The Role Of Gasdermin D In Staphylococcus Aureus Cutaneous Infection

Gasdermin D(GSDMD)is a member of Gasdermin protein family,as a executor of pyroptosis which is an inflammasome dependent cell death.The discovery is one of the important progress of natural immunity research field in recent years.The discovery gave rise to the enthusiasm of researchers about the role of GSDMD in inflammatory dependent and inflammatory non-dependent biological functions,as well as the role of GSDMD in infection,inflammation and other diseases.Staphylococcus aureus(S.aureus)is a gram-positive pathogens,which exists extensively in the nature,and it can cause a variety of diseases of animals and human,such as dermatitis,mastitis,arthritis,pneumonia,enteritis and severe infection can cause sepsis.In recent years,with the emergence of multi-drug resistant S.aureus,the high pathogenicity and morbidity have attracted much public attention..In order to find the control target of S.aureus from the host,we explore the role and mechanism of GSDMD in S.aureus cutaneous infection.Through the use of WT and GSDMD-/-mice to establish a mouse model of cutaneous infection,we first analyzed the effect of GSDMD in S.aureus cutaneous infection.The two genotypes of mice were detected as follows: the size of skin abscess was measured and calculated by using vernier caliper;The pathologic damage of skin tissue was detected by H&E;The bacterial burden in skin was detected by plate method;The secretion of inflammatory cytokines in skin tissues was assayed using ELISA kits;The infiltration of inflammatory cells in skin tissues was analyzed by immunohistochemical.The results showed that compared with WT mice,GSDMD-/-mice skin lesion size was significantly increased(2nd to 8th days,p<0.01;the 9th day,p<0.05),the tissue pathology damage was more serious,the bacteral colonization was more significantly increased(p<0.01),the secretion of CXCL1 was significantly increased(p<0.05),but the secretion of IL-1? was significantly decreased(p<0.05)and the number of neutrophils and macrophages was significantly increased.Results showed that GSDMD can significantly inhibit the skin inflammatory reaction caused by S.aureus infection.Subsequently,we investigated the regulating mechanism of GSDMD in S.aureus cutaneous infection.GSDMD can be cleaved by Caspase-1 and Caspase-11,the released N-terminal perforates in the cell membrane,mediating pyroptosis and the release of IL-1? and IL-18.Therefore,we compared the difference in response to GSDMD-/-mice by means of Caspase-1/11-/-mice in S.aureus cutaneous infection.As a result,the Caspase-1/11-/-mice and GSDMD-/-mice all exhibited the secretion of IL-1?,but their phenotypes were not identical: Caspase-1/11-/-mice was similar to WT mice,compared with WT mice,the Caspase-1/11-/-mice had slightly increased in the lession size,the bacterial colonization and the histopathologic damage,but there was no significant difference,while there was a significant difference between GSDMD-/-mice and WT mice.Interestingly,the pattern of CXCL1 secretion was consistent with the size of lession and bacterial colonization in the three genotype mice.Epidermal cells and infiltrated inflammatory cells are important components of the skin immune barrier.We then analyzed the expression of GSDMD in skin tissues by immunohistochemistry,and found that GSDMD was mainly expressed in inflammatory cells.We used mouse bone marrow-derived macrophages as a research model to analyze the effect of GSDMD on CXCL1 secretion and its regulatory pathways in vitro.Compared with WT BMDM,the secretion of IL-1? is significantly decreased(p<0.05)in GSDMD-/-BMDM,confirming that GSDMD participated in inflammasome activation.But more importantiy,the secretion of CXCL1 in GSDMD-/-BMDM is significantly increased(p<0.001),which was consistent with in-vivo experiments.The expression of Ik B-?,p-Ik B-? and p-NF-k B P65 protein was analyzed by Western blot,finding that the activation of NF-k B signal was enhanced after GSDMD gene deletion.Then,we analyzed the activation of NF-k B signaling pathway in the skin tissue,and the results were consistent with in-vitro experiments.The above results indicated that GSDMD may inhibit the secretion of CXCL1 by negatively regulating NF-k B signal activation.Whether the GSDMD gene deletion effect is mediated by the increased secretion of CXCL1,which is a question we need to further answer.We used the CXCL1 inhibitor SB225002 for validation.The results showed that CXCL1 inhibitor can significantly alleviated the histopathological damage in GSDMD-/-mice,and it can limit the colonization of S.aureus in cutaneous infection.In summary,the present study revealed that GSDMD can resist S.aureus cutaneous infection by inhibiting NF-k B-CXCL1 signaling.