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

## TL;DR

Removing TLR4 in intestinal cells of mice caused sex-specific changes in gut health and microbes, but these effects disappeared under an anorexia model.

## Contribution

First study to show sex-specific gut and microbiome changes due to TLR4 knockout in an anorexia model.

## Key findings

- TLR4 knockout in male mice reduced inflammation and tight junction proteins but increased specific gut microbes.
- Female mice showed increased inflammation markers and different microbial changes compared to males.
- ABA model reversed TLR4 knockout effects on gut barrier and Lactobacillus abundance in both sexes.

## Abstract

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 (TLR4IEC−/−) was subsequently associated with behavioral and energy balance changes in ABA mice. Our study aimed to assess the intestinal response to TLR4IEC−/− 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, TLR4IEC−/− 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 TLR4IEC−/− females showed increased expression of four inflammatory markers and one target involved in the gut barrier. The levels of the Bacillota phylum and the Deltaproteobacteria class and their subdivisions, up to the Desulfovibrio genus, increased in the control TLR4IEC−/− males compared to wt. In females, only an increase in the Alcaligenaceae genus, which ranks from the Betaproteobacteria phylum, was observed. Interestingly, in both males and females, these alterations were not observed in response to ABA model in TLR4IEC−/− mice. Similarly, ABA increased Tjp1 expression and Lactobacillus abundance, both of which were decreased by TLR4IEC−/−. Our study shows for the first time the impact of inducible TLR4IEC−/− on the intestinal response. TLR4IEC−/− 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.