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

细胞内nod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，导致病原体消除。nlr的激活导致炎性小体的组装，这是激活caspase-1所必需的，随后是IL-1ß前体加工成具有生物活性的IL-1ß。最近发现流感嗜血杆菌激活NLRP3炎性体；然而，所涉及的机制尚不清楚，也不清楚这是否对适应性免疫至关重要。虽然流感嗜血杆菌的荚膜是适应性免疫的主要靶点，但其在激活先天免疫中的作用尚不清楚。抗荚膜抗体激活补体-这导致细菌溶解；补体也是触发NLRP3炎性体激活的危险信号。因此，将胶囊识别为PAMP可能代表先天免疫和适应性免疫之间的联系。假设：流感嗜血杆菌胶囊促进NLRP3炎性小体激活，导致炎症反应，并为适应性免疫提供必要的共同刺激。***主要目的：探讨流感嗜血杆菌胶囊在炎性小体活化中的作用。* * * * * *具体目标:1。研究被荚膜与未被荚膜流感嗜血杆菌引起的NLRP3炎性体的激活。***为了测试胶囊/胶囊类型在炎性小体激活中的作用，将不同血清型（自然失去胶囊的突变体或通过bexA基因缺失产生的突变体）的荚膜或非荚膜流感嗜血杆菌感染原代人单核细胞（Mo）和小鼠骨髓源性巨噬细胞（BMDM）。炎性体的激活将通过量化成熟IL-1ß的产生、caspase-1活性和细胞死亡（焦亡）来评估。* * * 2。确定对流感嗜血杆菌的炎症反应是否取决于NLRP3炎性小体的激活。***为了测试炎症反应的激活是否依赖于炎性体，将研究caspase-1缺陷或野生型小鼠感染荚膜型或突变型流感嗜血杆菌株的bmms中促炎细胞因子的表达。为了将观察结果扩展到人类细胞，将使用caspase-1特异性抑制剂处理的THP-1细胞。确定炎症小体激活是否在流感嗜血杆菌的先天和适应性免疫反应之间提供了联系。***为了检验共刺激信号的产生是否依赖于胶囊，Mo将被胶囊化或突变型流感嗜血杆菌感染。可溶性和膜结合的b细胞活化因子BAFF和APRIL，以及其他b细胞活化因子和共刺激分子的产生将被量化。******提出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.***********