Understanding how perturbations in microbial mimicry promotes breakdown in tolerance to insulin

背景1型糖尿病（T1D）是一种自身免疫性疾病，由t细胞介导的产生胰岛素的胰岛细胞破坏引起。人白细胞抗原（HLA）ⅱ类分子多态性是T1D的主要遗传危险因素。最近，一种宿主-微生物相互作用解释了HLA II类的易感性。本文鉴定了交叉反应性T细胞，这些细胞识别胰岛素与易患t1d的II类分子DQ8结合的微生物模拟物。t1d保护性HLA-DQ6分子强烈结合微生物模拟胰岛素，与交叉反应调节性T细胞（Treg）在降低疾病风险中的作用一致。而易患t1d的HLA-DQ8分子与这些肽的结合更弱，有利于交叉反应效应T细胞（Teff）从胸腺逃逸，增强抗胰岛素自身免疫。大多数微生物模拟物属于转酮醇酶（TKT）超家族。TKT是一种参与加工膳食纤维的酶，在婴儿断奶期间上调2。这种从牛奶到固体食物的转变会引起肠道菌群的迅速扩张，是产生耐受性的关键时期。由于胰岛素的第一个自身抗体最常出现在断奶后，9-18个月大，这表明自身免疫性糖尿病的主要原因。我们假设微生物- tkt的异常表达破坏了免疫稳态，有利于激活交叉反应性Teffs，从而触发对胰岛素的自身免疫。这些扰动可能是由生态失调的微生物类群驱动的，它们表达了不适当的TKT量。研究设计通过GPPAD联盟4,5收集了670名T1D高风险儿童的纵向粪便和血液样本（基于遗传风险评分（GRS））6。自身抗体水平、血液代谢组和肠道微生物组的纵向变化将通过T1D-GRS、t1d自身抗体的存在和临床状态进行监测和分层。在我们的实验室中，从DMech-biome项目中收集了445名婴儿的唾液和粪便样本（8周龄和8个月龄）。由于这些参与者不是根据T1D-GRS选择的，他们可以作为人口控制队列。将分析微生物组组成、hla单倍型和其他遗传风险位点之间的相互作用。宏基因组分析的纵向比较将有助于深入了解断奶和早期生命期间肠道微生物组组成的变化。该分析可以推断微生物- tkt表达的变化，并确定可能促进交叉反应Teff激活的潜在生态不良微生物类群。HLA四聚体的开发将评估识别胰岛素和微生物- tkt肽的交叉反应T细胞的存在。将不同的HLA II类四聚体与微生物环境分子结合，再加上荧光激活的细胞分选进行评估，将有助于深入了解不同HLA单倍型如何促进交叉反应性Teffs的产生。这可以评估耐受性微生物环境分子是否可以通过易感HLA分子促进Treg的产生。影响：本项目旨在通过微生物- tkt的不恰当表达和呈现，评估生命早期发育的益生菌、促炎菌群是否会增加患T1D的风险。通过纵向评估，微生物群内的变化和微生物- tkt的表达可以与T1D自身抗体的发展和临床状况进行比较。微生物菌株的鉴定对于通过激活交叉反应Treg获得耐受性很重要，可以为预防性微生物组靶向治疗的开发提供信息。Garcia， A. R.等MedXiv 2022。齐格勒，A. G.等。Vatanen， T. et al . Nature 2018。齐格勒，A. G.等。al Nabhani， Z.等。雷东多

BackgroundType 1 diabetes (T1D) is an autoimmune disease, resulting from T-cell mediated destruction of insulin-producing pancreatic islet cells. Polymorphisms of human leukocyte antigen (HLA) class II molecules are major genetic risk factors for T1D. Recently, a host-microbiome interaction, explaining HLA class II susceptibility, has been characterised1. This paper identified cross-reactive T cells that recognised microbial-mimics of insulin bound to the T1D-predisposing class II molecule DQ8. The T1D-protective HLA-DQ6 molecule strongly bound microbial mimics of insulin, consistent with a role for cross-reactive regulatory T cells (Treg) in lowering disease risk. While the T1D-predisposing HLA-DQ8 molecule bound these peptides more weakly, favouring escape of cross-reactive effector T cells (Teff) from the thymus and increased anti-insulin autoimmunity. The majority of microbial-mimics belonged to the transketolase (TKT) superfamily. TKT is an enzyme involved in processing dietary fibre and is upregulated during infant weaning2. This transition, from milk to solid food, induces rapid expansion of gut microbiota, and is a critical period for developing tolerance3. As the first autoantibodies to insulin appear most often post-weaning, at 9-18 months of age, this suggests a primary cause of autoimmune diabetes.We hypothesise that aberrant expression of microbial-TKT disrupts immune homeostasis, favouring activation of cross-reactive Teffs, to trigger autoimmunity against insulin. These perturbations are likely to be driven by dysbiotic microbial taxa, expressing inappropriate amounts of TKT. Research DesignThrough the GPPAD consortium4,5, longitudinal stool and blood samples have been collected from 670 children, with a high risk of T1D based on their genetic risk score (GRS)6. Longitudinal changes in autoantibody levels, blood metabolomes, and gut microbiome will be monitored and stratified by T1D-GRS, presence of T1D-autoantibodies, and clinical status.In our lab, from the DMech-biome project, saliva and stool samples (at 8 weeks and 8 months of age) from 445 infants have been collected. As these participants have not been selected based on T1D-GRS, they can act as a population control cohort. Interactions between the microbiome composition, HLA-haplotypes, and other genetic risk loci will be analysed. Longitudinal comparisons of metagenomic analysis will allow insights into changes in gut microbiome composition across weaning and early life. This analysis could infer changes in microbial-TKT expression, and identify potential dysbiotic microbial taxa that may promote activation of cross-reactive Teff.Development of HLA tetramers will assess the presence of cross-reactive T cells recognising both insulin and microbial-TKT peptides. Assessment of different HLA class II tetramers in combination with microbial context-molecules, coupled with fluorescent-activated cell sorting, will allow insight into how different HLA-haplotypes favour production of cross-reactive Teffs. This could assess whether tolerogenic microbial context-molecules could promote Treg production despite presentation through predisposing HLA molecules.ImpactThis project aims to assess whether development of a dysbiotic, proinflammatory microbiome in early life can increase risk of developing T1D, through inappropriate expression and presentation of microbial-TKT. Through longitudinal assessment, changes within microbiota taxa and expression of microbial-TKT can be compared to development of T1D autoantibodies and clinical status. The identification of microbial strains, important for conferring tolerance through activation of cross-reactive Treg could inform development of preventative microbiome-targeted therapies.References1. Garcia, A. R. et al MedXiv 2022 4. Ziegler, A. G. et al BMJ Open 20192. Vatanen, T. et al Nature 2018 5. Ziegler, A. G. et al BMJ Open 20213. al Nabhani, Z. et al Immunity 2019 6. Redondo