- 學位論文
恙蟲病導致的細胞激素風暴:先天免疫調控機制之研究
Scrub typhus-induced cytokine storm:Mechanism of innate immunity regulation
摘要
恙蟲病是由一種絕對細胞內寄生菌，恙蟲病立克次體，所引起的。宿主的巨噬細胞一方面提供了本菌複製增生的環境，一方面又參與本菌的殲滅與清除，這種菌與宿主免疫細胞間的相互關係，影響了患者的最終臨床預後。嚴重併發症的病人，主要是由於免疫反應引起細胞激素的過度分泌，又稱為「細胞激素風暴」而導致，而造成這個原因的先天免疫機轉還未被解析明瞭。因此我們首先建立恙蟲病台灣最多的菌株TW-1感染人的巨噬細胞THP-1的體外研究模式，探討低菌量(模擬輕型感染或早期感染的狀態)與高菌量(模擬重型感染與感染後期的狀態)時，細胞激素與微小核醣核酸的交互調控機轉。在低菌量的情況下，巨噬細胞首先大量分泌細胞激素介白素10 (IL-10)，IL-10的產生是藉由細胞外激素調控激酶 (ERK) 路徑的活化，IL-10的增加進一步抑制了促炎性細胞激素 (TNF-α，IL-1, IL-6) 的產生，使得巨噬細胞的環境有利於病原體的胞內複製與增生。隨著病原體菌量的增加，IL-10的分泌逐漸減少，巨噬細胞透過活化核因活化B細胞輕鍊增強因子(NF-B)，轉而製造更多的促炎性細胞激素。然而，在高菌量的感染情況中，巨噬細胞會大量表現微小核醣核酸155 (miR-155)，miR-155會抑制NF-κB的訊息路徑，造成促炎性細胞激素的製造減緩，以避免細胞激素風暴的產生。因此，巨噬細胞分別藉由IL-10在低菌量與miR-155在高菌量時的敏感反應，有效的調控避免細胞激素的過度產生導致的細胞死亡與宿主傷害。ERK/IL-10/STAT3的反應路徑活化，可抑制NF-B/TNF-路徑的活化。在此雙重抑制的調控機轉下，我們發現IL-10是非常強而有力的miR-155抑制劑，在非常低劑量下即能有效抑制miR-155的產生，藉此避免過度壓制促炎性細胞激素的產生，使宿主維持有效的殲滅病原的免疫反應。我們進一步回朔性檢測臨床上曾患有細胞激素風暴的病人，以周邊血液單核細胞(PBMC)，在體外接受恙蟲病立克次體的感染的挑釁試驗，結果顯示具有細胞風暴易感性的病人，IL-10與miR-155的分泌顯著低於輕症患者。這樣的結果顯示IL-10與miR-155可作為恙蟲病感染患者的生物指標；唯有兩者有效的反應與交互調控，才能確保人體的防衛機制能有效的運作，並避免細胞激素風暴的產生。 在臨床診斷研究上，我們以被叮咬的皮膚組織為檢體，以恙蟲病立克次體47千道爾吞(KDa)的膜外蛋白序列，設計以探針基礎的定量聚合酶連鎖反應，發展新的診斷方法，以提高恙蟲病以血液為檢體的陽性診斷率。在我們的臨床研究中，在二十個病人中，六個疑似感染的病人以傳統方法包含血清學診斷、血液檢體的聚合酶連鎖反應確診的病人，我們發現若用皮膚組織來進行定量聚合酶連鎖反應可得到更高的致病菌去氧核醣核酸的訊號(十倍到一百倍)。其他六個病人，以傳統方法診斷均顯示偽陰性，但以皮膚組織來進行定量聚合酶連鎖反應卻可得到確診的反應。以受試者操作曲線下面積的統計方法來檢定，可以得到於使用皮膚檢體聚合酶連鎖反應呈現出100%準確率的結果，相較於血液檢體，顯示皮膚檢體顯著提升了診斷率(p<0.001)，特別是在沒有發燒症狀的患者。而在新的檢驗方法證明下，恙蟲病的皮膚表徵，不只是經典的焦痂，還顯示出多元的症狀，如紅斑、紅疹、水疱、潰瘍、紫斑、腐蝕或蜂窩性組織炎樣的紅腫等症狀。因此若使用恙蟲病立克次體47KDa序列，以探針基礎的定量聚合酶連鎖反應，來檢測皮膚患部，將可提供一個更為準確的診斷工具，特別是對於在早期恙蟲病的確診，細菌還未進入血液循環中，或是在後期的患者，，已經使用過抗生素的，更能增加診斷的正確性。
並列摘要
Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi. Macrophages are the host cells that provide an environment for its replication, and are involved in the killing and clearance of the scrub typhus organism. The interactive relationship between bacterium and host immune cells influences the final prognosis of patients. Severe complications in patients are mainly caused by over-production of pro-inflammatory cytokines, also known as a “cytokine storm”; nevertheless, the molecular mechanism for the occurrence remains obscure. Thus, we first established an in vitro model by using the most common stain of O. tsutsugamushi in Taiwan. We infected THP-1 induced macrophages to investigate the mechanism of cross-regulation between cytokines and microRNA (miRNA) in low dose infection (which mimics the mild type or early stage of infection) and high dose infection (which mimics the severe type or later stage of infection). During low dose infection, macrophages first produce high levels of IL-10, which are produced through the activation of the ERK pathway. IL-10 then inhibits the pro-inflammatory cytokine (TNF-α, IL-1, IL-6) production and facilitates pathogen replication and reproduction in macrophages. Increasing levels of the pathogen results in reduced levels of IL-10, and macrophages begin to generate high levels of pro-inflammatory cytokines through NF-κB activation. However, during a high dose infection, macrophages produce high levels of miR-155 to slow the production of pro-inflammatory cytokines and prevent the consequent cytokine storm. Therefore, macrophages effectively control pro-inflammatory cytokine production to prevent cell death and host injury by IL-10 in low dose infection and miR-155 in sensitive high dose infection, respectively. The activation of ERK/IL-10/STAT3 axis suppresses the activation of NF-κB-TNF-α pathway. In this double inhibited mechanism, we found miR-155 suppressed by a low dose of IL-10, which indicated IL-10 is strong miR-155 inhibitor to prevent over-suppression of the production of pro-inflammatory cytokines and sustains the an effective immune response for killing and clearing the pathogen. We further evaluated patient susceptibility to cytokine storms. Sampling of these patients’ peripheral blood mononuclear cells (PBMC) showed significantly lower IL-10 and miR-155 responses to O. tsutsugamushi challenge in vitro. This result indicates IL-10 and miR-155 can qualify as biomarkers for the scrub typhus patient, and with effective cross-regulation can ensure the defense system works properly in the human body to prevent the risk of cytokine storms. In clinical studies, we developed a novel method for clinical diagnosis by the skin tissue of the bit site with a probe-based qPCR modeled on the 47-kDa outer membrane protein sequence of O. tsutsugamushi. In our clinical investigation, six out of twenty suspected patients were confirmed to be infected using serological tests or blood PCR, and those blood sample-positive patients tested positive using skin qPCR analysis with a much higher pathogen DNA copy (10 to 100 times). Another 6 patients were confirmed using skin qPCR, but all of them had a negative blood analysis. The area under receiver operating characteristic (ROC) curve analysis showed 100% accuracy of skin qPCR compared to blood PCR, which indicates skin samples significantly increased the diagnosis rate (p<0.001), especially in patients without fever. This newly developed diagnosis method demonstrated that skin symptoms are not limited to the classic eschar, but also includes cutaneous manifestations of erythema, purpura, bulla, and cellulitis-type erosion. Skin lesions for probe-based qPCR modeled on the 47-kDa sequence of O. tsutsugamushi provide a more accurate tool for confirming diagnosis in the early stage of infection before dissemination of bacteria in the blood, and also increased sensitivity at a later stage after antibiotic treatments.