Chronic intestinal inflammation occurring in IBD induces persistent damage and enhanced mucosal permeability along the gastrointestinal tract, playing an important role in the development of colorectal cancer (CRC) 
. Manipulation of colonic bacteria with probiotics has been shown to be effective in the regulation of gut homeostasis in part through production of bacterial metabolites 
. VSL#3 probiotic bacteria can produce CLA locally in the gut, a compound that down-modulates inflammatory responses by activating PPAR γ 
. In addition, CLA prevents or ameliorates experimental IBD in mice and pigs 
. Moreover, PPAR γ agonists have shown clinical efficacy against human UC 
. Recently, we demonstrated that the probiotic mixture VSL#3 suppresses intestinal inflammation by producing CLA locally in the colon and activating PPAR γ in macrophages 
. This study aimed to investigate the mechanisms of CLA and VSL#3 in mouse models of colitis-associated CRC.
The clinical response in the AOM/DSS model had a bimodal distribution in which the first peak corresponded to acute inflammation due to the DSS challenge followed by a period of clinical recovery characterized by lower chronic inflammation. The second peak of disease appeared on day 48 corresponding to the tumor formation. This pattern is consistent with results of previous studies from our group that characterized the anti-carcinogenic properties of CLA 
. CLA and VSL#3 treated mice also showed lower macroscopic inflammation-related lesions and significantly improved colonic histopathology in both experimental models. Nevertheless, while VSL#3 was the compound that prevented tumor formation most effectively, it did not prevent leukocyte infiltration in comparison to the other groups in the azoxymethane-induced colorectal cancer model, suggesting a possible role of infiltrating leukocytes in the anti-carcinogenic actions of VSL#3. In a follow up study, using a different model of colitis-associated colonic carcinogenesis, IL-10−/− mice in a 129/SvEv background were infected with H. typhlonius
, thus accelerating the colonic inflammation that the IL10−/− develop spontaneously and promoting colonic carcinogenesis 
. In this model, both VSL#3 and CLA ameliorated disease severity but only the probiotic mixture was effective in reducing inflammation-related lesions in MLN, spleen and colon. This consistency of results in both models demonstrates that the efficacy of VSL#3 in restoring mucosal homeostasis and attenuating colitis-associated CRC is not model-dependent.
Our gene expression analyses showed an upregulation of colonic TNF-α in CLA and VSL#3-treated mice when compared to the untreated control group, which may be indicative of immunostimulation and enhanced epithelial healing ability. Most notably, TNF-α exerts potent antitumoral effects by stimulating immune responses, including upregulation of human leukocyte antigen antigens in tumor cell surfaces 
, enhanced cytotoxicity 
stimulation of cytotoxic T cells and natural killer (NK) cells 
as well as epithelial healing 
. In addition, CLA-treated mice showed a significantly reduced expression of cyclooxygenase-2 (COX-2), an enzyme involved in arachidonic acid cascade and prostaglandin-mediated inflammation. Previous studies have suggested the suppressive effects of CLA on colon carcinogenesis due to changes in this cascade and in the activation of PPAR γ, both mechanisms involving the inhibition of COX-2 expression and inducing apoptosis 
. The levels of angiostatin, a proteolytic fragment of plasminogen and an endogenous inhibitor of angiogenesis 
, were greater in the VSL#3 group when compared to control fed mice, suggesting that the anti-carcinogenic actions of VSL#3 may be mediated, in part, by suppressed angiogenesis. As shown in previous studies 
, this inhibitory behavior of angiostatin on angiogenesis could be indirectly related with anti-carcinogenic efficacy. Our findings extend the findings of a recent report demonstrating that probiotic VSL#3 can attenuate chronic inflammation in rats, delaying the transition from inflammation to dysplasia and cancer 
The ability of VSL#3 and CLA to modulate immune responses and prevent CRC was also assessed by examining the distribution of immune cell subsets at the colonic mucosa and systemically. VSL#3 treatment enhanced the percentages of IL-17-expressing CD4+ T cells in the mucosal inductive site (i.e., MLN) and Foxp3-expressing CD4+ T cells in the effector site in mice with CRC, suggesting a possible role in modulating the plasticity between Th17 and Treg in the MLN and colonic LP. Results from a recent study identified segmented filamentous bacteria in the gut commensal microbiota as inducers of the Th17 polarization in the gut mucosa 
. It is tempting to speculate that changes in the composition of the colon microbiome triggered by probiotic bacteria may exert similar effects. In conclusion, our data demonstrate the ability of CLA and VSL#3 to ameliorate inflammation-induced colorectal cancer through a mechanism involving modulation of mucosal CD4+ T cell polarization and modulation of gene expression.