Tonia Akoumianaki

Katerina Vaporidi

Eleni Diamantaki

Frédéric Pène

Remi Beau

Mark S Gresnigt

Marina Gkountzinopulou

Maria Venichaki

Elias Drakos

Jamel El-Benna

Frederic Pene

Jamel El- Benna

George Samonis

Kieu T T Le

Vinod Kumar

Dimitrios Georgopoulos

Frank L Van De Veerdonk

Mihai G Netea

Jean-Paul Latge

Georgios Chamilos
email: hamilos@imbb.forth.gr

Uncoupling of IL-6 signaling and LC3-associated phagocytosis drives immunoparalysis during sepsis

Keywords: Aspergillus fumigatus, LAP, LC3-associated phagocytosis, phagosome, IL-6, JAK, NADPH oxidase, extracellular-signal-kinase regulated (ERK), microtubules, sepsis, sepsis, immunoparalysis

Introduction

Sepsis, a complex and heterogeneous disease caused by a deregulated inflammatory response to an infectious insult, remains a leading cause of death worldwide [START_REF] Reinhart | Recognizing sepsis as a global health priority -a WHO reso-lution[END_REF][START_REF] Singer | The third international consensus definitions for sepsis and septic shock (Sepsis-3)[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. Numerous clinical trials on immunomodulation in sepsis have failed to improve patient outcome, emphasizing the need for deeper mechanistic insight on pathogenesis [START_REF] Arnold | News feature: the quest to solve sepsis[END_REF][START_REF] Peters Van Ton | Precision immunotherapy for sepsis[END_REF][START_REF] Rello | Towards precision medicine in sepsis: a position paper from the European so-ciety of clinical microbiology and infectious diseases[END_REF]. In recent years, it has been realized that many patients who survive the initial sepsis episode enter a prolonged state of immune deactivation, termed sepsis-induced immunoparalysis, accounting for treatment failures and death due to secondary infections by opportunistic pathogens [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF][START_REF] Hotchkiss | Sepsis-induced immuno-suppression: from cellular dysfunctions to immunotherapy[END_REF].

As opposed to "immunological tolerance," a physiological compensatory mechanism that confers a balanced inflammatory response to an infectious agent, sepsis-induced immunoparalysis is characterized by impaired activation of essential immune effector pathways in myeloid phagocytes [START_REF] Cheng | Broad defects in the energy metabolism of leuko-cytes underlie immunoparalysis in sepsis[END_REF]Domínguez-Andrés et al., 2019;[START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF] and loss of their microbicidal activity [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF][START_REF] Chiswick | Acute-phase deaths from murine polymicrobial sepsis are characterized by innate immune suppression rather than exhaustion[END_REF]Döcke et al., 1997;[START_REF] Hotchkiss | Sepsis-induced immuno-suppression: from cellular dysfunctions to immunotherapy[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. At the molecular level, supra-physiological induction of pathways regulating "immunological tolerance," results in cytokine hyporesponsiveness and is a central molecular event of immune deactivation in sepsis [START_REF] Hotchkiss | Sepsis-induced immuno-suppression: from cellular dysfunctions to immunotherapy[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. In particular, ROS-mediated super-induction of activating transcription factor 3 (ATF3) suppress innate cytokines, predominantly IL-6, and accounts for heightened susceptibility to secondary infection by opportunistic bacterial and fungal pathogens [START_REF] Hoetzenecker | ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression[END_REF]. Aberrant ATF3 expression in sepsis is also induced via metabolic reprogramming of myeloid phagocytes [START_REF] Bambouskova | Electrophilic properties of itaconate and derivatives regu-late the IkBz -ATF3 inflammatory axis[END_REF]Domínguez-Andrés et al., 2019). Furthermore, epigenetic modulation of myeloid progenitors induced by sepsis leads to persistent immune deactivation of professional phagocytic cells [START_REF] Wen | Epigenetic regu-lation of dendritic cell-derived interleukin-12 facilitates immunosuppression after a severe innate immune response[END_REF][START_REF] Zhang | Sepsis induces hematopoietic stem cell exhaustion and myelosuppression through distinct contributions of TRIF and MYD88[END_REF]. Immunotherapy with cytokines, such as IFN-γ and GM-CSF, partially reverses cytokine hypo-responsiveness and restores the microbicidal activity of sepsis monocytes [START_REF] Arnold | News feature: the quest to solve sepsis[END_REF]Döcke et al., 1997;[START_REF] Leentjens | Reversal of immunoparalysis in hu-mans in vivo: a double-blind, placebo-controlled, randomized pilot study[END_REF][START_REF] Peters Van Ton | Precision immunotherapy for sepsis[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. However, successful implementation of cytokine therapies in human patients with sepsis requires understanding the underlying signaling defects that account for immunoparalysis in the individual patient.

LC3 associated phagocytosis (LAP), a phagosome biogenesis pathway utilizing part of the autophagy machinery in response to certain pattern-recognition receptors (PRRs), promotes phagolysosomal fusion and killing of an expanding list of pathogens by macrophages (Heckmann and Green, 2019;[START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF][START_REF] Sanjuan | Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis[END_REF]. Besides, LAP regulates the antiinflammatory activity of macrophages [START_REF] Chu | Gene-microbiota interactions contribute to the pathogenesis of inflammatory bowel disease[END_REF]Heckmann and Green, 2019;Heckmann et al., 2019;[START_REF] Martinez | Microtubule-associated protein 1 light chain 3 alpha (LC3)-associated phagocytosis is required for the efficient clearance of dead cells[END_REF][START_REF] Martinez | Noncanonical autophagy inhibits the auto-inflammatory, lupus-like response to dying cells[END_REF]. LAP utilizes a distinct signaling pathway from canonical autophagy and is regulated by Rubicon [START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF]. In particular, NADPHoxidase-complex-mediated ROS production is a fundamental requirement for LC3 + phagosome (LAPosome) formation [START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF].

Currently, there is no single biological marker to define sepsis immunoparalysis, and the underlying molecular mechanisms remain at least in part obscure. Aspergillus fumigatus, an airborne saprophytic mold shaping immunological tolerance in human respiratory epithelia [START_REF] Bacher | Regulatory T cell specificity directs tolerance versus allergy against aeroantigens in humans[END_REF], has become a model pathogen to study immune deactivation in sepsis [START_REF] Benjamim | Septic mice are susceptible to pulmonary aspergillosis[END_REF][START_REF] Benjamim | Reversal of long-term sepsis-induced immunosuppression by dendritic cells[END_REF][START_REF] Hoetzenecker | ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression[END_REF]. Interestingly, life-threatening respiratory infections caused by this opportunistic fungus are increasingly encountered in patients recovering from bacterial and viral sepsis [START_REF] Bae | Invasive pulmonary aspergillosis in patients with severe fever With thrombocytopenia syndrome[END_REF][START_REF] Bartoletti | Epidemiology of invasive pulmonary aspergillosis among COVID-19 intubated patients: a prospective study[END_REF][START_REF] Bassetti | Intensive care medicine research agenda on invasive fungal infec-tion in critically ill patients[END_REF][START_REF] Colombo | Candida and invasive mould diseases in non-neutropenic critically ill patients and patients with haematological cancer[END_REF][START_REF] Dimopoulos | Disseminated aspergillosis in intensive care unit patients: an au-topsy study[END_REF][START_REF] Meersseman | Invasive aspergillosis in critically ill pa-tients without malignancy[END_REF]. We and others have shown the crucial role of LAP in protective immunity against Aspergillus and other human pathogens (Akoumianaki et al., 2016;[START_REF] Kyrmizi | Calcium sequestration by fungal melanin inhibits calcium-calmodulin signal-ling to prevent LC3-associated phagocytosis[END_REF][START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF][START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF][START_REF] Oikonomou | Noncanonical fungal autophagy inhibits inflammation in response to IFN-gamma via DAPK1[END_REF], [START_REF] De Luca | IL-1 receptor blockade restores autophagy and reduces inflam-mation in chronic granulomatous disease in mice and in humans[END_REF]. However, the molecular mechanisms of crosstalk of cytokine signaling and LAP are poorly understood. In addition, the role of LAP in the pathogenesis of sepsis is currently unknown.

Using Aspergillus as a model pathogen, we identify defective activation of LAP as a hallmark feature of immune deactivation of phagocytes in septic patients and mice. In particular, we discover a unique role of autocrine IL-6/JAK2/Ninein axis on the regulation of microtubuledependent ERK recruitment to the phagosome for LAPosome formation. Next, we demonstrate that loss of IL-6 signaling during sepsis selectively disrupts microtubule dynamics regulating ERK-dependent activation of LAP, leading to impaired control of bacterial and fungal pathogens by monocytes/macrophages. IL-6 specifically restores microbicidal activity of myeloid phagocytes in sepsis patients and mice, in a LAP-dependent way. Collectively, our findings link IL-6 signaling with LAP, with an important role in the development of immunoparalysis of sepsis, which can be explored as a therapeutic target.

Results

Defective activation of LAP in monocytes is a dominant feature of immunoparalysis in human patients with sepsis

Given the essential role of LAP in killing of Aspergillus by phagocytes (Akoumianaki et al., 2016;[START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF][START_REF] Kyrmizi | Calcium sequestration by fungal melanin inhibits calcium-calmodulin signal-ling to prevent LC3-associated phagocytosis[END_REF][START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF], we explored whether this pathway is modulated by sepsis. We obtained monocytes from 38 consecutive patients admitted to intensive care unit (ICU) with community-acquired septic shock. Functional studies were performed on the day of patient admission (day 1) and upon recovery from the infectious episode (day 7) (Figure 1A). We used melanin-deficient (albino) conidia of Aspergillus fumigatus ΔpksP mutant as model bioparticles that induce a robust activation of LAP (Akoumianaki et al., 2016;[START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF][START_REF] Kyrmizi | Calcium sequestration by fungal melanin inhibits calcium-calmodulin signal-ling to prevent LC3-associated phagocytosis[END_REF][START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF] and compared immune responses of monocytes from sepsis patients with those induced in monocytes of healthy individuals (controls). Importantly, killing of wild-type Aspergillus conidia is also dependent on activation of LAP upon cell wall melanin removal inside monocytes/macrophages (Akoumianaki et al., 2016;[START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF][START_REF] Kyrmizi | Calcium sequestration by fungal melanin inhibits calcium-calmodulin signal-ling to prevent LC3-associated phagocytosis[END_REF][START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF]. Therefore, we additionally assessed killing of the isogenic wild-type A. fumigatus clinical isolate (ATCC46645) by monocytes of sepsis patients versus controls. In parallel, we analyzed clinical and microbiological characteristics and the outcome of patients to explore whether classical features of sepsis immunoparalysis (e.g., secondary infections) are associated with impaired LAP responses (Tables S1-S3). LAP was assessed by counting the percentage of LC3 + Aspergillus-containing phagosomes in monocytes by confocal microscopy (Figure S1) (Akoumianaki et al., 2016;[START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF][START_REF] Kyrmizi | Calcium sequestration by fungal melanin inhibits calcium-calmodulin signal-ling to prevent LC3-associated phagocytosis[END_REF][START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF]. LAP responses following sepsis recovery (day 7; when patients are at increased risk for development of secondary infections) segregated patients in two distinct groups (Figure S1). When compared with healthy individuals (n = 19), group A included patients with intact LAP responses (n = 15, P = NS versus healthy control group), and group B included patients with defective activation of LAP (n = 23, p < 0. 0001 versus healthy control group and versus patients with intact LAP) (Figure 1B). These differences in LAP responses were less apparent on day 1 of sepsis (Figure S1). Because LAP is dependent on NADPH oxidase [START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF], we assessed NADPH oxidase activation in monocytes of sepsis patients by measuring p47phox translocation to the phagosome membrane. Notably, monocytes from patients with LAP defects displayed impaired recruitment of p47phox to the phagosome (Figures 1C andS1). Accordingly, we found evidence of phagosome maturation arrest in monocytes of patients with LAP defects following Aspergillus infection, suggested by the lack of acquisition of CD63 (a marker of phagolysosomal fusion and acidification) [START_REF] Meersseman | Invasive aspergillosis in critically ill pa-tients without malignancy[END_REF], (Figures 1D and S1).

Defective LAP responses in monocytes of patients recovering from sepsis (day 7) were significantly associated with impaired killing of conidia of a clinical isolate of Aspergillus (ATCC 46645) (Figures 1E andS1), and our melanin-deficient Aspergillus mutant used as a model for phagocytosis (Figure S1). In contrast, monocytes from patients with compromised LAP responses had no apparent defects on the level of phagocytosis of conidia of melanindeficient ΔpksP Aspergillus strain (Figure S1). In addition, there was no difference in the percentage of CD16 + monocytes and the level of CD163 expression between patients with intact versus defective LAP responses (Figure S2). Of interest, HLA-DR expression were decreased in LAP-defective monocytes (Figure S2).

An impaired cytokine response of monocytes following ex vivo re-stimulation with TLR agonists is a prominent feature of immune deactivation in sepsis [START_REF] Hotchkiss | Sepsis-induced immuno-suppression: from cellular dysfunctions to immunotherapy[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. Specifically, suppressed IL-6 production has a major causative role in the development of sepsis-induced immunoparalysis [START_REF] Cenci | Impaired antifungal effector activity but not inflammatory cell recruit-ment in interleukin-6-deficient mice with invasive pulmonary aspergillosis[END_REF][START_REF] Hoetzenecker | ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression[END_REF]. Monocytes of patients with LAP defect on day 7 had a significant decrease of IL-6 production following overnight stimulation with A. fumigatus when compared with control healthy monocytes and monocytes of patients with intact LAP responses, which was proportional to the severity of the underlying LAP defect (Figure 1F; Table S2); production of other proinflammatory cytokines was decreased in monocytes of patients with LAP defects versus those with intact LAP but was not statistically significant (Figure S2).

Development of secondary infections is a hallmark clinical feature of sepsis-induced immunoparalysis [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF][START_REF] Hotchkiss | Sepsis-induced immuno-suppression: from cellular dysfunctions to immunotherapy[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. Notably, patients with LAP defect had a higher clinical severity of sepsis on the day of admission in the ICU (APACHE II score) (Figure 1G) and a significant 3-fold increase in cumulative risk for secondary infections as compared with patients with intact LAP (Figure 1H; Tables S1-S3). Collectively, defective activation of LAP in monocytes of patients with sepsis is associated with classical immunological and clinical features of immunoparalysis.

Localized ERK1/2 signaling on the phagosome regulates NADPH oxidase-dependent activation of LAP and is inhibited in sepsis

Impaired phagosomal localization of p47phox was a striking upstream signaling defect in monocytes/macrophages of sepsis patients with a LAP defect (Figures 1B and1C). To gain further insights into defective LAP activation in sepsis, we explored the signaling pathway regulating p47phox phosphorylation. We observed that infection of monocytes with Aspergillus conidia induced early and selective phosphorylation of p47phox on Ser345 (Figures 2A andS3); in contrast, PMA, a classical PKC activator, induced the phosphorylation of p47phox on distinct serine sites (Ser315, Ser328) (Figures 2A andS3). Since phosphorylation of p47phox on Ser345 is regulated by ERK1/2 signaling in human neutrophils [START_REF] Boussetta | The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-alpha-induced priming of the NADPH oxidase in human neutrophils[END_REF][START_REF] Dang | A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites[END_REF][START_REF] El-Benna | p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases[END_REF][START_REF] Makni-Maalej | Zymosan induces NADPH oxidase activation in human neutrophils by inducing the phosphorylation of p47phox and the activation of Rac2: involvement of protein tyrosine kinases, PI3kinase, PKC, ERK1/2 and p38MAPkinase[END_REF], we evaluated the role of ERK1/2 signaling on NADPH oxidase-mediated activation of LAP by Aspergillus in monocytes. Importantly, ERK1/2 signaling in monocytes was rapidly activated within minutes of infection by Aspergillus (Figures 2B andS3). In addition, immunostaining revealed an endosomal pattern of distribution of ERK in monocytes, with early and sustained ERK recruitment and phosphorylation on the phagosome following Aspergillus infection (Figures 2C andS3). Importantly, inhibition of ERK1/2 phosphorylation upon monocyte treatment with UO126 (Figures 2AB andS3), a specific MEK1/2 inhibitor [START_REF] Favata | Identification of a novel inhibitor of mitogen-activated protein kinase kinase[END_REF], significantly reduced p47phox phosphorylation at Ser345 (Figure 2B), p47phox recruitment to the phagosome (Figure 2D), NADPH oxidase complex assembly-as demonstrated by inhibition of phagosomal localization of p22phox membrane subunit (Figure 2E)-intracellular ROS production (Figure 2F), and LAPosome formation (Figure 2G). Accordingly, inhibition of ERK1/2 signaling significantly decreased the killing of Aspergillus conidia by monocytes (Figure 2H). Importantly, pharmacological inhibition of Class III PI3K (VPS34) complex by wortmannin did not impair ERK recruitment to the phagosome (Figure S3). Collectively, these studies reveal an essential role of ERK signaling in NADPHoxidase-dependent activation of LAP by Aspergillus. Monocytes of two representative patients recovering from sepsis with LAP defect (Figure 2I) had impaired trafficking of p47phox to the phagosome (Figure 2J) and blockade on ERK-dependent phosphorylation of p47phox at Ser345 (Figures 2K andS3). Accordingly, ERK trafficking to the phagosome was impaired in patients with LAP defect compared with a healthy control (Figure 2K); while ERK1/2 phosphorylation was not always impaired (Pt-2; Figure S3). Collectively, these findings reveal a pronounced defect in ERK recruitment to the phagosome of sepsis patients with impaired activation of LAP.

Autocrine IL-6 signaling regulates ERK trafficking and LAPosome formation independent of ERK1/2 phosphorylation IL-6 production is defective in phagocytes during immunoparalysis in sepsis [START_REF] Hoetzenecker | ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression[END_REF]. We observed that defective IL-6 production in sepsis monocytes was significantly associated with LAP blockade (Figure 1F; Table S2). In contrast, inhibition of LAP was not associated with reduction in the expression of genes regulating ERK-dependent activation of LAP (Data S1). To explore the role of IL-6 on regulation of LAP, we evaluated phagosome responses in bone-marrow-derived macrophages (BMDMs), of IL-6 knockout (IL-6 -/-) and control (IL-6 +/+ ) mice infected with Aspergillus; BMDMs are typically used for studies of LAP in myeloid phagocytes of mice (Akoumianaki et al., 2016;[START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF][START_REF] Kyrmizi | Calcium sequestration by fungal melanin inhibits calcium-calmodulin signal-ling to prevent LC3-associated phagocytosis[END_REF][START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF][START_REF] Oikonomou | Noncanonical fungal autophagy inhibits inflammation in response to IFN-gamma via DAPK1[END_REF][START_REF] De Luca | IL-1 receptor blockade restores autophagy and reduces inflam-mation in chronic granulomatous disease in mice and in humans[END_REF]. We observed that NADPH-oxidasemediated activation of LAP was significantly impaired in IL-6 -/-BMDMs as compared with control IL-6 +/+ BMDMs. In particular, there was a significant decrease on localization of the membrane-associated (p22phox) (Figure 3A) and cytosolic (p47phox) (Figure 3B) subunits of NADPH oxidase complex to the phagosome, and defective LAPosome formation in the IL-6 -/- versus control (IL-6 +/+ ) BMDMs (Figure 3C). In addition, IL-6 -/-BMDMs had significant defects in acquisition of markers of phagosome acidification (Figure 3D), phagolysosomal fusion (Figures 3E and 3F), and in the ability to kill conidia of a wild-type clinical isolate of Aspergillus fumigatus (Figure 3G). Accordingly, pulmonary infection of IL6 -/-mice with wild-type Aspergillus conidia resulted in significant increase in fungal load in the lungs as compared with infection of control IL6 +/+ mice (Figures 3H andS4), without evidence of invasive fungal disease in histopathology (Figure S4).

Next, we assessed activation of ERK1/2 signaling by Aspergillus in BMDMs of IL-6 -/-versus IL-6 +/+ mice. Of interest, western blot analysis of protein lysates obtained from BMDMs of IL-6 -/-versus IL-6 +/+ revealed no appreciable differences in total ERK1/2 levels and the degree of ERK1/2 phosphorylation induced by Aspergillus (Figures 3I and3J). In sharp contrast, we found a significant defect in ERK1/2 phosphorylation and recruitment on Aspergillus-containing phagosomes of IL-6 -/-versus IL-6 +/+ BMDMs (Figure 3K), which was rapidly restored within 15 min of IL-6 supplementation in culture media (Figure 3L). Furthermore, in contrast to other cytokines, IL-6 supplementation selectively enhanced defective killing of Aspergillus conidia by IL-6 -/-BMDMs (Figure 3M). Collectively, these studies reveal a previously uncharacterized mechanism of action of IL-6 signaling on ERK trafficking to the phagosome and LAPosome formation.

Defects in autocrine IL-6/JAK2/Ninein axis disrupt microtubule dynamics regulating phagosomal trafficking of ERK and LAPosome formation

Next, we evaluated the mechanism of regulation of phagosomal trafficking of ERK by IL-6 signaling. The phagosome maturation process is regulated by microtubule dynamics [START_REF] Blocker | Molecular requirements for bi-directional movement of phagosomes along microtubules[END_REF][START_REF] Harrison | Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP[END_REF]. In particular, physical clustering of dynein motors into lipid micro-domains on the phagosome membrane leads to rapid directed transport of the phagosome toward microtubule minus ends to promote fusion with lysosomes and pathogen degradation [START_REF] Rai | Dynein clusters into lipid microdomains on phagosomes to drive rapid trans-port toward lysosomes[END_REF]. Therefore, we assessed whether the process of ERK recruitment to the phagosome is also regulated by microtubules. We found that the disruption of microtubule assembly via depolymerization induced by nocodazole or inhibition of microtubule dynamics following treatment of BMDMs with the microtubule-stabilizing agent taxol resulted in abolished ERK localization to Aspergillus-containing phagosomes (Figure 4A). Therefore, we specifically assessed the effect of IL-6 on organization of the microtubule network and phagosome transport. Notably, immunostaining of uninfected IL-6 -/-BMDMs for β-tubulin revealed major defects in the size of the microtubule organizing center (centrosome) at the steady state, as compared with control IL-6 +/+ BMDMs, a defect that was reversed within 15 min of IL-6 supplementation in IL-6 -/-BMDMs (Figure 4B). We also found that centripetal transport of Aspergillus-containing phagosomes from the cell periphery to the microtubule minus end (microtubule organization center) was inhibited in IL-6 -/-BMDMs, as compared with control IL-6 +/+ BMDMs, a defect that was restored within minutes of IL-6 supplementation (Figure 4C-4E; Videos S1 and S2). Importantly, IL-6 -/-BMDMs had no apparent defect in phagocytosis of different pathogens, including Aspergillus and Pseudomonas, and biotin beads (Figure S5).

To investigate the signaling pathway of IL-6-depended regulation of microtubule dynamics and phagosome trafficking we evaluated the effect of inhibition of JAK/STAT signaling pathway downstream of IL-6 receptor on phagosomal localization of ERK, phagosome movement and LAP. Of interest, inhibition of JAK2 with Ruxolitinib, a specific JAK1/2 inhibitor (Quintás-Cardama et al., 2010) resulted in a significant, dose-dependent inhibition of phagosome transport toward the microtubule minus end (Figure 4F), leading to blockade in phagosomal localization of ERK (Figure 4G), LAPosome formation (Figure 4H) and fungal killing (Figure 4I). In contrast, inhibition of the phosphorylation of STAT3, which is downstream of JAK2, with the use of a specific STAT3 inhibitor (S3I-201) [START_REF] Siddiquee | Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity[END_REF] had no apparent effect on ERK trafficking to the phagosome (Data S2).

To further gain mechanistic insight on regulation of microtubule organizing center by IL-6/JAK2 signaling, we explored γ-tubulin expression, a master regulator of the centrosome nucleation, in IL-6 -/-BMDMs. We found no differences in γ-tubulin perinuclear localization in the area of centrosomes of IL-6 +/+ versus IL-6 -/-BMDMs by immunostaining (Figures 4J and4K). Next, we focused on the pattern of perinuclear localization of Ninein, a master regulatory protein of microtubule anchoring to the centrosome that has been shown to be phosphorylated by JAK2 [START_REF] Jay | JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein Ninein[END_REF]. Of interest, we found a significant decrease in perinuclear expression of Ninein in IL-6 KO BMDMs, accompanied by loss of co-localization of ninein with JAK2, which was rapidly restored within min of IL-6 supplementation (Figures 4L and4M). Collectively, these findings demonstrate a previously uncharacterized role of the pathway IL-6/JAK2/Ninein signaling on microtubule cytoskeleton dynamics, which then regulates phagosome transport and ERK-mediated LAPosome formation.

Sepsis-induced susceptibility to fungal infections is associated with LAP blockade in mouse monocytes/macrophages

To further explore the underlying mechanisms of LAP defect in sepsis, we developed a physiologically relevant mouse model of polymicrobial sepsis (peritonitis) by inducing different degrees of disease severity (mild sepsis, <5% mortality rates; severe sepsis, >50% mortality rates) (Figures 5A and5B). Severe sepsis selectively compromised the ability of mice to control superinfection with Aspergillus, as evidenced by a significantly higher fungal load in the lungs of mice recovering from severe sepsis (day 7) (Figure 5C), when compared with mice recovering from mild sepsis or SHAM-operated mice; Aspergillus infection in mice recovering from severe sepsis (hereafter sepsis) was specifically associated with extensive inflammatory immunopathology (Figure S6) and invasive fungal growth (Figures 5D andS6). In line with the in vivo studies, defective activation of LAP following Aspergillus infection was induced in splenic monocytes, BMDMs, and alveolar macrophages (AMs) of mice recovering from sepsis, in contrast to SHAM-operated mice (Figures 5E,5F,and S7). Furthermore, sepsis-induced LAP defect in BMDMs was associated with diminished ROS production in response to Aspergillus (Figure 5G) and phagosome maturation arrest, as evidenced by (1) defective accumulation of fluorescein isothiocyanate-labeled dextran (FITC-Dextran) to the phagosome following labeling of lysosomes (Figures 5H and5I), (2) impaired phagosome acidification assessed by VATPase staining (Figure 5J), and (3) defective phagosomal localization of the lysosomal markers cathepsin-D (Figure 5K) and LAMP-1 (Figures 5L andS7). Finally, sepsis BMDMs displayed significant loss of fungicidal activity against conidia of wild-type Aspergillus (Figure 5M) and diminished IL-6 production upon re-stimulation with fungal conidia (Figure S7). Overall, these findings reveal that sepsis-induced immune deactivation in murine monocytes/macrophages is associated with defective activation of LAP.

Loss of IL-6 signaling in mice and human patients with sepsis selectively impairs ERK trafficking, LAPosome formation, and fungal killing

Next, we assessed whether LAP blockade in sepsis macrophages is mechanistically linked with defects in IL-6-dependent trafficking of ERK to the phagosome. We found a profound defect in centrosome organization and microtubule assembly at the baseline in BMDMs obtained from mice with severe sepsis, a phenotype reminiscent of microtubule cytoskeleton abnormalities in IL-6 -/-BMDMs (Figure 6A). Indeed, sepsis-induced defects in the size of microtubule organizing center in uninfected BMDMs were rapidly restored following IL-6 supplementation (Figure 6A). Accordingly, BMDMs obtained from mice with severe sepsis displayed significant defects on phagosome transport following infection with Aspergillus, which were restored within minutes of IL-6 treatment (Figure 6B).

In line with these results, ERK trafficking to Aspergillus-containing phagosomes was abolished in BMDMs of mice recovering from severe sepsis in contrast to control SHAM treated mice (Figures 6C and6D). Instead, impaired phagosomal ERK recruitment was not always accompanied by defective ERK phosphorylation by Aspergillus (Data S2). Of interest, IL-6 supplementation resulted in complete restoration of phagosomal localization of ERK (Figures 6C and6D) and LAPosome formation (Figure 6E) in BMDMs obtained from mice with severe sepsis. Furthermore, as opposite to other cytokines (TNF, IFN-γ), IL-6 treatment selectively enhanced defective killing of Aspergillus by sepsis BMDMs (Figure 6F). Similar to the findings in macrophages of mice following sepsis recovery, we found defects in microtubule anchoring and organization at the centrosome and microtubule-mediated phagosome transport in monocytes of patients with sepsis-induced inhibition of LAP (Figure 6G). Importantly, IL-6 supplementation in culture media of human monocytes with sepsis-induced inhibition of LAP fully restored defective killing activity against Aspergillus (Figure 6H); instead, IL-6 treatment had no effect in fungicidal activity of healthy control monocytes. These studies reveal a previously uncharacterized role of IL-6 signaling on ERK-mediated activation of LAP in monocyte/macrophage with direct pathophysiological and therapeutic implications for immunoparalysis in sepsis.

IL-6-induced restoration of microbicidal activity of myeloid phagocytes in sepsis depends on LAP

In order to establish a causative relationship between loss of IL-6 signaling, LAP blockade, and sepsis immunoparalysis, we explored the effect of IL-6 supplementation on microbicidal activity of sepsis macrophages in mice with genetic defect of Atg5. BMDMs from mice with genetic deletion of Atg5 (LysMCre +/+ ; Atg5flox/flox mice; hereafter Atg5 -/-BMDMs), an autophagy protein crucial for LAPosome formation (Akoumianaki et al., 2016;[START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF], had no apparent defects on microtubule cytoskeleton organization (Figure 7A); however, Atg5 -/- BMDMs displayed broad defects on phagosome biogenesis, comparable with those induced by severe sepsis (Figures 7B-7E). Notably, IL-6 supplementation resulted in full reversal of sepsisinduced defects in killing of Aspergillus conidia by Atg5 +/+ BMDMs but failed to restore killing defects in Atg5 -/-BMDMs following sepsis (Figure 7F).

Next, we evaluated the physiological importance of IL-6/LAPosome pathway in macrophage antimicrobial activity against Pseudomonas, a bacterial pathogen classically associated with secondary infections due to sepsis-induced immunoparalysis [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF] including our patient cohort (Table S1). We initially verified that killing of a reference clinical isolate of Pseudomonas aeruginosa by macrophages depends on IL-6 signaling (Figure 7G) and LAP (Figure 7H). Next, we tested the effect of IL-6 supplementation on bactericidal capacity of BMDMs differentiated from control versus LAP-defective mice recovering from severe sepsis. Importantly, IL-6 supplementation significantly improved killing of Pseudomonas by BMDMs obtained from Atg5 +/+ (control) BMDMs following recovery from severe sepsis (Figure 7I). In contrast, IL-6 failed to restore bactericidal activity of Atg5 -/- BMDMs following sepsis (Figure 7I). Collectively, these studies lead to a new mechanism of regulation of LAP by IL-6 signaling with physiological importance in pathogenesis of sepsis immunoparalysis.

Discussion

Integration of signals from PRRs and cytokines to phagocytosis has a crucial role in successful control of infection by macrophages [START_REF] Pauwels | Patterns, re-ceptors, and signals: regulation of phagosome maturation[END_REF]. In particular, PRR-mediated activation of the LAP pathway regulates phagosome maturation and confers protective immunity against a range of pathogens (Akoumianaki et al., 2016;Heckmann and Green, 2019;[START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF][START_REF] Kyrmizi | Calcium sequestration by fungal melanin inhibits calcium-calmodulin signal-ling to prevent LC3-associated phagocytosis[END_REF][START_REF] Martinez | Molecular character-ization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins[END_REF]. Likewise, specific cytokines transcriptionally induce the expression of antimicrobial effectors to enhance the microbicidal capacity of macrophages [START_REF] Forbester | Interleukin-22 promotes phagolysosomal fusion to induce protection against Salmonella enterica Typhimurium in human epithelial cells[END_REF][START_REF] Jung | IL-12 and IL-27 regulate the phagoly-sosomal pathway in mycobacteria-infected human macrophages[END_REF][START_REF] Shen | Macrophage activation by IFNgamma triggers restriction of phagosomal copper from intra-cellular pathogens[END_REF][START_REF] Subramanian Vignesh | Granulocyte macrophage-colony stimulating fac-tor induced Zn sequestration enhances macrophage superoxide and limits intracellular pathogen survival[END_REF][START_REF] Via | Effects of cytokines on mycobacterial phagosome matura-tion[END_REF]. Moreover, reciprocal interactions of cytokines and LAP are required for an optimal anti-inflammatory function of macrophages [START_REF] De Luca | IL-1 receptor blockade restores autophagy and reduces inflam-mation in chronic granulomatous disease in mice and in humans[END_REF][START_REF] Oikonomou | Noncanonical fungal autophagy inhibits inflammation in response to IFN-gamma via DAPK1[END_REF]. However, a mechanistic link between cytokine signaling and LAP has not yet been identified.

Herein, we employ sepsis as a model human disease to explore physiologically important interactions of cytokine signaling and LAP. Our studies in humans and mice "identify LAP blockade in phagocytes as a core immunological feature of sepsis-induced immunoparalysis." Moreover, we discover "a role for the autocrine IL-6/JAK2 axis in microbicidal activity of phagocytes via regulation of LAP, with direct implications in immune deactivation of sepsis."

In our initial studies, we found that defective LAP is a prominent feature of phagocyte dysfunction in patients recovering from sepsis, who display all classical features of immunoparalysis [START_REF] Hotchkiss | Sepsis-induced immuno-suppression: from cellular dysfunctions to immunotherapy[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. In view of the absence of biological measures of immunoparalysis induced by sepsis, inhibition of LAP should be regarded as an immunological feature of this pathological process. Apart from monocytes/macrophages, neutrophils are major effectors of innate immune response in sepsis [START_REF] Hotchkiss | Sepsis-induced immuno-suppression: from cellular dysfunctions to immunotherapy[END_REF][START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF]. Therefore, the role of LAP defects in immune deactivation of neutrophils in sepsis should be investigated.

We also identified a selective mechanism of p47phox phosphorylation at Ser345 in monocytes during fungal infection, a phosphorylation site regulated by MAPK signaling [START_REF] Dang | A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites[END_REF][START_REF] El-Benna | p47phox, the phagocyte NADPH oxidase/NOX2 organizer: structure, phosphorylation and implication in diseases[END_REF][START_REF] Makni-Maalej | Zymosan induces NADPH oxidase activation in human neutrophils by inducing the phosphorylation of p47phox and the activation of Rac2: involvement of protein tyrosine kinases, PI3kinase, PKC, ERK1/2 and p38MAPkinase[END_REF]. This reveals a major role of ERK1/2 signaling on NADPH oxidase-mediated activation of LAP both physiologically and in sepsis, independently of VPS-34 complex formation on the phagosome. Because cytokine deregulation has a major role in the pathogenesis of sepsis [START_REF] Van Der Poll | The immunopathology of sepsis and potential therapeutic targets[END_REF], we evaluated the direct role of cytokine signaling on ERK-dependent activation of LAP. We focused on IL-6, a signature cytokine of sepsis-induced immunosuppression and related susceptibility to aspergillosis [START_REF] Cenci | Impaired antifungal effector activity but not inflammatory cell recruit-ment in interleukin-6-deficient mice with invasive pulmonary aspergillosis[END_REF][START_REF] Hoetzenecker | ROS-induced ATF3 causes susceptibility to secondary infections during sepsis-associated immunosuppression[END_REF]. Surprisingly, we observed that although p47phoxmediated activation of NADPH oxidase was abolished in IL-6 knockout (KO) BMDMs during fungal infection, upstream ERK1/2 phosphorylation remained intact. In contrast, ERK1/2 recruitment on the phagosome was abolished in IL-6 KO BMDMs, which explained the downstream blockade in NADPH oxidase-dependent activation of LAP. These findings confirmed our hypothesis on distinctive pathways regulating ERK phosphorylation and trafficking on the phagosome and revealed a central regulatory role for IL-6 signaling on endosomal trafficking of ERK and LAPosome formation.

Importantly, we discovered that IL-6 KO BMDMs display profound disorganization of microtubule organizing center at steady state, associated with impaired LAP-dependent killing of pathogens without an apparent defect in phagocytosis. Subsequently, we found that microtubuledependent phagosome movement and ERK trafficking were regulated by JAK2, independently of STAT3. Of interest, both STAT3 [START_REF] Jay | JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein Ninein[END_REF][START_REF] Ma | Identification of tubulin as a substrate of Jak2 tyrosine kinase and its role in Jak2-dependent signaling[END_REF] and JAK2 [START_REF] Morris | Stat3 regulates centrosome clustering in cancer cells via stathmin/PLK1[END_REF][START_REF] Ng | Stat3 regulates microtubules by antagonizing the depolymerization activity of stathmin[END_REF] are involved in microtubule organization dynamics via interactions with distinct signaling pathways. Evermore, JAK2 phosphorylates and is negatively regulated by the centrosomal protein ninein [START_REF] Jay | JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein Ninein[END_REF]. In particular, Ninein is a master regulator of microtubule anchoring to the centrosome. Accordingly, inhibition of JAK2 has been shown to selectively impair the size of microtubule organizing center without affecting the process of centrosome nucleation, a function regulated by γ-tubulin [START_REF] Jay | JAK2 tyrosine kinase phosphorylates and is negatively regulated by centrosomal protein Ninein[END_REF]. Indeed, our findings on diminished Ninein expression and co-localization with JAK2 in perinuclear regions of IL-6 KO macrophages without an apparent defect in topology of γ-tubulin are in line with previous studies [START_REF] Ma | Identification of tubulin as a substrate of Jak2 tyrosine kinase and its role in Jak2-dependent signaling[END_REF][START_REF] Rai | Dynein clusters into lipid microdomains on phagosomes to drive rapid trans-port toward lysosomes[END_REF][START_REF] Via | Effects of cytokines on mycobacterial phagosome matura-tion[END_REF].

In proof-of-concept studies, we demonstrated that monocytes/macrophages isolated from septic conditions display defects in microtubule cytoskeleton organization, ERK trafficking, LAPosome formation, and pathogen killing. These are specifically related to loss of IL-6 signaling, as they were all restored within minutes of IL-6 supplementation. Furthermore, we revealed that IL-6 induced restoration of LAP defects in sepsis has broad implications in phagocyte immunity against major bacterial and fungal pathogens. Of interest, previous studies report on the restoration of sepsis-induced immune deactivation in monocytes following several days of immunotherapy with IFN-γ (Döcke et al., 1997). It would be important to investigate the effects of prolonged immunotherapy with IFN-γ on IL-6/LAP axis. Alternatively, other cytokines may regulate phagosome biogenesis or trigger canonical autophagy (e.g., xenophagy) responses against certain microbial pathogens (e.g., intracellular bacteria) independently of NADPH oxidase signaling and LAP [START_REF] Sokolovska | Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function[END_REF]. In addition, whether unique intracellular trafficking of IL-6 as compared with other cytokines may have a role on the specialized role of this cytokine on regulation of ERK-dependent activation of LAP deserves exploitation [START_REF] Murray | A role for the phagosome in cytokine secretion[END_REF]. Collectively, our study identifies an unexplored physiological pathway linking autocrine IL-6/JAK2/Ninein axis, cytoskeleton organization, and LAP with the microbicidal activity of myeloid phagocytes and offers mechanistic insight in immunoparalysis of sepsis.

Our findings have also broad implications for understanding emerging mechanisms of iatrogenic immunosuppression predisposing to opportunistic infections. In particular, recent clinical studies report an alarming increase in cases of severe bacterial and fungal diseases in patients receiving small molecule kinase inhibitors (SMKIs), in the absence of additional immunosuppressive conditions. Notably, SMKIs targeting ERK1/2 and JAK/STAT signaling have been recently associated with clinical cases of invasive aspergillosis (Chamilos et al., 2018a(Chamilos et al., , 2018b;;[START_REF] Dioverti | Infectious complica-tions in patients on treatment with Ruxolitinib: case report and review of the literature[END_REF][START_REF] Lussana | Ruxolitinib-associated infections: a systematic review and meta-analysis[END_REF]. Preclinical studies also demonstrate compromised killing capacity of phagocytes against several pathogens, including Aspergillus, following pharmacological or genetic inhibition of IL-6, JAK1/2, and ERK [START_REF] Cenci | Impaired antifungal effector activity but not inflammatory cell recruit-ment in interleukin-6-deficient mice with invasive pulmonary aspergillosis[END_REF][START_REF] Dubourdeau | Aspergillus fumigatus induces innate immune responses in alveolar macrophages through the MAPK pathway independently of TLR2 and TLR4[END_REF][START_REF] Espinosa | Type III interferon is a critical regulator of innate antifungal immunity[END_REF][START_REF] Taylor | JAK/STAT regulation of Aspergillus fumigatus corneal in-fections and IL-6/23stimulated neutrophil, IL-17, elastase, and MMP9 activity[END_REF]. Finally, IL-6 is an appealing therapeutic target for restoration of inflammatory immunopathology induced by cytokine storm in COVID 19 [START_REF] Moore | Cytokine release syndrome in severe COVID-19[END_REF]. Importantly, superinfections by bacterial and fungal pathogens are increasingly encountered in patients recovering from COVID-19 [START_REF] Bartoletti | Epidemiology of invasive pulmonary aspergillosis among COVID-19 intubated patients: a prospective study[END_REF]. Therefore, our findings provide novel mechanistic view on the emergence of secondary infections caused by opportunistic pathogens in patients with receiving immunomodulatory therapies with inhibitors of IL-6 and JAK/STAT signaling pathways. Furthermore, our findings lead to a model of phagosome biogenesis that explains the synergistic mechanism of immunosuppressive action of corticosteroid therapy in patients receiving SMKIs, as a result of dual inhibition of two major pathways regulating LAP, PRRs and cytokine signaling [START_REF] Kyrmizi | Corticosteroids block autophagy protein recruitment in Aspergillus fumigatus phagosomes via targeting dectin-1/Syk kinase signaling[END_REF]. Understanding genetic and epigenetic factors regulating LAP responses could have important implications on risk stratifications for susceptibility to infection of these patients. Overall, our work provides the scientific rationale for the development of personalized cytokine-based immunotherapies for sepsis and/or other conditions of immune deregulation, aiming at the restoration of LAP-dependent effector functions and anti-inflammatory pathways in phagocytes.

STAR Methods

Key

Data collection

Baseline data

Baseline data collection included demographics (age and physician-assigned gender), comorbidities, and immunosuppressive medications.

Characteristics of the primary infection

The primary site of infection and microbiological data on the pathogens isolated were recorded. We also recorded the APACHE II score, and the SOFA scores on the day of admission in the ICU and at Day 7.

Secondary infections

Secondary infection in septic shock patients was defined as a new infection acquired more than 48 hours after admission to the ICU. The diagnosis of a secondary infection was performed according to the criteria of the Centers for Disease Control and Prevention (CDC, 2008) [START_REF] Horan | CDC/NHSN surveillance definition of health care-associated infection and criteria for specific types of infections in the acute care setting[END_REF]. Infection was differentially diagnosed from colonization according to the CDC criteria and required one or more new antibiotics. The time, site and pathogen isolated for the secondary infection were recorded, and the analysis was restricted to the first episode of secondary infection at the same site.

Endpoints

Mortality, as well as hospital and ICU length of stay were collected.

Functional studies

Blood was immediately processed upon collection, CD14 + monocytes were isolated, stimulated with Aspergillus conidia and fixed on coverslips for immunostaining and analysis. Total RNA from 1 million unstimulated CD14 + monocytes lysed in Trizol reagent (Invitrogen) was kept in -80°C. Evaluation of LC3 + phagosomes and other markers of phagosome biogenesis was performed in batches of paired samples of each individual patient (Day 1, Day 7) as compared to the LAPosome formation in monocytes obtained from healthy volunteers (control) from the blood bank of the Hospital.

Mouse models

C57BL/6 mice (obtained from the IMBB Institute, Crete), IL6 -/-and IL6 +/+ mice of 129×C57BL/6 genetic background [START_REF] Venihaki | Corticotropin-releasing hormone regulates IL-6 expression during inflammation[END_REF]

Polymicrobial sepsis Model in mice

Pathogen-free, male C57BL/6, 8-10 weeks old (25-30 gr) were subjected to cecal ligation and puncture (CLP) polymicrobial sepsis model [START_REF] Benjamim | Septic mice are susceptible to pulmonary aspergillosis[END_REF][START_REF] Benjamim | Reversal of long-term sepsis-induced immunosuppression by dendritic cells[END_REF]. In brief, mice were anesthetized prior to surgery, by intraperitoneal injection of a ketamine/xylazine mixture. Under sterile conditions, a longitudinal skin midline incision was made and the cecum was exposed to the surface. The cecum was partially ligated (60% of the cecum in reference to the distal pole) with a 4-0 silk suture without disrupting the intestinal continuity. The ligated cecum was punctured through and through with a 21-gauge needle three times (six punctures in total) in order to achieve severe sepsis conditions. For mild sepsis, only one puncture was performed. Sham-operated mice underwent the same surgery but without cecal ligation and puncture and were used as controls. Six hours after induction of polymicrobial sepsis, sham or CLP operated mice were started on antibiotic treatment with 10mg/kg Imipenem/cilastatin. The antibiotic was administered every 12 hours for a period of three days. On Day 7 following recovery from sepsis, sham and CLP mice were challenged by intratracheal installation of 5x10 7 A. fumigatus conidia.

After 48 hours of infection, mice were euthanized, their lungs were harvested and homogenized in phosphate buffer saline. The fungal burden was evaluated by CFU plating of lung homogenates.

Microorganisms and culture conditions

All A. fumigatus strains used (ΔpksP and the isogenic wild type strain ATCC46645) have been described previously (Akoumianaki et al., 2016). All strains were grown on YAG agar plates for 3 days at 37°C. Fungal conidia (spores) were harvested by gentle shaking in the presence of sterile 0.1% Tween 20 in PBS, washed twice with PBS, filtered through a 40μm-pore-size cell strainer (Falcon) to separate conidia from contaminating mycelium, counted by a hemocytometer, and suspended at a concentration of 10 8 spores/mL. When indicated, the conidia were labeled with FITC or Alexa Fluor 633 succinimidyl ester dye (Invitrogen) as previously described (Akoumianaki et al., 2016). Briefly, freshly harvested conidia (5 × 10 7 /2 mL in 100mM NaHCO 3 , pH 8.3) were incubated with FITC (Sigma Aldrich, 0.1 mg/mL final concentration) or Alexa Fluor 633 succinimidyl ester dye (Invitrogen, 1.7 mg/mL) at 37°C for 2h and washed by centrifugation three times in PBS-0.1% Tween 20. Inactivation of fungal conidia was done by exposure to 4% FA (2 h, room temperature) following by treatment with glycine (100 mM in PBS) and three washes in PBS and verified by CFU plating. Importantly, we have previously validated that FA-inactivation of A. fumigatus conidia does not affect LAPosome formation and PRR signaling activation (Akoumianaki et al., 2016).

A single colony of Pseudomonas aeruginosa clinical isolate (ATCC 27853) isolated from LB plates was grown at a mid-logarithmic phase in LB liquid media the day of infection. Bacterial cells were washed with PBS, adjusted to 0.5 MacFarlane (10 8 bacteria/ml) and immediately used for infection experiments.

Method details

Isolation and stimulation of primary human monocytes

Healthy volunteers without any known infectious or inflammatory disorders donated blood.

Monocytes from sepsis patients or healthy controls were isolated from PBMCs using magnetic bead separation with anti-CD14 coated beads (MACS Miltenyi Germany) according to the protocol supplemented by the manufacturer. The monocytes were resuspended in RPMI 1640 culture medium supplemented with Hepes 10 mM, penicillin-streptomycin 1%, l-glutamine 1%, sodium pyruvate 1%, glucose 25 mM and fetal bovine serum 10%. The cells were counted, and their number was adjusted to 2 × 10 6 /mL. A total of 2 × 10 5 monocytes per condition in a final volume of 100 μL were allowed to adhere to sterile polylysine coated glass coverslips (Ø 13 mm) for 1 h followed by stimulation with A. fumigatus conidia at a multiplicity of infection (MOI) of 3:1 at 37°C for the indicated time point. After stimulation, cells were washed twice with PBS to remove medium and non-phagocytosed spores and cells were fixed for 15 min in 4% paraformaldehyde or 4% formaldehyde (w/o MeOH). Subsequently, the coverslips were washed with PBS followed by an extra step of fixation with ice-cold methanol for 10 min at -20°C in case of immunostaining for LC3, after which were stored in PBS at 4°C until immunofluorescence staining.

Immunofluorescence staining

For immunofluorescence imaging, fixed cells were blocked for 15 minutes with 2% BSA, 0.1% saponin in PBS, incubated for 1 h with the indicated primary antibody, washed twice in PBS, stained by the appropriate secondary AlexaFluor secondary Ab (Molecular Probes), followed by DNA staining with 1 μM TOPRO-3 iodide (642/661; Invitrogen). For ERK staining coverslips were incubated overnight with the primary antibody at 4°C. After the washing steps, slides were mounted in Prolong Gold antifade media (Molecular Probes). Images were acquired using a laser-scanning spectral confocal microscope (TCS SP8; Leica), LCS Lite software (Leica), and a 40× Apochromat 1.25 NA oil objective using identical gain settings. A low fluorescence immersion oil (11513859; Leica) was used, and imaging was performed at room temperature.

Serial confocal sections at 0.5 μm steps within a z-stack spanning a total thickness of 10 to 12 μm of the cell were taken, and 3D images were generated using the LCS Lite software to assess for internalized conidia contained within phagosomes. Unless otherwise stated, mean projections of image stacks were obtained using the LCS Lite software. Phagosomes surrounded by a rim of fluorescence of the indicated protein-marker were scored as positive, according to established protocols in our lab. At least 200 phagosomes were analyzed for each experimental condition in three independent experiments, in a double blinded fashion by the same investigator (T.A.). Assessment of phagolysosomal fusion was performed in BMDMs pre-loaded with FITC-Dextran. Briefly, BMDMs were pre-loaded with FITC-Dextran for 4h, media was replaced for 2h to allow for labeling of lysosomes, and phagolysosomal fusion was assessed following fungal infection based on the accumulation of FITC-Dextran at the phagosomes.

In addition, phagocytosis rate and phagocytosis index were assessed in primary human monocytes at 30 min of infection with FITC-labeled A. fumigatus (MOI 3:1). The phagocytic index was expressed with the following formula: (total number of engulfed cells/total number of counted macrophages) × (number of macrophages containing engulfed cells/total number of counted macrophages) × 100.

Live imaging

BMDMs were loaded with FITC-Dextran (5 μM final concentration, Invitrogen) for 4h washed with PBS cells counted and seeded in chambers for live imaging (627870 Greiner) in HBSS (w/o red phenol, 2 mM CaCl 2 , 1 mM MgCl 2 , 20 mM Hepes). After adhesion of the cells, infections with the indicated A. fumigatus strain were performed just before live imaging. The acquisition was performed in SP8 Leica converted microscope with 40× Apochromat 1.25 NA water objective using identical imaging setting, and high speed live imaging with the use of a resonant scanner.

Measurement of ROS production

ROS measurements were performed by means of a dichlorofluorescein assay (Akoumianaki et al., 2016). The Stock solution of dichlorofluorescein diacetate (DCFH-DA) was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mM. Human monocytes (2 × 10 5 /well) or mouse BMDMs (1 × 10 5 /well) were plated on 96-well round bottom plates, incubated at 37°C for 30 min and accordingly stimulated for 1h with A. fumigatus conidia (MOI 10:1) or zymosan (50 μg/ml) in the presence of DCFH-DA added to a final concentration of 10 μM during the last 30 min. After 30 min of exposure, the well content was transferred to vials and the fluorescence of the cells was measured by flow cytometry. Cells were acquired on a FACSCalibur (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Generation of murine BMDMS

BMDMs were generated by culturing bone marrow cells obtained from 8 to 12 weeks old female mice in DMEM, supplemented with L929 cell-conditioned medium (30%). The resulting cultures consisted of macrophages (>95% purity), as determined by staining for F4/80 and flow cytometry.

Isolation of splenic monocytes

Mouse monocytes were isolated from single cell suspension of spleens obtained from control and septic mice. Briefly, splenic cells were incubated for 30 min with a Ly6C biotinylated antibody and monocytes were isolated with magnetic streptavidin beads.

Killing of A. fumigatus by monocytes/macrophages

Primary human monocytes (2 x 10 5 /well) or BMDMs (1 x 10 5 /well) were plated onto 96-well round bottom plates for 1h, and subsequently infected with the indicated A. fumigatus strain at an MOI of 1:10 (conidia: target cell ratio) for 1 h at 37°C with or without the indicated concentration of JAK2 inhibitor (ruxolitinib) or STAT3 inhibitor (S3I-201) added 30 min before infection. Medium containing non-adherent, non-phagocytosed conidia was removed, wells were washed three times using warm PBS, and new media with or without the presence of the corresponding inhibitor were added. Monocytes were then allowed to kill conidia for 6h before intracellular conidia were harvested by lysis of monocytes with 0.5% Triton-X. The process of cellular lysis was confirmed by light microscopy and the killing of A. fumigatus conidia was assessed by CFU plating. Each condition was performed in quadruplicate with monocytes obtained from 4 different donors. In other experiments killing of FITC-labeled conidia by BMDMs was assessed by viability staining with Propidium Iodide (PI). Briefly, murine BMDMs were allowed to ingest FITC-labeled A. fumigatus conidia at an MOI of 1:1 for 1 h at 37°C in 24 well plates. BMDMs were allowed to kill conidia for 6 h before intracellular conidia were harvested by sonication (Akoumianaki et al., 2016). Next, cell lysates were stained with Propidium Iodide (PI) to assess the viability of A. fumigatus conidia. FITC labeled PFA-killed or live A. fumigatus conidia served as controls for PI staining.

Killing of Pseudomonas by BMDMs

BMDMs (2 × 10 5 per condition) from IL-6 -/-(KO) or control IL-6 +/+ mice were seeded overnight in 96 well flat bottom plates and infected with Pseudomonas aeruginosa (ATCC 27853 grown at a mid-logarithmic phase) in antibiotic free media at a MOI of 10:1 (target: effector ratio) with or without supplementation of culture media with IL-6 at a final concentration of 10 ng/ml. Cells were extensively washed at 1h of infection with warm PBS and a cell-impermeable antibiotic (gentamicin 300 μg/ml) was added in culture media for 30 min to kill remaining extracellular bacteria. The amount of phagocytosed bacteria at 1h of infection was assessed following cell lysis by CFU plating. Bacterial killing by BMDMs was assessed by CFU counts of intracellular bacteria at 12h of infection.

Western blot analysis

Primary human monocytes or murine BMDMs were stimulated with Aspergillus fumigatus conidia or PMA for the indicated time points at MOI 10:1 with or without the presence of the indicated inhibitor. Cells were washed once in PBS prior to lysis in 1% NP-40 containing RIPA buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0,25% sodium deoxycholate, 1 mM NaF, 1 mM Na 3 VO 4 , 1mM PMSF plus a mixture of protease inhibitors [Roche Molecular Biochemicals]). For analysis of p47phox phosphorylation, a mixture of phosphatase inhibitors (PhosSTOP; Roche Molecular Biochemicals) was added to the lysis buffer. Cell lysis was performed on ice for 30 minutes, and samples were centrifuged. After protein estimation of supernatants, addition of SDS sample buffer and boiling, samples were separated on SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Western blotting was performed according to the instructions of the manufacturer using the indicated primary antibodies: Secondary antibodies used in western blotting were purchased from Cell Signaling (anti-rabbit HRP, anti-goat HRP) as well as Millipore (anti-mouse HRP). The blots were developed using chemiluminescence (ECL; Thermo Scientific).

Measurement of cytokines

Supernatants of stimulated human monocytes were collected after overnight culture and were stored at -20°C until analysis. Cytokine levels in the supernatants were determined by using ELISA kits for human IL-1β, IL-1Ra, and TNF (R&D Systems) and IL-6 (Sanquin) according to the manufacturer's instructions.

RNA extraction

Monocytes isolated from different groups of sepsis patients stratified by LAP responses were lysed in Trizol reagent and stored at -80°C. Total RNA was extracted from cells using the QIAGEN RNeasy RNA extraction kit, using on-column DNaseI treatment. Ribosomal RNA was removed using the riboZero rRNA removal kit.

Library preparation for RNA-seq

RNA samples were shipped to the sequencing facility. RNA integrity was measured using Agilent TapeStation System. All samples had good qualities (RIN score >=8). RNA libraries were prepared to enrich for polyA tail-RNA using Bioscientific NEXTflex-Rapid-Directional mRNA-seq kits. RNA libraries were prepared with the Caliper SciClone system. The libraries were sequenced using the NextSeq500 High Output kit V2 (Illumina) for 75 cycles.

RNA-seq data analysis

Raw RNA-seq reads were aligned to the human genome version 37 using STAR (v2.5.1b) [START_REF] Dobin | STAR: ultrafast universal RNA-seq aligner[END_REF] HTSeq-count package (v0.61) was used to count and normalize the number of reads mapped to each gene [START_REF] Anders | HTSeq-a Python framework to work with highthroughput sequencing data[END_REF]. Differential expression was determined using DEseq2 (v.1.12.4) [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. For subsets of genes involved in autophagy, LAP, IL-6 signaling and ERK pathways (refer to Figure S7), normalized counts were log transformed and comparisons between patient groups was performed using One-way ANOVA and corrected for the number of genes tested (Bonferroni) (FDR<=0.05 and fold change >1.5). The RNAseq data have been deposited in the GEO database (GEO access number: GSE174183).

Quantification and statistical analysis

For in vivo results, each n value represents an individual experiment and mouse. For the human in vitro experiments, each n refers to a separate independent experiment. The number of experiments for each figure can be found in the figure legends. The data were expressed as mean ± SEM. Data were tested for Gaussian distribution using a Shapiro-Wilk normality analysis.

For normally distributed data two-sided unpaired Student's t test was used and for non-Gaussian data two sided unpaired Mann-Whitney tests were used to compute statistical significance between two groups. One-way ANOVA with the indicated post hoc test was used for multiple comparisons (P < 0.05 was considered statistically significant). Survival analysis and studies on the cumulative probability of infection in patients over time were performed by log-rank (Mantel-Cox) test. Analysis was performed using the GraphPad Prism software (version VII). All in vitro experiments were performed at least in triplicate and replicated at least twice.

Figure 1 .

 1 Figure 1. LAP blockade in monocytes is a hallmark of immunoparalysis in patients with sepsis. (A) Outline of the study protocol. (B) Monocytes obtained from healthy controls or patients upon sepsis recovery (day 7) were stimulated for 30 min with conidia of A. fumigatus melanin-deficient ΔpksP strain and analyzed for LC3+ phagosome (LAPosome) formation. Oneway ANO A, and Tukey's multiple comparisons. (C and D) Comparative analysis of (C) p47phox and (D) CD63 recruitment on the phagosome of monocytes stimulated as in (B). Oneway ANO A, Tukey's multiple comparisons. (E) Correlation between LAPosome formation and clearance of Aspergillus conidia (ATCC 46645, wild-type strain) by human monocytes obtained from sepsis patients (day 7; n = 9) and healthy controls (n = 5). Killing was assessed at 24 h of infection by CFU counting. Pearson correlation coefficient, two tailed. (F) IL-6 production in culture supernatants of monocytes following overnight stimulation with conidia of melanindeficient Aspergillus (ΔpksP strain). Student's t test. (G) Differences in Apache II score of patients with intact LAP versus defective LAP responses. Unpaired Student's t test. (H) Kaplan Mayer curves on the cumulative probability of secondary infections over time (n = 43 patients; n = 54 infectious episodes). Log-rank test. * * * p < 0.0001, * * p < 0.001, * p < 0.01.

Figure 2 .

 2 Figure 2. ERK signaling on the phagosome regulates NADPH oxidase-mediated activation of LAP. (A) Monocytes obtained from healthy individuals were left untreated or stimulated with PMA or conidia of Aspergillus ΔpksP strain (MOI 10:1) and phosphorylation of p47phox was determined in cell lysates by immunoblot analysis. (B) Human monocytes stimulated with Aspergillus as in (A) with or without the presence of the MEK1/2 inhibitor UO126 and phosphorylation of ERK1/2 and p47phox (Ser345) was determined in cell lysates by immunoblot analysis. (C) Kinetics of ERK recruitment and representative fluorescent image of ERK localization to the phagosome in monocytes at 30 min of infection with Aspergillus ΔpksP strain. Scale bar, 5 μm. (D and E) Data on quantification of p47phox + (D) and p22phox + (E) phagosomes in human monocytes stimulated with Aspergillus with or without UO126 as in (B). Unpaired Student's t test. (F) Human monocytes were left untreated or stimulated with Aspergillus for 1 h with or without UO126, and intracellular ROS production was determined. (G) Data on quantification of LC3 + phagosomes in human monocytes stimulated as in (D). Unpaired Student's t test. (H) Killing of Aspergillus ΔpksP conidia by human monocytes infected as in d for 24 h and assessed by CFU counts. Unpaired Student's t test. (I-K) (I) Data on quantification of LC3 + (I), p47phox + (J), and ERK + (K) phagosomes in monocytes obtained from a healthy individual (HC) and two patients following recovery from septic shock (day 7) stimulated as in (D). Unpaired Student's t test. Representative fluorescent images of ERK localization in phagosomes are shown. * * * p < 0.0001, * * p < 0.001, * * p < 0.01.

Figure 4 .

 4 Figure 4. IL-6/JAK2/ninein autocrine axis regulates microtubule-dependent ERK trafficking and LAPosome formation. (A) Data on quantification of ERK + phagosomes in BMDMs obtained from C57BL/6 (B6) mice left untreated or treated with nocodazole (5 μM) or taxol (5 μM). One-way ANO A, and Tukey's multiple comparisons. (B) Representative fluorescent images of β-tubulin in uninfected BMDMs from IL-6 -/-(KO), IL-6 +/+ (control), or IL-6 KO mice following IL-6 supplementation in culture media. Scale bar, 5 μm. (C) Representative fluorescent images of β-tubulin in BMDMs from IL-6 -/-(KO), IL-6 +/+ (control), or IL-6 KO mice following IL-6 supplementation, upon infection with Aspergillus ΔpksP strain (MOI 3:1, 30 min). Scale bar, 5 μm. (D) Representative time-lapse images of Aspergillus phagosome transport in BMDMs of IL-6 +/+ (control) or IL-6 -/-(KO) mice. Scale bar, 5 μm. (E) Data on quantification of the transport of Aspergillus-containing phagosomes from the minus end of microtubules to the organizing center of BMDMs analyzed from the experiment performed in (C). One-way ANOVA and Tukey's multiple comparisons. (F-I) Data on quantification of (F) Aspergillus-containing phagosomes transport, (G) ERK + phagosomes, (H) LAPosome formation, and (I) fungal killing, in BMDMs obtained from B6 mice left untreated, or treated with JAK2 inhibitor (ruxolitinib) and infected with Aspergillus ΔpksP strain. One-way ANO A and Tukey's multiple comparisons. (J) Data on quantification of γ-tubulin relative fluorescent intensity at the microtubule organizing center of uninfected BMDMs from IL-6 -/-(KO), IL-6 +/+ (control), or IL-6 KO mice following IL-6 supplementation in culture media for 15 min. (K) Data on quantification of ninein relative fluorescent intensity at the microtubule organizing center of uninfected BMDMs from IL-6 -/- (KO), IL-6 +/+ (control), or IL-6 (KO) mice following IL-6 supplementation. One-way ANOVA and Tukey's multiple comparisons. (L) Representative fluorescent images of γ-tubulin from experiment performed in (J). Scale bar, 5 μm. (M) Representative fluorescent images of immunostaining for ninein and JAK2 in uninfected BMDMs from IL-6 -/-(KO), IL-6 +/+ (control), or IL-6 (KO) mice following IL-6 supplementation. * * * p < 0.0001, * * p < 0.001, * p < 0.01.

Figure 5 .

 5 Figure 5. Increased susceptibility of mice recovering from sepsis to fungal disease is associated with LAP blockade in monocytes/macrophages. (A) Outline of the mouse model of bacterial peritonitis (CLP). (B) Survival rates of C57BL/6 (B6) mice subjected to different degree of severity of polymicrobial sepsis (CLP) or SHAM treated (control) mice. (C) Fungal loads of C57BL/6 (B6) mice infected via intratracheal administration of Aspergillus (ATCC46645 strain) following the recovery from mild, severe sepsis or SHAM treatment (day 7), assessed by CFU counts at 48 h. Mann-Whitney test. (D) Histopathology (GMS staining) from representative tissue sections of lungs from B6 mice on day 3 of infection with Aspergillus wild-type strain as in (C) following recovery from severe sepsis (hereafter sepsis) or SHAM treatment. Magnification ×400. (E) Representative immunofluorescent images of LAPosome formation in splenic monocytes from B6 and BMDMs from GFP-LC3 transgenic mice stimulated for 30 min with Aspergillus (melanin-deficient ΔpksP strain) following recovery from severe sepsis (sepsis) or SHAM (control). (F) Data on quantification of LC3 + phagosomes in monocytes, BMDMs, or alveolar macrophages (AMs) stimulated as in (E). One-way ANO A and Tukey's multiple comparisons. (G) Intracellular ROS production in BMDMs from mice following recovery from sepsis (day 7) or control SHAM treatment. (H) Assessment of phagolysosomal fusion in BMDMs pre-loaded with FITC-dextran and infected for 4 h as in (G). (I) Representative fluorescence images are shown in (I). Scale bar, 5 μm. (J-L) Data on quantification of VATPase + (J), cathepsin D + (K), and LAMP-1 + (L) phagosomes in BMDMs from B6 mice stimulated as in (E). Unpaired Student's t test. (M) Data on killing of Aspergillus (ATCC 46645 strain) by BMDMs obtained from B6 mice following recovery from sepsis or SHAM treatment. Unpaired Student's t test. * * * p < 0.0001, * * p < 0.001, * p < 0.01.

Figure 6 .

 6 Figure 6. Restoration of IL-6 signaling defects in mice and human patients recovering from sepsis reverses LAP blockade and impaired fungal clearance. (A) Representative fluorescent images of β-tubulin in uninfected BMDMs from B6 mice following SHAM treatment or recovery from sepsis with or without IL-6 supplementation. Scale bar, 5 μm. (B) Data on quantification of the transport of Aspergillus-containing phagosomes (ΔpksP strain) in BMDMs obtained from experiments performed as in (A). One-way ANO A and Tukey's multiple comparisons. (C)Representative images of ERK recruitment on the phagosome of BMDMs (30 min) obtained from experiments performed as in (B). Scale bar, 5 μm. (D and E) Data on quantification of ERK + (D) and LC3 + (E) phagosomes in BMDMs from experiment performed in (C). One-way ANO A and Tukey's multiple comparisons. (F) BMDMs from SHAM treated mice, or mice recovering from sepsis (day 7) were infected with the indicated Aspergillus strain, with or without supplementation with IL-6, TNF, or IFN-γ. Κilling of intracellular conidia was assessed by counting of viable (germinating) conidia. One-way ANOVA and Tukey's multiple comparisons. (G) Representative fluorescent images of β-tubulin in monocytes from a healthy control (HC) individual, and patients recovering from sepsis at 30 min of infection with Aspergillus ΔpksP strain. Scale bar, 5 μm. (H) Μonocytes obtained from sepsis patients and healthy controls (HC) were infected with conidia of Aspergillus fumigatus (ATCC 46645 strain), with or without IL-6 supplementation and killing of conidia was assessed by CFU counting at 24 h. Unpaired Student's t test. * * * p < 0.0001, * * p < 0.001, * p < 0.01.

resources table

	REAGENT or RESOURCE	SOURCE	IDENTIFIER
	Rabbit polyclonal
	REAGENT or RESOURCE p47 phox antibody anti-PhosphoSer315-	SOURCE Boussetta et al., 2010	IDENTIFIER N/A
	Antibodies Rabbit polyclonal
	Monoclonal Anti-beta-Tubulin antibody produced in anti-PhosphoSer328-p47 phox antibody Rabbit polyclonal	Boussetta et al., 2010 Sigma-Aldrich	N/A Cat# T4026; RRID: AB_477577
	mouse anti-PhosphoSer345-Mouse monoclonal Anti-Actin Antibody, clone C4 p47 phox antibody Mouse Ninein (F-5) antibody	Dang et al., 2006 Millipore Santa Cruz y Biotechnolog	N/A Cat# MAB1501; RRID: AB_2223041 Cat# sc-376420, RRID:AB_11151570
	monoclonal Anti-LC3 PerCP anti-human HLA-DR	Biolegend	Cat# RRID: AB_893574
	(microtubule-associated protein APC anti-human CD14 1 light chain 3B) antibody Deposited data	Nanotools Biolegend	Cat# 0231-100/LC3-5F10; RRID: AB_2722733 Cat# RRID: AB_830680
	Rabbit Anti-LC3 polyclonal, Unconjugated Original Wester Blot and FACS gating	Novus This study	Cat# NB 100-2220; RRID: AB_578334 https://data.mendeley.com/datasets/cgzsvpmpzc/draft?a=f204d5 e0-349e-4781-914c-96fd2a0f74d8
	antibody RNA-seq data	This study	GEO database (GEO access number: GSE174183)
	Rabbit polyclonal LC3B Antibody Chemicals	Novus	Cat# NB600-1384; RRID: AB_669581
	Rabbit mAb IL-6 (D5W4V) XP®	Cell Signaling	Cat#12912; RRID: AB_2798059
	Mouse	Anti-
	CD107a
	monoclonal Antibody,		BD Biosciences	Cat# 555798; RRID: AB_396132
	Unconjugated,
	Clone H4A3
	Mouse	Anti-
	CD63
	monoclonal Antibody,		BD Biosciences	Cat# 556019; RRID: AB_396297
	Unconjugated,
	Clone H5C6
	Mouse		Santa Cruz Cat#sc-17845; RRID: AB_627986

, peptides, and recombinant proteins

	REAGENT or RESOURCE	SOURCE	IDENTIFIER
	(Succinimidyl
	Ester)
	Nocodazole		Sigma-Aldrich	M1404; CAS: 31430-18-9
	Paxene
	(paclitaxel		IVAX	N/A
	6mg/ml)
	S3I-201		Sigma-Aldrich	SML0330; CAS: 501919-59-1
	Critical commercial assays
	Mouse	IL-6
	ELISA	MAX	BioLegend Cat:430304
	Deluxe kit
	Human	IL-
	6 Uncoated Elisa	Invitrogen	REF:88-7066-22
	kit
	Experimental models: Cell lines
	NCTC clone 929
	[L cell, L-929, derivative of	ATCC	CCL-1
	Strain L]
	Experimental models: Organisms/strains
	Aspergillus fumigatus		ATCC	ATCC 46645
	Ruxolitinib Mouse: IL6 +/+ of background 129×C57BL/6 genetic	CAYMAN CHEMICAL COMPANY (Venihaki et al., 2001)	11609; CAS: 941678-49-5 N/A
	Recombinant Murine IL-6 Mouse: IL6 -/-of 2′,7′-Dichlorofluoresci n diacetate 129×C57BL/6 genetic background U0126 Mouse: C57BL/6 (B6)	PEPROTEC H (Venihaki et al., 2001) Sigma-Aldrich Calbiochem 662005; CAS: 109511-58-2 216-16 N/A D6883; CAS: 4091-99-0 The Jackson Laboratory 000664
	Propidium Iodide Mouse: GFP-LC3	Sigma-Aldrich RIKEN BioResource	P4170; CAS: 25535-16-4 RBRC00806
	Fluorescein isothiocyanate-dextran Software and algorithms Center Sigma-Aldrich Alexa Fluor™ 633 NHS Ester ThermoFishe r Scientific GraphPad Prism 5 GraphPad Software	46945; CAS: 60842-46-8 A20005 https://www.graphpad.com/

Experimental model and subject details Human study subjects and clinical specimens

  solid organ transplantation, daily corticosteroid therapy greater than 0.5 mg/kg prednisone equivalent, cytokine-blocking agents, chemotherapy or any other immunosuppressive treatments. We enrolled healthy volunteers as controls for functional studies.

	Approval for the collection of clinical information and tissue samples from septic patients and
	control healthy individuals was obtained from the Ethics Committee of the University Hospital of
	Heraklion, Crete, Greece (5159/2014 and 10925/2018) from the appropriate Ethics Committee in
	France (CPP Ile-de France 3, ref #S.C. 3351). The patient or next of kin and each healthy
	volunteer provided written informed consent in accordance with the Declaration of Helsinki.
	Blood was collected on the day of sepsis diagnosis (Day 1) and later on following sepsis recovery
	(Day 7).
	All consecutive adult patients admitted to the ICU of a tertiary center Hospital (University
	Hospital of Crete) from April 2014 to December 2017 with the clinical diagnosis of community
	acquired septic shock, and a high index of disease severity (APACHE II score ≥ 8) were eligible
	for the study. Additional sepsis patients were enrolled between December 2019 and April 2020.
	The diagnosis of septic shock was based on Standardized Criteria (Singer et al., 2016) as follows:
	an identifiable site of infection and at least two of the signs of systemic inflammatory response
	syndrome (SIRS); and 2) arterial blood pressure of < 90 mmHg despite adequate fluid
	resuscitation and requiring vasopressor therapy. Additional patients with septic shock admitted to
	the medical ICU of Cochin Hospital (Paris, France) were enrolled in 2017.

Exclusion criteria were an age less than 18 years, pregnancy, do-not-resuscitate orders on admission, hematologic malignancy, immunodeficiency defined as HIV infection, presence of bone marrow or

  , GFP-LC3 mice (RIKEN BioResource Center), Atg5 conditional KO mice generated by crossing Atg5 flox/flox mice(RIKEN BioResource Center; (Akoumianaki et al., 2016)) and VavCre +/+ mice(Akoumianaki et al., 2016), were maintained in grouped cages in a High-Efficiency Particulate Air-filtered environmentally controlled virus-free facility (24°C, 12/12 h light/dark cycle), and fed by standard chow diet and water ad libitum. All experiments were approved by the local ethics committee of the University of Crete Medical School, Greece in line with the corresponding national and European Union legislation (animal protocols 07/05/14-6167 and 17/07/2017-147075).

  ). Oneway ANO A, Tukey's multiple comparisons. (E) Correlation between LAPosome formation and clearance of Aspergillus conidia (ATCC 46645, wild-type strain) by human monocytes obtained from sepsis patients (day 7; n = 9) and healthy controls (n = 5). Killing was assessed at 24 h of infection by CFU counting. Pearson correlation coefficient, two tailed. (F) IL-6 production in culture supernatants of monocytes following overnight stimulation with conidia of melanindeficient Aspergillus (ΔpksP strain). Student's t test. (G) Differences in Apache II score of patients with intact LAP versus defective LAP responses. Unpaired Student's t test. (H) Kaplan Mayer curves on the cumulative probability of secondary infections over time (n = 43 patients; n = 54 infectious episodes). Log-rank test. * * * p < 0.0001, * * p < 0.001,

* p < 0.01.

  Dextran + phagosomes in BMDMs from IL-6 -/-(KO) or control IL-6 +/+ mice infected with Aspergillus ΔpksP strain for 15 min (A-C) or 1 h (D-F). Unpaired Student's t test. (G) BMDMs from IL-6 -/-(KO) or control IL-6 +/+ mice were infected with conidia of Aspergillus wild-type strain (ATCC 46645) for 6 h and killing was assessed by counting the viability of intracellular conidia. Unpaired Student's t test. (H) Fungal loads in lung homogenates from IL-6 -/-(KO) (n = 9) or control IL-6 +/+ (n = 9) mice infected via intratracheal administration of wild-type Aspergillus fumigatus (ATCC 46645) assessed by CFU counts at 48 h of infection. Mann-Whitney test. (I) BMDMs from IL-6 -/-(KO) or control IL-6 +/+ mice were left untreated or stimulated with Aspergillus for 15 min and phosphorylation of ERK1/2 was determined in cell lysates by immunoblot analysis. (J) Quantification of relative density of p-ERK1/2 to total ERK1/2 in blots from four different experiment performed as in (I). Mann-Whitney test. (K) Representative fluorescent images of ERK phosphorylation (p-ERK) and ERK localization on the phagosome of BMDMs stimulated as in (J). Scale bar, 5 μm. (L) Data on quantification of ERK + phagosomes in BMDMs of IL-6 -/-(KO), IL-6 +/+ (control), or IL-6 (KO) mice following IL-6 supplementation in culture media upon infection with Aspergillus (30 min). One-way ANO A, Tukey's multiple comparisons. (M) BMDMs from IL-6 -/-(KO) or control IL-6 +/+ mice were infected with conidia of Aspergillus ΔpksP strain or wild-type strain (ATCC 46645) with or without supplementation of culture media with IL-6, TNF, or IFN-γ, and killing was assessed by counting the viability of intracellular conidia at the indicated time point. Oneway ANO A, Tukey's multiple comparisons. * * * p < 0.0001, * * p < 0.001,

	Unpaired Student's t test. (F) Human monocytes were left untreated or stimulated with
	Aspergillus for 1 h with or without UO126, and intracellular ROS production was determined.
	(G) Data on quantification of LC3 + phagosomes in human monocytes stimulated as in (D).
	Unpaired Student's t test. (H) Killing of Aspergillus ΔpksP conidia by human monocytes infected
	as in d for 24 h and assessed by CFU counts. Unpaired Student's t test. (I-K) (I) Data on
	quantification of LC3 + (I), p47phox + (J), and ERK + (K) phagosomes in monocytes obtained from
	a healthy individual (HC) and two patients following recovery from septic shock (day 7)
	stimulated as in (D). Unpaired Student's t test. Representative fluorescent images of ERK
	localization in phagosomes are shown. * * * p < 0.0001, * * p < 0.001, * * p < 0.01.

+ (D) and p22phox + (E) phagosomes in human monocytes stimulated with Aspergillus with or without UO126 as in (B). Figure 3. Autocrine IL-6 signaling regulates ERK trafficking to the phagosome for LAPmediated fungal killing. (A-F) Data on quantification of (A) p22phox + , (B) p47phox + , (C) LC3 + , (D) VATPase + , (E) cathepsin-D + , and (F) FITC-* p < 0.01.

Acknowledgments

The authors would like to thank Yiannis Talianidis and Agostinho Carvalho for helpful suggestions. T.A.'s work was supported by a grant from Hellenic Foundation for Research and innovation (HFRI) (#1787); G.C. was supported by grants from the Greek State Scholarship Foundation (I.K.Y.), the Hellenic General Secretariat for Research and Technology-Excellence program (ARISTEIA), an Advanced Research Grant from Institut Mérieux (#4719), H2020-SC1-BHC-2018-2020 (HDM-FUN 847507), and an ERC Consolidator Grant (iMAC-FUN-#864947); J.-P.L. was supported by European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement 260338 ALLFUN and ANR-10-BLAN-1309 hydrophobin, and the Association Vaincre La Mucoviscidose (RF20140501052/1/1/141). M.G.N. is supported by an ERC Consolidator grant (#310372) and a Spinoza grant of the Netherlands Organization for Scientific Research (NWO). This study was supported by a TOP grant (project number: 91214016) of the Netherlands Organization for Health Research and Development (ZonMw) (to M.G.N.).

ACKNOWLEDGMENTS

The authors would like to thank Yiannis Talianidis and Agostinho Carvalho for helpful suggestions. T.A.'s work was supported by a grant from Hellenic Foundation for Research and innovation (HFRI) (#1787); G.C. was supported by grants from the Greek State Scholarship Foundation (I.K. .), the Hellenic Gen-eral Secretariat for Research and Technology-Excellence

Materials availability

All the data that support the findings of this study will be made available upon request made to the Lead Contact.

Data and code availability

The published article includes all datasets generated or analyzed during this study. The RNAseq data have been deposited in the GEO database (GEO access number: GSE174183). Original FACS analysis and western blots data were deposited at Mendeley at: https://data.mendeley.com/datasets/cgzsvpmpzc/draft?a=f204d5e0-349e-4781-914c-96fd2a0f74d8

analyzed data and provided discussions and suggestions throughout the study; G.C. conceived and supervised the study, was involved in the design and evaluation of all of the experiments, and wrote the manuscript along with comments from co-authors.

Declaration of interests

The authors declare no competing interests. analyzed data and provided discussions and suggestions throughout the study; G.C. conceived and supervised the study, was involved in the design and evaluation of all of the experiments, and wrote the manu-script along with comments from co-authors.

AUTHOR CONTRIBUTIONS

DECLARATION OF INTERESTS

The authors declare no competing interests.


Evolene Premillieu
email: evolene.premillieu@colorado.edu

Simon Labouesse

Kristina Irsch

Rafael Piestun

Blind speckle illumination for aberration correction

We propose a computational optical technique based on speckle-pattern illumination of an object and a gradient descent algorithm, enabling aberration correction without requiring the addition of adaptive optics.

Introduction

Aberration correction in imaging systems is traditionally done with complex optical designs or adaptive optics. Unfortunately, in some circumstances it is not feasible to use aberration-corrected optics or set up an adaptive-optics sub-system. With the development of computational imaging methods, new algorithms offer solutions for postprocessing aberration correction and resolution improvement. Achieving both aberration correction and improved resolution is however not trivial. In recent years, novel algorithms have been developed to recover high-resolution images from a stack of shifted images [START_REF] Guo | 13-fold resolution gain through turbid layer via translated unknown speckle illumination[END_REF], [START_REF] Zhang | Near-field Fourier ptychography: super-resolution phase retrieval via speckle illumination[END_REF]. The principle consists of scanning a speckle pattern illumination over an area of interest and then recover a high-resolution object from the information encoded in the blurry images. These techniques originated from Blind Structured Illumination Microscopy (Blind SIM) with unknown speckle patterns [START_REF] Mudry | Structured illumination microscopy using unknown speckle patterns[END_REF]. Scanning a speckle illumination to acquire fluorescence images works quite well, as demonstrated by Kaikai Guo et al. [START_REF] Guo | 13-fold resolution gain through turbid layer via translated unknown speckle illumination[END_REF]. However, it is not always applicable to any imaging system. He Zhang et al. developed a way to perform coherent imaging with improved resolution using scanned speckle illuminations and phase retrieval [START_REF] Zhang | Near-field Fourier ptychography: super-resolution phase retrieval via speckle illumination[END_REF].

Here we propose a new approach using a simple interferometric set-up and requiring no prior knowledge of the system. We aim at correcting for aberrations and improving the resolution using a gradient descent algorithm. We demonstrate the correction of aberrations caused by a prescription lens and reach a resolution limited by the size of the speckles.

Methods

An image can be represented mathematically as the convolution between the point spread function of the system and the object times the illumination:

𝑦(𝑟, 𝑥) = ∫ ℎ(𝑟 -𝑟 ′ )𝜌(𝑟 ′ )𝐸(𝑟 ′ -𝑥)𝑑𝑟′
where y is the image, h the point spread function, ρ the object and E the illumination. When shifting the object with respect to the illumination we can recover enough information from the images to find an estimate for all the unknowns. We carry out the reconstruction through a gradient descent algorithm, which is expressed as the minimization problem of the functional J defined as:

𝐽 = ∑‖𝑦 𝑖 -ℎ 𝑘 ⨂(𝜌 𝑘 . 𝐼 𝑖 𝑘 )‖ 2 𝑖
We implement an alternating minimization of the criterion. The three gradients are expressed as follows:

𝛻 𝜌 𝐽 = 𝛻 𝜌 ∑ ‖𝑦 𝑖 -ℎ 𝑘 ⨂(𝜌 𝑘 . 𝐼 𝑖 𝑘 )‖ 2 𝑖 , 𝛻 𝐼 𝐽 = 𝛻 𝐼 ∑ ‖𝑦 𝑖 -ℎ 𝑘 ⨂(𝜌 𝑘 . 𝐼 𝑖 𝑘 )‖ 2 𝑖 and 𝛻 ℎ 𝐽 = 𝛻 ℎ ∑ ‖𝑦 𝑖 -ℎ 𝑘 ⨂(𝜌 𝑘 . 𝐼 𝑖 𝑘 )‖ 2 𝑖
. When the convergence is reached, the algorithm yields an object free of aberrations with a resolution dictated by the speckle size, and estimates of the transfer function of the system, of the illumination and of the actual shift positions. In order to experimentally acquire the data, we build a simple imaging interferometer. We use a He-Ne laser and use a random phase mask on a Spatial Light Modulator (SLM) that we Fourier filter to create the speckle illumination. The object is imaged onto CCD camera. We use motorized linear stages to shift the object with respect to the illumination. The data is acquired for N different steps with N=5 on a 30x30 position grid. The step size is set to be smaller than the speckle size (14.5 μm with speckle size 35 μm, for the data presented).

Results

To retrieve an aberration-free, high-resolution image we first generate an estimate of the shifted positions of the object. We also need to generate an initial guess of the object, the illumination and the point spread function of the system to start the gradient descent algorithm. As an example, we correct the aberrations induced by prescription glasses (spherical aberration of -1 D and astigmatism of 0.25 D) near the Fourier plane of a 4f imaging system. The object is a USAF resolution target, centered on groups 3, 4, 5, 6, and 7. The reconstructed aberration-free object with increased resolution is depicted in Figure 1.

Discussion

We introduce a new technique to correct for aberrations in a coherent imaging set-up. A advantage of our method is that it works in blind conditions where no prior knowledge of the system is required. All that is needed is a speckle illumination and a means to scan the object with respect to the illumination or vice-versa. We anticipate this method could find great use in improving imaging setups as an alternative to adaptive optics.

Fig. 1 .

 1 Fig. 1. Experimental results illustrating correction of aberrations from a prescription lens. a) USAF resolution target groups 3, 4, 5, 6, 7 imaged through the aberrated system with uniform illumination. b) USAF resolution target groups 3, 4, 5, 6, 7 imaged through the system with uniform illumination without any aberration induced by the prescription lens. The intensity profile shows that group 5 elements 4, 5, 6 are unresolved. c) Initial guess for the object obtained by averaging images acquired with speckle illumination. d) Reconstruction of the object obtained after 200 iterations of the algorithm. The intensity profile shows that group 5 elements 4 and 5 are resolved, while element 6 remains unresolved. e) Recovered coherent point spread function of the total imaging system composed of 2 imaging lenses and the prescription lens inserted in between. f) Recovered illumination incident on the object. g) Recovered shift positions; the initial estimate of the position grid is shown in blue, while the corrections made by the algorithm are marked in red.

FM5C.6.pdfFrontiers in Optics + Laser Science © Optica Publishing Group 2021