一個非結核分枝桿菌肺病相關的NLRP3基因單一核苷酸多型性位點之生化作用機制

Abstract

分枝桿菌屬（Mycobacterium）為可以被酸性染劑染色的耐酸菌，包含兩種會造成嚴重疾病的菌種：肺結核分枝桿菌（Mycobacterium tuberculosis）以及痲瘋分枝桿菌（Mycobacterium leprae）。除此之外，還有一些分枝桿菌也會造成臨床的症狀，並被統稱為非結核分枝桿菌（nontuberculous mycobacteria, NTM）。傳統上，人們認為感染NTM所引發的疾病只會發生在少數免疫缺乏的病患身上。然而，近十年來，NTM所造成的肺部疾病（nontuberculous mycobacterial lung disease, NTM-LD）在免疫力正常的族群中，發生率及盛行率皆有增加的趨勢。會有這樣的現象除了微生物學的檢驗技術進步之外，也代表著有些免疫功能正常的患者中，可能帶有其他的風險因子，讓宿主對於疾病具有易感性（susceptibility）。然而，宿主對NTM-LD易感性的機制目前仍舊不清楚。在東亞以及美國地區，最常見會造成NTM-LD的菌種為Mycobacterium avium complex（MAC）。而在宿主受到MAC感染時，會引發體內的巨噬細胞和單核球產生發炎體複合物。發炎體複合物包含不同的感測器（sensor），包括NLRP3，當細胞偵測到病原體相關分子樣式（PAMPs）時NLRP3會被活化。此外，由NLRP3所組成的發炎體已被證實，對於預防分枝桿菌的感染扮演重要的角色。先前的研究中發現，當NLRP3發炎體的表現減少會減弱巨噬細胞的免疫反應。同時也透過分析找到NLRP3基因上數個與MAC-LD有相關性的單一核苷酸多型性（SNP）位點，而rs34298354位點與女性MAC-LD病人群體具有顯著的相關性。為了探討此位點和宿主對於MAC感染的易感性之角色，我們首先在 HEK293T 細胞中表現rs34298354位點有不同等位基因的NLRP3蛋白質。結果顯示，在此位點為T等位基因的 NLRP3 蛋白質表現量有減少的情形。此外，在此位點為T等位基因的 NLRP3基因所轉錄的 RNA顯示出較低的穩定性，這說明NLRP3的rs34298354位點在RNA穩定性中非常重要。為了進一步研究 NLRP3 蛋白質表現量和巨噬細胞功能的相關性，目前正以 CRISPR-Cas9 基因組編輯系統建立在此位點具有兩個等位基因的 THP1 單核細胞白血病細胞株。同時，目前已經以同一系統成功建立在此位點具有兩個等位基因T的 HEK293T 細胞株。藉由建立的這些HEK293T 細胞株，驗證了 NLRP3 基因的 rs34298354 位點確實會影響 RNA 的穩定性和蛋白質的表現量。

Mycobacteria are acid-fast bacteria, including Mycobacterium tuberculosis and Mycobacterium leprae, both associated with serious diseases. In addition to those, nontuberculous mycobacteria (NTM) are another group of mycobacteria that cause clinical diseases. Traditionally, it has been thought that NTM infection-caused diseases only happened in immunocompromised people. However, the incidence and prevalence of NTM-lung disease (LD) increased in the last ten years in patients with normal immune systems. Besides the advancement of inspection techniques for microbiology, this increase is also attributed to other risk factors in patients without immune dysfunction. However, the mechanism of the host susceptibility to NTM-LD still remains unclear. Mycobacterium avium complex (MAC) is the most common pathogen associated with NTM-LD in the East Asia and the United States, and inflammasome complex is found to assemble during MAC infection in macrophages and monocytes. Inflammasome complex contains a variety of sensors, including NLRP3, which is induced when pathogen-associated molecular patterns (PAMPs) are detected. Besides, NLRP3-inflammasome plays an important role for preventing mycobacterial infection. It has been demonstrated that impaired expression of NLRP3-inflammasome in macrophages weakens inflammatory responses and several single nucleotide polymorphisms (SNPs) of NLRP3 gene are associated with the MAC-LD. Moreover, the SNP rs34298354 of NLRP3 is significantly associated with MAC-LD in female patients. To investigate the role of the SNP in host susceptibility to MAC infection, NLRP3 protein with different alleles at the SNP rs34298354 was over-expressed in HEK293T cells. The results revealed that the protein level of NLRP3 with T allele at the SNP rs34298354 was decreased. Besides, the transcript from NLRP3 gene with T allele at the SNP rs34298354 showed a significantly lower stability. It suggests that the SNP rs34298354 of NLRP3 plays an important role in RNA stability. To further investigate the association of NLRP3 protein level and macrophage functions, THP1 monocytic leukemia cell lines with two alleles at the SNP rs34298354 have being established via a CRISPR-Cas9 genome editing system. In parallel, HEK293T cell lines with two alleles at the SNP rs34298354 have being established successfully via the same system. Using the generated HEK293T cell lines, it has been validated that the SNP rs34298354 of NLRP3 has molecular effect on RNA stability and protein level.