Figure 31: Sex does not affect the immune effects of b 1 -AR. ..................................

Figure 32 :

 32 Figure 32: Graphical summary of the involvement of b 1 -AR in sepsis-induced

  Criteria for SIRS, sepsis, severe sepsis, and septic shock based on the 1991 ACCP/SCCM Consensus Conference. .....................................................Table 2: Criteria for sepsis and septic shock based on the Third International Consensus. ........................................................................................................ Table 3: Quick SOFA criteria. .................................................................................... Table 4: Epidemiology of septic shock in France from 1995 to 2020. ....................... Table 5: Effects on sepsis on CD4 T cells. ................................................................ Table 6: Classification, localisation, signalling, agonist, antagonist, hemodynamiceffects and inflammatory effects of main adrenoreceptors. ............................... Table 7: Summary of experimental studies ............................................................... Table 8: Summary of non-comparative clinical studies. ............................................ Table 9: Summary of comparative clinical studies. ................................................... Table 10: Primers sequence used for mouse genotyping. ........................................ Table 11: Number and reasons for failed measurements and excluded values. ...... Table 12: Despite reduced heart rate, b 1 -AR blockade did not alter hemodynamic characteristics in sepsis. .................................................................................. Table 13: Sepsis induces an increase in both pro-and anti-inflammatory mediators in β 1 +/+ and β 1 -/-mice. ............................................................................................

  . A study published by Fleishman et al. estimated the global burden of sepsis at 19.4 million incident sepsis cases and 5.3 million sepsis-related deaths annually in seven high-income countries for the years 1979 to 2015 [2]. More recently, Rudd et al. used data from 109 million individual death records to assess mortality related to sepsis in 2017. They estimated the incident cases of sepsis at 48.9 million and the number of sepsis-related deaths at 11.0 million, representing

  Simultaneously, patients urgently need an hemodynamic stabilisation. Initial resuscitation starts at the onset of diagnosis. At least 30 ml/kg of IV crystalloid fluid should be infused within the first 3 hours. Patients who are still hypotensive despite fluid resuscitation, vasopressors are requires vasopressors aiming to restore a mean arterial pressure (MAP) of 65 mmHg. Norepinephrine (NE) is the first recommended vasopressor choice. This a 1 and β 1adrenergic agent increases vascular resistance and inotropism with little change in heart rate (HR) compared to dopamine or adrenaline which are also a 1 , β 1 , and β 2 adrenergic agents.

(

  IL-1β) decreased myocardial cell contraction in vitro [102]. Another study using rabbit ventricular myocytes demonstrated in vitro that myocytes exposed to high doses of TNF-a showed decreased contractility [103]. Kumar et al. showed that TNF-α and IL-1β were responsible for early sepsis-induced cardiac dysfunction, while prolonged effects were associated with the excessive production of nitric oxide (NO) [103-105]. Nitric oxide Nitric oxide is a widely expressed intracellular messenger. It is a small lipophilic molecule produced through the metabolism of L-arginine by the activity nitric oxide synthase (NOS)[106]. The effects of NO are divided into direct and indirect effects Direct effects induce the activation or inhibition of haem enzymes. NO induces the activation of guanylate cyclase, a second messenger, that initiates several physiological effects resulting, for instance, in the relaxation of vascular smooth muscle cells, inhibition of platelet aggregation, and activation or inhibition of leukocyte adhesion to the vascular endothelium [107-109]. Indirect effects of NO occur with the release of oxygen species such as nitrous oxide. These specific molecules have a strong affinity for thiol groups and have inhibitory effects on glycolysis (inhibition of glyceraldehyde-3-phosphate dehydrogenase by nitrosylation) [106]. However, the major indirect effect of NO is the formation of peroxynitrite (ONOO -), produced by the interaction between NO and the superoxide anion (O 2 -). The peroxynitrite molecule, including during septic processes, has deleterious effects by inducing both necrosis and apoptosis and promoting pro-inflammatory cellular responses [110-112].

, Theory 3 )

 3 [170].

Figure 2 :

 2 Figure 2: Theories of the host immune response in sepsis.

Theory 2 :

 2 After the onset of sepsis, activation of both the pro-and anti-inflammatory response occurs.

Figure 4 :

 4 Figure 4: Pathogenic pathways involved in the activation of coagulation in sepsis.

  ]. Taken as a whole, these alterations are associated with an increased risk of death after septic shock[201].Multiple mechanisms contribute to sepsis-induced impairment of neutrophils migration[202]. For instance, proportionally to the severity of sepsis, pro-inflammatory cytokine release increases neutrophil cell membranes rigidity and reduces cellular deformability [203]. Thus, Lu et al. demonstrated a reduction in the neutrophil chemotaxis velocity to LPS in septic patients compared to healthy controls [204]. Furthermore, Raymond et al. showed a decrease in spontaneous neutrophil migration velocity in whole blood samples from septic adults [205]. One explanation might be the reduction in surface expression of chemokine (C-X-C motif) receptor 2 (CXCR2) and the adhesion molecule CD11b [205]. Interestingly, neutrophil phagocytosis capacity seemed to be preserved [206]. Monocytes/macrophages During sepsis, monocytes undergo phenotypic and functional re-programming that allows them to transit from an inflammatory to an immunosuppressive state. Two of the most well studied markers of monocyte dysfunction in sepsis are ex vivo LPS-induced TNF-ɑ production and monocyte cell surface expression of the human leukocyte antigen DR isotype (HLA-DR) [207]. Typically, monocytes from septic patients have a diminished capacity to release pro-inflammatory cytokines such as TNF-ɑ, IL-1α, IL-6, and IL-12, while the production of anti-inflammatory mediators such as IL-1ra and IL-10 is enhanced [208]. Moreover, during septic shock, monocytes are reprogrammed and develop a kind of tolerance to endotoxin [209]. HLA-DR expression by monocytes is reduced in septic patients and has become a hallmark of monocyte anergy and sepsis-induced immunosuppression [210, 211]. Decreased HLA-DR expression is associated with an increased in nosocomial infections and death [212, 213]. Re-expression of HLA-DR is a therapeutic strategy under investigation to reduce repeated super-infections in mechanically ventilated patients [214]. Dendritic cells DCs are antigen-presenting cells that play a major role in the activation of adaptive immunity. During sepsis, the number of DCs is decreased in both the spleen and the circulation [215-217]. This decrease in septic shock patients during the first 24 hours is associated with disease severity and could predict a poor outcome [218]. This loss of DCs may significantly impair B and T cell function and participate in sepsis-induced immunosuppression [89]. Indeed, DC populations derived from septic patient monocytes are not able to induce a robust effector T cell response, but rather induce either T cell anergy or Treg cell proliferation [219]. In addition, DCs from septic patients show reduced surface HLA-DR expression, reduced secretion of pro-inflammatory cytokines, and increased synthesis of IL-10 [220, 221].Natural killer lymphocytes NK cells are granular lymphocytes largely recognised for their role in tumour surveillance and in the host response to viral infections. A significant decrease in the proportion of NK cells to the total lymphocyte count is observed in septic patients and is associated with an increase in mortality[222][223][224]. Several studies have reported that both CD56 hi and CD56 low NK cell subsets are affected during sepsis[225, 226]. In addition, there is a significant decrease in NK cell cytotoxicity and a profound reduction in their INF-γ release capacity[227, 228]. The decrease in the production of IFN-γ by NK cells could contribute to the incidence of secondary infections[229]. γδ T cells γδ T cells are a distinct subset of lymphocytes that reside in the intestinal mucosa and display capacity of both innate and adaptive immune cells. Due to their location, γδ T cells may be responsible of the first line of defence against infection[230]. There is a marked reduction in γδ T cells in the circulation of septic patients (by around 80%), which translates into an increase in patient mortality[231, 232]. However, the consequences of the decreased number of γδ T cells are still not fully elucidated[233]. In γδ T cell-deficient mice, septic shock induces a decrease in inflammatory cytokines and an increase in mortality[234, 235]. One study suggested that the Vγ4+ subset of γδ T cells contributed to the protective immune response of septic mice and could delay mortality[235].

Figure 5 :

 5 Figure 5: Effects of septic shock on immune cell subsets. The immune system develops a state of leukocytosis for the first 2-4 days after the onset of septicaemia,

•

  Reduced ability to control new infec:ons • Innate immune cells : o Reduced cytokines produc:on and cytoly:c ac:vity o Decrease ability to orchestrate adap:ve responses • Adap:ve immune cells (B cells, CD4 & CD8 T cells): o B cells: decreased ability to generate Ag-specific plasma cells and an:bodies o CD8 T cells: reduced ability to proliferate and produce effectors cytokines, increased suscep:bility to exhaus:on, reduced effec:veness of peripheral responses o CD4+T cells: see table 5

  a pivotal role in both innate and adaptive immune responses. Their key role in the humoral response, i.e. immunoglobulin synthesis, is the best known. As antigen-presenting cells, B lymphocytes can activate T cells through the expression of major histocompatibility complex (MHC) class II [237]. B cells recognise a wide variety of antigens (such as proteins, lipids, polysaccharides, nucleic acids, and chemicals) that bind to surface immunoglobulin M (IgM) or IgD and cause their activation and cytokine production [238, 239]. Moreover, a subpopulation of regulatory B cells with immunoregulatory properties has been described [240]. In the specific setting of septic shock, the phenotype and function of B cells during the immunosuppressive phase have been poorly studied. Kelly-Scumpia et al. demonstrated that, in B cell deficient mice, the induction of septic shock decreased inflammatory cytokine and chemokine production and increased mortality rate [239]. In septic shock patients, in the immunosuppressive state, the absolute B cell number is reduced due to an increase in apoptosis, whereas the B cell proportion is increased compared to the whole lymphocyte count [225, 241, 242]. The functions of B cells are also altered. B cells from septic patients present decreased in HLA-DR expression, a deficiency in IgM synthesis, and a decrease in activation, proliferation, and IL-10 production capacities [242]. CD8 T lymphocytes CD8 T lymphocytes recognise and respond to pathogen through the MHC class I molecules expressed on infected cells and antigen-presenting cells. Activated CD8 T cells clear infected cells by cytolysis (via granzyme B and perforin production) or recruit and activate other immune cells (via IFN-γ and TNF-α secretion) [243, 244]. Following sepsis, alterations in the number and functions of CD8 T cells occur. Post-septic apoptosis affects both pathogenspecific naïve and memory CD8 T cells [245, 246]. The recovery of naive CD8 T cells following sepsis is slow and most often partial, resulting in the loss of some precursor specificities [247].

  mediated) and intrinsic (mitochondria-mediated) pathways [257]. The extrinsic pathway is initiated by the binding of inducers to surface death receptors. For instance, Ayala et al. demonstrated that survival was improved by the administration of Fas death receptormediated pathway inhibitors in polymicrobial sepsis [258]. Conversely, the intrinsic pathway is triggered by an inversion of the balance between pro-and anti-apoptotic proteins within mitochondria. Several studies have found that mice overexpressing the anti-apoptotic B-cell lymphoma 2 (Bcl-2) protein, localised on the mitochondrial membrane, were resistant to sepsis-induced apoptosis [259]. For most patients, a one-month delay is required for a post-septic recovery in absolute CD4 T cell numbers. The failure to normalise CD4 T cell functions is associated with a poor outcome [252, 253, 260]. In addition, advanced age has been shown to increase lymphopenia and to prolong CD4 T cell recovery in elderly septic patients [253]. Finally, Cabrera-Perez et al., demonstrated an incomplete recovery of the antigen-specific T cell repertoire after sepsis, secondary to the attrition of antigen-specific T cell receptor (TCR) diversity. This is associated with impaired CD4 T cell responses after sepsis [254]. CD4 T cell functional defects Many early studies have shown that cytokine production by CD4 T cells is altered, and suggest that septic shock induces a phenotypic change in the CD4 T cell balance from Th1 to Th2 [261-266]. A recent study published by Boomer et al. investigated freshly isolated postmortem spleen and lung cytokine production in patients who died from septic shock [170].

  be involved in decreasing T cell proliferation[278, 279].Stieglitz et al. demonstrated that specific depletion of Treg cells improved impaired T cell proliferation in septic mice, illustrating the participation of Treg in sepsis-induced immunosuppression [279]. In addition, IL-33, release during widespread tissue injury, has an essential function in the expansion of Treg cells; IL-33 induces the polarisation of M2 macrophages, thus enhancing expansion of the Treg cell population through IL-10 secretion and finally contributes to the development of longterm sepsis-induced immunosuppression [280]. Treg cells can also have a suppressive effect on innate immune cells. In the setting of LPS activation, Treg cells act on monocytes by inhibiting their survival through a pro-apoptotic mechanism involving the Fas/FasL pathway [281]. Furthermore, Treg cells inhibit IFN-γ production by γδ T cells in response to Mycobacterium tuberculosis [282].

  because of their impact on other cellular processes[236]. T Thereafter, research has been redirected towards investigation of immunostimulant strategies[287]. Among them, the four most studied immunomodulating agents are GM-CSF, INF-γ, PD.Meisel et al. demonstrated that treatment using GM-CSF is safe and restores monocytic immunocompetence in patients with sepsis. This tended to improve the severity of the disease, although 28-day mortality was not increased[289]. Two studies have shown that INFγ treatment had a beneficial effect on monocyte reactivation, characterised by an increase in HLA-DR expression[290, 291].Another approach to reversing immunosuppression in sepsis involves the blockade of the co-inhibitory molecules PD1 and PDL1. To date, there have been no randomised controlled trials on PD1 blockade in septic shock. Preliminary experimental studies showed that blockade of PD1-PDL1 signalling could improved survival in clinically-relevant animal models[292]. IL-7 is also a potential immunomodulating agent. Studies in mouse septic models and in T lymphocytes from humans showed that IL-7 administration might be able to restore T lymphocyte changes[293]. In addition, a phase II clinical trial conducted in patients with septic shock and severe lymphopenia also demonstrated that IL-7 reversed the marked loss of CD4 and CD8 T cells[294].

  The β-AR belongs to the G-protein-coupled seven-transmembrane domain receptor superfamily. There are three β-AR receptor subtypes: β 1 -AR, β 2 -AR, and β 3 -AR. The β 1 -AR gene is on chromosome 10, the β 2 -AR gene is on chromosome 5, and the β 3 -AR gene is on chromosome 8. β 1 -ARand β 2 -AR are widely expressed. In the heart, β 1 -AR is predominant (β 1 -AR: 75-80% of cardiac β-AR, β 2 -AR: 20-25%); conversely, in smooth muscles such as in the bronchi and vessels, β 2 -AR are predominant[297]. β 3 -AR is the least studied isotype; it is expressed in adipose tissue, but also in the myocardium, in the urinary tract, and in endothelial cells[299, 300].Following activation by specific agonists such as NE, epinephrine or dopamine, β 1 -AR binds to the G s protein. The G s protein activates adenylate cyclase, which causes an increase in the second messenger cyclic AMP (cAMP). Protein kinase A (PKA) is subsequently activated by cAMP, which phosphorylates numerous targets in cytoplasm. In cardiomyocytes, PKA activates L-type Ca 2+ channels, which increases the influx of Ca 2+ , phospholamban (PLB), and the phosphorylation of troponin I, strengthening cardiac contraction[301]. This modulation also favours the occurrence of pro-apoptotic processes. Thus, in response to β 1 -AR stimulation, an increase in intracellular calcium is observed, which activates the protein kinase calcium/calmodulin (CaMKII). This protein promotes the mitochondrial apoptotic pathway through the release of cytochrome C, leading to the activation of caspase-9 and cell death[302, 303].Unlike β 3 -AR, prolonged stimulation of the β 1 -AR or β 2 -AR is associated with desensitisation due to the phosphorylation of the C-terminal end of receptors.Adrenoreceptor desensitisation requires three steps: 1. phosphorylation of the receptor by PKA and β-AR kinase (β-ARK), leading to G s uncoupling; 2. sequestration of the receptor by translocation of the β-AR from the plasma membrane to a vesicular compartment deficient in G s ; 3. Downregulation of the receptor, resulting in its degradation through a lysosomal pathway[139, 304]. The β 3 -AR does not have these phosphorylation sites and is therefore not subjected to a desensitisation process[305]. The β 3 -AR signalling pathway is still poorly understood. Nevertheless, it is probably involved in regulation processes during sympathetic overstimulation[306].

Figure 7 .

 7 NE treatment of neutrophils, in vitro and in vivo, inhibits their migratory function, activation, and phagocytosis capacity [313-315]. Similarly, the consequences of β-AR stimulation on monocytes are usually anti-inflammatory. β-AR activation can also inhibit oxygen radical production, TNF-α production, and LPS-induced MIP-1, IL-18, and IL-12 production [316-319]. Under certain conditions (i.e. in unstimulated cells), β-AR stimulation may result in a pro-inflammatory response in monocytes, including an increased production of IL-18 or an increase in metalloproteinases (MMP-1) in both circulating monocytes and macrophages [320, 321]. NE and epinephrine decrease NK cell cytotoxicity and cytokine production [322-325]. Dendritic cells also express β-AR [326]. Several studies have demonstrated that β2-AR activation on bone marrow DCs impaired the production of TNF-α and IL-6, reduced the ability of DCs to present antigens, and enhanced IL-10 production by

  Th17, and Treg cells predominantly express β 2 -AR, while Th2 cells do not express β 2 -AR[330,[334][335][336]. However, catecholamines drive a Th2 shift at the level of both antigen presenting cells and Th1 cells. β 2 -AR stimulation on CD4 T cells inhibits IFN-γ production by Th1 cells, but does not change IL-4 production by Th2 cells, while β 2 -AR antagonists are able to restore Th1 cytokine production [295, 334, 337, 338]. While SNS-mediated modulation of CD4 T cell immune responses has been focused on the Th1/Th2 dichotomy, very little is known on the effect of adrenergic stimulation on other subsets such as Th17 and Treg cells. Liu et al. recently demonstrated that β 2 -AR are expressed on Th17 cells and that β 2 -AR/cAMP/PKA signalling in Th17 cells reduces their inflammatory response in the context of rheumatoid arthritis [339]. Another study showed a suppressive effect of NE on IL-17 and IFN-γ production by human leukocytes [340]. In acute stress, two studies showed a decrease in the number of peripheral of Treg cells, while Guereschi et al.

Figure 9 :

 9 Figure 9: Structural formula of esmolol.

Figure 10 :

 10 Figure 10: Flow diagram of researched studies.

  Of note, the clinical studies in Tables8, 9sought to assess the efficiency in reducing HR and tolerance for patients initially with tachycardia > 90/minutes. Prior to β 1 -AR blocker administration, in all studies, patients were optimally resuscitated and considered hemodynamically stable. The first work was published in 2006 by Gore et al. This prospective before-and-after study in six non-severe septic patients assessed the physiological effects of a 20% decrease in HR by esmolol. The authors demonstrated that the reduction in HR was associated with a decrease in cardiac output with an increase in systolic ejection volume without a hypoxic effect or a change in vascular resistance. They observed good tolerance to this treatment

Animals b 1 -

 1 AR (C57BL/6J, DBA/2, 129Sv) knockout mice ( b 1 -/-) and wild type littermates (b 1 +/+ ), obtained free of charge from the Institute of Pharmacology and Toxicology of Munich, Germany, were used for this study. The French Animal Care Committee, in accordance with European regulations, has approved all animal protocols (n°APAFIS#4636). Both males and females were used. β 1 -/-mice were generated by the department of molecular pharmacology of Roche Bioscience [395]. Briefly, the targeting vector of the endogenous β 1 -AR genomic locus was constructed using sequence that had been cloned from a 129sv mouse genomic library. The gene of the β 1 -AR was disrupted in the targeting vector by placing Escherichia coli lacZ gene encoding b-galactosidase and neomycin resistance gene. Embryonic stem (ES) cells were electroporated and selected for neomycin resistance. ES where homologous recombination occurred were injected into C57Bl/6 blastocysts to obtain germ line chimeras. Chimeric males were then mated to DBA/2 female mice to screen for germ line transmission of the ES cell DNA.

  TAT CAG GAC ATA GCG TTG G R: ATA GAG CCC ACC GCA TCC buprenorphine 0.2 mg/kg (Buprécare®, Axience, Pantin, France) in physiological saline solution (0.9% NaCl; 5ml/100g; Baxter, Guyancourt, France). The mouse was then returned in its cage and kept until recovery with water and food freely available.

Figure 12 :

 12 Figure 12: Anatomical locations for caecal ligation of mice.Schematic illustration of caecal ligation position to induce mid-grade sepsis (dotted green line) and highgrade sepsis (dotted red line). The yellow line represents the basis of the caecum, below the ileocaecal valve usedas a reference to ligature the caecum at the same length during each surgery. According to the reference:[396].

  immunophenotyped (n=74). Splenic CD4 T cell proliferation assays were also performed (n=44). In b 1 +/+ non-septic mice, Treg cells and CD4 T cells were isolated and co-cultured. A 5-day survival study was performed in 3 groups of mice: b 1 +/+ CLP (n=20), b 1 +/+ CLP + esmolol (n=20), and b 1 -/-CLP mice (n=20). The number and reasons for failed measurements and excluded mice are provided in Table

for 5

 5 minutes before the echocardiographic assessment. Ascending aortic diameter (AD) was obtained during systole from the left parasternal long axis view. The aortic velocity time integral (VTI) was measured in the aortic arch from suprasternal view. Stroke volume (SV), cardiac output (CO) and cardiac index (CI) were calculated with the following formulas: -Aortic root area: A=π x (Aortic root diam) 2 / 4 (cm 2 ) -Stoke volume: SV=VTI x A (ml) -Cardiac output: CO=VTI x HR x A (ml/min) -Cardiac index: CI=CO / animal weight (ml/min/g) -Cardiac power index: CPI=MAP x CI / 451 All measurements were averaged over three consecutive cardiac cycles. The Figure 13 represent a representative example of the VTI measurement.

Figure 13 :

 13 Figure 13: Representative measurement of Velocity Time Integral by echocardiography. The VTI was measured in the aortic arch from suprasternal view. Measurement was the averaged over three cardiac cycles between two breaths in Doppler mode.

  Stimulated cells (2 x 10 6 /tube) were stained 30 minutes at 4°C with 100 μl of PBS containing surface markers antibodies PerCP Cy5.5-CD3, BV421-CD4, PE-Cy7-CD8 (all from BD Pharmigen, BD Bioscience) and Fixable Viability Dye eFluor™ 506 (eBioscience TM , ThermoFisher Scientific) according to the manufacturer's instructions.Intracellular stainingCells were washed with PBS + 1% FBS and resuspended in 400 μl of Fixation/Permeabilization solution (BD Cytofix/ Cytoperm TM , BD Bioscience) during 20 minutes at 4°C and washed twice in BD Perm/Wash TM (BD Bioscience) buffer.Fixed/permeabilised cells were resuspended in 100 μl of BD Perm/Wash TM buffer containing intracellular antibodies AF700-INF-γ (Biolegend, San Diego, CA), APC-IL4 and PE-IL-17A (both from BD Pharmigen, BD Bioscience) and incubated at 4°C during 30 minutes in the dark. Cells were washed and resuspended in BD Perm/Wash TM (BD Bioscience) buffer prior to flow cytometric analysis.Flow cytometry was carried out using a Gallios analyzer (Bekman Coulter, Brea, CA).Blind analysis of the data was performed using the Flowlogic™ software (MiltenyiBiotec, Bergisch Gladbach, Germany). The gating strategy of the Th1, Th2, Th17 lymphocyte subpopulations are describe in the Figure 15. Purification of CD4+ CD25+ Treg and CD4+ CD25-T cells CD4 T cell effectors and Treg cells were isolated in a two-step procedure following the manufacturer's instructions (CD4+ CD25+ Regulatory T Cell Isolation Kit, MiltenyiBiotec) in b 1 +/+ non-septic mice. First CD4 T cells were negatively selected by depletion of underwanted cells. CD4-cells were magnetically labeled with a biotin-conjugated antibodies cocktail against CD8a, CD11b, CD45R, CD49b, and Ter-119 (10 μL per 10 7 cells in adapted buffer: PBS, 0,5% BSA, 2mM EDTA, pH 7,2) at 4°C for 10 min. Then cells were incubated at 4°C for 15 minutes in the dark with anti-biotin monoclonal antibodies conjugated to microbeads (20 μL per 10 7 cells) and anti CD25-PE antobody (10 μL per 10 7 cells). The magnetically labeled cells were flowed through LD MACS ® Column (MiltenyiBiotec), which is placed in the magnetic field of a MACS ® Separator (MiltenyiBiotec). Labeled CD4-cells were retained in the column and CD4 T cells passed through (Figure 17, panel A). Then CD4+ CD25+ Treg cells were positively selected from the pre-enriched CD4 T cells fraction. CD4 T cells was centrifuged and incubated with anti-PE microbeads (10 μL per 10 7 cells) at 4°C during 15 minutes in the dark. Cells were flowed through MD MACS ® Column placed in the magnetic field of a MACS ® Separator. The CD4+ CD25+ Treg cells were retained in the column, while the flow-through CD4+ CD25-T cells were collected. A sample of the two fractions were stained with CD4-BV421 (BD Pharmigen, BD Bioscience) and Fixable Viability Dye eFluor™ 506 (eBioscience TM , Thermo Fisher Scientific) antibodies and analyzed by flow cytometer to validate the isolation of Treg cells (Figure 17, panel B).

Figure 21 :

 21 Figure 21: b 1 -AR does not change monocytes proportions in sepsis.Splenocytes were immunophenotyped 18 hours after high-grade sepsis induction. The proportion of monocytes was determined. Data are expressed as mean ± SD.

b 1 -

 1 AR contributes to post-septic immunosuppressive phenotypeEighteen hours after surgery, the spleens of b 1 +/+ and b 1 -/-mice were harvested and immunophenotyped to study T lymphocyte subpopulations. Figure22, panel A shows that, compared to the b 1 +/+ sham group, the percentage of CD3 T cells decreased in the b 1 +/+ CLP (-24% vs. sham, p=0.002) and b 1 +/+ CLP + esmolol groups (-20% vs. sham, p=0.004). In b 1 -/-mice, the percentages of CD3 T cells were similar in the sham and CLP groups (-8% vs. sham, p=0.06).

Figure 22 ,(Figure 22 :

 2222 Figure 22, panel C shows that in b 1 +/+ mice, high-grade sepsis induced a significant decrease

Figure 24 :

 24 Figure 24: b 1 -AR does not change lymphocyte viability in sepsis.Splenocytes were immunophenotyped 18 hours after high-grade sepsis induction. The viabilities of T cells (A), CD8 T cells (B), CD4 T cells (C) and Treg cells (D)were determined in each group. Data are expressed as mean ± SD.

Figure 25 :

 25 Figure 25: b 1 -AR activation decreases CD4 T cell proliferation during sepsis. Splenocytes harvested 18 hours after sepsis induction were cultured for 72 hours with a mitogen (PHA) to assess their proliferative response. (A) Histograms of the proliferation marker in non-stimulated and PHA stimulated splenocytes from one representative b 1 +/+ sham and CLP mouse. Each peak has been fitted and colored by FlowLogic software. (B) Proliferation of CD4 T cells according to cell tracking dye staining. (C) Proportion of live cells in each group. Data are expressed as mean ± SD. * p<0.05; ** p<0.01; *** p<0.001.

Figure 30 :

 30 Figure 30: sex does not affect the hemodynamic effects of b 1 -AR. Heart rate (A,B), mean arterial pressure (C,D) and cardiac index (E,F) are presented according to the sex of the animals in the b 1 +/+ (A,C,E) and b 1 -/-(B,D,E) groups. Data are expressed as mean ± SD.

Figure 31 :

 31 Figure 31: Sex does not affect the immune effects of b 1 -AR. CD4 T cell (A,B) and Treg cell (C,D) proportions are presented according to the sex of the animals in the b 1 +/+ (A,C) and b 1 -/-(B,D) groups. Data are expressed as mean ± SD.

  adaptive immunity, characterised by a lymphopenia (inhibition of T CD4, T CD8 and B cell proliferations), a decreased production of pro-inflammatory cytokines such as TNF-a, an increased production of the anti-inflammatory cytokine IL-10, and an enhanced bacterial growth.[405][406][407][408][409]. However, no clinical study has specifically investigated the detrimental effects of norepinephrine-induced immunosuppression as this therapeutic agent is meanwhile the cornerstone of the hemodynamic stabilisation at the onset of the shock[385, 410].Several non-adrenergic vasopressors have been explored notably vasopressin orangiotensinII [411, 412]. Despite no overall outcome benefit compared to norepinephrine, low dose of vasopressin reduces norepinephrine requirement and may improve the survival rate of less severe shocked patients[413].

β 1 -

 1 AR and β 2 -AR. Their distribution vary according to organ and species[297]. Classically, β 2 -AR are predominant in vessels, and β 1 -AR are rarely found[429]. In all cases, excessive and persistent β-AR stimulation leads to vascular hyporesponsiveness to catecholamines, associated with AR desensitisation[430]. Nevertheless, β 3 -AR, which are widely present on endothelial cells, are not affected by this desensitisation process[159]. The activation of β 3 -AR is consistent during sepsis and seems to be associated with vasorelaxant effects.

Fig. 1

 1 Fig. 1 Diagramme de flux des études recherchées.

  d'efficacité et de tolérance de la réduction de la FC par βbloquants, chez des patients tachycardes « inadaptés » ou dysautonomiques. Dans toutes les études, les patients étaient considérés comme stables et réanimés de façon optimale sur le plan volémique. Le premier travail retracé n'a été publié qu'en 2006 par Gore et al. Il s'agissait d'un travail prospectif avant-après chez six patients septiques non sévères, qui évaluait les effets physiologiques de la diminution de 20 % de la FC par esmolol. Les auteurs démontraient que la réduction de la FC s'associait à une diminution du débit cardiaque avec une Tableau 2 protein kinase B ; Bax : bcl-2-associated X ; CAMP : cathelicidin antimicrobial peptide ; CLP : caecal ligation and puncture ; DC : débit cardiaque ; dP/dtmax : dérivée première de pression maximale ; dP/dtmin : dérivée première de pression minimale ; ECE-1 : endothelin converting enzyme-1 ; E. : esmolol ; ET-1 : endothelin-1 ; FC : fréquence cardiaque ; GSH : glutathion réduit ; HMGB-1 : high-mobility group box-1 ; HO-1 : hème oxygénase-1 ; IL : interleukine ; INF-γ : interféron-γ ; iNOS : inducible nitric oxide synthase ; IP : intrapéritonéal ; IV : intraveineux ; JNK : c-jun N-terminal protein kinase ;I T V:i n t é g r a l et e m p sv i t e s s e;L P S:l i p o p o l y s a c c h a r i d e;M C P -1:monocyte chemo-attractant protein 1 ; NADPH : nicotinamide adénine dinucléotide phosphate ; NF-κB: nuclear factor-kappa B ;P AM:pressionartériellemoyenne;p-eNOS:phosphorylated endothelial nitric oxide synthase ; SC : sous-cutané ; TNF-α : tumor necrosis factor-α ; VES : volume d'éjection systolique ; ì : augmentation ; î : diminution ; = : identique Définition de la sévérité : +++ : modèle de CLP avec ligature directement sous la valve iléocaecale, modèle non réanimé, PAM basse, mortalité > 5 0% à 24 h ; ++ : modèle de CLP avec ligature à 75 % du caecum, modèle réanimé, mortalité < 50 % à 24 h ; + : modèle de LPS ou CLP avec ligature à 30 % du caecum, temps de l'expérience court. a Expérience réalisée sur colonne de Langerdorff

  Liens d'intérêts : Antoine Kimmoun et Bruno Levy déclarent avoir reçu des rémunérations de Baxter pour des conférences, des financements de congrès et symposium par Baxter et Amomed et de l'assistance technique d'Amomed pour la recherche expérimentale. Manon Durand, Huguette Louis, Caroline Fritz déclarent ne pas avoir de liens d'intérêts.

Références 1 .Annexe 2 :F2

 12 Levy B, Fritz C, Tahon E, Jacquot A, Auchet T, Kimmoun A, (2018) Vasoplegia treatments: the past, the present, and the future. Crit Care 22: 52 2. Abraham E, Singer M, (2007) Mechanisms of sepsis-induced organ dysfunction. Crit Care Med 35: 2408-2416 The β 1 -adrenergic receptor contributes to sepsis-induced immunosuppression through modulation of regulatory T cell inhibitory function Corresponding author: Antoine Kimmoun, Université de Lorraine, CHRU de Nancy, Service de médecine intensive et réanimation Brabois, INSERM U116, F-CRIN-INI-CRCT, Vandoeuvre-lèsshock are a leading cause of intensive care unit admission and recognized by the World Health Organization as a major healthcare issue 1 . Data estimate the number of sepsis cases globally at 48.9 million, resulting in 11.0 million deaths in 2017 2 . Unfortunately, despite significant effort, all therapeutic research conducted over the last four decades has systematically failed 3 .

F

FF

  Shandong, China). Red blood cells were lysed using BD PharmLyse TM (BD Bioscience, San Jose, CA) solution. Splenocytes were then washed twice in PBS before being resuspended in RPMI 1640 medium (Gibco, ThermoFisher Scientific, San Diego, CA) supplemented with 10% FBS.ImmunophenotypingSplenocyte Fc receptors were blocked (CD16/CD32-purified antibodies, eBioscience TM , ThermoFisher Scientific) and cell surface markers used to identify total T cells (CD3+), CD4 T cells (CD3+ CD4+), CD8 T cells (CD3+ CD4-) and Treg cells (CD3+ CD4+ CD25 hi CD127 low ). Th1 (INF-γ+), Th2 (IL-4+) and Th17 (IL-17+) status was characterized by intracellular staining of CD4 T cells. Cell surface antigens were stained for 30 minutes in the dark. For intracellular staining, cells were stimulated for 4 hours at 37 °C with phorbol myristate acetate (PMA, 250 ng/10 6 cells) and ionomycin (500 ng/10 6 cells) (both Sigma Aldrich) and Brefeldin A (BD Bioscience), and then fixed and permeabilised with BD Cytofix/Cytoperm TM solution (BD Bioscience).The following monoclonal antibodies were used according to the manufacturer's recommendations: PerCP Cy5.5-CD3, BV421-CD4, PE-Cy7-CD8, PE-CD25, APC-CD127, APC-IL4, PE-IL-17A (all BD Pharmigen, BD Bioscience), and AF700-INF-γ (Biolegend, San Diego, CA). Dead cells were excluded using Fixable Viability Dye eFluor™ 506 (eBioscience TM , ThermoFisher Scientific). Flow cytometry was carried out using a Gallios analyzer (Beckman Coulter, Brea, CA). Blind analysis of the data was performed using Flowlogic™ software (MiltenyiBiotec, Bergisch Gladbach, Germany).Proliferation assaySplenocytes (1 x 10 6 cells/ml in PBS) were stained with Tag-it Violet TM Proliferation and Cell Tracking Dye (5 μM/10 6 cells; Biolegend) for 20 minutes at 37 °C. Staining was then quenched by addition of culture medium containing 10% FBS. Cells were then centrifuged (300g, 5 minutes), resuspended in RPMI 1640 medium (supplemented with 10% FBS, 2 mM L-glutamine, 20 U/ml penicillin and 20 μg/ml streptomycin) and incubated for 10 minutes at 37°C. Thereafter, splenocytes were seeded (2 x 10 5 cells per well) in 96-well plates and stimulated with phytohemagglutinin (PHA; 4μg/10 6 cells; Remel, ThermoFisher Scientific) for 72 hours at 37°C. Cells were stained with CD4-BV421 (BD Pharmigen) and propidium iodide(MiltenyiBiotec). The Tag-it Violet signal was assessed in live CD4 T cells by flow cytometry.Purification of CD4+ CD25-T cells and CD4+ CD25+ Treg cellsTreg cells were isolated in a two-step procedure following the manufacturer's instructions (CD4+ CD25+ Regulatory T Cell Isolation Kit, MiltenyiBiotec). Briefly, non-CD4 T cells were magnetically labelled with a biotin-conjugated antibody cocktail against CD8a, CD11b, CD45R, CD49b, and Ter-119, then flowed through a magnetic field. CD4-T cells were retained by the magnetic field and CD4 T cells collected. CD4+ CD25+ Treg cells were then magnetically labelled with a biotin-conjugated anti-CD25 antibody. The flow-through containing CD4+ CD25-T cells, while CD4+ CD25+ Treg cells were retained by the magnetic field and then were collected (FigureS2).

F 11 Echocardiography

 11 Transthoracic echocardiography was performed 18 hours after CLP with a VEVO770 (FUJIFILM VisualSonics, Toronto, Canada) and a 30 MHz mechanical probe. The aortic diameter, and velocity integral time were averaged over three consecutive cardiac cycles and used to calculate all parameters : stroke volume (SV), cardiac index (CI), and cardiac power index (CPI). Mean arterial pressure (MAP) was measured by a blood pressure transducer (Emka, Paris, France) inserted through the left carotid artery. Details of the in vivo hemodynamic procedure are provided as supplementary data.

Figure 1 ,

 1 panel A shows that in 1 +/+ mice, sepsis induced a significant decrease in CD4 T cell proportions (-34% vs. sham, p<0.001) counteracted by 1 -AR blockade (+37% vs. CLP, p=0.03). In 1 -/-mice, sepsis significantly decreased the proportion of CD4 T cells (-17% vs. sham, p=0.02), however no change in CD8 T cells proportion was observed between groups (Figure 1, panel B). With respect to Treg cells (CD25 hi CD127 low ), in 1 +/+ mice, sepsis induced an increase in Treg cell proportions (+65% vs. sham, p <0.001), inhibited by 1 -AR blockade (-19% vs. CLP, p=0.04) (Figure 1, panels C, D). In 1 -/-septic mice, the proportion of Treg cells was unchanged (+7% vs. sham, p=0.68). Of note, 1 -AR blockade in 1 +/+ sham mice had no impact on CD4 T cell and Treg cell proportions (Figure S4, panels A, B). No modification was observed in other CD4 T cells subsets (Th1 (INF-+), Th2 (IL-4+

F 1 - 1 -

 11 did not decrease the proliferative capacities of CD4 T cells (-4% vs. sham, p=0.71) (Figure2, panel B). These results were not caused by variations in cell viability among groups (FigureS7). Of note, 1 -AR blockade in 1 +/+ sham mice had no impact on CD4T cell proliferation (FigureS4, panel C). AR stimulation of Treg cells increases their immunosuppressive capacities. The direct effect of 1 -AR stimulation on the immunosuppressive functions of Treg cells was assessed. CD4 T cell counts tended to decrease after five days of co-culture with Treg cells pre-treated with dobutamine, in comparison to untreated Treg cells (p=0.08, Figure 3, panel A). This lower cell count, noted when co-culture was performed with dobutamine pretreated Treg cells, was not caused by an increase in cell death (Figure 3, panel B). Similar results were found when co-culture assays of CD4 T cells were performed in the presence of norepinephrine and phenylephrine pre-treated Treg cells (Figure S8). AR blockade, despite a reduced heart rate, did not alter hemodynamic characteristics in sepsis.

F

F

  . The benefits of β 1 -AR blockade on immune and cardiovascular function should improve the survival rate. However, in the present study, and without any treatment (antibiotics, fluid or

F 22 AK

 22 Figure S1 -S10

F

Figure 1 : 1 -

 11 Figure 1: 1 -AR contributes to the post-septic immunosuppressive phenotype, which is

Figure 2 : 1 -

 21 Figure 2: 1 -AR activation decreases CD4 T cell proliferation during sepsis. Splenocytes

Figure 3 : 1 -

 31 Figure 3: 1 -AR stimulation of Treg cells increases their immunosuppressive capacities.Treg cells isolated from β 1 +/+ mice were treated with dobutamine or PBS for 30 minutes. Pre-

F 32 Figure 4 : 1 -

 3241 Figure 4: 1 -AR modulates the level of both pro-and anti-inflammatory circulatory

Figure 5 : 1 - 1 +

 511 Figure 5: 1 -AR blockade modulates survival during sepsis. Survival curve of 1 +/+ CLP mice,

Figure 6 :F

 6 Figure 6: Graphical summary of the involvement of 1 -AR in sepsis-induced dysautonomia

Figure 1 F

 1 Figure 1

Figure 2 F 36 Figure 4 F

 2364 Figure 2

Figure 5 F

 5 Figure 5

  Figure 6:

  ..........................................................................

Figure S1 :

 S1 Figure S1: Esmolol at 18 mg/kg/h decrease heart rate by 20% in septic mice. ..............................

Figure S2 :

 S2 Figure S2: Isolation of CD4 T cells and Treg cells. .........................................................................

Figure S3 :

 S3 Figure S3: Co-culture of CD4 T cells and Treg cells pre-treated with catecholamines. ...................

Figure S4: 1 -

 1 Figure S4: 1 -AR blockade in sham treated mice does not affect lymphocyte proportions and functions. ....................................................................................................................................

Figure S5: 1 -

 1 Figure S5: 1 -AR does not affect the CD4 T, Th1, Th2, Th17 subpopulation proportions in sepsis.

Figure S6: 1 -

 1 Figure S6: 1 -AR does not affect lymphocyte viability in sepsis. .................................................

Figure S7: 1 -

 1 Figure S7: 1-AR does not affect splenocyte viability after a proliferation assay. .......................

Figure

  Figure S8: Treg cell stimulation by catecholamines increases their immunosuppressive capacities. .................................................................................................................................

Figure S9 :

 S9 Figure S9: Sex does not affect the hemodynamic effects of 1 -AR. .............................................

Figure S10 :

 S10 Figure S10: Sex does not affect the immune effects of 1 -AR. ....................................................

Figure S1 :BFigure S2 :

 S1S2 Figure S1: Esmolol at 18 mg/kg/h decrease heart rate by 20% in septic mice. Esmolol infusion

Figure S3 :

 S3 Figure S3: Co-culture of CD4 T cells and Treg cells pre-treated with catecholamines. Treg cells

Figure S4: 1 -

 1 Figure S4: 1 -AR blockade in sham treated mice does not affect lymphocyte proportions and

Figure S5: 1 -

 1 Figure S5: 1 -AR does not affect the Th1, Th2, Th17 subpopulation proportions in sepsis.

Figure S6: 1 -

 1 Figure S6: 1 -AR does not affect lymphocyte viability in sepsis. Splenocytes were

Figure S7: 1 -

 1 Figure S7: 1-AR does not affect splenocyte viability after a proliferation assay. Eighteen hours

Figure S8 :

 S8 Figure S8: Treg cell stimulation by catecholamines increases their immunosuppressive capacities. Treg cells isolated from β 1 +/+ mice were treated with phenylephrine,

Figure S9 :

 S9 Figure S9: Sex does not affect the hemodynamic effects of 1 -AR. Heart rate (A,B), mean

Figure S10 :

 S10 Figure S10: Sex does not affect the immune effects of 1 -AR. CD4 T cell (A,B) and Treg cell

  1 -AR chez les souris en choc septique, qu'il soit pharmacologique ou génétique, préserve les proportions physiologiques de lymphocytes T CD4 et de Tregs dans la rate. Ceci étant associé à un maintien des capacités prolifératives des T CD4 chez les souris traitées suggérant ainsi que l'activation β 1 -AR participe à

	Limitations de l'étude
	Si des études expérimentales suggèrent que l'administration précoce de β 1 -AR
	bloquant est bénéfique en termes hémodynamique, inflammatoire et pour la survie, les
	études cliniques traitant des patients en choc septique avec des β 1 -AR bloquants sont
	néanmoins peu concluantes ou méthodologiquement fragiles.
	Dans notre modèle, les animaux septiques n'ont pas été réanimés et aucun
	antibiotique n'a été administré, contrairement aux recommandations habituelles pour les
	les animaux β 1 -AR bloqués la FC, déterminant majeur de la consommation en oxygène des
	tissus, était réduite en comparaison avec les animaux non-traités, notre étude suggère ainsi
	une meilleure efficience cardiaque lorsqu'un traitement β 1 -AR bloquant est administré.

l'immunosuppression induite par le sepsis. Le blocage β 1 -AR pourrait donc contrer l'immunosuppression induite par le sepsis et favoriser le passage d'un état immunosuppressif à un état homéostatique.

Le blocage β 1 -AR maintient l'hémodynamique Hormis l'impact immunitaire de la modulation β 1 -AR observé lors d'un choc septique expérimental, il est important de rappeler que le blocage β 1 -AR a également des effets cardiovasculaires majeurs, principalement dus à ses effets chronotropes négatifs. En accord avec la plupart des études expérimentales publiées précédemment, la réduction de la FC chez les souris septiques β 1 -AR bloquée n'a pas modifié l'index cardiaque ni la pression artérielle

[25, 26]

. De fait, il a été constaté que la puissance cardiaque, un marqueur du travail cardiaque externe, était similaire dans les groupes traités et non traités avec un β 1 -AR bloquant. Chez patients en choc septique. L'objectif était de limiter l'administration d'agonistes adrénergiques qui, bien que recommandée dans le traitement du choc septique, aurait interféré avec nos objectifs.

Les études expérimentales reportées sur l'administration de β 1 -AR bloquant sont principalement réalisées sur de petits animaux, souris ou rats, pour lesquels la FC au repos est ente 300 et 400 par minute. Le volume et le débit cardiaque sont dans ces modèles, étroitement dépendant de la FC. Cependant, dans notre étude et dans bien d'autres, tous les modèles murins de sepsis ne sont pas tachycardes

Table 1 : Criteria for SIRS, sepsis, severe sepsis, and septic shock based on the 1991 ACCP/SCCM Consensus Conference.

 1

Table 2 : Criteria for sepsis and septic shock based on the Third International Consensus.

 2

Table 3 : Quick SOFA criteria.

 3

INNATE ADAPTIVE Time (days)

		Late deaths due to
		intractable
		inflamma5on
		induced organ injury
	Early deaths due to
	overwhelming
	inflamma5on
	INNATE
	5	10
	Recovery	Time (days)
	ADAPTIVE
		Late deaths due to
		persistent
		immunosuppressio
		n and recurrent
		infec5ons
	Theory 1: After the onset of sepsis, the hyper-inflammatory response dominates; this state is called the
	'cytokine storm'. In the second step, an anti-inflammatory compensatory syndrome occurs and may cause
	immunosuppression. If patient does not achieve homeostasis, he frequently dies from a secondary infection.

Table 5 : Effects on sepsis on CD4 T cells. According

 5

to the reference:

[236]

Table 6 : Classification, localisation, signalling, agonist, antagonist, hemodynamic effects and inflammatory effects of main adrenoreceptors.

 6 According to the references:[296 , 297 , 298]

	Adreno-	Localis	Signalling				Agonist	Antagonist Hemodynamic	Inflammatory
	receptor	ation								effects	effects
	α 1	Heart	Gα q protein				Phenylephrine	Prazosin	Vasoconstricti	Pro-
		Blood	→ ↗PLC				Methoxamine	Doxazosin	on	inflammator
		vessels	→ ↗Ca 2	+ ↗PKC			Terazosin	Smooth	y
		Lung	→ ↗ Eicosanoids,	muscle
		Digestive	↗ Ca	2+ channels,	contraction
		system	↗MAPK signals,
			Regulation of K	+
			channels
	α 2	Blood	Gα l		and	Gα o	Clonidine	Yohimbine	Vasoconstricti	Pro-
		vessels	protein					Idazoxan	on	inflammator
		Digestive	→ ↘AC					Atipamezole	y
		system	→ ↘cAMP, ↘PKA,
		Nervous	↗ K	+	channel
		system	activity
	β 1	Heart	Gα s protein				Isoproterenol	Propranolol	Positive	Pro-
		Kidney	→ ↗AC					Dobutamine	Metoprolol	inotropic and	inflammator
		Adipose	→	↗	cAMP,	Atenolol	chronotropic	y
		cells	↗PKA, ↗ Ca	2+	Esmolol	effects
			channel activity			Landiolol
	β 2	Heart	Gα s and G α l	Isoproterenol	Propranolol	Vasodilatation	Anti-
		Blood	protein					Terbutamine	Butaxamine	Smooth	inflammator
		vessels	→ ↗AC					Albuterol	muscle	y
		Lung	→	↗	cAMP,	Salmeterol	relaxation
		Digestive	↗PKA, ↗ Ca	2+
		system	channel activity
		Liver
		Urinary
		system
		Reproduc
		tive
		system
		Nervous
		system
	β 3	Adipose	Gα s		and	Gα l	Isoproterenol	Propranolol	Negative	Anti-
		cells	protein					Mirabegron	inotropic	inflammator
		Heart	→ ↗AC					Amibegron	effects	y
			→ ↗PKA, ↗ Ca ↗ cAMP, 2+	Solabegron
			channel activity

Abbreviations: AC: adenylate cyclase; cAMP: cyclic adenosine monophosphate; MAPK: mitogen-activated protein kinase; PLC: phospholipase C; PKA: protein kinase A; PKC: protein kinase C; →: induced; ↗: increased; ↘: decreased.

  Nevertheless, noncardioselective β-AR blockers, i.e. β 1 /β 2 -AR blockers, are probably less suitable than cardioselective β 1 -AR blockers. Indeed, β 2 -AR inhibition is counterintuitive since β 2 -AR activation has a key role in the inflammatory balance [295]. Both experimental and clinical

  minutes and an elimination half-life of 9 minutes[351] [352]. Esmolol-induced β 1 -AR blockade can be maintained as long as the infusion is continued. This molecule is hydrolysed in blood by esterases and metabolised in the liver by forming its main metabolites, which are methanol and propionic acid. The metabolites, cleared by the renal system, have 1500-fold less β 1blocking activity than esmolol. Thus, no further effect is detected 30 minutes after treatment has stopped[353]. The concentration of methanol, which is a metabolite of esmolol, remains within the range of the normal endogenous level[354].

	[TW] OR 'adrenergic beta receptor blockaders' [TW] OR 'blockers, beta-adrenergic' [TW] OR
	'beta adrenergic receptor blockaders' [TW] OR 'beta adrenergic blocking agents' [TW] OR
	'beta adrenergic blockers' [TW] OR 'adrenergic beta-blockers' [TW] OR 'beta-adrenergic
	blockers' [TW] OR 'adrenergic beta-receptor blockaders' [TW] OR 'beta-adrenergic blocking
	agents' [TW] OR 'beta-adrenergic receptor blockaders' [TW] OR 'adrenergic beta antagonists'
	'septicaemic shock' [TW] OR 'sepsis-associated hypotension' [TW] OR 'septicaemic shock'
	[TW])) OR ('sepsis' [MH] OR ('sepsis' [TW] OR 'blood poisoning' [TW] OR 'septicaemias' [TW]
	OR 'blood poisonings' [TW] OR 'sepsis, severe' [TW] OR 'pyohaemia' [TW] OR 'septicaemia'

Search and selection of studies investigating the effect of β 1 -AR blockers on septic shock We conducted a systematic literature search of experimental and clinical studies on the hemodynamic and inflammatory effects of β 1 -AR blocker treatment in septic shock in the period ranging from January 1, 1980 through September 30, 2020. The following MeSH terms were applied on the PubMed website: 'shock, septic' [MH] OR ('shock, septic' [TW] OR 'septic shock' [TW] OR 'toxic shock' [TW] OR 'endotoxic shock' [TW] OR 'toxic shock syndromes' [TW] OR 'syndrome, toxic shock' [TW] OR 'toxic shock syndrome' [TW] OR 'septic shock' [TW] OR 'septic shock (disorder)' [TW] OR [TW] OR 'pyaemia' [TW] OR 'severe sepsis' [TW] OR 'pyaemia' [TW] OR 'poisoning, blood' [TW] OR 'pyaemias' [TW] OR 'septicaemia' [TW] OR 'unspecified septicaemia' [TW] OR 'unspecified septicaemia' [TW] OR 'bacteraemia' [TW] OR 'SIRS' [TW] OR 'systemic inflammatory response syndrome' [TW])) AND ('adrenergic beta-antagonists' [MH] OR ('adrenergic beta-antagonists' [TW] OR 'beta blockers' [TW] OR 'beta-adrenergic antagonist' [TW] OR 'beta-blocker poisoning' [TW])) NOT Review [ptyp] AND ('1990/01/01' [PDAT]: '2020/09/30' [PDAT])

Table 7 : Summary of experimental studies

 7

	Author	Model	Severity	β-blocker	Time of		Survival	Assessment: cardiac	Assessment:	Lactate	Assessment: immunity function
						assessment		function	vasoreactivity		Systemic		Tissue
		SMALL ANIMAL STUDIES
	Suzuki	et	Rat	+++	E. (10 mg/kg/h or 20 mg/kg/h)	24h	after	NA	In vivo:	NA	=	↘		NA
	al.,	2005	Male		to reduce HR	induction		↘ MAP for E. 10 mg/kg/h			Markers: TNF-α ↘, no
	[36]		CLP						= MAP for E. 20mg/kg/h			effect on white blood
					Injection: during CLP				Ex vivo*:			cells count
									↗ CO
									↗ SV
									↗ dP/dtmax, dP/dtmin
	Schmitz et	Mice	++	Propranolol (0.5 mg/k, SC,	48h	after	↘	NA	NA	NA	NA		↗
	al.,	2007	Male		every 2h)	induction							Markers:	apoptosis	↗
	[355]		CLP											proliferation↘, IL-6 ↗
					Injection: 20 min before CLP									and INF-γ ↘ in splenocytes
	Hagiwara et	Rat	+	Landiolol (0.1 mg/kg/min) to	12h	after	NA	In vivo:	NA	NA	↘		↘
	al.,	2009	Male		reduce HR by 25%	induction		= MAP			Markers:	peak	Markers: inflammatory cells
	[356]		LPS						Ex vivo*:			circulating level of	infiltration↘, oedema↘, lung
					Injection: before LPS				↗ dP/dtmax, dP/dtmin			TNF-α, IL-6 and HMGB-	DNA binding activity of NF-κB
												1 ↘		↘
	Ackland et	Rat	+	Metoprolol (100 mg/kg, IP),	24 to 48h	↗	In vivo:	NA	NA	↘		↘
	al.,	2010	Male		Atenolol (6 mg/kg, IP) to	after			= CO			Marker: IL-6 ↘		Markers: hepatic cytokines ↘
	[34]		CLP	or	reduce HR by 20%	induction		= SV					(TNF-α, IL-1β, IL-6, INF-γ)
			LPS						↗ MAP					cardiac	inflammation	↘
					Injection: 2 days before				↗ dP/dtmax, dP/dtmin					(MCP-1, IL-18)
	Mori et al.,	Rat	++	E. (15 mg/kg/h) to reduce HR	18h	after	↗	In vivo:	NA	=	NA		↘
	2011 [357]	Male		by 20%	induction		= MAP					Markers: intraperitoneal TNF
			CLP											α ↘, injury score of the gu
					Injection: 1h after CLP									mucosa ↘
	Wilson et	Rat	+	Propranolol (10 mg/kg, IP) to	24h	after	↗	In vivo:	NA	=	↘		↘
	al.,	2013	Male		reduce	induction		= MAP			Marker: TNF-α ↘		Marker: lung HO-1 expression
	[358]		CLP											↗
					Injection: 30 min before CLP
					then every 8h
	Ibrahim-	Mice	+	E. (6.67 μg/kg/min)	48h	after	↗	In vivo:	NA	NA	NA		↘
	Zada et al.,	Male			induction		= VTI					Marker:	CAMP	gene
	2014 [359]	LPS		Injection: 24 h before LPS									expression ↗
	Seki et al.,	Rat	+	Landiolol (100 μg/kg/min)	3h	after	NA	In vivo:	NA	↘	=		↘
	2014 [360]	Male			induction		= MAP			Markers: no difference	Marker:	endothelin
			LPS		Injection: 15 min before LPS							on TNF-α, IL-6 and ET-	converting enzyme ECE-1 ↘
												1
	Abdel et al.,	Rat	+++	Carvedilol (2 mg/kg, IP)	18h	after	↗	In vivo:	NA	NA	↘		↘
	2015 [361]	Male			induction		= MAP			Markers: TNF-α et
			CLP		Injection: 30 min before CLP							IL-6↘

Abbreviations: AKT: protein kinase B; Bax: bcl-2-associated X; CAMP: cathelicidin anti-microbial peptide; CLP: caecal ligation and puncture; CVP: Central venous pressure; CO: cardiac output; dP/dtmax: derivative of pressure over time maximum; dP/dtmin: derivative of pressure over time minimum; ECE-1: endothelin converting enzyme-1; E.: esmolol; ET-1: endothelin-1; GSH: reduced glutathione; HMGB-1: high-mobility group box-1; HO-1: haem oxygenase-1; HR: heart rate; IL: interleukin; INF-γ: interferon-γ; iNOS: inducible nitric oxide synthase; IP: intraperitoneal; IV: intravenous; JNK: c-Jun N-terminal protein kinase; LPS: lipopolysaccharide; MAP: mean arterial pressure; MCP-1: monocyte chemo-attractant protein 1; NADPH: nicotinamide adenine dinucleotide phosphate; NF-κB: nuclear factor-kappa B; p-eNOS: phosphorylated endothelial nitric oxide synthase; SC: subcutaneous; SV: stroke volume; TNF-α: tumour necrosis factor-α; VIT: velocity integral time; ↗: increased; ↘: decreased; =: equal. *Experiment performed with a Langendorff isolated heart perfusion apparatus.

Table 8 : Summary of non-comparative clinical studies.

 8

	Author	Type of study	Method		Time of	Patients	Heart rate (/min)	Stroke	Vascular	Mortality	Tissue	Additional results
		design			assessment	number	mean±SD or	volume and	resistance		perfusion
							median	cardiac
							[interquartile]	output
	Gore et al.,	Prospective,	Sepsis without shock	Before and	6	114± 15	SV: ↗	=	NA	=	Not very severe patients
	2006 [375]	interventional,			after 3 h of E.		vs.					No anabolic effect on energy
		monocentric,	E. to reduce HR by 20%	administration		91± 12	CO: ↘				metabolism
		before-after
	Schmittinger	Retrospective,	Septic shock	Before	40	101± 18	SV: ↗	=	33% at	=	Cardiac output decrease in 7
	et al., 2008	monocentric			metoprolol		vs.			days		patients with milrinone
	[376]		Metoprolol per os to	administration		84± 13	CO: =				administration
			reduce HR <95/min	and at 6,12,
			+/-		24, 72 and 96
			milrinone if CI <2.5	h
			l/min/m	2
	Balik et al.,	Prospective,	Bolus of E. followed by	Before E.	10	142± 11	SV: =	=	10% at	=	Very mild patients
	2012 [377]	monocentric	continuous 24h infusion	administration		vs.			days
			to reduce HR by 20%	and at 2,6, 12,		112± 9	CO: =
					24 and 30 h
	Chen et al.,	Prospective,	Severe sepsis	Before E.	16	114 ± 15	SV: =	=	NA	=
	2013 [378]	monocentric,			administration		vs.
		before-after	Continuous infusion of E.	and at 3 h		91 ± 13	CO: ↘
			to reduce HR by 20%
	Morelli et	Prospective,	Septic shock	At 24 h	25	117 [112-126]	SV: =	NA	NA	=	Improvement of microvascular
	al., 2013	interventional,					vs.					flow index
	[374]	monocentric,	Continuous infusion of E.			86 [80-89]	CO: ↘				Norepinephrine requirements
		before-after	to reduce HR between 80								were significantly reduced
			and 94/min								with E. therapy
	Morelli et	Prospective,	Septic shock	Before E.	45	115 ± 11	SV: =	↘	51% at	=	Ventricular-arterial decoupling
	al., 2016	monocentric,			administration		vs.			days		improvement
	[379]	before-after	Continuous infusion of E.	and at 4 h		88 ± 9	CO: ↘
			to reduce HR between 80
			and 94/min
	Du et al.,	Prospective,	Septic shock	Before E.	63	107.8 ± 8.7	SV: ↗	NA	6.3% at 28	=	Very low mortality
	2016	monocentric,	hemodynamically stable	administration		vs.			days
	[380]	before-after	for at least 3h with HR	and 2 h after		86.2 ± 10.2	CO: ↘				Target blood pressure higher
			>100/min	achieving the							than recommended in
					targeted HR.							surviving sepsis campaign
			Continuous infusion of E.
			to reduce HR by 10-15%

Abbreviations: CI: cardiac index; CO: cardiac output; E.: esmolol; HR: heart rate; SV: stroke volume; vs.: versus; ↗: increased; ↘: decreased; =: same.

Table 9 : Summary of comparative clinical studies.

 9

	Author	Type of study	Goal		Inclusion		Method	Patients	Main result	Secondary results	Remark
			design							number
	Morelli et	Randomised,	HR reduction	Stabilised		2 groups:E. vs. C.	154 (77 in	HR (/min)*	Increased in E., vs. C. group:	The	need	for
	al., 2013	open-label,		between 80 et	septic shock	Levosimandan administration if	each group)	E.: -28 [-37--21]	SV* and SVR*	levosimendan	rescue
	[25]		comparative,	94 /min		for 24 hours	SvO2<65%		vs.	28 day mortality*	therapy did not differ
			monocentric,			with	HR	Assessment before and at 24, 48,		C.: -6 [-14 -0]	E.: 49.4%	between groups (E.: 49.9%
			phase II				>95/min		72, 96 h			vs.C.: 80.5%	vs. C.: 40.3%)
	Yang et	Randomised,	Not	clearly	Septic shock	Randomisation within 6 h	41	HR (/min)*	The ScvO2 was not decrease in both group	Target blood pressure
	al., 2014	controlled,		defined.				2 groups:	E.: 21	E.: 93 ± 4	higher	than
	[381]		monocentric	General				E. vs. C.	vs.	vs.	Troponin I significantly decrease in E. group vs.	recommended in surviving
					hemodynamic				C.: 20	C.: 118 ± 13	C. group	sepsis campaign
					assessment			HR reduction <100/min within 2 h
									after inclusion in the E. group.		CI (l/min/m	2 )*
											E.: 3.3 ± 8
									Assessment before and at 12, 24,		vs.
									48 and 72 h		C.: 4.5 ± 6
	Wang et	Randomised,	HR reduction	Sepsis with	3 groups:	90 (30 in	Successful	HR	Increase in E. -milrinone vs. C. group: SV* and
	al., 2015	controlled,		between 75 et	HR>95/min	C. vs. milrinone vs. E. -milrinone	each group)	control*:	CI*
	[382]		open-label,		94 /min						C.: 43.3%	Decrease in E.-milrinone vs. C. group: TNF-a
			monocentric,					Bolus of milrinone followed by		vs.	and IL-6
			phase II						continuous administration		milrinone: 53.3%	28 day mortality*:
											vs.	C.: 73%
									Continuous administration of E.		E. -milrinone: 100%	vs. milrinone: 67%
												vs. E. -milrinone: 40%
									Assessment before and at 12, 24,
									48 and 72 h
	Xinqiang	Randomised,	28	day	Stabilised		E. continuous administration to	48 (24 in	28 day mortality *:	Decrease at 24 h in E. vs. C. group: HR* and at
	et	al.,	controlled,		mortality and	septic shock	obtain a HR <100/min	each group)	E.: 25%	48 hours: lactate*
	2015		double-blinded,	length of stay in	for 6 hours			vs.C.: 62.5%	Increase at 24 in E. vs. C. group: SVR*, ScvO2*
	[383]		monocentric	ICU		with		Assessment before and at 24, 48		Length of stay in ICU	No difference at 24 h in E. vs. C. group: arterial
							HR>95/min	and 72 h		(days)*:	blood pressure, central venous pressure, CI
											E.: 13.75 ± 8.68
											vs. C.: 21.7 ± 6.06
	Shang et	Prospective,	Not	clearly	Severe sepsis	E. continuous administration or	151	At 24 h	At 24 hours:	No randomisation in this
	al., 2016	cohort,	non-	defined.		with		natural saline in C. group	E.: 75	HR (/min)*:	ScvO2 *:	work
	[384]		randomised	General		HR>100/min		vs.	E.: 101.96 ± 7.36	E.: 0.8 ± 0.05	Low mortality (no septic
			comparison	hemodynamic			Assessment before and at 24, 48	C.: 76	vs.	vs. C.: 0.76 ± 0.05	shock)
			between C. and	assessment			and 72 h		C.: 110.12 ± 8.59
			E.									28 day mortality:
												E.: 5.3%vs. C.: 7.9%
	Abbreviations: C.: control; CI: cardiac index; E: esmolol; HR: heart rate; IL: interleukin; Svc02: central venous oxygen saturation; SV: stroke volume; SVR: systemic vascular resistance; TNF-a: tumour necrosis factor-a;
	vs.: versus; * p<0.05.

Table 10 : Primers sequence used for mouse genotyping.

 10

Table 11 : Number and reasons for failed measurements and excluded values.

 11

	Experiment	n=	Measure failed	Reason	Excluded value	Reason
	T cells		11	Reagent expired	4	No staining
	Immunophenotyping
	Treg cells		11	Reagent expired	1	No staining
	Echocardiography	74	NA		NA
	Mean arterial pressure measurement		4	Catheterization failed	NA
	Plasma level of inflammatory mediators		NA		37
	Proliferation assay	44	NA		NA
	CD4 T and Treg cells co-culture	4	1 well	Contaminated	NA
						Mouse dead
	Survival study	60	NA		1	before the first
						count

* Due to covid-19 pandemia, Magnetic Luminex® Performance Assay -Mouse XL Discovery Premixed kit (Bio-Techne, Minneapolis, MN) distribution was stopped by the manufacturer. Thus 37 plasma samples were analyzed. * Not measured

Table 12 : Despite reduced heart rate, b 1 -AR blockade did not alter hemodynamic characteristics in sepsis.

 12 Hemodynamic characteristics were measured by echocardiography 18 hours after surgery. Data are expressed as mean ± SD.

	β 1	+/+
	Variable

Table 13 : Sepsis induces an increase in both pro-and anti-inflammatory mediators in β 1 +/+ and β 1 -/-mice.

 13

	Data are expressed as mean ±SD.
	Cytokine	β 1	+/+
	(pg/ml)

  The role of adrenoceptor-mediated signals in the modulation of lymphocyte function. AdvNeuroimmuno 5: 283-298 395. Rohrer DK, Desai KH, Jasper JR, Stevens ME, Regula DP, Jr., Barsh GS, Bernstein D, Kobilka BK, (1996) Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects. Proc Natl Acad Sci U S A93: 7375-7380 396. Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA, (2009) Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Prot 4: 31-36 397. Osuchowski MF, Ayala A, Bahrami S, Bauer M, Boros M, et al., (2018) Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS): An International Expert Consensus Initiative for Improvement of Animal Modeling in Sepsis. Shock 50: 377-380 398. Hoffman M, Kyriazis ID, Lucchese AM, de Lucia C, Piedepalumbo M, Bauer M, Schulze PC, Bonios MJ, Koch WJ, Drosatos K, (2019) Myocardial Strain and Cardiac Output are Preferable Measurements for Cardiac Dysfunction and Can Predict Mortality in Septic Mice. J Am Heart Assoc 8: e012260 399. Ruiz S, Vardon-Bounes F, Merlet-Dupuy V, Conil JM, Buléon M, Fourcade O, Tack I, Minville V, (2016) Sepsis modeling in mice: ligation length is a major severity factor in cecal ligation and puncture. Intensive Care Med Exp 4: 22 400. Coakley JD, Breen EP, Moreno-Olivera A, Al-Harbi AI, Melo AM, O'Connell B, McManus R, Doherty DG, Ryan T, (2019) Dysregulated T helper type 1 (Th1) and Th17 responses in elderly hospitalised patients with infection and sepsis. PloS One 14: e0224276 401. Xue M, Xie J, Liu L, Huang Y, Guo F, Xu J, Yang Y, Qiu H, (2019) Early and dynamic alterations of Th2/Th1 in previously immunocompetent patients with communityacquired severe sepsis: a prospective observational study. J Transl Med 17: 57 402. Lemaire LC, de Kruif MD, Giebelen IA, Levi M, van der Poll T, Heesen M, (2006) Dobutamine does not influence inflammatory pathways during human endotoxemia.

	Crit Care Med 34: 1365-1371
	403. Levy B, Fritz C, Tahon E, Jacquot A, Auchet T, Kimmoun A, (2018) Vasoplegia
	treatments: the past, the present, and the future. Crit Care 22: 52
	404. Andreis DT, Singer M, (2016) Catecholamines for inflammatory shock: a Jekyll-and-
	Hyde conundrum. Intensive Care Med 42: 1387-1397
	405. Freestone PP, Lyte M, Neal CP, Maggs AF, Haigh RD, Williams PH, (2000) The
	mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial
	growth in the presence of transferrin or lactoferrin. J bacteriol 182: 6091-6098
	406. Freestone PP, Haigh RD, Lyte M, (2007) Blockade of catecholamine-induced growth
	by adrenergic and dopaminergic receptor antagonists in Escherichia coli O157:H7,
	Salmonella enterica and Yersinia enterocolitica. BMC microbiol 7: 8
	407. Huang HW, Tang JL, Han XH, Peng YP, Qiu YH, (2013) Lymphocyte-derived
	catecholamines induce a shift of Th1/Th2 balance toward Th2 polarization.
	Neuroimmunomodulation 20: 1-8
	408. Kohm AP, Sanders VM, (2001) Norepinephrine and beta 2-adrenergic receptor
	stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo.
	Pharmacol Rev 53: 487-525
	409. Prass K, Meisel C, Höflich C, Braun J, Halle E, et al., (2003) Stroke-induced
	immunodeficiency promotes spontaneous bacterial infections and is mediated by
	sympathetic activation reversal by poststroke T helper cell type 1-like
	immunostimulation. J Exp Med 198: 725-736

  Tableau 2 Résumé des études expérimentales

	Auteurs	Modèle Sévérité ß-bloquant	Moment	Survie Évaluation : fonction	Évaluation :	Lactate Évaluation : fonction immunitaire
					de l'évaluation		cardiaque	vasoréactivité
										Systémique	Tissulaire
	Étude sur le petit animal
	Suzuki et al.	Rat	+++	E. (10 mg/kg/h	24 h après	NA	In vivo :	NA	=	î	NA
	2005 [34]	Mâle		ou 20 mg/kg/h)	induction		î PAM pour E. 10 mg/kg/h			Marqueurs :
		CLP		pour réduire la FC			= PAM pour E. 20 mg/kg/h			TNF-α î, nombre
				Injection : lors de la CLP			Ex vivo a :			de globules blanc
							ì DC			ne varie pas
							ì VES
							ì dP/dt max , dP/dt min
	Schmitz et al.	Souris	++	Propranolol (0,5 mg/k	48 h après	î	NA	NA	NA	NA	ì
	2007 [35]	Mâle		en SC tous les 2 h)	induction						Marqueurs : apoptose ì,
		CLP		Injection : 20 min							prolifération î, IL-6 ì et
				avant CLP							INF-γ î
											dans les splénocytes
	Hagiwara et al.	Rat	+	Landiolol (0,1 mg/kg/	12 h après	NA	In vivo :	NA	NA	î	î
	2009 [36]	Mâle		min) pour réduire	induction		= PAM			Marqueurs :	Marqueurs : infiltration
		LPS		de 25 % la FC			Ex vivo a :			pic de TNF-α, IL-6	des cellules inflammatoires
				Injection : avant les LPS			ì dP/dt max , dP/dt min			et HMGB-1 î	î, oedème î, liaison NF-
											κB à ADN î
											dans les poumons
	Ackland et al.	Rat	+	Métoprolol (100 mg/kg	24 à 48 h après	ì	In vivo :	NA	NA	î
	2010 [37]	Mâle		en IP), Aténolol	induction		= DC			Marqueur :
		CLP		(6 mg/kg en IP)			= VES			IL-6 î
		ou LPS		pour réduire de 20 %			ì PAM
				la FC			ì dP/dt max , dP/dt min
				Injection : 2 jours avant

  Tableau 3 Résumé des études cliniques non comparatives

	Auteurs	Type d'étude Méthode	Moment de l'évaluation augmentation du volume d'éjection systolique sans effet Nombre de patients FC (/min) moyenne ± ET ou médiane [interquartile] Volume d'éjection systolique et débit cardiaque Résistances vasculaires hypoxique associé ni modification des résistances vasculaires.	Mortalité Perfusion tissulaire	Résultats additionnels	Méd. Intensive Réa
	Gore	Prospective,	Sepsis sans choc	Avant et après	6	114 ± 15	VES : ì	=	NA	=	Patients peu sévères
	et Wolfe	intervention-	E. pour réduire la FC	3 heures		vs.	DC : î				Aucun effet
	2006 [55]	nelle,	de 20 %	d'administration		91 ± 12					anabolique
		monocentri-		d'E.							sur le métabolisme
		que, avant-									énergétique
		après
	Schmittin-	Rétrospective,	Choc septique	Avant	40	101 ± 18	VES : ì	=	33 %	=	Diminution du débit
	ger et al.	monocentrique	Métoprolol per os	l'administration		vs.	DC : =		à 28 jours		cardiaque
	2008 [56]		pour réduire	de métoprolol		84 ± 13					chez 7 patients
			la FC < 95/min	puis à 6, 12, 24,							avec administration
			±	72 et 96 heures							de milnirone
			Milrinone si
			IC < 2,5 l/min/m 2
	Balik et al.	Prospective,	Bolus d'E. puis	Avant	10	142 ± 11	VES : =	=	10 %	=	Patients très peu
	2012 [57]	monocentrique	perfusion continue	l'administration		vs.	DC : =		à 28 jours		sévères
				d'E. puis à 2,6,		112 ± 9
				12, 24 et
				30 heures
	Chen et al.	Prospective,	Sepsis sévère	Avant	16	114 ± 15	VES : =	=N A	=
	2013 [58]	monocentri-	E. en perfusion	l'administration		vs.	DC : î
		que, avant-	continue pour réduire	d'E. puis		91 ± 13
		après	la FC de 20 %	à 3 heures
	Morelli	Prospective,	Choc septique	À 24 heures	25	117 [112-126]	VES : =	NA	NA	=	Amélioration
	et al. 2013	intervention-	E. en perfusion			vs.	DC : î				de l'index de débit
	[54]	nelle,	continue pour réduire			86 [80-89]					microvasculaire
		monocentri-	la FC entre 80 et 94/								en OPS
		que, avant-	min								Diminution sous E.
		après									de la posologie
											de noradrénaline
	Morelli	Prospective,	Choc septique	Avant	45	115 ± 11	VES : =	î	51 %	=	Amélioration
	et al. 2016	monocentri-	E. en perfusion	l'administration		vs.	DC : î		à 28 jours		du couplage
	[59]	que, avant-	continue pour réduire	d'E. puis		88 ± 9					ventriculoartériel
		après	la FC entre 80 et	à 4 heures
			94/min
											(Suite page suivante)

Table 1 : Despite reduced heart rate, 1 -AR blockade did not alter hemodynamic characteristics in high-grade sepsis.

 1 Hemodynamic characteristics were measured by echocardiography 18 hours after surgery. Data are expressed as mean ± SD.

	30

Résumé Contexte :

 Contexte La dysautonomie sympathique est une complication sepsis à l'origine d'une altération des fonctions cardiovasculaires, d'une immunosuppression et d'une surmortalité. Lors d'une septicémie, la modulation du système sympathique par le blocage des β1-adrénorécepteurs protège les fonctions cardiovasculaires. En revanche, l'effet du blocage β1-adrénergique sur la réponse immunitaire adaptative n'a que très peu été étudiée. Objectifs : Cette étude vise à étudier les effets de la modulation β1-AR sur les dysfonctions lymphocytaires T CD4 (LT CD4) dans un modèle murin sévère de sepsis. Méthodes et résultats : La septicémie est induite par ligature et ponction du caecum chez des souris knock-out pour le β1-adrénorecepteur et des souris sauvages traitées ou non par un β1-AR bloquant (esmolol). Dix-huit heures après la chirurgie, les souris présentaient toutes les caractéristiques d'un sévère sepsis et notamment une diminution des proportions des LT CD4 et de leurs capacités prolifératives, ainsi qu'une augmentation des proportions des LT régulateurs (Tregs), trois marqueurs caractéristiques de l'immunoparalysie. Le blocage pharmacologique et génétique des β1-adrenorecepteurs a permis de rétablir des proportions de LT CD4 et de Treg similaires aux souris non-septiques, mais également de maintenir les capacités prolifératives des LT CD4. Le blocage β1-adrénergique entraîne également une pondération globale des cytokines inflammatoires circulantes ainsi qu'une amélioration de l'efficience myocardique in vivo.Conclusion :Le renforcement des fonctions inhibitrices des Tregs survenant lors du sepsis est réduit pour le blocage β1-adrénergique. Dès lors, bloquer ces récepteurs pourrait avoir un réel intérêt thérapeutique pour contrer l'immunosuppression post-septique en rétablissant l'homéostasie des LT CD4.

L'histoire naturelle d'une infection grave est un continuum qui débute par le sepsis simple et qui évolue en choc septique. Schématiquement, pour la forme la plus sévère de l'infection, l'invasion de l'organisme par un agent pathogène est associée à une réponse inflammatoire exagérée à l'origine d'une vasodilatation et d'une coagulopathie prothrombotique. L'ensemble de ces phénomènes est responsable des dysfonctions d'organes de l'état de choc septique. Les réponses de l'organisme face à cette agression visent à assurer, en premier lieu, le transport en oxygène. Les structures impliquées pour le maintien de ce transport sont nombreuses, mais incluent principalement les systèmes : nerveux sympathique et parasympathique, corticosurrénalien, posthypophysaire antidiurétique et rénine-angiotensine-aldostérone[1].En l'absence de traitement, l'évolution vers le choc septique se caractérise par l'importance de la réponse proinflammatoire initiale qui va sidérer, à différents degrés, les systèmes neuroendocriniens et endocriniens périphériques précités et aggraver l'hémodynamique[2].L'activation du système nerveux sympathique, en libérant de la noradrénaline et de l'adrénaline, est une réponse essentielle à l'agression à la phase initiale de tout processus septique. Ces effets sont une augmentation de l'inotropisme, une tachycardie et une vasoconstriction, qui contrecarrent les effets toxiniques et/ou inflammatoires initiaux du processus infectieux. Néanmoins, la persistance de cette activation dans le temps à un haut niveau d'intensité peut aussi être délétère et entraîner un syndrome de dysautonomie sympathique[3]. M. Durand • C. Fritz • B. Levy • A. Kimmoun (*) Inserm U1116, équipe 2, défaillance circulatoire aiguë et chronique, faculté de médecine de Nancy, F-54500 Vandoeuvre-lès-Nancy, France e-mail : a.kimmoun@chru-nancy.fr H. Louis Inserm U1116, équipe 1, défaillance circulatoire aiguë et chronique, faculté de médecine de Nancy, F-54500 Vandoeuvre-lès-Nancy, France C. Fritz • A. Kimmoun Service de réanimation et médecine intensive Brabois, CHRU de Nancy, F-54511 Vandoeuvre-lès-Nancy, FranceMéd. Intensive RéaDOI 10.3166/rea-2019-0095

Méd. Intensive Réa

Remerciements

Acknowledgments:

The authors wish to thank IBSLor facility for the flow cytometer and Biorender.com for the creation of the diagrams. The authors are grateful to Stefan Engelhardt (Pharmacology and Toxicology of Munich, Germany) for providing 1 -/-mice and to François Husson (Agrosup, France) for the help in performing the statistical analysis.

Availability of supporting data.

Raw data and statistical analyses of all presented results are available at the following: https://osf.io/nz5u9/ or DOI 10.17605/OSF.IO/NZ5U9. MD takes the responsibility of the integrity of the data.

Sources of Funding:

This work was supported by the Fondation pour la Recherche Médicale (FRM grant number

ECO20170637495 to MD)

Disclosures:

Abbreviations

Survival study

Survival was studied in b 1 +/+ CLP mice, b 1 +/+ CLP + esmolol (18 mg/kg/h) mice, and b 1 -/-CLP mice. Census of animals was undertaken twice a day for 5 days. Administration of esmolol (Esmocard®, Amomed) was started 3 hours post-CLP and continued for a total infusion period of 24 hours. The one animal that died before the first count was considered a technical failure and excluded from the study.

Statistical analysis

Results are expressed as mean and standard deviation (mean ± SD). Results in b 1 -/-mice were compared using a Mann-Whitney test while the Kruskal-Wallis test was used to compare b 1 +/+ groups. When the Kruskal-Wallis test was significant, post hoc comparisons were performed between sham, CLP, and CLP + esmolol groups using Dunn's multiple comparisons and a Bonferroni correction.

Variations were calculated as follows: 100-((mean group 1/mean group 2) x100).

To analyze the effect of catecholamines on Treg cells co-cultured with CD4 T cells, we firstly assessed the interaction between mouse and catecholamine at each time point. Considering the mouse has a random effect, the p-value of the test for the treatment effect was calculated.

Only the last p-value (obtained at 132 hours) is shown.

For measurement of plasma inflammatory mediators, groups were compared using the Mann-Whitney test. A Benjamini-Hochberg adjustment was performed for multiple comparisons.

Kaplan-Meier curves were drawn. Significance was assessed using the log-rank test.

P values <0.05 were considered statistically significant.

Statistical analysis was performed using R, version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria), using Graph-Pad Software 6.0, (San Diego, CA) and using NCSS Statistical Software 9 (East Kaysville, UT).

Annexes

Annexe 1: β-bloquants dans la prise en charge du choc septique

Detailed Methods

Echocardiography: Mice were placed on a prewarmed (38 °C) electric heat pad, anaesthetized with 1.5% isoflurane and kept at rest for 5 minutes before the echocardiographic assessment.

Ascending aortic diameter (AD) was obtained during systole from the left parasternal long axis view. The aortic velocity time integral (VTI) was measured from the aortic arch.

Stroke volume (SV) and cardiac output (CO) were calculated with the following formulas:

The CO was divided by the weight of the mouse to define the cardiac index (CI). The cardiac power index (CPI) was calculated with the following formula: CPI = MAP x CI / 451. All measurements were averaged over three consecutive cardiac cycles. Mean arterial pressure (MAP) was measured by a blood pressure transducer (Emka, Paris, France) inserted through the left carotid artery.

Supplemental Tables