Sex-dependent effects of intestinal epithelial TLR4 deletion induced before activity-based anorexia

RationaleA role for the microbiota-gut-brain axis in the pathophysiology of anorexia nervosa (AN) has emerged in the last decade. An alteration of intestinal Toll-like receptor type 4 (TLR4) has been reported in the activity-based anorexia (ABA) model with an increase in its expression at the cell surface of colonic epithelial cells. In addition, inducible TLR4 invalidation in intestinal epithelial cells (IECs) was associated with behavioral and energy balance changes in ABA mice. The aim of this study was to assess the intestinal response, e.g. inflammation, gut barrier function and gut microbiota composition, to TLR4 invalidation in IEC in ABA mice. MethodsMale and female Villin-CreERT2-TLR4 LoxP C57Bl/6 mice were injected with tamoxifen to induce a specific invalidation of TLR4 in IECs (TLR4IEC-/- mice). Then, wild-type (wt) and TLR4IEC-/- mice were subjected or not to the ABA protocol which combines an access to a running wheel and a progressively limited access to food. After 12 days, colon samples were collected and the expression of 44 mRNAs encoding proteins involved in inflammatory response, gut barrier function and homeostatic regulation was measured by qPCR. Results were compared by a two-way ANOVA (ABA x TLR4IEC-/-). Gut microbiota composition was analysed by 16S rRNA Illumina sequencing. ResultsIn both male and female ABA TLR4IEC-/- mice, the kinetics of body weight loss was slowed down. In addition, male and female ABA TLR4IEC-/- mice showed an increase and a decrease in food intake, respectively. In males, TLR4 invalidation in IEC was associated with a reduction of Tlr2, Ticam1, Myd88, Tnf, I{kappa}B, Irf3, Cxcr3 and Tgf{beta} mRNA expression and fecal calprotectin levels under control conditions but not in response to the ABA model. In females, Myd88, Il6, Cxcl1 and Ccl2 mRNA levels were increased by TLR4IEC invalidation in control mice but not in ABA, except for Ccl2. TLR4 invalidation also affected the expression of genes involved in gut barrier function in control and ABA mice in a sex-dependent manner. Male mice exhibited more marked alterations. For instance, male CT TLR4IEC-/- showed a decrease of numerous targets (Ocln, Marveld2, F11r, Tjp1, Cldn7, Cldn12, Cldn15). ABA TLR4IEC-/- mice did not exhibit this decrease but other changes were observed such as an increase in Cldn3 and Cldn7 mRNA levels. Finally, TLR4IEC invalidation in control mice, but not in ABA, altered the gut microbiota in a sex dependent manner with an increase in the abundance of Parasutterella and Desulfovibrio genera in females and males, respectively. Interestingly, the ABA model per se induced an increase in the abundance of the Lactobacillus genus in both sexes, which was not observed in ABA TLR4IEC-/-. ConclusionsOur study shows for the first time the impact of inducible TLR4 invalidation in IEC on the intestinal response. We highlighted numerous colonic alterations regarding epithelial permeability, mucosal inflammation and gut microbiota composition, in control and ABA conditions: all were partially reversed in ABA TLR4IEC-/-. TLR4 invalidation in IEC also induced changes in energy homeostasis in response to the ABA model both in female and male mice. Further studies are warranted to deeply evaluate the underlying mechanisms.