摘要:經由inflammasome 活化而釋放IL-1β的過程對於宿主對抗病原體和細菌而言是基本且需要的。然而過量的IL-1β經常會擴大發炎的反應而造成發炎性損害的疾病。因此了解在發炎性疾病發病期間如何調節IL-1β這個發炎性細胞激素的產生是相當重要的。最近的研究顯示調節inflammasome 是與細胞的特異性有關,但是真正的機制仍不清楚。更進一步的,目前的資訊對於如何調節NLRP3 inflammasome 的過程也很有限,尤其蛋白激酶訊息傳遞調節NLRP3 inflammasome 的相關資訊也仍未知。PKC 是一個屬於serine/threonine 類的蛋白質激酶,在許多細胞的反應過程扮演著相當重要的角色。然而PKC 在免疫調節inflammasome 活化伴隨IL-1β產生方面的機制依然未知。HMG-CoA 還原酶抑制劑(例如:statins 類藥物)可以經由干擾蛋白的isoprenylation 而產生抗發炎的作用,除此之外,它也有降低膽固醇合成的效用。我們先前的研究也顯示:在人類單核球細胞株(THP-1)中,fluvastatin 和lovastatin 可以經由活化NLRP3inflammasome 而致使IL-1β 的產生, 而這個作用是藉由蛋白isoprenylation 的路徑而使得ATP 釋放與P2X7 活化而來。在這個階段,我們仍需要探討isoprenylation 過程與ATP 釋放相關的分子機制。相同地,也需了解ROS 的產生、溶酶體(lysosome)伴隨cathepsin B 釋出和PKC 活化是否也參與statins 類藥物的作用過程。初步的研究顯示:調節IL-1β的釋放可能與細胞的種類與生物種類有關。因此我們有興趣闡述從鼠類和人類而來的單核球(monocytes)、巨噬細胞(macrophages)以及神經膠質細胞(microglia) 在調節IL-1β 釋放上的差異。我們將使用murine bonemarrow-derived macrophages (BMDM), murine bone marrow-derived monocyte(BMM),human THP-1 monocytes, human THP-1 derived macrophages 以及murine BV2microglia。這個研究中我們將了解,這些細胞對PKC 所傳遞(以PMA 當刺激劑)和statins類(fluvastatin 和lovastatin)刺激而產生的IL-1β的情形。我們也將闡述在不同種細胞中產生IL-1β maturation 的機制,特別是P2X7、溶解酶的不穩定性、ROS、蛋白激酶以及isoprenylation modification 在PMA 和statins 刺激下致使inflammasome 活化的角色。我們也將提供證據來支持蛋白isoprenylation 和ATP 釋放之間的關聯性。在研究當中,也將利用P2X7 缺失的老鼠細胞來驗證P2X7 和內生性的ATP 如何調控inflammasome 的活化。總結,在這個計劃中我們預期的目標是:(1).探討在不同種細胞中,PMA 和statins如何產生IL-1β。(2).了解在statins 刺激下,產生ATP 和IL-1β的機制。(3).PMA 所誘發IL-1β釋放的訊息傳遞路徑。在這個計劃當中,我們的研究將可以提供極具潛在性新的治療策略。
Abstract: IL-1β secretion via inflammasome activation is essential for host protection againstpathogens and bacterial clearance. However, excess IL-1β release, which often amplifiesthe inflammatory response, leads to lesion progression of inflammatory diseases. Thusunderstanding the molecular events that regulate IL-1β production is important formodulating this potent proinflammatory cytokine during inflammatory diseases. Recentstudy regarding the cell-type specific regulation of inflammasome has been suggested, butthe molecular details remain unclear. Moreover, currently very limited information has beenobtained for the regulation of NLRP3 inflammasome. Specifically the roles of variousprotein kinases-mediated signaling cascades essential for regulating NLRP3 inflammasomeactivation is still a mystery.PKC is a serine-threonine protein kinase with important roles in multiple cellular processes.However, much less is known about the immunoregulatory role of PKC ininflammasome-associated IL-1β production. HMG-CoA reductase inhibitors (i.e. statins),which interfere with protein isoprenylation, were shown to present pleiotropicanti-inflammatory actions in addition to their lipid-lowering action. In our previous studywe have observed the stimulating effects of fluvastatin and lovastatin on NLRP3-mediatedIL-1β secretion in human THP-1 monocytes, and suggest this action relies on a proteinisoprenylation pathway leading to ATP release/P2X7 activation. At this stage, we still needto explore the molecular relationship between isoprenylation process and ATP release, aswell as the possible involvement of ROS production, lysosome-associated cathepsin B andPKC activation in this event.Given preliminary understanding that differences and complications in regulation of IL-1βrelease might depends on cell types and species, we interest to pursue studies onmonocytes, macrophages and microgila from mouse and human, including murine bonemarrow-derived macrophages (BMDM), murine bone marrow-derived monocytes (BMM),human THP-1 monocytes, human THP-1 derived macrophages, and murine BV-2microglia. In this study we propose to understand PKC-mediated (by taking phorbol esterPMA as a stimulus) and statin (fluvastatin and lovastatin)-stimulated IL-1β production inthese cell types. We will delineate molecular basis underlying IL-1β maturation in variouscell types; especially how P2X7, lysosome instability, ROS, protein kinases andisoprenylation modification contribute either independently or in a coordinate manner toPMA- and statin-elicited inflammasome activation. We will further elucidate the molecularmechanisms and provide evidence to support the relationship between proteinisoprenylation and ATP release. Studies in P2X7-/- murine cells can help us to highlight thecrucial functions of P2X7 and endogenous ATP in modulating inflammasome activation.In summary, the specific aims of this project include (1) Determine PMA’s and statin’seffects on IL-1β production in various cell types; (2) Dissect mechanisms underlyingstatin-induced ATP release and IL-1β secretion; (3) Dissect signaling pathways forPMA-induced L-1β release. Our study of this project is believed to provide cues fordevising novel therapeutic strategies.