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

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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.***********