P164 EPITHELIAL TLR4-INDUCED DYSBIOSIS INCREASES SUSCEPTIBILITY TO TUMORIGENESIS
Authors: Burgueno Juan , Fritsch Julia , Santander Ana , Brito Nivis , Fernandez Irina , Pignac-Kobinger Judith , Conner Gregory , Abreu Maria 
Published: January 2020

Abstract

Background
Toll-like receptor 4 (TLR4), the receptor for gram negative lipopolysaccharide, is upregulated in biopsies of ulcerative colitis and colorectal cancer (CRC) patients, particularly in intestinal epithelial cells (IECs). We have demonstrated that activation of TLR4 promotes redox activity in IECs and induces susceptibility to colitis, colitis-associated cancer (CAC) and CRC. Furthermore, we have shown that TLR4-induced susceptibility to colitis is transmissible by simple cohousing, suggesting that the microbiota plays a key role in TLR4-driven effects. In this study, we aimed to determine the involvement of the redox potential and the microbiota in TLR4-induced susceptibility to tumorigenesis. We hypothesized that TLR4-induced dysbiosis and redox activity are responsible for the pro-tumorigenic effects of this receptor.

Methods
Villin-TLR4 mice (which express a constitutively active form of TLR4 in IECs) and their WT littermates underwent a model of CAC by administration of azoxymethane followed by 2 cycles of 1.5–3% dextran sulfate sodium. Mucosa-associated microbiota of villin-TLR4 and WT mice in steady state conditions was used to colonize WT germ-free mice. Three weeks after engraftment, WT recipient mice underwent the same model of CAC with a supplementary 3rd cycle of dextran sulfate sodium. Additional villin-TLR4 mice and their WT littermates were rederived into germ-free conditions and underwent a model of CRC by repeated administration of azoxymethane. Tumor burden, size and epithelial H2O2 production (Amplex red) were measured at the end of each experiment.

Results
Activation of epithelial TLR4 in villin-TLR4 mice enhanced the formation of tumors in the CAC model, which was accompanied by a significant increase in the epithelial production of H2O2 when compared to that of steady state conditions and that of their WT littermates. Recipient mice of villin-TLR4 mucosa-associated microbiota developed an increased number of tumors of bigger size when compared to recipient mice receiving a WT microbiota. Consistently, IECs isolated from non-involved areas demonstrated higher release of H2O2 in villin-TLR4 microbiota-recipient mice, suggesting that dysbiosis increases susceptibility to CAC by promoting redox activity in the mucosa. 100% of villin-TLR4 mice (5/5) undergoing the CRC model developed tumors when housed in conventional conditions, whereas only 12.5% of villin-TLR4 mice (1/8) housed in germ-free conditions did. Epithelial production of H2O2 was slightly higher in germ-free villin-TLR4 mice. No WT mice developed tumorigenesis in this model.

Conclusions
These results demonstrate a key role for the TLR4-induced microbiota in tumor development. Furthermore, they suggest that increased epithelial redox activity does not play a direct pro-tumorigenic role, but instead might participate in modifying the microbial community in villin-TLR4 mice. Our results suggest that overexpression of TLR4 during CRC contributes to dysbiosis and therefore facilitates tumor initiation and progression.