AIM2 and IFI16 Innate Immune Sensors for Cytosolic DNA in Prostatic Diseases

描述（由申请人提供）： 研究表明，前列腺炎症与年龄相关性前列腺疾病（如良性前列腺增生（BPH）和前列腺癌（PC））的发展之间存在密切联系。然而，导致前列腺炎症的分子机制在很大程度上仍然未知。研究已经确定了胞质双链DNA（dsDNA）传感器蛋白在引发导致慢性炎症的先天免疫应答中的作用。这些蛋白质包括NALP 3和干扰素（IFN）诱导型AIM 2和IFI 16。在感测胞质dsDNA时，AIM 2和NALP 3蛋白募集衔接蛋白ASC以形成炎性体，其促进促炎细胞因子如IL-1和IL-18的分泌。然而，在感测胞质dsDNA时，IFI 16蛋白募集干扰素基因刺激物（STING）蛋白以刺激IFN-γ的表达，而在感测核dsDNA时，该蛋白募集ASC蛋白以形成炎性小体。基于我们的初步观察，我们假设在前列腺上皮细胞（PrEC）中通过AIM 2和IFI 16蛋白感测dsDNA触发先天免疫应答，其有助于促炎细胞因子的产生增加。此外，我们假设PrEC中AIM 2和IFI 16蛋白之间的物理和功能相互作用激活NF-B的转录活性，从而促进促炎细胞因子和趋化因子的表达，从而导致慢性前列腺炎症。目标一：我们试图确定I型和II型IFN调节AIM 2蛋白表达水平以及AIM 2和IFI 16蛋白在人正常PrEC和癌细胞系中的亚细胞定位的分子机制。这些方法包括凝胶迁移率变动分析、启动子-报告基因分析、STAT 1表达敲低、突变分析和染色质免疫沉淀分析（ChIP）。目标二：表征AIM 2和其他DNA传感器在人类正常PrEC、前列腺癌细胞系和前列腺病变中胞质DNA触发的先天免疫应答中的作用。具体而言，我们建议：（i）研究胞质dsDNA是否触发PrEC和前列腺癌细胞系中的AIM 2和其他炎性小体激活;和（ii）检查炎症相关前列腺疾病中AIM 2和其他DNA传感蛋白的表达水平和亚细胞定位。将使用免疫组织化学、微阵列和定量实时PCR。目的#3：研究AIM 2和IFI 16蛋白之间的相互作用如何促进PrEC中NF-B活性的调节。将使用生物化学和分子细胞生物学方法，包括ChIP。重要性：拟议的研究可能会确定AIM 2和IFI 16先天免疫传感器dsDNA有助于前列腺炎症和炎症相关前列腺疾病的发展的分子机制。

DESCRIPTION (provided by applicant): Studies indicate close links between prostatic inflammation and the development of aging-associated prostatic diseases such as benign prostate hyperplasia (BPH) and prostate cancer (PC). However, the molecular mechanisms that contribute to prostatic inflammation remain largely unknown. Studies have identified a role for cytosolic double-stranded DNA (dsDNA) sensor proteins in initiating innate immune responses that contribute to chronic inflammation. These proteins include NALP3 and the interferon (IFN) - inducible AIM2 and IFI16. Upon sensing cytosolic dsDNA, the AIM2 and NALP3 proteins recruit adaptor protein ASC to form an inflammasome, which promotes the secretion of pro-inflammatory cytokines such as IL-1¿ and IL-18. However, upon sensing cytosolic dsDNA, the IFI16 protein recruits the stimulator of interferon genes (STING) protein to stimulate the expression of IFN-¿, whereas upon sensing the nuclear dsDNA the protein recruits ASC protein to form an inflammasome. Based on our preliminary observations, we hypothesize that sensing of dsDNA by AIM2 and IFI16 proteins in prostate epithelial cells (PrECs) triggers innate immune responses that contribute to an increased production of proinflammatory cytokines. Additionally, we postulate that the physical and functional interactions between the AIM2 and IFI16 proteins in PrECs activate the transcriptional activity of NF-¿B that promotes the expression of the proinflammatory cytokines and chemokines; thus, leading to the chronic prostatic inflammation. Aim # 1: We seek to identify the molecular mechanisms by which type I and type II IFNs regulate the expression levels of AIM2 protein and subcellular localization of the AIM2 and IFI16 proteins in human normal PrECs and cancer cell lines. The approaches, including gel-mobility shift assays, promoter-reporter assays, knockdown of STAT1 expression, mutational analyses, and chromatin immunoprecipitation assays (ChIPs) will be used. Aim # 2: Characterize the role of AIM2 and other DNA sensors in cytosolic DNA-triggered innate immune responses in human normal PrECs, prostate cancer cell lines, and the prostatic lesions. Specifically, we propose to: (i) investigate whether cytosolic dsDNA triggers the AIM2 and other inflammasome activation in PrECs and prostate cancer cell lines; and (ii) examine expression levels and subcellular localization of the AIM2 and other DNA sensor proteins in inflammation-associated prostatic diseases. Immunohistochemistry, microarray, and quantitative real-time PCR will be used. Aim # 3: Investigate how interactions between the AIM2 and IFI16 proteins contribute to the modulation of NF-¿B activity in PrECs. Biochemical and molecular cell biology approaches including ChIPs will be used. Significance: Proposed studies are likely to identify the molecular mechanisms through which the AIM2 and IFI16 innate immune sensors for dsDNA contribute to prostatic inflammation and the development of inflammation-associated prostatic diseases.