A.G. Ma

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)

Sustained Suppression of IL-13 by a Vaccine Attenuates Airway Inflammation and Remodeling in Mice

We previously reported that a recombinant IL-13 peptide-based virus-like particle vaccine significantly suppressed murine acute airway allergic inflammatory responses. The impact of this strategy on the development of chronic airway inflammation and remodeling has not been investigated. We evaluated whether the vaccine-mediated sustained suppression of IL-13 attenuates features of chronic airway inflammation and remodeling in mice repeatedly challenged with allergen. BALB/c mice received two intraperitoneal sensitizing injections of ovalbumin (OVA) and alum, followed by six consecutive 2-day intranasal OVA challenges at 12-day intervals and then a 4-week recovery period. Anti-IL-13 antibodies were induced with a vaccine before (preventive experiments) or after (interventional experiments) the OVA challenge commenced. Respiratory mechanics were assessed using low-frequency forced oscillation with a small animal ventilator. Cytokine concentrations in bronchoalveolar lavage fluid (BALF) and lung histology were also assessed. In the preventive experiments, vaccination significantly suppressed IL-13 concentrations, the accumulation of inflammatory cells in BALF, lung mucus production, and collagen deposition. Furthermore, vaccination significantly attenuated OVA challenge-induced increases in airway resistance, tissue resistance, and tissue elastance, both acutely and after a 4-week recovery from allergen challenges. In the interventional experiments, vaccination decreased IL-13, TGF-β1, and IL-12p40 concentrations in BALF, as well as mucus production and collagen deposition. Chronic inflammation and sustained airway hyperresponsiveness were not significantly reversed. The persistent suppression of IL-13 with a vaccine inhibits chronic airway inflammation and the development of several key components of airway remodeling, and this intervention is more effective at early stages than later during chronic inflammation.

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.

Employing an IL-23 p19 vaccine to block IL-23 ameliorates chronic murine colitis

BACKGROUND Overexpression of IL-23 has been implicated in the pathogenesis of Crohns disease. Using vaccines to block overexpressed endogenous cytokines has emerged as a new therapeutic strategy for the long-term treatment of the disease. AIM We sought to develop peptide-based vaccines specific to IL-23 and evaluate their effects in colitis mice. MATERIALS & METHODS The vaccine was developed by inserting a peptide derived from mouse IL-23 p19 into the carrier protein, hepatitis B core antigen, using molecular engineering methods. One vaccine against IL-23 p19 was obtained that induced high-titered and long-lasting antibodies to IL-23 without the use of adjuvants. The inhibitory effect of vaccine-immunized serum was subsequently evaluated in vitro. To evaluate the in vivo effects, mice were subcutaneously injected with the vaccine, carrier or saline three times. Two weeks after the last injection, chronic colitis was induced in mice by seven weekly administrations with 2,4,6-trinitrobenzene sulfonic acid. RESULTS In vitro studies revealed that serum IL-23 p19-specific IgG significantly suppressed IL-23-induced IL-17 production by splenocytes. In vivo evaluation of the effect of the vaccine in mice with chronic colitis indicated that vaccine-immunized mice exhibited a decrease in colon inflammation, collagen deposition and levels of IL-23 and IL-12 cytokines, compared with control groups. CONCLUSION IL-23 p19 vaccine is capable of downregulating inflammatory responses in chronic murine colitis, providing a novel therapeutic approach in Crohns disease.

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.