The Role Of Nestin+Mesenchymal Stem Cells(MSCs) In Bone Marrow Chronic Graft-Versus-Host Disease

Background and ObjectivesAllogeneic hematopoietic cell transplantation(allo-HSCT)is a common therapy for hematological malignancies,autoimmune diseases and metabolic disorders.Chronic Graft-versus-host disease(cGVHD)is a major cause of mortality and morbidity after allo-HSCT,and restricts the efficacy of allo-HSCT.The development of cGVHD is affected by many factors,such as stem cell source,HLA matching,donor source,patients' age.Skin,lung,liver and intestine are regarded as the principal target organs of cGVHD.However,the alteration of BM caused by cGVHD have not been reported yet.BM is the origin of hematopoietic cells as well as the site of the origin and progression of hematological malignancies.BM,as one of the targets of acute GVHD(aGVHD),is mainly characterized by the reduction of hematopoietic stem and progenitor cells and the abnormal of stromal cells.Alloantigens are presented by antigen-presenting cells(APCs).Donor T cells are activities via interaction with APC and exert their effector function through both cell-contact-dependent cytotoxic or soluble factors.Hematopoietic cells are cleared after conditioning,while stromal cells survive because of better tolerance to chemoradiotherapy.Systemic inflammation lead to residual stromal cells delepted and upregulation of MHCII,CD40 in stromal cells.Furthermore,vessel walls get more permeable,which facilitate activation and entrance of alloreactive lymphocytes into BM.However,the BM cGVHD is rarely reported and the mechanism for BM cGVHD is still unclear.Nestin+mesenchymal stem cells(MSCs)are capable of differentiating into cells of multiple cell lineages and involved in the formation of fibrotic lesions.To explore whether Nestin+MSCs play an important role in the development of BM cGVHD,we performed a study by using MHC-mismatched murine model.MethodsThe cGVHD model of allo-HSCT was established by C57BL/6? BALB/c mice.The deposition of collagen fibers and reticular fibers in bone marrow was detected by masson staining and silver staining.The infiltration of T lymphocytes in BM was observed by flow cytometry and immunohistochemistry.The changes and transformation of nestin+MSC were detected by immunofluorescence,as well as the level of TGF-?1 around the Nestin+MSC and TGF-?RII expressed in the Nestin+MSC.The alterations of BM macrophages were detectd by flow cytometry and immunofluorescence staining.Results1.Establishing a C57BL/6? BALB/c cGVHD murine model:The survival of cGVHD mice was decreased,and cGVHD mice began to appear weight loss,hair loss,archback and decreased activity.The hematoxylin and eosin staining of target organs meets the cGVHD pathology.2.Detecting myelofibrosis and T lymphocyte infiltration in cGVHD group:Compared with mice in TCD-BM group,the deposition area of collagen fibers and reticular fibers in BM was significantly increased in cGVHD group;The frequencies and counts of CD4+and CD8+T lymphocytes were also significantly increased in BM of cGVHD group.3.Detecting expansion and transformation of Nestin+MSC in cGVHD group:Compared with mice in TCD-BM group,the counts of Nestin+MSCs and surrounding collagen I deposition area were significantly increased in cGVHD group.Nestin+aSMA+ double positive cells in BM of cGVHD group were significantly higher than those in TCD-BM group.4.Detecting expansion of BM macrophages in cGVHD group:It was found via immunofluorescence that the counts of F4/80+macrophages were significantly increased in cGVHD group compared with the TCD-BM group and the level of TGF?1 around F4/80+ macrophages were also increased significantly.In addition,it was detected by flow cytometry that the frequencies and counts of Ly6GLy6ClowF4/80+CD 115-SSClow macrophages and Ly6G-Ly6Clow F4/80+CD 115SSClowCD 169+ macrophages in the cGVHD group were higher than those in the TCDBM group.ConclusionsBone marrow is one of the targets of cGVHD,which is characterized by increased deposition of collagen or reticular fibers,and lymphocyte infiltration.In the BM microenvironment of cGVHD mice,Nestin+MSC expanded abnormally and transformed to Nestin+?-SMA+double positive cells,and the level of TGF-?1 and TGF-?RII expressed on the surface of Nestin+MSC were increased.In addition,macrophages in BM of the cGVHD group was increased,which may contributed to the development of BM cGVHD.