| Myocardial overload,infection,inflammation and ischemia lead to myocardial injury,myocardial fibrosis,cardiac structure and function damage,causing myocarditis,cardiomyopathy,myocardial infarction and even lead to heart failure.It is of great significance to elucidate the role of regulatory cells or factors in cardiac injury model for the prevention and treatment of cardiovascular disease.B10 cells（IL-10 producing regulatory B cells,B10 cells）is a heterogeneous group,characterized by their ability to produce IL-10,an inhibitory cytokine,regardless of their cell surface phenotype.Previous studies have shown that changes in the number and function of B10 cells are different in different disease.However,dynamic changes of B10 cells in heart injury disease have not been well discussed,and their biological functions cells remain unclear.Objective:Viral myocarditis（VMC）,experimental autoimmune myocarditis（EAM）and Myocardial infarction model were established to explore the dynamic changes of B10cells in the heart injury,underlying mechanisms and the role of B10 cells in heart disease,especially anti-fibrosis function.Methods:1.Exploring the changes of B10 cells and their suppressive role in myocardial injury models.VMC models were induced in mice by intraperitoneal injection of CVB3,the heart sections were examined by hematoxylin-eosin（H&E）staining.The mice were weighted every day before being sacrificed.RT-q PCR was used to detect viral RNA.IL-10expression in B cells from spleen and heart of VMC and control mice was analyzed by flow cytometry（FCM）.Splenic B10 cells were purified from VMC mice after 7 days of infection.Non-B10 or B10 cells were immediately transferred into the VMC mice.Heart sections were examined by HE staining.The frequency and number of Th17 cells were measured by FCM.The proportion of B cell subsets in spleen,peritoneal cavity and bone marrow of CD19-/-mice was detected by FCM.Meanwhile,the proportion of B10 cells in spleen and heart of CD19-/-mice were detected by FCM.The level of TNF-α,IL-1β,and IL-10 in B220+cells purified from spleen of CD19-/-mice were measured by ELISA.Probability of CD19-/-and WT mice after viral injection with or without B10 cells transferred into the appropriate recipient mice were calculated to draw survival curve.Mice were immunized with peptide derived fromα-myosin heavy chain to induce the EAM model.Heart sections were examined by HE staining.The number of B10 cells in the spleen and heart from each group was analyzed by flow cytometry.The MI model was established by coronary artery ligation.The changes of B10 cells in spleen and heart were examined during MI.2.Exploring the underlying mechanism of B10 expansion in cardiac injury.TNF-αwas used to induce the apoptosis of cardiomyocytes（CM）.Apoptotic CM and its supernatant were co-cultured with B cells,and then the number of B10 cells was examined by FCM and the level of IL-10 was detected by ELISA.The level of PGE2 in Apoptotic CM and activated cardiac fibroblasts（CF）were detected by ELISA.PGE2levels in hearts of VMC mice were measured by ELISA.Purified spleen B cells from normal mice were treated by PGE2,IL-17,IL-22,HMGB1,ANG II and IL-33,and the percentage of B10 cells was analyzed by intracellular cytokine staining assay.Purified spleen CD19+B cells were treated with different PGE2 concentrations.B10 cell frequencies are examined by FCM,and IL-10 levels in the supernatant were measured by ELISA.PGE2 receptors（EPs）expression was analyzed by RT-q PCR.B cells were pre-treated with specific EP receptor antagonists and then stimulated with PGE2.After 48h treatment,cells were harvested to detect B10 cell frequency.Spleen B cells were treated with PGE2,and phosphorylation of ERK,P38,AKT,and GSK3α/βwas measured by western blot.The expression of c-Jun and C/EBP-βwas assessed by western blot.Ah R expression in the cytoplasm and nuclei in treated and non-treated cells was measured by western blot and immunofluorescence staining.IL-10 in the supernatant was detected by ELISA and B10 cells were determined by FCM after B cells were pre-exposed to SB203580 or Loureirin A,and then treated with PGE2.The frequency of B10 cells was determined by FCM after B cells were pre-exposed to CH223191.PGE2 or PBS was intravenously administered to VMC mice,after which spleens and hearts were harvested.Heart sections were assessed by HE staining.The percentage of B10 cells in the spleen was assessed by flow cytometry,and the RNA copy number of CVB3 was measured by q PCR and normalized relative to GADPH.Na（?）ve CD4+T cells activated with anti-CD3,anti-CD28,IL-6,IL-23,and TGF-βwere differentiated in co-culture with non-B10 cells,B10 cells,or PGE2-pretreated B10cells for 24 h.The frequency of Th17 cells were analyzed by FCM.PATs in MI mice were weighed and the number and frequency of B10 cells in PATs of MI mice were analyzed by FCM.The MI model was established after removing pericardial adipose tissue,and the changes of B10 cells in cardiac tissue were detected in MI mice at day 28.3.Exploring the anti-fibrosis properties of B10 cells in the heart disease.CFs were co-cultured with non-B10 cells and B10 cells under TGF-βinduced fibroblast activation conditions with or without pre-exposure to IL-10R1 antibody.α-SMA and collagenⅠexpression were measured by western blot.Cell migration was assayed by the scratch wound healing assay.CFs were treated by IL-10.Pericellular HA coat was visualized by the exclusion of sheep erythrocytes,and HA level in the supernatant was detected by ELISA.HA level in B10 cells purified and co-cultured with cardiac fibroblast for 24 h with or without pre-exposure to IL-10R1 antibody.After adoptive transfer of B10 or non-B10 cells to EAM mice,Sirius red staining was used to detect the deposition of collagen fibers in cardiac tissue,ELISA was used to detect serum HA levels.CD19-/-mice were used to established MI model.Sirius red staining was used to detect collagen deposition in cardiac tissue and serum HA level was examined by ELISA.HA levels in EAM mouse sera on days 28 and 35 postimmunization.Concentration of HA was examined by ELISA.Level of inflammation and fibrosis assessed by H&E and Sirius red staining,respectively,in the hearts of EAM mice following intragastric administration of 4 MU.CollagenⅠandⅢexpression analyzed by RT-q PCR.Fibrosis level assessed by Sirius red staining following intravenous HA or PBS administration on EAM mice.Result:1.B10 cells transiently increased and play an immunosuppressive function in heart injury disease.VMC models were induced in 6-week-old mice by intraperitoneal injection of CVB3.High inflammatory cell infiltration was observed in the VMC group compared with the control group.However,no inflammatory cell infiltration was detected after day 14 of model induction.The CVB3 gene in the heart was validated by RT-q PCR on days 3,5,and 7.Interestingly,the total number and frequency of B10 cells in the spleen expanded to a peak on day 3 and then gradually.Similar changes were observed in the heart;however,B10 reached the highest level on day 7.Adoptive transfer of B10 cells significantly attenuated inflammatory cell infiltration and decreased Th17 infiltration compared with the non-B10 cell group.Deletion of CD19 had no deleterious effects on the generation of B cells in the bone marrow,but there was a significant reduction in the number of B1 cells,especially B1a,in peritoneum and spleen.B1a cells were the predominant source of B10 cells.In CD19-/-mice,the percentage of B220+IL-10+B cells in the spleen and heart are dramatically reduced.Wild type（WT）and CD19-/-mice were treated i.p.with CVB3 to induce VMC.The concentration of IL-10 was decreased in CD19-/-mice compared with WT mice before and after viral infection.Whereas,the inflammatory cytokines IL-1βwas increased.Adoptive transfer of B10 cells significantly prolong the survival time compared with the non-B10 cell group.The number of B10cells in spleen and heart of EAM mice was increased on days 21,28 and 35 during which the number of B10 cells reached to a peak on days 21 and 28 and then declined gradually.MI mouse model was successfully established.The number of B10 cells in spleen of MI mice was increase on day 7.After this time,the ratio of heart to body weight increased.The number of B10 cells in the heart tissue significantly increased on day 28 of the MI.2.Apoptotic cardiomyocytes promote the amplification of B10 cells by secreting PGE2.TNF-αinduce CM apoptosis.The apoptotic CM and its supernatant can promote the expansion of B10 cells and the secretion of IL-10.The secretion of PGE2 were evaluated in apoptotic CM.Purified B cells were cultured with IL-17,IL-22,IL-33,ANG II,or HMGB1.Only ANG II and PGE2 induced an increase in B10 cells,especially PGE2,which induced about a 1.5-fold change at 48h.EPs m RNA were upregulated following PGF2 treatment.Only EP4 antagonist ONO-AE3-208 abolished B10 cell expansion.Enhanced activation of ERK,p38-MAPK and AKT was observed in PGE2-stimulated purified B cells.The expression of c-Jun and Ah R was significantly increased in purified B cells stimulated by PGE2.Furthermore,western blot and immunofluorescence staining also demonstrated that Ah R was translocated from the cytoplasm to nuclei.Inhibiting P38-MAPK and AKT phosphorylation and Ah R translocation decreased B10 cells expansion and IL-10 secretion.PGE2enhanced the immunosuppressive ability of B10cells on Th17 differentiation.The frequency and total number of Th17 cells were decreased in the PGE2-pretreated B10 group compared with the non-pre-treated group.In response to acute MI,significantly more donor B10 cells infiltrated LVs accompanied by a reduction of these cells in PATs.The changes of B10 cells in cardiac tissue were decreased in mice removing PATs before MI induction.3.B10 cells alleviate the fibrosis of the late phase of heart damage disease.The expression level ofα-SMA and collagenⅠwere decreased in the group treated with B10 cells compared with the group treated with non-B10 cells treated group.Anti-fibrotic role of B10 cells was abolished upon supplementation with a neutralizing anti-murine IL-10R1 antibody.B10 cells impeded cardiac fibroblast migration through IL-10 secretion.Further,IL-10 also accelerated HA synthesis by fibroblasts,which produce a large PCM.B10 cells also facilitated HA production via IL-10.Adoptive transfer of B10 cells significantly attenuated collagen deposition and upregulated the level of sera HA in EAM mice.the areas of collagen deposition in CD19-/-mice with MI were larger than those WT mice.Furthermore,the level of HA was significantly reduced in CD19-/-mice with MI.Our data demonstrated that the concentration of HA was increased on day 28,which subsequently decreased on day 35 in the EAM model.In the4-MU treated group,cardiac injury persisted and cardiac fibrosis was drastically aggravated compared with the EAM group.HMW-HA（>950 k Da）is the main form of HA in a healthy heart.Hence HMW-HA was injected intravenously into the EAM mice on day 14.Fibrosis in HA treated mice was ameliorated significantly compared with the EAM mice.Conclusion:The frequency and number of B10 cells were transiently increased during heart injury.Apoptotic cardiomyocytes derived PGE2 drove B10 cell expansion via the MAPKs/AKT-AP1 axis or Ah R.B10 cells potentially exert dual anti-inflammatory and anti-fibrotic effect via producing IL-10 and mediating HA secretion. |
PDF Full Text Request