Recommendations for Improved Use of the Murine TNBS-Induced Colitis Model in Evaluating Anti-inflammatory Properties of Lactic Acid Bacteria: Technical and Microbiological Aspects

Abstract

Probiotic bacteria have been shown to exert promising beneficial effects in different types of intestinal disorders, including chronic inflammation. In this context, animal models of inflammatory bowel disease are useful in studying the possible prophylactic role of candidate probiotic strains. This study aimed at evaluating the critical technological and microbiological parameters as well as the robustness of the murine trinitrobenzene sulfonic acid (TNBS)-induced model of colitis, after intragastric administration of lactic acid bacteria (LAB) preparations. A standardized methodology was applied to assess the protective effect achieved by various bacterial concentrations and culture conditions of the reference strain Lactobacillus plantarum NCIMB 8826. Not only was protection found to vary in function in different levels of colitis, but also repeated experiments showed a clear bacterial dose-dependent attenuation of colitis. The physiological stage of bacteria was shown to impact as well, with substantial, mild, or reduced improvement of inflammatory scores for exponentially growing, stationary-phase, or killed bacteria, respectively. A recombinant strain, secreting murine interleukin-10 (IL-10) and previously reported to successfully treat colitis in two different models of murine colitis (dextran sulfate sodium [DSS] and IL-10-deficient mice), was used to validate the final experimental conditions. In conclusion, we identified and optimized some of the key parameters that need to be controlled in order to ensure reliable comparison of results generated over a long period of time or independent experiments. The recommendations for an improved model presented here will prove to be helpful for reproducible, independent comparison of the anti-inflammatory potential of wild-type or recombinant candidate probiotic strains, whether administered as pure cultures or as blends.

Anti-Inflammatory Properties of Streptococcus salivarius, a Commensal Bacterium of the Oral Cavity and Digestive Tract

“…Induction of colitis and inflammation scoring. A standardized murine TNBS colitis model was used in which moderate and high levels of inflammation were induced as previously described (31). Briefly, a 50-l solution of 100 or 130 mg/kg body weight TNBS (Sigma) in 50% ethanol was administered in the colon.…”

“…Three days after induction of colitis, the mice were killed, and blood samples were immediately taken and stored in heparinized tubes. After mouse dissection, 2 independent observers blindly scored the macroscopic inflammation of the colon following the Wallace scale as described previously (31). Two-centimeter-long fragments of the distal colon were collected and frozen at Ϫ80°C for histological analysis; paraffin-embedded 5-m sections stained with May-Grünwald-Giemsa stain were examined under a microscope, and tissue lesions were scored according to the Ameho criteria (32).…”

Anti-inflammatory activity of probiotic Bifidobacterium: Enhancement of IL-10 production in peripheral blood mononuclear cells from ulcerative colitis patients and inhibition of IL-8 secretion in HT-29 cells

“…IL-10 plays a key role in the control of inflammatory responses to enteric organisms [9][10][11][12] . More recently, it has been shown in animal models that probiotic strains displaying an in vitro potential to induce higher levels of the anti-inflammatory cytokine IL-10 and lower levels of the inflammatory cytokine IL-12, offer the best protection against in vivo colitis in the model [13] . A genetically engineered Lactococcus lactis thy12 producing IL-10 ameliorated colitis in two models of experimental colitis, providing proof of principal that topically delivers IL-10, can be therapeutically efficacious [14] and a recent proof-or-principle experiment using this transgenic bacterium expressing IL-10 in 10 patients with Crohn's disease showed efficacy [15] .…”

Changes in intestinal Toll-like receptors and cytokines precede histological injury in a rat model of necrotizing enterocolitis

“…Differences in the expression of TLRs may alter a host's response to a commensal or pathogenic microorganism. TLR2 signaling, produced by treatment with Lactobacillus lactis, may provide protective signaling, for example in models of inflammatory colitis (15).…”