Michela Miani

Julie Le Naour

Emmanuelle Waeckel-Enée

Subash Chand Verma

Marjolene Straube

Patrick Emond

Bernhard Ryffel

Peter Van Endert

Harry Sokol

Dr Julien Diana
email: julien.diana@inserm.fr

Marjolène Straube

Gut Microbiota-Stimulated Innate Lymphoid Cells Support β-defensin 14 Expression in Pancreatic Endocrine Cells Preventing Autoimmune Diabetes

Introduction

Antimicrobial peptides (AMPs) also called host defense peptides, are evolutionarily conserved molecules of the immune system found in almost all plants and animals. AMPs, mainly cathelicidins, defensins and regenerating islet-derived proteins, are mainly expressed at the epithelial surfaces and due to their microbicide ability, AMPs play a critical role in fighting infection and in regulating the microbiota [START_REF] Bevins | Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis[END_REF][START_REF] Hancock | The immunology of host defence peptides: beyond antimicrobial activity[END_REF][START_REF] Ostaff | Antimicrobial peptides and gut microbiota in homeostasis and pathology[END_REF][START_REF] Zasloff | Antimicrobial peptides of multicellular organisms[END_REF][START_REF] Zhang | Antimicrobial peptides[END_REF]. In the last decades, the ability of AMPs to modulate pro-and anti-inflammatory immune responses has been increasingly appreciated [START_REF] Hancock | The immunology of host defence peptides: beyond antimicrobial activity[END_REF][START_REF] Zhang | Antimicrobial peptides[END_REF]. Not surprisingly, dysregulated expression of AMPs has been associated with autoinflammatory and autoimmune diseases such as atherosclerosis, systemic lupus erythematous, rheumatoid arthritis or type 1 diabetes (T1D) [START_REF] Diana | Crosstalk between neutrophils, B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes[END_REF][START_REF] Frasca | Role of defensins and cathelicidin LL37 in auto-immune and auto-inflammatory diseases[END_REF][START_REF] Kahlenberg | Little peptide, big effects: the role of LL-37 in inflammation and autoimmune disease[END_REF]. Recently, we have revealed the protective role of cathelicidin against autoimmune diabetes in the NOD mouse model. In diabetes-resistant mouse strains, cathelicidin is constitutively expressed by pancreatic endocrine cells and owing to its immunoregulatory abilities, prevents pancreatic inflammation.

We have further showed that pancreatic expression of cathelicidin is regulated by gut microbiota-derived metabolites and that, due to dysbiosis in NOD mice, pancreatic expression of cathelicidin is defective allowing the development of autoimmune diabetes [START_REF] Sun | Pancreatic beta-Cells Limit Autoimmune Diabetes via an Immunoregulatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota[END_REF].

While the recent literature highlights the role of cathelicidin in autoimmunity, how β-defensins may also modulate autoimmune diseases remains poorly investigated. A recent study demonstrates the protective role of the β-defensin 14 (mBD14) against experimental autoimmune encephalomyelitis (EAE) a mouse model of multiple sclerosis [START_REF] Bruhs | Prevention and Mitigation of Experimental Autoimmune Encephalomyelitis by Murine beta-Defensins via Induction of Regulatory T Cells[END_REF].

We hypothesized that mBD14 may also play a role in the development of autoimmune diabetes in NOD mice. Here, we reveal that mBD14 is a part of a complex interplay between the gut microbiota, innate immune cells and pancreatic endocrine cells preserving immune tolerance in the pancreas and preventing autoimmune diabetes.

Results

Pancreatic endocrine cells express mBD14 in non-autoimmune but not in NOD mice.

β-defensins are known to be expressed by epithelial cells in the gut, skin or lung but their expression in the pancreas remains unknown [START_REF] Ganz | Defensins: antimicrobial peptides of innate immunity[END_REF]. Using confocal microscopy, we observed that mBD14 was expressed by pancreatic endocrine cells (glucagon + α-cells and insulin + β-cells) with a lower expression in NOD mice compared with BALB/c and C57BL/6 mice (Figure 1A and S1A). Expression of human β-defensin 3 (hBD3), the orthologue of mBD14, was observed in pancreatic endocrine cells from healthy donors, in accordance with the evolutionary conserved nature of AMPs (Figure 1B). Interestingly, the expression of mBD14 mRNA in pancreatic islets was lower in NOD female mice compared with nonautoimmune female mice and lower in female NOD mice compared with male NOD mice (Figure 1C,D). These results suggest that mBD14 may play a protective role against autoimmune diabetes since male NOD mice are partially protected against the disease. These data prompted us to investigate whether mBD14 modulates the development of autoimmune diabetes in female NOD mice.

MBD14 is protective against autoimmune diabetes in NOD mice.

We investigated whether administration of mBD14 modulates the development of autoimmune diabetes in NOD mice. RNAseq analysis revealed significant transcriptome changes in pancreatic islets of NOD mice after mBD14 administration. Noticeably, many genes related to inflammation were identified among down-regulated genes in mBD14-treated NOD mice (Figure S2A-B). Flow cytometry analysis confirmed the anti-inflammatory effect of mBD14 in pancreatic islets as administration of mBD14 but not of scrambled peptide (scmBD14) reduced the frequency of pancreatic IFNγ + CD8 + effector T cells specific for the β-cell antigen islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP)206-214 (Figure 1E).

Accordingly, treatment of prediabetic NOD mice with mBD14 but not with scmBD14 reduced the immune infiltration of the pancreatic islets and the incidence of autoimmune diabetes (Figure S1C and1F). Additionally mBD14-treatment of hyperglycemic NOD mice resulted in a transient decrease of their glycaemia (Figure S1B). To decipher how mBD14 regulated the diabetogenic immune response, we analyzed pancreatic immune cells from mBD14-treated NOD mice. MBD14 treatment significantly increased the number of B cells and macrophages, while the number of dendritic cells (cDCs) and T cells remained unchanged (Figure 1G and Figure S1D). No significant difference was observed in the pancreatic lymph nodes (PLN) or in the spleen (Figure S1E-F). But the number of B cells in the PLN had a tendency to increase suggesting that mBD14 may also impact B cells in this organ. Together these data support a protective effect of mBD14 against autoimmune diabetes in NOD mice via the regulation of the diabetogenic response.

MBD14 induces pancreatic regulatory B cells.

To decipher the immunoregulatory effect of mBD14, we analyzed pancreatic B cells as their number significantly increased in mBD14-treated NOD mice. Although the precise role of B cells in T1D remains vague, it is believed that cytokine production and auto-antigen presentation by autoreactive B cells to self-reactive T cells play a significant role in the pathogenesis of T1D [START_REF] Bloem | The elusive role of B lymphocytes and islet autoantibodies in (human) type 1 diabetes[END_REF]. Additionally, Breg cell induction in pancreatic islets dampens the local inflammation and prevents disease development [START_REF] Boldison | Immune and Pancreatic beta Cell Interactions in Type 1 Diabetes[END_REF]. We observed that mBD14 treatment induced pancreatic B cell proliferation as demonstrated by increased Ki67 expression (Figure 2A) and B cell recruitment likely from PLN as showed by increased expression of CXCL13 mRNA (Figure S2C). Functionally, mBD14-induced B cells showed an increased expression of the regulatory molecules IL-4, active transforming growth factor β (TGFβ) (LAP) and Indoleamine 2,3-dioxygenase (IDO) while IL-10 expression was not significantly increased (Figure 2B). About 25% of B cells were double positive for IDO and IL-4 with cells also expressing LAP. The induction of IL-4 + B cells by mBD14 in pancreatic islets was dose-dependent and not observed with scmBD14 (Figure S2D), and not observed in the PLN or the spleen (Figure S2E). Using an IL-4-neutralizing mAb, we demonstrated that IL-4 was required for the protection against diabetes conferred by mBD14 treatment (Figure 2C). Regulatory B (Breg) cells are classically characterized by the expression of CD5 and other surface markers depending on the context studied [START_REF] Mauri | The expanding family of regulatory B cells[END_REF]. IL-10 + CD138 + plasma cells were also described to be protective against experimental autoimmune encephalomyelitis (EAE) [START_REF] Fillatreau | B cells regulate autoimmunity by provision of IL-10[END_REF]. Here, IL4 + mBD14-induced B cells expressed CD5, CD1d, B220, CD21 and CD24 but were negative for CD138 (Figure 2D, E and Figure S2F-G). Next, using in vitro system, we wanted to determine the receptor for mBD14 at the surface of pancreatic B cells. In vitro, mBD14 induced IL-4 and LAP expression in isolated B cells in a dose dependent manner (Figure 2F). Using B cells from myd88 -/-NOD mice, we found that mBD14 acted via TLRs and more particularly via the TLR1/2 heterodimer as demonstrated by the use of anti-TLR2 blocking antibody and of the TLR1/2 heterodimer antagonist CU-CTP22 (Figure 2G). Noticeably, pancreatic CD5 + B cells expressed a higher level of TLR2 compared with CD5 -B cells, partially explaining why CD5 + B cells were a preferential target for mBD14 (Figure S2H). Together our data support that mBD14 targets pancreatic B cells via TLR2 inducing the expression of regulatory cytokines.

MBD14-induced B cells promote regulatory macrophages and T cells.

Pancreatic mBD14-induced B cells were characterized by their high expression of IL-4, a cytokine known to drive macrophage polarization to a regulatory phenotype [START_REF] Mantovani | New vistas on macrophage differentiation and activation[END_REF]. After mBD14 treatment, we observed an increased frequency of pancreatic macrophages likely due to the increased expression of the macrophage-recruiting chemokines CCL3 and CCL5 in pancreatic islets (Figure S3A). Interestingly mBD14 treatment decreased the ratio between inflammatory CD11c + CD206 -macrophages and regulatory CD206 + CD11c - macrophages and this decrease was dependent on IL-4 and B cells as demonstrated by the use of an IL-4-neutralizing mAb and anti-CD20 depleting mAb (Figure 3A). Functionally, mBD14 treatment switched the expression of cytokines in pancreatic macrophages from an inflammatory profile (TNFα, IL-12) towards a regulatory profile (IL-4, TGFβ) (Figure 3B) and induced the mRNA expression of arginase I and Ym1/Ym2, two markers of regulatory macrophages (Figure S3A). In vitro, we did not observed a direct effect of mBD14 on bone marrow-derived macrophages (Figure S3B) suggesting that the induction of regulatory macrophages by mBD14 was only dependent on IL-4-secreting B cells. DCs, the other main antigen-presenting cells in pancreatic islets were also affected by mBD14 treatment with a reduction of IL-12 expression (Figure S3C). Both macrophages and DCs have the potential to induce regulatory T (Treg) cells necessary for a long-term protection against autoimmune diabetes [START_REF] Tang | The Foxp3+ regulatory T cell: a jack of all trades, master of regulation[END_REF]. Ten days after mBD14 treatment, the frequency and number of Foxp3 + Treg cells increased in pancreatic islets but not in the PLN or the spleen (Figure 3C and Figure S3D,E). Using neutralizing antibodies, we demonstrated that induction of Treg cells by mBD14 was dependent on IL-4 and TGFβ, two cytokines produced by mBD14induced B cells and macrophages (Figure 3D). To determine the contribution of each cell type in Treg cell induction we used an in vitro system. Isolated pancreatic myeloid cells (CD11b + cells) or B cells (CD19 + cells) from vehicle-or mBD14-treated NOD mice were cultured with monoclonal naïve (CD62L + ) autoreactive BDC2.5 CD4 + T cells devoid of Treg cells (CD25 -). Myeloid cells but not B cells were able to induce Treg cells regardless their source. However, myeloid cells from mBD14-treated NOD mice had a significantly higher potency to induce Treg cells compared to their counterparts from vehicle-treated NOD mice (Figure 3E and Figure S3F). We did not observe an induction of a regulatory phenotype in BDC2.5 T cells cultured alone with mBD14 excluding a direct effect of the peptide on these T cells (Figure S3G). Together, our data support that mBD14 induces an immunoregulatory cascade in pancreatic islets from B cells to Treg cells.

IL-22 promotes pancreatic expression of mBD14

We next sought to determine the pathway governing pancreatic expression of mBD14 and why this pathway was defective in NOD mice. In the gut, but also in other tissues including the pancreas, IL-22 has emerged as a key cytokine in tissue homeostasis partially through its ability to promote the production of AMPs [START_REF] Aujla | IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia[END_REF][START_REF] Hill | The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes[END_REF][START_REF] Liang | Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides[END_REF][START_REF] Mulcahy | Interleukin-22 regulates antimicrobial peptide expression and keratinocyte differentiation to control Staphylococcus aureus colonization of the nasal mucosa[END_REF][START_REF] Sonnenberg | Innate lymphoid cells promote anatomical containment of lymphoid-resident commensal bacteria[END_REF][START_REF] Wolk | IL-22 increases the innate immunity of tissues[END_REF]. The IL-22 receptor is highly expressed by pancreatic islets and β-cells [START_REF] Shioya | Interleukin 22 receptor 1 expression in pancreas islets[END_REF][START_REF] Wolk | IL-22 increases the innate immunity of tissues[END_REF], and we hypothesized that IL-22 may induce mBD14 expression in pancreatic endocrine cells. We observed that IL22 mRNA expression was reduced in pancreatic islets from female NOD mice compared with female non-autoimmune mice (Figure 4A) and male NOD mice (Figure S4A).

The IL-22 level in the serum was also reduced in female NOD mice (Figure S4B). Importantly the administration of IL-22Fc to NOD mice induced the expression of mBD14 mRNA in pancreatic islets (Figure 4B). In vitro, rIL-22 induced mBD14 by targeting directly mouse and human pancreatic islets (Figure 4C,D). Moreover, rIL-22 induced the expression of mBD14 in the Min6 β-cell line (Figure S4C). As previously described [START_REF] Hill | The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes[END_REF][START_REF] Kinnebrew | Interleukin 23 production by intestinal CD103(+)CD11b(+) dendritic cells in response to bacterial flagellin enhances mucosal innate immune defense[END_REF], IL-22Fc also stimulated the expression of the protective AMP Reg3γ in pancreatic islets and ileum (Figure S4D,E). Together, these data support that IL-22 induces the expression of mBD14 by pancreatic endocrine cells.

Type 3 innate lymphoid cells are the main source of IL-22 in pancreatic islets.

Both type 3 innate lymphoid cells (ILC3s) and conventional T cells can produce IL-22 [START_REF] Sabat | Therapeutic opportunities of the IL-22-IL-22R1 system[END_REF]. By flow cytometry, we investigated the ILC population in pancreatic islets from NOD, C57BL/6 and BALB/c mice. ILCs (CD127 + CD90 + Lin -) were observed in pancreatic islets of all mouse strains in equal number (Figure 4E). We confirmed these data using rag -/- mice to exclude a potential contamination with conventional T cells in our gating strategy (Figure S4F). Analyzing pancreatic ILC subtypes, we observed a reduction in the frequency of RORγt + ILC3s in NOD mice compared with non-autoimmune mice (Figure 4F and Figure S4G). Accordingly, the expression of IL-22 by pancreatic ILCs was reduced in NOD mice compared with non-autoimmune mice (Figure 4G andS4J). Conventional α/β and γ/δ T cells did not significantly expressed IL-22 in pancreatic islets from all mouse strains (Figure S4H-J). Using confocal microscopy, we observed the presence of IL-22 + CD5 -cells and IL-22 - CD5 + cells inside the pancreatic islets of C57BL/6 mice (Figure 4H and S5A). To confirm that only ILCs and not T cells were needed as an IL-22 source to induce mBD14 in the pancreatic islets, we treated C57BL/6 rag -/-mice with the IL-22 inducer IL-23. We observed that IL-23 efficiently increased mRNA expression of IL-22 and mBD14 in the pancreatic islets and ileum of these T-cell deficient mice (Figure S5B-E). The reduced frequency of ILC3s in NOD mice was not observed in pancreatic or mesenteric LN (Figure S6A-B). Furthermore, despite a high frequency of ILC3s in pancreatic or mesenteric LN of NOD mice, ILCs in these organs did not expressed higher level of IL-22 compared with non-autoimmune strains, but rather expressed significantly higher level of IFNγ and TNFα (Figure S6C-D). Together our data support that IL-22 production was defective in NOD mice and that pancreatic ILC3s were the source of IL-22 promoting mBD14 expression in pancreatic islets of non-autoimmune mice.

Gut microbiota controls IL-22 and mBD14 expression in pancreatic islets.

It is recognized that microbiota controls the production of IL-22 by ILC3s in the gut [START_REF] Klose | Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis[END_REF]. To evaluate how gut microbiota may control IL-22 and consequently mBD14 expression in pancreatic islets, we treated BALB/c mice with a cocktail of broad-spectrum antibiotics (ABX) to reduce the gut microbiota. As described in the skin [START_REF] Zanvit | Antibiotics in neonatal life increase murine susceptibility to experimental psoriasis[END_REF], ABX-treatment decreased IL-22 protein level in the serum and mRNA expression of IL22 in pancreatic islets (Figure 5A,B). In parallel, ABX-treatment decreased the expression of mBD14 mRNA in pancreatic islets suggesting that the gut microbiota controlled the expression of mBD14 in pancreatic islets via the induction of IL-22 (Figure 5C). To confirm these data, we performed gut microbiota-transfer experiments. Expression of mBD14 and IL22 mRNA was increased in pancreatic islets from NOD mice that received gut microbiota from BALB/c mice or C57BL/6 mice compared with NOD mice supplied with NOD gut microbiota (Figure 5D andS7A). Together, these data support that the gut microbiota controls the expression of mBD14 in pancreatic islets via IL-22 induction.

Gut microbiota drives pancreatic IL-22 expression via AHR ligands and IL-23.

In the gut, microbiota controls the expression of IL-22 by ILC3s via different pathways [START_REF] Zhou | AHR Function in Lymphocytes: Emerging Concepts[END_REF]. Specific bacteria species produce aryl hydrocarbon receptor (AHR) ligands that directly activate ILC3s to produce IL-22. Other bacteria species produce metabolites that promote IL-23 secretion by intestinal phagocytes and IL-23 activates ILC3s to produce IL-22.

Consequently, we hypothesized that one of these pathways or both may be at play in pancreatic islets to stimulate the production of IL-22. To evaluate the ability of AHR ligand to stimulate mBD14 expression in pancreatic islets, we administrated the AHR ligand 6-Formylindolo-[3,2b]-carbazole (FICZ) to BALB/c mice. FICZ administration induced mRNA expression of Cyp1a1 in pancreatic islets confirming the activation of AHR + cells (Figure 6A) and increased the expression of IL22 and mBD14 mRNA in pancreatic islets (Figure 6B,C). A similar induction of mBD14 was observed after injection of the AHR ligand indole-3-aldehyde, a tryptophan metabolite naturally produced by the gut microbiota (Figure 6C). As systemic AHR ligand administration may induce mBD14 in pancreatic islets via indirect pathways, we treated isolated pancreatic islets in vitro with FICZ and we observed an increased expression of Cyp1a1, IL22 and mBD14 mRNAs (Figure 6D-F). The induction of pancreatic mBD14 by FICZ was dependent on IL-22 as demonstrated by the use of neutralizing mAb (Figure 6F) and by the fact that FICZ did not induce mBD14 expression in Min6 β-cell line excluding a direct role of AHR ligand on β-cells (Figure 6G). Using a reporter assay, we failed to observe a significant defect in AHR ligand activity in the feces or the serum of NOD mice compared with non-autoimmune mice (Figure 6H,I). Similarly, using mass spectrometry, we did not detect significant difference in the concentration of various AHR ligands in the feces or the serum of NOD mice compared with non-autoimmune mice (Figure S7C). These data made unlikely that a defective AHR ligand production in NOD mice may explain the defect in mBD14 expression in pancreatic islets. Nevertheless, NOD strain expresses a low affinity AHR d allele contrary to BALB/c or C57BL/6 strains carrying the AHR b allele [START_REF] Kerkvliet | Activation of aryl hydrocarbon receptor by TCDD prevents diabetes in NOD mice and increases Foxp3+ T cells in pancreatic lymph nodes[END_REF][START_REF] Okey | Toxicological implications of polymorphisms in receptors for xenobiotic chemicals: the case of the aryl hydrocarbon receptor[END_REF].

Accordingly, by analyzing the expression of Cyp1a1 mRNA, we found that pancreatic islet cells from NOD mice were hyporesponsive to FICZ compared with BALB/c islet cells (Figure 6J). These data support that AHR ligands stimulated IL-22-secreting AHR + cells in pancreatic islets inducing mBD14 expression but in NOD mice, due to the presence of a low affinity AHR allele, the level of AHR ligands may be insufficient to promote IL-22 and mBD14 expression.

Then, we evaluated how IL-23 may also be implicated in the induction of mBD14 in pancreatic islets. Expression of IL23 mRNA was reduced in pancreatic islets from NOD mice compared with BALB/c and C57BL/6 mice (Figure 7A) and IL23 mRNA expression was increased in pancreatic islets from NOD mice that received gut microbiota from BALB/c mice or C57BL/6 mice compared with NOD mice supplied with NOD gut microbiota (Figure S7B). Flow cytometry analysis of pancreatic phagocytes confirmed a defective IL-23 expression in NOD mice compared with non-autoimmune strains (Figure 7B). In non-autoimmune mice, pancreatic IL-23 expression was mainly supported by macrophages (Figure S7D).

Administration of rIL-23 to BALB/c mice increased the level of IL-22 in the serum associated with an increased expression of IL22 and mBD14 mRNA in pancreatic islets (Figure 7C-E).

Then we attempted to determine why pancreatic phagocytes failed to produce IL-23 in NOD mice. A recent study and our present data (Figure S7E) demonstrated the effect of butyrate, a microbiota-derived metabolite, in upregulating IL-23 expression in DCs and macrophages [START_REF] Berndt | Butyrate increases IL-23 production by stimulated dendritic cells[END_REF]. Additionally, our previous study demonstrated that female NOD mice harbored reduced level of butyrate in the serum and the gut compared with non-autoimmune mice [START_REF] Sun | Pancreatic beta-Cells Limit Autoimmune Diabetes via an Immunoregulatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota[END_REF]. Thus, we hypothesized that butyrate may stimulate the expression of IL-23 by pancreatic phagocytes and a defective production of butyrate in NOD mice may lead to reduced expression of pancreatic IL-23. By 16s rDNA sequencing from feces of NOD, BALB/c and C57BL/6 mice, we observed that gut microbiota in NOD mice harbored a higher Bacteroidetes/Firmicutes ratio with a lower abundance of Clostridiales Lachnospiraceae, Ruminococcaceae and Erysipelotrichaceae (Figure 5E-G). These bacteria families are butyrogenic [START_REF] Riviere | Bifidobacteria and Butyrate-Producing Colon Bacteria: Importance and Strategies for Their Stimulation in the Human Gut[END_REF] and their low abundance in NOD mice is in line with the low level of butyrate observed in this strain. Administration of butyrate to BALB/c mice increased the level of IL-23 in the serum and the expression of IL23, IL22 and mBD14 mRNA in pancreatic islets (Figure 7F-I), however we failed to observe a significant increase in the expression of mBD14 mRNA in butyrate-treated NOD mice (Figure S7F). In vitro, butyrate directly stimulated pancreatic islet cells to induce IL23, IL22 and mBD14 mRNA expression (Figure 7J-L). This induction of mBD14 by butyrate was dependent on IL-22 as demonstrated by the use of neutralizing mAb (Figure 7L). In vivo, we observed that butyrate increased the expression of IL-23 by pancreatic phagocytes (Figure 7M). Since butyrate failed to induce mBD14 expression in Min6 β-cell line (Figure S7G), we conclude that butyrate induced the expression of mBD14 in pancreatic islets via the stimulation of IL-23 + phagocytes.

Discussion

The immunomodulatory role of AMPs is increasingly recognized and their involvement in the immunopathology of autoimmune diseases is progressively identified [START_REF] Kahlenberg | Little peptide, big effects: the role of LL-37 in inflammation and autoimmune disease[END_REF][START_REF] Mansour | Host defense peptides: front-line immunomodulators[END_REF]. Many studies have revealed the deleterious role of cathelicidin in autoinflammatory and autoimmune diseases including atherosclerosis, small vessel vasculitis, psoriasis and systemic lupus erythematous [START_REF] Gupta | The role of neutrophils and NETosis in autoimmune and renal diseases[END_REF][START_REF] Kahlenberg | Little peptide, big effects: the role of LL-37 in inflammation and autoimmune disease[END_REF][START_REF] Pinegin | Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity[END_REF]. In sterile condition, cathelicidin can be aberrantly expressed in the tissues by infiltrating neutrophils stimulating the uncontrolled production of type I IFNs, which are important contributors to autoimmunity [START_REF] Hall | Type I interferons: crucial participants in disease amplification in autoimmunity[END_REF]. We have shown that this pathway is also at play during the initiation of autoimmune diabetes in NOD mice [START_REF] Diana | Crosstalk between neutrophils, B-1a cells and plasmacytoid dendritic cells initiates autoimmune diabetes[END_REF], however we have further demonstrated that cathelicidin may be also protective against autoimmune diabetes [START_REF] Sun | Pancreatic beta-Cells Limit Autoimmune Diabetes via an Immunoregulatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota[END_REF]. Indeed, in non-autoimmune-prone mice but not in NOD mice, cathelicidin is produced by pancreatic endocrine cells and this endocrine cathelicidin exerts an immunoregulatory effect on macrophages thereby preserving immune tolerance in the pancreas. Conversely, a recent study shows that the cathelicidin-like peptide FhHDM-1 from the helminth Fasciola hepatica regulates inflammatory macrophages, thus dampening the development of diabetes in NOD mice and the severity of EAE, a mouse model of multiple sclerosis [START_REF] Lund | A parasite-derived 68-mer peptide ameliorates autoimmune disease in murine models of Type 1 diabetes and multiple sclerosis[END_REF]. Collectively, these observations indicate that cathelicidin aberrantly produced from neutrophils is deleterious, while cathelicidin constitutively produced by non-immune cells or exogenous cathelicidin is protective against autoimmune diabetes. These opposite effects of cathelicidin from different cellular source may be due to distinct posttranslational modifications which affect its function [START_REF] Wang | Post-translational Modifications of Natural Antimicrobial Peptides and Strategies for Peptide Engineering[END_REF].

Like cathelicidin, β-defensins exert diverse immunomodulatory activities including receptormediated chemotaxis or modulation of immune cells via TLR regulating both innate and adaptive immunity [START_REF] Lehrer | Primate defensins[END_REF][START_REF] Semple | beta-Defensins: multifunctional modulators of infection, inflammation and more?[END_REF]. Studies in the Dorin laboratory of hBD3 have demonstrated that this defensin inhibits the production of pro-inflammatory cytokines by LPS-primed macrophages in vitro and reduces the level of pro-inflammatory cytokines in the serum of LPS-treated mice when administrated in vivo [START_REF] Semple | Human beta-defensin 3 affects the activity of pro-inflammatory pathways associated with MyD88 and TRIF[END_REF][START_REF] Semple | Human beta-defensin 3 has immunosuppressive activity in vitro and in vivo[END_REF]. Studies from the group of Schwarz have shown the ability of mBD14 to induce Treg cells in vitro and in vivo, reducing the severity of EAE [START_REF] Bruhs | Prevention and Mitigation of Experimental Autoimmune Encephalomyelitis by Murine beta-Defensins via Induction of Regulatory T Cells[END_REF][START_REF] Navid | Induction of regulatory T cells by a murine beta-defensin[END_REF]. Our present study reveals the direct effect of mBD14 on B cells via the TLR1/2 heterodimer that is highly expressed on pancreatic CD5 + B cells. Consistent with our finding, hBD3 activates human monocytes via the TLR1/2 heterodimer [START_REF] Funderburg | Human -defensin-3 activates professional antigenpresenting cells via Toll-like receptors 1 and 2[END_REF].

Consequently, we cannot totally rule out a direct effect of mBD14 on pancreatic TLR2expressing phagocytes. Together, the literature and our data support that β-defensins exert immunomodulatory function on different immune cell types ensuring an efficient protection against tissue inflammation and autoimmunity.

Our data support that the protective role of mBD14 against autoimmune diabetes is mediated by induction of IL-4-producing B cells. In line with our results, transgenic expression of IL-4 by pancreatic β-cells efficiently prevents the development of diabetes in NOD mice [START_REF] Mueller | Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic (NOD) mice[END_REF]. Using B cell-deficient NOD mice and depleting CD20 antibody, the pathogenic effect of B cells in autoimmune diabetes has been demonstrated [START_REF] Xiu | B lymphocyte depletion by CD20 monoclonal antibody prevents diabetes in nonobese diabetic mice despite isotype-specific differences in Fc gamma R effector functions[END_REF][START_REF] Yang | Development of insulitis and diabetes in B cell-deficient NOD mice[END_REF]. However, various Breg subtypes play a protective role against several immune-related diseases including systemic lupus erythematous, rheumatoid arthritis and autoimmune diabetes [START_REF] Rosser | Regulatory B cells: origin, phenotype, and function[END_REF]. Reconciling these observations, the protective effect of B cell depletion against autoimmune diabetes is associated with the induction of IL-10-producing Breg cells in the reconstituted B cell population and the subsequent induction of Treg cells (Di [START_REF] Caro | Involvement of suppressive B-lymphocytes in the mechanism of tolerogenic dendritic cell reversal of type 1 diabetes in NOD mice[END_REF].

We show that IL-22 stimulates pancreatic β-cells to express mBD14 and that IL-22 expression is defective in pancreatic islets of NOD mice. IL-22 is known to induce AMPs including β-defensins in different tissues (e.g. the gut, skin and lung) preventing tissue damage [START_REF] Aujla | IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia[END_REF][START_REF] Lindroos | IL-23-mediated epidermal hyperplasia is dependent on IL-6[END_REF][START_REF] Mulcahy | Interleukin-22 regulates antimicrobial peptide expression and keratinocyte differentiation to control Staphylococcus aureus colonization of the nasal mucosa[END_REF][START_REF] Wolk | IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis[END_REF][START_REF] Zheng | Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens[END_REF].

Pancreatic β-cells highly express IL-22R [START_REF] Shioya | Interleukin 22 receptor 1 expression in pancreas islets[END_REF] and IL-22 is protective against metabolic disorders by improving β-cell proliferation and function and by decreasing inflammation [START_REF] Hasnain | Glycemic control in diabetes is restored by therapeutic manipulation of cytokines that regulate beta cell stress[END_REF][START_REF] Wang | Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes[END_REF]. Additionally, IL-22 stimulates the expression of the AMPs Reg2 and Reg3 by pancreatic acinar cells preventing tissue damage in a mouse model of pancreatitis [START_REF] Aggarwal | Acinar cells of the pancreas are a target of interleukin-22[END_REF][START_REF] Xue | Aryl hydrocarbon receptor regulates pancreatic IL-22 production and protects mice from acute pancreatitis[END_REF]. In NOD mice, IL-22 promotes the expression of Reg1 and Reg2 by β-cells preventing β-cell loss in an autocrine manner [START_REF] Hill | The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes[END_REF]. Together, IL-22 induces different AMPs in pancreatic islets promoting β-cell function, regulating inflammation and preventing autoimmune response. The therapeutic use of IL-22 may be of interest against autoimmune diabetes. However, IL22 -/- NOD mice did not display an increased incidence of autoimmune diabetes [START_REF] Ishigame | Excessive Th1 responses due to the absence of TGF-beta signaling cause autoimmune diabetes and dysregulated Treg cell homeostasis[END_REF] but as showed here, NOD mice are characterized by a constitutively low level of pancreatic IL-22 so that genetic complete abrogation may not have a significant impact on the development of the disease. A recent study shows that IL-22 treatment of prediabetic NOD mice does not reduced the incidence of diabetes [START_REF] Borg | The effect of interleukin-22 treatment on autoimmune diabetes in the NOD mouse[END_REF]. However, as mentioned by the authors, a more pronounced effect may be obtained by increasing the dose, the frequency of administration and by the use of a more metabolically stable Fc-based fusion protein as used against metabolic diseases [START_REF] Wang | Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes[END_REF] and in our present study. Finally, the endogenous production of the neutralizing IL-22 binding protein may limit the therapeutic use of IL-22 at a lower dose [START_REF] Martin | Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid[END_REF].

We reveal for the first time the presence of IL-22-secreting ILC3s within pancreatic islets and their defect in NOD mice. In a mouse-model of pancreatitis, AHR-reactive conventional T cells are a source of IL-22 in the whole pancreas [START_REF] Xue | Aryl hydrocarbon receptor regulates pancreatic IL-22 production and protects mice from acute pancreatitis[END_REF]; however, in the pancreatic islets we observe that α/β or γ/δ T cells did not express significant levels of IL-22. The role of ILCs in autoimmune diseases is an emerging field of research and to our knowledge, the role of ILCs in autoimmune diabetes remains unknown. A deleterious role of IFNγ-producing ILC1s has been proposed in inflammatory bowel diseases while IL-17-producing ILC3s may participate to rheumatic diseases, psoriasis or multiple sclerosis [START_REF] Shikhagaie | Innate lymphoid cells in autoimmunity: emerging regulators in rheumatic diseases[END_REF]. As autoimmune diabetes is not an IL-17-mediated disease [START_REF] Bedoya | Th17 cells in immunity and autoimmunity[END_REF], and due to the protective nature of IL-22, pancreatic ILC3s may play a protective role against autoimmune diabetes.

Interestingly despite a high number of ILC3s, ILCs in PLN or MLN of NOD mice did not expressed high level of IL-22, but rather expressed significantly levels of IFNγ and TNFα. These data support that the inflammatory environment in the PLN and MLN in NOD mice may influence the cytokine expression of ILCs towards an inflammatory profile regardless of the subtype of ILCs as already described in the gut (Melo-Gonzalez and Hepworth, 2017).

Finally, we show that the expression of IL-22 in pancreatic islets is under the control of the gut microbiota-derived molecules AHR ligands and butyrate. NOD mice such as the DBA/2 strain carry a low affinity AHR d genotype contrary to BALB/c or C57BL/6 mice carrying the AHR b allele. Consequently, AHR d mice require at least a tenfold higher dose of AHR ligand than AHR b mice to induce the same effects [START_REF] Kerkvliet | Activation of aryl hydrocarbon receptor by TCDD prevents diabetes in NOD mice and increases Foxp3+ T cells in pancreatic lymph nodes[END_REF][START_REF] Okey | Toxicological implications of polymorphisms in receptors for xenobiotic chemicals: the case of the aryl hydrocarbon receptor[END_REF]. These studies and our data support that AHR + cells in NOD mice are hyporesponsive to endogenous AHR ligands.

Nevertheless, treatment of NOD mice with high dose of AHR ligand reduces insulitis and the incidence of diabetes via the induction of regulatory AHR + DCs and Treg cells [START_REF] Kerkvliet | Activation of aryl hydrocarbon receptor by TCDD prevents diabetes in NOD mice and increases Foxp3+ T cells in pancreatic lymph nodes[END_REF]. The anti-inflammatory effect of AHR ligands is also mediated by the induction of IL-22 in various tissues including the gut, lung and pancreas [START_REF] Qiu | The aryl hydrocarbon receptor regulates gut immunity through modulation of innate lymphoid cells[END_REF][START_REF] Simonian | gammadelta T cells protect against lung fibrosis via IL-22[END_REF][START_REF] Xue | Aryl hydrocarbon receptor regulates pancreatic IL-22 production and protects mice from acute pancreatitis[END_REF]. Together, our data and the literature support a protective role for AHRdependent IL-22 expression in the pancreas via the induction of AMPs promoting β-cell regeneration and controlling inflammation.

As described in the gut [START_REF] Abraham | IL-23 and autoimmunity: new insights into the pathogenesis of inflammatory bowel disease[END_REF], we show that IL-23 induces IL-22 expression by ILC3s in pancreatic islets. We observed that IL-23 expression is reduced in pancreatic islets of NOD mice as described in the serum of T1D patients [START_REF] Roohi | Serum IL-17, IL-23, and TGF-beta levels in type 1 and type 2 diabetic patients and age-matched healthy controls[END_REF]. Under the influence of gut microbiota derived factors, resident intestinal phagocytes secrete IL-23 inducing, via IL-22, the expression of AMPs by epithelial cells [START_REF] Caballero | Microbiota-mediated inflammation and antimicrobial defense in the intestine[END_REF][START_REF] Honda | The microbiota in adaptive immune homeostasis and disease[END_REF]. Here, we demonstrate that butyrate, a short chain fatty acid classically produced by Clostridia species in the gut microbiota, stimulates the expression of IL-23 by pancreatic phagocytes as previously described [START_REF] Berndt | Butyrate increases IL-23 production by stimulated dendritic cells[END_REF]. We have previously demonstrated that NOD mice harbor a reduced level of circulating butyrate [START_REF] Sun | Pancreatic beta-Cells Limit Autoimmune Diabetes via an Immunoregulatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota[END_REF] and we presently show that the gut microbiota of NOD mice is defective in butyrogenic bacteria confirming previous reports in NOD mice and consistent with findings in children with β-cell autoimmunity [START_REF] Brown | Prolonged antibiotic treatment induces a diabetogenic intestinal microbiome that accelerates diabetes in NOD mice[END_REF][START_REF] De Goffau | Fecal microbiota composition differs between children with beta-cell autoimmunity and those without[END_REF][START_REF] Endesfelder | Towards a functional hypothesis relating anti-islet cell autoimmunity to the dietary impact on microbial communities and butyrate production[END_REF][START_REF] Giongo | Toward defining the autoimmune microbiome for type 1 diabetes[END_REF]. Together, these data support that butyrate stimulates the production of immunoregulatory AMPs by pancreatic endocrine cells preventing autoimmune diabetes. A recent study also demonstrated the protective role of butyrate against autoimmune diabetes in NOD mice through the expansion of colonic and splenic Treg cells [START_REF] Marino | Gut microbial metabolites limit the frequency of autoimmune T cells and protect against type 1 diabetes[END_REF].

Considering the role of butyrate in promoting gut integrity and immune tolerance [START_REF] Rooks | Gut microbiota, metabolites and host immunity[END_REF], this gut microbiota-derived metabolite appears as an attractive therapeutic candidate against T1D. Our studies and the literature highlight the beneficial relationships between the pancreas and the gut and this increasing knowledge should lead to identification of new approaches for the treatment of T1D [START_REF] Tilg | Beyond Digestion: The Pancreas Shapes Intestinal Microbiota and Immunity[END_REF].

Limitation of the Study

Our study describes a complex interplay between innate immune cells, pancreatic endocrine cells and the intestinal microbiota participating to the prevention of autoimmune diabetes in NOD mice. Our data support that gut microbiota-stimulated ILCs, via the production of IL-22, promote the expression of immunoregulatory mBD14 by pancreatic endocrine cells. As the number of pancreatic ILCs is low, it would be important to confirm the presence of IL-22producing ILCs into the pancreatic islets using reporter mice. The role of ILCs in autoimmune diseases is emerging with both protective and deleterious roles described. To strengthen the protective role of pancreatic ILCs against autoimmune diabetes, transfer experiments would be helpful, although these experiments may be challenging due to the difficulty to obtain a sufficient number of ILCs from donor mice. Our group and others have described various pathways used by the gut microbiota to prevent autoimmune diabetes. Before transfer these findings to the clinic, it would be important to determine the relative importance of each of these pathways in the prevention of the disease. Finally, as the well-characterized microbiome of laboratory mouse strain differs from human microbiome continuously facing environmental factors, it is critical to perform human studies to translate our findings to the human disease and envisage future therapies based on our conclusions.

STAR METHODS

Detailed methods are provided in the online version of this paper and include the following: Data are the median +/-interquartile of 5 to 6 independent mice per group. (G) Min6 β-cells were treated with increasing doses of FICZ or IL-22 (200 ng.ml -1 ) for 24 h. The expression of mBD14 mRNA was determined by RT-qPCR. Data are the median +/-interquartile of 5 independent experiments. (H, I) AHR ligand activity was determined in the serum (H) and feces (I) using reporter bioassay. Data are the median +/-interquartile of 5 to 9 independent mice per group. (J) mRNA expression of Cyp1a1 and mBD14 were determined in islets cultured for 48 h with growing dose (ng.ml -1 ) of FICZ. Data are the median +/-interquartile of 4 to 5 independent experiments with 4 mice pooled per group per experiment. See also Figure S7.

• KEY RESOURCES TABLE • CONTACT FOR

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, J. Diana (julien.diana@inserm.fr).

EXPERIMENTAL MODEL DETAILS

Mice and treatments

Female BALB/c, C57BL/6J, NOD, NOD rag -/-, C57BL/6J rag -/-, C57BL/6J IL22 -/-and NOD myd88 -/-mice between 10 and 12 weeks of age were used, bred and housed in specific pathogen-free conditions. In some experiments, male mice were used as indicated in the figure. indole (Sigma-Aldrich) 100 µg in 200 µl PBS for 12 h; sodium butyrate (Sigma-Aldrich) daily for 5 days with 1 mg per day in 200 µl PBS. For antibiotic treatment, mice were given 1 g.l -1 metronidazole (Sigma-Aldrich), 0.5 g.l -1 vancomycin (Sigma-Aldrich), 1 g.l -1 ampicillin (Sigma-Aldrich), 1 g.l -1 gentamicin (Sigma-Aldrich) and 1 g.l -1 neomycin (Sigma-Aldrich) by daily oral gavage of 200 µL in antibiotic solution (100% H20). In parallel with the antibiotic treatment, mice were treated per os with 200 mg l -1 amphotericin B of 100 µL in solution (100% H20) to avoid occasional overgrowth of Candida spp. For microbiota transfer experiment, the gut microbiota was transferred between groups of mice by initially depleting the native microbiota with a single oral dose of streptomycin (20 mg) 24 h prior the transplantation. Fresh fecal pellets (120 mg) from 5 donor mice were collected and placed in 1 ml of transfer buffer (pre-reduced sterile cold PBS containing 0.05% cysteine HCl (Sigma-Aldrich)). The fecal pellets were homogenized, centrifuged at 800 g for 2 min, and 50 µl fecal supernatant was orally inoculated to recipient mice over the subsequent 15 days for a total of 6 times. All animal experimental protocols were approved by the ethic committee for animal experimentation (APAFIS#3535-2015092416202090).

Spontaneous diabetes incidence

Eight-weeks-old NOD female mice received one injection of mBD14 or scmBD14 (10 µg) per week during two weeks or similar injections of vehicle (PBS-1% H2O). In some conditions, mice were treated (200 µg per injection at day -1, 1, 3, 6 and 9 post mBD14 treatment) with anti-IL4 (11B11) or relevant isotype control. Overt diabetes was defined as two positive urine glucose tests, confirmed by a glycemia >200 mg.dl -1 . Glukotest kit was purchased from Roche.

Glucose tests and measure of glycemia were performed in a blind fashion.

METHOD DETAILS Preparation of pancreatic islets

Pancreata were perfused with a solution of collagenase P in HBSS-1% HEPES (0.75 mg.ml -1 , Roche), then dissected free from surrounding tissues. Pancreata were digested at 37°C for 8 min. Digestion was stopped by adding HBSS-10% FCS-1% EDTA followed by extensive washes. For flow cytometry analysis, islets were isolated on a discontinuous Ficoll® PM400 gradient (Sigma-Aldrich) and then cells were released from the islets by incubation at 37°C for 6 min in non-enzymatic cell dissociation solution (Sigma-Aldrich). For islets culture, cytology and RT-qPCR, to avoid potential contamination by exocrine tissue, islets were purified by handpicking in 3 consecutive baths of HBSS-10% FCS supplemented with 1% DNAse 1.

RT-qPCR

Total RNA was isolated using the Nucleospin RNA XS kit (Macherey-Nagel) from a minimum of 100 handpicked islets per mouse. RNA was reverse transcribed to synthesized cDNA using the high capacity cDNA reverse transcription kit (ThermoFisher) and measurements were performed by qPCR using Takyon™ ROX SYBR® MasterMix dTTP blue (Eurogentec) on a 7900HT Fast System (Applied Biosystems). Resulting levels of fluorescence were submitted to relative quantification by normalization against a housekeeping gene (GAPDH) and expressed as 2 -(∆CT) values.

Flow cytometry

Single cell suspensions were prepared from various tissues and were stained for 30 min at 4°C

after FcγRII/III blocking with anti-CD16/CD32 (Biolegend, 93) monoclonal antibody (mAb).

Staining buffer was PBS containing 2% FCS, 0.5% EDTA and 0.1% sodium azide. Surface staining was performed with the following mAbs against: CD45 (eBioscience, 30-F11), CD11b

(eBioscience, M1/70), F4/80 (eBioscience, BM8), CD11c (eBioscience, N418), TCRβ (eBioscience, H57-597), TCRγ/δ (eBioscience, UC7-13D5), CD19 (eBioscience, 1D3), CD206

(Biolegend, C068C2), CD5 (Miltenyi, 53-7.3), CD1d (Biolegend, K253), CD21 (Biolegend, 7E9), CD24 (Biolegend, 30-F1), B220 (Biolegend, RA3-6B2), CD138 (Biolegend, 281-2), TLR2 (Biolegend, CB225), CD127 (Biolegend, A7R34), -CD90.2 (eBioscience, 53-2.1). Lin markers are a mix of anti-CD19, -CD5, -CD3ε , -B220, -CD11b, and -CD11c mAbs. For measurement of active TGFβ, cells were surface stained with anti-LAP mAb (eBioscience, TW7-16B4). For cytokine expression, cell suspensions were incubated 5 h at 37°C with the relevant stimulus, if required, in the presence of a protein transport inhibitor cocktail (eBioscience), surface stained, fixed and then intracellularly stained using the intracellular staining kit (Biolegend). For macrophages and DCs, cell suspension was incubated with LPS

(1 µg.ml -1 , Sigma-Aldrich) and stained with anti-TNFα (eBioscience, MP6-XT22), -IL-12 (eBioscience, C17.8), -IL-4 (eBioscience, 11B11), -IL-10 (eBioscience, JES5-16E3) and -IL-23 (eBioscience, fc23-cpg). For innate lymphoid cells, cell suspension was incubated with rmIL-23 (10 ng.ml-1, Miltenyi) plus PMA/ionomycin (both 500 ng.ml -1 , Sigma-Aldrich) and stained with anti-IL-22 pAb (Biolegend, poly5164) or control isotype (goat IgG, Biolegend, poly24030). For diabetogenic CD8 + T cells, cell suspension was incubated with bone-marrow derived DCs loaded with IGRP206-214 peptide and stained with anti-CD8α (BD, 53-6.7) and anti-IFNγ mAbs (eBioscience, XMG1.2). For B cells, cell suspension was incubated without additional stimulus and stained with anti-IL-4 (eBioscience, 11B11), -IL-10 (eBioscience, JES5-16E3) and -IDO (Biolegend, 2E2/IDO1) mAbs. For regulatory T cell detection, cells were surface stained with anti-TCRβ, -CD4 (BD, RM4-5), fixed and then stained for Foxp3

(eBioscience, FJK-16s) expression, using the Foxp3 staining kit (eBioscience). For staining of transcription factors RoRγt, GATA3 (eBioscience, B2D and 16E10A23) and ki67 (eBioscience, SolA15), cells were stained using the True-Nuclear™ Transcription Factor kit (Biolegend). In all experiments dead cells were excluded using Fixable Viability Dye (eBioscience). Stained cells were analyzed on a Becton Dickinson Fortessa flow cytometer. Data were analyzed with Flowjo™ v10 software.

Immunocytology

Handpicked pancreatic islets were seeded on SuperFrost Gold Plus microscope slide. For βdefensin staining, islets were fixed, blocked with goat serum, permeabilized, and stained with anti-insulin pAb (Abcam, ab7842), anti-glucagon mAb (Abcam, ab10988) and anti-mBD14 pAb (Mybiosource, MBS1490249) or anti-hBD3 pAb (Abcam, ab19270), overnight at 4°C.

After washing, second-step reagents were applied: anti-guinea pig-AlexaFluor488, anti-mouse-Alexa647 and anti-rabbit-AlexaFluor555 pAbs (Invitrogen). For IL-22 staining, isolated islets were incubated 5 h at 37°C with rIL-23 (10 ng.ml-1, Miltenyi) and PMA/ionomycin (both 500 ng.ml -1 , Sigma-Aldrich) in the presence of a protein transport inhibitor cocktail. Then islets were fixed, blocked with donkey serum, permeabilized and stained with anti-insulin pAb (Abcam), anti-CD5 mAb (Biolegend, 53-7.3) and anti-IL-22 mAb (Biolegend, poly5164), overnight at 4°C. After washing, second-step reagents were applied: anti-guinea pig-AlexaFluor488, anti-rat-Alexa555 and anti-goat-AlexaFluor647 pAbs (Invitrogen). Nuclei were stained with DAPI. Image acquisition was performed on Necker Institute Imaging Facility using a Leica SP8 confocal microscope.

In vitro B cell culture B cells were magnetically isolated using MACS cell separation system (anti-biotin beads, Miltenyi). CD19 + B cells (2x10 5 cells per well) from pancreatic lymph nodes or pancreatic islets of 10-weeks-old female WT or myd88 -/-NOD mice were incubated for 4 days in complete IMDM with growing doses (0.1 to 10 µg.ml -1 ) of mBD14 or vehicle. Blocking reagents were added as indicated: neutralizing anti-TLR2 mAb (5 µg.ml -1 , Invivogen), TLR1/2 heterodimer antagonist CU-CTP22 (10 µM, Tocris).

In vitro Treg cell induction

All cells were magnetically isolated using MACS cell separation kit and system (Miltenyi).

CD62L + CD4 + CD25 -BDC2.5 T cells (4x10 4 cells per well) from splenocytes of BDC2.5 TCR transgenic NOD mice were incubated with 2x10 3 myeloid cells (CD11b + ) and or 2x10 3 B cells (CD19 + ) obtained from pancreatic islets of NOD mice treated with mBD14 (10 µg.ml -1 ) or vehicle 4 days earlier. Cell culture was performed for 4 days in complete IMDM with 20 ng.ml -1 of peptide 1040-51, a mimotope of BDC2.5 T cells.

In vitro pancreatic islet culture

Mouse and human (Tebu-bio) pancreatic islets isolated by handpicking were cultured in complete IMDM (10% FBS, 0.6 g.l -1 L-glutamine, 100 U.ml -1 penicillin streptomycin, 100 µM 2-mercaptoethanol, 4.5 g.l -1 D-Glucose). In some experiments, islets were treated with: rmIL-22 (100 ng.ml -1 , Biolegend) for 4 h; FICZ (50 ng.ml -1 , Tocris) for 12 h; rmIL-23 (20 ng.ml -1 , Miltenyi) for 18 h; sodium butyrate (1 mg.ml -1 , Sigma-Aldrich) for 48 h with or without neutralizing anti-IL-22 mAb (1 µg.ml -1 , IL22JOP, eBioscience).

Cytokine quantification

The level of IL-23 and IL-22 in the serum was determined using commercial ELISA kit (Biolegend).

RNAseq gene expression profiling

Total RNA were isolated from pancreatic islets (>150) using the RNeasy Kit (QIAGEN) including a DNAse treatment step. RNA quality was assessed using RNA Screen Tape 6000

Pico LabChips with the Tape Station (Agilent Technologies) and RNA concentration was measured by spectrophometry using the Xpose (Trinean). RNAseq libraries were prepared starting from 1 µg of total RNA using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) as recommended by the manufacturer. Half of the oriented cDNA produced from the poly-A + fraction was PCR amplified (11 cycles). The RNAseq libraries were sequenced on an Illumina HiSeq2500 (Paired-End sequencing 130x130 bases, High Throughput Mode). A mean of 23 million of paired-end reads was produced per library sample (between 21 to 25 million of passing filter reads). The generated data were analyzed using the Ingenuity Pathway Analysis software (Qiagen).

AHR ligand activity measurement

The BioDetection Systems Company performed experiments to determine AHR ligand activity in the serum using a reporter assay system (PAH-CALUX bioassay) [START_REF] Pieterse | PAH-CALUX, an optimized bioassay for AhR-mediated hazard identification of polycyclic aromatic hydrocarbons (PAHs) as individual compounds and in complex mixtures[END_REF].

AHR activity in stool samples was determined in the laboratory using the H1L1.1c2 cell line, containing a stably integrated dioxin-response element (DRE)-driven firefly luciferase reporter plasmid pGudLuc1.1, as described previously [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF][START_REF] Zhao | Common commercial and consumer products contain activators of the aryl hydrocarbon (dioxin) receptor[END_REF].

Analysis of AHR ligand composition

The concentrations of Indole-3-Lactic acid, Indole-3-aldehyde, Indole-3-Acetic acid in the serum and the caecal content of mice were determined by a specific method using HPLCcoupled to high resolution mass spectrometry. This quantitative method has been validated analytically on both types of samples in order to meet the criteria FDA Guidance for Industry.

16S rDNA gene sequencing

Murine stools were frozen at -80°C immediately after emission and stored until further processing. Then, as previously described [START_REF] Lamas | CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands[END_REF], feces samples were weighed and then resuspended for 10 min at room temperature in 250 μl of 4 M guanidine thiocyanate in 0.1 M Tris (pH 7.5) (Sigma-Aldrich) and 40 μl of 10% N-lauroyl sarcosine (Sigma-Aldrich).

The RNA-seq data reported in this paper is published in Mendeley Data (http://dx.doi.org/10.17632/4g2jdjxcff.1).

  REAGENT AND RESOURCE SHARING • EXPERIMENTAL MODEL DETAILS o Mice and treatments o Spontaneous diabetes incidence • METHOD DETAILS o Preparation of pancreatic islets o RT-qPCR o Flow cytometry o Immunocytology o In vitro B cell culture o In vitro Treg cell induction o In vitro pancreatic islet culture o Cytokine quantification o RNAseq gene expression profiling o AHR ligand activity measurement o Analysis of AHR ligand composition o 16S rDNA gene sequencing • QUANTIFICATION AND STATISTICAL ANALYSIS o 16S rDNA gene sequence analysis o Statistical analysis • DATA AND SOFTWARE AVAILABILITY SUPPLEMENTAL INFORMATION Supplemental Information includes eleven figures.

Figure legends Figure 1

 legends1 Figure legends

Figure 2

 2 Figure 2 MBD14 induces pancreatic regulatory B cells via TLR2. (A, B and D, E) NOD mice were treated with mBD14 or vehicle (d-3). B cells (CD19 + CD11b -) from islets, lymph nodes

Figure 3

 3 Figure 3 MBD14 induces pancreatic regulatory macrophages and Treg cells. (A) Macrophage

Figure 4

 4 Figure 4 IL-22 stimulates pancreatic mBD14 expression and is produced by pancreatic innate

Figure 5

 5 Figure 5 Gut microbiota controls pancreatic mBD14 expression. (A-C) Six-weeks-old BALB/c

Figure 6

 6 Figure 6 AHR ligands stimulates pancreatic mBD14 expression. (A, B) Expression of Cyp1a1

Figure 7

 7 Figure 7 Butyrate induces IL-23 expression in the pancreas stimulating mBD14 expression.

  and provided after endotoxin removal processing. Peptides were administrated intraperitoneally (i.p.) at the dose of 10 µg diluted in 200 µl of vehicle (PBS-1% H2O). For in vivo blocking experiments, mice were treated (200 µg per i.p. injection at day -1, 1 and 3 post mBD14 treatment) with anti-IL4 (11B11) or anti-TGFβ (1D11) mAbs (BioXcell) or relevant isotype controls. For in vivo depleting experiments, mice were treated (200 µg per i.p. injection at day 0 and 1 post mBD14 treatment) with anti-CD20 (SA271G2) mAb (Biolegend) or relevant isotype controls. In some experiments, mice were i.p. injected with: rmIL-22Fc (Adipogen), 200 µg in 200µl PBS for 3 h; rmIL-23 (Biolegend), 2 µg in 200µl PBS for 2 h; 6-Formylindolo-[3,2-b]-carbazole (FICZ, Tocris) 2 µg in 200 µl PBS for indicated time;

  Figure 1 A NOD

  Figure 5

E

  treated mice. Solid lines represent direct interactions and dashed lines indirect interactions. (C-H) Analysis of B cell population after mBD14 treatment in female NOD mice. (C) Expression of chemokine mRNAs was analyzed by RT-qPCR in pancreatic islets from NOD mice treated with mBD14 or vehicle (d-4). Data are the median +/-interquartile range of 5 independent mice per group. (D, E) NOD mice were treated with mBD14 (10 µg/mouse in E or growing dose in D) or vehicle (d-3). B cells (CD19 + CD11b -) from pancreatic islets (D), lymph nodes and spleen (E) were analyzed by flow cytometry. Cells were incubated for 5 h in the presence of a protein transport inhibitor cocktail before staining. Results show the frequency of positive cells or the MFI of IL-4 among B cell population as indicated. Data are representative (E) or are the median +/-interquartile range (D) of 5 independent mice per group. (F-H) B cells (CD45 + CD19 + CD11b -) from the pancreatic islets of mBD14-treated NOD mice were analyzed by flow cytometry for surface expression of the indicated markers. Data are representative (F, G) or are the median +/-interquartile range (H) of 6 independent mice.

Figure S3 .

 S3 Figure S3. Related to Figure 3. Effect of mBD14 on macrophages and dendritic cells and on Treg cell induction. (A) Expression of arginase I and Ym1/Ym2 mRNAs was analyzed by RT-qPCR in pancreatic macrophages magnetically isolated (F4/80 + ) from NOD mice treated with mBD14 or vehicle (d-4). Data are the median +/-interquartile range of 5 independent mice per group. (B) Bone-marrow derived macrophages were differentiated in presence of M-CSF during 8 days and then treated with vehicle or mBD14 at the indicated dose for 24 h. Then cells were stained for LAP expression on CD11b + F4/80 + cells. Data are representative and are the median +/-interquartile range of 6 independent experiments. (C) Cytokine intracellular expression in pancreatic dendritic cells was analyzed in NOD mice treated with mBD14 or vehicle (d-4). Cells were incubated for 5 h in the presence of LPS and a protein transport inhibitor cocktail before staining. Results show the MFI of indicated marker among CD11b +

Figure S5 .

 S5 Figure S5. Related to Figure 4. (A) Pancreatic islets from C57BL/6 WT or IL22 -/-female mice were cultured for 5 h in presence of rmIL23 plus PMA/ionomycin in presence of a protein transport inhibitor cocktail and stained for insulin (green), CD5 (red), IL-22 (blue) and DNA (grey). Data are representative of 3 independent experiments. (B-E) mRNA expression of IL22 and mBD14 was determined in pancreatic islets (B, C) and ileum (D, E) from C57BL/6 Rag -/- female mice treated with rmIL-23 (2 µg, i.p., h-2). Data are the median +/-interquartile range of 6 independent mice per group.

Figure S6 .

 S6 Figure S6. Related to Figure 4. Analysis of innate lymphoid cells in lymphoid tissues and small intestine. (A-B) Innate lymphoid cells (ILCs) were analyzed in the pancreatic (A) and mesenteric (B) lymph nodes from the indicated mouse strains. Data show the frequency of ILCs (CD127 + CD90 + Lin (CD5, CD19, CD11b, CD11c) -) among CD45 + immune cells and the

ACKNOWLEDGMENTS

The authors greatly acknowledge Valérie Gaboriau-Routhiau (Inserm U1163, France), Marie Cherrier (Inserm U1151, France), Gaetan Barbet (Cornell University, USA) and Nicolas Serafini (Inserm U668, France) for fruitful discussions. The authors acknowledge Antoine Lefevre from the PST facility "Analyse des Systèmes Biologiques" (Tours, France). The authors acknowledge the SFR Necker facilities including Meriem Garfa-Traoré and Nicolas Goudin of the Imaging Facility, Christine Bole of the genomics facility, Nicolas Cagnard of the

AUTHOR CONTRIBUTIONS

MM designed, performed and analyzed experiments, with general assistance of JL, EWE and SV for confocal microscopy, histology and cell culture. BR provided the IL22 -/-C57BL/6 mice. PE performed the mass spectrometry experiments. MS and HS performed gut microbiota analysis and AHR ligand analysis. PvE and HS provide intellectual inputs. JD designed the project, performed experiments, interpreted data and wrote the paper.

DECLARATION OF INTERESTS

The authors declare no competing interests. After the addition of 500 μl of 5% N-lauroyl sarcosine in 0.1 M phosphate buffer (pH 8.0), the 2-ml tubes were incubated at 70 °C for 1 h. One volume (750 ml) of a mixture of 0.1-and 0.6mm-diameter silica beads (Sigma-Aldrich) (previously sterilized by autoclaving) was added, and the tube was shaken at 6.5 m/s three times for 30 s each in a FastPrep (MP Biomedicals) apparatus. Polyvinylpolypyrrolidone (15 mg) was added to the tube, which was then vortexed and centrifuged for 5 min at 20,000g. After recovery of the supernatant, the pellets were washed with 500 μl of TENP (50 mM Tris (pH 8), 20 mM EDTA (pH 8), 100 mM NaCl, 1% polyvinylpolypyrrolidone) and centrifuged for 5 min at 20,000g, and the new supernatant was added to the first supernatant. The washing step was repeated two times. The pooled supernatant (about 2 ml) was briefly centrifuged to remove particles and then split into two 2-ml tubes.

STAR METHODS

KEY RESOURCES TABLE

DNA was isolated from the feces of the various mouse strains using the Nucleospin DNA stool (Macherey-Nagel). Microbial diversity was determined for each sample by targeting a portion of the ribosomal genes. A 16S rDNA gene fragment comprising the V3 and V4 hypervariable regions (16S (sense) 5′-TACGGRAGGCAGCAG-3′ and (antisense) 5′-CTACCNGGGTATCTAAT-3′) was amplified using an optimized and standardized 16Samplicon-library preparation protocol (Metabiote, GenoScreen). Briefly, 16S rDNA gene PCR was performed using 5 ng genomic DNA according to the manufacturer's protocol (Metabiote) using 192 bar-coded primers (Metabiote MiSeq Primers) at final concentrations of 0.2 μM and an annealing temperature of 50 °C for 30 cycles. The PCR products were purified using an Agencourt AMPure XP-PCR Purification system (Beckman Coulter), quantified according to the manufacturer's protocol, and multiplexed at equal concentrations. Sequencing was performed using a 300-bp paired-end sequencing protocol on an Illumina MiSeq platform (Illumina) at GenoScreen. Raw paired-end reads were subjected to the following process: (1) quality-filtering using the PRINSEQ-lite PERL script38 by truncating the bases from the 3′ end that did not exhibit a quality <30 based on the Phred algorithm;

(2) paired-end read assembly using FLASH39 (fast length adjustment of short reads to improve genome assemblies) with a minimum overlap of 30 bases and a 97% overlap identity; and (3) searching and removing both forward and reverse primer sequences using CutAdapt, with no mismatches allowed in the primers sequences. Assembled sequences for which perfect forward and reverse primers were not found were eliminated.

QUANTIFICATION AND STATISTICAL ANALYSIS 16S rDNA gene sequence analysis

The sequences were demultiplexed, quality-filtered using the 'quantitative insights into microbial ecology' (QIIME, version 1.8.0) software package [START_REF] Caporaso | QIIME allows analysis of high-throughput community sequencing data[END_REF], and the forward and reverse Illumina reads were joined using the fastq-join method (http://code.google.com/p/ea-utils). The sequences were assigned to OTUs using the UCLUST algorithm [START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF] with a 97% threshold of pairwise identity and classified taxonomically using the Greengenes reference database [START_REF] Mcdonald | An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea[END_REF]. Rarefaction was performed (30,000 sequences per sample) and used to compare the abundances of OTUs across samples.

Statistical analysis

Diabetes incidence was plotted according to the Kaplan-Meier method. Incidences between each group were compared with the log-rank test. Reported values are median +/-interquartile range as indicated. Comparison between each group was performed using the non-parametric Mann-Whitney U-test. P values < 0.05 were considered statistically significant. All data were analyzed using GraphPad Prism v6 software.

DATA AND SOFTWARE AVAILABILITY