Toll-like receptors (TLRs) are innate pattern recognition receptors 6
involved in host defense7
, control over commensal bacteria and the maintenance of tissue integrity8, 9
. The role of TLRs involvement in organ-specific autoimmunity is not clear. The MyD88 adaptor protein is used by multiple TLRs (except TLR4 and TLR3, which can or must signal via TRIF, respectively) and other receptors (e.g. interleukin-1 receptor, IL-1R). To test the contributions of these receptors to development of T1D in NOD mice, we examined the effect of MyD88 gene disruption on disease incidence and progression. Two MyD88 knockout (KO) NOD strains were independently derived at the Jackson Laboratory (J) and at Yale University (Y). Both showed a loss of diabetes development during 30 week observation periods when housed under normal specific-pathogen free (SPF) conditions with continuous monitoring for the presence of murine pathogens (). Because multiple TLRs signal through the MyD88 adaptor, follow-up studies were conducted in NOD mice lacking individual TLRs (TLRKO). We found that TLR2 and TLR4 (as well as TLR3, data not shown) were dispensable for development of T1D (or protection from it by complete Freund's adjuvant; Supplemental Fig. 1
) when deleted individually
, in contrast to the effect of complete protection from diabetes associated with loss of MyD88 ().
MyD88-negative (MyD88KO) mice are completely protected from development of type 1 diabetes
These findings suggested that signaling through receptors which use the MyD88 adaptor is critical for T1D development, and that the autoimmune T cells would likely be affected systemically in NOD.MyD88KO mice. Two types of experiments were performed to examine this hypothesis. First
, splenocytes from pre-diabetic MyD88-sufficient and NOD.MyD88KO mice were transferred into immunodeficient NOD.SCID animals. All recipients of control MyD88-sufficient splenocytes (n=5), and 4 out of 5 recipients of NOD.MyD88KO splenocytes became diabetic, arguing against profound systemic tolerance of T cells in NOD.MyD88KO mice. Second
, an Elispot analysis of interferon-γ (IFN-γ) production by T cells in response to four peptides known to be recognized by diabetogenic T cells10-13
was performed (). T cells from spleens, mesenteric and pancreatic lymph nodes (MLN and PLN, respectively) were analyzed. Spleens and MLN of NOD.MyD88KO mice contained activated precursors of the diabetogenic T cells, whereas their numbers were clearly reduced in PLN (). Since individual mice vary in their responses to different peptides14
, the overall reactivity to all four peptides is shown in (see Supplemental Fig. 2
for primary data). The responses to a prevalent diabetes-associated peptide recognized by the 8.3 CD8+
T cell clone 12, 15
, were found to be attenuated in the PLN of NOD.MyD88KO mice in a statistically significant manner (). In addition, adoptively transferred carboxy-fluorescein succinimidyl ester (CFSE)-labeled naïve CD4+
T cells from mice carrying the diabetogenic T cell receptor BDC2.5 proliferated in the PLN but not in other (mesenteric or skin-draining) lymph nodes of the NOD mice, however, their proliferation was clearly attenuated in the PLN of MyD88 KO mice ().
MyD88 deficiency leads to local tolerance to pancreatic antigens
Since the anti-diabetogenic effect of MyD88 deficiency was localized to PLN, it became clear that there was no systemic suppression of immune activation in SPF NOD.MyD88KO mice, making initial conclusions about the requirement of MyD88 signaling for initiation of T1D an oversimplification.
PLN drain both the pancreas and the intestine, and they are an important compartment where islet-specific T cells are activated16, 17
. Because MyD88 signaling could be critical for the interactions of the host with the gut microbiota, we explored the hypothesis that the T1D resistance of NOD.MyD88KO could be driven by their intestinal microbiota. First, we treated SPF NOD.MyD88KO mice with a broad-spectrum antibiotic (Sulfatrim) throughout their lifetime. Antibiotic-treated NOD.MyD88KO animals developed T1D at higher rates compared to antibiotic-free NOD.MyD88KO mice ( versus ), although they did not quite reach the incidence observed in control NOD.MyD88KO/+
mice. Thus, to fully eliminate any residual microbes, we re-derived NOD and NOD.MyD88 KO animals as germ-free (GF). T1D development in our GF MyD88-sufficient NOD mice was similar to previously reported18
and not dramatically different from mice raised in our SPF facilities (; Supplemental Figs. 3 and 4
). Thus, initiation
of autoimmunity is genetically programmed and not affected by the presence of microbiota in immunocompetent SPF NOD animals in high-health-standard facilities.
MyD88-negative NOD mice are protected from diabetes by the gut microbiota
Most importantly, in contrast to our finding that NOD.MyD88KO mice raised under SPF conditions did not develop T1D, GF NOD.MyD88KO mice robustly developed diabetes ( and Supplemental Fig. 3
). This finding clearly shows that neither IL-1R nor MyD88-dependent TLRs are required for activation of an anti-islet T cell response [similar to MyD88KO, autoimmune regulator (Aire)-deficient mice19
]. The efficient T cell priming observed in GF NOD.MyD88KO mice does not confirm a previous report that suggested that TLR2 signaling, which is MyD88-dependent, is required for T cell priming in NOD mice20
To directly show that T1D development in GF NOD.MyD88 KO mice was indeed a consequence of the lack of a microbiota, adult GF NOD.MyD88KO/+
mice were colonized with a consortium of bacterial species contained in the Altered Schaedler Flora (ASF)21
and intercrossed. After introducing the ASF cocktail, PCR assays revealed that 6 of the 8 species colonized the animals (based on sampling of cecal contents, Supplemental results and Supplemental Fig. 5
). The ASF-colonized NOD.MyD88KO mice exhibited a significant reduction in the incidence of diabetes (): only 34% of males became diabetic at 30 weeks of age as compared to >80% of GF NOD.MyD88KO males (), whereas 70% of ASF-colonized NOD.MyD88KO/+
males developed diabetes with kinetics similar to disease development in SPF NOD.MyD88KO/+
mice ( versus ).
A histological analysis of pancreata from SPF, GF and ASF-colonized NOD.MyD88KO mice was performed to compare the effects of the microbiota on T cell-mediated destruction of the islets of Langerhans. The islets of SPF NOD.MyD88KO mice were less infiltrated compared to islets of SPF NOD.MyD88KO/+
mice. Moreover, GF NOD.MyD88KO mice had considerably increased islet infiltration, which was moderated by the introduction of the ASF (, histological grading shown in Supplemental Fig. 6
). Although, the overall effect of ASF on diabetes development was not fully penetrant, these results suggest that bacterial lineages normally present in the gut can modify T1D progression.
We found that MyD88 signaling is critical for development of T1D, and postulated that it is needed for control over component(s) of the microbiota that otherwise (in MyD88KO mice) can protect against development of T1D. To test how MyD88-dependent innate immunity shapes the composition of the gut microbiota, we used a culture-independent, 16S rRNA gene sequence-based approach to characterize the microbial communities of SPF NOD.MyD88KO/+
and NOD.MyD88KO littermates. Because MyD88 deficiency affects T1D development at the early stages (Supplemental Fig. 7
), experimental female mice were housed individually and killed at 8 weeks of age. DNA was isolated from their cecal contents and bacterial 16S rRNA genes were amplified by PCR, and the resulting full-length amplicons sequenced (n=36 mice; total of 7,223 16S rRNA gene sequences; average of 201 sequences/animal; average sequence length of 1,310 nucleotides).
In concordance with previous findings in mice and humans22,23,24
two bacterial divisions (phyla) - the Firmicutes (F) and the Bacteroidetes (B) dominated the distal gut (cecal) microbiota of mice from all groups (80.7% and 16.9% of all sequences, respectively). The remainder of the microbiota was composed of divisions commonly encountered at lower abundance in the mouse and human gut: Verrucomicrobia, Proteobacteria, Actinobacteria and the candidate phylum TM725
. Furthermore, close relatives of ASF strains were detected in SPF NOD mice.
Analysis of the cecal microbiota of NOD.MyD88KO/+
versus NOD.MyD88KO mice showed significant differences. Antibiotic-free NOD.MyD88KO mice had, on average, a significantly lower F/B ratio when compared to all other groups (one-tailed t-test t=-2.31, p=0.013) (). Antibiotic treatment of animals eliminated the statistically significant difference in F/B ratio (). A change of F/B ratio may be important by itself because it can influence the efficiency of processing of otherwise indigestible complex polysaccharides in the diet 26,27
. Diet-related changes in diabetes in GF NOD mice have been observed 28
; these effects could be related to the components of the diet per se or attributed to the presence of microbial products in the feed.
MyD88 deficiency leads to specific changes in the composition of intestinal microbiota
To further characterize the changes in microbiota imposed by MyD88 deficiency, 16S rRNA genes were classified taxonomically to the family level (Ribosomal Database Project Classifier29
). The proportion of sequences in each family was determined for individual mice, averaged and compared across treatments. The representation of three bacterial families were increased significantly in the microbiota of antibiotic-free SPF NOD.MyD88KO mice compared to the SPF NOD animals: the Lactobacillaceae (Firmicutes), Rikenellaceae and Porphoromadaceae (both Bacteroidetes) (). Interestingly, the VSL3 probiotic mix, containing four species of Lactobacillaceae affects diabetes30
in NOD mice.
Gut microbial communities are known to be inherited from the mother22
: this was also the case in these experiments, where clustering of 16S rRNA genes was strongly influenced by shared mothers ().
To show that the changes in the intestinal microbiota of MyD88KO animals were responsible for attenuation of T1D development, the newborn progeny of GF NOD mice were exposed to SPF NOD.MyD88 KO females and allowed to mature to 8 weeks of age, after which time their pancreata were removed and analyzed histologically (). Islet infiltration was significantly reduced in GF NOD animals exposed to microbiota from SPF MyD88 KO mouse compared to GF NOD mice [increased percentage of intact islets (p=0.001) and reduced percentage of infiltrated islets (p=0.0006)].
Although the precise mechanism of induction of local tolerance by the microbiota remains to be elucidated (see Supplemental Fig. 8
), the finding that the normal intestinal microbiota can alleviate progression of autoimmune diabetes in a MyD88-independent manner provides a different perspective about disease pathogenesis. Knowledge-based use of live microbial lineages, or microbial products could present new therapeutic options for T1D in the future.