Ifi204/p204, a new piece in the sepsis puzzle

Innate immunity is the first line of defense against microbial infection. The host cells are equipped with cellular sensors, which detect various components of microbes to mount rapid and early antimicrobial immune responses (1-3). The Toll-like receptors (TLRs) are transmembrane proteins, located on the cell surface or the endosome, to recognize the extracellular microbial components, and trigger pro-inflammatory and antimicrobial responses (1,3-5). Among the thirteen known TLRs, TLR4 is expressed both on the plasma, and the endosomal, membranes to detect the extracellular bacterial lipopolysaccharides (LPS) (6,7). LPS recognition by the plasma membrane-bound TLR4 triggers intracellular signaling pathways by recruiting MyD88, a cytoplasmic adaptor protein. The TLR4-MyD88 complex activates, via a series of signaling proteins and kinases, the transcription factor NF-κB to induce the pro-inflammatory cytokines (8,9). Extracellular LPS is also recognized by the endosomal TLR4, which recruits the cytoplasmic adaptor protein TRIF to activate NF-κB as well as IRF3, another transcription factor (8,10). TLR4-TRIF signaling induces, in addition to the NF-κB-dependent pro-inflammatory cytokines, Type-I IFNs, e.g., IFN-β by the coordinated action of both NF-κB and IRF3 (Figure 1). The uncontrolled expression of TLR4-induced genes leads to a pathological state known as sepsis (6,11).

Figure 1 Ifi204/p204 forms TLR4 dimers to activate downstream signaling. LPS stimulates TLR4 on the cell surface and the endosome. TLR4-mediated downstream signaling is dependent on receptor dimerization, which is mediated by the pyrin domains (PYD) of Ifi204/p204. TLR4 signaling triggers the induction of pro-inflammatory cytokines and IFN-β. Unregulated expression of TLR4-induced genes leads to lethal septic shock.

Bacterial sepsis is detrimental to the host and, therefore, the discovery of new proteins that regulate TLR4 signaling is highly significant. In a recent study, Liu and his colleagues described a new molecular mechanism by which Ifi204/p204 contributes to TLR4 signaling and subsequently promotes sepsis in mice (12). Ifi204/p204 is a member of the p200 family of proteins, which are IFN-stimulated gene (ISG) products containing the Pyrin (PYD) and HIN-200 domains (13,14). The p200 family of proteins are involved in a variety of functions including cellular differentiation, antiviral defense, and inflammasome activation (13-17). These proteins bind directly to the cytoplasmic nucleic acids to activate intracellular signaling pathways. Among the human p200 family of proteins, IFI16 recognizes cytoplasmic double-stranded (ds) DNA to induce Type-I IFN (18). AIM2, another human p200 protein, is involved in inflammasome activation in virus or bacteria-infected cells (15,19). Ifi204/p204, the murine homolog of human IFI16, co-operates with cGAS, a cytoplasmic DNA sensor, to induce Type-I IFN in response to bacterial infection (20). However, the in vivo role of p204 remained unclear due to the absence of p204^-/- animals. Yi et al. reported the first generation of p204^-/- mice using the traditional gene-editing technology (12). Using cells derived from the p204^-/- mice, the authors demonstrated that p204 is essential for LPS-activated TLR4 signaling and gene induction in the macrophages, which are critical cells that contribute to sepsis. The p204^-/- murine macrophages exhibit a strong reduction in LPS-induced pro-inflammatory cytokines, e.g., TNF-α, IL-6, and IL-1β as well as Type-I IFNs, e.g., IFN-β. The p204^-/- mice, when exposed to LPS, display a significant reduction in TLR4-induced genes. As a result of the reduced cytokine levels in the sera, the p204^-/- mice are significantly resistant to LPS-induced sepsis. These results revealed a new function of p204 in LPS-induced sepsis both ex vivo and in vivo. LPS, in addition to stimulating TLR4, is also internalized by the macrophages to activate the intracellular LPS sensors. However, p204^-/- macrophages are not deficient in inflammasome activation in response to intracellular LPS. Therefore, p204 is neither a sensor for intracellular LPS nor a component of the intracellular LPS-activated inflammasome pathway. These results led to the identification of a direct role of p204 in TLR4 activation. TLR4, upon binding to LPS, undergoes dimerization, a step that is essential for the recruitment of MyD88 or TRIF. The p204^-/- cells display a strong reduction in LPS-induced TLR4 dimerization. The PYD of p204 directly interacts with TLR4 to facilitate TLR4 dimerization. Furthermore, the PYD contains a binding motif (RKR), which is essential for p204-TLR4 interaction and LPS-induced TLR4 dimerization. As a result, the p204^-/- macrophages are defective in TLR4-mediated activation of NF-κB and IRF3, the transcription factors responsible for the induction TNF-α, and IFN-β, respectively. The authors propose a model, in which p204 binds to TLR4 using its PYD to trigger LPS-mediated receptor dimerization and downstream signaling (Figure 1).

How the host mounts and regulates the innate immune responses is an area of intense investigation. The new mechanisms allow the successful design of therapeutics to target specific components of the innate signaling pathways (11). TLR4 signaling, on the one hand, contributes to the antimicrobial defense by triggering the synthesis of pro-inflammatory and antimicrobial genes. However, unregulated TLR4 activation leads to the pathological septic shock response. Moreover, TLR4 signaling in the Kupffer cells, activated by gut bacteria-derived LPS, contributes to the alcoholic liver diseases (21). Work through our research revealed that EGFR activity is required specifically for the endosomal TRIF-dependent, but not the cell surface MyD88-dependent, branch of TLR4 signaling (7). As a result, EGFR inhibitors protect against LPS-induced bacterial sepsis in mice (7,22). EGFR activity is also required for the activation of TLR3, and TLR9, the endosomal TLRs (23,24). In addition to EGFR, we have uncovered new components of TLR4 signaling; using knockout cells and mice, we showed that HDAC6 and β-catenin are critical for LPS/TLR4 signaling (25). Ifi204/p204 is a new component of LPS/TLR4 signaling in macrophages (12). In the future, studies will be directed to understand the role of p204 in other TLRs, which activate similar downstream signaling pathways. Because dimerization is essential for all TLRs, it would be interesting to investigate the generality or the specificity of p204 involvement. The identification of a domain of TLR4 that interacts with p204 will help address these questions. It will be mechanistically significant to determine whether IFI16, the human homolog of murine p204, also similarly participates in TLR4 signaling. Future studies are required, using macrophage-specific p204^-/- mice, to explore the cell type-specific role of p204 in bacterial sepsis. These studies will help develop therapeutics by targeting TLR4/p204 interaction for the treatment against sepsis.

Acknowledgements

Funding: Research in the authors’ laboratories is supported by the National Institutes of Health grant AA026017 (SC), American Heart Association Grants 15SDG25090212 (SC), 15SDG2308025 (RC), and the University of Toledo College of Medicine and Life Sciences startup funds (SC).

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

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