Steven M. Mortin-Toth

Sex Differences in the Gut Microbiome Drive Hormone-Dependent Regulation of Autoimmunity

Mighty Male Microbes Both genetic and environmental factors contribute to an individuals susceptibility to autoimmune disease, but the specific environmental influences are not well characterized. Markle et al. (p. 1084, published online 17 January; see the Perspective by Flak et al.) explored how microbial factors, in particular the gut microbiota, influence susceptibility to type 1 diabetes in mice. In the non-obese diabetic (NOD) mouse model of type 1 diabetes, female mice are significantly more susceptible to disease than males; however, this difference was not apparent under germ-free conditions. Transfer of cecal contents from male NOD mice to female NOD mice prior to disease onset protected against pancreatic islet inflammation, autoantibody production, and the development of diabetes and was associated with increased testosterone in female mice. Blocking androgen receptor activity abrogated protection. Thus, the microbiota may be able to regulate sex hormones and influence an individuals susceptibility to autoimmunity. In mice, the gut microbiota influences levels of sex hormones and the development of autoimmune disease. [Also see Perspective by Flak et al.] Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipients microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.

Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells

Graft failure in the transplantation of hematopoietic stem cells occurs despite donor-host genetic identity of human leukocyte antigens, suggesting that additional factors modulate engraftment. With the nobese diabetic (NOD)–severe combined immunodeficiency (SCID) xenotransplantation model, we found that the NOD background allowed better hematopoietic engraftment than did other strains with equivalent immunodeficiency-related mutations. We used positional genetics to characterize the molecular basis for this strain specificity and found that the NOD Sirpa allele conferred support for human hematopoiesis. NOD SIRP-α showed enhanced binding to the human CD47 ligand, and its expression on mouse macrophages was required for support of human hematopoiesis. Thus, we have identified Sirpa polymorphism as a potent genetic determinant of the engraftment of human hematopoietic stem cells.

Two Genetic Loci Regulate T Cell–Dependent Islet Inflammation and Drive Autoimmune Diabetes Pathogenesis

Insulin-dependent diabetes mellitus (IDDM) is a polygenic disease caused by progressive autoimmune infiltration (insulitis) of the pancreatic islets of Langerhan, culminating in the destruction of insulin-producing beta cells. Genome scans of families with diabetes suggest that multiple loci make incremental contributions to disease susceptibility. However, only the IDDM1 locus is well characterized, at a molecular and functional level, as alleleic variants of the major histocompatibility complex (MHC) class II HLA-DQB1, DRB1, and DPB1 genes that mediate antigen presentation to T cells. In the nonobese diabetic (NOD) mouse model, the Idd1 locus was shown to be the orthologous MHC gene I-Ab. Inheritance of susceptibility alleles at IDDM1/Idd1 is insufficient for disease development in humans and NOD mice. However, the identities and functions of the remaining diabetes loci (Idd2-Idd19 in NOD mice) are largely undefined. A crucial limitation in previous genetic linkage studies of this disease has been reliance on a single complex phenotype-diabetes that displays low penetrance and is of limited utility for high-resolution genetic mapping. Using the NOD model, we have identified an early step in diabetes pathogenesis that behaves as a highly penetrant trait. We report that NOD-derived alleles at both the Idd5 and Idd13 loci regulate a T lymphocyte-dependent progression from a benign to a destructive stage of insulitis. Human chromosomal regions orthologous to the Idd5 and -13 intervals are also linked to diabetes risk, suggesting that conserved genes encoded at these loci are central regulators of disease pathogenesis. These data are the first to reveal a role for individual non-MHC Idd loci in a specific, critical step in diabetes pathogenesis-T cell recruitment to islet lesions driving destructive inflammation. Importantly, identification of intermediate phenotypes in complex disease pathogenesis provides the tools required to progress toward gene identification at these loci.

SIRPα polymorphisms, but not the prion protein, control phagocytosis of apoptotic cells

The regulation of phagocytosis previously ascribed to prion protein PrPC is found to be controlled by the linked locus encoding SIRPα.

Sex-Specific Effect of Insulin-Dependent Diabetes 4 on Regulation of Diabetes Pathogenesis in the Nonobese Diabetic Mouse

Many human autoimmune diseases are more frequent in females than males, and their clinical severity is affected by sex hormone levels. A strong female bias is also observed in the NOD mouse model of type I diabetes (T1D). In both NOD mice and humans, T1D displays complex polygenic inheritance and T cell-mediated autoimmune pathogenesis. The identities of many of the insulin-dependent diabetes (Idd) loci, their influence on specific stages of autoimmune pathogenesis, and sex-specific effects of Idd loci in the NOD model are not well understood. To address these questions, we analyzed cyclophosphamide-accelerated T1D (CY-T1D) that causes disease with high and similar frequencies in male and female NOD mice, but not in diabetes-resistant animals, including the nonobese diabetes-resistant (NOR) strain. In this study we show by genetic linkage analysis of (NOD × NOR) × NOD backcross mice that progression to severe islet inflammation after CY treatment was controlled by the Idd4 and Idd9 loci. Congenic strains on both the NOD and NOR backgrounds confirmed the roles of Idd4 and Idd9 in CY-T1D susceptibility and revealed the contribution of a third locus, Idd5. Importantly, we show that the three loci acted at distinct stages of islet inflammation and disease progression. Among these three loci, Idd4 alleles alone displayed striking sex-specific behavior in CY-accelerated disease. Additional studies will be required to address the question of whether a sex-specific effect of Idd4, observed in this study, is also present in the spontaneous model of the disease with striking female bias.

Molecular Genetic Analysis of the Idd4 Locus Implicates the IFN Response in Type 1 Diabetes Susceptibility in Nonobese Diabetic Mice

High-resolution mapping and identification of the genes responsible for type 1 diabetes (T1D) has proved difficult because of the multigenic etiology and low penetrance of the disease phenotype in linkage studies. Mouse congenic strains have been useful in refining Idd susceptibility loci in the NOD mouse model and providing a framework for identification of genes underlying complex autoimmune syndromes. Previously, we used NOD and a nonobese diabetes-resistant strain to map the susceptibility to T1D to the Idd4 locus on chromosome 11. Here, we report high-resolution mapping of this locus to 1.4 megabases. The NOD Idd4 locus was fully sequenced, permitting a detailed comparison with C57BL/6 and DBA/2J strains, the progenitors of T1D resistance alleles found in the nonobese diabetes-resistant strain. Gene expression arrays and quantitative real-time PCR were used to prioritize Idd4 candidate genes by comparing macrophages/dendritic cells from congenic strains where allelic variation was confined to the Idd4 interval. The differentially expressed genes either were mapped to Idd4 or were components of the IFN response pathway regulated in trans by Idd4. Reflecting central roles of Idd4 genes in Ag presentation, arachidonic acid metabolism and inflammation, phagocytosis, and lymphocyte trafficking, our combined analyses identified Alox15, Alox12e, Psmb6, Pld2, and Cxcl16 as excellent candidate genes for the effects of the Idd4 locus.

The Idd4 Locus Displays Sex-Specific Epistatic Effects on Type 1 Diabetes Susceptibility in Nonobese Diabetic Mice

The nonobese diabetic (NOD) mouse recapitulates many aspects of the pathogenesis of type 1 diabetes in humans, including inheritance as a complex trait. More than 20 Idd loci have been linked to type 1 diabetes susceptibility in NOD mice. Previously, we used linkage analysis of NOD crossed to the nonobese diabetes-resistant (NOR) strain and NOD congenic strains to map susceptibility to both spontaneous and cyclophosphamide-accelerated type 1 diabetes to the Idd4 locus on chromosome 11 that displayed a sex-specific effect on diabetes susceptibility. Here, we elucidate the complex genetic architecture of Idd4 by analysis of congenic strains on the NOD and NOR backgrounds. We previously refined Idd4.1 to 1.4 Mb and demonstrated an impact of this interval on type 1 interferon pathways in antigen-presenting cells. Here, we identify a second subregion, the 0.92 Mb Idd4.2 locus located telomeric to Idd4.1. Strikingly, Idd4.2 displayed a sex-specific, epistatic interaction with Idd4.1 in NOR.NOD congenic females that was not observed in syngenic males. Idd4.2 contains 29 genes, and promising candidates for the Idd4.2 effect on type 1 diabetes are described. These data demonstrate sex-dependent interaction effects on type 1 diabetes susceptibility and provide a framework for functional analysis of Idd4.2 candidate genes.

Polymorphism in the Innate Immune Receptor SIRPα Controls CD47 Binding and Autoimmunity in the Nonobese Diabetic Mouse

The signal regulatory protein (SIRP) locus encodes a family of paired receptors that mediate both activating and inhibitory signals and is associated with type 1 diabetes (T1D) risk. The NOD mouse model recapitulates multiple features of human T1D and enables mechanistic analysis of the impact of genetic variations on disease. In this study, we identify Sirpa encoding an inhibitory receptor on myeloid cells as a gene in the insulin-dependent diabetes locus 13.2 (Idd13.2) that drives islet inflammation and T1D. Compared to T1D-resistant strains, the NOD variant of SIRPα displayed greater binding to its ligand CD47, as well as enhanced T cell proliferation and diabetogenic potency. Myeloid cell–restricted expression of a Sirpa transgene accelerated disease in a dose-dependent manner and displayed genetic and functional interaction with the Idd5 locus to potentiate insulitis progression. Our study demonstrates that variations in both SIRPα sequence and expression level modulate T1D immunopathogenesis. Thus, we identify Sirpa as a T1D risk gene and provide insight into the complex mechanisms by which disease-associated variants act in concert to drive defined stages in disease progression.

Donor SIRPα polymorphism modulates the innate immune response to allogeneic grafts

Detection of nonself SIRPα by CD47 triggers an innate immune response to allografts in mice that lack T, B, and NK cells. Looking beyond MHCs in transplant rejection Mice engineered to lack T, B, and NK cells generate mature dendritic cells in response to allogeneic transplants. Precisely how these mice recognize allografts to be “nonself” has remained a mystery. Using an elegant positional cloning approach, Dai et al. have identified polymorphisms in the mouse gene encoding signal regulatory protein α (SIRPα) to be key in this innate self-nonself recognition. They show that SIRPα receptor CD47 binds SIRPα variants with distinct affinities and propose this affinity sensing to be the mechanism that triggers dendritic cell maturation, the first step in the initiation of the alloimmune response. Given that the SIRPα gene is also polymorphic in humans, it remains to be seen whether human SIRPα variations influence transplantation success. Mice devoid of T, B, and natural killer (NK) cells distinguish between self and allogeneic nonself despite the absence of an adaptive immune system. When challenged with an allograft, they mount an innate response characterized by accumulation of mature, monocyte-derived dendritic cells (DCs) that produce interleukin-12 and present antigen to T cells. However, the molecular mechanisms by which the innate immune system detects allogeneic nonself to generate these DCs are not known. To address this question, we studied the innate response of Rag2−/−γc−/− mice, which lack T, B, and NK cells, to grafts from allogeneic donors. By positional cloning, we identified that donor polymorphism in the gene encoding signal regulatory protein α (SIRPα) is a key modulator of the recipient’s innate allorecognition response. Donors that differed from the recipient in one or both Sirpa alleles elicited an innate alloresponse. The response was mediated by binding of donor SIRPα to recipient CD47 and was modulated by the strength of the SIRPα-CD47 interaction. Therefore, sensing SIRPα polymorphism by CD47 provides a molecular mechanism by which the innate immune system distinguishes between self and allogeneic nonself independently of T, B, and NK cells.

Identification of the Inflammasome Nlrp1b as the Candidate Gene Conferring Diabetes Risk at the Idd4.1 Locus in the Nonobese Diabetic Mouse

Type 1 diabetes in the NOD mouse model has been linked to >30 insulin-dependent diabetes (Idd) susceptibility loci. Idd4 on chromosome 11 consists of two subloci, Idd4.1 and Idd4.2. Using congenic analysis of alleles in NOD and NOD-resistant (NOR) mice, we previously defined Idd4.1 as an interval containing >50 genes that controlled expression of genes in the type 1 IFN pathway. In this study, we report refined mapping of Idd4.1 to a 1.1-Mb chromosomal region and provide genomic sequence analysis and mechanistic evidence supporting its role in innate immune regulation of islet-directed autoimmunity. Genetic variation at Idd4.1 was mediated by radiation-sensitive hematopoietic cells, and type 1 diabetes protection conferred by the NOR allele was abrogated in mice treated with exogenous type 1 IFN-β. Next generation sequence analysis of the full Idd4.1 genomic interval in NOD and NOR strains supported Nlrp1b as a strong candidate gene for Idd4.1. Nlrp1b belongs to the Nod-like receptor (NLR) gene family and contributes to inflammasome assembly, caspase-1 recruitment, and release of IL-1β. The Nlrp1b of NOR was expressed as an alternative spliced isoform that skips exon 9, resulting in a premature stop codon predicted to encode a truncated protein. Functional analysis of the truncated NOR Nlrp1b protein demonstrated that it was unable to recruit caspase-1 and process IL-1β. Our data suggest that Idd4.1-dependent protection from islet autoimmunity is mediated by differences in type 1 IFN– and IL-1β–dependent immune responses resulting from genetic variation in Nlrp1b.