Fibronectin Regulates Chronic Graft-versus-host Disease By Influencing Infiltration Of Alternatively Activated Macrophages

Objectives:To investigate the role of fibronectin(FN)in chronic graft-versus-host disease(cGVHD),the therapeutic effect of exogenous FN peptides in cGVHD and the mechanism how FN peptides alleviate cGVHD,so as to provide new ideas and theoretical basis for the treatment of cGVHD.Methods:FN KO mice were identified via extracting DNA,PCR and gel electrophoresis.Mice plasma FN level was detected by ELISA to verify the knockout(KO)efficiency induced by tamoxifen.After TBI conditioning,recipients were injected with a certain dose of bone marrow cells and spleen cells from donors,then acuteGVHD(aGVHD)and cGVHD murine models were established,including BALB/c to C57BL/6 and DBA/2 to BALB/c cGVHD murine models.The clinical manifestations ofGVHD were observed and scored.HE staining and masson’s trichrome staining were used to score pathologicalGVHD.GVHD severity between FN KO mice and FN lox P+/+,Cre-mice were compared to investigate the effect of FN onGVHD.Different cGVHD models,including BALB/c to C57BL/6,and DBA/2 to BALB/cGVHD models were established.Then exogenous FN peptides were injected into recipients via tail vein injection and intraperitoneal injection to rise FN level in vivo.TheGVHD severity of mice receiving FN peptides was compared with that receiving PBS,so as to investigate the effect of FN onGVHD and the therapeutic effect of FN peptides onGVHD.Finally,flow cytometry and immunofluorescence were used to investigate the mechanism how FN peptides regulate cGVHD.Using flow cytometry analysis,the proportion of B cells,F4/80~+macrophages in spleen were analyzed.Multiplex cytometric bead array was used to detect IL-6,IL-10,MCP-1,TNF,IFN levels.By immunofluorescence staining technique,infiltration of F4/80+macrophages,infiltration of F4/80+CD206+M2macrophages and antibody deposition in recipients’target tissues,including the skin,liver,lung,kidney were detected.Results:In BALB/c to C57BL/6 murine model,aGVHD was induced by injecting5*10^6 BALB/c donor bone marrow cells and 3*10^6 spleen cells after 11Gy TBI,and cGVHD was induced by 3*10^6 donor bone marrow cells and(0.1-1.5)*10^6 donor spleen cells after 11Gy TBI.In DBA/2 to BALB/c murine model,recipients received1*10^6 bone marrow cells and(10-15)*10^6 spleen cells from DBA/2 donors after 8Gy TBI to induce cGVHD.Recipients with cGVHD showed clinical manifestations,including weight loss,hair loss,hunch back and skin scleroderma.Inflammatory cells infiltration,tissue damage and fibrosis could be detected pathologically.Compared with the control group,down-regulation of FN level with FN KO mice had no significant effect on the survival of recipients with aGVHD,but the clinical and pathological manifestations of cGVHD in FN KO mice were more severe significantly.The up-regulation of FN levels using exogenous FN peptides had no significant effect on the survival of aGVHD.While FN peptides which was given via tail vein injection in vivo prolonged survival of mice with cGVHD.Besides,in cGVHD models,including both BALB/c to C57BL/6 mice model and DBA/2 to BALB/c mice model,recipients receiving exogenous FN peptides via intraperitoneal injection showed milder cGVHD severity than that receiving PBS.The level of MCP-1 in receipents receiving exogenous FN peptides decreased significantly.Infiltration of M2 macrophages in target tissues decreased,and the deposition of antibodies in target organs also decreased.Conclusion:FN had no significant effect on aGVHD,and exogenous FN peptides had also no significant effect on aGVHD.However,FN showed a protective effect on cGVHD.FN KO aggravated cGVHD.While up-regulation of FN level by exogenous FN peptides could weaken the severity of cGVHD,and FN peptides showed a therapeutic effect on c GHVD which is not model-dependent.FN peptides may inhibit the migration and infiltration of M2 macrophages in target tissues by reducing MCP-1,which may contribute to reduction of TGF-βsecreted by M2 macrophages in target tissues,and antibodies’deposition in target organs also decreased.As a result,cGVHD could be attenuated.