Function And Mechanism Of Gpr84 In Septic Mice

Sepsis is a disease of systematic inflammation and has a high rate of morbidity and mortality.At present,the treatment of sepsis is mainly antimicrobial mediation,but it can not accurately diagnose the pathogens sometimes,so there are some limitations on this treatment.The use of anti-inflammatory drugs for immunotherapy can block the exaggerated inflammatory response of patients with sepsis,and it is considered to be an effective treatment.However,due to the lacks of effective studies on long-term inflammatory response in sepsis and its regulatory mechanism,this immunotherapy has not yet achieved the desired therapeutic effect.The tests of the blood from patients of sepsis in children with septic shock showed that the expression of GPR84 was obviously higher compared with healthy ones,which hints that GPR84 may be involved in the regulation of the sepsis process.Free fat acids receptor GPR84 is a member of the Free fatty acids receptor family which contains a typical seven transmembrane helix structure of G protein coupled receptors.Studies have suggested that Gpr84 is a kind of pro-inflammatory receptor which plays an important role in immune response in vivo.Recently it was found that Gpr84 may regulate the functions of macrophages during the inflammation and was expressed highly in bone marrow macrophage induced by LPS.The agonist of Gpr84 can enhance the level of LPS-induced IL-12beta p40 on raw 264.7 cell lines.To further study the function of Gpr84 in septic mice,we conducted the following experiments.In our previous experiment,we used CRISPR/Cas9 system to construct Gpr84 knockout mice and DNA sequencing analysis was carried out on the gene of Gpr84 in the knockout mice.The Gpr84 knockout mice lost 172bp in the length of the base sequence in one exon of Gpr84.Meanwhile,the expression of Gpr84 in the levels of mRNA and protein in mice BMM were tested and no any expression were shown.Therefore,we built the Gpr84 knockout mice successfully and the mice could be used to perform the experiments about the involvement of Gpr84 in septic inflammation through regulating the functions of macrophages both in vitro and in vivo.To explore the mechanism of Gpr84 in septic inflammation,we construct sepsis model by intraperitoneally injected with LPS.The siurvival rate of Gpr84-/-sepsis mice increased.The levels of TNF-a,IL-6 and IL-12? p40 declined in the serum of Gpr84 knockout mice compared with those in WT mice.And the concentration of IL-6 and IL-12? p40 in the lavage fluid of Gpr84-/-mice decreased as well.Next,to investigate the effect of Gpr84-/-macrophage in inflammation,the bone marrow cells in wild-type(WT)and Gpr84 knockout mice were extracted respectively to be induced to macrophages in vitro.The found of higher expression of Gpr84 in mice BMM and PM than other Free fatty acids receptors after stimulated by LPS,which has been analyzed by RT-PCR and Q-PCR,are consistent with the results of Wang J,et(2006).And the peritoneal macrophages of WT mic and Gpr84 knockout mice were extracted directly after the intraperitoneal injection of thioglycollate medium.The results showed that after LPS treatment,the mRNA levels of TNF-a,IL-6,IL-12ap35 and IL-12?p40 in macrophages derived from Gpr84 knockout mice were significantly lower than those in the wild type mice.The phagocytosis of GFP-E.coli decreased in Gpr84-/-macrophages.Gpr84 had no significant effects on the phagocytosis of GFP-E.coli in PM.Gpr84 was not involved in the release of chemokines in macrophages and the chemotaxis abilities induced by the LPS.To summarize,as a pro-inflammatory receptor,Gpr84 up-regulated the transcription of TNF-?,IL-6 and IL-12? p40 in macrophages to enhance the inflammatory response.The deficiency of Gpr84 could down-regulate the concentration of TNF-?,IL-6 and IL-12? p40 in serum and IL-6 and IL-12? p40 in the lavage fluid in mice,and rescued lethal inflammation in septic mice by inhibiting the release of pro-inflammatory cytokines of macrophages.So our results contribute to provide a potential therapeutic target of Gpr84 on prevention and treatment to sepsis.