질편모충에 의한 전립선 상피세포주의 IL-1β생산에서 NLRP3 inflammasome의 역할

Abstract

질편모충 (Trichomonas vaginalis)은 주로 성적 접촉에 의해 사람의 비뇨생식계에 감염되는 기생 원충으로 성병을 일으키는 병원체 중 가장 흔한 원인이 된다. 선천면역의 구성성분인 inflammasome은 pro-IL-1β를 활성형 IL-1β로 성숙시키는데 관여하는 복합체이다. NLRP3 inflammasome은 세포내 병원체가 대식세포에 감염되었을 때 선천면역에서 중요한 역할을 한다고 알려졌으나, 질편모충에 감염된 인체 전립선 상피세포주 (prostate epithelial cell line: RWPE-1)에서 분비되는 IL-1β생산의 관여에 대해서는 알려진 바가 없다. 이전의 연구에서 질편모충으로 전립선 상피세포주를 자극시켰을 때 IL-1β 생산이 증가하는 것이 확인되었다. 이 연구에서는 질편모충으로 자극시킨 전립선 상피세포주에 의한 IL-1β의 생산에서 NLRP3 inflammasome이 관여하는지 알아보았다.

1. 전립선 상피세포주에 살아있는 질편모충과 반응시켰을 때 IL-1β mRNA 및 단백질 생산이 농도와 시간에 의존적으로 증가하였다. 2. 살아있는 질편모충에서 유래된 LTB4가 IL-1β생산에 미치는 영향을 알아보았다. 살아있는 질편모충에서 LTB4가 생산되었고, arachidonic acid나 5-lipoxygenase (5-LO) 억제제를 처리한 질편모충에서 LTB4 생산의 증감을 확인하였다. 질편모충과 함께 LTB4를 전립선 상피세포주와 반응시켰을때 IL-1β생산이 유의하게 증가하였다. 따라서 LTB4가 IL-1β 생산에 관여함을 알수있었다. 3. 질편모충에 의한 전립선 상피세포주에서 NLRP3 inflammasome을 구성하는 ASC mRNA발현이 증가하였으며 NLRP3와 caspase-1 mRNA 및 단백질 이 증가하였다. 또한 caspase-1 억제제를 전립선 상피세포주에 전 처리 하였 을 때 IL-1β생산이 감소되었다. 4. siRNA를 이용한 NLRP3 및 caspase-1 유전자 발현 억제는 질편모충에 의해 유도된 IL-1β생산을 유의하게 감소시켰다. 5. NLRP3 inflammasome 활성에 영향을 주는 ROS 생산은 질편모충과의 반응 시간에 의존적으로 증가되었고, ROS, K+efflux 및 ATP-sensitive K+channels에 대한 억제제(DPI, KCl 및 Glibenclamide)를 전립선 상피세포주 에 처리하였을 때 대조군에 비해 IL-1β 및 caspase-1의 생산이 유의하게 감 소되었다. 6. NF-κB 억제제를 처리 하였을 때 NLRP3, pro-IL-1β 및 IL-1β mRNA가 감소되었으며, caspase-1 및 IL-1β 단백질 생산이 유의하게 감소하였다.

이상의 결과를 종합할 때 질편모충에 의한 인체 전립선 상피세포주의 IL-1β 생산에서 NLRP3 inflammasome의 역할을 알아보고자 질편모충을 전립선상피세포와 반응시켰을 때 ROS 생산과 K+efflux에 의해 NLRP3 inflammasome이 활성화되어 caspase-1을 활성화시켜 IL-1β를 생산하고, NF-κB가 NLRP3 inflammasome활성에 영향을 줌을 알 수 있었다. 따라서 이 연구에서는 질편모충에 의한 전립선 상피세포주의 IL-1β생산에 NLRP3 inflammasome이 관여함을 실험적으로 확인하였다.| Trichomonas vaginalis is a sexually transmitted protozoan parasite that causes vaginitis in women, and urethritis and prostatitis in men. Inflammasomes have been characterized in monocytes and macrophage extensively, but not in epithelial cell lineages, which are the preferred host cells for many pathogens. Prostate epithelium is known to have powerful innate immune system that protects male reproductive organs from various infections. Interleukin-1β (IL-1β) plays an major role in inflammation by orchestrating pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 which mediated by a multi-protein complex known as inflammasome. It has been previously demonstrated that T. vaginalis induces IL-1β release through activation of TLR4/MAPK/NF-κB in prostate epithelial cells. In this study, we investigated the inflammatory response of a prostate epithelial cell line (RWPE-1) to T. vaginalis and specifically, its capacity to trigger activation of an NLRP3 inflammasome. When prostate epithelial cell line was stimulated with live T. vaginalis, IL-1β, NLRP3 and caspase-1 protein levels as well as mRNA level of each component were increased. Moreover, silencing of NLRP3 and caspase-1 by small interfering RNA attenuated T. vaginalis-induced IL-1β. Production of reactive oxygen species (ROS) and K+efflux have been known to act as common activators of NLRP3 inflammasome. NADPH oxidase inhibitor DPI and high extracellular K+ suppressed secretion of IL-1β and caspase-1 in prostate epithelial cell line stimulated with T. vaginalis. However, two signals are required for IL-1β production. NF-κB has a role as first signal resulting in production of pro-IL-1β. In this study, we investigated whether NF-κB may affects on activation of NLRP3 inflammasome as second signal for IL-1β production. NF-κB inhibitor, Bay 11-7082 shown to inhibit IL-1β production, caspase-1 activation and NLRP3 gene expression. Taken together, our results indicated that T. vaginalis induces NLRP3 inflammasome via ROS and K+efflux resulting in IL-1β production in human prostate epithelial cell line. NF-κB affects not only activation of NLRP3 inflammasome but also production of pro-IL-1β. In conclusion, it is suggested that NLRP3 inflammasome may involved in IL-1β production by human prostate epithelial cell line RWPE-1 stimulated with live Trichomonas vaginalis.; Trichomonas vaginalis is a sexually transmitted protozoan parasite that causes vaginitis in women, and urethritis and prostatitis in men. Inflammasomes have been characterized in monocytes and macrophage extensively, but not in epithelial cell lineages, which are the preferred host cells for many pathogens. Prostate epithelium is known to have powerful innate immune system that protects male reproductive organs from various infections. Interleukin-1β (IL-1β) plays an major role in inflammation by orchestrating pro-inflammatory response. IL-1β is synthesized as an immature pro-IL-1β form. It is cleaved by activated caspase-1 which mediated by a multi-protein complex known as inflammasome. It has been previously demonstrated that T. vaginalis induces IL-1β release through activation of TLR4/MAPK/NF-κB in prostate epithelial cells. In this study, we investigated the inflammatory response of a prostate epithelial cell line (RWPE-1) to T. vaginalis and specifically, its capacity to trigger activation of an NLRP3 inflammasome. When prostate epithelial cell line was stimulated with live T. vaginalis, IL-1β, NLRP3 and caspase-1 protein levels as well as mRNA level of each component were increased. Moreover, silencing of NLRP3 and caspase-1 by small interfering RNA attenuated T. vaginalis-induced IL-1β. Production of reactive oxygen species (ROS) and K+efflux have been known to act as common activators of NLRP3 inflammasome. NADPH oxidase inhibitor DPI and high extracellular K+ suppressed secretion of IL-1β and caspase-1 in prostate epithelial cell line stimulated with T. vaginalis. However, two signals are required for IL-1β production. NF-κB has a role as first signal resulting in production of pro-IL-1β. In this study, we investigated whether NF-κB may affects on activation of NLRP3 inflammasome as second signal for IL-1β production. NF-κB inhibitor, Bay 11-7082 shown to inhibit IL-1β production, caspase-1 activation and NLRP3 gene expression. Taken together, our results indicated that T. vaginalis induces NLRP3 inflammasome via ROS and K+efflux resulting in IL-1β production in human prostate epithelial cell line. NF-κB affects not only activation of NLRP3 inflammasome but also production of pro-IL-1β. In conclusion, it is suggested that NLRP3 inflammasome may involved in IL-1β production by human prostate epithelial cell line RWPE-1 stimulated with live Trichomonas vaginalis.