Functional characterization of myeloid-derived suppressor cell subpopulations during the development of experimental arthritis.

Recent evidence indicates the existence of subpopulations of myeloid-derived suppressor cells (MDSCs) with distinct phenotypes and functions. Here, we characterized the role of MDSC subpopulations in the pathogenesis of autoimmune arthritis in a collagen-induced arthritis (CIA) mouse model. The splenic CD11b(+) Gr-1(+) MDSC population expanded in CIA mice, and these cells could be subdivided into polymorphonuclear (PMN) and mononuclear (MO) MDSC subpopulations based on Ly6C and Ly6G expression. During CIA, the proportion of splenic MO-MDSCs was increased in association with the severity of joint inflammation, while PMN-MDSCs were decreased. MO-MDSCs expressed higher levels of surface CD40 and CD86 protein, but lower levels of Il10, Tgfb1, Ccr5, and Cxcr2 mRNA. PMN-MDSCs exhibited a more potent capacity to suppress polyclonal T-cell proliferation in vitro, compared with MO-MDSCs. Moreover, the adoptive transfer of PMN-MDSCs, but not MO-MDSCs, decreased joint inflammation, accompanied by reduced levels of serum cytokine secretion and the frequencies of Th1 and Th17 cells in draining lymph nodes. These results suggest that there could be a shift from potently suppressive PMN-MDSCs to poorly suppressive MO-MDSCs during the development of experimental arthritis, which might reflect the failure of expanded MDSCs to suppress autoimmune arthritis.

Th17 cells play a critical role in the development of experimental Sjögren's syndrome.

OBJECTIVE: Although Th17 cells have been increasingly recognised as an important effector in various autoimmune diseases, their function in the pathogenesis of Sjögren's syndrome (SS) remains largely uncharacterised. This study aims to determine the role of Th17 cells in the development of experimental SS (ESS). METHODS: The ESS was induced in wildtype and IL-17A knockout (IL-17 KO) C57BL/6 mice immunised with salivary glands (SG) proteins. Phenotypic analysis of immune cells in the draining cervical lymph nodes (CLN) and SG was performed by flow cytometry and immunofluorescence microscopy. To determine the role of Th17 cells in ESS, immunised IL-17 KO mice were adoptively transferred with in vitro-generated Th17 cells and monitored for SS development. The salivary flow rate was measured, whereas inflammatory infiltration and tissue destruction in SG were assessed by histopathology. RESULTS: SG protein-immunised mice developed overt SS symptoms with increased Th17 cells detected in CLN and within lymphocytic foci in inflamed SG. Notably, immunised IL-17 KO mice were completely resistant for SS induction, showing no evidence of disease symptoms and histopathological changes in SG. Adoptive transfer of Th17 cells rapidly induced the onset of ESS in immunised IL-17 KO mice with markedly reduced saliva secretion, elevated autoantibody production and pronounced inflammation and tissue damage in SG. CONCLUSIONS: Our findings have defined a critical role of Th17 cells in the pathogenesis of ESS. Further studies may validate Th17 cell as a potential target for treating SS.

Blockade of Notch signaling ameliorates murine collagen-induced arthritis via suppressing Th1 and Th17 cell responses.

Recent studies have demonstrated that Notch signaling is critically involved in the regulation of immune response and contributes to autoimmune pathogenesis. Here, Notch signaling was found to be activated in CD4(+) T cells and synovial tissue from collagen-induced arthritis mice. In vivo administration of the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) substantially reduced the severity of arthritic symptoms and joint damage in collagen-induced arthritis mice. Notably, DAPT treatment significantly suppressed Th1- and Th17-cell responses in spleen and lymph nodes and reduced IFN-γ and IL-17 levels in plasma. In polarization culture, DAPT treatment markedly reduced Th17 cell expansion from naïve T cells, whereas fusion protein of the Notch receptor ligand delta-like 3 significantly increased the frequency and absolute number of Th17 cells. These results suggest a novel therapeutic strategy for treatment of human rheumatoid arthritis by targeting Notch signaling using γ-secretase inhibitors.

A Trichosanthin-derived peptide suppresses type 1 immune responses by TLR2-dependent activation of CD8(+)CD28(-) Tregs.

A group of 15-aa-long Trichosanthin-derived peptides was synthesized and screened based on their differential abilities to induce low-responsiveness in mouse strains with high and low susceptibility. One of them was conjugated to form a homo-tetramer Tk-tPN. At concentrations of 0.1-50 µg/ml, Tk-tPN activated CD8(+)CD28(-) Tregs in vitro to induce immune suppression as effectively as the native Trichosanthin but did not exhibit cytotoxicity. In EAE mice which were pre-treated with Tk-tPN or Tk-tPN-activated CD8(+) T cells, a marked attenuation of clinical scores was recorded together with an expansion of the CD8(+)CD28(-) Treg from 2.2% to 36.1% in vivo. A pull-down assay and signal transduction analyses indicated that the ability of Tk-tPN to convert the CD8(+)CD28(-) Treg-related cytokine secretion pattern from type 1 to type 2 depends on the TLR2-initiated signaling in macrophages. The high production of IL-4/IL-10 by the Tk-tPN-activated CD8(+)CD28(-) Treg suggests the value of using Tk-tPN as a therapeutic reagent for Th1-dominant immunological diseases.

The expression of Toll-like receptor 8 and its relationship with VEGF and Bcl-2 in cervical cancer.

BACKGROUND: Cervical cancer is one of the most common cancers in women worldwide, often associated with the infection of human papillomavirus (HPV). Toll-like receptor 8 (TLR8), a pattern recognition receptor, is involved in viral nucleic acid sensing. Recently TLR8 has been shown to be expressed in cancer cells, and it has been suggested that it may help cancer cell growth and tumor development. The objective of this study is to investigate the expression of TLR8 expression and its relationship with Bcl-2 and VEGF in cervical cancer cells. METHODOLOGY/PRINCIPAL: The mRNA expression levels of Bcl-2, VEGF and TLR-7,-8,-9 in newly diagnosed cervical cancer patients were detected by quantitative real-time PCR (qRT- PCR). Epifluorescence microscope was used to determine the presence of TLR8 protein in Hela cells. The cell cycle and apoptosis were analyzed by flow cytometer, and the cell proliferation was measured by MTT assay. Our data showed the increased mRNA levels of TLR8 in human cervical cancer samples as well as in HeLa cells, a cell line derived from a human cervical cancer. In addition, there was a positive correlation between the expression levels of TLR8 and Bcl-2 and VEGF in cervical cancer patients. When Hela cells were treated with TLR8 agonist CL075, the percentage of cells in G2/M +S was remarkably increased, accompanied by increased COX-2, BCL-2 and VEGF mRNA levels. CONCLUSIONS/SIGNIFICANCE: The mRNA expression level of TLR8 in the patients with cervical cancer and Hela cells were up-regulated, it consistent with the increased expression of VEGF and Bcl-2. The results suggest that TLR8 may be an interesting therapeutic target in cervical cancer.

Glucocorticoid-induced tumor necrosis factor receptor family-related protein exacerbates collagen-induced arthritis by enhancing the expansion of Th17 cells.

Rheumatoid arthritis (RA), a chronic autoimmune form of inflammatory joint disease, progressively affects multiple joints with pathological changes in the synovia, cartilage, and bone. Numerous studies have suggested a critical role for glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) in the pathogenesis of autoimmune arthritis by modulating both innate and adaptive immune reactions, but the underlying mechanisms by which GITR activation promotes arthritic progression remain largely unclear. In this study, we found that collagen-induced arthritis mice treated with the ligand of GITR (GITRL) displayed an earlier onset of arthritis with a markedly increased severity of arthritic symptoms and joint damage, in which significantly increased Th17 cells in both spleen and draining lymph nodes were observed. Notably, results showed that a marked expansion of Th17 cells with increased RORγt mRNA expression was induced from naïve CD4(+) T cells when cultured with GITRL. Consistently, normal mice that were treated with GITRL were found to display a substantial expansion of splenic Th17 cells. Furthermore, we detected elevated serum levels of GITRL in patients with RA, which were positively correlated with an increase in interleukin-17 production. Taken together, the results from this study have revealed a new function of GITRL in exacerbating autoimmune arthritis via the enhancement of the expansion of Th17 cells.

IL-10-producing regulatory B10 cells ameliorate collagen-induced arthritis via suppressing Th17 cell generation.

IL-10-producing CD1d(hi)CD5(+) B cells, also known as B10 cells, have been shown to possess a regulatory function in the inhibition of immune responses, but whether and how B10 cells suppress the development of autoimmune arthritis remain largely unclear. In this study, we detected significantly decreased numbers of IL-10-producing B cells, but increased IL-17-producing CD4(+) T (Th17) cells in both spleen and draining lymph nodes of mice during the acute stage of collagen-induced arthritis (CIA) when compared with adjuvant-treated control mice. On adoptive transfer of in vitro expanded B10 cells, collagen-immunized mice showed a marked delay of arthritis onset with reduced severity of both clinical symptoms and joint damage, accompanied by a substantial reduction in the number of Th17 cells. To determine whether B10 cells directly inhibit the generation of Th17 cells in culture, naive CD4(+) T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were co-cultured with B10 cells. These B10 cells suppressed Th17 cell differentiation via the reduction of STAT3 phosphorylation and retinoid-related orphan receptor γt (RORγt) expression. Moreover, Th17 cells showed significantly decreased proliferation when co-cultured with B10 cells. Although adoptive transfer of Th17 cells triggered the development of collagen-induced arthritis in IL-17(-/-)DBA/1J mice, co-transfer of B10 cells with Th17 cells profoundly delayed the onset of arthritis. Thus, our findings suggest a novel regulatory role of B10 cells in arthritic progression via the suppression of Th17 cell generation.

IL-17 contributes to cardiac fibrosis following experimental autoimmune myocarditis by a PKCß/Erk1/2/NF-κB-dependent signaling pathway.

Myocarditis is a common clinical cardiovascular disease, and some patients progress to dilated cardiomyopathy (DCM) with chronic heart failure. Common viral infections are the most frequent cause of myocarditis, but other pathogens and autoimmune diseases have also been implicated. T(h)17 cells are novel IL-17-producing effector T helper cells that play an important role in the development of autoimmune myocarditis. Furthermore, IL-17 is also important in post-myocarditis cardiac remodeling and progression to DCM. However, the mechanisms whereby IL-17 and IL-17-producing cells promote the progression of cardiac fibrosis remain unclear. We therefore investigated whether IL-17 directly induced cardiac fibrosis in experimental autoimmune myocarditis (EAM) and explored the possible molecular mechanisms. The EAM model was induced and serum IL-17 level was detected by ELISA; western blot, immunofluorescence and sirius red staining were used to analyze the collagen expression. PCR was used to assay the IL-17RA and IL-17RC. The results indicated that IL-17 induced cardiac fibrosis both in vitro and in vivo. The protein kinase C (PKC)ß/Erk1/2/NF-κB (Nuclear Factor κappa B) pathway was involved in the development of myocardial fibrosis and IL-17 contributed to cardiac fibrosis following EAM via this pathway. These results provide the first direct evidence for the involvement of the PKCß/Erk1/2/NF-κB signaling pathway in IL-17-induced myocardial fibrosis.

Wetland ecological index and assessment of spatial-temporal changes of wetland ecological integrity.

Long-term, quantitative, and dynamic monitoring of regional ecological integrity using remote sensing can provide powerful decision-making support for sustainable regional development. However, existing methods are unable to accurately evaluate the quality of the surface ecological integrity because they do not consider vegetation saturation and salinization of wetlands. In addition, the ecological fragility of wetlands is characterized by a high frequency of changes in ecological conditions over time, leading to a lack of directionality in the analysis of ecological changes over long time series. To accurately assess the surface ecological integrity, this study integrates environmental salinity (Baseline-based Soil Salinity Index, BSSI) and a new vegetation element (Improved Hyperspectral Image-based Vegetation Index, IHSVI), and proposes the wetland ecological index (WEI) for the ecological integrity assessment system. Combined with the annual ecological integrity assessment using the WEI, the Mann-Kendall test was used to obtain the nodes of long-term changes. The WEI-Mann Kendall (WEI-MK) framework indicates the direction of analysis and realizes clear long-term series change monitoring. In this study, we analyzed the spatial and temporal changes in ecological integrity in the Yellow River Delta from 1991 to 2020 based on the WEI-MK framework. The results showed that: 1) Compared with Remote Sensing-based Ecological Index (RSEI), the WEI improved the accuracy of wetland integrity evaluation to 89 %. The WEI also improved accuracy of assessments in other typical regions by approximately 10 %. 2) The selection of nodes based on the WEI-MK framework clarified the direction of environmental change analysis. The results show that although the quality of the terrestrial ecological environment has improved over the past 30 years in the Yellow River Delta, that of the marine ecological environment has gradually declined. In particular, the state of the marine ecological environment after 2016 should be of concern.

PTD-hFOXP3 protein acts as an immune regulator to convert human CD4(+)CD25(-) T cells to regulatory T-like cells.

Regulatory T cells (Tregs) are critical for maintaining self-tolerance and homeostasis, and have potential application in clinical disease therapy, such as autoimmune diseases and transplant rejection, but their numbers are limited. FOXP3 is a key transcription factor controlling Tregs development and function. Although transfection of CD4(+)CD25(-) lymphocytes with the FOXP3 gene can convert them to Treg-like cells, there is the risk of insertional mutagenesis and thus an alternative to genetic intervention is sought. The protein transduction domain (PTD) from the HIV transactivator of transcription is a useful tool to deliver protein to the cytoplasm and nucleus. In this study, we generated a fusion protein linking the human FOXP3 to PTD (PTD-hFOXP3), and explored its function in T cells. The results showed that the PTD rapidly and effectively delivered the hFOXP3 protein into cells where it localized not only in the cytoplasm, but also to the nucleus. PTD-hFOXP3-transduced Jurkat cells (human T lymphoma cell line) and CD4(+)CD25(-) T cells failed to proliferate and produce IL-2 and IFN-γ, but produced large amounts of the cytokines IL-4, IL-10, and TGF-ß, in response to TCR stimulation in vitro. PTD-hFOXP3-transduced CD4(+)CD25(-) T cells also expressed high levels of CTLA-4 and low levels of CD25 after stimulation. Most importantly, PTD-hFOXP3-transduced T cells inhibited the proliferation of activated CD4(+)CD25(-) T cells. Furthermore, chromatin immunoprecipitation assays demonstrated that PTD-hFOXP3 can bind with the IL-2 gene promoter and repress the expression of IL-2. These results indicate that PTD-hFOXP3 has the capability to convert conventional T cells to Treg-like cells.

HMGB1 blockade attenuates experimental autoimmune myocarditis and suppresses Th17-cell expansion.

High-mobility group box 1 (HMGB1), a non-histone nuclear protein, has been implicated in cardiovascular diseases. Dilated cardiomyopathy (DCM), one of the leading causes of heart failure, is often caused by coxsackievirus B3-triggered myocarditis and promoted by the post-infectious autoimmune process. Th17 cells, a novel CD4(+) T subset, may be important in the pathogenesis of autoimmune myocarditis. In the present study, we attempted to block HMGB1 function with a monoclonal antibody specific for HMGB1 B box and investigated the effects of the blockade on Th17 cells and experimental autoimmune myocarditis (EAM). After induction of EAM, HMGB1 protein levels were significantly elevated both in the heart and blood. Administration of an anti-HMGB1 B box mAb attenuated cardiac pathological changes and reduced the number of infiltrating inflammatory cells in the heart during EAM. These protective effects of HMGB1 blockade correlated with a reduced number of Th17 cells in local tissues and lower levels of IL-17 in the serum. Furthermore, in vitro, studies demonstrated that HMGB1 promoted Th17-cell expansion. Therefore, we speculate that HMGB1 blockade ameliorates cardiac pathological changes in EAM by suppressing Th17 cells.

Increased IL-17-producing CD4(+) T cells in patients with esophageal cancer.

Increased interleukin-17 (IL-17)-producing Th (Th17) cells have been described in a variety of human carcinoma cases, however, the mechanism of Th17 cells' accumulation in a tumor microenvironment remains elusive. This study was designed to investigate whether Th17 cells were involved in the development of esophageal cancer. We found that the proportion of Th17 cells increased within the peripheral blood and tumor tissues of esophageal cancer patients. Furthermore, the proportion of circulating Th17 cells was higher in advanced esophageal cancer patients than that in early esophageal cancer patients. In addition, the Th17 cells differentiation-related cytokines (IL-23, IL-1ß, and IL-6) and accumulation-related chemokines (CCL22 and CCL20) were present in a tumor microenvironment. Therefore, the findings may partly explain the cause for the increased proportion of Th17 cells and indicate a potential prognostic marker of Th17 cells in esophageal cancer.

Phenotypic alterations of dendritic cells are involved in suppressive activity of trichosanthin-induced CD8+CD28- regulatory T cells.

The nature and differentiation of regulatory CD8(+)CD28(-) T cells are poorly understood. In this study, we demonstrate that native Ag trichosanthin (Tk), a highly purified linear peptide isolated from a Chinese medicinal herb, is able to induce strong suppression of OVA-specific lymphoproliferation at low concentrations via activation of IL-4/IL-10-secreting CD8(+)CD28(-) regulatory T cells (Tregs). To elucidate the underlying mechanisms, we firstly identified two types of mouse inbred strains, high susceptible (HS) and low susceptible, for the Tk-related suppression. They are H-2(d) (or H-2(b)) and H-2(k), respectively. The suppression is evoked only if bone marrow-derived dendritic cells (BDCs) instead of purified T cells are treated with Tk in an OVA-specific T-BDC interaction. Moreover, a special pattern of cytokine/transcription factors (IL-4(+)IL-10(+)IFN-gamma(-)Gata3(+)T-bet(-)) during suppressed OVA-specific T cell proliferation was observed in HS C57BL/6 but not in low-susceptible C3H/He mice. Consistently, the percentage of CD8(+)CD28(-) Tregs preferentially expanded from 5.5 to 26.1% in the presence of Tk, an occurrence that was also detected only in HS C57BL/6 mice. These expanded Tregs were able to induce a strong inhibition of one-way MLCs, which indicated that the Tk-induced hyporeaction and the activation of CD8(+)CD28(-) Tregs might be under the influence of different genetic backgrounds. Additionally, obvious alterations of phenotypic parameters of BDCs after Tk stimulation were also identified, including enhanced production of IL-10, decreased secretion of IL-12, and detection of Jagged1, a Notch ligand on BDCs. Collectively, our data suggest that the changed APC-related factors are essential, at least in part, for the activation and differentiation of Tk-induced CD8(+)CD28(-) Tregs.

Notch signaling mediates TNF-α-induced IL-6 production in cultured fibroblast-like synoviocytes from rheumatoid arthritis.

It has been reported that Notch family proteins are expressed in synovium tissue and involved in the proliferation of synoviocyte from rheumatoid arthritis (RA). The aim of this paper was to investigate whether Notch signaling mediated TNF-α-induced cytokine production of cultured fibroblast-like synoviocytes (FLSs) from RA. Exposure of RA FLSs to TNF-α (10 ng/ml) led to increase of Hes-1, a target gene of Notch signaling, and a marked upregulation of Notch 2, Delta-like 1, and Delta-like 3 mRNA levels. Blockage of Notch signaling by a γ-secretase inhibitor (DAPT) inhibited IL-6 secretion of RA FLSs in response to TNF-α while treatment with recombinant fusion protein of Notch ligand Delta-like 1 promoted such response. TNF-α stimulation also induced IL-6 secretion in OA FLSs; however, the Hes-1 level remained unaffected. Our data confirm the functional involvement of Notch pathway in the pathophysiology of RA FLSs which may provide a new target for RA therapy.

Enhanced HMGB1 expression may contribute to Th17 cells activation in rheumatoid arthritis.

Rheumatoid arthritis(RA) is a common autoimmune disease associated with Th17 cells, but what about the effect of high-mobility group box chromosomal protein 1 (HMGB1) and the relationship between Th17-associated factors and HMGB1 in RA remains unknown. In the present study, we investigated the mRNA levels of HMGB1, RORγt, and IL-17 in peripheral blood mononuclear cells (PBMCs) from patients with rheumatoid arthritis by quantitative real-time PCR (RT-qPCR), and the concentrations of HMGB1, IL-17, and IL-23 in plasma were detected by ELISA. And then, the effect of HMGB1 on Th17 cells differentiation was analyzed in vitro. Our clinical studies showed that the mRNAs of HMGB1, RORγt, and IL-17 in patients were higher than that in health control (P < 0.05), especially in active RA patients (P < 0.05). The plasma HMGB1, IL-17, and IL-23 in RA patients were also higher than that in health control (P < 0.05); there was a positive correlation between the expression levels of HMGB1 and the amount of CRP, ERS, and RF in plasma. In vitro, the IL-17-produced CD4(+)T cells were increased with 100 ng/mL rHMGB1 for 12h, which indicated that the increased HMGB1 might contribute to Th17 cells activation in RA patients.

Increased frequency of circulating follicular helper T cells in patients with rheumatoid arthritis.

Follicular helper T (Tfh) cells are recognized as a distinct CD4(+) helper T-cell subset, which provides for B-cell activation and production of specific antibody responses, and play a critical role in the development of autoimmune disease. So far, only one study investigated the circulating Tfh cells increased in a subset of SLE patients. Since relatively little is known about the Tfh cells in rheumatoid arthritis (RA) patients, in this study, Tfh-cell frequency, related cytokine IL-21, and transcription factor Bcl-6 were investigated in 53 patients with RA and 31 health controls. Firstly, we found that the frequency of CD4(+)CXCR5(+)ICOS(high) Tfh cells was increased significantly in the peripheral blood of RA patients, compared with that in healthy controls. It is known that Tfh cells are critical for directing the development of an antibody response by germinal centers B cells; secondly, we observed that the Tfh-cell frequency is accompanied by the level of anti-CCP antibody in RA patients. Furthermore, expression of Bcl-6 mRNA and plasma IL-21 concentrations in RA patients was increased. Taken together, these findings have shown that the increased frequency of circulating Tfh cells is correlated with elevated levels of anti-CCP antibody, indicating the possible involvement of Tfh cells in the disease progression of RA.

The Th17/Treg imbalance and cytokine environment in peripheral blood of patients with rheumatoid arthritis.

Broken Th17/Treg balance has been reported contributing to several inflammatory autoimmune diseases. The objective of the study was to investigate whether the Th17/Treg balance was impaired in the peripheral blood of patients with rheumatoid arthritis (RA). The frequencies of Treg cells and Th17 cells and mRNA expression of transcription factor RORγt and FoxP3 in peripheral blood of RA patients (n = 37) and healthy controls (n = 30) were determined by flow cytometry and real-time PCR, respectively. Eleven serum cytokines were analyzed by using cytometeric bead array (CBA). The results demonstrated that active RA patients exhibited increased peripheral Th17 cells, Th1- and Th17-related cytokines and RORγt expression while decreased Treg cells and FoxP3 expression. In addition, Th17/Treg ratios were positively correlated with serum concentrations of Th1- and Th17-related cytokines. In conclusion, our results indicated that Th17/Treg balance was broken in peripheral blood, which may play an important role in the development of RA.

Increased expression of mGITRL on D2SC/1 cells by particulate ß-glucan impairs the suppressive effect of CD4+CD25+ regulatory T cells and enhances the effector T cell proliferation.

ß-Glucans have been shown to enhance immune responses for centuries, which contributes to their anti-tumor property. However, their mechanisms of action are still elusive. Dectin-1, the C-type lectin receptor for ß-glucan, is expressed abundantly on dendritic cells (DCs). Activation of DCs via Dectin-1 can lead to the maturation of DC, inducing both innate and adaptive immune responses against tumor development and microbial infection. In this study, we found that particulate yeast-derived ß-glucans could induce the maturation of murine dendritic cell line D2SC/1 cells and increase the expression of mGITRL on D2SC/1 cells via Dectin-1/Syk pathway in a dose dependent manner. Furthermore, we demonstrated that the increased mGITRL on D2SC/1 cells could impair the suppressive activity of CD4(+)CD25(+) regulatory T cells (Tregs) and enhance the proliferation of CD4(+)CD25(-) effector T cells (Teffs). These findings suggest that particulate ß-glucan can be used as immunomodulator to stimulate potent T cell-mediated adaptive immunity while down-regulate immune suppressive activity, leading to a more efficient defense mechanism against tumor development or infectious diseases.

MHC class II transactivator represses human IL-4 gene transcription by interruption of promoter binding with CBP/p300, STAT6 and NFAT1 via histone hypoacetylation.

In addition to its property of enhancing major histocompatibility complex (MHC) class II expression, the class II transactivator (CIITA) was recently demonstrated to be involved in T helper type 1/type 2 (Th1/Th2) differentiation by regulating interleukin-4 (IL-4) gene transcription. There was however, controversy regarding whether CIITA promotes or suppresses IL-4 expression in the experiments with transgenic mice. To clarify the discrepancy by using simpler experimental systems, human Jurkat T cells that express IL-4 but not interferon-gamma, even if stimulated with phorbol 12-myristate 13-acetate plus ionomycin, were used for CIITA transfection. Significant suppression of IL-4 gene expression was demonstrated. Simultaneously, histones H3 and H4 in the IL-4 promoter were hypoacetylated. The suppression could be totally reversed by the histone deacetylatase inhibitor trichostatin A. Furthermore, the IL-4 expression was determined in primarily established human Th1/Th2 cells to which CIITA small interference RNA (siRNA) had been introduced. A substantially increased level of IL-4 was recorded in the CIITA siRNA-transfected Th1 cells, which was in parallel with significantly enhanced acetylation in histone H3 of the IL-4 promoter. Chromatin immunoprecipitation analysis indicated that CIITA abrogated the binding of coactivator CBP/p300 and transcription factors STAT6/NFAT1 to IL-4 promoter in the CIITA-transfected cells. In conclusion, CIITA was active in the repression of transcription activation of human IL-4 gene in both the T-cell line and the primary human CD4 T cells by preventing transcription factors from binding to IL-4 promoter through histone hypoacetylation. Our data confirm a potential significant role of CIITA in controlling Th1/Th2 differentiation via modulation of IL-4 gene activation.

Up-regulation of IL-10 expression in dendritic cells is involved in Trichosanthin-induced immunosuppression.

We report here that Trichosanthin (Tk), a primary active component isolated from a Chinese traditional medicinal herb, Trichosanthes kirilowii, potently inhibits lymphocyte proliferative response in vitro. We found that Tk treatment increased production of the interleukins IL-4 and IL-10, while production of IL-2 and interferon-gamma (IFN-gamma) decreased in the allogeneic antigen-induced immune response. Moreover, up-regulation of IL-10 and IL-4 contributed to the inhibitory activities of Tk. Tk induced immunosuppression through an antigen presenting cell dependent way. Dendritic cells (DCs) are the most potent of the antigen presenting cells, which play a critical role in initiation and regulation of immune responses. We found that Tk could stimulate bone marrow-derived dendritic cells (BMDC) to express IL-10. In addition, pre-exposure of BMDC to Tk produced increased levels of IL-10, but decreased levels of IL-12, following subsequent lipopolysaccharide (LPS) stimulation. Using BMDC obtained from IL-10 deficient mice, we provided evidence that it was IL-10 derived from DCs that initiated the Tk-induced immunosuppression. Furthermore, we found that Tk activated c-Jun N-terminal kinase (JNK) of BMDC and that JNK and p38 mitogen-activated protein kinase (MAPK) activations were associated with Tk-induced IL-10 up-regulation. These data suggest that Tk acts on the function of DCs to change the ratio of IL-10 to IL-12 production and, thus, predominantly inhibits Th1 responses.