IL-22 Negatively Regulates Helicobacter pylori-Induced CCL20 Expression in Gastric Epithelial Cells

Helicobacter pylori is a Gram-negative bacterium that infects the human gastric mucosa and causes various gastric diseases. H. pylori infection induces the production of inflammatory chemokine CCL20 in gastric mucosa and leads to gastric inflammation. Given that the IL-22/IL-22R axis plays a critical role in the regulation of homeostasis and inflammation of epithelial cells at barrier surfaces, we investigated the effect of IL-22 on CCL20 expression induced by H. pylori. We demonstrated that H. pylori infection of the gastric epithelia-derived AGS cells significantly induced CCL20 expression and the induction was inhibited by IL-22. Functional analysis of the CCL20 promoter revealed that the H. pylori-induced CCL20 expression required the activation of NF-κB, and that IL-22 inhibited the induction by attenuating NF-κB activation. Knockdown of endogenous STAT3 by either short interfering RNAs or a short hairpin RNA significantly reduced the inhibitory effect of IL-22. Furthermore, STAT3 phosphorylation elicited by IL-22 was crucial for the inhibition of H. pylori-induced CCL20 expression. Consistent with the in vitro data showing that IL-22 negatively regulated H. pylori-induced CCL20 expression in gastric epithelial cells, studies on the tissue sections from patients with H. pylori infection also revealed an inverse association of IL-22 expression and CCL20 expression in vivo. Together, our findings suggest that IL-22 plays a role in the control of overproduction of the inflammatory chemokine and thus may protect the gastric mucosa from inflammation-mediated damage.     

Vaccine-induced immunity against Helicobacter pylori in the absence of IL-17A

Background: Helicobacter pylori (H. pylori) is a gram negative bacterium that can cause diseases such as peptic ulcers and gastric cancer. IL‐17A, a proinflammatory cytokine that can induce the production of CXC chemokines for neutrophil recruitment, has recently been shown to be elevated in both H. pylori‐infected patients and mice. Furthermore, studies in mouse models of vaccination have reported levels significantly increased over infected, unimmunized mice and blocking of IL‐17A during the challenge phase in immunized mice reduces protective immunity. Because many aspects of immunity had redundant or compensatory mechanisms, we investigated whether mice could be protectively immunized when IL‐17A function is absent during the entire immune response using IL‐17A and IL‐17A receptor knockout (KO) mice immunized against H. pylori. Materials and Methods: Gastric biopsies were harvested from naïve, unimmunized/challenged, and immunized/challenged wild type (WT) and KO mice and analyzed for inflammation, neutrophil, and bacterial levels. Groups of IL‐17A KO mice were also treated with anti‐IFNγ or control antibodies. Results: Surprisingly, all groups of immunized KO mice reduced their bacterial loads comparably to WT mice. The gastric neutrophil counts did not vary significantly between IL‐17A KO and WT mice, whereas IL‐17RA KO mice had on average a four‐fold decrease compared to WT. Additionally, we performed an immunization study with CXCR2 KO mice and observed significant gastric neutrophils and reduction in bacterial load. Conclusion: These data suggest that there are compensatory mechanisms for protection against H. pylori and for neutrophil recruitment in the absence of an IL‐17A‐CXC chemokine pathway.     

Stromal Cells Induce Th17 during Helicobacter pylori Infection and in the Gastric Tumor Microenvironment

Gastric cancer is associated with chronic inflammation and Helicobacter pylori infection. Th17 cells are CD4⁺ T cells associated with infections and inflammation; but their role and mechanism of induction during carcinogenesis is not understood. Gastric myofibroblasts/fibroblasts (GMF) are abundant class II MHC expressing cells that act as novel antigen presenting cells. Here we have demonstrated the accumulation of Th17 in H. pylori-infected human tissues and in the gastric tumor microenvironment. GMF isolated from human gastric cancer and H. pylori infected tissues co-cultured with CD4⁺ T cells induced substantially higher levels of Th17 than GMF from normal tissues in an IL-6, TGF-β, and IL-21 dependent manner. Th17 required interaction with class II MHC on GMF for activation and proliferation. These studies suggest that Th17 are induced during both H. pylori infection and gastric cancer in the inflammatory milieu of gastric stroma and may be an important link between inflammation and carcinogenesis.     

IL-17A Induces Pendrin Expression and Chloride-Bicarbonate Exchange in Human Bronchial Epithelial Cells

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.     

B Cells Contribute to Heterogeneity of IL-17 Producing Cells in Rheumatoid Arthritis and Healthy Controls

Secretion of the proinflammatory cytokine Interleukin-17A (IL-17A) is the hallmark of a unique lineage of CD4 T cells designated Th17 cells, which may play a crucial role in the pathogenesis of rheumatoid arthritis (RA) and many autoimmune diseases. Recently, IL-17-producing cells other than T cells have been described, including diverse innate immune cells. Here, we show that the cellular sources of IL-17A in RA include a significant number of non-T cells. Multicolour fluorescence analysis of IL-17-expressing peripheral blood mononuclear cells (PBMC) revealed larger proportions of IL-17⁺CD3^- non-T cells in RA patients than in healthy controls (constitutive, 13.6% vs. 8.4%, and after stimulation with PMA/ionomycin 17.4% vs. 7.9% p < 0.001 in both cases). The source of IL-17 included CD3^-CD56⁺ NK cells, CD3^-CD14⁺ myeloid cells as well as the expected CD3⁺CD4⁺ Th17 cells and surprisingly a substantial number of CD3^-CD19⁺ B cells. The presence of IL-17A-expressing B cells was confirmed by specific PCR of peripheral MACS-sorted CD19⁺ B cells, as well as by the analysis of different EBV-transformed B cell lines. Here we report for the first time that in addition to Th17 cells and different innate immune cells B cells also contribute to the IL-17A found in RA patients and healthy controls.     

TH17细胞相关细胞因子IL-17和IL-23在系统性红斑狼疮中的表达及其意义

目的:探究SLE的发病机制,通过测定SLE患者及正常人外周血浆中的IL-17以及IL-23的表达水平,研究SLE患者体内的IL-17以及IL-23水平是否异常,并探讨其在SLE疾病中的作用.通过研究有望为SLE患者的治疗在细胞因子方面提供方向.方法:SLE组33例;健康对照组20例.采用酶联免疫吸附试验测定血浆中IL-17、IL-23的水平,收集整理SLE患者的临床资料及实验室数据.按照SLE患者疾病活动度、有无狼疮肾炎和抗ds-DNA阳性与否以及补体水平进行分组.结果:活动期SLE患者血浆中的IL-17以及IL-23水平明显高于对照组(P＜0.01),与SLE非活动组相比,也有明显差异,其具有统计学意义(P＜0.01),但非活动组与正常对照组间无统计学意义.狼疮肾炎组和非狼疮肾炎组患者血浆中的IL-17和IL-23水平均高于正常对照组(P＜0.01),但IL-17和IL-23的水平在肾炎组和非肾炎组间无明显统计学差异.抗ds-DNA抗体阳性组与阴性组间IL-17和IL-23水平也无明显统计学差异.补体C3、C4水平高值组与正常组间IL-17和IL-23水平也无明显统计学差异.结论:IL-17和IL-23表达水平在SLE患者活动期血浆中表达有明显增高,提示IL-17和IL-23可能参与了SLE疾病的发生发展过程,可能与疾病的活动度有密切的关系.是否有肾脏损害以及抗ds-DNA抗体阳性与否、补体水平的高低与否可能对SLE患者血浆中IL-17和IL-23的表达水平影响较小.通过此研究我们可以在SLE的细胞因子治疗方面有进一步突破,进一步明确SLE的发病机制,为SLE患者带来更大的福音.     

DEC205在幽门螺杆菌感染胃上皮细胞中的表达

目的：探讨幽门螺杆菌及其热休克蛋白60（H．pylori—HSP60）感染与胃上皮细胞表面DEC205受体的关系。方法：分别用H．pylori、H．pylori-HSP60及E．coliLPS刺激胃上皮细胞KATOIII,利用免疫荧光染色技术观察KATOIII细胞表面DEC205蛋白的表达变化,再利用RT—PCR技术,观察细胞中DEC205mRNA对上述抗原刺激后的变化。结果：H．pylori、H．pylori—HSP60及E．coliLPS的刺激明显引起细胞表面DEC205蛋白的表达以及细胞内DEC205mRNA的产生。结论：H．pylori感染与胃上皮细胞表面的胞吞受体DEC205有着密切的关系。     

DEC205在幽门螺杆菌感染胃上皮细胞中的表达

目的:探讨幽门螺杆菌及其热休克蛋白60(H.pylori-HSP60)感染与胃上皮细胞表面DEC205受体的关系。方法:分别用H.pylori、H.pylori-HSP60及E.coli LPS刺激胃上皮细胞KATOⅢ,利用免疫荧光染色技术观察KATOⅢ细胞表面DEC205蛋白的表达变化,再利用RT-PCR技术,观察细胞中DEC205mRNA对上述抗原刺激后的变化。结果:H.pylori、H.pylori-HSP60及E.coli LPS的刺激明显引起细胞表面DEC205蛋白的表达以及细胞内DEC205 mRNA的产生。结论:H.pylori感染与胃上皮细胞表面的胞吞受体DEC205有着密切的关系。     

Differential Capacity of Human Skin Dendritic Cells to Polarize CD4+T Cells into IL-17, IL-21 and IL-22 Producing Cells

Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c⁺CD14⁻ and CD14⁺ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4⁺ or CD8⁺T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c⁺CD14⁻DDCs were able to differentiate naïve CD4⁺T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4⁺T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4⁺ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.     

C5a Regulates IL-12+DC Migration to Induce Pathogenic Th1 and Th17 Cells in Sepsis

Objective

It is well known that complement system C5a is excessively activated during the onset of sepsis. However, it is unclear whether C5a can regulate dentritic cells (DCs) to stimulate adaptive immune cells such as Th1 and Th17 in sepsis.

Methods

Sepsis was induced by cecal ligation and puncture (CLP). CLP-induced sepsis was treated with anti-C5a or IL-12. IL-12⁺DC, IFNγ⁺Th1, and IL-17⁺Th17 cells were analyzed by flow cytometry. IL-12 was measured by ELISA.

Results

Our studies here showed that C5a induced IL-12⁺DC cell migration from the peritoneal cavity to peripheral blood and lymph nodes. Furthermore, IL-12⁺DC cells induced the expansion of pathogenic IFNγ⁺Th1 and IL-17⁺Th17 cells in peripheral blood and lymph nodes. Moreover, IL-12, secreted by DC cells in the peritoneal cavity, is an important factor that prevents the development of sepsis.

Conclusion

Our data suggests that C5a regulates IL-12⁺DC cell migration to induce pathogenic Th1 and Th17 cells in sepsis.     

利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的pBlueBacHis2A转移载体中,将重组质粒pBlueBacHis2A-CagA与亲本病毒Bac-N-blue DNA共转染Sf9细胞,以空斑法纯化获得的重组杆状病毒.经PCR法鉴定后进行扩增培养,SDS-PAGE和Western bolt检测结果证实所表达的蛋白为CagA蛋白,间接ELISA分析表明,表达产物可与Hp感染者血清发生特异性的免疫反应.     

利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的 pBlueBacHis2A转移载体中 ,将重组质粒 pBlueBacHis2A CagA与亲本病毒Bac N blueDNA共转染Sf9细胞 ,以空斑法纯化获得的重组杆状病毒。经PCR法鉴定后进行扩增培养 ,SDS PAGE和Westernbolt检测结果证实所表达的蛋白为CagA蛋白 ,间接ELISA分析表明 ,表达产物可与Hp感染者血清发生特异性的免疫反应     

Expression and regulation of IL-22 in the IL-17-producing CD4＋ T lymphocytes

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4＋ helper T lymphocytes （TH） were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFI3 and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-l 7 signaling pathways in tissue inflammation and autoimmune diseases.     

Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

Human respiratory syncytial virus (HRSV) is a leading cause of severe lower respiratory tract infection in children under five years of age. IL-17 and Th17 responses are increased in children infected with HRSV and have been implicated in both protective and pathogenic roles during infection. Bovine RSV (BRSV) is genetically closely related to HRSV and is a leading cause of severe respiratory infections in young cattle. While BRSV infection in the calf parallels many aspects of human infection with HRSV, IL-17 and Th17 responses have not been studied in the bovine. Here we demonstrate that calves infected with BRSV express significant levels of IL-17, IL-21 and IL-22; and both CD4 T cells and γδ T cells contribute to this response. In addition to causing significant morbidity from uncomplicated infections, BRSV infection also contributes to the development of bovine respiratory disease complex (BRDC), a leading cause of morbidity in both beef and dairy cattle. BRDC is caused by a primary viral infection, followed by secondary bacterial pneumonia by pathogens such as Mannheimia haemolytica. Here, we demonstrate that in vivo infection with M. haemolytica results in increased expression of IL-17, IL-21 and IL-22. We have also developed an in vitro model of BRDC and show that co-infection of PBMC with BRSV followed by M. haemolytica leads to significantly exacerbated IL-17 production, which is primarily mediated by IL-17-producing γδ T cells. Together, our results demonstrate that calves, like humans, mount a robust IL-17 response during RSV infection; and suggest a previously unrecognized role for IL-17 and γδ T cells in the pathogenesis of BRDC.