Zhikang Peng

AMPK agonist downregulates innate and adaptive immune responses in TNBS-induced murine acute and relapsing colitis

AMP-activated protein kinase (AMPK), a cellular energy sensor, has been reported to participate in modulating inflammatory responses, but its role in intestinal inflammation remains unclear. IBD has been characterized by excessive innate and adaptive immune responses. Here, the roles of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an agonist of AMPK, in regulating immune responses of experimental colitis were investigated. In vitro effects of AICAR on LPS-induced macrophage activation and Th1 and Th17 differentiation, as well as in vivo effects of AICAR in mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, were explored. In acute colitis, daily AICAR treatment commenced 2 days after TNBS delivery (day 1), while in relapsing colitis, AICAR treatment commenced after three weekly TNBS administrations. Colon inflammation, production of proinflammatory cytokines and NF-κB activation in colon tissues, and Th1 and Th17 cell populations in lamina propria mononuclear cells (LPMCs) and mesenteric lymph node cells (MLNs) were assayed. Results show that AICAR significantly inhibited in vitro LPS-induced macrophage activation and Th1 and Th17 cell differentiation. Administration of AICAR was therapeutically effective in ameliorating acute and relapsing experimental colitis, as shown by reduced body weight loss and significant attenuation in colon histological inflammation. Moreover, this treatment inhibited NF-κB activation in macrophages, and reduced levels of TNF, Th1- and Th17-type cytokines, and Th1 and Th17 cell populations in LPMCs and MLNs. AICAR-initiated AMPK activation may act as a central downregulator in ongoing innate and adaptive immune responses of murine colitis, providing a novel therapeutic approach in the treatment of IBD.

Novel Anti-Inflammatory Action of 5-Aminoimidazole-4-carboxamide Ribonucleoside with Protective Effect in Dextran Sulfate Sodium-Induced Acute and Chronic Colitis

AMP-activated protein kinase (AMPK) is an important cellular energy sensor that is responsible for maintaining systemic and cellular energy balance. Its role in intestinal inflammation remains unclear. Recent studies indicate that AMPK activation initiated by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) participates in modulating inflammatory responses. Inflammatory bowel disease (IBD) has been characterized by sustained intestinal mucosa inflammation, caused mainly by excessive macrophage activation and T helper type 1 (Th1) and Th17 immune responses. Thus, we sought to determine the effect of AICAR on inflammatory responses of murine models of IBD. Mice with acute or chronic colitis induced by dextran sulfate sodium (DSS) were treated with or without AICAR. Body weight and colon inflammation were evaluated, and production of proinflammatory cytokines in colon tissues was determined. Nuclear factor κB (NF-κB) activation in colon tissues was assayed, and Th1 and Th17 cell responses were also evaluated. By inducing AMPK activation, AICAR had a therapeutic effect in ameliorating acute and chronic DSS-induced murine colitis as shown by reduced body weight, loss and significant attenuation in clinical symptoms, and histological inflammation. Moreover, AICAR treatment inhibited NF-κB activation in macrophages, reduced levels of Th1- and Th17-type cytokines in colon tissues, and down-regulated Th1 and Th17 cell responses during the progress of acute and chronic experimental colitis. AICAR acts as a central inhibitor in immune responses of experimental colitis. Our data show that AICAR-initiated AMPK activation may represent a promising alternative to our current approaches to suppress intestinal inflammation in IBD.

Targeting TGF‐β1 by employing a vaccine ameliorates fibrosis in a mouse model of chronic colitis

Background: Intestinal fibrosis and stricture formation are major complications of inflammatory bowel disease (IBD), for which there are currently few effective treatments. We sought to investigate whether targeting transforming growth factor‐beta1 (TGF‐&bgr;1), a key profibrotic mediator, with a peptide‐based virus‐like particle vaccine would be effective in suppressing intestinal fibrosis by using a mouse model of 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced chronic colitis. Methods: The vaccine was prepared by inserting a peptide derived from mouse TGF‐&bgr;1 into a carrier hepatitis B core antigen using gene recombination methods. Chronic colitis was induced in BALB/c mice by 8 weekly TNBS administrations. Mice were subcutaneously injected with vaccine, carrier, or phosphate‐buffered saline (PBS) in 2 separate studies: either before or after acute inflammatory responses commenced. Results: Sera from vaccinated mice exhibited significantly elevated levels of TGF‐&bgr;1‐specific immunoglobulin G (IgG), which inhibited TGF‐&bgr;1‐induced luciferase production in mink lung epithelial cells. In the chronic colitis model, mice receiving vaccine showed improved body weight gain and significantly reduced colonic collagen deposition. Hematoxylin and eosin staining and semiquantitative scoring indicated that vaccination even ameliorated colonic inflammation. Cytokine profile analysis revealed that levels of TGF‐&bgr;1, interleukin (IL)‐17, and IL‐23 in vaccinated mouse colon tissues were decreased, and that percentages of IL‐17‐expressing CD4+ lymphocytes in mesenteric lymph node cells were reduced. Furthermore, Smad3 phosphorylation, a key event in TGF‐&bgr; signaling, was decreased in colonic tissue in vaccinated mice. Conclusions: This TGF‐&bgr;1 peptide‐based vaccine, which suppressed excessive TGF‐&bgr;1 bioactivity, may prevent the development of intestinal fibrosis and associated complications, presenting a novel approach in the treatment of IBD. (Inflamm Bowel Dis 2010)

Development of recombinant vaccines against IL-12/IL-23 p40 and in vivo evaluation of their effects in the downregulation of intestinal inflammation in murine colitis.

Overexpression of IL-12 and IL-23, which share the p40 subunit, has been implicated in the pathogenesis of Crohns disease. Targeting these cytokines with monoclonal antibodies has emerged as a new and effective therapy, but one with adverse reactions. In this study, we sought to develop p40 peptide-based virus-like particle vaccines that elicit autoantibodies to IL-12 and IL-23 for a long-term treatment of the disease. Three vaccines (named C, D and F) against the p40 were developed by inserting peptides derived from p40 into the carrier, hepatitis B core antigen, using molecular engineering methods. Immunization with the vaccines, without the use of adjuvants, induced high titered and long-lasting antibodies to IL-12, IL-23 and p40. The three vaccines were evaluated in vivo in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced chronic murine colitis. Mice were immunized with a vaccine three times, followed by weekly intrarectal administrations of TNBS. Vaccine groups, especially groups C and F, showed reduced expression of IL-12/IL-23p40, less inflammation, and decreased collagen deposition in colon tissues when compared with controls. We concluded that IL-12/IL-23p40 vaccines may be a potential therapeutic approach in the treatment of Crohns disease and other autoimmune diseases.

Enhancement of IL-10 bioactivity using an IL-10 peptide-based vaccine exacerbates Leishmania major infection and improves airway inflammation in mice.

IL-10 is a regulatory cytokine that plays important roles in promoting disease progression in cutaneous leishmaniasis and suppressing allergic responses in asthma. We sought to develop an IL-10 peptide-based vaccine for the control of IL-10-related diseases. To break self-tolerance, hepatitis B core antigen (HBcAg) was used as a carrier. The vaccine was prepared by inserting a peptide derived from mouse IL-10 into the carrier using gene recombination methods. This vaccine presented as virus-like particles, bound to polyclonal anti-IL-10 antibodies, and induced high titers of IL-10-specific IgG. The in vivo effects of the vaccine were investigated in a murine model of cutaneous leishmaniasis. Unexpectedly, vaccinated mice developed larger cutaneous lesions, harbored significantly more parasites, and cells from lymph nodes produced higher amounts of parasite-specific IL-4, IL-10 and IFN-gamma in cultures. Further in vitro studies showed that serum IL-10-specific IgG from vaccinated mice significantly enhanced IL-10 bioactivity dose-dependently. This enhancing effect was confirmed in OVA-induced asthmatic mice. Vaccinated mice exhibited a significant decrease in airway eosinophils, lung inflammation, goblet hyperplasia, and levels of serum OVA-specific IgE, compared to control mice. We concluded that the IL-10 vaccine enhances the bioactivity of IL-10 in vitro and in vivo, providing a potential therapeutic approach in diseases associated with insufficient IL-10 production.

An IL-17 peptide-based and virus-like particle vaccine enhances the bioactivity of IL-17 in vitro and in vivo

AIMSnTo develop an IL-17 peptide-based virus-like particle vaccine that elicits autoantibodies to IL-17 and to evaluate the effects of the vaccine in mice with experimental colitis.nnnMATERIALS & METHODSnRecombinant IL-17 vaccines were constructed by inserting selected peptides derived from mouse IL-17 into the carrier protein, hepatitis B core antigen, using molecular engineering methods. To evaluate the in vivo effects of the vaccine, mice with 2,4,6-trinitrobenzene sulfonic acid-induced chronic colitis were injected three times with the vaccine, carrier or saline after the second delivery of 2,4,6-trinitrobenzene sulfonic acid. Colon inflammation and fibrosis were evaluated by histological examination. Serum IL-17-specific IgG and colon-tissue cytokine levels were measured by ELISA. In vitro inhibition tests of sera from vaccine-immunized mice were performed using IL-17-induced IL-6 production by NIH 3T3 cells and IL-17-induced TNF production by macrophages.nnnRESULTSnImmunization with the vaccine without the use of adjuvants induced high-titered and long-lasting antibodies to IL-17. Unexpectedly, vaccinated mice exhibited increases in colon inflammation, collagen deposition, levels of TNF and IL-17 cytokines compared with carrier and saline groups. Furthermore, in vitro study revealed that serum IL-17-specific IgG from vaccine-immunized mice significantly enhanced IL-17-induced IL-6 production and IL-17-induced TNF production dose-dependently.nnnCONCLUSIONnThe IL-17 peptide-based vaccine enhances the bioactivity of IL-17 in vitro and in vivo, providing a potential immunotherapy for treatment of diseases associated with insufficient IL-17 production, such as hyper-IgE syndrome.

The Potential Protective Role of Caveolin-1 in Intestinal Inflammation in TNBS-Induced Murine Colitis

Background Caveolin-1 (Cav-1) is a multifunctional scaffolding protein serving as a platform for the cell’s signal-transduction and playing an important role in inflammation. However, its role in inflammatory bowel disease is not clear. A recent study showed that Cav-1 is increased and mediates angiogenesis in dextran sodium sulphate-induced colitis, which are contradictory to our pilot findings in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. In the present study, we further clarified the role of Cav-1 in TNBS-induced colitis. Methods In BALB/c mice, acute colitis was induced by intra-rectal administration of one dose TNBS, while chronic colitis was induced by administration of TNBS once a week for 7 weeks. To assess the effects of complete loss of Cav-1, Cav-1 knockout (Cav-1−/−) and control wild-type C57 mice received one TNBS administration. Body weight and clinical scores were monitored. Colon Cav-1 and pro-inflammatory cytokine levels were quantified through ELISAs. Inflammation was evaluated through histological analysis. Results Colon Cav-1 levels were significantly decreased in TNBS-induced colitis mice when compared to normal mice and also inversely correlated with colon inflammation scores and proinflammatory cytokine levels (IL-17, IFN-γ and TNF) significantly. Furthermore, after administration of TNBS, Cav-1−/− mice showed significantly increased clinical and colon inflammatory scores and body weight loss when compared with control mice. Conclusions and Significance Cav-1 may play a protective role in the development of TNBS-induced colitis. Our findings raise an important issue in the evaluation of specific molecules in animal models that different models may exhibit opposite results because of the different mechanisms involved.

S1713 IL-12/IL-23 P40 Peptide-Based Vaccine Ameliorates TNBS-Induced Murine Chronic Colitis By Down-Regulation of TH1 and TH17 Cytokines

BACKGROUND: The aetiology of crohns disease (CD) is poorly understood at present, but an immune pathology involving the CD4+T-cell phenotypes is suspected to be a major factor for the development and the perpetuation of CD. In line with this thinking, recently, we found that in patients with ulcerative colitis, the peripheral level of the regulatory CD4+CD25High+ T-cell phenotype was compromised, while the CD4+ T-cells lacking CD28 (CD4+CD28-) had expanded (Dig Dis Sci 2007;52:2725-31). We have now extended our work to patients with CD. METHODS: The study subjects included 31 patients with active CD (aCD) who were to be treated with infliximab (IFX). At baseline, the mean values of CD activity index (CDAI) and the duration of CD were 200.9±84.8 and 5.7±7.0 years, respectively. Sixteen patients with CD in remission (rCD, CDAI≤150) and 19 healthy control (HC) subjects were also included in this study. Fifteen patients received IFX as initial (top down) therapy, 7 received IFX as an episodic (step up) therapy, and the other 9 received IFX after operation. Peripheral blood samples were obtained before and 12 hours after each IFX infusion. Lymphocytes were stained with anti CD4, anti CD25 and anti CD28 antibodies for analyses by fluorescence-activated cell sorter (FACS) in flow cytometry. RESULTS: The aCD group had significantly lower level of CD4+CD25High+ T-cell expression as compared with the rCD group (P<0.05) or the HC group (P<0.05). A significant up-regulation of CD4+CD25High+ T-cell phenotype was associated with the post first IFX infusion as compared with the pre first IFX infusion (P<0.01). Conversely, patients with aCD had higher % CD4+CD28T-cells as compared with the rCD group (P<0.05) or the HC group (P<0.05). A significant down-modulation of the CD4+CD28T-cell phenotype was associated with post first IFX infusion vs pre first IFX infusion (P<0.01) CONDLUSIONS: IFX therapy was associated with a marked improvement of patients quality of life, but our clinical observations indicated that the efficacy of IFX diminishes after frequently administration. However, in this study, the clinical efficacy of IFX was associated with a significant up modulation of the regulatory CD4+CD25High+ T-cell phenotype and unexpectedly, some normalization of peripheral CD4+CD28T-cell phenotype. We believe that a likely impact of IFX on key cells of the immune system is potentially very significant and warrants to be substantiated in future studies.

Reversing Ongoing Chronic Intestinal Inflammation and Fibrosis by Sustained Block of IL-12 and IL-23 Using a Vaccine in Mice

Interleukin (IL)-12 and IL-23 that share subunit p40 are important cytokines in the pathogenesis of inflammatory bowel disease. We reported that mouse p40 peptide-based vaccines ameliorated intestinal inflammation in the prevention of trinitrobenzene sulfonic acid (TNBS)-induced murine colitis model. Here, we evaluated whether administration of the vaccine after establishment of colitis would be effective in modifying both TNBS-induced and dextran sulfate sodium (DSS)-induced chronic colitis and the underlying immune mechanisms. We further examined whether vaccination could exacerbate infections. Chronic colitis was developed by either intrarectally administrating TNBS or drinking 4% DSS water. Vaccination started after two TNBS administrations or 7 days of DSS treatment. Results showed that administrating p40 vaccine induced high tittered antibodies to IL-12 and IL-23, improved clinical scores, reduced intestinal inflammation and fibrosis, and down-regulated proinflammatory cytokine productions in colon tissue, compared with control mice. Furthermore, in lamina propria mononuclear cells and/or mesenteric lymph nodes, mice immunized with p40 peptide vaccine exhibited high ratios of Treg/Th1 and Treg/Th17 cells and increased IL-10 expression in CD11c+IL-10+cells. In mice infected with lung chlamydia, in which the protective role of Th1/Th17 is well documented, vaccine immunization did not increase lung bacterial burden. We conclude that p40 vaccine may provide a potential and safe approach for treatment of IBD. 10.1093/ibd/izy142_videoizy142.video5785979965001.

368 Recombinant Tumor Necrosis Factor Alpha (TNFα) Peptide-Based Vaccine Ameliorates Acute and Chronic Murine Colitis

Background: Interleukin (IL)-12 and 23 are heterodimeric cytokines composed of a common p40 subunit and p35 and p19 subunits, respectively. IL-23 is strongly implicated in the pathogenesis of inflammatory bowel disease. Aim: The aim of this study is to characterize the molecular regulation of Il23a gene expression and promoter activation in murine macrophages and experimental colitis. Results: Interferon-γ (IFN-γ) inhibits LPS-induced Il23a mRNA and IL-23 protein expression in murine bone marrow-derived macrophages (BMMs). A conserved nucleotide sequence across multiple species was identified in the promoter of the Il23a gene that contains an interferon stimulated response element (ISRE). In LPS and IFN-γ activated BMMs, chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrate that interferon regulatory factors (IRFs) interact with this ISRE. Using a 1.8 kb murine Il23a luciferase reporter plasmid, LPS induces and IFN-γ inhibits LPSinduced p19 promoter activity in BMMs. Mutations in the ISRE abrogate IFN-γ inhibition of LPS-induced promoter activity. To understand the contribution of specific IRFs, IRF-1 was inhibited in BMMs by siRNA and experiments were performed in IRF-1 deficient (-/-) macrophages. IRF-1 knockdown and IRF-1-/BMMs demonstrate increased LPS-induced IL-23 expression. Primary response genes have promoters that either exist in an open chromatin structure or undergo rapid nucleosomal remodeling. In contrast, secondary response genes with delayed induction kinetics require new protein synthesis prior to transcription initiation. Il23a expression in LPS-activated BMMs demonstrates rapid induction kinetics and unlike Il12b (established secondary response gene) is not dependent on new protein synthesis. Interestingly, LPS plus IFN-γ activated BMMs demonstrate loss of the IFN-γ inhibitory effect, suggesting IFN-γ mediated regulation of Il23a requires new protein synthesis. Il23a regulation was then studied in murine colitis models. Following intrarectal administration of TNBS, IRF-1-/mice exhibit increased inflammation and increased colonic IL-23 expression compared to wild type (WT) mice. Additionally, we demonstrate mucosal regulation of IL-23 by the enteric microbiota using germ-free and conventionalized WT and colitis-prone IL-10-/mice. Enteric bacteria induce significantly higher intestinal IL-23 expression in IL-10-/-mice compared withWTmice. Conclusions: IFN-γ and IRF-1 negatively regulate Il23a in murine macrophages and experimental colitis. Regulation of intestinal Il23a by the enteric microflora is a significant event in the initiation of chronic intestinal inflammation.