The Effect And Mechanism Of IL-33 In Murine Experimental Colitis The Mechanism Of Early Rejection Promoted By HMGB1 In Murine Cardiac Transplantation

Crohn's disease (CD) is characterized by chronic intestinal inflamation frequently relapsing with clinical manifestations including diarrhea, blood in the stool, abdominal pain, and weight loss. The etiology of Crohn disease is so far unknown but epidemiological and linkage studies suggest a genetic predisposition, the involvement of environmental factors and the immunological dysregulation play key roles in the development of the disease. Recently, imbalance of the development and function of Th1/Thl7 and regulatory T cell (Treg) has been shown to play a critical role in CD.IL-33, also known as NF-HEV and IL-1F11, has been identified as a novel member of IL-1 family. Previous studies have been shown IL-33 appears highly reminiscent of IL-1αand HMGB1, two dual-function proteins that also play important roles as both intracellular nuclear proteins and extracellular cytokines. IL-33 has been identified to be the special ligand for the receptor ST2L, which is a selective marker of Th2 cells but not Thl cells. Recent studies have shown that IL-33 plays a deleterious role associated with the activation and production of type II cytokines. Although IL-33 levels are elevated in human Crohn's disease, the functional relevance of increased IL-33 production during intestine inflammation remains unclear.Here, we assess the effects and mechanisms of IL-33 on the trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis that mimics the human CD.1. Production of recombinant mouse IL-33 protein and polyclonal anti-mouse IL-33 antibody and assessment of their activityThe plasmid rIL-33-PGEX-4T-2 was previously constructed in our laboratory. rIL-33 protein was expressed by E. coli induced by isopropylβ-D-thiogalactoside and purified by glutathione-affinity chromatography. IL-33-GST fusion protein was cleaved with thrombin, followed by polymyxin B column. The purity and activity of rIL-33 was determined.A polyclonal anti-mouse IL-33 antibody (anti-IL-33) was raised in rabbits immunized with purified rIL-33.2. The role of IL-33 in murine experimental colitis2.1. IL-33 is up-expressed in Thl/Th17 mediated murine TNBS-induced colitisIn this study, a markedly increased expression of IL-33, IFN-y and IL-17 expression in colonic tissue were observed in TNBS-induced colitis when compared with control group by immuneblot analysis. The increased expression of rIL-33 was further confirmed by real-time PCR. In addition, accompanied with severe colonic tissues destruction in TNBS-treated mice, a specific abundance of IL-33 expressed in the cytoplasm of infiltrated F4/80+macrophages in the lamina propria (LP) and submucosa of TNBS-treated mice. In vitro, IL-33 mRNA and protein were both present in the cell line RAW 264.7 cells and primary peritoneal macrophage.2.2. IL-33 reduces the development of experimental colitis in mice rIL-33, PBS, anti-IL-33 antibody, or control IgG was given intraperitoneally daily at the time of TNBS administration respectively. Compared with the PBS, antibody, and IgG group, rIL-33-treated mice display markedly lower loss of original weight. In addition, the antibody group exhibited more severe weight loss than IgG group. Moreover, a lower morphologic scores and a reduced inflammatory cell infiltration of mucosa and submucosa were observed in rIL-33-treated mice. Next, a therapeutic role of IL-33 was observed in TNBS-induced colitis by treating with rIL-33 or PBS day 2 after TNBS inoculation.3. IL-33 inhibits Thl and promotes Th2/Treg profile in TNBS-induced colitis3.1. rIL-33 switch Thl/Th2 immune responseWe analyzed the cytokines of sera and MLN cells from colitis mice treated with PBS or rIL-33. As expected, TNBS-treated mice receiving rIL-33 exhibited markedly decreased expression of IFN-y and increased Th2-type cytokines production of IL-5 and IL-13 as compared with PBS controls. Consistently, similar results were observed in the colonic tissues by real-time PCR and immunoblot analysis. ST2L+CD4+T cells, which are thought to play an important role in the generation of Th2-type cytokines, were higher in MLN and LPMC from rIL-33-treated mice.3.2. IL-33 induces regulatory T cellsWe also detected the Foxp3 and IL-10 mRNA, the key transcript factor and effector molecule of Treg, were significantly higher in rIL-33 treated mice with TNBS-induced mice when compared with PBS control group. Consistently, Treg differentiation induced by rIL-33 was further confirmed by flow cytometry and immunoblot analysis. Unexpected, IL-17 expression was not changed between two groups.3.3 IL-33-mediated reduction of experimental colitis development is mainly relied on Tregs expansionThe role of Th2 deviation and Tregs expansion induced by IL-33 treatment in the TNBS-induced colitis mice were explored. An anti-IL-4 or anti-CD25 antibody was used to neutralize IL-4 activity and deplete CD25+Tregs respectively. Specifically, Tregs depletion evidently abolished the protective role of IL33. Furthermore, anti-IL-4 antibody treatment partly reversed IL-33 effect.4. The mechanism of Treg differentiation promoted by rIL-334.1. rIL-33 expands CD103+IDO+tolerogenic DCs Previous studies have shown that tolerogenic CD103+DCs exhibit a key role in gut homeostasis and maintenance of tolerance. MLN lymphocytes and LPMCs were harvested to be analyzed. Our data shown that CD103+DCs, the primary source of IDO in the gut and involved in Foxp3+Treg cells development via IDO, was remarkably increased in MLN lymphocytes and LPMCs of IL-33-treated mice.4.2. IL-33 indirectly promotes development of tolerogenic CD103+DC via activating intestine epithelial cellsTo further support our contention that the induction of CD103+DC might be mediated by IL-33. Unexpectedly, rIL-33 has no direct effect on the promotion of CD103+CD11c+tolerogenic DCs. Previous studies have shown that IECs promote gut homeostasis and maintenance of tolerance via educating DC to tolerogenic CD103+ DC phenotype by TSLP, RA, and TGFβ1. Therefore, we hypothesized that IL-33 may indirectly promote the development of CD103+DC through activating intestine epithelial cells. As expected, ST2L was expressed on mice primary IECs, and the IECs from rIL-33-treated mice with TNBS-induced colitis displayed highly increased mRNA expressions of TSLP and ALDH1A1 (aldehyde dehydrogenase 1A1). Furthermore, the supernatants of primary IECs from TNBS-treated mice with IL-33 administration significantly promoted the proportion of CD103+DC differentiation.4.3. Supernatant of IL-33-activated IEC from colitis mice markedly promotes development of CD103+DC, which favors inhibitory Treg differentiation.We isolated colonic epithelial cells from mice with TNBS-induced colitis and incubated them with rIL-33. IL-33-treated IECs remarkably increased expression of TSLP and ALDH1A1 in vitro, and the supernatants significantly augmented the CD103+DCs when compared with PBS group. As expected, BMDC educated by the supernatants of IL-33-treated IECs markedly promoted Tregs differentiation and inhibited T cells proliferation.Conclusion1. IL-33 is highly expressed by infiltratory macrophages in Thl/Thl7 mediated TNBS-induced colitis.2. IL-33 administration significantly ameliorates the development of TNBS-induced colitis.3. IL-33 inhibits Thl and promotes Th2/Treg profile in TNBS-induced colitis4. IL-33-mediated reduction of experimental colitis development is mainly relied on Treg expansion.5. IL-33 indirectly promotes development of tolerogenic CD103+DC and Treg via activating intestine epithelial cells High mobility group box 1 (HMGB1) was originally characterized as a nuclear protein implicated in facilitating DNA binding. Recent studies have now consistently demonstrated that it also acts as a critical mediator to initiate innate immune response upon inflammatory or other insults. HMGB1 can be either secreted by activated immune cells such as dentritic cells and macrophages or passively released by necrotic or damaged cells.Previously, we demonstrated a role for HMGB1 in the initiation and progression of allograft rejection. Th17 cells are a recently identified T helper subset implicated in various inflammatory responses and in the pathogenesis of many autoimmune diseases. They are characterized as preferential producers of interleukin-17A (IL-17A), IL-17F, IL-21, and IL-22. Naive CD4+T cells can be polarized into Th17 cells upon antigen receptor ligation in the presence of IL-6 and TGF-β. The orphan nuclear receptor RORyt has been suggested to be responsible for the transcription of genes encoding IL-17 family cytokines. IL-23 can expand differentiated Th17 cells or maintains IL-17 production. It is noteworthy that IL-6 controls Thl7 immunity by inhibiting the conversion of naive CD4+T cells into Foxp3+regulatory T cells (Tregs).In the present study, we sought to explore the role of Thl7 cells in allograft, and dissect the role of HMGB1 in de novo generation of Thl7 during acute allograft rejection.1. Th17 implicates in allograft rejection response1.1. Kinetics for IL-17+ and IFN-γ+ alloreactive T cell production during acute allograft rejectionWe first sought to determine the kinetics for IL-17+ and IFN-γ+ alloreactive T cell production during acute allograft rejection. A murine cardiac allograft transplantation model was employed to address the question. Splenic T cells were isolated from recipient mice at indicated time after transplantation and then subjected to analysis of IL-17+ and IFN-γ+ alloreactive T cells. A time-dependent increase for Thl alloreactive response was observed characterized by a steady increase of IFN-γ+ cells in CD4+ and CD8+T cells. In contrast, IL-17-producing CD4+with the strongest response noted at day 3 of posttransplantation. Ex vivo studies further indicated that the expansion of IL-17+cells in total splenic CD4+and CD8+T cells is alloantigen-specific.We performed quantitative real-time PCR analysis of HMGB1, IL-6, IL-17 and IFN-γin the cardiac allografts. We observed a time-dependent increase of HMGB 1 and IFN-γmRNA in allogeneic cardiac grafts. In agreement, the highest expression levels for IL-17 and IL-6 within the grafts was noted at early stage of acute allograft rejection. 1.2. Differentiation of Th17 in vitroHMGB1 is potent to stimulate BMDCs secretion of IL-6.We established MLC assays by using BMDCs derived from donor mice and splenic T cells originated from recipient mice. BMDCs were cocultured with recipient-derived splenic T cells in the presence of HMGB1, TGF-(3 and/or IL-6. Cultures in the presence of LPS were used as positive controls. As expected, the addition of TGF-(3 and IL-6 significantly promoted the generation of CD4+IL-17+ alloreactive T cells. Of note, HMGB1 showed high potency to promote the production of both CD4+and CD8+IL-17-producing cells as compared to that of cells cultured with TGF-βonly. HMGBl is also potent to augment the production of CD4+IFN-y+ and CD8+IFN-γ+T cells. To tackle the underlying mechanism, an IL-6 neutralizing antibody was added into the culture system. Remarkably, blockade of IL-6 abolished the regulatory effect of HMGB1 on IL-17+T cell production. A similar result of cytokines in cultural supernatanants was obtained.2. HMGB1 promotes early acute allograft rejection by enhancing Th17 alloreactive response2.1. HMGB1 promotes allo-reactive Th17 immune responseWe now sought to address the role of HMGB1 in IL-17-producing alloreactive T cell response during acute allograft rejection. For this purpose, an HMGB1 neutralizing Ab was i.p. injected into cardiac allograft recipient mice. Blockade of HMGB1 significantly suppressed Thl7 response as manifested by the decrease of CD4+IL-17+alloreactive T cells after day 3 of transplantation, while blockade of HMGB1 showed very minor effect on CD8+IL-17+alloreactive T cells. Consistently, quantitative RT-PCR analysis of IL-6 and IL-17 in the cardiac allografts also revealed a remarkable decrease of mRNA levels. In contrast to controls, the infiltration of neutrophils in cardiac allograft was dramatically attenuated upon administration of HMGB1 blocking antibody. Recipients treated with anti-HMGB1 Abs had a significant longer allograft survival time and a significantly decreased percentage of splenic alloreactive IFN-y-producing CD8+T cells.2.2. IL-17-producing T cells mediated allograft rejection through neutrophil recruitmentTo further address the involvement of CD4+IL-17-producing T cells in allograft rejection, we performed immunostaining of cardiac allograft sections. CD4+IL-17+T cells were detected in the grafts at day 3 of posttransplantation, while these cells were absent from sections originated from recipients after day 7 of transplantation. Next, we examined inflammatory cell infiltration in the cardiac allografts. It was found that Gr-1+neutrophils were the major infiltrates at the early stage of allograft rejection. To directly examine a link between IL-17 and neutrophil recruitment in cardiac allograft rejection, a neutralizing antibody against IL-17A was administered to each recipient. Recipients received same amount of control IgG were served as controls. Blockade of IL-17A significantly prolonged allograft survival time. In line with this result, IL-17A neutralizing antibody treatment significantly attenuated neutrophil infiltration.Conclusion1. HMGB1 promotes Th17 differentiation via IL-6-dependent pathway2. Th17 palys a critical role in the early stage of allograft rejection through neutriphil recruitment3. Blockage of HMGB1 could inhibit rejection response mediated by Th17...