Host Immune Responses To Intracellular Bacterial Infection Mediated By Activation Of Pattern Recognition Receptor Signaling Pathway

Upon pathogen infection, immune cells can recognize such molecules referred to as PAMPs (or inducers) through one or more the pattern-recognition receptors (PRRs), and in turn activate a wide range of immune responses, including the innate and adaptive immune responses. On one hand, rapid and efficient mechanisms for detection of invading pathogens and triggering of proinflammatory signaling are of pivotal importance for the initial containment and control of infection. On the other hand, pathogen recngnition by PRRs is a perquisite for the activation and shaping of adaptive immune responses through maturation of antigen-presenting cells and activation of antigen-specific T lymphocytes to resolve the infection and induce immunological memory. Therefore, the molecular mechanisms of signal transduction pathways elicited by a given pathogen and the interactions between host and pathogen serve to illustrate and reveal important aspects of how the immune system fuction in defense and disease. The majority of pathogens can be detected by conserved and unique structural microbial components such as flagellin, lipopolysaccharides, polysaccharides and polynucleotides that differ little from one pathogen to another but are not found in the host. PRR families include the family of the Toll-like receptors (TLRs), C-type lectins (CTLs), the nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs), RNA-sensing RIG-Like helicases (RLHs), DNA sensors, as well as Caspase-11, which was proven to be a direct intracellular LPS receptor. The same repertoire of PRRs can also recognize DAMPs originating from dying cells, such as heat shock proteins (HSPs), thioredoxin (TRX), adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1) etc.Caspases are a family of cysteine proteases that play important roles in development and homeostasis, as well as inflammation and host defense against microbial pathogens. The canonical inflammasomes NLRP3, NLRC4 and AIM2 recruit the common adaptor ASC to activate Caspase-1, leading to the secretion of mature IL-1β and IL-18 as well as a pro-inflammatory form of cell death termed pyroptosis. In contrast to Caspase-1, activation of another class of inflammatory caspases including mouse Caspase-11 induces pyroptosis by the noncanonical inflammasome independently of NLRP3, NLRC4, ASC and Caspase-1. The noncanonical inflammasome triggered by intracellular lipopolysaccharide (LPS) is critical for the cytosolic sensing of several Gram-negative pathogens as well as induction of mouse mortality by LPS, which is independent of TLR4. Activation of Caspase-11 also relies on guanylate binding proteins (small IFN-inducible GTPases) that mediate phagosomal lysis, allowing bacterial LPS to be sensed in the cytosol. In addition to pyroptosis, the noncanonical inflammasome can indirectly promote IL-1β secretion by triggering the canonical NLRP3 inflammasome, but the mechanism remains unclear. Recent studies have revealed that activation of the noncanonical inflammasome is induced by direct binding of LPS to the mouse Caspase-11 and related human Caspase-4 and Caspase-5. However, the signaling events that mediate pyroptosis downstream of Caspase-11 remain unknown.In this work, firstly, we work on resolving the immune responses of zebrafish induced by the attenuated Edwardsiella tarda strain (WED). We performed a comparative gene transcription analysis of livers from PBS-moked and WED-stimulated zebrafish using RNA-seq technology to investigate their differential transcriptsomic profile. Through DEGseq software to analyze the RNA-seq raw data, we found 4565 genes were significant up- or down-expressed in WED-stimulated zebrafish liver. After Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) clustering enrichment of the significant expressed genes, the TLR5 signal cascades, endoplasmic reticulum protein export, phagosome and antigen processing and presentation pathways were significant enriched. Especially, the highly up-regulation of TLR5 signal cascade was detected during the early stage of E. tarda infection in zebrafish. Furthermore,12 genes enriched in major histocompatibility complex (MHC) antigen processing revealed an activated MHC I pathway and an inhibited MHC II pathway during infection. It was prompted that WED conferred a robust innate immune responses in zebrafish, and suggested that this pathogen could elicite an effective cellular immunity via the MHC-I pathway. Based on the transcriptsomic profile results above, we established a 5-week immunization-challenge model to thoughly understand the adaptive immune responses signal pathway elicited by E. tarda. The results reveled that the pattern recognition receptor, TLR5, expression was significantly increased in the zebrafish liver and spleen immediately upon infection, whereas TLR2 and 4, together with other TLRs revealed by the RNA-seq data, were not induced, which suggested that WED could activate the fish innate immune system through the recognition of the E. tarda flagellin through TLR5. Furthermore, in contrast to the significant co-upregulation of MHC I antigen processing and presentation pathway and the CD8+ cytotoxic T lymphocyte (CTL) responses, the MHC Ⅱ antigen processing pathway and the markers of CD4+ T lymphocyte activation were down-regulated, and IgM transcription and specific IgM antibody titers were not significantly induced following WED-stimulation. During the 1-week challenge phase, the induction of MHC I and CTL responses and the inhibition of MHC Ⅱ and CD4+ T cell responses were similarly observed in WED-stimulated zebrafish following challenge with wild E. tarda. With the 5-week immunization and challenge model, our data suggest the basic mechanism that underlying the long-lasting protective immunity elicited by WED in zebrafish. This mechanism involved the induction of the TLR-5 signaling pathway, the MHC I antigen processing pathway and CTL effector function, and CTL function seems play amajor role in the protection against E. tarda infection in zebrafish.Secondly, we work on interpreting the caspase 11-mediated noncanonical inflammasome signaling pathway activated by intracellular LPS. We provide evidence of a linear signaling pathway of the noncanonical inflammasome, in which caspase-11 cleaves and activates the Pannexin-1 channel to induce ATP release, which in turn activates the purinergic P2X7 receptor to induce cytotoxicity and susceptibility to endotoxic shock. Consistently, P2x7-/- and Panx1-/- mice are protected from lethality induced in the cecal ligation and puncture sepsis model. Cellular demise by P2X7 receptor activation is mediated by robust K+ efflux leading to collapse of intracellular ion gradients, organelle damage and cell death. The Caspase-11/Pannexin-1 pathway bifurcates at Pannexin-1 that also regulates the activation of the canonical NLRP3 inflammasome through the induction of K+ efflux independently of P2X7 receptor. Although clinical sepsis is a highly complex disorder in which multiple mediators are involved in morbidity, our studies suggest that inhibitors of the Pannexin-1 channel and P2X7 may benefit patients with Gram-negative sepsis.