The therapeutic effect of glatiramer acetate in a murine model of inflammatory bowel disease is mediated by anti-inflammatory T-cells

Abstract

Inflammatory bowel diseases (IBDs) are complex multifactorial immunological disorders characterized by dysregulated immune reactivity in the gut and imbalance between pro-inflammatory and anti-inflammatory reactivity. The therapeutic effect of the immunomodulatory drug glatiramer acetate (GA, Copaxone, copolymer 1) has been established in several IBD models, including trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS)-induced colitis, as well as in a spontaneous colitis model. In the present study we investigated the mechanism of action of GA and cells specifically induced by it. Immunization of naive mice by GA, generated a lymphocyte population of the Th2/3 subtype, that drastically reduced disease manifestations upon their adoptive transfer to mice with DSS colitis. This was demonstrated by the substantial decrease in weight loss, intestinal bleeding and diarrhea, as well as by the prevention of macroscopic and microscopic colonic damage. In contrast, adoptive transfer of control lysozyme-specific cells did not induce any beneficial effect on the disease. Moreover, GA-specific short-term T-cell lines, either exogenously labeled or genetically marked, adoptively transferred by the intraperitoneal route to colitis-induced mice, localized in the inner layers of the colon and secreted in situ the regulatory cytokine TGF-β. These results demonstrate the accumulation of GA-specific Th2/3 cells secreting regulatory cytokines in the injured colon, and thus draw a direct linkage between the therapeutic effect of GA in IBD and an immunomodulatory effect at the site in which the pathological process occurs.

Introduction

Inflammatory bowel diseases (IBDs) are severe gastrointestinal disorders including ulcerative colitis (UC) and Crohn's disease (CD). In the latter case, a major factor in the pathogenesis is the uncontrolled detrimental immune response towards local microorganisms, involving mainly CD4⁺ T-helper Th1 cells. This is manifested in the findings that inflammatory mediators such as tumor necrosis factor-α (TNF-α) produced by infiltrating CD4⁺ T cells and macrophages, exacerbate the disease; in contrast, regulatory cytokines such as transforming growth factor-β (TGF-β) and interlukin-10 (IL-10) secreted by Th2/3 cells, provide a beneficial effect [1], [2]. Accordingly, the level of these cytokines in the intestine may ultimately determine whether an immune response to a gut antigen is detrimental or innocuous. Thus, an imbalance between pro-inflammatory and anti-inflammatory reactivity plays a pivotal role in the pathogenesis of IBD [3].

Although the detrimental immune response in IBD is directed at an exogenous antigen (an as yet unknown intestinal microflora), it is to a great extent similar to the autoimmune response in experimental autoimmune encephalomyelitis (EAE), induced by the injection of myelin proteins. In that case as well, it has been established that the disease is mediated by pro-inflammatory Th1 cells and its course may be altered by agents which lead to a shift from the Th1 to Th2 phenotype [4], [5].

The synthetic copolymer glatiramer acetate (GA, Copaxone, copolymer 1), an approved drug for the treatment of multiple sclerosis, is very well tolerated with a high safety profile and is highly effective in the prevention and suppression of EAE [6]. Studies on the mechanism of action of GA revealed that it exerts its therapeutic activity by immunomodulating the immune response at different levels of specificity. Thus, it has been demonstrated that GA binds promiscuously with high affinity to various class II major histocompatibility (MHC) molecules of murine and human origin and can even displace antigens from the MHC antigen-binding groove [7]. This competition for MHC binding can hinder the presentation of other antigens and consequently lead to inhibition of various pathological effector functions. In addition, GA was shown to be a potent inducer of Th2/3 cells that secrete high amounts of regulatory substances, such as IL-10 and TGF-β [8]. Moreover, GA treatment leads to deviation of the immune reactivity from a Th1- to Th2-biased cytokine profile in both experimental animals and humans [9].

In view of these immunomodulating activities of GA and the Th1-related immunopathological nature of IBD, we have investigated whether GA can be effective in the suppression of IBD animal models. Indeed, we have recently demonstrated that GA treatment ameliorates very significantly the various pathological manifestations in several models of IBD, including trinitriobenzene sulfonic acid (TNBS)-induced colitis [10], dextran sulfate sodium (DSS)-induced colitis of different severity levels and a spontaneous IBD model in transgenic mice [11]. In these IBD models it was demonstrated that the detrimental pro-inflammatory response manifested by cell proliferation as well as TNF-α and IFN-γ expression was modulated by GA, whereas the regulatory anti-inflammatory TGF-β and IL-10 response was elevated.

Our more recent studies on the mechanism of action of GA in the suppression of EAE have revealed that GA-specific T-cells can be isolated from the brains of mice treated with this agent [12], [13]. Furthermore, its beneficial effect on EAE can be achieved not only by GA treatment but also by adoptive transfer of specific T-cells originating in mice immunized with GA. Upon transfer to EAE-induced mice, these cells migrated specifically to the target organ, namely the brain, and secreted in situ the regulatory beneficiary cytokines IL-10 and TGF-β [14]. In view of the therapeutic consequence of GA in colitis models, it was of interest to explore whether a similar mechanism of action is effective in IBD, and whether in this case adoptive transfer of GA-specific cell would lead to their homing to the relevant target organ, namely the colon. In the present study we report that indeed, GA-induced lymphocytes drastically reduced disease manifestations. Furthermore, GA-specific short-term cell lines, either exogenously labeled or genetically marked, adoptively transferred intraperitoneally to colitis-inflicted mice, undergo localization into the inner layers of the colon and secrete in situ the anti-inflammatory cytokine TGF-β, thus inducing immunomodulation at the site in which the pathological process occurs.