Mice used in this study were housed in specific pathogen-free (SPF) facilities with a 12-hour-dark/light cycle at Yale University. NOD mice, BDC2.5 ⁺ NOD mice, Il-10 ^-/- NOD mice and NOD.scid mice were originally obtained from the Jackson Laboratory and maintained at Yale University. BDC2.5 ⁺ Il-10 ^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were generated by breeding BDC2.5 ⁺ NOD mice with Il-10 ^-/- NOD mice, followed by intercrossing the BDC2.5 ⁺ Il-10 ^+/- NOD mice. The use of animals in this study was approved by the Institutional Animal Care and Use Committee of Yale University (approval number 2016-07911). Except for the experiments to observe diabetes incidence and adoptive transfer experiments using splenocytes from diabetic mice, all the other experiments were performed using 4-5 week-old non-diabetic mice. The detail of the number of animals used and the number of replicates are included in the figure legends for each experiment and also see Table S1 .

BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice (both sexes) were observed for spontaneous diabetes development by screening for glycosuria (Bayer Diastix) weekly and diabetes onset was confirmed by blood glucose ≥ 250 mg/dl (13.9 mmol/l).

Pancreata removed from 4-week-old BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were agitated in a 37°C shaking water bath after addition of 1.5 mg/ml collagenase (Sigma) and 62.5 units/ml DNase-I (Sigma). Collagenase activity was stopped by adding complete RPMI-1640 media after digestion. Islets were hand-picked under a light microscope, and subsequently incubated in a 37°C water bath for 6 minutes in the presence of 500 μl Cell Dissociation Buffer (Gibco), for immune cell isolation. After washing, isolated immune cells were filtered and re-suspended in complete RPMI-1640 media before staining.

BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were fasted overnight prior to intraperitoneal injection with 20% glucose (2 g/kg). Blood glucose was measured before glucose injection and at different time points after glucose injection.

CD4⁺ T cells, antigen-presenting cells, Treg cells and neutrophils were each purified from the splenocytes of 4-week-old BDC2.5⁺Il-10^+/+ NOD mice, BDC2.5 ⁺ Il-10 ^-/- NOD mice, or wild type NOD mice. Splenic CD4⁺ T cells were purified by depletion, of CD8⁺ T cells (clone T1B105), MHC class II⁺ cells (clone 10.2.16), and B cells (anti-mouse IgM and IgG), incubating the cells with monoclonal antibody (mAb) hybridoma supernatants, followed by magnetic bead separation. For splenic antigen-presenting cell (APC) isolation, anti-Thy1 (Y19) mAb hybridoma supernatant and complement was used to remove Thy1⁺ T cells. The supernatants of different mAb hybridomas were kindly provided by the late Charles Janeway (Yale University). Magnetic beads conjugated with goat anti-mouse IgG, goat anti-mouse IgM, or goat anti-rat IgG were purchased from QIAGEN. Treg cells were isolated using MojoSort™ Mouse CD4⁺ T cell Isolation Kit (BioLegend), followed by CD25 positive isolation using a PE-anti-mouse CD25 antibody (Clone, PC61, BioLegend) and MojoSort™ Mouse anti-PE Nanobeads (BioLegend). The remaining CD4⁺CD25^- T cells were used as effector CD4⁺ T cells. Neutrophils were isolated according to the manufacturer’s instructions using MojoSort™ Mouse Neutrophil Isolation kit (BioLegend).

Splenic CD4⁺ T cells (1 × 10⁵/well) purified from 4-week-old BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were stimulated with different concentrations of mimotope peptide in the presence of mitomycin-c-treated APCs for 3 days at 37°C. CD4⁺CD25⁺ Tregs (5 × 10⁴/well) purified from 4-week-old BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD were co-cultured with effector CD4⁺ T cells (CD4⁺CD25^- T cells, 1 × 10⁵/well) from either BDC2.5⁺Il-10^+/+ NOD mice or BDC2.5 ⁺ Il-10 ^-/- NOD mice, in the presence of 5 ng/ml mimotope peptide (amino acid sequence RTRPLWVRME) and mitomycin-c-treated APCs (5 × 10⁴/well), which were purified from wild-type NOD mice. Cells were incubated for 3 days at 37°C. Purified splenic neutrophils (5 × 10⁴/well) from BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were co-cultured with purified splenic CD4⁺ T cells (1 × 10⁵/well) isolated from either BDC2.5⁺Il-10^+/+ NOD mice or BDC2.5 ⁺ Il-10 ^-/- NOD mice in the presence of mitomycin-treated APCs (5 × 10⁴/well) from wild type NOD mice and mimotope peptide (5 ng/ml). Cell proliferation was determined by ³H-thymidine incorporation over 16 hours, with supernatants collected prior to ³H-thymidine addition.

Total splenocytes (10 × 10⁶) from diabetic BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were injected (i.v.) into irradiated 4-week-old wild-type NOD mice. Total splenocytes (10 × 10⁶), or purified splenic CD4⁺ T cells (7 × 10⁶) from non-diabetic BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were transferred (i.v.) into 4-week-old NOD.scid mice. Recipients were monitored for glycosuria (Bayer Diastix) weekly and diabetes was confirmed by blood glucose ≥ 250 mg/dl (13.9 mmol/l).

Fecal pellets collected from 4-week-old BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were resuspended in 300 μl Tris-EDTA buffer (10 mM Tris and 1 mM EDTA, pH8) containing 7.5 μl 0.5% SDS and 3 μl Proteinase K (200 μg/ml). The samples were then incubated at 37°C for 1 h and homogenized in solution, containing one volume of phenol/chloroform/isoamyl alcohol (25:24:1), 200 μl 20% SDS and 0.3 g zirconium silica beads, with a mini-bead-beater (BioSpec) for 2 minutes. Phenol/chloroform/isoamyl alcohol was then added to the samples prior to centrifugation (4°C, 12000 g, 15 mins), with the upper aqueous layer, containing DNA, transferred to a new tube. Bacterial DNA was subsequently precipitated with isopropanol, washed with 70% ethanol, air-dried, and resuspended in 100 μl of sterile water.

The V4 region of the bacterial 16S rRNA gene was amplified from each DNA sample by PCR using barcoded broadly-conserved primer pairs (5’-GTGCCAGCMGCCGCGGTAA-3’) and (5’-GGACTACHVGGGTWTCTAAT-3’). The PCR products were purified using gel extraction kits (QIAGEN) with DNA concentration quantified on a Nanodrop spectrophotometer. Equimolar amounts of each sample were pooled for pyrosequencing using the Ion Torrent Personal Genome Machine (PGM) sequencing system (Life Technologies). The sequencing results were analyzed with the Quantitative Insights Into Microbial Ecology (QIIME) software package (version 1.8) and UPARSE pipeline (version 7.0). β-diversity was analyzed to compare differences between microbial communities, and the data are shown as a Principal Coordinate Analysis (PCoA). Taxonomy assignment was performed at various levels using representative sequences of each operational taxonomic unit (OTU).

For depletion of endogenous commensal microbiota, BDC2.5 ⁺ Il-10 ^+/- NOD breeders were treated with an antibiotic cocktail containing 0.5 g/l vancomycin, 1 g/l ampicillin, 1 g/l metronidazole, and 1 g/l neomycin added in drinking water, from one week before delivery to 4 weeks after birth.

Murine IL-17A, IL-10 and IFN-γ from the serum and gut flush from different sections of the intestine were measured using the Mouse ELISA kits (BioLegend), following the manufacturer’s instructions. Serum samples were diluted 1:100 in PBS before measurement. Gut flush was obtained by infusing 10 ml PBS to the gut lumen, after termination of the mice, and removal of the intestine. The collected fluid used for ELISA, after removing the solid material by centrifugation (12,000 g, 5 min, RT).

Four-week-old BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were fasted overnight for 13 hours. Food was resupplied to the mice two hours post-gavage with 0.6 mg/g FITC-dextran (MW 3,000-5,000, Sigma). Blood samples were collected from the mice two hours after food restoration and were centrifuged (2300 g, 5 min, RT) for serum separation. Serum samples were diluted 1:1 in PBS with the FITC-dextran concentration determined using a fluorescence spectrophotometer (Perkin Elmer). Standard curves were generated using known concentrations of FITC-dextran, diluted in serum from untreated NOD mice. The concentrations in serum from FITC-dextran gavaged BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were determined using linear regression.

Fresh stool samples, collected from 4-week-old BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice, were resuspended at 1 g/mL in sterile PBS, and homogenized by vortexing vigorously for 30 secs. The samples were then centrifuged (300 g, 1 min, RT) to remove large debris and subsequently further centrifuged at 12,000 g for 5 minutes to pellet the bacteria. Bacteria from BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice were re-suspended in sterile PBS, heat-inactivated at 95°C for 30 min, and co-cultured overnight (10⁸ CFU) with 2 million total splenocytes or purified splenic neutrophils from BDC2.5 ⁺ Il-10 ^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice. Stimulated splenocytes or neutrophils were further analyzed by flow cytometry and real-time qPCR.

RNA from purified neutrophils (un-stimulated or stimulated with gut microbiota) or small intestinal tissue was extracted using Trizol reagent and an RNeasy mini plus kit (QIAGEN). After quantification, RNA was used for cDNA synthesis using the iScript cDNA synthesis kit (Invitrogen). Samples were analyzed on an iCycler qPCR machine (Bio-rad). Gene expression level was determined using the 2^−ΔΔCt method and normalized with the housekeeping gene, Gapdh. Primers sequences are listed in Table 1 . Each sample was assayed in duplicate and the experiments were repeated at least twice.

0.5 - 1 × 10⁶ single-cell suspensions from different lymphoid tissues were incubated with Fc block at room temperature for 20 min, before cell surface staining. For intracellular cytokine staining, cells were incubated at 37°C for 4 h in the presence of 10 ng/ml phorbol myristate acetate (PMA, Sigma), 500 ng/ml of ionomycin (Sigma) and 1 μl of Golgi plug™ (BD Bioscience), followed by cell surface staining, washing, fixation and permeabilization and intracellular cytokine staining. For intranuclear staining, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (Tonbo Biosciences). After incubation, the cells were stained with an anti-Foxp3 antibody (clone: FJK-16s, eBioscience) and T-bet (clone:4B10, BioLegend). Cells were stained with mAbs to the following surface markers: CD45 (clone: 30-F11), TCR-β (clone: H57-597), CD4 (clone: GK1.5), CD8 (clone: 53-6.7), CD11b (clone: M1/70), CD62L (clone: MEL-14), ICOS (clone: 15F9), CD69 (clone: H1.2F3), CD25 (clone: 3C7), CD19 (clone: 6D5), all from BioLegend, and Ly6G (clone: 1A8) from BD Biosciences. mAbs to intracellular cytokines include TNF-α (clone: MP6-XT22), IFN-γ (clone: XMG1.2) and IL-17A (clone: TC11-18H10.1) were purchased from Biolegend. Samples were analyzed on a BD LSRII flow cytometer and subsequently analyzed by FlowJo 8.8.6 software (Tree star). Immune cells were gated based on their FSC-A/SSC-A properties. Single cells and subsequent live cells were gated on their FSC-A/FSC-H properties and live/dead staining, respectively.

Diabetes incidence was compared using a log-rank test for survival. Insulitis scores were analyzed using a Chi-square test. Statistical analysis of microbial β-diversity was conducted using an analysis of similarities (ANOSIM). Differences between microbial species were determined following analysis using multiple t-tests with Bonferroni correction. Data from experiments in vitro were assessed for normality and subsequently analyzed using a two-tailed Student’s t test (if data were normally distributed), a two-tailed Mann-Whitney test (if data were not normally distributed), or a two-way ANOVA. P < 0.05 was considered statistically significant.

To assess the role of IL-10 in diabetes protection observed in BDC2.5⁺ NOD mice, we first monitored the natural history of type 1 diabetes development in BDC2.5⁺Il-10^+/+ NOD mice and BDC2.5 ⁺ Il-10 ^-/- NOD mice. Interestingly, both female and male BDC2.5 ⁺ Il-10 ^-/- NOD mice developed accelerated diabetes at a very young age ( Figures 1A, B ). In line with islet β cell destruction, BDC2.5 ⁺ Il-10 ^-/- NOD mice displayed impaired glucose tolerance compared with BDC2.5⁺Il-10^+/+ NOD mice ( Figure 1C ). We found no significant difference in severity of insulitis between BDC2.5⁺Il-10^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figures S1A, B ); however, BDC2.5 ⁺ Il-10 ^-/- NOD mice had more CD45⁺ immune cells, especially CD11b⁺Ly6G⁺ neutrophils, infiltrating the islets than BDC2.5 ⁺ Il-10 ^+/+ NOD mice, but no significant differences in the proportion of CD4⁺ T cells, CD8⁺ T cells, B cells, macrophages, and dendritic cells ( Figure 1D and data not shown). Our data showed that in the absence of IL-10, BDC2.5⁺ NOD mice develop accelerated diabetes at a very young age.

We observed an increased frequency of neutrophils infiltrating the islets of BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figure 1D ). To investigate the crosstalk between IL-10 and neutrophils, we assessed the neutrophil population in both the bone marrow and peripheral tissues of BDC2.5⁺Il-10^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice. Consistent with the results from the islets, we also found a higher percentage of neutrophils in the spleen, bone marrow and blood of BDC2.5 ⁺ Il-10 ^-/- NOD mice, compared to BDC2.5⁺Il-10^+/+ NOD mice ( Figures 2A–E and S2A, B ). Moreover, there was a higher proportion of IFN-γ-producing splenic neutrophils in BDC2.5⁺Il-10^-/- NOD mice compared with BDC2.5 ⁺ Il-10 ^+/+ NOD mice ( Figures 2F, G ). The expression levels of cytokines in the neutrophils from the blood and islets in BDC2.5⁺Il-10^+/+ NOD mice were the same as those in BDC2.5⁺Il-10^-/- NOD mice ( Figures S3A–G ). Additionally, we found that neutrophils from the islets of the BDC2.5⁺Il-10^+/+ NOD mice had lower levels of CD62L ( Figure 2H ). We then purified neutrophils and found no significant differences in the gene expression of Inos, Nos2, Arg1, and Caspase9 in the spleen ( Figures S4A–D ); however, we found increased Nos2 and Il-1β in purified neutrophils from the bone marrow of BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figures 2I, J ), but no significant differences in the expressions of Inos, Arg1, Caspase 9, and Mmp9 ( Figures S4E–H ). Next, we assessed the phagocytotic ability of neutrophils and found that neutrophils from BDC2.5 ⁺ Il-10 ^-/- NOD mice were not different from those from BDC2.5⁺Il-10^+/+ NOD mice ( Figures S5A, B ). Taken together, our data suggested that Il-10 deficiency altered neutrophil homeostasis in BDC2.5 ⁺ NOD mice.

Next, we assessed the phenotype and function of different T cell populations in BDC2.5 ⁺ Il-10 ^-/- NOD mice. Interestingly, unlike the composition of the immune cells seen in the islet infiltrate ( Figure 1D ), BDC2.5 ⁺ Il-10 ^-/- NOD mice had a higher proportion of splenic CD4⁺ T cells when compared with BDC2.5⁺Il-10^+/+ NOD mice ( Figure 3A ), while no significant difference was found in the percentage of CD8⁺ T cells ( Figure 3A ). The proportions of both CD4⁺ and CD8⁺ T cells were increased in the pancreatic lymph node (PLN) of BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figure 3B ). We found that splenic CD4⁺ T cells from BDC2.5 ⁺ Il-10 ^-/- mice expressed a higher proportion of ICOS, T-bet and CD69 ( Figure 3C ) while splenic CD8⁺ T cells expressed higher level of T-bet only ( Figure 3D ). Moreover, splenic CD4⁺ T cells from BDC2.5 ⁺ Il-10 ^-/- mice showed increased expressions of inflammatory cytokines including IFN-γ and IL-17A in the spleen ( Figure 3E ) and more TNF-α in CD4⁺ T cells from PLN ( Figure 3F ). Additionally, splenic CD8⁺ T cells in BDC2.5 ⁺ Il-10 ^-/- NOD mice expressed more TNF-α ( Figure 3G ). Interestingly, splenic CD4⁺ T cells from BDC2.5 ⁺ Il-10 ^-/- NOD mice showed stronger proliferative responses to their antigenic peptide (mimotope) compared to CD4⁺ T cells from BDC2.5⁺Il-10^+/+ splenocytes ( Figure 3H ). No significant differences were found in the proportion of CD8⁺ T cells expressing IFN-γ, TNF-α, or IL-17A in the PLN between BDC2.5 ⁺ Il-10^+/+ and BDC2.5⁺Il-10 ^-/- NOD mice ( Figures S6A–C ). To determine the effect of IL-10 on diabetogenicity of the T cells from BDC2.5⁺Il-10 ^-/- NOD mice in vivo, we adoptively transferred total splenocytes from diabetic BDC2.5⁺Il-10 ^-/- or BDC2.5 ⁺ Il-10^+/+ NOD mice into 4-week-old irradiated wild-type female NOD mice. Supporting the in vitro data, splenocytes from diabetic BDC2.5 ⁺ Il-10 ^-/- NOD mice showed more potent diabetogenicity by inducing a higher incidence of diabetes in the recipients, compared with the splenocytes from BDC2.5⁺Il-10^+/+ NOD mice ( Figure 3I ). Moreover, when we transferred total splenocytes from non-diabetic BDC2.5 ⁺ Il-10 ^-/- or BDC2.5⁺Il-10^+/+ NOD mice into NOD.scid mice, splenocytes from BDC2.5 ⁺ Il-10 ^-/- mice induced rapid diabetes in the NOD.scid recipients as early as 3-days after transfer ( Figure 3J ). We further confirmed the enhanced diabetogenicity by BDC2.5 CD4⁺ T cells by adoptive transfer with purified splenic CD4⁺ T cells from non-diabetic BDC2.5 ⁺ Il-10 ^-/- or BDC2.5⁺Il-10^+/+ NOD mice into NOD.scid recipients. Similar to the adoptive transfer with total splenocytes, CD4⁺ T cells from BDC2.5 ⁺ Il-10 ^-/- NOD mice induced rapid onset of diabetes compared to their counterparts from BDC2.5⁺Il-10^+/+ NOD mice ( Figure 3K ). To determine the effects of neutrophils on CD4⁺ T cells, we performed criss-cross co-culture in which purified splenic neutrophils from BDC2.5 ⁺ Il-10 ^+/+ or BDC2.5 ⁺ Il-10 ^-/- NOD mice were cultured with purified splenic CD4⁺ T cells from BDC2.5 ⁺ Il-10 ^-/- or BDC2.5 ⁺ Il-10 ^+/+ NOD mice. Interestingly, neutrophils from both BDC2.5 ⁺ Il-10 ^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice effectively inhibited the proliferation of CD4⁺ T cells from BDC2.5 ⁺ Il-10 ^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice when stimulated by antigenic peptide ( Figures S7A, B ). However, there was no significant difference in the inhibitory effect of neutrophils from BDC2.5 ⁺ Il-10 ^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figures S7A, B ). When we transferred purified splenic CD4⁺ T cells alone or purified splenic neutrophils together with CD4⁺ T cells from BDC2.5 ⁺ Il-10 ^-/- NOD mice into Rag ^-/- NOD mice, no significant difference in the diabetes incidence was found, indicating that the altered neutrophils in BDC2.5 ⁺ Il-10 ^-/- NOD mice may contribute to the disease development, but not by direct effects on CD4⁺ T cells ( Figure S7C ). We examined the Treg cells in BDC2.5 ⁺ Il-10 ^-/- NOD mice. Surprisingly, there were no significant differences in both the proportion and the suppressive function of Treg cells between BDC2.5⁺Il-10^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figures S8A–C ). Taken together, our data showed that in the absence of Il-10, CD4⁺ T cells in BDC2.5 ⁺ NOD mice were more activated and highly pathogenic. However, Tregs did not appear to be directly affected by the absence of Il-10.

To study whether Il-10 deficiency also alters the gut bacteria of TCR transgenic BDC2.5 ⁺ NOD hosts, we investigated the gut bacterial composition of BDC2.5 ⁺ Il-10 ^+/+ and BDC2.5 ⁺ Il-10 ^-/- NOD mice. Il-10-deficient BDC2.5 ⁺ NOD mice had an altered composition of gut bacteria, indicated by β-diversity of the gut microbiota shown in the Principal Coordinate Analysis (PCOA; Figure 4A ), compared to Il-10-sufficient BDC2.5 ⁺ NOD mice. Further analysis also revealed differences at the phylum, class, and species levels. At the phylum level, BDC2.5 ⁺ Il-10 ^-/- NOD mice had increased levels of Proteobacteria compared with BDC2.5 ⁺ Il-10 ^+/+ NOD mice ( Figures 4B, C ), with increased abundance of Epsilonproteobacteria and Gammaproteobacteria ( Figure 4D ). Moreover, the relative abundance of Escherichia from Proteobacteria was significantly different between the two groups ( Figure 4E ). We also assessed gut permeability in vivo to determine whether the altered gut bacteria affected the gut barrier in Il-10-deficient BDC2.5 ⁺ NOD mice. Indeed, BDC2.5 ⁺ Il-10 ^-/- NOD mice exhibited increased gut permeability compared to BDC2.5 ⁺ Il-10 ^+/+ NOD mice as demonstrated by the increased concentration of serum FITC-dextran ( Figure 5A ). We found decreased expression of the intestinal tight junction protein Zonulin1 in BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figure 5B ), but not Claudin2 or Occludin (data not shown). Moreover, when we assessed the inflammatory cytokines in the gut lumen, we found an increased level of IL-17A ( Figure 5C ) in BDC2.5 ⁺ Il-10 ^-/- NOD mice. Additionally, the intestinal expression of Myd88, which plays an essential role in the activation of innate immunity, was also increased in BDC2.5 ⁺ Il-10 ^-/- NOD mice when compared with that in BDC2.5 ⁺ Il-10 ^+/+ NOD mice ( Figure 5D ). Interestingly, BDC2.5 ⁺ Il-10 ^-/- NOD mice showed increased expression of intestinal antimicrobial peptide genes, including Crp, Defcr6, Reg3γ, and Reg3β ( Figures 5E–H ), which could be induced to defend against the altered bacteria in the gut. Additionally, we found that the percentages of TCR-γδ⁺ cells and NKP46⁺ (one of the ILC markers) cells decreased in BDC2.5 ⁺ Il-10 ^-/- NOD mice, but no significant differences in CD11c⁺ cells, IFN-γ⁺CD4⁺ (Th1) cells, IL-17⁺CD4⁺ (Th17) cells, and CD117⁺ (one of the ILC markers) cells between the two groups ( Figures S9A–F ). Taken together, Il-10 deficiency in BDC2.5 ⁺ NOD mice not only altered the gut bacterial composition, but also changed the intestinal immune responses of the host.

To determine whether the altered gut bacteria in BDC2.5 ⁺ Il-10 ^-/- NOD mice contribute to the increased frequency of neutrophils, we first depleted the endogenous commensal bacteria of BDC2.5 ⁺ Il-10 ^+/- NOD breeders by treating the mice with a cocktail of antibiotics (0.5 g/L vancomycin, 1 g/L ampicillin, 1 g/L metronidazole, and 1 g/L neomycin) in drinking water and investigated the changes in neutrophils. Our results revealed that the antibiotic treatment significantly decreased the proportions of neutrophils in the islet, bone marrow, spleen and peripheral blood ( Figures 6A–H ). We also had the wildtype mice (BDC2.5 ⁺ Il-10 ^+/+) as controls, treated with or without antibiotic, and found that neutrophils were only decreased in the spleen in BDC2.5 ⁺ Il-10 ^+/+ NOD mice ( Figure S10 ). To further confirm the role of gut microbiota from BDC2.5 ⁺ Il-10 ^-/- NOD mice in modulating neutrophil homeostasis, we co-cultured BDC2.5 ⁺ Il-10 ^-/- NOD splenocytes in vitro with heat-inactivated gut bacteria from either BDC2.5 ⁺ Il-10 ^+/+ or BDC2.5 ⁺ Il-10 ^-/- NOD mice. Interestingly, we found that gut microbiota from BDC2.5 ⁺ Il-10 ^-/- NOD further expanded the neutrophil population significantly ( Figures 6I, J ). Next, we purified splenic neutrophils from BDC2.5 ⁺ Il-10 ^-/- NOD mice and stimulated them with heat-inactivated gut bacteria from either BDC2.5 ⁺ Il-10 ^+/+ or BDC2.5 ⁺ Il-10 ^-/- NOD mice, followed by assessment of gene expression. Our qPCR results showed that BDC2.5 ⁺ Il-10 ^-/- NOD gut microbiota promoted Ifn-γ gene expression ( Figure 6K ), which was consistent with the intracellular cytokine staining results seen in BDC2.5 ⁺ Il-10 ^-/- NOD mice ( Figures 2F, G ). Similar to the results using fresh ex-vivo splenic neutrophils ( Figures S4A, B ), there were no significant differences in the gene expressions of Inos and Nos2 in the neutrophils after in vitro stimulation ( Figures 6L, M ). Taken together, our data suggested that altered gut bacteria from BDC2.5 ⁺ Il-10 ^-/- NOD mice modulated neutrophil homeostasis, which play a role in the development of type 1 diabetes.