The colibactin-producing Escherichia coli alters the tumor microenvironment to immunosuppressive lipid overload facilitating colorectal cancer progression and chemoresistance.

Intratumoral bacteria flexibly contribute to cellular and molecular tumor heterogeneity for supporting cancer recurrence through poorly understood mechanisms. Using spatial metabolomic profiling technologies and 16SrRNA sequencing, we herein report that right-sided colorectal tumors are predominantly populated with Colibactin-producing Escherichia coli (CoPEC) that are locally establishing a high-glycerophospholipid microenvironment with lowered immunogenicity. It coincided with a reduced infiltration of CD8+ T lymphocytes that produce the cytotoxic cytokines IFN-γ where invading bacteria have been geolocated. Mechanistically, the accumulation of lipid droplets in infected cancer cells relied on the production of colibactin as a measure to limit genotoxic stress to some extent. Such heightened phosphatidylcholine remodeling by the enzyme of the Land's cycle supplied CoPEC-infected cancer cells with sufficient energy for sustaining cell survival in response to chemotherapies. This accords with the lowered overall survival of colorectal patients at stage III-IV who were colonized by CoPEC when compared to patients at stage I-II. Accordingly, the sensitivity of CoPEC-infected cancer cells to chemotherapies was restored upon treatment with an acyl-CoA synthetase inhibitor. By contrast, such metabolic dysregulation leading to chemoresistance was not observed in human colon cancer cells that were infected with the mutant strain that did not produce colibactin (11G5∆ClbQ). This work revealed that CoPEC locally supports an energy trade-off lipid overload within tumors for lowering tumor immunogenicity. This may pave the way for improving chemoresistance and subsequently outcome of CRC patients who are colonized by CoPEC.

The biotherapeutic Clostridium butyricum MIYAIRI 588 strain potentiates enterotropism of Rorγt+Treg and PD-1 blockade efficacy.

Immune checkpoint inhibitors (ICI) have been positioned as a standard of care for patients with advanced non-small-cell lung carcinomas (NSCLC). A pilot clinical trial has reflected optimistic association between supplementation with Clostridium butyricum MIYAIRI 588 (CBM588) and ICI efficacy in NSCLC. However, it remains to be established whether this biotherapeutic strain may be sufficient to heighten the immunogenicity of the tumor draining lymph nodes to overcome resistance to ICI. Herein, we report that supplementation with CBM588 led to an improved responsiveness to antibody targeting programmed cell death protein 1 (aPD-1). This was statistically associated with a significant decrease in α-diversity of gut microbiota from CBM588-treated mice upon PD-1 blockade. At the level of the tumor-draining lymph node, such combination of treatment significantly lowered the frequency of microbiota-modulated subset of regulatory T cells that express Retinoic Orphan Receptor gamma t (Rorγt+ Treg). Specifically, this strongly immunosuppressive was negatively correlated with the abundance of bacteria that belong to the family of Ruminococcaceae. Accordingly, the colonic expression of both indoleamine 2,3-Dioxygenase 1 (IDO-1) and interleukin-10 (IL-10) were heightened in mice with greater PD-1 blockade efficacy. The CBM588-induced ability to secrete Interleukin-10 of lamina propria mononuclear cells was heightened in tumor bearers when compared with cancer-free mice. Conversely, blockade of interleukin-10 signaling preferentially enhanced the capacity of CD8+ T cells to secrete Interferon gamma when being cocultured with CBM588-primed lamina propria mononuclear cells of tumor-bearing mice. Our results demonstrate that CBM588-centered intervention can adequately improve intestinal homeostasis and efficiently overcome resistance to PD-1 blockade in mice.

Loss of NOD2 in macrophages improves colitis and tumorigenesis in a lysozyme-dependent manner.

Background: Crohn's disease (CD) is a complex and poorly understood myeloid-mediated disorder. Genetic variants with loss of function in the NOD2 gene confer an increased susceptibility to ileal CD. While Nod2 in myeloid cells may confer protection against T-cell mediated ileopathy, it remains unclear whether it may promote resolution of the inflamed colon. In this study, we evaluated the function of Nod2 in myeloid cells in a model of acute colitis and colitis-associated colon cancer (CAC). Methods: To ablate Nod2 specifically within the myeloid compartment, we generated LysMCre/+;Nod2fl/fl mice. The role of NOD2 was studied in a setting of Dextran Sodium Sulfate (DSS)-induced colitis and in azoxymethane (AOM)/DSS model. Clinical parameters were quantified by colonoscopy, histological, flow cytometry, and qRT-PCR analysis. Results: Upon DSS colitis model, LysMCre/+;Nod2fl/fl mice lost less weight than control littermates and had less severe damage to the colonic epithelium. In the AOM/DSS model, endoscopic monitoring of tumor progression revealed a lowered number of adenomas within the colon of LysMCre/+;Nod2fl/fl mice, associated with less expression of Tgfb. Mechanistically, lysozyme M was required for the improved disease severity in mice with a defect of NOD2 in myeloid cells. Conclusion: Our results indicate that loss of Nod2 signaling in myeloid cells aids in the tissue repair of the inflamed large intestine through lysozyme secretion by myeloid cells. These results may pave the way to design new therapeutics to limit the inflammatory and tumorigenic functions of NOD2.

Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

NOD2 in monocytes negatively regulates macrophage development through TNFalpha.

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.

A Protective Role of NOD2 on Oxazolone-induced Intestinal Inflammation Through IL-1ß-mediated Signalling Pathway.

BACKGROUND AND AIMS: NOD2 has emerged as a critical player in the induction of both Th1 and Th2 responses for potentiation and polarisation of antigen-dependent immunity. Loss-of-function mutations in the NOD2-encoding gene and deregulation of its downstream signalling pathway have been linked to Crohn's disease. Although it is well documented that NOD2 is capable of sensing bacterial muramyl dipeptide, it remains counter-intuitive to link development of overt intestinal inflammation to a loss of bacterial-induced inflammatory response. We hypothesised that a T helper bias could also contribute to an autoimmune-like colitis different from inflammation that is fully fledged by Th1 type cells. METHODS: An oedematous bowel wall with a mixed Th1/Th2 response was induced in mice by intrarectal instillation of the haptenating agent oxazolone. Survival and clinical scoring were evaluated. At several time points after instillation, colonic damage was assessed by macroscopic and microscopic observations. To evaluate the involvement of NOD2 in immunochemical phenomena, quantitative polymerase chain reaction [PCR] and flow cytometry analysis were performed. Bone marrow chimera experimentation allowed us to evaluate the role of haematopoietic/non-hematopoietic NOD2-expressing cells. RESULTS: Herein, we identified a key regulatory circuit whereby NOD2-mediated sensing of a muramyl dipeptide [MDP] by radio-resistant cells improves colitis with a mixed Th1/Th2 response that is induced by oxazolone. Genetic ablation of either Nod2 or Ripk2 precipitated oxazolone colitis that is predominantly linked to a lack of interferon-gamma. Bone marrow chimera experiments revealed that inactivation of Nod2 signalling in non-haematopoietic cells is causing a biased M1-M2 polarisation of macrophages and a decreased frequency of splenic regulatory T cells that correlates with an impaired activation of CD4 + T cells within mesenteric lymph nodes. Mechanistically, mice were protected from oxazolone-induced colitis upon administration of MDP in an interleukin-1- and interleukin-23-dependent manner. CONCLUSIONS: These findings indicate that Nod2 signalling may prevent pathological conversion of T helper cells for maintenance of tissue homeostasis.

The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult mouse gut.

The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. The signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. We set out to systematically compare the effect of two ECM classes, the interstitial matrix and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all cell types present in both cultures, albeit at different ratios. The collagen cultures contained a subset of cells enriched in fetal-like markers. In contrast, laminin increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture resembled gut development in vivo. The dramatic ECM remodelling was accompanied by a local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. Importantly, deletion of laminin in the adult mouse resulted in a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.

Long-Term Overconsumption of Fat and Sugar Causes a Partially Reversible Pre-inflammatory Bowel Disease State.

Nutrition appears to be an important environmental factor involved in the onset of inflammatory bowel diseases (IBD) through yet poorly understood biological mechanisms. Most studies focused on fat content in high caloric diets, while refined sugars represent up to 40% of caloric intake within industrialized countries and contribute to the growing epidemics of inflammatory diseases. Herein we aim to better understand the impact of a high-fat-high-sucrose diet on intestinal homeostasis in healthy conditions and the subsequent colitis risk. We investigated the early events and the potential reversibility of high caloric diet-induced damage in mice before experimental colitis. C57BL/6 mice were fed with a high-fat or high-fat high-sucrose or control diet before experimental colitis. In healthy mice, a high-fat high-sucrose diet induces a pre-IBD state characterized by gut microbiota dysbiosis with a total depletion of bacteria belonging to Barnesiella that is associated with subclinical endoscopic lesions. An overall down-regulation of the colonic transcriptome converged with broadly decreased immune cell populations in the mesenteric lymph nodes leading to the inability to respond to tissue injury. Such in-vivo effects on microbiome and transcriptome were partially restored when returning to normal chow. Long-term consumption of diet enriched in sucrose and fat predisposes mice to colitis. This enhanced risk is preceded by gut microbiota dysbiosis and transcriptional reprogramming of colonic genes related to IBD. Importantly, diet-induced transcriptome and microbiome disturbances are partially reversible after switching back to normal chow with persistent sequelae that may contribute to IBD predisposition in the general population.

NOD2 Influences Trajectories of Intestinal Microbiota Recovery After Antibiotic Perturbation.

BACKGROUND & AIMS: Loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) impair the recognition of the bacterial cell wall component muramyl-dipeptide and are associated with an increased risk for developing Crohn's disease. Likewise, exposure to antibiotics increases the individual risk for developing inflammatory bowel disease. Here, we studied the long-term impact of NOD2 on the ability of the gut bacterial and fungal microbiota to recover after antibiotic treatment. METHODS: Two cohorts of 20-week-old and 52-week-old wild-type (WT) C57BL/6J and NOD2 knockout (Nod2-KO) mice were treated with broad-spectrum antibiotics and fecal samples were collected to investigate temporal dynamics of the intestinal microbiota (bacteria and fungi) using 16S ribosomal RNA and internal transcribed spacer 1 sequencing. In addition, 2 sets of germ-free WT mice were colonized with either WT or Nod2-KO after antibiotic donor microbiota and the severity of intestinal inflammation was monitored in the colonized mice. RESULTS: Antibiotic exposure caused long-term shifts in the bacterial and fungal community composition. Genetic ablation of NOD2 was associated with delayed body weight gain after antibiotic treatment and an impaired recovery of the bacterial gut microbiota. Transfer of the postantibiotic fecal microbiota of Nod2-KO mice induced an intestinal inflammatory response in the colons of germ-free recipient mice compared with respective microbiota from WT controls based on histopathology and gene expression analyses. CONCLUSIONS: Our data show that the bacterial sensor NOD2 contributes to intestinal microbial community composition after antibiotic treatment and may add to the explanation of how defects in the NOD2 signaling pathway are involved in the etiology of Crohn's disease.

Lactobacillus reuteri 5454 and Bifidobacterium animalis ssp. lactis 5764 improve colitis while differentially impacting dendritic cells maturation and antimicrobial responses.

Crohn's disease is linked to a decreased diversity in gut microbiota composition as a potential consequence of an impaired anti-microbial response and an altered polarization of T helper cells. Here, we evaluated the immunomodulatory properties of two potential probiotic strains, namely a Bifidobacterium animalis spp. lactis Bl 5764 and a Lactobacillus reuteri Lr 5454 strains. Both strains improved colitis triggered by either 2,4,6-trinitrobenzenesulfonic acid (TNBS) or Citrobacter rodentium infection in mice. Training of dendritic cells (DC) with Lr 5454 efficiently triggered IL-22 secretion and regulatory T cells induction in vitro, while IL-17A production by CD4+ T lymphocytes was stronger when cultured with DCs that were primed with Bl 5764. This strain was sufficient for significantly inducing expression of antimicrobial peptides in vivo through the Crohn's disease predisposing gene encoding for the nucleotide-binding oligomerization domain, containing protein 2 (NOD2). In contrast, NOD2 was dispensable for the impact on antimicrobial peptide expression in mice that were monocolonized with Lr 5454. In conclusion, our work highlights a differential mode of action of two potential probiotic strains that protect mice against colitis, providing the rational for a personalized supportive preventive therapy by probiotics for individuals that are genetically predisposed to Crohn's disease.

The regenerating family member 3 ß instigates IL-17A-mediated neutrophil recruitment downstream of NOD1/2 signalling for controlling colonisation resistance independently of microbiota community structure.

OBJECTIVE: Loss of the Crohn's disease predisposing NOD2 gene results in an intestinal microenvironment conducive for colonisation by attaching-and-effacing enteropathogens. However, it remains elusive whether it relies on the intracellular recruitment of the serine-threonine kinase RIPK2 by NOD2, a step that is required for its activation of the transcription factor NF-κB. DESIGN: Colonisation resistance was evaluated in wild type and mutant mice, as well as in ex-germ-free (ex-GF) mice which were colonised either with faeces from Ripk2-deficient mice or with bacteria with similar preferences for carbohydrates to those acquired by the pathogen. The severity of the mucosal pathology was quantified at several time points postinfection by using a previously established scoring. The community resilience in response to infection was evaluated by 16S ribosomal RNA gene sequence analysis. The control of pathogen virulence was evaluated by monitoring the secretion of Citrobacter-specific antibody response in the faeces. RESULTS: Primary infection was similarly outcompeted in ex-GF Ripk2-deficient and control mice, demonstrating that the susceptibility to infection resulting from RIPK2 deficiency cannot be solely attributed to specific microbiota community structures. In contrast, delayed clearance of Citrobacter rodentium and exacerbated histopathology were preceded by a weakened propensity of intestinal macrophages to afford innate lymphoid cell activation. This tissue protection unexpectedly required the regenerating family member 3ß by instigating interleukin (IL) 17A-mediated neutrophil recruitment to the intestine and subsequent phosphorylation of signal transducer and activator of transcription 3. CONCLUSIONS: These results unveil a previously unrecognised mechanism that efficiently protects from colonisation by diarrhoeagenic bacteria early in infection.

Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens.

Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-κB and subsequent activity of the JAK/STAT signaling pathway. While NLRP12 deficiency renders septic mice highly susceptible towards MDP, a sustained sensing of MDP through NOD2 is observed among monocytes lacking NLRP12. This loss of tolerance in monocytes results in greater colonization resistance towards Citrobacter rodentium. Our data show that this is a consequence of NOD2-dependent accumulation of inflammatory mononuclear cells that correlates with induction of interferon-stimulated genes. Our study unveils a relevant process of tolerance towards the gut microbiota that is exploited by an attaching/effacing enteric pathogen.

Innate lymphoid cells sustain colon cancer through production of interleukin-22 in a mouse model.

Patients with inflammatory bowel disease (IBD) have an increased risk of colon cancer. However, the immune cells and cytokines that mediate the transition from intestinal inflammation to cancer are poorly understood. We show that bacteria-induced colon cancer is accompanied by differential accumulation of IL-17(+)IL-22(+) colonic innate lymphoid cells (cILCs), which are phenotypically distinct from LTi and NK-22 cells, and that their depletion in mice with dysplastic inflammation blocks the development of invasive colon cancer. Analysis of the functional role of distinct Type 17 cytokines shows that although blockade of IL-17 inhibits some parameters of intestinal inflammation, reduction in dysplasia and colorectal cancer (CRC) requires neutralization of IL-22 indicating a unique role for IL-22 in the maintenance of cancer in this model. Mechanistic analyses showed that IL-22 selectively acts on epithelial cells to induce Stat3 phosphorylation and proliferation. Importantly, we could detect IL-22(+)CD3(+) and IL-22(+)CD3(−) cells in human CRC. Our results describe a new activity of IL-22 in the colon as a nonredundant mediator of the inflammatory cascade required for perpetuation of CRC, highlighting the IL-22 axis as a novel therapeutic target in colon cancer.

Identification of a genetic locus controlling bacteria-driven colitis and associated cancer through effects on innate inflammation.

Chronic inflammation of the intestine has been associated with an elevated risk of developing colorectal cancer. Recent association studies have highlighted the role of genetic predisposition in the etiology of colitis and started to unravel its complexity. However, the genetic factors influencing the progression from colon inflammation to tumorigenesis are not known. We report the identification of a genetic interval Hiccs that regulates Helicobacter hepaticus-induced colitis and associated cancer susceptibility in a 129.RAG(-/-) mouse model. The 1.7-Mb congenic interval on chromosome 3, containing eight genes and five microRNAs, renders susceptible mice resistant to colitis and reduces tumor incidence and multiplicity. Bone marrow chimera experiments showed that resistance is conferred by the hematopoietic compartment. Moreover, the Hiccs locus controls the induction of the innate inflammatory response by regulating cytokine expression and granulocyte recruitment by Thy1(+) innate lymphoid cells. Using a tumor-promoting model combining chronic Helicobacter hepaticus infection and the carcinogen azoxymethane, we found that Hiccs also regulates the frequency of colitis-associated neoplasia. Our study highlights the importance of innate immune cells and their genetic configuration in driving progression from inflammation toward cancer and opens the door for analysis of these pathways in human inflammatory disorders and associated cancers.

Pathogenic and protective roles of MyD88 in leukocytes and epithelial cells in mouse models of inflammatory bowel disease.

BACKGROUND & AIMS: Toll-like receptors (TLR) are innate immune receptors involved in recognition of the intestinal microflora; they are expressed by numerous cell types in the intestine, including epithelial cells, myeloid cells, and lymphocytes. Little is known about the relative contributions of TLR signaling in distinct cellular compartments to intestinal homeostasis. We aimed to define the roles of TLR signals in distinct cell types in the induction and regulation of chronic intestinal inflammation. METHODS: We assessed the roles of the shared TLR signaling adaptor protein, MyD88, in several complementary mouse models of inflammatory bowel disease, mediated by either innate or adaptive immune activation. MyD88-deficient mice and bone marrow chimeras were used to disrupt TLR signals selectively in distinct cellular compartments in the intestine. RESULTS: MyD88-dependent activation of myeloid cells was required for the development of chronic intestinal inflammation. By contrast, although epithelial cell MyD88 signals were required for host survival, they were insufficient to induce intestinal inflammation in the absence of an MyD88-competent myeloid compartment. MyD88 expression by T cells was not required for their pathogenic and regulatory functions in the intestine. CONCLUSIONS: Cellular compartmentalization of MyD88 signals in the intestine allow the maintenance of host defense and prevent deleterious inflammatory responses.

TLR2-independent induction and regulation of chronic intestinal inflammation.

Interactions between the intestinal microflora and host innate immune receptors play a critical role in intestinal homeostasis. Several studies have shown that TLR2 can modulate inflammatory responses in the gut. TLR2 signals enhance tight junction formation and fortify the epithelial barrier, and may play a crucial role in driving acute inflammatory responses towards intestinal bacterial pathogens. In addition, TLR2 agonists can have direct effects on both Th1 cells and Treg. To define the role of TLR2 in the induction and regulation of chronic intestinal inflammation we examined the effects of TLR2 deletion on several complementary models of inflammatory bowel disease. Our results show that TLR2 signals are not required for the induction of chronic intestinal inflammation by either innate or adaptive immune responses. We further show that TLR2(-/-) mice harbor normal numbers of Foxp3(+) Treg that are able to suppress intestinal inflammation as effectively as their WT counterparts. We also did not find any intrinsic role for TLR2 for pathogenic effector T-cell responses in the gut. Thus, in contrast to their role in acute intestinal inflammation and repair, TLR2 signals may have a limited impact on the induction and regulation of chronic intestinal inflammation.

Mapping non-class II H2-linked loci for type 1 diabetes in nonobese diabetic mice.

Major histocompatibility complex (MHC) plays a largely predominant role in the genetic predisposition to type 1 diabetes, in both humans and rodents. While class II loci have long been recognized as essential, they do not fully explain the MHC-linked genetic component of type 1 diabetes. In the present study, using new NOD congenic strains harboring defined chromosomal segments from C57BL/6 mice, we circumscribed three distinct loci influencing murine type 1 diabetes and tightly linked to but separated from the class II region. Our findings might guide the search for additional HLA-linked loci in human type 1 diabetes.

An interval tightly linked to but distinct from the H2 complex controls both overt diabetes (Idd16) and chronic experimental autoimmune thyroiditis (Ceat1) in nonobese diabetic mice.

The major histocompatibility complex (MHC) has long been associated with predisposition to several autoimmune diseases, including type 1 diabetes and autoimmune thyroiditis. In type 1 diabetes, a primary role has been assigned to class II genes, both in humans and in the nonobese diabetic (NOD) mouse model. However, an involvement of other tightly linked genes is strongly suspected. Here, through two independent sets of experiments, we provide solid evidence for the existence of at least one such gene. First, using a new recombinant congenic NOD strain, R114, we definitively individualized the Idd16 locus from the MHC in a 6-cM interval proximal to H2-K. It affords almost complete protection against diabetes and is associated with delayed insulitis. Second, by genome scan, we mapped non-H2 genes associated with the highly penetrant form of chronic experimental autoimmune thyroiditis (EAT) that is elicited in NOD and NOD.H2(k) mice by immunization with thyroglobulin. We identified one major dominant locus, Ceat1, on chromosome 17, overlapping with Idd16. Most importantly, R114 recombinant congenic mice challenged with thyroglobulin did not develop chronic EAT. This new major region defined by both Idd16 and Ceat1 might thus concur to the unique strength of the MHC in autoimmune susceptibility of NOD mice.

Genetic analysis of autoimmune sialadenitis in nonobese diabetic mice: a major susceptibility region on chromosome 1.

The nonobese diabetic (NOD) mouse strain provides a good study model for Sjögren's syndrome (SS). The genetic control of SS was investigated in this model using different matings, including a (NOD x C57BL/6 (B6))F(2) cross, a (NOD x NZW)F(2) cross, and ((NOD x B6) x NOD) backcross. Multiple and different loci were detected depending on parent strain combination and sex. Despite significant complexity, two main features were prominent. First, the middle region of chromosome 1 (chr.1) was detected in all crosses. Its effect was most visible in the (NOD x B6)F(2) cross and dominated over that of other loci, including those mapping on chr.8, 9, 10, and 16; the effect of these minor loci was observed only in the absence of the NOD haplotype on chr.1. Most critically, the chr.1 region was sufficient to trigger an SS-like inflammatory infiltrate of salivary glands as shown by the study of a new C57BL/6 congenic strain carrying a restricted segment derived from NOD chr.1. Second, several chromosomal regions were previously associated with NOD autoimmune phenotypes, including Iddm (chr.1, 2, 3, 9, and 17, corresponding to Idd5, Idd13, Idd3, Idd2, and Idd1, respectively), accounting for the strong linkage previously reported between insulitis and sialitis, and autoantibody production (chr.10 and 16, corresponding to Bana2 and Bah2, respectively). Interestingly, only two loci were detected in the (NOD x NZW)F(2) cross, on chr.1 in females and on chr.7 in males, probably because of the latent autoimmune predisposition of the NZW strain. Altogether these findings reflect the complexity and heterogeneity of human SS.