The Preliminary Study Of Th17Cell Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation And Mechanism Of IRF8Regulating Th17Cell Differentiation

Part I Primary Study on Clinical Significance of Th17Cell Reconstitution after Allogeneic Hematopoietic Stem Cell TransplantationBackground and ObjectiveThe recent study showed that interleukin-17-producing T helper type17(Thl7) played important roles in autoimmune diseases and inflammatory bowel disease(IBD.This induced many scholars further interest in exploring the roles of graft-versus-host disease(GVHD) after allogeneic hematopoietic stem cell transplantation, allo-HSCT). Some studies found that cytokine storm played important roles on pathopoiesis of acute GVHD.And anti-thymocyte globulin(ATG) was the main immunosuppressant for allogeneic hematopoietic stem cell transplantation pretreatment, and was used for the prevention of acute and chronic GVHD. Previous studies mainly focused on that Thl cells with the characteristic cytokine IFN-γ mediated the pathophysiological process of acute GVHD.Recent studies showed that Thl and Th17cells were not the only one effector T cell causing GVHD.Meanwhile,pathogenesis of IL-17and Th17in GVHD remained controversial. In this study, we explored the relationships of conditioning regeimen with or without anti-thymocyte globulin (ATG) and Th17cells reconstruction,Thl7cells reconstruction and acute graft-versus-host disease (aGVHD) or cytomegalovirus (CMV) reactivation, at early stage post allogeneic haematopoietic stem cell transplantation for haematologic diseases.MethodsIn this study,78patients with haematological diseases were analyzed.At early stage posttransplantation,the T lymphocyte subtypes and regulatory T (Treg) cells were detected in peripheral blood mononuclear cells using multicolor flow cytometry and Th17/Treg cell-associated cytokines in serum were examined by enzyme linked immunosorbent assay(ELISA). Indirect immunofluorescence was used to detect cytomegalovirus antigen pp65.Heanthy volunteers (n=10) were severed as control.And then we performed these studies as follow.①The absolute counts of lymphocytes,T cell subtypes of CD3+CD4+, CD3+CD4-, Thl, Th17, Th1/17, Non-Th1/17in peripheral blood were detected and compared for patients who were administrated conditioning regimens with or without ATG, without aGVHD within30-60days, with or without CMV viral reactivation within100days after transplantation respectively.②The relationships between different degree of aGVHD and percentage of regulatory T (Tregs)(CD25+CD4+Foxp3+) cells and Th17(IL-17+CD4+) cells or ratio of Th17to Treg cells were analyzed.③Th17-associated cytokines including IL-17, IL-23, IL-21, IFN-γ, TGF-βl and IL-22were detected,when patients complicated with aGVHD. Meanwhile, cyokines above were dynamicaly tracked after effective treatment for aGVHD.Results1. At early stage within30-60days posttranstation, the absolute counts of lymphocytes, T cell subtypes of CD3+CD4+, CD3+CD4-,Th17, Non-Thl/17in peripheral blood in Non-ATG group(n=12) were lower than health control group (n=10)(p<0.05). The absolute counts of lymphocytes in ATG group was lower than Non-ATG group,but there was not statistical difference(p>0.05).The absolute counts of Thl, CD3+CD4+and Thl7, Non-Thl/17cells in ATG group were lower than in Non-ATG group (p<0.05). Even though the absolute counts of Th1/17(×103/L)in ATG group were lower than Non-ATG group(2.12±1.42vs4.45±2.70), but there was not statistical difference (p>0.05).2. The absolute counts of CD3+CD4-、Th1、Th17、Th1/17in cytomegalovirus(CMV) reactivation group(n=8) were significantly higher than that in Non-CMV reactivation group (p<0.05), but absolute counts of Non-Th17cells and CD3+CD4+cells was not statistical difference (p>0.05).3. As the severity of aGVHD increased, the percentages of Treg cells in peripheral blood gradually decreased:heathy control group(n=10):4.80±1.14, aGVHD degree0(n=10):4.80±1.14, degree1-2(n=12):2.47±0.59,degree3-4(n=8):1.44±0.34.There was not not statistical difference between aGVHD of0degree and heathy control (p>0.05).The percentage of Treg cells in aGVHD degree1-2group were lower than in heathy control group (p<0.05). The percentage of Treg cells in aGVHD degree3-4group were lower than in aGVHD degree1-2group (p<0.05)The proportion of Th17cell gradually increased(healthy control group:1.27±0.35, degree01.46±0.28, degree1-21.94±0.31, degree3-43.24±0.96).There was statistical difference between healthy control group and degree0group (p>0.05). The proportion of Th17cells in degree1-2group was higher than healthy control group and aGVD degree0group (p<0.05), and the The proportion of Th17cells in degree3-4group was higher than other groups (p<0.05)Accordingly, along with gradually increased the ratios of Th17/Tregs (healthy control group:0.25±0.11,degree0:0.38±0.22, degree1-2:0.78±0.4,degree3-4:2.34±0.79). The ratio of Thl7/Tregs in aGVHD degreel-2group were higher than in healthy group (p<0.05). The ratio of Thl7/Tregs in degree3-4group was higher than other groups (p<0.05) 4. The serum cytokine levels (pg/ml)of patients with aGVHD(n=12) were significantly higher compared with health control group(n=10) and No-aGVHD group(n=14)(27.88±10.07vs9.85±3.72or10.38±6.62) of IL-17,(201.67±66.54vs56.34±11.18or88.10±28.37) of IL-23,(201.67±66.54vs56.34±11.18or132.43±48.99) of IL-21,(42.56±22.47vs23.42±8.78or21.78±6.93) of IFN-y(p <0.05).On contrary, The TGF-β1level(pg/ml)(1156.61±458.04) was significantly lower than the health control group(2978.34±1647.71) and Non-aGVHD group (2541.62±1805.16)(p<0.05)(p<0.05). Even though IL-21,IL-22and IL-23were higher in Non-aGVHD group compared with healthy control group(p<0.05), but there was not statistical difference about other cytokines between healthy control group and Non-aGVHD group(p>0.05).After treatment, the cytokine levels above in10cases of effective treatment were recovered to considerable extent,and there was significant difference between before and after effective treatment (IL-17:11.85±3.81vs6.48±1.89;IL23:46.35±11.11vs29.42±7.14;IL-21:280.20±66.86vs148.78±49.28;IFN-y:47.46±12.83vs36.31±6.66; TGF-β1:183.52±29.86vs223.32±29.86)(p<0.05),but the IL-22showed no significant difference between before and after effective treatment (22.61±1.27vs25.40±4.41)(p>0.05)。Conclusions①ATG in conditioning regimen delayed early immune reconstitution of Thl and Th17cells.②CMV reactivation promotes Th17immune response.③Th17/Treg balance in peripheral blood indicates the severity of aGVHD.④Th17/Treg cell-associated cytokines (IL-17, IL-23, IL-21, IFN-y, TGF-β1) in serum were assocoated with pathogenesis and the development, and treatment efficacy of aGVHD, whereas acute GVHD is no relationship with the IL-22in serum. Part II Transcription factor IRF8inhibit Th17cell differentiation and alleviate T cell immune-mediated colitis in miceBackground and ObjectiveTh17cell recently were identified as a unique subset of T helper cells(Th) that played vital roles in the pathogenesis of autoimmune and inflammatory disorders and played important pathogenic roles in graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. It is well known that although transcription factor RORyt is necessary for the development of Th17cells, the molecular mechanisms underlying the differentiation of Th17cells are not completely elucidated. The interferon regulatory factor (IRF) families such as IRF4are closely related to Th17cells development. In addition, Interferon regulatory factor-8(IRF8) is one of IRF family, which existed in a variety of cells and are induced by interferon-y. It regulate development of B cells, dendritic cells and macrophages, and play a variety of roles in innate and adoptive immunity.However, IRF8expression in T cells was poorly understood. This study will investigate the IRF8impact and its mechanism for Th17cell differentiation, as well as infulence on T-cell immune-mediated experimental colitis in mice.MethodsEffects of IRF8gene knockout on differentiation of mouse Th17cells1. Experimental animals:mice which IRF8gene knockout in conventional(IRF8-/-) or special T-cell(Lck-Cre+Irf8fl/fll) were used as research subjects for the study(5mice every group).2. The naive CD4+(CD4+CD62L+CD44low) T cells were sorted by flow cytometry, and immune beads were used to sort CD4+CD25+regulatory T cells (Tregs).3. The naive CD4+T cells polarization culture:Cells stimulated under neutral conditions were defined as ThO cell; likewise, supplementation with IL-12and anti-IL-4as Thl; supplentmentation with IL-4and anti-IFN-y as Th2; For Thl7cell differentiation,cells were stimulated with transforming growth factor-β1(TGF-β1), IL-6, IL-23, in presence or absence of anti-IL-4, anti-IFN-γ. 4. Some technologies including ELISA, quantitative PCR (qPCR), flow cytometry, were used to detect protein levels of cytokine, and gene of and transcription factor and cytokines in which results were normalized based on the expression of ubiquitin, and T cell subsets, respectively.5. Impact on Treg cells development for IRF8gene knockoutThe naiveCD4+T cells were sorted from wt or IRF8-/-mice and then stimulated by supplenment with IL-6(10ng/ml) and diffent concentrations of TGF-β (0.1,1,5ng/ml), then CD4+Foxp3+T cells were detected by flow cytometry.6. Impact on Th17cell differentiation of naive CD4+T cells for IRF8gene knockout①TGF-β combined with different concentrations of IL-6The naive CD4+T cells wt or IRF8-/-mice were stimulated by supplenment with TGF-β(10ng/ml) and different concentrations of IL-6(0.1,1,5ng/ml) for three days,and then the cells were stimulated with PMA/Ion, and the cells were stained intracellular IFN-y and IL-17.Flow cytometry was used to detect IFN-y+CD4+and IL-17+CD4+T cells.②IL-17gene expression of naive CD4+T cells under Th17ploarizationThe naiveCD4+T cells were sorted from WT or IRF8-/-mice and then stimulated with IL-6,IL-23and TGF-p for24,48and72h, then RNA was extracted, IL-17mRNA and IL-17FmRNA levels were detected by qPCR.③Impact on producing-IL-17cells and IL-17under different combinations of cytokonesThe naiveCD4+T cells were sorted from WT or IRF8-/-mice and then stimulated with IL-23alone,or incombination with IL-23,IL-6and TGF-β, and the flow cytometry was used for IL-17+CD4+T cells,ELISA for IL-17concentration in supernatant.④Impact on genes associated with Thl7diffenentiation under different combinations of cytokoines.The naiveCD4+T cells from wt or IRF8-/-mice were stimulated with TGF-β/IL-6or TGF-β/IL-21,and the RNA was extracted.IL-17A, IL-22, IL-23R,RORyt and CCR6genes were detected by qPCR.Antagonistic role of the transcription factor IRF8on ROR y t inhibited IL-17promoter activity1. IRF8expression in activated T cellsThe naive CD4+T cells from spleen and lymph nodes were cultured under polarizing conditions of Th1,Th2, and Thl7,or TGF-β stimulation without TCR stimulation,and then cell lysates were analyzed by western-blot assay. The WT or IRF8-/-mouse naiveCD4+T cells were cultured for60hours under Th17-polarization, Co-immunoprecipitation(Co-IP) was used to examine the interaction of IRF8and ROR y t protein.2. The RORyt and IRF8plasmids were transfected in293T cells by calcium phosphate method.The binding state of RORyt and IRF8protein were detected by western-blot assay and Co-IP.3. Transfected IRF8and RORyt plasmids in293T cells influence IL-17promoter activityFirstly,293T cells were transfected with IL-17promoter containing6-kb luciferase by Liposomes method,and then the293T cells divided into three groups according trasfected plasmid:①different dose of RORyt plasmid②different dose of IRF8plasmid and RORyt plasmid③RORyt plasmid and Foxp3plasmid④RORyt plasmid and IRF8truncated mutant structure (1-390,1-305,1-253).And then β-galactosidase reporter gene structure were co-transfected for normalizing test and then detected the luciferase activity4. Transtected IRF8gene in naive CD4T cell inhibit IL-17-producing cellsThe naiveCD4+T cells from WT mice were transfected with IRF8-IRES-GFP or vector,and cultured under Th17polarization for4days,and then flow cytometry was used for detecting IL-17+CD4+T cells.5. Interaction of RORyt and IRF8family①293T cells were co-transfeted with RORyt plasmid and IRF family gene(IRF8,or IRF4,or IRF1),or IRF8truncated mutant,and the Co-IP was used to examine the interaction of RORyt and IRF8or its amino acid residues of truncated mutant.②Impact of IRF8on IRF4genne in naiveCD4+T cellsThe naiveCD4+T cells from wt or IRF8-/-mice were stimulated with TGF-β/IL-6, and the qPCR was used for detecton of IRF4mRNA level.③Interaction of RORyt and IRF8protein in naive CD4+T cellsThe naiveCD4+T cells fromWT or IRF8-/-mice were cultured for60h,and the cell lysates were extracted,the anti-IRF8antibody was used immunoprecipitation,anti-RORyt and anti-IRF8antibody were used for western-blotting assay.Establishment of T cells immune-mediated experimental colitis modelThe experiment colitis models were establisted according to different groups. The CD4+CD45RBhiT cells from WT or IRF8-/-mice were intraperitoneally injected into RAG1-/-mice alone, or combined with CD4+CD25+Treg cells from WT or IRF8-/-mice into RAG1-/-mice, respectively(5mice per group).The changes of the body weight in the experimental model mice were recorded every week,and the mice were sacrificed ath the fifth week for evaluating the colitis pathological score and T lymphocyte subtypes in mesenteric lymph nodes.ResultsIRF8gene knockout promote Th17cell differentiation1. Impact of IRF8gene knockout on Thl7cell differentiation①Naive CD4+T cells from spleen and lymph nodes of WT or IRF8-/-mice,were cultured under polarized conditions of Th1, Th2and Th17. IRF8gene knockout significantly increased the percentage of IL-17+CD4+(Th17) cells in CD4+T cells (36.2±2.46vs12.12±0.96) compared with WT mice (p<0.05), but did not infulenced the percentage of Thl or Th2cells(p>0.05) and concentrations of IL-4or IFN-y in supernatant (p>0.05) under Thl or Th2polarization.②Lamina propria lymphocytes from IRF8-/-or WT mice were cultured under Th17-polarization.Producing-IL-17+cells were significantly higher compared with WT group(33.90±1.75vs15.60±1.29)(p=0.000).Similarily,The cells were stimulated with PMA/Ion again, the percentage of IL-17+CD4+cell in IRF8-/-group was significantly higher compared with WT group((10.24±0.28vs2.60±0.14)(p=0.000).2. polarization culture of special T cell IRF8gene knockout naive CD4+T cellsThe naiveCD4+T cells from Lck-Cre+Irf8fl/fl or Lck-Cre+Irf8wt/w tmice spleen were cultured under Th17-polarization.The pecentage of IL-17+CD4+T cells was significantly higher in Lck-Cre+Irf8fl/fl mice compared with Lck-Cre+Irf8wt/wt mice(13.62±0.30vs5.12±0.21)(p=0.000), and the IL-17concentration in supernatant was also significantly higher compared with WT group((6.17±0.10vs2.70±0.10)(ng/ml)(p=0.000).3. The cytokine gene expression in naiveCD4+T cells under Th17-polarizationThe IL-17,IL-17F and IL-22mRNA levels were significantly higher in IRF8-/-mice compared with WT mice (108076.00±96.77vs6769.00±106.13,93207.00±144.98vs6663.61±240.61,3122.6±33.8vs372.4±16.9)(p=0.000).But IL-4(5529.26±63.45vs5562.61±583.61)and INF-y(40375.00±433.69vs40333.00±102.04) mRNA levels were not statistic differences between two groups(p>0.05).4. Impact of IRF8gene knockout on Treg cells developmentNaive CD4+T cells from WT or IRF8-/-mice were stimulated with IL-6(10ng/ml) and different concentrations of TGF-P(0.1,1,5ng/ml). Along with the increased concentration of TGF-β,the percentage of IL-17+CD4+T cells in IRF8-/-group showed rising trend, and was higher compared with WT group (p<0.01),but Foxp3+cells was not statistic difference between two groups (p>0.05).5. The impact of IRF8gene knockout on Th17cell differentiation under IL-6/TGF-β stimulationNaive CD4+T cells from WT or IRF8-/-mice were stimulated with TGF-β(5ng/ml) and different concentrations of IL-6(5,10,50,100ng/ml) for3days.The percentages of two groups showed rising trend,and IRF8-/-group was significantly higher compared with WT group(p<0.01),but the percentage of INF-y+CD4+cells was not statistic difference between two groups(p>0.05). 6. IL-17gene expression under Th17polarizationNaive CD4+T cells from WT or IRF8-/-mice were stimulated with TGF-p,IL-6and IL-23.The levels of IL-17mRNA and IL-17FmRNA were significantly higher in IRF8-/-group compared to WT group at24h(584.40±97.74vs146.10±7.88and2800.00±38.36vs1200.66±29.12),48h (3798.80±42.63vs487.0±41.04and8400.31±652.80vs2332.88±205.35) and72h(6233.80±11.65vs1899.40±93.05and8000.02±392.93vs3732.76±553.17)(p=0.000).7. Different cytokine combination impact Th17cell differentiation The percentages in IRF8-/-group were significant higher compared with WT group when naive CD4+T cells from WT or IRF8-/-mice were stimulated with IL-23alone(13.80±0.86vs5.78±0.12) or combination of TGF-β,IL-6and IL-23(72.20±4.13vs32.70±2.42)(p<0.05)8. Impact of cytokine on gene associated with Th17cell differentiation Naive CD4+T cells from WT or IRF8-/-mice were stimulated with TGF-β/IL6or TGF-β/IL-21for48h.The gene expression levels of IL-17A、IL-22、IL-23R、RORy and CCR6in IRF8-/-group were significantly higher compared with WT group(p<0.05), but the Foxp3mRNA did not obviously change and was no statistic difference between two groups(P>0.05).Antagonistic role of the transcription factor IRF8on ROR y t inhibited IL-17transcription1. IRF8expression in activated T cellsThe naive CD4+T cells from spleen and lymph nodes were cultured under polarizing conditions of Th1,Th2, and Thl7,or TGF-β stimulation without TCR stimulation. Western-blot assay showed that IRF8protein expressed in naiveCD4+T cells and was more stable under Th17-polarization.The WT or IRF8-/-mouse naiveCD4+T cells were cultured for60hours under Th17-polarization, Co-IP displayed endogenous interaction between IRF8and ROR γ t proteins in CD4+T cells. 2. IRF8interaction with RORyt inhibit IL-17transcription in293T cellsIn293T cells,IRF8ihibited IL-17promoter activity at pattern of dose-dependent. Inaddition,Foxp3and IRF8truncated mutants((Full、1-390、1-305、1-253) inhibited IL-17promoter activity.3. Interaction of IRF8and RORyt IRF8and RORyt plamids was transfected in293T cells,and Co-IP showed that RORyt binded to IRF8protein,which did not involve DNA. Amino acid residues230-190of IRF8protein was main site binding to RORyt protein.4. Interaction of IRF8family interaction and RORyt293T were transfected with RORyt and IRF family genes(IRF1、IRF4、 IRF8).Co-IP showed that RORyt interacted with IRF8and IRF4,but IRF1did not binding to RORyt.The IRF4mRNA level in naive CD4+T cells from WT or IRF8-/-mice which were stimulated in appearance or absence of TGF-β/IL-6for0and24h were not statistic difference between two groups(p>0.05)5. Transfected IRF8gene inhibit producing-IL17cells for naiveCD4+T cellsNaive CD4+T cells from C57BL/6WT mice were transfected with IRF8-IRES-GFP or vector under Th17-polarization for4days.Transfected IRF8gene significantly inhibit IL-17+CD4+cells compared with control group(4.20±0.31vs15.42±2.34)(P＜0.001)IRF8control Th17differentiation and alleviate experimental colitis in mice1. The body weight of RAG1-/-mice decreased significantly(p<0.05) and colon pathology or clinical scores of the RAG-/-mice transferred with CD4+CD45RBhiT cells from IRF8-/-mice were higher than that of the RAG-/-A mice transferred with CD4+CD45RBhiT from WT mice (p<0.05).Meanwhile, The percentage of IL-17+CD4+cells in mesenteric lymph nodes in IRF8-/-group was also higher than that of in WT mice(8.07±0.21vs2.44±0.17, p=0.000), but the percentage of IFN-y+CD4+(6.58±1.05vs5.83±0.31), Foxp3+CD4+cells(0.30±0.05vs0.39±0.02) were no statistic different between the two groups(p>0.05). 2. CD4+CD25+Treg cells from WT or IRF8-/-mice showed similar impact on body weight of RAG-/-mice(p>0.05), in which the CD4+CD45RBhiT cells from the WT mice were tranfered to the RAG1-/-mice.Conclusion:①Th17polarization promote naive CD4+T cells express IRF8protein.IRF8protein inhibits IL-17transcription and differentiation of Th17cells through direct binding to RORyt protein.IRF8and RORyt are antagonistic to each other and regulate Th17cells differentiation②Transcription factor IRF8do not take effects on Th1, Th2and Tregs differentiation. B cells, dendritic cells and macrophages do not involve in the role of IRF8on Th17differentiaton.③IRF8controls the differentiation of Th17cells in vivo, consequently,exacerbates immune-mediated experimental colitis,which is related to that knockout of transcription factor IRF8gene promote the reconstruction of Th17cells. IRF8-/-Treg cells display normal T cell effector suppressive activity in the process of immune mediated experimental colotis.