分枝桿菌肺部感染的宿主免疫調節

Abstract

背景: 分枝桿菌肺部感染，仍然是全世界相當重要的感染症之一，而對感染的宿主免疫調節是其中值得研究的方向，但目前仍有許多課題的研究是較不足而需要努力的。其中一個是肺外結核的免疫診斷，雖然目前肺結核有逐漸被控制的趨勢，但肺外結核仍然是不容易診斷的，而因為其診斷的延遲而會造成預後的不良。可能的困難診斷原因包括了表現較不典型、檢體的不易取得等因素，而結核胸腔積水 (tuberculous pleural effusion) 則是肺外結核中最常見的表現之一，但其死亡率並不罕見。除了通過微生物學診斷的傳統診斷，胸腔積水中免疫學診斷輔助是可行的，雖然胸腔積水中的一些發炎指標已知有助於診斷，但仍有局限，且尚未有研究關於胸腔積水中抗發炎細胞激素和細胞毒性T淋巴細胞效應分子的研究，整體的多因子調查將免疫反應應用到臨床診斷是缺乏的。另一個隨著肺結核的下降的課題，是非結核分枝桿菌肺部感染 (Nontuberculous mycobacteria lung disease, NTM-LD) 的發生卻是正在增加的，而宿主免疫調節失調是可能的因素之一。NTM-LD中，鳥型分枝桿菌 (Mycobacterium avium complex, MAC) 正是最常見的菌種，因為它是環境中就存在的伺機性菌種，且痰液中培養出非結核分枝桿菌的病人卻只有一小部份最後被認定為有疾病，故推論宿主的免疫易感受性是一個可能的原因，但目前對於MAC-LD感染當下的宿主免疫調節並沒有好的了解，需要進一步探究。故我們希望研究這兩個尚未清楚宿主免疫調節反應但卻重要的分枝桿菌肺部感染議題。 方法: 我們在這項前瞻性研究中收案95名具有淋巴細胞為主 (≥50%) 的滲出性胸腔積水，並分析其發炎、抗發炎細胞激素和細胞毒性T淋巴細胞的效應分子，分析其作結核胸腔積水的預測評估。並招募了50名MAC-LD和30名對照組，採集受試者的周邊血液單核細胞，並處理為淋巴細胞和巨噬細胞，用於後續MAC抗原刺激，研究其宿主免疫反應。 結果: 在淋巴細胞為主的滲出性胸腔積水中，35名為患有結核胸腔積水，其胸腔積水中的IFN-γ、腺苷脫氨酶 (Adenosine deaminase, ADA）、誘餌受體 (Decoy receptor, DcR) 3、單核細胞化學引誘蛋白（monocyte chemo-attractant protein, MCP）-1，干擾素誘導蛋白（interferon-induced protein，IP）-10，顆粒酶 (granzyme) A和穿孔素 (perforin) 的結果，都是高於46位惡性和14位其它原因引起的胸腔積水的，通過logistic多變數回歸分析，IFN-γ ≥75pg / ml、ADA ≥40IU / ml、DcR3≥9.3ng / ml和可溶性腫瘤壞死因子受體1（TNF-sR1）≥3.2ng / ml是結核性胸腔積水相關的獨立因素，基於四個預測因子的預測機率在ROC曲線下面積為0.920，在四因子預測臨界值為0.303之下，可具有82.9％的靈敏度和86.7％的特異性。在結核胸腔積水病人中，具有陽性胸腔積水或胸膜培養為結核菌陽性的患者，具有比其它培養陰性的病人有較高的IFN-γ、MCP-1、IP-10和穿孔素。在MAC-LD的病人，PBMC接受MAC桿菌刺激試驗下，與對照組相比較是具有較低的腫瘤壞死因子-α (tumor necrosis factor-alpha, TNF-α) 和丙型干擾素 (interferon-gamma, IFN-γ) 反應。這些反應在MAC治療後有觀察到改善。另外，MAC-LD患者的淋巴細胞上的程序性細胞死亡受體-1 (programmed cell death-1, PD-1) 、程序性細胞死亡配體-1 (PD-1 ligand-1, PD-L1) 和細胞凋亡 (apoptosis) 表達，都比對照組更高。在MAC-LD患者中，通過直接的MAC桿菌或間接MAC活化的巨噬細胞來刺激淋巴球胞下，T淋巴細胞上的PD-1和細胞凋亡表現都有顯著增加。在使用PD-1和PD-1配體有的拮抗性抗體來阻斷PD-1路徑下，可觀察到在上述刺激測定中的IFN-γ產生有顯著的增加，並能減少T淋巴細胞的細胞凋亡現象。 結論: 在不易診斷的淋巴球為主胸腔積水中，雖然IFN-γ和ADA是診斷結胸腔積水的常見免疫指標，但同時測量抗發炎的DcR3和TNF-sR1，可以提高免疫指標模組的診斷效力，減少遺漏的病人。在MAC-LD的患者中，可發現減弱的淋巴細胞免疫，這可能與淋巴細胞上增加表現的PD-1和PD-1配體有關，進一步抑制淋巴細胞的活化過程，阻斷PD-1相關路徑可以改善淋巴細胞的IFN-γ產生並減少其細胞凋亡，未來可能可以應用在難治的MAC-LD病人。

Background: Mycobacterial pulmonary infection is still one of the most important infectious diseases. The host immune regulation is worthy of study, but there are many topics needed further investigation. On of them is immune-diagnosis for extra-pulmonary tuberculosis (TB). Although pulmonary TB has been gradually controlled, the diagnosis of extrapulmonary TB remains difficult because of less typical manifestations, difficultly collecting specimens, and low yield rate in mycobacteria culture. Among the extrapulmonary TB, tuberculous pleural effusion (TPE) is a common type but the mortality rate is not uncommon. In addition to the traditional microbiology, the immune-diagnosis is feasible but lacks an integrated study. Anti-inflammatory cytokines and effector molecules of cytotoxic T lymphocytes in PE has rarely been studied and might be helpful. In addition, the occurrence of nontuberculous mycobacteria lung disease (NTM-LD) is increasing, and MAC (Mycobacterium avium complex) is one of the most common causing species. Because it exists in the environment, and only a small part of those with sputum isolating MAC is identified as MAC-LD, the immune susceptibility is a probable cause, but there is lack of understanding. Therefore, we studied the two unsolved issues regarding the understanding and applicaton of the host immune regulation in TPE and MAC-LD. Methods: We recruited 95 cases with lymphocyte predominant exudative PE. Inflammatory and anti-inflammatory cytokines as well as effector molecules of the cytotoxic T lymphocytes were measured in the PE and were analyzed for the predictive value for TPE. In addition, 50 MAC-LD patients and 30 controls were enrolled, and we collected and isolated the peripheral blood mononuclear cells (PBMC), lymphocytes and macrophages for MAC antigen stimulation. Results: The 35 patients with TPE had higher interferon (IFN)-γ, adenosine deaminase (ADA), Decoy receptor (DcR) 3, monocyte chemo-attractant protein (MCP)-1, interferon-induced protein (IP)-10, granzyme A, and perforin than those with malignant PE (n=46) or other PE (n=14). By logistic regression analysis, IFN-γ ≥75 pg/ml, ADA ≥40 IU/ml, DcR3 ≥9.3 ng/ml and soluble tumor necrosis factor receptor 1 (TNF-sR1) ≥3.2 ng/ml were independent factors associated with TPE. The predicted probability based on the four predictors had an area under ROC curve of 0.920, with 82.9% sensitivity and 86.7% specificity under the cut-off value of 0.303. In the TPE group, patients with positive PE/pleura culture for Mycobacterium tuberculosis had higher pleural IFN-γ, MCP-1, IP-10, and perforin than those with positive sputum but negative PE culture. With regards to MAC-LD, patients had lower tumor necrosis factor-α and IFN-γ responses compared to the healthy controls in PBMC stimulation assays with MAC bacilli. These responses improved after MAC treatment. The PD (programmed cell death)-1 and PD-1 ligand expressions and apoptosis were higher in the lymphocytes of the patients with MAC-LD compared to the controls. Both PD-1 and apoptosis on T lymphocytes were significantly increased in the patients with MAC-LD, either by direct MAC or by MAC-priming macrophage activation. Partially blocking PD-1 and the PD ligand with antagonizing antibodies in the stimulation assay significantly increased the cytokine production of IFN-γ and decreased the apoptosis on T lymphocytes. Conclusions: For patients with lymophocye predominant pleural effusion, while pleural IFN-γ and ADA are conventional inflammatory markers for suspecting TPE, simultaneously measuring DcR3 and TNF-sR1 can improve diagnostic efficacy. The patients with MAC-LD have attenuated lymphocyte immunity, which might be associated with increasing activation of PD-1 pathway. Blocking such activation might improve the lymphocytic secretion of IFN-γ and reduce apoptosis.