Does post-transcriptional control of NLRP3 inflammasome activity impact development of type 1 diabetes?

人类基因组包含大量顺式和反式作用调节元件，它们在从基因转录到翻译的过程中的不同点调节基因活性。全基因组关联研究 (GWAS) 中，基因组非编码或调控区域的改变与自身免疫 1 型糖尿病 (T1D) 有关，这表明基因或基因产物表达的变化可能在功能性免疫失调中发挥重要作用。我们在 NLRP3（一种代谢功能障碍的胞质先天免疫传感器）的 3Ꞌ-UTR（非翻译区）中发现了多个转录后调节元件，这些元件影响 NLRP3 mRNA 的稳定性并与 T1D 风险相关。 NLRP3 激活会导致称为炎症小体的大型寡聚信号复合物的组装，促进一种称为焦亡的炎症细胞死亡形式，并产生生物活性 IL-1β（一种与 T1D 病理生理学有关的促炎细胞因子）。在 NOD（非肥胖糖尿病）小鼠模型中，NLRP3 缺陷通过 IL-1β 的产生受损和致病性 T 细胞向胰岛的迁移减少来防止 T1D 的发展，这表明 NLRP3 炎性体在 T1D 发病机制中发挥着关键作用。虽然这组相关事件已得到很好的描述，但人们对控制 NLRP3 表达并设定炎症体激活阈值以及与 T1D 发展相关的病理事件的转录后机制知之甚少。我们的遗传数据表明，人类 NLRP3 基因 3Ꞌ-UTR 中的单核苷酸多态性（功能性增加 NLRP3 mRNA）与 T1D 风险显着相关，表明 NLRP3 的增加可能在疾病发展中发挥作用。我们发现NLRP3基因也有两个聚腺苷酸化位点，它们要么编码稳定的短3Ꞌ-UTR，要么编码不太稳定的较长3Ꞌ-UTR。较长的 3Ꞌ-UTR 包含多个顺式作用调节基序，例如富含 AU 的元件、miRNA 结合位点和翻译元件的 γ 干扰素抑制剂，已知这些元件可抑制蛋白质表达。基于这些数据，我们假设优先使用短 3'-UTR 而不是长 3'-UTR 会增加 NLRP3 的基因剂量，从而使细胞对 NLRP3 激活剂敏感并降低 T1D 中 NLRP3 炎性体激活的阈值。在这笔资助中，我们将生成在 NOD 背景上携带 NLRP3 短与长 3'-UTR 变体的小鼠，目的是通过交替使用 3'-UTR 来剖析 NLRP3 基因剂量对体内 T1D 发生和进展的影响。我们的研究将生成基础小鼠模型来研究转录后调控在 T1D 发展中的作用，并将对理解遗传变异引起的表达变化如何影响复杂的自身免疫性疾病（如 T1D）的发展产生更广泛的影响。

The human genome contains a large number of cis- and trans-acting regulatory elements that modulate gene activity at different points in the progression from gene transcription to translation. Alterations in non-coding or regulatory regions of the genome are linked to autoimmune type 1 diabetes (T1D) in Genome-wide association studies (GWAS), suggesting that changes in gene or gene product expression might play an important role in functional immune dysregulation. We have identified multiple post-transcriptional regulatory elements in the 3Ꞌ-UTR (untranslated region) of NLRP3, a cytosolic innate immune sensor of metabolic dysfunction, that impact NLRP3 mRNA stability and are associated with T1D risk. NLRP3 activation results in assembly of a large oligomeric signaling complex called the inflammasome that promotes a form of inflammatory cell death called pyroptosis and production of bioactive IL-1β, a pro-inflammatory cytokine implicated in the pathophysiology of T1D. In the NOD (non-obese diabetic) mouse model, NLRP3 deficiency protects from development of T1D through impaired production of IL-1β and reduced migration of pathogenic T cells to the pancreatic islets, suggesting a critical role for the NLRP3 inflammasome in T1D pathogenesis. While this set of linked events is well described, little is known about the post-transcriptional mechanisms that control the expression NLRP3 and set the threshold for inflammasome activation and the pathological events associated with development of T1D. Our genetic data document that a single nucleotide polymorphism in the 3Ꞌ-UTR of the human NLRP3 gene that functionally increases NLRP3 mRNA is significantly associated with T1D risk suggesting that an increase in NLRP3 may play a role in disease development. We have found that the NLRP3 gene also has two polyadenylation sites, which either encode for a stable short 3Ꞌ-UTR or a less stable longer 3Ꞌ-UTR. The longer 3Ꞌ-UTR harbors several cis acting regulatory motifs, such as AU-rich elements, miRNA binding sites and gamma interferon inhibitor of translation elements, which are known to suppress protein expression. Based on these data we hypothesize that preferential usage of the short 3'-UTR as opposed to the long 3'-UTR increases the gene dosage of NLRP3 thereby sensitizing cells to NLRP3 activators and lowering the threshold for NLRP3 inflammasome activation in T1D. In this grant we will generate mice carrying the short vs. long 3'-UTR variants of NLRP3 on the NOD background with the goal of dissecting the impact of gene dosage of NLRP3 via alternate 3Ꞌ-UTR usage on the development and progression of T1D in vivo. Our study will generate foundational mouse models to study the role of post-transcriptional regulation in development of T1D and will have broader implications for understanding how expression changes caused by genetic variation impact the development of complex autoimmune diseases like T1D.