NOD1-IL-32경로에 의한 Orientia tsutsugamushi 감염의 염증 유발 기전

Abstract

연구목적: Orientia tsutsugamushi (OT)는 세포 내 감염을 통해 가을철 열성 질환의 하나인 쯔쯔가무시병을 일으킨다. 이러한 쯔쯔가무시병의 병인에는 혈관 염증이 주요 기전으로 알려져 있고, 본 연구에서는 이러한 혈관 염증에 NOD1-IL-32 경로가 관여하는지 알아보고자 하였다. 실험재료 및 방법: 쯔쯔가무시병의 병인으로 중요한 OT의 세포 내 염증반응 기전을 확인하기 위해, 사람 혈관 내피세포인 ECV304에 OT를 감염시킨 후, 분비 되는 사이토카인들을 측정하였다. OT의 세포 내 염증반응에서 NOD1을 경유하는 신호전달과 IL-32가 내피세포의 사이토카인 분비를 조절할 것이라 생각하고, siRNA로 NOD1 발현의 감소를 유도하여 OT를 감염시키고 IL-32 및 IL-1β, IL-6, IL-8, ICAM-1 분자의 발현 정도를 확인하였다. IL-32의 역할을 확인하기 위하여 IL-32를 인위적으로 주입하여 IL-1β, IL-6, IL-8, ICAM-1의 발현 변화를 관찰하였다. 결과: OT에 감염된 ECV304세포에서는 CCL5, CCL17, IL-1α, IL-6, IL-8, IL-10, IL-15, TNF-α, TNF-β의 분비가 증가하였다. 또한 IL-6와 IL-8의 유도를 조절하는 것으로 알려진 IL-32가 OT에 감염된 세포에서 많이 증가되었는데, 이때 NOD1 활성을 동반하였다. OT를 감염시킨 ECV304 세포에서 siRNA를 사용하여 NOD1의 발현을 감소시키자 IL-32가 감소하였고, 또한 IL-1β, IL-6, IL-8, ICAM-1도 감소하였다. 이러한 감소는 IL-32를 첨가하면, NOD1 발현 억제에 의하여 감소한 IL-1β, IL-6, IL-8, ICAM-1는 회복되었다. 결론: NOD1 경로의 활성화를 통해 IL-32의 분비를 유도하며, 그 결과 IL-1β, IL-6, IL-8, ICAM-1이 증가하였다. 그러므로 NOD1-IL-32의 경로가 OT감염증에서 내피세포 염증반응을 활성화시키는 역할을 하는 것으로 생각된다.;BACKGOUND: Orientia tsutsugamushi (OT), the causative agent of scrub typhus, is an obligate intracellular bacterium. During scrub typhus infection, OT locates in endothelial cells in all the intenal organs and cause vascular injury. The pathogenesis of inflammation in scrub typhus involves the inflammatory mediators including cytokines, prostaglandins, leukotrienes, and kinins from infected cells. To verify the inflammatory responses involved in the pathogenesis of scrub typhus, human ECV304 cell line was infected with OT and the cytokine profilewas assessed. METHOD: Human ECV304 cells were infected with OT and the cytokine profile was assessed with Cytokine Array. To evaluate the effect of IL-32 in OT-infected ECV304 cells, IL-32 level was measured by ELISA with various doses of the inoculum and time dependent manner. OT infected cells produced more IL-6, IL-8, and ICAM-1. To prove the role of NOD1 pathway for the production of IL-32, siRNA was used. To check the NOD1 knockdown effect, the expression levels of IL-6, IL-8 and ICAM-1 in ECV304 cells were compared with the control si RNA transfection. In addition IL-32 was treated to reverse the effects of NOD1 knockdown on the expression of IL-1b, IL-6, IL-8, and ICAM-1. RESULT: It was noted that CCL5, CCL17, IL-1α, IL-6, IL-8, IL-10, IL-15, TNF-α and TNF-β were strongly induced in response to OT. Additionally, IL-32, the candidate modulator for the induction of IL-6 and IL-8, was increased significantly with OT infection and these increases were observed along with NOD1 pathway activation. Thus, it was hypothesized that NOD1 pathway and IL-32 might act on cytokine release in endothelial cells as a modulator of the inflammation caused by OT infection. NOD1 knockdown siRNA resulted in a reduction of IL-32 levels, and also reduced IL-1β, IL-6, IL-8, and ICAM-1 expression in OT-infected ECV304 cells. These changes in IL-1β, IL-6, IL-8, and ICAM-1 induced by NOD1 knockdown were reversed after IL-32 treatment. This study suggested that OT infection activated the NOD1 pathway followed by IL-32 secretion, thus resulting in the production and expression of IL-1β, IL-6, IL-8, and ICAM-1. Therefore, IL-32 might perform a role upstream of the inflammatory reaction in endothelial cells of OT infection. CONCLUSION: It was determined that intracellular OT infection led to the activation of NOD1 and subsequently induction of IL-32, propagating inflammation via the induction of IL-1β, IL-6, IL-8, and ICAM-1 in endothelial cells. These results means that the NOD1-IL-32 pathway may function as a modulator of endothelial immune response in OT infection This study may contribute to further perception of the inflammatory process of the endothelium in scrub typhus.