The immunomodulator AS101 abrogates DSS-induced Colitis

Inflammatory bowel diseases (IBD) are a group of idiopathic, chronic immune-mediated diseases characterized by an aberrant immune response, including imbalances of inflammatory cytokine production and activated innate and adaptive immunity. Selective blockade of leukocyte migration into the gut is a promising strategy for the treatment of IBD. This study explored the effect of the immunomodulating tellurium compound, ammonium trichloro (dioxoethyleneo,o') tellurate (AS101) on DSS-induced murine colitis. Both oral and i.p. administration of AS101 significantly reduced clinical manifestations of IBD. Colonic inflammatory cytokine levels (IL-17, IL-1β) were significantly downregulated by AS101 treatment, whereas IFN-γ was not affected. Neutrophil and α4β7 + macrophage migration into the tissue was inhibited by AS101 treatment. Adhesion of Mesenteric Lymph Node (MLN) cells to mucosal addressin cell adhesion molecule (MAdCAM-1), the ligand for α4β7 integrin, was blocked by AS101 treatment both in vitro and in vivo. DSS-induced destruction of colonic epithelial barrier/integrity was prevented by AS101, via upregulation of colonic Glial-Derived Neurotrophic Factor (GDNF), which was found previously to regulate the intestinal epithelial barrier through activation of the PI3K/AKT pathway. Indeed, the upregulation of GDNF by AS101 was associated with increased levels of colonic pAKT and BCL-2, and decreased levels of BAX. Furthermore, AS101 treatment reduced colonic permeability to Evans Blue, and decreased colonic TUNEL + cells. Our data revealed multifunctional activities of AS101 in the DSSinduced colitis model via anti inflammatory and anti apoptotic properties. We suggest that treatment with the small, non toxic molecule, AS101, may be an effective early therapeutic approach for controlling human IBD. Introduction Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of chronic inflammatory bowel disease (IBD). Studies in humans and in experimental models of IBD have revealed impaired mucosal barrier function, activated innate immunity, altered production of Th1 and Th2 http://www.jbc. org/cgi/doi/10.1074/jbc.M113.536664 The latest version is at JBC Papers in Press. Published on April 24, 2014 as Manuscript M113.536664 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M arch 4, 2020 hp://w w w . jb.org/ D ow nladed from The immunomodulator AS101 abrogates DSS-induced Colitis 2 cytokines, and the activation of CD4 + T cells in the pathogenesis of IBD (1-3). T regulatory cells play an essential role in the maintenance of intestinal immune homeostasis and were also shown to be capable of preventing and reversing established colitis (4). The etiologies of these diseases remain unclear. The most widely held hypothesis regarding the pathogenesis of IBD is that aberrant activation or defective downregulation of innate immunity, and overly aggressive acquired (T cell) immune responses to a subset of commensal enteric bacteria develop in genetically susceptible hosts, and environmental factors precipitate the onset or reactivation of disease (1,2,5). Cytokines provide key signals in the intestinal immune system, and were shown to have a central role in the pathophysiology of IBD (6). It is evident now that both innate cell populations (e.g. γδ T cells, LTi-like cells, and monocytes/macrophages) and adaptive T cells (Th17 cells) can produce IL-17, which has a crucial role in inflammatory and autoimmune diseases (7-10). IL-17 was found to be elevated in peripheral blood and intestinal tissue of IBD patients (11,12). In addition to IL-17, IL-1 is important in the pathogenesis of IBD due to its pro-inflammatory activities. Peripheral blood mononuclear cells obtained from patients with CD were shown to produce high levels of IL-1 in vitro compared with normal control cells (13), and enhanced secretion of IL-1β was shown in colonic biopsies from patients with inflammatory bowel disease (12,14). Based on these findings, several cytokine-based therapies have been developed and tested for the treatment of IBD patients (6,15). Macrophages have been implicated in the pathogenesis of a variety of chronic and autoimmune diseases including IBD. The classically activated macrophages are key producers of many cytokines (e.g. IL-1, IL-6 and TNFα) and reactive metabolites of oxygen and nitrogen (e.g. nitric oxide) that have been implicated in the development of IBD (16,17). Feeding mice with polymeric Dextran Sodium Sulfate (DSS) in their drinking water induces acute or chronic colitis characterized by bloody diarrhea, ulceration, loss of body weight, and infiltration with granulocytes or mononuclear cells, reflecting IBD symptoms (18). This widely used murine colitis model is helpful for studying the effect of novel therapeutic agents for the management of IBD (19). Integrins are transmembrane cell adhesion receptors composed of noncovalently associated α and β subunits that bind to cell-surface ligands, soluble ligands and extracellular matrix proteins (20). These adhesive interactions are essential for leukocyte recirculation, migration into inflammatory sites, recognition of foreign antigens, and survival and proliferation (21-23). Circulating T cells that bear α4β7 integrins bind to Mucosal Addressin Cell Adhesion Molecule (MAdCAM), which is constitutively expressed on high endothelial venules of Peyer's patches (PP) and Mesenteric Lymph Nodes (MLN), as well as on postcapillary venules of gut lamina propia (LP) (2,24). MAdCAM-1 expression is upregulated on inflamed venules in chronic inflammatory diseases such as in IBD, diabetes, chronic relapsing experimental autoimmune encephalomyelitis etc. Of note, α4β7 integrin is also expressed on stimulated monocytes and macrophages (25). Monoclonal antibody to integrin-α4 (natalizumab), which binds both integrin-α4β7 (the MAdCAM-1 ligand) and integrin-α4β1 (the Vascular Cell Adhesion Molecule-1(VCAM-1) ligand), is effective in treating Crohn’s disease (26), while a humanized anti-integrin-α4β7 antibody has been used to treat active ulcerative colitis (27). The neurotrophin Glial-Derived Neurotrophic Factor (GDNF) has a strong effect on neurite outgrowth and differentiation and is able to protect neurons from apoptosis under various conditions. Interestingly, it has been shown that ablation of enteric glia leads to a fulminant hemorrhagic jejunoileitis in mice (28), suggesting a central role of the enteric glia in the maintenance of gut mucosal integrity. GDNF is upregulated in IBD, and has strong anti-apoptotic properties in colonic epithelial cells via activation of MAPK and PI3K/AKT pathways (29). AS101, is a small non toxic tellurium-IV compound (Fig. 1), currently being evaluated in PhaseII clinical trials in psoriasis patients (unpublished data) and in prevention of bone marrow toxicity induced by chemotherapy in cancer patients (30). This compound is a potent immunomodulator (in-vitro and in-vivo) with a variety of potential therapeutic applications (3133). Accumulated evidence suggests that much of the biological activity of tellurium compounds is by gest on M arch 4, 2020 hp://w w w . jb.org/ D ow nladed from The immunomodulator AS101 abrogates DSS-induced Colitis 3 directly related to their specific chemical interactions with endogenous thiols. If the reacting thiol is a cysteine residue, the reaction product may alter the biological activity of the target protein (34). The Te(IV)-thiol chemical bond may lead to conformational change or disulfide bond formation, possibly resulting in a loss of biological activity, if the thiol residue is essential for a particular function. Indeed, we demonstrated that AS101 specifically inactivates cysteine proteases while exhibiting no effect on the other families of serine-, aspartic-, and metallo-proteases, in good agreement with the predictions of its unique Te (IV)-thiol chemistry. Furthermore, the proteolytic activity of the inactivated cysteine proteases could be recovered by reducing agents such as NaBH4, further supporting the suggestion that the inactivation process involves oxidation of the catalytic thiol to a disulfide (34). In light of its chemical activities, AS101 has been shown to exert anti inflammatory and anti apoptotic effects in different in vivo models (35-38), through inhibition of caspase 1, 3 and 8 activities and by its ability to induce GDNF production (38,39). In the present study, we tested the effect of AS101 on a DSS-induced murine colitis model. Our findings revealed anti-inflammatory and antiapoptotic properties of AS101 through the downregulation of colonic inflammatory cytokine levels, prevention of inflammatory cell migration into the tissue, and inhibition of colonic apoptotic