The Roles Of Chemokine CXCL10 And Its Receptor CXCR3 In Chronic Nonbacterial Prostatitis And Mechanism Research

Background:Chronic prostatitis/chronic pelvic pain syndrome(CP/CPPS)is a common urinary disease that seriously affects the health and quality of life of patients.The main features of CP/CPPS are pain and lower urinary tract irritation.Pain can occur in the pelvis,perineum,scrotum,rectum,testicles,penis and back.The etiology of CP/CPPS is unclear,and chronic inflammation as a cause of CP/CPPS has been well studied.Accumulating evidence suggests that CP/CPPS may be caused by inflammation resulting from autoimmune disorders directed against prostate antigen(PAg).A large amount of evidences based on CP/CPPS patients and animal models suggest that CP/CPPS patients have autoimmune dysfunction;T?cell immune imbalance and macrophages play an important role in the development of CP/CPPS.The causative factor may trigger chronic inflammation in the form of prostate autoimmunity,promoting prostate recruitment by T cells and different other white blood cells(including macrophages),leading to inflammation of prostate tissue and chronic pelvic pain.The changes of local inflammatory microenvironment and the secretion of inflammatory mediators may induce neurosensitization and lead to the occurrence of chronic pelvic pain.Although changes in the composition of inflammatory immune cells and their pathogenic role in CP/CPPS have been identified,more research is urgently needed to determine the factors and mechanisms that influence the composition of inflammatory immune cells.Chemokine CXCL10 and its receptor CXCR3 are involved in the regulation of immune and inflammatory responses.However,the role of chemokine CXCL10 and its receptor CXCR3 in CP/CPPS remains unclear.Based on the establishment of experimental immune prostatitis(EAP)model,this study investigated the role of chemokine CXCL10 and its receptor CXCR3 in CP/CPPS,as well as the effect and mechanism of macrophage chemotaxis,macrophage polarization and Th1 cell differentiation.Methods:1.Detection of CXCL10 in CP/CPPS patients and EAP modelThe changes of CXCL10 in serum of CP/CPPS patients and healthy volunteers were detected by enzymelinked immunosorbent assay(ELISA).The EAP model was successfully constructed uing twice immunization modeling method.The changes of CXCL10 in serum or prostate tissue of EAP model were detected by real?time quantitative polymerase chain reaction(RT?QPCR),immunohistochemical staining(IHC)and ELISA.2.Detection of CXCR3 in prostate tissue and EAP model of patients with benign prostatic hyperplasiaAccording to the degree of tissue inflammation,prostate tissues of BPH patients were divided into different levels of inflammation,and the expression changes of CXCR3 were detected by IHC.The expressions of CXCR3 in prostate tissues of EAP model were detected by RT?q PCR and IHC.3.Source analysis of CXCL10 cells in prostate tissue of EAP modelImmunohistochemical fluorescence staining(IF)was used to detect the cell localization of CXCL10 in prostate tissue of EAP model.4.The production and secretion of CXCL10 increased after activation of prostatic stromal cellsIn vitro,prostatic stromal cells were stimulated with cytokines IFN?? and IL?17A to simulate the microenvironment of prostate tissue in EAP model.Changes of CXCL10 were detected by RT?q PCR and ELISA.5.Effects of chemokine CXCL10 and its receptor CXCR3 on inflammation and pelvic pain of EAP mice in vivoEAP model mice were treated with CXCL10 sh RNA and CXCR3 antagonist(AMG487).H?E staining was used to detect the degree of inflammation in prostate tissue,behavioral evaluation for the reactivity to pain stimulation,and ELISA was used to detect the changes of inflammatory cytokines.6.Chemokine CXCL10 and its receptor CXCR3 promote macrophage migration to prostate tissue in vivoEAP mice were treated with CXCL10 sh RNA and CXCR3 antagonist(AMG487).Flow cytometry and IF were used to detect the changes of macrophages in prostate tissues.7.Chemokine CXCL10 and its receptor CXCR3 can promote macrophage chemotaxis in vitroTranswell chamber and ??slide were used to detect the effects of rm CXCL10 protein and AMG487 on macrophage migration,and western blot(WB)was used to detect changes in phosphorylation levels of ERK1/2 or P38 MAPK.8.Chemokine CXCL10 and its receptor CXCR3 had no effect on macrophage viability in vitroCCK8 assay was used to detect the effects of rm CXCL10 protein and AMG487 on macrophage viability in vitro.9.Effect of chemokine receptor CXCR3 on macrophage polarization in vivoIF was used to detect the changes of M1?type macrophages in prostate tissue of EAP model.10.Effect of chemokine receptor CXCR3 on macrophage polarization in vitroIF was used to detect the effect of AMG487 on polarization of M1?type macrophages,and WB was used to detect the changes of phosphorylation levels of ERK1/2,P38 MAPK and NF??B.11.Effect of chemokine receptor CXCR3 on Th1 cell differentiation in vivoIF and flow cytometry were used to detect the proportion of Th1 cells in EAP model prostate tissue.12.Effect of chemokine receptor CXCR3 on Th1 cell differentiation in vitroThe proportion of Th1 cells was detected by flow cytometry,and the phosphorylation level of STAT4 was detected by WB.Result1.CXCL10 expression was elevated in CP/CPPS patients,and positively correlated with disease severity.CXCL10 expression was elevated in serum and prostate tissue of EAP model.2.CXCR3 is highly expressed in the prostate tissue with inflammation,and is positively correlated with the degree of inflammatory cell infiltration.CXCR3 expression was elevated in EAP model prostate tissue.3.CXCL10 can co?locate with macrophages,CD4+ T cells and prostatic stromal cells.The fluorescence intensity of CXCL10 in CD4+T cells,macrophages and prostate stromal cells in EAP model mice was higher than that in the normal group.4.Cytokines,including IFN?? and IFN?? ? IL?17A,could stimulate prostatic stromal cells to secrete CXCL10,but IL?17A alone stimulation could not induce CXCL10 secretion.5.Interference of CXCL10 expressions and inhibition of CXCR3 activity can reduce the degree of inflammation and pelvic pain in EAP mice,and reduce the expression of pro?inflammatory factors IL?6 and MCP1 in EAP mice.6.CXCL10/CXCR3 axis can promote macrophage migration in vivo and in vitro.CXCL10/CXCR3 axis promotes macrophage migration by activating downstream Erk1/2 and P38 MAPK signaling pathways.CXCL10/CXCR3 axis promotes M1?type polarization of macrophages in vivo and in vitro through LPS/NF??B P65,p38 MAPK and ERK1/2 signaling pathways.CXCL10/CXCR3 axis promotes Th1 differentiation in vivo and in vitro through IL12?STAT4 signaling pathways.ConclusionsIn CP/CPPS disease and EAP model,chemokine CXCL10 and its receptor CXCR3 are important mediators in the occurrence and development of chronic prostatitis and participate in inflammatory infiltration of prostate tissue and pain symptoms.Chemokine CXCL10 promotes macrophage migration and M1?type polarization through CXCR3?mediated activation of ERK1/2 and p38 MAPK.In addition,Chemokine receptor CXCR3 could activate IL?12?STAT4 pathway to promote Th1 cell differentiation and secrete inflammatory mediators leading to disease progression of EAP.