IL-27 Is Involved With The Occurrence Of Tuberculous Pleural Effusion By Activating Tim-3/Gal-9 Pathway In NK Cell

Background: Natural killer(NK)cells play an important role in maintaining the stability of innate immunity and the activation of adaptive immunity,serving as the first line of defense against tuberculosis infection.Since the immune system is unable to rapidly eradicate the pathogen of tuberculosis,immune cells are chronically stimulated by the antigen,which in turn highly express inhibitory receptors,leading to cellular dysfunction.At present,many immunoregulatory receptors and their regulatory effects have been confirmed in tumors and chronic infectious diseases.However,the regulation of the expression of NK cell surface immunoregulatory receptor Tim-3 and its effect on NK cell function in patients with tuberculosis are not clear.Purpose: 1.To investigate the expression of NK cell Tim-3 and its ligand Gal-9 in tuberculous pleural effusion(TPE).2.To investigate the potential factors affecting the expression of Tim-3 in NK cells in the thoracic microenvironment.3.To investigate the effect of Tim-3/Gal-9 on NK cell function in TPE.Method: 1.Subjects Experimental group: Peripheral blood samples: Peripheral venous blood samples were collected from 22 patients with TPE,including 17 males and 5 females,in Xiangya Hospital of Central South University.Hydrothorax samples: The hydrothorax of 39 cases of tuberculous pleural effusion(TPE),19 cases of malignant pleural effusion(MPE)and 15 cases of transudate(TE)were collected in Xiangya Hospital of Central South University.Healthy control group: 25 cases who underwent routine physical examination in Xiangya Hospital and were not accompanied with other acute and chronic diseases as healthy controls.Nine females and 16 males were included in the study.The peripheral venous blood samples of the control group were collected.2.NK cell Tim-3 expression detection Peripheral mononuclear cells were extracted from peripheral blood(2–5 m L)of subjects;PFCs were extracted from hydrothorax(50 m L–100 m L);and Tim-3 on the surface of NK cells was labeled with the fluorescent antibodies CD45-PE-Cy7,CD3-FITC,CD56-PE,and Tim-3-BV421.3.the determination of plasma and pleural effusion Gal-9The plasma levels of Gal-9 were determined by ELISA in 22 TPE patients and 25 healthy controls.The levels of Gal-9 in pleural effusion of 39 cases of TPE,19 cases of MPE and 15 cases of TE were detected.4.effect of il-27 on that expression and function of Tim-3 in NK cells PBMC from fresh whole blood were inoculated in petri dishes and stimulated with ESAT-6,IL-27,and Gal-9,respectively or in combination,and the untreated cells served as the negative control,which were incubated for 24 hours.At the end of the culture,the expression of Tim-3 and CD107 and the secretion of INF-γ and TNF-α in NK cells of each group were detected by flow cytometry.Result: 1.The expression of NK cell Tim-3 in peripheral blood of patients with TPE was significantly increased as compared with that in the control group(P < 0.001),and the serum level of Gal-9(157±72.2 pg/m L)was higher than that in the control group(88.1 35.7 pg/m L)(P < 0.001).2.The expression of NK cell Tim-3 in TPE was significantly increased as compared with that in the TE group(P < 0.001),and the level of Gal-9 in TPE(121.9 50.7 pg/m L)was also higher than that in the TE group(81.8 26.5 pg/m L)(P < 0.001).3.There was no correlation between NK cell Tim-3 and hydrothorax albumin levels(r =-0.151,P=0.758,n=39),LDH(r =-0.025,P=0.877,n=39),AHDBH(r =-0.014,P=0.932,n=39)and ADA levels(r =-0.063,P=0.703,n=39)in TPE.There was no correlation between NK cell Tim-3 and ESR(r =-0.103,P=0.648,n=22),serum albumin level(r =-0.338,P=0.527,n=22),AST(r=0.048,P=0.832,n=22),ALT(r=0.182,P=0.418,n=22),and serum creatinine level(r=0.172,P=0.443,n=22)in peripheral blood of patients with TPE.4.In 4.TPE,there was no significant correlation between Gal-9 level and hydrothorax LDH(r =-0.111,P=0.503,n=39),AHDBH(r =-0.105,P=0.531,n=39)and ADA level(r=0.038,P=0.816,n=39).Gal-9 levels positively correlated with hydrothorax albumin levels(r=0.382,P=0.016,n=39).In PTE peripheral blood,Gal-9 levels positively correlated with serum albumin levels(r=0.482,P=0.023,n=22),and there was no correlation with ESR(r =-0.129,P = 0.568,n=22),AST(r =-0.027,P=0.906,n=22),ALT(r =-0.113,P=0.615,n=22),and serum creatinine levels(r =-0.008,P=0.917,N = 22).5.TPE showed a positive correlation between NK cell Tim-3 expression and Gal-9 level(r=0.351,P=0.028,n=39),and no correlation was observed in peripheral blood(r=0.177,P=0.428,n=22).6.In vitro experiments,stimulated by ESAT-6,the expression of Tim-3 in NK cells was increased as compared with that in the control group.After the addition of IL-27,the expression of Tim-3 was further increased as compared with that in the ESAT-6 group(P < 0.001).7.Under the stimulation of ESAT-6 and IL-27,the expression of CD107 a and the secretion of IFN-γ and TNF-α in NK cells were increased compared with those in the control group(P < 0.001).After the addition of Gal-9,the degranulation function of NK cells was inhibited and the secretion of IFN-γ and TNF-α was decreased(P < 0.001).Conclusion: 1.The expression of NK cell Tim-3 and the level of ligand Gal-9 in pleural effusion and peripheral blood of TPE patients were increased.2.In TPE,IL-27 can promote the expression of Tim-3 in NK cells,and the latter can inhibit the cell killing effect and secretion function of NK cells after binding to Gal-9.