Preliminary Study Of CLEC18A In Regulating Inflammatory Response Of Macrophage In Sepsis

Objectives CLEC18A(C-type lectin domain family 18,member A)is a new C-type lectin family member widely expressed on monocytes,macrophage,T cells,B cells,and granulocytes.Previous studies have found that the expression of CLEC18 A can be detected in the culture supernatant of macrophages,while its specific role in sepsis and iniflammatory responses of macrophage has not been reported.The present study aims to explore the function of CLEC18 A in septic inflammation and its potential regulation and potential mechanism in inflammatory responses of macrophage.Methods 1.Expression of CLEC18 A in septic patients and LPS-stimulated macrophage 1)Clinical samples: Blood sample from patients diagnosed by sepsis,non-sepsis patients underwent same surgery and healthy volunteer were collected.The serum level of CLEC18 A and m RNA level in peripheral blood mononuclear cell(PBMC)were analyzed;PBMC from healthy volunteer were collected and stimulated with LPS.CLEC18 A in the supernatant of cell culture were analyzed.2)In vitro experiments: mouse PM were extracted.The m RNA and protein level in cells and culture supernatant after LPS stimulation were analyzed respectively.2.In vivo and in vitro effects of exogenous CLEC18 A to septic mice and inflammatory reponse of macrophage 1)In vivo experiment: mice were randomly divided into the blank control group,r CLEC18A(80?g/kg)group,LPS group(10mg/kg,intraperitoneal injection)and LPS+r CLEC18 A group(20,40,80?g/kg).After 6-hour treatment,the serum IL-6 and TNF-? levels were detected.2)In vitro experiments: the PM and mouse bone marrow-derived macrophage(BMDM)were extracted,and cells were divided as: NC group,LPS(100ng/ml)group,r CLEC18A(40?g/ml)group,LPS+r CLEC18A(5?10?20?40?g/ml)for 6h.Then,IL-6 and TNF-? in the supernatant were analyzed.3.Verification of function of CLEC18 A via CLEC18A-knockdown and CLEC18ALyz-cre knockout mice 1)In vivo experiments: Sepsis model of cecal ligation & puncture(CLP)and LPS peritoneal injfection model was applied to CLEC18 A Lyz-cre and littermate mice(WT).Survival rate was monitored in CLP model and serum IL-6 and TNF-? were analyzed in 6 hours or 12 hours of LPS and CLP model were analyzed respectively.2)In vitro experiments: mouse PM were extracted,and si RNA with liposome transduction was applied to interfere the expression of CLEC18 A.Then cells were stimulated with LPS at 100ng/ml 1h,3h or 6h to analyze the m RNA in 1h and 3h,and protein level of IL-6 and TNF-? in 6h respectively;meanwhile,exogenous r CLEC18 A was used in CLEC18A-interfered PM.And IL-6 and TNF-? in supernatant were analyzed;PM of CLEC18 A lyz-cre mice were extracted and stimulated with 100ng/ml LPS for 6 hours.IL-6 and TNF-? levels in the cell culture supernatant were analyzed.4.Preliminary investigation on expression and anti-inflammatory mechanism of CLEC18 A of macrophage 1)Regulatory mechanism: PM were treated with the specific inhibitors of JNK,ERK,p38-mapk,and p65.Cells were then stimulated with LPS for 3h and 6h for the analysis of m RNA level of CLEC18 A or 1h and 3h for the analysis of protein level of CLEC18 A in culture supernatant.2)Anti-inflammation mechanism: PM with CLEC18A-interference,or from CLEC18A Lyz-cre mice,were stimulated with LPS.The phosphorylation of JNK,ERK,p38,p65 were analyzed;PM treated with r CLEC18 A were stimulated with LPS and the phosphorylation of above molecules were analyzed.Results 1.CLEC18 A was elevated in serum of sepsis patients as well as the LPS-stimulated PBMC from healthy volunteers;m RNA level of CLEC18 A in PM and BMDM was peaked in 1h and CLEC18 A in supernatant was elevated and accumulated.2.Exogenous administration of CLEC18 A recombinant protein can reduce the levels of LPS-induced IL-6 and TNF-? in serum in mice and lower the levels of IL-6 and TNF-? in macrophages stimulated by LPS.3.CLEC18 A lyz-cre knockout mice showed decreased survival rate and elevated serum IL-6 and TNF-? at 12 h in CLP-induced sepsis model;CLEC18A lyz-cre mouse showed elevated IL-6 and TNF-? level at 6h in LPS-induced inflammation;the CLEC18 Ainterfered or CLEC18A-knockout macrophage produced elevated IL-6 and TNF-? level after LPS stimulation.4.Pre-treatment of the inhibitor of ERK and JNK led to the reduced m RNA and protein level of CLEC18 A in LPS-stimulated macrophage;The phosphorylation of JNK,ERK,p38,and p65 were elevated in CLEC18A-knockdown and CLEC18A-knockout PM whereas the exogenous r CLEC18 A decreased the level of the phosphorylation of above molecules.Conclusion CLEC18 A is elevated in patients with sepsis and could inhibite the inflammatory responses of macrophage and shows protective effects in septic mice.CLEC18 A may be mainly up-regulated by JNK/ERK signaling pathway after LPS activation and suppress the phosphorylation of MAPK/NF-k B signaling in macrophage,suppressing the inflammatory responses.This study suggested the negative regulation of CLEC18 A in septic inflammation and potential related mechanism,expanding the understanding of the septic inflammation.