The Effects And Mechanisms Of IFI204 Protecting Against Staphylococcus Aureus Infection

Staphylococcus aureus is not only a component of the symbiotic bacteria,but also a major cause of invasive infections.Despite intense research in understanding the pathogenesis and host-pathogen interaction,the mechanisms by which Staphylococcus is cleared from the host are largely unclarified,thereby impeding the development of novel strategies for control of this infection.The innate immune system plays a key role in the early recognition and elimination of invading pathogens.Sensing pathogen through PRRs enables innate immune cells to mobilize defense mechanisms.Characterized as a DNA sensor,interferon-inducible protein 204?IFI204,its human ortholog IFI16?is one member of PRRs.The research on IFI16/IFI204 participating in immune response focuses on the recognition of viral genome and inhibition of viral replication.There are also several reports on its mediation of recognition and reaction of several intracellular bacteria.These studies are based on gene knockdown in vitro.There is little knowledge regarding the role of IFI16/IFI204 in response to extracellular bacterial infection.Although Staphylococcus can also grow and spread intracellularly,it is generally considered to be an extracellular pathogen.Here,using IFI204-deficient mice we explored the action of IFI204 in host defense against Staphylococcus.When Staphylococcus pulmonary infection occurs,significantly more bacteria were detected in the lungs,BALF?bronchoalveolar lavage fluid?and blood of IFI204^-/-?IFI204-dificient?mice and less neutrophils and macrophages accumulation in the airways of IFI204^-/-mice compared with WT?wild-type?mice.There were more severe lung injury in IFI204^-/-mice compared with WT mice.When Staphylococcus systemic infection occurs,IFI204^-/-mice demonstrated a significantly lower survival rate than their WT counterparts.As compared with WT mice,IFI204^-/-mice had more severe kidney injury and higher bacterial burdens in blood and kidney.Additionally,bacterial challenge dramatically induced the expression of cytokines?IL-6,IL-1?,IFN-??and chemokines?KC/CXCL1,CXCL2,CXCL10?in lung homogenates,BALF or blood from WT mice.However,the production of these cytokines/chemokines in IFI204^-/-mice were attenuated compared with WT mice.These data suggest that IFI204 is essential for the host immune defense against Staphylococcus infection,inhibits the proliferation of Staphylococcus,reduces histopathological damage and mortality,and promotes the secretion of inflammatory cytokines and the recruitment of inflammatory cells in the early stage of infection.IFI204 was mainly located at the recruited inflammatory cells in the lungs of mice infected with Staphylococcus.It was also found that IFI204 expressed in macrophages was mainly located in the nucleus,and could significantly up-regulated by Staphylococcus challenge.Therefore,mouse macrophages were used as cell models in vitro.Staphylococcus dramatically induced IFN-?and KC expression in WT cells.However,the levels of IFN-?and KC were significantly reduced in IFI204^-/-macrophages compare with their WT counterparts.A similar effect of IFI204 on KC and IFN-?production triggered by Staphylococcus-derived genomic DNA was found.The results provided in vitro evidence that IFI204 mediates inflammatory cytokine secretion induced by Staphylococcus.To further dissect the pathway of this,we analyzed STING-IRF3 signaling which plays a pivotal role in DNA-triggered induction of IFN-?,and NF-?B signaling which plays a key role in cellular inflammatory response and immune response.The activation of STING and IRF3 induced by Staphylococcus or its genomic DNA were significant decreased in IFI204^-/-macrophages compared with WT macrophages,and the NF-?B signaling was also largely impaired.The results in vivo are consistent with this,indicating that IFI204 promotes the activation of STING-IRF3 and NF-?B activation induced by Staphylococcus infection.In addition,bacterial proliferation in lung tissue of mice with Staphylococcus pulmonary infection were not inhibited by recombinant cytokines rKC and rIFN-?.Blockade of IFN signaling using anti-IFNAR1 MAb significantly decreased the mortality induced by Staphylococcus systemic infection.Therefore,inflammatory cytokines KC and IFN-?do not mediate the protective effect of IFI204 against Staphylococcus infection.In order to clarify the mechanism of IFI204 against Staphylococcus infection,the phagocytosis,intracellular and extracellular killing capacity of IFI204^-/-phagocytes were studied to explore the effect of IFI204 on the bactericidal ability of phagocytes.IFI204^-/-macrophages infected with Staphylococcus internalized similar numbers of FITC-labelled live or heat-killed bacteria to WT macrophages.The gentamicin protection assay showed that the number of recovered viable Staphylococcus was comparable in macrophages from both genotypes.However,the number of extracellular viable Staphylococcus in IFI204^-/-macrophages was significantly higher than that in WT macrophages.These suggest that IFI204 does not affect the phagocytosis and intracellular killing of Staphylococcus by macrophages,but promotes its extracellular bactericidal activity.Extracellular traps?ETs?provides an extracellular site for microbial killing in the innate immune defense.The formation of ETs and extracellular bactericidal activity of IFI204^-/-macrophage or neutrophils were significantly reduced compare with those of WT phagocytes after Staphylococcus infection.The absence of IFI204 also inhibits PMA-induced extracellular DNA release.Hence,these results suggest that IFI204 promotes ETs formation in phagocytes and enhances their extracellular bactericidal activity.To further confirm,the Staphylococcus pulmonary infection was carried out after bone marrow cells transplantation in mice.Compare with IFI204^-/-recipient mice that received IFI204^-/-bone marrow cells,IFI204^-/-recipient mice that received WT bone marrow cells had less bacterial burden,less lung injury,more recruitment of neutrophil and macrophages,stronger MPO activity.The proinflammatory cytokines?KC,IL-1?,IL-6?in lung showed slight similar patterns.These results suggest that bone marrow cells transplantation of WT mice could restore neutrophil and macrophages recruitment in lung tissue and save lung injury caused by Staphylococcus infection in IFI204-deficient mice,indicating that the protective effect of IFI204 against Staphylococcus infection depends on bone marrow-derived immune cells such as phagocytes.In summary,our studies demonstrate that IFI204 is involved in host immune defense against Staphylococcus infection,inhibits the proliferation of Staphylococcus,reduces histopathological damage,so then protects the host,and promotes the secretion of inflammatory cytokines and the recruitment of inflammatory cells in the early stage of infection.However,the protective effect of IFI204 against Staphylococcus infection depends on bone marrow-derived immune cells such as phagocytes,rather than on inflammatory cytolines.IFI204 promotes ETs formation in phagocytes after staphylococcus challenge and enhances their extracellular bactericidal activity.These observations document a novel and physiologically important role for IFI204 in host defense against Staphylococcus infection.