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

人类基因组包含大量的顺式和反式调控元件，它们在基因转录到翻译的不同阶段调节基因活性。在全基因组关联研究（GWAS）中，基因组非编码或调控区域的改变与自身免疫性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。我们已经在NLRP3的3Ꞌ-UTR（非翻译区）（代谢功能障碍的胞质先天免疫传感器）中发现了多个转录后调控元件，这些元件影响NLRP3 mRNA的稳定性并与T1D风险相关。NLRP3的激活导致称为炎症小体的大寡聚信号复合物的组装，炎症小体促进炎症细胞死亡的一种形式，称为焦亡，并产生生物活性IL-1β，一种与T1D病理生理有关的促炎细胞因子。在NOD（非肥胖糖尿病）小鼠模型中，NLRP3缺乏通过破坏IL-1β的产生和减少致病性T细胞向胰岛的迁移来保护T1D的发展，这表明NLRP3炎症小体在T1D的发病过程中起着关键作用。虽然这组相关事件被很好地描述，但对控制NLRP3表达、设置炎性体激活阈值和与T1D发展相关的病理事件的转录后机制知之甚少。我们的遗传数据证明，人类NLRP3基因3Ꞌ-UTR的单核苷酸多态性在功能上增加NLRP3 mRNA，与T1D风险显著相关，这表明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化位点，它们编码一个稳定的短3Ꞌ-UTR或一个不太稳定的长3Ꞌ-UTR。较长的3Ꞌ-UTR包含几个顺式调控基序，如富au元件、miRNA结合位点和翻译元件的γ干扰素抑制剂，已知这些基序可抑制蛋白质表达。基于这些数据，我们假设优先使用短的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。在本次资助中，我们将在NOD背景下产生携带NLRP3短3'-UTR变体和长3'-UTR变体的小鼠，目的是通过3Ꞌ-UTR的交替使用来解剖NLRP3基因剂量对体内T1D发生和进展的影响。我们的研究将产生基础小鼠模型来研究转录后调控在T1D发展中的作用，并将对理解由遗传变异引起的表达变化如何影响T1D等复杂自身免疫性疾病的发展具有更广泛的意义。

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.