Serine protease inhibitors protect better than IL-10 and TGF-β anti-inflammatory cytokines against mouse colitis when delivered by recombinant lactococci

Bermdez-Humarn et al. Microbial Cell Factories Serine protease inhibitors protect better than IL-10 and TGF- anti-inflammatory cytokines against mouse colitis when delivered by recombinant lactococci Luis G Bermdez-Humarn 0 1 Jean-Paul Motta Camille Aubry 0 1 Pascale Kharrat 0 1 Laurence Rous-Martin Jean-Michel Sallenave Cline Deraison Nathalie Vergnolle Philippe Langella 0 1 0 AgroParisTech , UMR1319 Micalis, F-78350 Jouy-en-Josas , France 1 INRA, Commensal and Probiotics-Host Interactions Laboratory , UMR 1319 Micalis, F-78350 Jouy-en-Josas , France Background: Different studies have described the successful use of recombinant lactic acid bacteria (recLAB) to deliver anti-inflammatory molecules at the mucosal level to treat Inflammatory Bowel Disease (IBD). Methods: In order to identify the best strategy to treat IBD using recLAB, we compared the efficacy of different recombinant strains of Lactococcus lactis (the model LAB) secreting two types of anti-inflammatory molecules: cytokines (IL-10 and TGF-1) and serine protease inhibitors (Elafin and Secretory Leukocyte Protease Inhibitor: SLPI), using a dextran sulfate sodium (DSS)-induced mouse model of colitis. Results: Our results show that oral administration of recombinant L. lactis strains expressing either IL-10 or TGF-1 display moderate anti-inflammatory effects in inflamed mice and only for some clinical parameters. In contrast, delivery of either serine protease inhibitors Elafin or SLPI by recLAB led to a significant reduction of intestinal inflammation for all clinical parameters tested. Since the best results were obtained with Elafin-producing L. lactis strain, we then tried to enhance Elafin expression and hence its delivery rate by producing it in a L. lactis mutant strain inactivated in its major housekeeping protease, HtrA. Strikingly, a higher reduction of intestinal inflammation in DSS-treated mice was observed with the Elafin-overproducing htrA strain suggesting a dose-dependent Elafin effect. Conclusions: Altogether, these results strongly suggest that serine protease inhibitors are the most efficient anti-inflammatory molecules to be delivered by recLAB at the mucosal level for IBD treatment. Elafin; Proteases; Inflammation; Colitis; Inflammatory bowel disease; Lactococcus lactis; Probiotics - Background Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders that cause inflammation of the digestive tract. The two major forms of IBD are Crohns Disease (CD) and Ulcerative Colitis (UC) and they are characterized by an uncontrolled inflammatory response to lumenal content [1]. Despite the fact that several antiinflammatory molecules have been tested in preclinical and clinical models for IBD treatment, their therapeutic potential and clinical application have been frequently hampered by different obstacles such as successful delivery or even toxic side-effects [1]. In the last 10 years, an increasing number of clinical and experimental studies have proven that probiotic bacteria may counteract the chronic inflammatory process. This effect is achieved by stabilizing the gut microbial environment and permeability barrier functions and by modulating the microbiota composition [2,3]. In addition, the successful use of food-grade Lactic Acid Bacteria (LAB) for the oral delivery of anti-inflammatory molecules to the inflamed intestine in preclinical experiments, as well as clinical trials have been reported [4-10]. This approach is based on the local synthesis and delivery of therapeutic molecules by viable recombinant LAB (recLAB) in situ. The pioneer use of such recLAB for the prevention and treatment of experimental IBD was performed by Steidler et al. [11] who developed a recombinant strain of Lactococcus lactis (the LAB model) secreting biologically active anti-inflammatory cytokine IL-10. Interestingly, the authors showed that daily oral administration of L. lactis IL-10 in mice resulted in ~50% reduction in dextran sulfate sodium (DSS)-induced colitis [11]. The beneficial effect of L. lactis IL-10 strain was dependent on the in situ secretion of IL-10 by recombinant live lactococci. Steidler et al. have then developed the first biocontainment system for L. lactis IL-10 strain to start the first human clinical study using it [12]. A phase I clinical trial was then conducted with this biocontained L. lactis IL-10 strain in Crohns disease patients, showing that the containment strategy was effective [13]. Following this, a phase IIA trial was performed and a press release was published in 2009 revealing that all three primary endpoints have been met: i) safety and tolerability; ii) environmental containment and iii) assessment of biomarkers associated with the strain (data from ActoGeniX press release). Unfortunately, the clinical results did not reveal a statistically significant difference in mucosal healing with L. lactis IL-10 versus placebo. In view of these results, one can wonder whether IL-10 was the righ (...truncated)