炎性小体中凋亡相关斑点样蛋白抗结核分枝杆菌感染作用的研究

Protection Role of ASC in Host Resistance against Mycobacterium tuberculosis Infection

目的分析核苷酸结合寡聚化结构域样受体家族含pyrin结构域蛋白3（NLRP3）炎性小体在结核分枝杆菌（Mycobacterium tuberculosis，MTB）感染小鼠中的免疫保护作用。方法选取无免疫缺陷的野生型（widetype，WT）C57BL／6小鼠、半胱天冬酶-1^-/-小鼠、ASC^-/-小鼠及NLRP3^-/-小鼠用于体内MTB感染实验，各种系小鼠骨髓和腹腔巨噬细胞用于体外MTB感染实验。体内感染实验小鼠分为2组：第1组中4种小鼠各感染30只，用于细菌载量分析、细胞因子检测及生存期评价；第2组中4种小鼠各感染15只，用于组织病理学分析及肺组织细胞流式分析；实验第1、2、3、4周时各种系小鼠各取1只未感染小鼠作为对照。检测感染小鼠的生存率及肺组织匀浆中细菌载量；采用酶联免疫吸附试验（ELISA）法，测定未感染野生型小鼠、感染小鼠肺组织匀浆中的肿瘤坏死因子-α（TNF—α）、白细胞介素-12p40（IL-12p40）、IL-1p和γ-干扰素（IFN-γ）水平；应用流式细胞术分析小鼠感染后肺组织中的浸润细胞组分及细胞坏死情况，并以ELISA法分析支气管灌洗液和血清中坏死相关因子高迁移率族蛋白1（HMGB-1）和IL-1α的分泌情况；对感染肺组织进行组织病理学分析；采用蛋白质免疫印迹检测半胱天冬酶-1的成熟情况；采用ELISA法测定巨噬细胞培养上清中的IL-1β水平。结果感染MTB24h后，野生型小鼠骨髓巨噬细胞培养上清中IL．1B水平明显高于半胱天冬酶-1^-/-小鼠、ASC^-/-小鼠及NLRP3^-/-小鼠[分别为：（316．29±4．64）pg／ml、（21．30±2．37）pg／ml、（22．99±0．46）pg／ml、（32．61±0．22）pg／ml；F=30．53，P〈0．01]；野生型小鼠腹腔巨噬细胞培养上清中IL-1β水平也明显高于半胱天冬酶-1^-/-小鼠、ASC-/-小鼠及NLRP3^-/-。小鼠[分别为：（2970．36±11．53）pg／ml、（130．48±2．52）pg／ml、（120．24±3．43）pg／ml、（121．66±2.48）pg／ml；F=549．92，P〈0．01]．与野生型小鼠相比，半胱天冬酶‘和NLRP3。小鼠未表现出对MTB抵抗力的降低，感染29周后生存率分别为72．2％（13／18）、66．7％（12／18）、61．1％（11／18），但ASC。小鼠对MTB高度易感，感染4～12周后全部死亡．感染4周时，ASC^-/-小鼠肺脏细菌载量较野生型小鼠、半胱天冬酶-1^-/-及NLRP3^-/-小鼠升高约100倍（菌落计数分别为0．93×10^7±426、5．07×10^5±221、5．32×10^5±209、5．26×10^5±606；F=87．74，P〈0．01），显微镜检表现为急性坏死性肺炎，坏死细胞数量明显增高，伴有大量中性粒细胞浸润，且全肺细胞计数明显高于野生型小鼠、半胱天冬酶-1^-/-小鼠及NLRP3^-/-小鼠[分别为（10342．64±103．56）×10^5、（3014．26±22．87）×10^5、（2919．28±36．75）×10^5、（2974．77±17．35）×10^5；F=349．11，P〈0．01）；ASC^-/-小鼠中与细胞坏死相关的血清HMGB-1支气管灌洗液HMGB-1和支气管灌洗液中IL-1α水平较野生型小鼠明显升高[血清HMGB-1水平分别为（155893．36±168．30）pg／ml、（5112．56±43．62）pg／ml；t=-206．98，P〈0．01；支气管灌洗液HMGB-1水平分别为（137．80±1．67）ng／ml、（75．16±1．47）ng／ml；t=-21．71，P〈0．01；支气管灌洗液IL-1水平分别为（848．17±4．40）ng／ml、（197．04±2．93）ng／ml；t=-54．78，P〈0．01]。结论在MTB感染中，ASC分子具有独立于炎性小体NLRP3和半胱天冬酶-1的关键保护性作用。ASC^-/-小鼠中肺组织病理异常与增强的细胞坏死相关，细胞过度坏死引起组织损伤，可能造成的细菌载量升高及过度的免疫反应都可能导致宿主死亡。

Objective To investigate the role of NLRP3 （NOD-like receptor family, pyrin domain-containing protein 3） inflammasome in host protective immunity against Mycobacterium tuberculosis （MTB） infection. Methods Wild type （WT） C57BL/6 mice, mice deficient in either caspase-1,ASC or NLRP3 and the peritoneal macrophages （PECs） and bone-marrow derived macrophages （BMDMs） from corresponding mice were used to perform in vitro infection experiments with MTB. Mice were divided into 2 groups.In the first group, each type 30 mice were infected with MTB to analyze the bacterial loads, the concentration of cytokine and survival of mice. In the second group, 15 mice from each type were also infected with MTB for pathological examination and flow cytometry. At the first, second, third and fourth week after infection, one uninfected mouse from each type was used as control. The survival of mice and bacterial loads was determined from the lung homogenates aider challenge of infection by MTB. The level of tumor necrosis factor α （TNF-α）, interleukin-12p40 （IL-12p40）, IL-1β and interferon γ （IFN-γ ） from lung homogenates was determined by enzyme-linked immunosorbent assay （ELISA）. Lung inflammatory cells were analyzed by flow cytometric analysis and the lung cells necrosis was tested respectively. ELISA determined the level of high-mobility group box 1 （HMGB-1） and IL-1 α from bronchial veolar lavage fluid （BALF） and serum.Histopathological analysis was performed.Western blotting was performed to determine the mature of caspase-1 after infected with MTB. The productions of IL-1β in BMDMs and PECs obtained from WT, caspase-1^-/-, ASC^-/- and NLRP3 ^-/-mice infected by MTB were determined. Results The levels of IL-1β produced by BMDMs and PECst which were obtained from WT mice was significantly higher than those obtained from caspase^-/-, ASC^-/- and NLRP3^-/- mices infected with MTB post infection by 24 hour （BMDMs： （316.29 ± 4.64） pg/ml, （21.30 ± 2.37） pg/ml, （22.99 ± 0.46） pg/ ml, （32.61 ± 0.22） pg/ml ; F=30.53, P 〈 0.01 ; PECs： （2970.36 ± 11.53） pg/ml, （130.48 ± 2.52） pg/ml, （120.24 ± 3.43） pg/ml, （121.66 ± 2.48） pg/ ml; F=549.92, P 〈 0.01）. When compared with WT mice, mice deficient in caspase^-/- and NLRP3^-/-exhibited no decrease in the resistance to MTB infection, and the survival rates after infection of 29 weeks were 72.2% （13/18）, 66.7% （12/18） and 61.1% （11/18）, respectively. In contrast, the mice deficient in ASC^-/- showed high susceptibility to MTB infection, which resulted into 100% of mortality rate during 4-12 weeks after infection.By the forth week after infection, the bacterial burden in the lungs of mice deficient in ASC^-/-（0.93 × 10^7 ± 426） was 100 times higher than those of WT mice （5.07 ×10^5 ± 221）, mice deficient in caspase^-/-（5.32 ×10^5 ± 209） and NLRP3/（5.26 ×10^5± 606）, respectively （F=87.74, P 〈 0.01）.Morphological analysis revealed that the number of the lung cells in the lungs of ASC^-/- mice （（10 342.64 ± 103.56） ×10^5） were increased significantly than that from WT mice （（3014.26 ± 22.87） ×10^5）, caspase-1^-/- （（2919.28 ± 36.75） ×10^5） and NLRP3^-/- group （（2974.77 ± 17.35）×10^5） after the infection by 4 weeks, respectively （F=349.11, P 〈 0.01）. The concentrations of HMGB-1 and IL-1α associated with necrosis were further detected by ELISA, and our data revealed that the concentrations of HMGB-1 in serum （155 893.36 ± 168.30） pg/ml） and BALF （137.80 ± 1.67 ng/ml） and IL-la in BALF （848.17 ± 4.40 ng/ml） of mice deficient in ASC^-/- were significantly higher than those of WT mice （5112.56 ± 43.62 pg/ml, t=-206.98, P 〈 0.01 for HMGB-1 in serum; 75.16± 1.47 ng/ml, t=21.71, P 〈 0.01 for HMGB-I in BALF; and 197.04±2.93 ng/ml; t=54.78, P 〈 0.01 for IL-1α in BALF）, respectively. Conclusion Our findings demonstrate that ASC molecule plays a protective role against MTB infection, which is independent from NLRP3 and caspase-1.The unusual presentations in the lungs of mice deficient in ASC^-/- are associated with the increased necrosis. The excessive tissue damage may result in the increased bacterial burden and immune response, thereby leading to the mortality of hosts deficient in ASC^-/-.