The Role Of IL-17C In Acute Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation

Part ?: The function of IL-17 C in acute graft-versus-host disease afterallogeneic hematopoietic stem cell transplantationObject: The current study aimed to investigate the function of IL-17 C in murine a GVHD after allogeneic hematopoietic stem cell transplantation.Methods: The BALB/C(H-2d)mice,wild type(WT)C57BL/6(H-2b)mice and IL-17C(interleukin-17C)knockout(IL-17-/-)mice in B6 background were used as donor mice,and BALB/C(H-2d)mice were used as recipients.BALB/C recipients received lethal irradiation of 650 c Gy by X-Ray and were injected intravenously with 1x10^7 bone marrow(BM)cells and 5 x10^6 splenocytes(SP)from BALB/C mice or C57BL/6 mice respectively.Total body irradiation(TBI)was divided into 2 doses separated by 4 hours to reduce the gastrointestinal toxicity.Total RNA from GVHD target tissues including spleen,liver,lung and intestine were extracted and reverse transcribed into c DNA at day 0,3,7,12 after transplantation.Quantitative real-time PCR was performed to detect the relative IL-17 C expression.BALB/C recipients received lethal irradiation of 650 c Gy by X-Ray and were injected intravenously with 1x10^7 bone marrow(BM)cells and 5 x10^6 splenocytes(SP)from C57BL/6 mice or IL-17C-/-mice respectively.Mice survival and GVHD scores were monitored and recorded.Systemic GVHD score was assessed by a cumulative scoring system.For histology examination,2 weeks after transplantation,tissue was fixed and embedded for hematoxylin and eosin(H&E)staining.The histopathology score was assessed by a semi-quantitative scoring system.BALB/C recipients were lethally irradiated with 650 c Gy by X-Ray and transplanted with either C57BL/6 WT or IL-17C-/-allografts at the same cell numbers as previously described.Survival was monitored and recorded.Results:(1)Compared to syngeneic controls,IL-17 C expression was reduced in spleen and lung in allogeneic transplantation,as well as the expression of IL-17 C receptor(IL-17RE).(2)Donor IL-17 C deficiency significantly augmented a GVHD pathogenesis and promoted a GVHD-related mortality.Histologic assessment revealed that tissue damage was enhanced in the liver,lung,small intestine,as well as skin in the IL-17C-/-recipients(3)Recipients with both IL-17C-/-bone marrows and IL-17C-/-splenocytes exhibited the highest mortality.Recipients with WT allografts showed best survival.Recipients with either IL-17C-/-BM or IL-17C-/-spleen grafts developed a more severe a GVHD than WT controls,but had prolonged survival compared with the mice that received double deficiency grafts.Conclusion: IL-17 C expression was significantly decreased in tissues after allogeneic transplantation when compared to those after syngeneic transplantation.IL-17 C deficiency aggravated murine a GVHD and shortened survival.Both bone marrow cells and splenocytes derived IL-17 C exhibited a protective effect on a GVHD after hematopoietic stem cell transplantation.Part II: The mechanism of IL-17 C in acute graft-versus-host disease afterallogeneic hematopoietic stem cell transplantationObject: We attempted to investigate the mechanism of IL-17 C in the regulation of murine a GVHD after allogeneic hematopoietic stem cell transplantation.Methods: Wild type(WT)C57BL/6(H-2b)mice and IL-17 C knockout(IL-17-/-)mice in C57BL/6 background were used as donor mice,and BALB/C(H-2d)mice were used as recipients.BALB/C recipients received lethal irradiation with 650 c Gy by X-Ray and were injected intravenously with 1x10^7 bone marrow(BM)cells and 5 x10^6 splenocytes(SP)from C57BL/6 or IL-17-/-mice respectively.Total body irradiation(TBI)was divided into 2 doses separated by 4 hours to reduce the gastrointestinal toxicity.Lymphocytes in spleen,liver,lung and intestine were isolated and detected for allo-reactive T cell responses by flow cytometry 2 weeks after transplantation.To further validate the function of IL-17 C in a GVHD development,we constructed IL-17C-overexpression plasmids.Recipient BALB/C mice injected with empty vector plasmid or IL-17C-overexpression plasmid respectively by hydrodynamic gene transfer(HGT)3 days before transplantation.All recipient mice were transplanted with 1x10^7 IL-17C-/-BMs and 5x10^6 IL-17C-/-splenocytes.Survival was monitored.Lymphocytes in a GVHD target tissue were isolated and further assessed T cell responses to confirm the function of IL-17 C in a GVHD development and its role on T cell responses.Mice were delivered FITC-dextran via gavage to examined intestine permeability.RNA from intestine was extracted and reversed into c DNA.Inflammatory cytokines expression including IFN-?,IL-6,IL-1 ? and TNF-? were detected by real-time PCR.The function of IL-17 C on intestine permeability and inflammation was confirmed by IL-17 C overexpression.Populations of Treg cells in WT and IL-17C-/-recipients in a GVHD target tissues were detected by flow cytometry 2 weeks after transplantation.To further investigate the role of IL-17 C on the regulation of Treg cells,we overexpressed IL-17 C.Treg cell differentiation was detected by in vivo Ed U proliferation assay.In vitro studies also revealed the effect of IL-17 C on Treg cell differentiation by CD3/CD28 and allo-antigen stimulation.We depleted donor-derived Treg cells by using anti-CD25 antibody to investigate whether IL-17 C regulated a GVHD in a Treg cell dependent manner.Results:(1)Compared to recipients of WT grafts,activation of CD4+ and CD8+ T cells in recipients with IL-17C-/-grafts were significantly increased.(2)Populations and absolute numbers of IFN-? producing CD4+ and CD8+ T cells were markedly elevated in recipients with IL-17C-/-grafts.(3)Overexpression of IL-17 C significantly prolonged murine survival.(4)Overexpression of IL-17 C effectively inhibited CD4+ and CD8+ T cells activation.(5)Overexpression of IL-17 C also inhibited IFN-? production by CD4+ and CD8+ T cells.(6)Intestine permeability and inflammatory cytokines IFN-? and IL-6 production were increased in IL-17C-/-recipients.(7)Intestine permeability and inflammatory cytokines IFN-? and IL-6 production were decreased after IL-17 C overexpression.(8)Treg cells were decreased in IL-17C-/-recipients,whilst increased after IL-17 C overexpression.(9)Treg cell proliferation was reduced in IL-17 C deficient mice.In vitro polarizing assay also indicated IL-17 C deficiency affected Treg cell differentiation.(10)The effect of IL-17 C in a GVHD was diminished after depletion of donor Treg cells.Conclusion: CD4+ and CD8+ T cells activation and inflammatory cytokine IFN-? production were increased in IL-17 C deficient mice,while overexpressing IL-17 C inhibited allo-reactive T cell responses.Moreover,IL-17 C ameliorated a GVHD by maintaining intestinal permeability and inhibiting intestinal inflammation.Both in vivo and in vitro studies suggested that IL-17 C promoted Treg cell differentiation.IL-17 C regulated murine a GVHD in a Treg cell dependent manner.Part III: The predictive value of IL-17 C in human acute graft-versus-hostdisease after allogeneic hematopoietic stem cell transplantationObject: We examined the serum IL-17 C expression level in human patients and evaluated its predictive value in GVHD after allogeneic hematopoietic stem cell transplantation.Methods: In this study,we collected 68 patients underwent allogeneic hematopoietic stem cell transplantation in the First Affiliated Hospital of Soochow University in 2012.We examined the expression of IL-17 C by enzyme-linked immunosorbent assay(ELISA)and analyzed its clinical significance.Results:(1)Serum IL-17 C production was significantly decreased in grade II-IV a GVHD compared to grade 0-I a GVHD patients.However,IL-17 C expression level was comparable between c GVHD patients and non-c GVHD patients.(2)IL-17 C expression could be used to predict grade II-IV a GVHD(AUC=0.668,95% CI=0.535-0.802,P=0.019).(3)Cumulative incidences of grade II-IV a GVHD were significantly lower in IL-17 C high patients(IL-17C?106.32 ng/ml)that those of IL-17 C low(IL-17C<106.32 ng/ml).Patients with high IL-17 C high expression in serum have a prolonged survival than IL-17 C low expression patients(P=0.086).(4)We performed univariate analysis and found that IL-17 C expression level <106.32 ng/ml(HR=3.974,95% CI=1.391-11.356,P=0.010)and high risk of disease status(HR=3.000,95% CI=1.022-8.810,P=0.046)were significantly associated with higher a GVHD incidences.(5)Multivariate analysis further confirmed that low IL-17 C level(HR=1.480,95% CI=0.276-12.003,P=0.012)was the strongest parameter associated with grade II-IV a GVHD.Conclusion: IL-17 C expression level might be an independent risk factor for predicting the incidence of grade II-IV a GVHD.