Nadia Nathan
email: nadia.nathan@aphp.fr

Chiara Sileo
email: chiara.sileo@aphp.fr

Alain Calender
email: alain.calender@chu-lyon.fr

Yves Pacheco
email: yves.pacheco@chu-lyon.fr

Paul-André Rosental

Catherine Cavalin

Odile Macchi

Dominique Valeyre
email: dominique.valeyre@aphp.fr

Annick Clement
email: annick.clement@aphp.fr

Paediatric sarcoidosis

Keywords: sarcoidosis, children, granuloma, interstitial lung disease

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Sarcoidosis, also called Besnier-Boeck-Schaumann disease, was first described in the 19 th century as a systemic granulomatous disorder involving predominantly the lungs and the lymphatic system (1). More than a century after, its pathogenesis remains unknown and its pathophysiology remains controversial [START_REF] Hunninghake | ATS/ERS/WASOG statement on sarcoidosis. American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and other Granulomatous Disorders. Sarcoidosis Vasc Diffuse Lung Dis Off[END_REF][START_REF] Iannuzzi | Sarcoidosis[END_REF]. The current hypothesis is a multifactorial disease associating a genetic susceptibility and an environmental exposure that triggers or enhances the inflammatory and granulomatous process. Sarcoidosis is mainly an adult disease, children being affected around 10 times less frequently. Its phenotypic spectrum is wide, from a sub-clinical form with only two organs involved to a severe multi-organic disease [START_REF] Shetty | Sarcoidosis in children[END_REF][START_REF] Shetty | Childhood sarcoidosis: A rare but fascinating disorder[END_REF]. A pathologic confirmation after rigorous investigation for excluding alternative diagnoses, particularly immune deficiencies, is particularly indispensable in children in whom paediatric sarcoidosis remains a "diagnosis of exclusion".

The aim of this review is to provide an overview on sarcoidosis in children, and to propose ways to improve its understanding, diagnosis and management. This work is based on the literature on paediatric sarcoidosis that is extremely limited. Most of the studies refer to Blau syndrome and "early onset sarcoidosis", which are now recognized as specific genetic entities different from sarcoidosis. This inflammatory disorder, that is not discussed in this review is related to NOD2/CARD15 gene mutations.

Literature information and epidemiology

In recent decades, a maximum of 22 manuscripts per year have been published with reference (in the title or in the abstract) to paediatric sarcoidosis (compared to more than 900 for adult sarcoidosis), more than half of them being case reports, and the majority of the remaining articles not being focused on sarcoidosis. This could be due to the rarity of the disease in the paediatric population, but also to the lack of centralized management of orphan diseases in the majority of the countries that does not help to gather these rare cases. Considering these limitations, cohort studies can be an unreachable challenge. However, in the recent years, a few studies that will be discussed reported cohorts of patients and showed that paediatric sarcoidosis can be of importance in the comprehension of sarcoidosis pathophysiology.

Only 3 retrospective limited case series have been described since 2004: 48 Danish patients (that we will further call the "DK series"), 41 French patients (the "FR series") and 27 American patients from Louisiana (the "US-LA series") respectively [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF][START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF]. Their main characteristics are summarized in Table 1. The DK series was conducted on the national registries between 1979 and 1994 and reviewed 48 patients among which 2 twins were finally diagnosed with Blau syndrome [START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF]. A pathologic confirmation of the diagnosis was available for 33 of the 46 patients. The management and the follow-up of these children was secondarily reported until 2006 [START_REF] Milman | Childhood sarcoidosis: long-term follow-up[END_REF]. The FR series reported 41 patients registered since 2008 in the national reference network for rare lung diseases. All of them had lung involvement and a histologically proven diagnosis [START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF]. The US-LA series reported 27 patients diagnosed in a single rheumatologic department between 1992 and 2013 with paediatric sarcoidosis (22 patients), or Blau syndrome (5 patients). A histologic confirmation was obtained in 18 (67%) [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF].

The incidence of sarcoidosis is difficult to assess but the DK series estimated an incidence of 0.29 per 100,000 children, and the FR series 0.4-0.8 per 100,000 children. Excluding Blau syndrome, the incidence seems to increase with age, from 0.06 in children <4 years of age to 1.02 in children aged 14-15 years, but children of all ages were affected (Figure 1) [START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Pulmonary sarcoid-like granulomatous disease in an 11-month-old girl[END_REF]. However, paediatric figures are far lower than in adults as recently shown in Seine Saint Denis area (France) with an incidence of 4.9/100,000 [START_REF] Duchemann | Prevalence and incidence of interstitial lung diseases in a multi-ethnic county of Greater Paris[END_REF]. The mean age at diagnosis was 11 to 13 years-old in the 3 studies and sarcoidosis was encountered equally in males and females. A predominance of black children was noticed in the US-LA series (n=20, 74%) and the FR series (n=36, 88%) contrasting to only 33.8% of adult patients in the same area in France, and to none of the patients of the DK series [START_REF] Mirsaeidi | Racial Difference in Sarcoidosis Mortality in the United States[END_REF][START_REF] Rybicki | Racial differences in sarcoidosis incidence: a 5-year study in a health maintenance organization[END_REF].

Pathophysiology

Knowledge of the pathophysiology of sarcoidosis is still limited. The current hypothesis is an aberrant granulomatous and inflammatory reaction following the combination of 2 complementary factors: a genetic predisposition, and an exposure to an organic or a mineral antigen. Studies involving twins suggested that genetic background accounts for more than half of the risk factors for sarcoidosis onset, but they were limited studies and the relative contributions of both genetic and environmental aspects to sarcoidosis evolution remains to be ascertained [START_REF] Sverrild | Heredity in sarcoidosis: a registry-based twin study[END_REF].

Granuloma constitution

Sarcoidosis granulomas have a typical pathologic aspect of a central follicle constituted by activated macrophages named epithelioid cells, and T CD4 lymphocytes (L) mainly of the Helper subtype (Th1). A lymphocytic ring of CD8 TL and BL surrounds the central follicle.

The granuloma contains no necrotic aspects (Figure 2) [START_REF] Nunes | Pulmonary sarcoidosis[END_REF]. During the granuloma formation process, a Th17 (T-reg) TL influx is observed and could be locally responsible for the perpetuation of the granulomatous response despite the eventual removal of the causative agent and, at the peripheral level, could be responsible for the negative tuberculosis skin test of the patients (16).

Genetics and sarcoidosis

A genetic background of sarcoidosis is supported by the observations of familial forms of sarcoidosis in 3% to 5% of the cases [START_REF] Rybicki | Familial aggregation of sarcoidosis. A case-control etiologic study of sarcoidosis (ACCESS)[END_REF]. An additional effect of a limited number of genes, such as common variants, presenting in more than 1% of the general population (single nucleotide polymorphisms, SNP) seems a more realistic model than a monogenic transmission. Recent whole genome association studies highlighted the implication of various genes or loci in the development of the disease. These genes have in common their role in regulation, activation and differentiation of TL and macrophages. Among them, the most studied are BTNL2 (Butyrophilin Like 2), Anxa11 (Annexine A11), HLA-DRB1, CCDC88B (Coiled-Coil Domain Containing 88B), and XAF1 (XIAP Associated Factor 1) [START_REF] Fischer | Granuloma genes in sarcoidosis: what is new?[END_REF][START_REF] Pacheco | Pathogenesis of sarcoidosis[END_REF][START_REF] Pacheco | Sarcoidosis and genetics[END_REF].

BTNL2 encodes a cofactor of the TL cluster of differentiation (CD) 86. A splice variant, rs2076530, that truncates the BTNL2 protein, has been initially associated with an increased risk of sarcoidosis [START_REF] Lin | BTNL2 gene polymorphism and sarcoidosis susceptibility: a meta-analysis[END_REF]. However, this association seems weak with an odds ratio (OR) of 2 in the development of familial and sporadic forms of sarcoidosis [START_REF] Pacheco | Comparative phenotype and BTNL2 polymorphism in familial and sporadic sarcoidosis in the French SARCFAM cohort[END_REF][START_REF] Pacheco | Familial vs. sporadic sarcoidosis: BTNL2 polymorphisms, clinical presentations, and outcomes in a French cohort[END_REF]. Anxa11 plays an important role in cellular division, intra-cellular trafficking and apoptosis issues. The ''C'' allele of SNP rs1049550 seems associated with an increased risk of sarcoidosis, again with a limited OR around 1.5, whereas the SNP rs2573346 and rs2789679 seem to confer a protection against sarcoidosis [START_REF] Zhou | The Association between ANXA11 Gene Polymorphisms and Sarcoidosis: a Meta-Analysis and systematic review. Sarcoidosis Vasc Diffuse Lung Dis Off[END_REF]. Recently, the hypertrophy and proliferation of the macrophages resulting in the granuloma formation were shown to be enhanced by the activation of the checkpoint kinase mTORC1 in macrophages in vivo [START_REF] Linke | Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression[END_REF]. The role of autophagy pathways was also supported by a national study on a large cohort of paediatric and familial cases of sarcoidosis that revealed with the mean of trio analysis (one child with sarcoidosis and his two healthy parents), that numerous genes involved in autophagy seem to play a major role in the development of the disease (26).

Exposome and sarcoidosis

The role of environmental exposures in sarcoidosis has been reinforced in recent years. Organic exposure, mainly to mycobacteria, were first reported, followed by inorganic, mainly mineral exposures [START_REF] Gupta | Molecular evidence for the role of mycobacteria in sarcoidosis: a meta-analysis[END_REF][START_REF] Agrawal | Tuberculosis or sarcoidosis: Opposite ends of the same disease spectrum?[END_REF][START_REF] Mortaz | Sarcoidosis: Role of non-tuberculosis mycobacteria and Mycobacterium tuberculosis[END_REF][START_REF] Heffner | The cause of sarcoidosis: the Centurial enigma solved[END_REF]. Various studies have revealed an increased incidence of sarcoidosis after uncommon exposures to silica, aluminium, and inorganic dusts [START_REF] Newman | Occupational causes of sarcoidosis[END_REF]. This observation has been reinforced by the significant increase of sarcoidosis cases in individuals exposed to the World Trade Centre debris in 2001 [START_REF] Girvin | Radiologic Features of World Trade Center-related Sarcoidosis in Exposed NYC Fire Department Rescue Workers[END_REF]. To date, the impact of inorganic particles on inflammatory stimuli seems accepted in sarcoidosis but needs further confirmation [START_REF] Vincent | From the definition of silicosis at the 1930 Johannesburg conference to the blurred boundaries between pneumoconioses, sarcoidosis, and pulmonary alveolar proteinosis (PAP)[END_REF][START_REF] Pacheco | Granulomatous lung inflammation is nanoparticle type-dependent[END_REF]. This hypothesis is the basis of a national study on the exposome in the French paediatric cohort [START_REF] Nathan | Inorganic exposome in pediatric sarcoidosis: The PEDIASARC study[END_REF]. A larger adult and paediatric project studying the mineral exposome in sarcoidosis and other inflammatory diseases is currently undergoing: the SILICOSIS project founded by the European Research Council (2012-2017) http://www.sciencespo.fr/silicosis/fr.

Diagnosis

In children, sarcoidosis is an exclusion diagnosis that is suggested by clinical manifestations and needs to be confirmed by the observation of a typical granuloma at the histologic examination of a biopsied tissue. Other causes of granuloma should be excluded: Blau syndrome, tuberculosis and other mycobacteria, immune deficiency such as combined immunodeficiency, eosinophilic granuloma, Crohn's disease, tumours, and rarely drug induced granulomatosis.

The investigations need to be extensive as paediatric sarcoidosis is identified as a multi-organ disorder in 70% of the US-LA patients and 85% of the FR patients, with a mean of 3.8 to 5.1 involved organs at diagnosis (6,8).

Clinical presentation

In the 3 main paediatric cohorts, general signs were at the forefront (98% in the DK series, 78% in the FR series and 66% in the US-LA series). They included mainly fever (38%, 44%, 48% in the DK, FR, US-LA series respectively), but also marked fatigue, weight loss or lack of weight gain [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF][START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF][START_REF] Kapoor | Sarcoidosis-associated fatigue: an often forgotten symptom[END_REF]. The families frequently reported severe fatigue in the weeks or months preceding the diagnosis.

The most frequent presentations of sarcoidosis in children were related to lung and mediastinal disease [START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF][START_REF] Baughman | Clinical characteristics of patients in a case control study of sarcoidosis[END_REF]. Respiratory symptoms were reported in the US-LA series in 3 of the 7 patients (43%) with a lung involvement and in 56% of the FR patients, despite all of them having lung sarcoidosis. When present, the lung manifestations were mainly dry cough, dyspnoea and chest pain. They were respectively reported by 65%, 29% and 12% of the DK patients. Extra-respiratory manifestations were often present. These should be actively sought at diagnosis: hepatomegaly and/or splenomegaly (50%, 49%, 30% in the DK, FR, US-LA series respectively); a decrease in visual acuity, an eye pain, a red eye, or any other visual anomaly that can evocate a posterior or anterior uveitis (29%, 39%, 74% respectively); peripheral lymphadenopathy (40%, 29%, 30% respectively); skin anomalies (42%, 19%, 18% respectively); or articular pain or inflation (14%, 15%, 30% respectively). Other clinical signs were less frequently described (ENT, cardiac, neurological, gastrointestinal, parotiditis) but have to be considered at diagnosis, as well as indirect signs of haematologic involvement (pallor, ecchymosis, purpura) (6-8). Finally, an anergic tuberculin skin test for tuberculosis was usually noted.

Paraclinical tests

In the reported children with lung sarcoidosis, the chest X-ray could be normal (stage 0 of the Scadding classification), as reported in 39% of the patients included in the DK, FR and US-LA series, even if respiratory signs were present [START_REF] Scadding | Calcification in sarcoidosis[END_REF]. Hilar and mediastinal lymphadenopathies without lung involvement (stage I) were reported in 34% of the patients.

A lung infiltration without fibrosis (stages II and III) was reported in 10% and 15% respectively. No patient presented with a stage IV lung disease. High-resolution computed tomography (HRCT) revealed with greater sensibility and specificity the mediastinal and lung involvement. It highlighted features of an interstitial lung disease with peri-lymphatic nodules or micro-nodules involving pleural surfaces and fissures and peri-bronchovascular interstitium, but also ground glass opacities, alveolar consolidations and thickened septa. The irreversible parenchymal abnormalities were less common (Figure 3). Hilar with or without mediastinal, non-compressive and non-necrotizing, roughly symmetrical, lymphadenopathy were usually associated, similarly to adults, as reported in a recent study on 18 patients retrieved from the FR series [START_REF] Sileo | Sarcoidosis in children: HRCT findings and correlation with pulmonary function tests[END_REF]. The flexible bronchoscopy with bronchoalveolar lavage (BAL) documented the lung involvement and was performed, as long as it was possible, before any corticosteroid therapy. The macroscopic aspect was normal, and in most situations, the BAL cell count was characterized by a moderate lymphocytosis, seen in 35 out of 37 FR patients, with a mean percentage of 40% (±22) and a mean CD4+/CD8+ ratio of 4 (±4).

However, a normal BAL cell count does not exclude sarcoidosis diagnosis. Lung function tests (LFT) were detailed in the DK and the FR series in 8 and 37 patients respectively. A restrictive pattern with a significant decrease in FVC was documented in 20% and 65% respectively, and an airflow obstruction was seen in 37% of the cases in the DK patients [START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF].

DLCO impairment was reported in 80% and 54% respectively. Interestingly, in the FR series, the lung function was more impaired in the oldest than in the youngest (<10 years-old)

patients.

An abdominal ultrasound (US) or an abdominal CT scan was often useful to confirm abdominal involvement, or to guide hepatic biopsy. The abdominal US and CT-scans of 22 patients from the FR series were recently analysed [START_REF] Morel | Ultrasonography and Computed Tomographic Manifestations of Abdominal Sarcoidosis in Children[END_REF]. Eleven (50%) of them had radiological abnormalities. Hepatomegaly was the most frequent sign, mostly homogeneous and rarely nodular, homogeneous lymph node enlargement was noted in 6 patients, and splenomegaly in 4 patients. All 11 patients had simultaneous thoracic involvement, despite a lack of any clinical respiratory signs in some of them. The authors concluded that a thoracic CT-scan should be performed in all patients with radiologic abdominal sarcoidosis. In all patients, an electrocardiogram and a cardiac US, as well as a specialized ophthalmologic examination are also considered mandatory.

Biologic blood tests are not specific. Stigmata of inflammation could be observed with a moderate increase of the sedimentation rate (median 36mm, 44mm, 46mm in the DK, FR and US-LA series respectively), an inflammatory anaemia (54% of the US-LA patients), an hypergammaglobinaemia (36% and 65% respectively in the DK and US-LA series), and an inconsistent lymphocytosis or lymphopenia [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF][START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF]. Alternatively, a non-regenerative anaemia or a thrombocytopenia is suggestive of a splenic or a bone marrow involvement (that should be confirmed by a bone marrow biopsy). Increased transaminases could be a sign of liver sarcoidosis. Serum angiotensin converting enzyme [ACE] dosage's significance was restricted to positive cases (63% of the patients included in the 3 series). Blood and urinary calcium were also tested and a mean of 16% of the patients suffered from hypercalcaemia at diagnosis [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF][START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF]. Other biologic tests were guided by the organs involved. In all cases, a DNA biobank specimen should be discussed with expert centres, in both familial cases and sporadic cases.

Pathologic examination of the biopsied organs is critical to assess the diagnosis of sarcoidosis in children. Biopsies are usually difficult and invasive in children, and corticosteroids can mask granuloma after only a few days or weeks of treatment. They should be performed, when possible, before any corticosteroids are started. In the DK series, 73% of the patients benefited from a histologic diagnosis and peripheral lymph nodes, mediastinal lymph nodes and skin lesions were the most common biopsy sites [START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF]. In the FR series, the accessory salivary glands, the liver and the lung were the most informative biopsy sites, but more than one organ biopsy were often needed to highlight a typical granuloma. The lung biopsies were performed by bronchial or transbronchial biopsy during a flexible bronchoscopy, but this procedure was difficult in infants and young children below the age of 6 years old because of the size of the bronchoscopes [START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF]. Surgical lung biopsies were an alternative and were performed through video-assisted thoracoscopy or open lung biopsy with a usually good safety [START_REF] Van Niekerk | Paediatric thoracoscopy: state of the art[END_REF].

Therapeutic management

The management of paediatric sarcoidosis should be overseen by multidisciplinary specialized reference centres with the aim of gathering these rare cases and improving the patients' care. The management and evolution of the DK, FR and US-LA series are summarized in Table 2.

In children, therapeutic abstention [no treatment] was almost never discussed as a management option, except in the rare asymptomatic cases, as reported in the DK series [START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF].

Corticosteroids were the mainstay of the treatment in the published cases. They were given orally, starting with high doses (1-2 mg/kg/d) and/or by intravenous pulses (300 to 500 mg/m 2 /d for 3 days) in 50%, 97% and 89% of the patients in the DK, FR and US-FR series respectively. Corticosteroids were given for a mean duration of 15.6, 23.9 and 9 months respectively, with extreme durations of 1 month to 23 years [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF][START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF][START_REF] Milman | Childhood sarcoidosis: long-term follow-up[END_REF]. Besides corticosteroids, the caloric supplementation and the usual D vitamin intake was adapted, depending on the calcium dosage. Various immunosuppressive drugs were used as a second line of therapy in a highly variable proportion of patients, depending on the local prescribing preferences (4%, 36% and 85% respectively) [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF][START_REF] Milman | Childhood sarcoidosis: long-term follow-up[END_REF]. Methotrexate was the most frequently used in the 3 studies (total 22 (19%) of the overall 114 reported patients), followed by mycophenolate mofetil (7 patients), hydroxychloroquine (4 patients), infliximab (4 patients), cyclophosphamide (4 patients), ethanercept (2 patients), azathioprine (1 patient), and tacroilimus (1 patient) [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF][START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF][START_REF] Milman | Childhood sarcoidosis: long-term follow-up[END_REF]. The authors did not provide any explanations for the choice of the immunosuppressive drugs in these 3 studies. In adults, recent data on sarcoidosis treatments highlighted that low-dose methotrexate or azathioprine were the most used and that ethanercept, tacrolimus and thalidomide did not prove any efficacy [START_REF] Valeyre | Current Medical Therapy for Sarcoidosis[END_REF][START_REF] Droitcourt | A randomized, investigator-masked, double-blind, placebo-controlled trial on thalidomide in severe cutaneous sarcoidosis[END_REF]. However, there is no available study evaluating these drugs in paediatric patients.

Aside from drugs, a nutritional support is frequently needed in children presenting an initial weight loss, and has to be adapted to the corticosteroid regimen. A paediatric nutritionist

[dietician] can be a precious resource when managing the dietary needs depending on the age of the children and the on-going therapies. A social management plan is crucial in paediatric sarcoidosis, including adapting school time as long as fatigue is present.

Evolution and prognosis

The follow-up management reported in the 3 main paediatric studies was not consistent and seemed adapted to each individual situation (Table 2). Following the patients' description at diagnosis, the DK series provided a long-term report (mean follow-up duration of 23 years (4-30)) on the follow-up of 46 patients, based on clinical evaluation and national registry data [START_REF] Milman | Childhood sarcoidosis: long-term follow-up[END_REF]. At a median of 0.7 (0.6-5.9) years after diagnosis, 65% of the patients experienced a full recovery; and 78% at follow-up. Recovery with sequelae was noted in 2 additional patients (unilateral loss of vision and persistent radiographic stage II disease with normal spirometry), and 5 patients presented with active disease at follow-up. Three patients died: two from central nervous system sarcoidosis and one from secondary acute leukemia. The same authors evaluated the quality of life of 34 adult survivors of paediatric sarcoidosis (30 who had recovered, and 4 with persistent sarcoidosis). They found no differences of the QoL scores compared to a reference Danish population and concluded that the majority of the patients had a health status that was similar to the reference population [START_REF] Milman | Health-related quality of life in adult survivors of childhood sarcoidosis[END_REF]. The FR series was followed for 18 months for 38 (93%) patients, and for 4-5 years for 29 (70%) [START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF]. A full recovery, defined as an asymptomatic patient on no treatment, was observed in 45% of the patients at follow-up, 14 additional patients were stable under treatment, and 41% experienced 1 to more than 10 relapses, mainly with an extra-thoracic localization. In this study, the <10 years-old group who had a lighter lung disease at diagnosis but received more steroid pulses and less immunosuppressive drugs than the >10 years-old group had a better outcome. The US-LA series reported a mean follow-up of 4 years (0.1-14) [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF]. At a mean follow-up of 3 months (1-9), 50% of the patients were in remission, defined as asymptomatic patients under treatment.

At follow-up, 1 to 4 relapses were encountered by 40% of the patients, mainly with ocular involvement. Four (58%) patients experienced side-effects from the corticosteroids. The morbidity included iritis (n=4), joint sequelae (n=3), skin lesions (n=2), uveitis (n=1) and restrictive lung disease (n=1). One patient died after kidney transplantation for granulomatous nephritis. Prognostic factors are not yet established in children. However, the FR series raised the hypothesis that the disease extension, evaluated by the number of affected organs at diagnosis, could be associated with a higher rate of mid-term and long-term relapses [START_REF] Nathan | Lung sarcoidosis in children: update on disease expression and management[END_REF]. The age at diagnosis nor the Scadding radiological stage were not found to be associated with the disease evolution, perhaps in part because of the small number of children with stage IV disease; in contrast to adults with lung fibrosis and pulmonary hypertension factors being most associated with mortality [START_REF] Walsh | The ILD cohort is developed in collaboration with the Rare Cohort Disease (RaDiCo)-ILD project (ANR-10-COHO-0003), the FP7-305653-child-EU project and the COST Action European network for translational research in children's and adult interstitial lung disease (COST-ILD) project (CA16125). Funding sources Data included in this work were collected with the support of grants from the Institut National de la Santé et la Recherche Médicale (INSERM)[END_REF].

There is no guideline in children regarding the optimal follow-up schedule. Considering the variable outcomes of the disease, a repeated clinical evaluation seems necessary life-long and emphasizes the importance of a close collaboration between paediatricians and adult pulmonologists. In the DK series, 39 patients had a radiographic evaluation at follow-up, and that found that 80% had normal chest X-rays (vs 9% at diagnosis), whilst only 1 patient that evolved toward lung fibrosis [START_REF] Milman | Childhood sarcoidosis: long-term follow-up[END_REF]. In the US-LA patients, 37% had an abnormal chest X-ray at follow-up, and 68% had impaired spirometry [START_REF] Gedalia | Childhood sarcoidosis: Louisiana experience[END_REF]. In an evaluation of 18 of the FR patients, the spirometry at follow-up was shown to be well-correlated with the lung imaging appearance and so could be used to reduce the frequency of imaging and thereby lower the irradiation exposure in children [START_REF] Sileo | Sarcoidosis in children: HRCT findings and correlation with pulmonary function tests[END_REF].

Conclusion and future research directions

Regarding the 3 main patient series of paediatric sarcoidosis, it seems that the disease is severe and long-lasting. Children of all ages can be affected. It is a multi-organic disorder presenting with non-specific general symptoms. Despite an initial aggressive treatment, the evolution may be marked by relapses. There is a crucial need for future large international studies, and for long-term studies assessing the evolution of the disease in adult age range: these goals may be achieved by the launch of European and other international networks for rare lung diseases and by a better paediatric and adult physician cooperation.

Educational aims

The reader will come to appreciate:

• Paediatric sarcoidosis is a rare granulomatous disease of uncertain aetiology that can affect all organs, in patients of all ages.

• At diagnosis, fever and weight loss are often at the forefront of the symptoms, respiratory signs being inconstant despite usual thoracic involvement.

• In children, pathologic evidence is mandatory, highlighting a typical epithelioid non-necrotizing granuloma.

• Oral or pulses of intravenous corticosteroids are the mainstay of the treatment.

• Relapses are frequently observed, mainly extra-thoracic: even in cases of initial remission, so long-term follow-up is required.

Figure 2 :

 2 Figure 2: Sarcoidosis granuloma (10).

Figure 3 :

 3 Figure 3: Chest high resolution computed tomography (HRCT) in pulmonary paediatric

  Figure 1

Figure 3

 3 Figure 3

Table 1 :

 1 Characteristics at diagnosis of the patients included in the 3 main pediatric sarcoidosis series (Hoffman et al. 2004, Nathan et al. 2015, Gedalia et al. 2016 (6-8))

		DK series (7)	FR series (8)	US-LA series (6)	Total
	EPIDEMIOLOGY
	Dates	1979-1994	2008-2014	1992-2013
	Total number of included patients, n	48	41	27	116
	-Including patients with Blau syndrome, n	2	0	5	7
	-Including patients with sarcoidosis, n	46	41	22	109
	Pathologic confirmation of sarcoidosis, n (%)	33 (69%)	41 (100%)	18 (67%)	92 (84%)

Table 2 :

 2 Management and evolution of the patients included in the 3 main pediatric sarcoidosis series[START_REF] Milman | Childhood sarcoidosis: long-term follow-up[END_REF], Nathan et al. 2015, Gedalia et al. 2016 (6,8,9)) Age at sarcoidosis diagnosis of the children included in the Danish experience (DK series[START_REF] Hoffmann | Childhood sarcoidosis in Denmark 1979-1994: incidence, clinical features and laboratory results at presentation in 48 children[END_REF]) and the French experience (FR series (8)).The Danish cohort is composed of 46 patients with sarcoidosis (after exclusion of 2 patients with Blau syndrome) of European origin aged 8 months to 15 years (median age 13 years) at diagnosis. The French cohort includes 41 patients, mainly Afro-Caribbean and Sub-Saharan children aged 13 months to 15.75 years at diagnosis (median age 11.8). In both cohorts, the majority of the patients are teenagers, but it seems important to notice that 20 to 30% of the patients are below 10 years of age at diagnosis.

	DK series (9)	FR series ((8)	US-LA series (6)	Total

Abbreviations: DK: Danish; FR: French; US-LA: Louisiana; NA: not available * data available for 38 patients; ** data available for 29 patients

Legends to figures

Figure 1:

Acknowledgements

We wish to thank the French sarcoidosis group (GSF) (https://splf.fr/groupes-de-

Conflict of interest statement

The authors have no conflict of interest to disclose Word count: 3770


Alejandro Cabezas Cruz
email: cabezasalejandrocruz@gmail.com

Pedro Espinosa

Pilar Alberdi

José De

La Fuente

Alejandro Cabezas-Cruz

José De La Fuente
email: jose_delafuente@yahoo.com

Tick-pathogen interactions: The metabolic perspective

Keywords: Ixodes, Anaplasma phagocytophilum, Borrelia spp., metabolism, Tick-pathogen interactions

2

Tick-pathogen metabolic interactions at the center of the infectious storm

Metabolism is key to cellular function [START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Green | Cell biology. Metabolic control of cell death[END_REF]. In consequence, all major molecular pathways in living cells are interconnected to and regulated by the availability and levels of metabolites [START_REF] Ganeshan | Metabolic regulation of immune responses[END_REF][START_REF] Green | Cell biology. Metabolic control of cell death[END_REF]. Recent studies in mammalian cells showed that interactions between intracellular bacterial pathogens and the host cells can lead to physiological changes in both interacting members [START_REF] Eisenreich | Metabolic Adaptations of Intracellullar Bacterial Pathogens and their Mammalian Host Cells during Infection ("Pathometabolism")[END_REF]. The metabolic adaptations in mammalian cell-pathogen systems promote proliferation or elimination of the pathogen within the host cells [START_REF] Eisenreich | Metabolic Adaptations of Intracellullar Bacterial Pathogens and their Mammalian Host Cells during Infection ("Pathometabolism")[END_REF]. Tick-pathogen interactions, however, involve a more complex array of outcomes which include conflict and cooperation that ultimately benefit both ticks and pathogens [START_REF] De La Fuente | Tick-host-pathogen interactions: conflict and cooperation[END_REF]. This is not surprising considering that in vector-borne pathogen systems the survival of the vector is essential for the completion of the life cycle of the pathogen. In addition, tick-borne pathogen infection increases tick performance in challenging environmental conditions [START_REF] Cabezas-Cruz | Tick-pathogen ensembles: do molecular interactions lead ecological innovation? Front[END_REF][START_REF] Busby | Expression of heat shock proteins and subolesin affects stress responses, Anaplasma phagocytophilum infection and questing behaviour in the tick, Ixodes scapularis[END_REF]. Therefore, in some cases, vector tolerance to pathogen infection is an advantageous life trait in arthropod vectors [START_REF] Shaw | Vector Immunity and Evolutionary Ecology: The Harmonious Dissonance[END_REF].

Tick metabolism modulation by tick-borne pathogens is a result of coevolution and adaption to a considerable number of tick and reservoir host species [START_REF] De La Fuente | Tick-host-pathogen interactions: conflict and cooperation[END_REF][START_REF] De La Fuente | Flying ticks: anciently evolved associations that constitute a risk of infectious disease spread[END_REF][START_REF] De La Fuente | Anaplasma phagocytophilum Uses Common Strategies for Infection of Ticks and Vertebrate Hosts[END_REF]. The Ixodes scapularis genome, the first for a medically important chelicerate, is the only tick genome available so far [START_REF] Gulia-Nuss | Genomic insights into the Ixodes scapularis tick vector of Lyme disease[END_REF]. The sequencing of the I. scapularis genome was an essential step towards the understanding of the molecular processes that support the parasitic lifestyle of the tick and its success as a vector of multiple pathogens including bacteria, viruses, protozoa, and helminths which constitute a growing burden for human and animal health worldwide [START_REF] De La Fuente | Tick-pathogen interactions and vector competence: identification of molecular drivers for tick-borne diseases[END_REF].

This review focuses on recent research that provided insights on the finely tuned metabolic changes that the model pathogen Anaplasma phagocytophilum induces in its tick vector I. scapularis. For comparative purposes, tick metabolic changes induced by other pathogens including mainly Borrelia spp., flaviviruses and fungi will be also included (Table 1). Results suggest that tick-borne pathogens use tick metabolism as a hub to modulate tick physiology and tick cell processes including immunity and apoptosis among others.

Nutritional immunity and iron metabolism in ticks

various metabolic processes. Therefore, iron holds a central position in host-pathogen interactions [START_REF] Núñez | Innate Nutritional Immunity[END_REF]. While host evolved mechanisms to deprive pathogens from iron, pathogens coevolved mechanisms to acquire host iron. Host mechanisms involved in limiting pathogens access to iron were termed 'nutritional immunity' [START_REF] Núñez | Innate Nutritional Immunity[END_REF]. During feeding, ticks are exposed to the large amounts of iron present in the blood meal. Excess iron can react with H 2 O 2 which yields hydroxyl radical, a potent biological oxidant [START_REF] Núñez | Innate Nutritional Immunity[END_REF]. Therefore, a balanced regulation of iron levels is essential for ticks [START_REF] Galay | Iron metabolism in hard ticks (Acari: Ixodidae): the antidote to their toxic diet[END_REF], and potentially, pathogen survival. Two ferritin (FER) proteins, FER1 and FER2, are the primary iron storage and transporter, respectively, in ticks [START_REF] Galay | Iron metabolism in hard ticks (Acari: Ixodidae): the antidote to their toxic diet[END_REF]. In addition of controlling the production of reactive oxygen species from iron, like in other model organism [START_REF] Núñez | Innate Nutritional Immunity[END_REF][START_REF] Galay | Iron metabolism in hard ticks (Acari: Ixodidae): the antidote to their toxic diet[END_REF], ferritins may also contribute to nutritional immunity in ticks by depriving pathogens from essential iron supply. Interestingly, Escherichia coli infection induces the expression of a fer gene in Dermacentor variabilis and silencing of fer genes in Haemaphysalis longicornis decreases tick survival following a challenge with E. coli [START_REF] Galay | Iron metabolism in hard ticks (Acari: Ixodidae): the antidote to their toxic diet[END_REF].

The tick-borne pathogen Ehrlichia ruminantium sense iron levels in mammalian cells and respond to iron starvation by upregulating virulence genes such as those involved in Type IV Secretion System (T4SS), which in turn may participate in iron acquisition by the pathogen [START_REF] Moumène | Iron starvation conditions upregulate Ehrlichia ruminantium type IV secretion system, tr1 transcription factor and map1 genes family through the master regulatory protein ErxR[END_REF]. A similar mechanism may be at play during pathogen infection in ticks. An iron-binding protein expressed in the infective stage of A. phagocytophilum [START_REF] Kahlon | Anaplasma phagocytophilum Asp14 is an invasin that interacts with mammalian host cells via its C terminus to facilitate infection[END_REF], and hypothesized to be secreted by the T4SS, was found in tick salivary glands and tick cells infected with these bacteria [START_REF] Villar | Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector, Ixodes scapularis[END_REF]. Bacterial iron-binding proteins may sequester iron to overcome nutritional immunity in ticks. An interesting mechanism to scape nutritional immunity is that of Borrelia burgdorferi which requires none or very low amounts of iron to survive [START_REF] Troxell | Borrelia burgdorferi, a pathogen that lacks iron, encodes manganese-dependent superoxide dismutase essential for resistance to streptonigrin[END_REF]. In addition, recent research provides evidence that A. phagocytophilum modulates tick heme metabolism. Host hemoglobin is cleavage to heme by three sequential set of reactions involving several hemoglobinolytic enzymes in ticks [START_REF] Villar | The intracellular bacterium Anaplasma phagocytophilum selectively manipulates the levels of vertebrate host proteins in the tick vector Ixodes scapularis[END_REF]. Due to the high redox potential of heme, this molecule is toxic and in excess can be bacteriostatic [START_REF] Sachla | The crimson conundrum: heme toxicity and tolerance in GAS[END_REF]. Quantitative proteomics analysis revealed that A. phagocytophilum modulates the levels of hemoglobinolytic enzymes which affects the levels of host hemoglobin in tick midguts and salivary glands [START_REF] Villar | The intracellular bacterium Anaplasma phagocytophilum selectively manipulates the levels of vertebrate host proteins in the tick vector Ixodes scapularis[END_REF]. This finding suggests that A. phagocytophilum infection may regulate the levels of heme in a tissue-specific manner with potential effects for pathogen and vector survival.

Tick hemocyte metabolism in response to tick-borne pathogen infection

Despite mammalian macrophages possess specialized effector functions to eliminate invading microorganism, many intracellular bacteria infect and replicate inside this immune cells [START_REF] Price | The macrophage paradox[END_REF]. Globally, macrophages can be divided into inflammatory (M1) and antiinflammatory (M2) macrophages [START_REF] Price | The macrophage paradox[END_REF]. Both macrophage types M1 and M2 can be infected by intracellular bacteria; however, the metabolic profiles of these macrophages in response to pathogenic stimulus are substantially different [START_REF] Price | The macrophage paradox[END_REF]. After stimulation with lipopolysaccharide (LPS), macrophages M1 uptake more glucose via glucose transporters (GLUTs, see Glossary) and increase the glycolytic flux and the production of lactate, a process named aerobic glycolysis. In contrast, macrophages M2 have the metabolic profile comparable to that of unstimulated cells, but with higher TCA-cycle and oxidative activity [START_REF] Price | The macrophage paradox[END_REF].

Cellular immunity in ticks is mostly mediated by hemocytes, which are involved in the phagocytosis of different microbes [START_REF] Hajdušek | Interaction of the tick immune system with transmitted pathogens[END_REF]. After establishing a primary infection in tick midguts, A. phagocytophilum migrates to the salivary glands [START_REF] De La Fuente | Tick-pathogen interactions and vector competence: identification of molecular drivers for tick-borne diseases[END_REF]. A secreted tick protein, P11, enables A. phagocytophilum to infect tick hemocytes, which are required for the migration of A. phagocytophilum from the gut to the salivary glands [START_REF] Liu | Ixodes scapularis salivary gland protein P11 facilitates migration of Anaplasma phagocytophilum from the tick gut to salivary glands[END_REF]. Isolation and in vitro culture of tick hemocytes has remained elusive. Therefore, the study of tick hemocytes metabolism in response to A. phagocytophilum infection has been based on the I. scapularis ISE6 cell line. Based on the transcriptional response to A. phagocytophilum, ISE6 cells were proposed to constitute a model of tick hemocytes involved in pathogen infection and immune response [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF][START_REF] Alberdi | Tissue-specific signatures in the transcriptional response to Anaplasma phagocytophilum infection of Ixodes scapularis and Ixodes ricinus tick cell lines[END_REF]. Comparison of the metabolic profiles of macrophages M1 with that of A. phagocytophilum-infected ISE6 cells suggests that the metabolic profile of tick hemocytes in response to pathogen infection is very different to that of inflammatory macrophages M1 (Fig. 1, Key Figure). Two major differences can be summarized: (i) ISE6 cells infected by A. phagocytophilum do not display the aerobic glycolysis observed in mammalian macrophages M1 and (ii) hypoxia-inducible factor 1-alpha (HIF-1α) activation and stabilization in ISE6 cells is not dependent of succinate accumulation and aerobic glycolysis (Fig. 1). This suggests that the metabolic reprogramming associated with macrophage M1 activation does not occurs in tick hemocytes infected with A. phagocytophilum. Interestingly, while 2-deoxyglucose (2DG), an inhibitor of glycolysis, inhibits HIF-1α activation in macrophages M1 [START_REF] Nonnenmacher | Biochemistry of proinflammatory macrophage activation[END_REF], this compound has no effect on HIF-1α activation in ISE6 cells [26].

Instead of aerobic glycolysis and succinate accumulation, HIF-1α activity in A. phagocytophilum-infected ISE6 cells appears to be mediated phosphoinositide 3-kinase (PI3K) pathway activation (Fig. 1). In agreement with this, the PI3K inhibitor LY294002 decreased HIF-1α DNA binding activity in infected ISE6 cells [26]. A metabolic property shared by macrophages M1 and ISE6 cells is, however, an overall decrease in tricarboxylic acid cycle (TCA cycle) activity upon bacterial infection [START_REF] Nonnenmacher | Biochemistry of proinflammatory macrophage activation[END_REF]26].

The absence of immunogenic molecules such as LPS and diaminopimelic-type peptidoglycans (DAP-PGN) in A. phagocytophilum and other bacteria transmitted by I. scapularis was previously suggested to contribute to vector tolerance to tick-borne pathogens [START_REF] Shaw | Vector Immunity and Evolutionary Ecology: The Harmonious Dissonance[END_REF]. The lack of these immune activators in tick-borne pathogens may help these microbes colonize ticks without being eliminated [START_REF] Shaw | Vector Immunity and Evolutionary Ecology: The Harmonious Dissonance[END_REF]. Interestingly, lipids 1-palmitoyl-2-oleoyl-sn-glycero-3phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) found in A. phagocytophilum activate the immune deficiency (IMD) pathway in ticks [START_REF] Shaw | Infection-derived lipids elicit an immune deficiency circuit in arthropods[END_REF]. Immune priming with POPG and PODAG reduces the A. phagocytophilum burden in infected ISE6 cells [START_REF] Shaw | Infection-derived lipids elicit an immune deficiency circuit in arthropods[END_REF]. This shows that tolerance to infection can be associated with immunity and resistance to tick-borne pathogens in ticks. This is expected considering that unchecked bacterial multiplication within host cells could affect vector, and therefore, tick-borne pathogen survival. Remarkably, recent research shows that IMD is involved in glucose and lipid metabolism regulation in Drosophila [START_REF] Kamareddine | The Drosophila immune deficiency pathway modulates enteroendocrine function and host metabolism[END_REF] and constitutive activation of IMD induces a significant reduction in the expression of genes involved in glycolysis, gluconeogenesis and TCA cycle in flies [START_REF] Davoodi | The Immune Deficiency Pathway Regulates Metabolic Homeostasis in Drosophila[END_REF]. Whether IMD activation by A. phagocytophilum regulates tick hemocyte metabolism remains to be tested.

Molecular mechanisms involved in tick metabolism modulation by tick-borne pathogens

Tick metabolism modulation by tick-borne pathogens results mainly from the regulation of the levels of metabolic enzymes. Although it remains a challenge to explain how pathogen infection modify the level of thousands of tick genes and hundreds of tick proteins [START_REF] Ayllón | Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis[END_REF], the molecular mechanisms behind this have been partially characterized using the model bacteria A. phagocytophilum. To induce transcriptional reprograming in infected cells, A. phagocytophilum hijacks the transcriptional regulatory machinery of ticks by inducing the activation of signaling pathways [START_REF] Sultana | Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks[END_REF] and the recruitment of transcription factors [START_REF] Khanal | Arthropod transcriptional activator protein-1 (AP-1) aids tickrickettsial pathogen survival in the cold[END_REF], metabolites [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF], and histone modifying enzymes [START_REF] Cabezas-Cruz | Anaplasma phagocytophilum increases the levels of histone modifying enzymes to inhibit cell apoptosis and facilitate pathogen infection in the tick vector Ixodes scapularis[END_REF] (Fig. 2). A. phagocytophilum may also induce the degradation of tick proteins by tick proteasome which in turn decreases the levels of targeted metabolic enzymes [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF]. In addition, A. phagocytophilum infection induces a high proportion of alternatively spliced transcript events (ASEs), which increases the predicted gene ontology processes related to lipids, lipoproteins, and cholesterol/sterol metabolism and other processes in human HL-60 cells infected by this pathogen [START_REF] Dumler | Alternative splicing of differentiated myeloid cell transcripts after infection by Anaplasma phagocytophilum impacts a selective group of cellular programs[END_REF]. The occurrence of ASEs has not been tested in A. phagocytophilum-infected ticks or tick cell lines yet. However, there is no reason to rule out the hypothesis that ASEs also occur in ticks in response to A. phagocytophilum infection.

Activation of tick signaling pathways by A. phagocytophilum involves G-protein-coupledreceptor (GPCR) that stimulates Ixodes p21-activated kinase (IPAK1) through the activation of PI3K which results in actin-mediated signaling [START_REF] Sultana | Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks[END_REF]. Activation of IPAK1 results in actin phosphorylation and accumulation of actin in the nucleus which mediates binding of RNA polymerase II (RNAPII) and TATA box-binding protein (TBP) to the promoter of the gene salp16 inducing its expression [START_REF] Sultana | Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks[END_REF][START_REF] Sukumaran | An Ixodes scapularis protein required for survival of Anaplasma phagocytophilum in tick salivary glands[END_REF]. PI3K activation was also suggested to induce the expression of the genes hif-1α, hif-1β and acetyltransferase p300 upon A. phagocytophilum infection in tick cells [26] (Fig. 2). Mobilization of HIF-1α, HIF-1β and p300 at HIF target genes may induce HIF-mediated transcriptional activation of glycolytic genes [26].

Among the transcription factors (TFs) activated by A. phagocytophilum infection is the tick transcriptional activator protein-1 (AP-1) which induces the expression of the genes iafgp (I. scapularis antifreeze glycoprotein) and kat (kynurenine aminotransferase) in infected cells [START_REF] Khanal | Arthropod transcriptional activator protein-1 (AP-1) aids tickrickettsial pathogen survival in the cold[END_REF]. KAT is an enzyme involved in the production of the tryptophan metabolite xanthurenic acid [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF]. Results suggests that xanthurenic acid acts as a co-factor of an uncharacterized TF that regulates the expression of one organic anion transporting polypeptide (oatp) gene in A. phagocytophilum-infected ISE6 cells [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF]. The upregulation of oatp and kat was proposed to be associated with increased levels of intracellular xanthurenic acid, which in turn favors A. phagocytophilum replication in tick salivary glands [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF]. Conversely, RNAi-mediated silencing of oatp and kat expression decrease A. phagocytophilum levels in tick cells [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF]. A general mechanism is then proposed by which A. phagocytophilum nucleomodulins recruit tick metabolites and TFs to activate target genes (Fig. 2).

The capacity of A. phagocytophilum to downregulate gene expression in mammalian neutrophils was associated with host histone deacetylase 1 (HDAC1) recruitment to the promoters of target genes by the bacterial nucleomodulin ankyrin repeat protein AnkA [START_REF] Rennoll-Bankert | Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression[END_REF].

Upon A. phagocytophilum infection, AnkA is secreted through T4SS, enters the granulocyte nucleus, binds stretches of AT-rich DNA and recruits host HDAC1 to its binding sites.

A. phagocytophilum infection also induces HDAC1 binding and deacetylation of histone H3 (H3) which in turn results in downregulation of many host defense genes including CYBB, which encodes a component (i.e. NOX2) of the NADPH oxidase [START_REF] Rennoll-Bankert | Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression[END_REF]. Downregulation of CYBB reduces superoxide anion production by NADPH oxidase which is essential for A. phagocytophilum survival in neutrophils [START_REF] Rennoll-Bankert | Chromatin-bound bacterial effector ankyrin A recruits histone deacetylase 1 and modifies host gene expression[END_REF]. Homologs of mammalian HDAC1 and H3 were identified in the I. scapularis genome [START_REF] Cabezas-Cruz | Anaplasma phagocytophilum increases the levels of histone modifying enzymes to inhibit cell apoptosis and facilitate pathogen infection in the tick vector Ixodes scapularis[END_REF]. Whether A. phagocytophilum AnkA is secreted and recruits HDAC1 to regulate gene transcription in infected ticks is currently unknown. However, Ixodes HDAC1 is overrepresented in A. phagocytophilum-infected tick salivary glands and chemical inhibition of this protein decreases A. phagocytophilum burden in tick cells [START_REF] Cabezas-Cruz | Anaplasma phagocytophilum increases the levels of histone modifying enzymes to inhibit cell apoptosis and facilitate pathogen infection in the tick vector Ixodes scapularis[END_REF]. This shows that the expression and activity of HDAC1 is critical for A. phagocytophilum survival in both mammalian and tick cells. Notably, in mammalian cells, HDAC1 controls the expression of the metabolic enzyme fructose-1,6-bisphosphatase (FBP), a rate-limiting enzyme in gluconeogenesis [START_REF] Yang | Inhibiting histone deacetylases suppresses glucose metabolism and hepatocellular carcinoma growth by restoring FBP1 expression[END_REF]. Interestingly, high HDAC1 protein levels [START_REF] Cabezas-Cruz | Anaplasma phagocytophilum increases the levels of histone modifying enzymes to inhibit cell apoptosis and facilitate pathogen infection in the tick vector Ixodes scapularis[END_REF] were associated with low FBP protein levels in A. phagocytophilum-infected tick salivary glands [26]. Conversely, low HDAC1 protein levels [START_REF] Cabezas-Cruz | Anaplasma phagocytophilum increases the levels of histone modifying enzymes to inhibit cell apoptosis and facilitate pathogen infection in the tick vector Ixodes scapularis[END_REF] correlated with high mRNA and protein levels of fbp and FBP, respectively, in A. phagocytophilum-infected tick midguts [26].

The negative correlation between the protein levels of HDAC1 and that of fbp and FBP strongly suggests that HDAC1 may be recruited by A. phagocytophilum nucleomodulins to act as a repressor of fbp expression in tick salivary glands (Fig. 2). A decrease in FBP protein levels may contribute to the inhibition of gluconeogenesis that A. phagocytophilum induces in infected tick cells (see below) [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF].

Tissue-specific changes of tick carbohydrate metabolism in response to infection

Glucose is the most abundant carbohydrate in the hemolymph of adult ticks and it is required in tick embryonic development [START_REF] Martins | The dynamics of energy metabolism in the tick embryo[END_REF]. The regulation of glucose metabolic pathways in ticks is essential for A. phagocytophilum survival in the vector [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF]26]. Notably, A. phagocytophilum infection inhibits gluconeogenesis by decreasing the protein levels of phosphoenolpyruvate carboxykinase-cytosolic (PEPCK-C) which results in lower levels of glucose in A. phagocytophilum-infected ISE6 cells [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF]26]. PEPCK-C is also a key regulatory enzyme in glyceroneogenesis [START_REF] Saponaro | The subtle balance between lipolysis and lipogenesis: a critical point in metabolic homeostasis[END_REF] and thus, low levels of PEPCK-C were associated with reduced glycerol 3-phosphate production in infected tick cells [START_REF] Cabezas-Cruz | Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine[END_REF]. Low PEPCK-C activity and decreased glucose anabolism inhibits apoptosis which is essential for A. phagocytophilum survival in tick cells and tissues [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF][START_REF] Ayllón | Systems biology of tissue-specific response to Anaplasma phagocytophilum reveals differentiated apoptosis in the tick vector Ixodes scapularis[END_REF]. Interestingly, glycerol 3-phosphate levels were also reduced in tick nymphs infected by Borrelia mayonii (Table 1). Low levels of glycerol 3phosphate may have a significant impact in fatty acids metabolism, a poorly explored facet in tick-pathogen interactions. Tissue-specific differential regulation of apoptosis pathways was observed in adult female midguts and salivary glands in response to A. phagocytophilum infection [START_REF] Alberdi | Anaplasma phagocytophilum manipulates host cell apoptosis by different mechanisms to establish infection[END_REF]. Likewise, the transcriptional reprograming induced by A. phagocytophilum affects the levels of carbohydrate metabolism genes and proteins in a tissue-specific manner [26]. For example, the protein levels of all glycolytic enzymes increase in A. phagocytophilum-infected tick midguts, except for the cofactor-independent phosphoglycerate mutase (iPGM) and pyruvate kinase (PK) that despite gene up-regulation, the proteins were under-represented in this tissue [26]. In addition, gene and protein levels of phosphofructokinase (PFK) did not change in tick midguts in response to infection [26]. However, in salivary glands, the glycolysis enzymes fructose-bisphosphate aldolase A (ALDA), PFK and hexokinase (HXK) were under-represented while the rest of the glycolytic enzymes were over-represented. To accurately assess the metabolic impact of the differential protein representation in tick midguts and salivary glands, a tissue-specific metabolomics approach is required. However, the lower levels of PK and HXK, key regulatory enzymes in glycolysis, in infected tick midguts and salivary glands, respectively, suggest that the production of glycolytic intermediates may be affected in a tissue-specific manner. It would be also important to assess the enzymatic activity of key enzymes which may vary in A. phagocytophilum-infected cells and tissues. For example, it was reported that the activity of HXK decreases more than two-folds in A. phagocytophilum-infected ISE6 cells [START_REF] Ayllón | Anaplasma phagocytophilum inhibits apoptosis and promotes cytoskeleton rearrangement for infection of tick cells[END_REF]. The transcriptional activation of tick glycolytic genes was proposed to be mediated by tick HIF-1α [26], which contains structural domains conserved in model organisms and the gene hif-1α was significantly upregulated in infected tick midguts [26]. Another important gene targeted by HIF-1α is pyruvate dehydrogenase kinase 1 (pdk1) which encodes the protein PDK1 that controls the enzymatic activity of pyruvate dehydrogenase E1 (PDE1) which transforms pyruvate into Acetyl-CoA, the first step in the TCA cycle. PDK1 mRNA and protein levels were higher in A. phagocytophilum-infected tick salivary glands suggesting that this pathogen inhibits the TCA cycle in infected salivary glands [26].

Crosstalk between tick amino acid and carbohydrate metabolism during pathogen infection

Several recent studies have shown that amino acids are central to host-pathogen metabolic interaction [START_REF] Eisenreich | Metabolic Adaptations of Intracellullar Bacterial Pathogens and their Mammalian Host Cells during Infection ("Pathometabolism")[END_REF][START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF][START_REF] Cabezas-Cruz | Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine[END_REF][START_REF] Olive | Metabolic crosstalk between host and pathogen: sensing, adapting and competing[END_REF]. When digested, the protein-rich diet of ticks generates large amounts of amino acids. Therefore, amino acid metabolism is of great interest in ticks. For example, tyrosine-associated toxicity is lethal for blood-sucking arthropods which points out the significance of tyrosine degradation pathways. Inhibition of tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), the first two enzymes of the phenylalanine/tyrosine degradation pathway, caused the death of mosquitoes, kissing bugs and ticks after a blood meal [START_REF] Sterkel | Tyrosine detoxification is an essential trait in the life history of blood-feeding arthropods[END_REF].

Infection by A. phagocytophilum rewires the network of tick vector cell processes and changes the relative importance of some biological pathways including those involved in metabolism [START_REF] Estrada-Peña | Use of graph theory to characterize human and arthropod vector cell protein response to infection with Anaplasma phagocytophilum[END_REF]. This is expected considering that A. phagocytophilum has a small genome and cannot actively carry out several metabolic processes, and therefore requires and hijacks tick cell resources for survival within the vector [START_REF] Dunning Hotopp | Comparative genomics of emerging human ehrlichiosis agents[END_REF]. For example, out of twenty amino acids, the A. phagocytophilum genome encodes only for the enzymes responsible of the biosynthesis of four amino acids (proline, glutamine, glycine and aspartate) and the glycolysis enzymes are reduced to those that produce glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (DHAP) from phosphoenolpyruvate (PEP) [START_REF] Dunning Hotopp | Comparative genomics of emerging human ehrlichiosis agents[END_REF]. In consequence, A. phagocytophilum changes the levels of amino acid metabolism enzymes [START_REF] Cabezas-Cruz | Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine[END_REF], which in turn affects the intracellular levels of several amino acids and metabolic intermediates [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF][START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF]. Interestingly, tick cells use tyrosine as a fuel to synthetize PEP during early A. phagocytophilum infection [START_REF] Cabezas-Cruz | Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine[END_REF]. This process is achieved by activating the tyrosine/oxaloacetate (OAA)/PEPCK-M/PEP node by which A. phagocytophilum infection may decrease the tyrosine pool, which in turn may protect ticks against tyrosine-induced toxicity in vivo [START_REF] Cabezas-Cruz | Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine[END_REF].

Tick-borne flaviviruses are known to affect protein processing and amino acid metabolism in tick cells [START_REF] Grabowski | RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells[END_REF][START_REF] Grabowski | Changes in the proteome of Langat-infected Ixodes scapularis ISE6 cells: metabolic pathways associated with flavivirus infection[END_REF]. Particularly, infection by Langat virus (LGTV) increased protein levels of acetyl-CoA acetyltransferase 1 (ACAT1), aldehyde dehydrogenase (DP5CD), glutamate dehydrogenase (GLUD1) and fumarylacetoacetase (FAH) in ISE6 cells [START_REF] Grabowski | Changes in the proteome of Langat-infected Ixodes scapularis ISE6 cells: metabolic pathways associated with flavivirus infection[END_REF]. Knockdown of the gene acat1 was associated with decreased LGTV genome replication and LGTV particle release, while knockdown of the gene fah decreased only LGTV particle release in ISE6 cells [START_REF] Grabowski | RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells[END_REF]. Interestingly, FAH catalyzes the last step in tyrosine degradation suggesting that flaviviruses infection may, similarly to A. phagocytophilum, contribute to tyrosine detoxification in ticks.

Tick lipid metabolism is exploited by different tick-associated microorganisms

Starvation and desiccating conditions that ticks have to endure during off-host periods affect the energy reserves of the tick [START_REF] Rosendale | Dehydration and starvation yield energetic consequences that affect survival of the American dog tick[END_REF]. For example, starvation of Dermacentor variabilis ticks in optimal humidity conditions is known to result in up to 60% losses in lipid reserves [START_REF] Rosendale | Dehydration and starvation yield energetic consequences that affect survival of the American dog tick[END_REF]. The parietal fat body, the main lipid reserve organ for triacylglycerol and cholesterol, is gaining increasing interest for the study of infections in insects and ticks [START_REF] Angelo | Neutral lipid composition changes in the fat bodies of engorged females Rhipicephalus microplus ticks in response to fungal infections[END_REF][START_REF] Charroux | Drosophila immune response: From systemic antimicrobial peptide production in fat body cells to local defense in the intestinal tract[END_REF]. However, lipid metabolism signaling pathways are still rarely studied in ticks.

Fatty acids play important roles as energy sources for several microorganisms including pathogenic bacteria [START_REF] Barisch | Breaking fat! How mycobacteria and other intracellular pathogens manipulate host lipid droplets[END_REF]. If a pathogen can prevent the use of energy reserves by the host, this will affect its immune system, therefore promoting pathogen infection and persistence. B. burgdorferi is not able to synthesize fatty acids and cholesterol, thus relying on the vertebrate host cell or the blood meal of the tick for lipids supply [START_REF] Hoxmeier | Metabolomics of the tick-Borrelia interaction during the nymphal tick blood meal[END_REF]. Similarly, host cholesterol and other lipids are required by A. phagocytophilum for infection and multiplication in human cells [START_REF] Truchan | Breaking in and grabbing a meal: Anaplasma phagocytophilum cellular invasion, nutrient acquisition, and promising tools for their study[END_REF]. Perilipin (PLIN), a major adipocyte lipid droplet-associated phosphoprotein that plays a central role during lipolysis and cholesterol synthesis, is differentially expressed in HL-60 cells in response to infection with A. phagocytophilum [START_REF] Manzano-Roman | Expression of perilipin in human promyelocytic cells in response to Anaplasma phagocytophilum infection results in modified lipid metabolism[END_REF].

The bacteria are able to alter PLIN levels in infected cells, thus modifying the lipid metabolism of the host to facilitate bacterial infection [START_REF] Manzano-Roman | Expression of perilipin in human promyelocytic cells in response to Anaplasma phagocytophilum infection results in modified lipid metabolism[END_REF]. This phosphoprotein is likely to play a similar role in the metabolism of the tick although its role has not been characterized so far.

Other obligate intracellular pathogens such as viruses need to hijack the cellular machinery for the synthesis of lipids to complete their replication. Flavivirus life cycle is very closely associated to host cell lipids, and they can rearrange intracellular membranes and reorganize the host cell lipid metabolism of infected cells to ensure viral replication [START_REF] Martín-Acebes | Lipids and flaviviruses, present and future perspectives for the control of dengue, Zika, and West Nile viruses[END_REF]. Dengue virus infection can alter metabolic pathways in humans such as fatty acid biosynthesis and βoxidation [START_REF] Chotiwan | Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes[END_REF]. Exosomes are small extracellular vesicles that appear to play a central role in cell-to-cell communication [START_REF] Chotiwan | Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes[END_REF]. Exosomes transport bioactive molecules, such as messenger RNA (mRNAs) and microRNA (miRNAs) that can be transferred in active form to adjacent cells or to distant organs thus being important vehicles for metabolic cross-talk [START_REF] Yu | Exosomes as miRNA Carriers: Formation-Function-Future[END_REF]. Recent research has shown that Flaviviruses use arthropod-derived exosomes secreted by tick cells as a mean for viral RNA and metabolite transmission from the vector, and the vertebrate exosomes for dissemination within the host [START_REF] Zhou | Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells[END_REF]. The knockdown of transcripts for a glycosylphosphatidylinositol (GPI)-anchored protein (VNN) was associated with decreased

LGTV genome replication and LGTV release in ISE6 tick cells [START_REF] Grabowski | RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells[END_REF]. VNN is involved in coenzyme-A (CoA) metabolism [START_REF] Kinoshita | Biosynthesis of GPI-anchored proteins: special emphasis on GPI lipid remodeling[END_REF], which is critical for lipid metabolism.

Fungal infections can also modulate the lipid metabolism of the host [START_REF] Sá | Metarhizium anisopliae s.l. modulation of lipid metabolism References Glycolysis ISE6 tick cells Anaplasma phagocytophilum Decreased levels and activity of HXK Increased levels of PEP Decreased levels of glucose and lactate[END_REF]. Recent research with different isolates of Metarhizium anisopliae showed that infection of Rhipicephalus microplus ticks with entomopathogenic fungi can modulate the lipid content thus inducing changes in important metabolic pathways [START_REF] Sá | Metarhizium anisopliae s.l. modulation of lipid metabolism References Glycolysis ISE6 tick cells Anaplasma phagocytophilum Decreased levels and activity of HXK Increased levels of PEP Decreased levels of glucose and lactate[END_REF]. However, lipid metabolism modulation during M. anisopliae infection in ticks was independent of AMP-activated protein kinase (AMPK) and extracellular signal-regulated protein kinase (ERK) pathways [START_REF] Sá | Metarhizium anisopliae s.l. modulation of lipid metabolism References Glycolysis ISE6 tick cells Anaplasma phagocytophilum Decreased levels and activity of HXK Increased levels of PEP Decreased levels of glucose and lactate[END_REF] which are the major pathways involved in the breakdown of triacylglycerol into fatty acids in mammals [63,64]. This suggested that tick lipid metabolism modulation by M. anisopliae occurs through alternative and uncharacterized lipolysis pathway.

Concluding Remarks

Interactions between ticks, hosts and pathogens involve many metabolic pathways such as carbohydrate, protein, lipid and redox that are affected to produce conflict and cooperation between them. These interactions at the metabolic level lead to mutual beneficial effects of the tick-hosts-pathogen molecular interactions. Among them, amino acids are central to the host-pathogen metabolic interaction. Nevertheless, synthesis and degradation of amino acids is narrowly connected to carbohydrate metabolism, and the later with the lipid metabolism.

Despite recent advances in the study of tick-host-pathogen molecular interactions, most of the pathways and molecules involved in these interactions are not fully characterized. Furthermore, the regulatory mechanisms affecting the metabolic pathways affected by these interactions are only partially characterized (see Outstanding Questions). Future directions should include the application of omics technologies together with data integration algorithms [START_REF] Estrada-Peña | Use of graph theory to characterize human and arthropod vector cell protein response to infection with Anaplasma phagocytophilum[END_REF] to the investigation of tick interactome, regulome, miRNAome, epigenome and metabolome and their interactions to advance the metabolic perspective on tick-pathogen interactions.

The identification of the metabolic adaptations during tick-pathogen interactions that support tick survival, spread and pathogen infection and transmission could provide new therapeutic targets for the prevention and control of tick-borne diseases. Studies of the interactions between the pathogen and the host lipid metabolism can help to identify potential targets for the development of vaccines or treatments against health threatening pathogens. These strategies could include the inhibition of lipid biosynthesis. However, manipulation of this major metabolic pathway can also be detrimental for the host. For instance, Lovastatin treatment, an inhibitor of cholesterol synthesis, appears to delay infection and increases survival rates in AG129 mice infected with Dengue Virus Serotype 2 while it also tends to increase viremia which could affect disease progression [65]. A better understanding of the interactions of pathogens with the host cell lipid metabolism and other metabolic pathways could provide new therapeutic targets to control infections by tickborne pathogens. Furthermore, as recently proposed the application of machine learning and big data analysis to the tick-host-pathogen interactions datasets may also lead to the high throughput identification of candidate vaccine protective antigens [66].

Funding

PA was funded by the Junta de Comunidades of Castilla-La Mancha (Spain).

Glossary

Tricarboxylic acid cycle (TCA cycle): a central route for oxidative phosphorylation in cells. In the TCA cycle, acetyl-CoA is oxidized into carbon dioxide and reducing factors (NADH and FADH 2 ), that contribute to synthesis of ATP during oxidative phosphorylation, are produced.

The TCA cycle is a crossroad of cellular metabolism in which carbohydrate, fatty acids and amino acid metabolisms are interconnected.

β-Oxidation: the major pathway for the degradation of fatty acids and is essential to produce energy from the generation of acetyl-CoA, NADH and FADH 2 when glucose supply is limiting.

Gluconeogenesis: a metabolic pathway that during a longer-term fast or starvation results in de novo glucose synthesis from non-carbohydrate carbon substrates, mainly lactate and glycerol.

Glyceroneogenesis: a metabolic pathway that is activated during glucose deprivation to synthesize glycerol 3-phosphate, a building block of triglyceride and fatty acid. In this pathway, glycerol 3-phosphate is synthetized from pyruvate, alanine, glutamine and TCA cycle intermediates.

Glycolysis: a major catabolic pathway in which one molecule of glucose is transformed into two molecules of pyruvate releasing in the process two net molecules of ATP and two of NADH. The expression of glycolytic genes is regulated by HIF-1α.

Aerobic glycolysis:

A term frequently used to name a metabolic profile similar to that of the Warburg effect in which a cell, even under normoxic conditions, generates high amount of energy by using the glycolytic pathway, notably with high production of lactate. The levels and activity of tick HXK decreased (red), and the levels of tick PFK did not change (black), in response to infection which was associated with lower levels of lactate in infected ISE6 cells [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF]26,[START_REF] Ayllón | Anaplasma phagocytophilum inhibits apoptosis and promotes cytoskeleton rearrangement for infection of tick cells[END_REF]. HIF-1α activation and stabilization (green), that enhances the expression of glycolytic genes, is mediated by aerobic glycolysis and succinate accumulation from glutamine in macrophages M1 [START_REF] Nonnenmacher | Biochemistry of proinflammatory macrophage activation[END_REF]. In consequence, blocking aerobic glycolysis with 2deoxyglucose (2DG) inhibits HIF-1α activation in these macrophages [START_REF] Nonnenmacher | Biochemistry of proinflammatory macrophage activation[END_REF]. In contrast, 2DG does not inhibit HIF-1α activation (green) in ISE6 cells, an effect that is achieved by LY294002, an inhibitor of the phosphoinositide 3-kinase (PI3K) pathway [26]. This suggests that HIF-1α activity in tick hemocytes infected with A. phagocytophilum is regulated by PI3K and not aerobic glycolysis. Macrophages M1 and ISE6 cells share a low TCA cycle activity [START_REF] Nonnenmacher | Biochemistry of proinflammatory macrophage activation[END_REF]26]. Vertical arrows represent decrease (red) and increase (green) in metabolite levels. induce the expression of salp16 [START_REF] Sultana | Anaplasma phagocytophilum induces actin phosphorylation to selectively regulate gene transcription in Ixodes scapularis ticks[END_REF], and hypoxia-inducible factor (HIF) hif-1α in ticks [26] (sequential steps and tick molecular factors colored in violet). Secondly, A. phagocytophilum recruits tick transcription factors (TF), such as transcriptional activator protein-1 (AP-1) [START_REF] Khanal | Arthropod transcriptional activator protein-1 (AP-1) aids tickrickettsial pathogen survival in the cold[END_REF] and metabolites, such as xanthurenic acid [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF], to regulate the expression of the metabolic genes oatp [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF] and kat [START_REF] Khanal | Arthropod transcriptional activator protein-1 (AP-1) aids tickrickettsial pathogen survival in the cold[END_REF] (sequential steps and tick molecular factors colored in blue).

Phosphoenolpyruvate carboxykinase (PEPCK

Other tick metabolic genes may be regulated by the same mechanism in which we propose that A. phagocytophilum uncharacterized nucleomodulins may participate in the recruitment of tick TFs. Thirdly, the recruitment of histone deacetylase 1 (HDAC1) to the promoters of target genes through the activity of nucleomodulins may contribute to the repression of tick genes, e.g. fructose-1,6-bisphosphatase (fbp) (sequential steps and tick molecular factors colored in green). Lastly, although the mechanism is unknown, A. phagocytophilum also targets tick proteins to the proteasome which in turn may result in the underrepresentation of targeted metabolic enzymes [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF]. Abbreviations are as follow: Ixodes p21-activated kinase (IPAK1), RNA polymerase II (RNAPII), TATA box-binding protein (TBP), acetyltransferase p300 (p300). Closed and open chromatin were represented as red and green double-stranded DNA, respectively.

Anaplasma phagocytophilum

Significant changes in the levels of genes and proteins of hemoglobinolytic enzymes affecting host hemoglobin levels in tick tissues [START_REF] Villar | The intracellular bacterium Anaplasma phagocytophilum selectively manipulates the levels of vertebrate host proteins in the tick vector Ixodes scapularis[END_REF] * Full name of enzymes are in the main text except for phosphoglycerate kinase 1 (PGK1), enolase (ENOL), glycerol-3-phosphate dehydrogenase (GPDH), triosephosphate isomerase (TPI). ** Only the levels of tick metabolites in response to Borrelia spp. infection at day 4 of feeding were considered [START_REF] Hoxmeier | Metabolomics of the tick-Borrelia interaction during the nymphal tick blood meal[END_REF].

9 Figure legends Figure 1 .

 9legends1 Figure legends

Figure 2 .

 2 Figure 2. Molecular mechanisms potentially implicated in tick metabolism modulation by the model pathogen A. phagocytophilum. Molecular mechanisms proposed to participate in the transcriptional reprograming and proteome modulation induced by A. phagocytophilum infection in tick cells. Firstly, A. phagocytophilum activates the phosphoinositide 3-kinase (PI3K) signaling pathway in a G-protein-coupled-receptor (GPCR)-dependent manner to

Table 1 . Tick metabolic pathways affected by tick-borne pathogens Metabolic pathways Experimental system (in vitro vs. in vivo)

 1

	Affected by
	(name of pathogen	Effect on tick metabolism*
	species)

Amino Acids

ISE6 tick cells

Anaplasma phagocytophilum

Decreased levels of alanine, glycine, glutamine, glutamate and methionine Increased levels of serine, proline, tyrosine and phenylalanine Tyrosine is used as fuel to synthetize PEP [START_REF] Villar | Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells[END_REF][START_REF] Cabezas-Cruz | Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine[END_REF] Upregulation of gene oatp Xanthurenic acid acts as a co-factor for unknown transcription factor(s) that control the expression of oatp KAT regulates the expression of oatp [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF] Ixodes scapularis adults midguts and salivary glands Changes in the levels of genes and proteins involved in amino acid synthesis and degradation [START_REF] Cabezas-Cruz | Ixodes scapularis tick cells control Anaplasma phagocytophilum infection by increasing the synthesis of phosphoenolpyruvate from tyrosine[END_REF] Ixodes scapularis nymphs salivary glands

Upregulation of genes oatp and kat

Xanthurenic acid acts as a co-factor for unknown transcription factor(s) that control the expression of oatp [START_REF] Taank | Human rickettsial pathogen modulates arthropod organic anion transporting polypeptide and tryptophan pathway for its survival in ticks[END_REF] Ixodes scapularis nymphs**

Borrelia mayonii

Decreased levels of alanine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, glutamate, glutamine and glycine [START_REF] Hoxmeier | Metabolomics of the tick-Borrelia interaction during the nymphal tick blood meal[END_REF]

Borrelia burgdorferi

Increased levels of tryptophan and glutamine Decreased levels of alanine, isoleucine, leucine, phenylalanine, proline, threonine, valine and glycine