Intestinal epithelial TLR4 knock out induces sex-specific effects on gut barrier and microbiome in an activity-based anorexia model

The role of the microbiota‒gut‒brain axis in the pathophysiology of anorexia nervosa has emerged in recent decades. Increased expression of Toll-like receptor 4 (TLR4) has been reported in the intestinal epithelial cells (IEC) of activity-based anorexia (ABA) mice. The inducible TLR4 knockout in IEC (TLR4^IEC^-/-) was subsequently associated with behavioral and energy balance changes in ABA mice. Our study aimed to assess the intestinal response to TLR4^IEC^-/- in both male and female ABA mice by focusing on three components: inflammation, the gut barrier, and the gut microbiota composition. After 12 d of undernutrition with free wheel access, the colonic expression of 43 markers was measured by RT-qPCR. The gut microbiota composition was analyzed by Illumina sequencing of the 16S rRNA gene. First, TLR4^IEC^-/- was associated with more marked alterations in male control mice compared to females. Indeed, a reduction in the mRNA expression of eight inflammatory factors, seven tight junction proteins and fecal calprotectin levels was observed in males. Control TLR4^IEC^-/- females showed increased expression of four inflammatory markers and one target involved in the gut barrier. The levels of the <i>Bacillota</i> phylum and the <i>Deltaproteobacteria</i> class and their subdivisions, up to the <i>Desulfovibrio</i> genus, increased in the control TLR4^IEC^-/- males compared to wt. In females, only an increase in the <i>Alcaligenaceae</i> genus, which ranks from the <i>Betaproteobacteria</i> phylum, was observed. Interestingly, in both males and females, these alterations were not observed in response to ABA model in TLR4^IEC^-/- mice. Similarly, ABA increased <i>Tjp1</i> expression and <i>Lactobacillus</i> abundance, both of which were decreased by TLR4^IEC^-/-. Our study shows for the first time the impact of inducible TLR4^IEC^-/- on the intestinal response. TLR4^IEC^-/- induced sex-specific colonic alterations and changes in the gut microbiota, which disappeared after the ABA model. Further studies are warranted to decipher the underlying mechanisms.