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

细胞内NOD样受体（NLR）感知病原体相关分子模式（PAMP）和“危险信号”，从而介导宿主反应，导致病原体消除。NLR的活化导致半胱天冬酶-1活化所需的炎性体的组装，随后IL-1 β前体加工成生物活性IL-1 β。最近发现H.流感病毒激活NLRP3炎性体;然而，所涉及的机制尚不清楚，也不知道这是否是适应性免疫所必需的。虽然H.流感病毒是获得性免疫的主要靶点，其在先天免疫激活中的作用尚不清楚。抗荚膜抗体激活补体-这导致细菌溶解;补体还充当触发NLRP 3炎性小体激活的危险信号。因此，将包膜识别为PAMP可能代表先天免疫和适应性免疫之间的联系。 假设：H.流感病毒胶囊促进NLRP3炎性体活化，其导致炎症反应并为适应性免疫提供必要的共刺激。 主要目的：探讨H.流感胶囊在炎性小体激活中的作用。 具体目标： 1.研究由囊化与非囊化的H.流感。 为了测试被膜/被膜型在炎性小体活化中的作用，原代人单核细胞（Mo）和骨髓衍生的小鼠巨噬细胞（BMDM）将用被膜化或未被膜化的H.不同血清型的流感病毒（突变体，其天然失去荚膜或通过bexA基因缺失而产生的突变体）。将通过定量成熟IL-1 β的产生、半胱天冬酶-1活性和细胞死亡（焦亡）来评估炎性小体活化。 2.确定是否对H.流感病毒依赖于NLRP3炎性体激活。 为了测试炎症反应的激活是否依赖于炎性体，将在来自感染有囊化或突变体H.流感病毒株。为了将观察扩展到人细胞，将使用用半胱天冬酶-1特异性抑制剂处理的THP-1细胞。 3.确定炎性小体激活是否提供了先天性和适应性免疫反应之间的联系H。流感。 为了检测共刺激信号的产生是否依赖于包膜，Mo将感染包膜或突变的H。流感病毒株。 将定量可溶性和膜结合B细胞活化因子BAFF和APRIL以及其他B细胞活化细胞因子和共刺激分子的产生。 拟议的研究计划将显着有助于了解宿主防御一个重要的人类病原体表现出多种策略的免疫逃避。

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.