The Role Of Soluble Fibrinogen-like Protein2in Renal Allograft Recipients With Acute Rejection And Its Mechanism

Object Acute rejection (AR), the major complication after kidney transplantation, hampers renal allograft survival. Tubular epithelial cells (TEC) are the primary target in AR. TEC apoptosis contributes to premature graft loss, while the mechanism has not been clarified. Soluble fibrinogen-like protein2(sFGL2), a novel effector of regulatory T cells (Treg), is known to mediate tissue injury through apoptosis induction. In this study, we explored the role of Treg-secreted sFGL2in AR patients, its activators, its impact on TECs and the underlying mechanism via both in vivo and in vitro investigations.Methods Forty renal transplant recipients were collected, and divided into the AR group or the stable group according to their allograft biopsy results. The AR group was further divided into subsets of grade I T cell-mediated rejection (n=8), grade II T cell-mediated rejection (n=6) and antibody-mediated rejection (n=6), based on Banff07criteria. Fifteen healthy volunteers were enrolled as the control group. The serum level of sFGL2, TNF-a and IFN-y was detected by ELISA and the proportion of CD4+CD25+Foxp3+Treg in the peripheral blood was measured by flow cytometry. In vitro, the isolated human CD4+T cells were stimulated with TNF-α (62.5,125,250,500,1000U/ml), IFN-γ (62.5,125,250,500,1000U/ml) or PBS for24,48and72h. sFGL2in the supernatant and MAPK signaling proteins in the activated CD4+T cells were detected by ELISA and Phosflow flow cytometry respectively. Moreover, human TEC were stimulated with10μg/ml sFGL2,10ng/ml TNF-a as positive control or PBS as negative control for48h, and investigated for apoptosis with Annexin V-FITC/PI staining by flow cytometry. Apoptosis-associated genes in TEC were further assessed by RT-qPCR Approval for this study was obtained from the Ethics Committee of Fudan University.Results Peripheral sFGL2increased in renal allograft recipients with acute rejection to an extent dependent upon the pathological type and severity of the response. Serum level of sFGL2was significantly higher in AR patients (64.84±10.36ng/ml, n=20) than those with stable allografts (37.82±3.56ng/ml, n=20, p<0.05) and healthy volunteers (31.25±4.08ng/ml, n=15, p<0.05), while no significant difference was observed between recipients with stable allografts and healthy volunteers. The sFGL2level also remarkably elevated in patients with antibody-mediated AR than those with T cell-mediated AR (118.80±20.28vs.41.71±4.39ng/ml, p<0.001), and was higher in patients with grade Ⅱ T cell-mediated rejection than those with grade I (51.87±7.81vs.34.10±3.27ng/ml p<0.05). Moreover, the level of TNF-α (114.90±10.20vs.67.21±8.27pg/ml,p<0.001), IFN-γ (257.69±28.60vs.88.15±10.85pg/ml, p<0.001) and the percentage of Treg (4.05±0.08vs.2.39±0.06%, p<0.001) were significantly increased in the peripheral blood of renal allograft recipients with AR and associated with serum level of sFGL2(Spearman correlation coefficient0.962,0.863and0.739respectively, all p<0.001), compared to those with stable renal function. In vitro, stimulation with1000U/ml (0.947±0.102ng/ml) or62.5U/ml IFN-γ (0.947±0.091ng/ml) for48h provided an optimal condition for CD4+T cells to secrete sFGL2. Under this condition, phosphorylated (p-) JNK was remarkably up-regulated in the activated CD4+T cells compared to PBS (INF-α:429.33±54.49vs.131.67±45.58, p<0.05; IFN-y:520.33±36.57vs.131.67±45.58, p<0.01), while no significant changes were found in p-p38MAPK or p-ERK1/2expression. Furthermore, inhibition of JNK significantly reduced sFGL2secretion by CD4+T cells (with TNF-α, IFN-γ or PBS stimulation:0.550±0.081vs.0.977±0.108ng/ml, p<0.05;0.548±0.105vs.1.010±0.127ng/ml, p<0.05;0.278±0.050vs.0.514±0.054ng/ml, p<0.05). In addition, compared with PBS, sFGL2remarkably induced TEC apoptosis (33.13±0.10vs.12.91±0.35%, p<0.001), both early (10.19±0.13vs.4.05±0.07%, p<0.001) and late (22.82±0.41vs.9.44±0.06%, p<0.001). Compared to TNF-α, sFGL2induced higher proportion of early apoptosis (9.74±0.07%, p<0.05), but lower proportion of late and total apoptosis (30.63±0.52%,40.34±0.24%, both p<0.001) in TEC. What’s more, sFGL2promoted a significant up-regulation of pro-apoptotic genes, such as CASP-3(p<0.05), CASP-8(p<0.05), CASP-9(p<0.05), CASP-10(p <0.05), TRADD (p<0.001), TNFSF10(p<0.01), FADD (p<0.001), FAS (p<0.001), FASLG (p<0.05), BAK1(p<0.05), BAD (p<0.05), BAX(p<0.001) and NF-KB1(p <0.01), compared to negative control, while no significant changes were observed in the expression of anti-apoptotic genes, including CARD-18, NAIP, BCL2, IKBKB and TBK1.Conclusion Serum level of sFGL2significantly increased in renal allograft recipients with acute rejection to an extent dependent upon the pathological type and severity of the response, associated with the level of TNF-α, IFN-γ and the percentage of Treg in the peripheral blood. In vitro, sFGL2secretion by CD4+T cells can be induced with TNF-α and IFN-γ stimulation through enhanced JNK phosphorylation. Moreover, sFGL2promotes TEC apoptosis by up-regulating pro-apoptotic genes without down-regulating anti-apoptotic genes. Our study suggests sFGL2a crucial mediator in the pathogenesis of allograft rejection.