Mechanisms Of Peripheral Blood-derived Mesenchymal Stem Cells Regulating The Polarization Of Macrophages Via The IL-10/STAT3 Pathway

Objective: 1.To investigate the effect of the IL-10/STAT3 pathways in the process of Peripheral blood-derived mesenchymal stem cells(PBMSCs)regulating macrophages polarization.2.To investigate the difference between PBMSCs and their conditioned medium in regulating the polarization of macrophages in vitro.Methods:1.Isolation,culture and identification of rat PBMSCs: The blood of the Sprague Dawley(SD)rats was collected after stimulated by G-CSF,then the monocyte layer of the blood samples was collected using Ficoll.The collected cells were then seeded,cultured and subcultured.The P3 generation of cultured cells was used to identify whether they are PBMSCs using flow cytometry(FCM;CD29,CD90,CD44,CD45,CD79 and CD11b),immunocytochemical staining(ICC;CD34,CD73,CD105 and HLA-DR)and mesoderm multilineage differentiation examination.2.Isolation,culture and identification of rat M0 macrophages:(1)L929 conditioned medium was prepared with L929 cells.(2)Flushing fluid of the SD rats’ femoral cavities was collected and centrifuged to acquire target cells,after seeded and cultured with macrophage medium(containing 20% of L929 conditioned medium)for six days,the cells were then identified for phenotypes by FCM.3.Co-culture of PBMSCs with M0 macrophages: The experiment was divided into 5groups: PBMSCs group,macrophage(M0)group,PBMSCs conditioned medium and M0 conditioned medium mixture(P-CM+M-CM)group,P-CM and M0 co-cultured(P-CM+M0)group,PBMSCs and M0 co-cultured(PBMSCs+M0)group.Among which,the M0 group,P-CM+M0 group and PBMSCs+M0 group were then dived into 3 groups:Control group,Ab9969(neutralizing antibody of IL-10)group and Stattic(inhibitor of STAT3)group.The supernatant and macrophages of the various groups were collected at the time points of 6h,2d and 4d for the detection of the density and gene expressions of relevant cytokines;the macrophages on 2d were collected for the tests of the relevant cytokine pathway and polarization.4.Relevant tests of the effect of IL-10/STAT3 pathway on the polarization of macrophages:real-time quantitative polymerase chain reaction(RT-q PCR)and enzyme-linked immunosorbent assay(ELISA)techniques were used to examine the density and expressions of IL-10,TNF-α and IL-1β;WB was used to detect the phosphorylation state of JAK and STAT3(JAK/p-JAK and STAT3/p-STAT3);An immunofluorescence technique was used to examine the distribution of STAT3;Flow cytometry(CD86 and CD206)was used to detect the polarization state of the macrophages.Results:1.Cells collected from peripheral blood: uniform and fibroblasts-like morphology,adherent growth;High expression of CD29,CD90,CD44,CD73 and CD105,no expression or weak expression of CD75,CD79,CD11 b,CD34 and HLA-DR.Calcium nodules,glycosaminoglycan and lipid droplets were induced after culture.2.Cells collected from femoral cavities: irregular in shapes,had pseudopodia,adherent growth;high expression of CD 68 and CD11 b.3.ELISA and RT-q PCR showed:(1)At different time points,the density and expression of IL-10 decreased gradually with the three time points in M0 group,P-CM+M-CM group and P-CM+M0(P<0.05);whereas IL-1β and TNF-α increased gradually(P<0.05)(the density and expression of TNF-αshowed no significant difference on 2d compared with 4d(P>0.05)).Then density or expression of IL-10,IL-1β and TNF-α in PBMSCs+M0 showed no significant difference at the three time points(P>0.05).(2)In various co-cultured groups,the density and expression of IL-10 decreased at the three time points in P-CM+M0 group and PBMSCs+M0 group compared with M0/P-CM+M-CM group respectively(P<0.01)(the expression of IL-1β at 6h in PBMSCs+M0 group and P-CM+M0 group increased(P>0.05));The very effect mentioned above was more intense in PBMSCs+M0 group compared with P-CM+M0 group(P<0.05).(3)The density and expression of IL-10 in Ab9969 group and Stattic group decreased at the three time points despite various co-cultures(P<0.05);The density or expression of IL-1βand TNF-α increased at the three time points despite various co-cultures(P<0.05).4.WB,immunofluorescence and flow cytometry showed: increased phosphorylation states of JAK1 and STAT3(P<0.05),increased distribution of STAT3 in the nucleus(P<0.05)and increased in the ratio of the M2 surface marker CD206 positive cells,decreased in the ratio of M1 surface marker CD86 positive cells(P<0.05)in P-CM+M0 group and PBMSCs+M0group compared with M0 group,as well as in P-CM+M0 group compared with PBMSCs+M0 group.Conclusion:1.Both PBMSCs and P-CM can promote the expression and secretion of the M2macrophages’ symbolized anti-inflammatory cytokine IL-10,while inhibit the expression and secretion of M1 macrophages’ symbolized pro-inflammatory cytokines TNF-α and IL-1β.2.Both PBMSCs and P-CM can activate the IL-10/STAT3 pathway to regulate macrophage polarization towards the M2 direction,to promote the expression and secretion of the anti-inflammatory cytokine,and inhibit the pro-inflammatory cytokines.3.There are differences between PBMSCs and P-CM in regulating the expression and secretion of inflammatory cytokines in macrophages as well as in regulating the polarization state.Among them,the control of PBMSCs on the expression and release of inflammatory cytokines in macrophages has been maintained at a relatively high level,while the regulation effect of P-CM decreases with time extension,showing a time-dependent manner.