# Butyrate Enhances Antimicrobial Defence in Chicken Macrophages Through Reactive Oxygen Species Generation and Autophagy Activation

## TL;DR

Sodium butyrate boosts chicken macrophages' ability to fight bacteria by increasing reactive oxygen species and autophagy.

## Contribution

This study reveals that butyrate enhances antimicrobial defense in chickens via ROS and autophagy activation.

## Key findings

- Butyrate priming increased bacterial clearance by 1 log in gentamicin protection assays.
- Butyrate caused a 30% ROS increase in HD11 cells and a 12% increase in BMDMs.
- Autophagy activation via mTOR inhibition was linked to butyrate's antimicrobial effects.

## Abstract

Sodium butyrate has been documented to support gut function and help control pathogens in the gastrointestinal tract. However, the precise mechanisms of dietary sodium butyrate’s control over enteric pathogens in chickens remain unclear. Our study demonstrated that priming chicken bone marrow-derived macrophages (BMDMs) or the HD11 cell line with 1 mM sodium butyrate significantly enhanced their antimicrobial capacity against key bacterial pathogens (Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, and Staphylococcus aureus) in gentamicin protection assays (p < 0.05; ≥1 log reduction in CFU/mL). This in vitro enhancement was associated with increased production of reactive oxygen species (ROS), as detected by DCFH-DA assays, showing approximately a 30% increase in HD11 cells and a 12% increase in BMDMs. Butyrate priming was observed to result in autophagy activation, potentially through mTOR pathway inhibition, evidenced by changes in related gene expression using RT-qPCR assay and a 2.5-fold increase in GFP-LC3B accumulation. Supporting this, pharmacological inhibition of ROS using the ROS scavenger N-acetyl-L-cystine (NAC) or autophagy with chloroquine reduced the butyrate-enhanced bacterial clearance. Furthermore, the mTOR inhibitor rapamycin synergized with butyrate priming, whereas the mTOR activator L-leucine counteracted enhanced antimicrobial activity. These findings offer crucial insights for improving host defence against bacterial infections and developing novel therapeutic strategies in chickens.