Protein Phosphatase 2A Is Involved In The Pathophysiology Of Acute Graft Versus Host Disease After Allogenic Hematopoietic Stem Cell Transplantation Through Th17 Differentiation

BackgroundAllogenic hematopoietic stem cell transplantation?allo HSCT?remains to be a potentially curative therapy for many malignant hematologic diseases and other life-threatening hematological disorders.However,the efficacy is partially discounted by graft versus host disease?Gv HD?,which is the main cause for non-relapse mortality after allo HSCT.Current insights on the role of alloreactive T cell immunity have led to an improved understanding of Gv HD pathophysiology.With the tissue damage following conditioning therapy,the cells releases inflammatory cytokines,chemokines and adhesin.In addition,antigen-presenting cells expressing human leukocyte antigen and costimulatory molecules stimulate the proliferation and differentiation of donor derived T cells into effector T cells,which causes immune damages to target tissues.Th17 cells are characterized by production of interleukin-17?IL-17?.A variety of cytokines promote Th17 development.The regulation of Th17 differentiation occurs at translational and post-translational levels.The role of Th17 and its related cytokines in Gv HD remains controversial.There are controversial results concerning the role of Th17cells in Gv HD pathophysiology.Some report that high dose of in vitro differentiated Th17cells mediated severe cutaneous and pulmonary lesions.Others report that il17 knock-out T cells induced high Th1 differentiation and severe acute Gv HD?aGvHD?.This indicated that Th17 was sufficient but dispensable for Gv HD.Results also showed that Th1 cell preferentially mediated Gv HD tissue damage in the gut and liver,while Th17 exacerbated tissue damage in the lung and skin.Other studies suggested that Th17 cells were involved in the early phase of Gv HD.Protein phosphatase 2A?PP2A?is a serine/threonine phosphatase ubiquitously expressed in eukaryotic cells.It plays an important role in cellular proliferation,transformation,apoptosis and metabolism.It was found that PP2A regulates the differentiation and proliferation of Th17 cells in autoimmune diseases,including systemic lupus erythematosus and rheumatoid arthritis.PP2A regulates the differentiation of T cell subsets,and facilitates the Th17 differentiation in autoimmune diseases.However,the role of PP2A in the pathophysiology of aGvHD is still unknown.And the underlying association of PP2A and Th17 differentiation in the context of aGvHD has not been reported yet.In the present study,we aimed to explore the correlation between PP2A and Th17 cell differentiation in aGvHD.Our results would provide a new rationale for the pathophysiology of aGvHD.MethodsFifty-four allo HSCT patients were included in this study,with a median age of 37years?range:16?64 years?.The patients were divided into Gv HD group?aGvHD group:n=14;chronic Gv HD[c Gv HD]group:n=9?and non-Gv HD group?n=31?.12 healthy donors were included as the controls.Peripheral blood?PB?samples were collected from all patients on 15 days before transplantation and at pre-determined time points after transplantation.The frequency of Th17 cells was detected by flow cytometry.The levels of Th17 signature cytokines in PB were detected by enzyme linked immunosorbent assay.The relative expression of PP2A_C,ROR?t,and IL-17A were detected by polymerase chain reaction.Western blot was applied for the expression levels of PP2A_C,p-Smad2,p-Smad3,p-STAT3and ROR?t in mononucleated cells of patients with and without aGvHD,respectively.Co-immunoprecipitation?Co-IP?was conducted for binding dectection of ROR?t with p-Smad2,p-Smad3 and p-STAT3.Results1.The levels of inflammatory cytokines in PB samples were significantly increased in the aGvHD group,including TNF-?,IL-1?,IL-2R,IL-6,reg3?,and skin-specific elafin.2.The frequency and absolute number of Th17 cells were increased in the aGvHD group when compared with those in the non-Gv HD group.The frequency of Th17 cells in c Gv HD and non-Gv HD groups were similar.3.Th17 related cytokines?TGF-?1,IL-6,IL-17A and IL-23?were significantly increased in the aGvHD group when compared with non-Gv HD group.4.The expression level of PP2A catalytic subunit?PP2A_C?gene was significantly increased in aGvHD group.The expression levels of Th17 signature genes?IL-17A,TGF-?1,IL-6 and IL-23?and the key transcription factor ROR?t were also increased.5.The expression level of PP2A_C protein in mononucleated cells of patients with aGvHD was higher than that of non-Gv HD group.The phosphorylation state of STAT3(Tyr⁷⁰⁵)and Smad2(Ser^465/467)and the expression level of ROR?t proteins were significantly increased for aGvHD patients.The phosphorylation state of Smad3(Ser^423/425)was decreased in patients with aGvHD.6.In mononucleated cells of patients with aGvHD,Co-IP experiments revealed that p-Smad2(Ser^465/467)and p-STAT3(Tyr⁷⁰⁵)binded with ROR?t.Conclusions1.Th17 cells participate in the pathophysiology of aGvHD.2.PP2A participates in the pathogenesis of aGvHD through regulating the Th17differentiation.