Polyglandular autoimmune inflammation accompanies type 1 diabetes (T1D) in NOD mice, affecting organs like thyroid and salivary glands. Although commensals are not required for T1D progression, germ-free (GF) mice had a very low degree of sialitis, which was restored by colonization with select microbial lineages. Moreover, unlike T1D, which is blocked in mice lacking MyD88 signaling adaptor under conventional, but not GF, housing conditions, sialitis did not develop in MyD88−/− GF mice. Thus, microbes and MyD88-dependent signaling are critical for sialitis development. The severity of sialitis did not correlate with the degree of insulitis in the same animal and was less sensitive to a T1D-reducing diet, but it was similar to T1D with regard to microbiota-dependent sexual dimorphism. The unexpected distinction in requirements for the microbiota for different autoimmune pathologies within the same organism is crucial for understanding the nature of microbial involvement in complex autoimmune disorders, including human autoimmune polyglandular syndromes.

Type 1 diabetes (T1D) develops spontaneously in humans and in rodents (NOD mice and biobreeding [BB] rats). In addition to leukocytic infiltration in the pancreas, autoimmune responses against other glands (thyroid, adrenal, submandibular, and lacrimal) occur frequently. This makes NOD mice very similar to a subset of human patients with T1D who develop autoimmune polyglandular syndrome (APS) types 2–4 (types are assigned based on the organs affected) (13). The rare APS type 1 and the more common type 2 are linked to AIRE (4) and MHC genes (5), respectively. Loss of tolerance and immunodeficiency in these patients results in autoimmune Addison’s disease that is typically accompanied by hypoparathyroidism (APS1) or hypothyroidism and/or T1D (APS2). The most prevalent APS type 3 is characterized by the same features as APS2, except that adrenal defects are lacking. Instead, it is often accompanied by nonorgan-specific (systemic) lupus erythematosus, rheumatoid arthritis, or Sjögren’s syndrome, which is similarly evident in type 4 APS, which is characterized by associations of autoimmune endocrine disorders that do not fulfill criteria for APS1–3 (6). It is not known whether these organ-specific autoimmune manifestations have common (or unique) mechanisms of regulation by extrinsic factors. Indirect evidence based on changes in predisease-onset microbiota, use of antibiotics, and testing of germ-free (GF) rodent models of autoimmune diseases suggests that the disease onset and/or severity are influenced by gut microbiota (7, 8). Although a GF state keeps the incidence of diabetes similar to the levels of T1D in specific pathogen–free (SPF) mice (9, 10) or even enhances it (11, 12), it clearly reduces inflammation in other autoimmune diseases (13). In this study, we used the NOD mouse model to compare microbial influences on the development of diabetes and sialitis.

NOD/ShiLtJ mice (The Jackson Laboratory, Bar Harbor, ME) were housed in an SPF and GF facility at The University of Chicago Animal Resource Center and used in accordance with institutional guidelines for animal welfare. The Biological Sciences Division Institutional Review Board at the University of Chicago approved all animal studies. GF status was monitored by aerobic and anaerobic fecal cultures and PCR amplification of bacterial 16S rRNA genes from fecal DNA. All mice were killed at 13 wk of age or when diagnosed with diabetes by testing for glycosuria using a urine dipstick (Diastix; Bayer, Elkhart, IN), as indicated in the text. Littermates were used where possible.

Gonads were excised from 5-wk-old males anesthetized with a ketamine (100 mg/kg) and xylazine (5 mg/kg) combination using the Change-A-Tip handheld cauterizer (Bovie Medical, Clearwater, FL), and the incision was sealed with EZ Clips (Stoelting, Wood Dale, IL). Sham-operated mice were anesthetized, and a scrotal incision was made and sealed by the application of wound clips.

Mice were fed JL Rat and Mouse/Auto 6F 5K67 chow diet (LabDiet, St. Louis, MO) or an AIN-93G modified diet with 20% hydrolyzed casein (HC) (Harlan Laboratories, Madison, WI). Diets were autoclaved and microbiologically tested before GF use.

GF mice were colonized by introduction of specific microbial taxa via gastric gavage to the breeding pairs in separate isolators. Bacteria were transferred to the offspring naturally from the mother, and the colonization was confirmed by 16S rDNA PCR for genes specific for the lineages (14, 15). The bacterial community in VSL#3 enriched for bifidobacteria, lactobacilli, and Streptococcus salivarius spp., and culturing and classification of a Proteobacteria, similar to Escherichia coli and Shigella (SECS), was described by Yurkovetskiy et al. (14). Akkermansia muciniphila was a generous gift from Dr. Dennis Sandris Nielsen (Department of Food Science, University of Copenhagen) and was introduced by gavage of 2 × 108 CFU.

Leukocytic inflammatory foci in salivary glands were scored in a blinded fashion, using 5-μm H&E-stained paraffin sections of wax-embedded salivary glands. Eight sequential sections of each gland, with 40-μm intervals, were scored by counting the number of focal infiltrates > 50 cells (16) and measuring the mean size of all foci/section. Foci size was measured with a Leica graticule with a 10 × 10-mm grid, and the sialitis score was calculated: score = average size of the foci per section (mm2) × mean number of foci per section.

Insulitis was scored on ≥100 islets/pancreas using 5-μm, H&E-stained sections with 40-μm intervals and graded as follows: 1, no visible infiltration; 2, peri-insulitis; 3, insulitis with <50% islet infiltration; and 4, insulitis with >50% islet infiltration. The percentage of islets with insulitis (score 3 and 4) was used for the analyses.

The results were analyzed for statistical probabilities of significance between two groups using the two-tailed unpaired Student t test. For analysis of more than two groups, significance was found by one-way ANOVA with the Tukey post hoc test. Diabetes incidence at 30 wk of age was analyzed by the Fischer exact test. The p values < 0.05 were considered significant. Graphs were generated and statistical analyses were performed using GraphPad Prism version 5.02 (GraphPad, San Diego, CA). Results are displayed as mean ± SEM.

To test the contribution of intestinal microbes to the development of autoimmune sialitis in NOD mice, we examined the severity of inflammation (number and extent of inflammatory foci) in histological sections of the salivary glands in GF and SPF mice. The sialitis score (derived by multiplying the mean number of lesions per eight histological sections by their mean size) was significantly lower in GF females compared with SPF females at 13 wk of age prior to the onset of overt diabetes (Fig. 1A). In contrast, T1D in GF female NOD mice was similar to the incidence in SPF NOD mice (10) or was increased, with the incidence close to 100% (11). The insulitis progressed similarly in our SPF and GF NOD mice and, therefore, had a GF/SPF ratio close to 1.0, whereas the sialitis score in GF NOD mice was only 0.33 ± 0.06 of the score in SPF NOD mice (Fig. 1B). In addition, nondiabetic 13-wk-old male mice had only very mild infiltration, if any, in SPF and GF conditions (Fig. 1A). This also contrasts with the development of autoimmune diabetes, during which GF males lose the protection mediated by the gut microbiota (14).

FIGURE 1.

Commensal microbes are required for the development of sialitis but not insulitis. (A) Histopathology scores of salivary glands collected from SPF and GF 13-wk-old nondiabetic female NOD mice (n = 8 and n = 10, respectively), diabetic females with glucosuria (n = 7 and n = 6, respectively), and 13-wk-old nondiabetic male NOD mice (n = 8 and n = 15, respectively). To determine the sialitis score, leukocytic inflammatory foci were scored on 8–10 sequential sections of each gland by counting the number of focal infiltrates and measuring the mean size of all foci/section. Mean values are shown. (B) Ratio of sialitis (n = 10) and insulitis (n = 13) scores in GF and SPF nondiabetic 13-wk-old female NOD mice. Mean values ± SEM. (C) Correlation between sialitis and insulitis scores from the same SPF nondiabetic 13-wk-old female NOD mice (n = 21). (D) Correlation between sialitis and insulitis scores in organs collected from the same GF nondiabetic 13-wk-old female NOD mice (n = 9). Representative images of pancreatic islets (arrows) from SPF (E) and GF (F) 13-wk-old nondiabetic female mice. Representative images of foci of inflammation in salivary glands (arrows) from SPF (G) and GF (H) 13-wk-old nondiabetic female mice. H&E staining of paraffin sections. *p < 0.05. NO, nondiabetic; ns, nonsignificant (p > 0.05); +, diabetic.

FIGURE 1.

Commensal microbes are required for the development of sialitis but not insulitis. (A) Histopathology scores of salivary glands collected from SPF and GF 13-wk-old nondiabetic female NOD mice (n = 8 and n = 10, respectively), diabetic females with glucosuria (n = 7 and n = 6, respectively), and 13-wk-old nondiabetic male NOD mice (n = 8 and n = 15, respectively). To determine the sialitis score, leukocytic inflammatory foci were scored on 8–10 sequential sections of each gland by counting the number of focal infiltrates and measuring the mean size of all foci/section. Mean values are shown. (B) Ratio of sialitis (n = 10) and insulitis (n = 13) scores in GF and SPF nondiabetic 13-wk-old female NOD mice. Mean values ± SEM. (C) Correlation between sialitis and insulitis scores from the same SPF nondiabetic 13-wk-old female NOD mice (n = 21). (D) Correlation between sialitis and insulitis scores in organs collected from the same GF nondiabetic 13-wk-old female NOD mice (n = 9). Representative images of pancreatic islets (arrows) from SPF (E) and GF (F) 13-wk-old nondiabetic female mice. Representative images of foci of inflammation in salivary glands (arrows) from SPF (G) and GF (H) 13-wk-old nondiabetic female mice. H&E staining of paraffin sections. *p < 0.05. NO, nondiabetic; ns, nonsignificant (p > 0.05); +, diabetic.

Close modal

About 55% of human patients with T1D have Sjögren’s syndrome, particularly during hyperglycemic phases (17). As one of the most prevalent multiorgan autoimmune diseases, Sjögren’s syndrome is characterized (among other things) by dry mouth (xerostomia) and dry eyes (keratoconjunctivitis) (18). NOD mice similarly manifest several features of Sjögren’s syndrome (lymphocytic infiltrations and progressive destruction of salivary and lachrymal glands) along with T1D, making them a good model for APS. Therefore, the connection between overt autoimmune diabetes and the development of salivary gland lesions was investigated. As mice progressed toward overt diabetes (Fig. 1A, 1E, 1F), the sialitis scores also increased; however, GF mice still lagged behind their SPF counterparts (Fig. 1A, 1G, 1H). We then plotted insulitis versus sialitis scores for SPF and GF NOD females. No correlation was found between these two processes in either condition, although sialitis was more pronounced in SPF mice, as expected (Fig. 1C, 1D). Thus, the two pathologies develop independently of each other and are under different pressure from the microbiota.

T1D in NOD mice is known to be sexually dimorphic (1), similar to the 3:1 female bias of T1D in APS2–4 patients (19) and unlike the 1:1 gender ratio of T1D in human patients with no other endocrine autoimmune manifestations (20). At the same time, the predominance of T1D in female NOD mice is lost in GF conditions (1012). Protection of males depends on male sex hormones and on the microbiota: castrated males, which change their gut microbiota toward a female profile, developed insulitis and diabetes at a higher rate, comparable to that of females (10, 14). Several microbial taxa were shown to be protective for the males (14). Thus, we investigated the sexual dimorphism of sialitis and microbiota’s contribution to it. Sialitis was very mild in males in SPF and GF conditions (Fig. 1A). Castration significantly increased the sialitis score (Fig. 2A). The fold change in the sialitis score that was induced by castration (7.4 ± 1.6) was even higher (p < 0.001) than for insulitis (2.5 ± 0.3), but it never reached the severity of sialitis in females (Fig. 2B). Thus, sex hormones regulate insulitis and sialitis in NOD mice and that could be related to the shaping of the microbiota by sex hormones (10, 14). However, because male castration leads to only mild increases in the severity of inflammation of salivary glands, sialitis may be affected by other nonhormonal gender differences (21).

FIGURE 2.

Sexual dimorphism of sialitis development. (A) Histopathology scores of salivary glands from SPF 13-wk-old nondiabetic female (n = 10), intact male (n = 8), and castrated male (n = 5) NOD mice. Mean values ± SEM of one experiment are shown. (B) Ratio of sialitis and insulitis scores in SPF sham-operated (n = 8 and n = 7, respectively) or castrated (Cast.) (n = 5 and n = 12, respectively) nondiabetic 13-wk-old male NOD mice/female NOD mice. Mean values ± SEM of one experiment are shown. **p < 0.01, ***p < 0.001.

FIGURE 2.

Sexual dimorphism of sialitis development. (A) Histopathology scores of salivary glands from SPF 13-wk-old nondiabetic female (n = 10), intact male (n = 8), and castrated male (n = 5) NOD mice. Mean values ± SEM of one experiment are shown. (B) Ratio of sialitis and insulitis scores in SPF sham-operated (n = 8 and n = 7, respectively) or castrated (Cast.) (n = 5 and n = 12, respectively) nondiabetic 13-wk-old male NOD mice/female NOD mice. Mean values ± SEM of one experiment are shown. **p < 0.01, ***p < 0.001.

Close modal

The microbiota is quite sensitive to dietary variations (22). Some dietary interventions lead to attenuation of autoimmunity (2325). Semipurified diets with modified protein fractions (e.g., by basing it solely on HC) can drastically reduce diabetes incidence in NOD mice and BB rats (26, 27). HC intervention is associated with changes in the gut microbiota, including increased levels of lactobacilli and decreased levels of Bacteroides spp. (28). To test whether dietary changes affect sialitis to the same degree as they do diabetes (29), we compared disease development in mice fed regular chow and an HC diet. The HC diet only showed a trend toward a reduction in the sialitis score in SPF mice; the scores in GF mice remained low, irrespective of the diet (Fig. 3A). Similarly, the hypoallergenic diet Pregestimil was not protective against salivary-infiltrating mononuclear cells in NOD mice (30). Thus, any changes in the microbiota associated with the HC diet are not sufficient to reduce sialitis. This result raises the question of whether specific bacterial lineages affect sialitis, or can a rather broad range of microbes support salivary gland inflammation? The issue was addressed by colonizing GF mice with defined microbial consortia. Previous reports suggested that A. muciniphila (in association with more abundant Proteobacteria) attenuated diabetes development in SPF NOD mice that were treated with antibiotics (8, 24). However, colonization of GF mice with a combination of A. muciniphila and Proteobacteria SECS isolated from NOD mice (14) did not reduce the severity of the histopathology in pancreatic islets (Fig. 3B). At the same time, salivary glands of gnotobiotic NOD mice colonized with these bacteria exhibited inflammation that had a significantly higher score than did that of GF NOD mice (Fig. 3A). A very different mix of microbes, VSL3 probiotic (31), did not reduce the development of insulitis in colonized gnotobiotic mice (14). It also failed to increase the severity of sialitis in VSL3-colonized female NOD mice over that observed in GF mice (Fig. 3A). Thus, randomly chosen commensals can either promote sialitis or not. Moreover, the proinflammatory activity of A. muciniphila/Proteobacteria combination was weaker than similar activity of the SPF microbiota. Thus, adding specific commensals supports sialitis in the GF mouse, but the effect of the tested colonizers was small, suggesting that other bacteria are necessary to fully augment salivary gland inflammation.

FIGURE 3.

Distinct effects of dietary and specific microbial challenges on sialitis and insulitis. (A) Histopathology of salivary glands from SPF and GF 13-wk-old nondiabetic female NOD mice on regular chow (C; n = 15 and n = 10, respectively) or 20% HC diet (n = 10 and n = 3, respectively) and GF mice colonized with A. municiphila (AM) + SECS, or VSL3 (n = 5). Mean values of one experiment are shown. *p < 0.05, **p < 0.01. ns (nonsignificant) indicates p > 0.05. (B) Histopathology of the pancreas from GF (n = 15) or GF colonized with AM + SECS (n = 11) nondiabetic 13-wk-old female NOD mice. Mean values of one experiment are shown.

FIGURE 3.

Distinct effects of dietary and specific microbial challenges on sialitis and insulitis. (A) Histopathology of salivary glands from SPF and GF 13-wk-old nondiabetic female NOD mice on regular chow (C; n = 15 and n = 10, respectively) or 20% HC diet (n = 10 and n = 3, respectively) and GF mice colonized with A. municiphila (AM) + SECS, or VSL3 (n = 5). Mean values of one experiment are shown. *p < 0.05, **p < 0.01. ns (nonsignificant) indicates p > 0.05. (B) Histopathology of the pancreas from GF (n = 15) or GF colonized with AM + SECS (n = 11) nondiabetic 13-wk-old female NOD mice. Mean values of one experiment are shown.

Close modal

Deletion of MyD88 adaptor protein in SPF NOD mice led to their complete protection from autoimmune diabetes, but it did not affect T1D development in GF NOD mice (11). These findings led to the conclusion that, in the absence of signaling through MyD88-dependent innate immune receptors, the microbiota induces negative signaling, reducing both anticommensal and autoimmune reactivity (11, 32). To test whether the same rules apply to the development of sialitis in the absence of MyD88, we examined salivary gland lesions in MyD88-knockout (KO) NOD mice. Similarly to the effect of MyD88 KO on T1D, sialitis was reduced significantly in MyD88 SPF mice (Fig. 4). However, sialitis in MyD88-KO GF mice was minimal and even further reduced compared with wild-type (WT) GF NOD mice (Fig. 4).

FIGURE 4.

MyD88-dependent signaling is required for sialitis development. (A) Histopathology scores of the inflammation of salivary glands from SPF and GF nondiabetic 13-wk-old MyD88-sufficient WT (n = 8 and n = 10, respectively) and MyD88-deficient (KO; n = 4 and n = 5, respectively) female NOD mice. Mean values ± SEM of one experiment are shown. (B) Ratio of sialitis and insulitis scores in MyD88- deficient (KO) SPF (n = 4 and n = 8, respectively) or GF (n = 8 and n = 6, respectively) nondiabetic 13-wk-old female NOD mice/MyD88-sufficient (WT) NOD mice. Mean values ± SEM of one experiment are shown. **p < 0.01, ***p < 0.001. ns, nonsignificant (p > 0.05).

FIGURE 4.

MyD88-dependent signaling is required for sialitis development. (A) Histopathology scores of the inflammation of salivary glands from SPF and GF nondiabetic 13-wk-old MyD88-sufficient WT (n = 8 and n = 10, respectively) and MyD88-deficient (KO; n = 4 and n = 5, respectively) female NOD mice. Mean values ± SEM of one experiment are shown. (B) Ratio of sialitis and insulitis scores in MyD88- deficient (KO) SPF (n = 4 and n = 8, respectively) or GF (n = 8 and n = 6, respectively) nondiabetic 13-wk-old female NOD mice/MyD88-sufficient (WT) NOD mice. Mean values ± SEM of one experiment are shown. **p < 0.01, ***p < 0.001. ns, nonsignificant (p > 0.05).

Close modal

These findings show that microbe-induced sialitis is dependent on MyD88, as well as that something besides live bacteria can induce low-grade sialitis in GF mice in a MyD88-dependent manner. Such an effect may be mediated by the presence of autoclave-resistant pathogen-associated molecular patterns in the diet (33). Another possibility is that the presence of endogenous viruses contributes to activation of adaptive immune responses against self-antigens. GF animals are free of viruses with the exception of endogenous retroviruses, which were shown to stimulate the MyD88 pathway through TLR7 (34). In SPF mice, other viruses may contribute to sialitis, even in a MyD88-independent manner: stimulation of MyD88-independent TLR3, which is highly expressed on the surface of salivary gland epithelial cells from Sjögren’s syndrome patients (35), by viral dsRNA is considered a likely contributor to Sjögren’s syndrome (36).

In sum, our study shows that, in complex autoimmune diseases, the microbiota can affect various manifestations of the pathological processes very differently. We find that gut microbiota is dispensable for the induction of T1D but not for the development of severe sialitis. Autoimmune responses initiated by the innate sensing, Ag presentation, and costimulatory activation of adaptive immunity were classified according to the regulatory effect of gut microbiota on disease outcome found in GF and gnotobiotic rodents (37). Diabetes NOD mice and BB rats belongs to the group of diseases to which GF and conventional animals are equally susceptible. However, attenuated development of sialitis in the same mouse model in GF isolators places this pathological manifestation in the group of diseases that develop independently of microbes but in which microbes amplify the disease. This group also includes models of other autoimmune disorders, such as rheumatoid arthritis and systemic lupus erythematosus (7, 38). The specific mechanisms that the microbiota uses to regulate different autoimmune diseases are not well understood. Although specific microbial lineages were shown to contribute to induction of autoimmunity (7), it is more likely that the salivary gland inflammation in NOD mice was induced by multiple members of the microbiota. Regardless, the basis for commensal involvement seems to lie in the communication between innate and adaptive immune responses, which are likely to be sensitive to regulation by the microbiota (37). Our findings revealed an additional complexity to multiorgan autoimmunity that needs to be taken in consideration during investigation of such regulation.

This work was supported by the Carlsberg Foundation and the Aase og Ejnar Danielsens Foundation, Denmark (to C.H.F.H.). L.A.Y. was supported by National Institutes of Health Grant T32 GM007183. A.V.C. was supported by National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases Digestive Diseases Research Core Center Grant DK42086, National Institutes of Health Grant AI0842418, and the JDRFI Grant 17-2011-519.

Abbreviations used in this article:

APS

autoimmune polyglandular syndrome

BB

biobreeding

GF

germ free

HC

hydrolyzed casein

KO

knockout

SECS

similar to Escherichia coli and Shigella

SPF

specific pathogen free

T1D

type 1 diabetes

WT

wild-type.

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The authors have no financial conflicts of interest.