A new therapeutic association to manage relapsing experimental colitis: Doxycycline plus Saccharomyces boulardii

Abstract

Immunomodulatory antibiotics have been proposed for the treatment of multifactorial conditions such as inflammatory bowel disease. Probiotics are able to attenuate intestinal inflammation, being considered as safe when chronically administered. The aim of the study was to evaluate the anti-inflammatory effects of doxycycline, a tetracycline with immunomodulatory properties, alone and in association with the probiotic Saccharomyces boulardii CNCMI-745. Doxycycline was assayed both in vitro (Caco-2 epithelial cells and RAW 264.7 macrophages) and in vivo, in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis and the dextran sodium sulfate (DSS) model of mouse colitis. In addition, the anti-inflammatory effect of the association of doxycycline and the probiotic was evaluated in vitro and in vivo in a DSS model of reactivated colitis in mice. Doxycycline displayed immunomodulatory activity in vitro, reducing IL-8 production by intestinal epithelial cells and nitric oxide by macrophages. Doxycycline administration to TNBS-colitic rats (5, 10 and 25 mg/kg) ameliorated the intestinal inflammatory process, being its efficacy comparable to that previously showed by minocycline. Doxycycline treatment was also effective in reducing acute intestinal inflammation in the DSS model of mouse colitis. The association of doxycycline and S. boulardii helped managing colitis in a reactivated model of colitis, by reducing intestinal inflammation and accelerating the recovery and attenuating the relapse. This was evidenced by a reduced disease activity index, colonic tissue damage and expression of inflammatory mediators. This study confirms the intestinal anti-inflammatory activity of doxycycline and supports the potential use of its therapeutic association with S. boulardii for the treatment of inflammatory bowel diseases, in which doxycycline is used to induce remission and long term probiotic administration helps to prevent the relapses.

Introduction

Different antibiotics have been described to comprise within their therapeutic effects the modulation of the immune response [1]. This is of increasing interest in the pharmacological treatment of diseases where infectious and inflammatory factors converge [2], [3], [4], [5]. However, the potential of immunomodulatory antibiotics in managing intestinal conditions, such as inflammatory bowel disease (IBD) [6], [7], [8], is poorly documented.

IBD pathogenesis is characterized by a genetic predisposition and an aberrant intestinal immune response to environmental factors [9]. Among the latter, an imbalance on the intestinal microbiota (dysbiosis) seems to play a key role [8], [10], [11], [12], [13], [14], [15], [16]. Despite this, IBD therapy has been mainly focused on suppressing the immune system rather than on restoring the composition of the altered microbiota, which can be achieved by the administration of probiotics, prebiotics or antibiotics [8]. Since no causative pathogen has been specifically identified, broad-spectrum antibiotics have shown the best results [17].

Semi-synthetic second generation tetracyclines, such as doxycycline and minocycline, have been reported to exert immunomodulatory activities in addition to their antimicrobial properties [2], [18]. Both compounds have been shown to possess anti-apoptotic, immunosuppressive and anti-inflammatory properties in different inflammatory diseases [19]. The mechanisms behind these effects comprise inhibition of different enzymes involved in inflammatory processes, such as nitric oxide synthase, phospholipase A2 and matrix metalloproteinases; scavenging of oxygen free radicals, and regulation of immune cell activation and proliferation [20]. Considering all this, second generation tetracyclines seem a promising approach for the treatment of IBD. In fact, minocycline's intestinal anti-inflammatory effects have been previously well described in different models of experimental colitis [21], [22], [23]; however, doxycycline has not been fully tested in these intestinal conditions [24].

The aim of this study was to determine if the intestinal anti-inflammatory activity ascribed to minocycline is a property shared by doxycycline, thus broadening the therapeutic choice for the treatment of IBD. For this purpose, we first compared the immunomodulatory properties of doxycycline to those displayed by minocycline in vitro. Likewise, the in vivo intestinal anti-inflammatory effect of doxycycline was tested in comparison with minocycline in the TNBS model or rat colitis [25] and confirmed in the DSS model in mice [26].

Despite the usefulness of antibiotics for the treatment of IBD [27], several studies have reported that discontinuation of antibiotic therapy results in a high relapse rate, and long-term antibiotic therapy is associated to increased risk of drug side effects and antibiotic resistance [28], [29]. In this case, remission could be maintained by the administration of probiotics, which have been previously reported to exert beneficial effects in IBD due to their immunomodulatory properties [30], [31]. Yeasts are commonly used in combination with doxycycline, remaining their growth, phenotype or functions unaffected by the antibiotic [32]. In particular, the probiotic Saccharomyces boulardii CNCMI-745 has been shown to modulate the gut immune response, inducing intestinal homeostasis, which supports its effectiveness in intestinal inflammatory states [33], [34]. For this reason, we evaluated the association of doxycycline and S. bourlardii both in vitro and in vivo, in a model of reactivated colitis in mice, as a therapeutic strategy aimed at inducing remission and preventing the onset of symptoms after antibiotic discontinuation [23].