# Effect of antibiotics on Kupffer cell immunometabolism relative to intracellular killing of S. aureus using NAD(P)H fluorescence lifetime imaging

## TL;DR

This study explores how different antibiotics affect liver macrophages' metabolism and ability to kill Staphylococcus aureus bacteria inside cells.

## Contribution

The study reveals how antibiotics modulate Kupffer cell immunometabolism and intracellular bacterial clearance, offering new insights for improving treatment outcomes in S. aureus bloodstream infections.

## Key findings

- Ceftobiprole induces a pro-inflammatory M1 profile and shortens NAD(P)H lifetime in uninfected Kupffer cells.
- Daptomycin and tedizolid most effectively clear intracellular S. aureus and alter metabolic signatures.
- Vancomycin fails to control intracellular infection and does not alter Kupffer cell metabolism.

## Abstract

Staphylococcus aureus bacteremia (SAB) remains a significant clinical burden with high mortality rates. Kupffer cells (KCs) are integral to clearing Staphylococcus aureus (SA) from the bloodstream yet also serve as a pathogenic reservoir, driving persistence and treatment failure. KCs utilize their metabolic plasticity to coordinate successful antimicrobial responses, and this process may be therapeutically targeted. We investigated the immunometabolic effects of antistaphylococcal antibiotics (vancomycin, ceftobiprole, daptomycin, and tedizolid) on KCs relative to clearance of intracellular SA through gene expression analysis and optical metabolic imaging using fluorescence lifetime imaging microscopy. We observed differential effects of antibiotics on KC polarization and metabolic signatures, with ceftobiprole inducing the strongest pro-inflammatory M1 expression profile and shortest mean NAD(P)H fluorescence lifetime in KCs in the absence of infection, and a retained M1 expression profile but longest NAD(P)H lifetime during infection. Daptomycin and tedizolid most effectively cleared intracellular SA, induced significantly longer NAD(P)H lifetimes, and exhibited decreased M1 gene expression. Vancomycin failed to control intracellular infection showing blunted M1 expression without altering NAD(P)H lifetimes. Stratification of NAD(P)H lifetime signals revealed daptomycin significantly increased binding to PDH-like enzymes and nitric oxide synthase 2 compared to other antibiotics. These findings reveal novel insights into antibiotic modulation of KC immunometabolism during SA infection, which may influence treatment outcomes in SAB.

Staphylococcus aureus bloodstream infection is a leading cause of sepsis and is associated with up to 30% mortality. Despite treatment with guideline-recommended antibiotics, persistent bacteremia develops in one in three patients, which may be attributed in part to survival of bacteria inside liver-resident macrophages known as KCs. Using gene expression and optical metabolic profiling, we demonstrated that antistaphylococcal antibiotics differentially affect KC metabolism and function, thereby contributing to the overall killing of intracellular bacteria. Daptomycin, ceftobiprole, and tedizolid exert distinct effects on KC metabolism that correspond to effective intracellular killing and prompt resolution of inflammatory response. Vancomycin, however, did not affect KC metabolism and was unable to control bacterial growth inside the cells. These findings suggest that choosing antibiotics based on direct antimicrobial activity as well as indirect effects on host immune function could improve treatment outcomes for patients with S. aureus bloodstream infection.