Role of NOD-like receptors and inflammasome activation in innate immunity to pathogenic bacteria

细胞内结节样受体(NLRs)感知病原体相关分子模式(PAMPs)和“危险信号”，从而介导宿主反应，从而消除病原体。NLRs的激活导致caspase-1激活所需的炎性小体的组装，随后IL-1？前体加工成具有生物活性的IL-1？最近发现流感嗜血杆菌可激活NLRP3炎症体，但其作用机制尚不清楚，也不清楚这是否对获得性免疫是必需的。虽然流感嗜血杆菌的衣壳是获得性免疫的主要靶标，但其在天然免疫激活中的作用尚不清楚。抗囊膜抗体激活补体--这会导致细菌溶解；补体也是触发NLRP3炎症小体激活的危险信号。因此，识别胶囊为PAMP可能代表了天然免疫和获得性免疫之间的联系。*假设：流感嗜血杆菌胶囊促进NLRP3炎症小体的激活，从而导致炎症反应，并为获得性免疫提供必要的共同刺激。*主要目的：研究流感嗜血杆菌胶囊在炎症小体激活中的作用。*特定目的：*1.研究包膜与非包膜流感嗜血杆菌对NLRP3炎症体的激活作用。*为检测包膜/包膜类型在炎症体激活中的作用，原代人单核细胞(Mo)和小鼠骨髓来源的巨噬细胞(BMDM)将感染不同血清型的包膜或非包膜流感杆菌(突变体，即自然失去包膜的突变体或通过BexA基因缺失而产生的突变体)。炎性小体的激活将通过量化成熟的IL-1的产生、caspase-1的活性和细胞死亡(下垂)来评估。*2.确定对流感嗜血杆菌的炎症反应是否依赖于NLRP3炎症体的激活。*为了测试炎症反应的激活是否依赖于炎症体，我们将研究caspase-1缺陷或野生型小鼠感染包膜或突变的流感嗜血杆菌菌株的BMDM中促炎症细胞因子的表达。为了将观察扩大到人类细胞，将使用caspase-1特异性抑制剂处理的THP-1细胞。*3.确定炎症体激活是否在对流感嗜血杆菌的先天免疫反应和获得性免疫反应之间提供了联系。*为了检查共刺激信号的产生是否依赖于被膜，将用包膜或突变的流感嗜血杆菌菌株感染Mo。产生可溶的和膜结合的B细胞激活因子BAFF和APRIL，以及其他B细胞激活的细胞因子和共刺激分子将被量化。*建议的研究计划将大大有助于理解宿主对一种重要的人类病原体的防御，这种重要的人类病原体表现出多种免疫逃避策略。

Intracellular NOD-like receptors (NLRs) sense pathogen-associated molecular patterns (PAMPs) and "danger signals", and consequently mediate host response leading to pathogen elimination. Activation of NLRs results in the assembly of inflammasomes required for the activation of caspase-1 followed by IL-1ß precursor processing into the biologically active IL-1ß. It was recently discovered that H. influenzae activates the NLRP3 inflammasome; however, the involved mechanisms are unclear and it is unknown whether this is essential for adaptive immunity. Although the capsule of H. influenzae is the major target of adaptive immunity its role in the activation of innate immunity is unknown. Anti-capsular antibodies activate complement - this leads to bacteriolysis; complement also acts as a danger signal triggering NLRP3 inflammasome activation. Hence, recognition of the capsule as a PAMP may represent a link between innate and adaptive immunity.***Hypothesis: H. influenzae capsule facilitates NLRP3 inflammasome activation that results in inflammatory response and provides essential co-stimulation towards adaptive immunity. ***Major objective: Investigate the role of H. influenzae capsule in inflammasome activation. ***Specific objectives:***1. Study activation of the NLRP3 inflammasome caused by capsulated vs non-capsulated H. influenzae.***To test the role of capsule/capsular type in inflammasome activation, primary human monocytes (Mo) and bone marrow derived mouse macrophages (BMDM) will be infected with capsulated or non-capsulated H. influenzae of different serotypes (mutants, which naturally lost the capsule or a mutant developed via bexA gene deletion). The inflammasome activation will be assessed via quantifying production of mature IL-1ß, caspase-1 activity, and cell death (pyroptosis). ***2. Determine if inflammatory response to H. influenzae depends on the NLRP3 inflammasome activation.***To test if the activation of inflammatory response depends on inflammasome, pro-inflammatory cytokines' expression will be studied in BMDMs from caspase-1 deficient or wild-type mice infected with either capsulated or mutant H. influenzae strains. To expand the observations to human cells, THP-1 cells treated with a caspase-1 specific inhibitor will be used.***3. Determine if inflammasome activation provides a link between innate and adaptive immune response to H. influenzae.***To examine whether generation of co-stimulatory signals depends on the capsule, Mo will be infected with capsulated or mutant H. influenzae strains.  Production of soluble and membrane-bound B-cell activating factors BAFF and APRIL, along with other B-cell activating cytokines and co-stimulatory molecules will be quantified.******The proposed research program will significantly contribute to the understanding of host defense against an important human pathogen exhibiting multiple strategies of immune evasion.***********