PPARα exacerbates Salmonella Typhimurium infection by modulating the immunometabolism and macrophage polarization

Abstract

Salmonella enterica serovar Typhimurium (STm) is a causative pathogen for robust inflammatory gastrointestinal disease and can lead to systemic infection. Eicosanoids, bioactive lipid mediators, play a crucial role in modulating both the induction and resolution of inflammatory responses during an infection. A subset of eicosanoids activates PPARs, nuclear receptor/transcription factors that regulate fatty acid metabolism, lipid body formation, and macrophage function. In this study, we determined that mice lacking PPARα exhibited reduced inflammatory hallmarks of STm infection, including lower inflammatory gene expression, cecal inflammation, and bacterial dissemination, along with a significant increase in cecal eicosanoid metabolism compared to wildtype C57BL/6 mice. In macrophages, STm favored M2b-polarized macrophages for intracellular infection, leading to reduced arachidonic acid and ceramide production. Inhibition of fatty acid oxidation via Etomoxir in STm-infected macrophages reduced bacterial burdens and promoted cell death. In Etomoxir-treated wildtype mice, STm infection increased ceramide production, decreased inflammatory gene expression in the cecum, and increased the number of STm-containing M1 macrophages in mesenteric lymph nodes. These findings revealed a novel role for the lipid-immune signaling axis in Salmonella infections, providing significant insights into the lipid-mediated regulation of inflammation during bacterial infections in the gut.

Keywords: Salmonella Typhimurium; eicosanoids; fatty acids; lipidomic analysis; macrophages.

Figure 1.

Ppara^—/— mice exhibit increased resistance to STm-induced inflammation. Male mice (n = 4-7 per group) were infected with 10⁸ GFP-expressing STm (or LB media only) via oral gavage 24-hrs following oral streptomycin treatment. Cecum, liver, and spleenwere collected for analysis 48 hours post infection. (a) Colony forming units (CFU) were quantified in the liver and spleen of infected animals. Bar graph represents the mean and standard error of the mean (SEM). (b) RNA was extracted from cecum homogenate and TaqMan Primer/Probe sets were used for q-PCR. Relative gene expression represents normalization to the housekeeping gene Eif1a. (c&d) Histopathology with H&E staining and inflammatory pathology scores of formalin-fixed cecum tissue. (e) Flow cytometry analysis of M1 and M2 macrophages from MLN single-cell suspensions. Bar graphs represent the mean ± SEM. 2-way ANOVA was performed to determine statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < .0001). Data are representative of three independent experiments.

Figure 2.

Eicosanoid profiling shows increased lipid metabolism in Ppara^—/— mice during STm infection. Male mice (n = 4-7 per group) were infected with 10⁸ GFP-expressing STm (or LB media only) via oral gavage 24-hrs following oral streptomycin treatment. Cecum was collected and analyzed 48-hrs post-infection. Eicosanoids were extracted from cecum homogenate via solid-phase extraction and analyzed by LC-MS. (a) Heat map of eicosanoids organized by enzyme families (COX, Cyclooxygenase; LOX, Lipoxygenase; CYP, cytochrome P450; DHA, docosahexaenoic acids; EPA, eicosapentaenoic acids; Lino, linoleic acids). Due to the variability in concentrations across different metabolites, the heatmap displays the mean normalized levels, with each lipid species scaled to its individual sample maximum (normalized to 100). (b) Average relative intensity of individual eicosanoid species under different conditions. Bar graphs represent the mean ± SEM. Relative intensity represents concentration. Statistical significance was determined using 2-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < .0001). Figures are representative of three independent experiments.

Figure 3.

STm infection in M2b-polarized macrophages influences bacterial burden, lipid body production, and eicosanoid production. Bone marrow-derived macrophages from C57BL/6 and Ppara^—/— mice were treated with DMSO (naïve, M0) or polarized to M2b for 48hrs, followed by infection with STm for 90 minutes and subsequent gentamicin treatment for 90 minutes. (a-c) Fluorescence microscopy analysis (a, b) and representative images (c) showing internalized STm (a) and lipid body count (b) per macrophage. (d) Average relative intensity of individual arachidonic acid and fatty acid species under different conditions. Bar graphs represent the mean ± SEM. Statistical significance was determined using 2-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < .0001). Data are representative of three independent experiments.

Figure 4.

Etomoxir induces cell death, reduces bacterial burden, and enhances anti-bacterial activity in STm-infected wildtype macrophages. (a) Schematic representation of PPARα-mediated long-chain fatty acid (LCFA) uptake leading to fatty acid oxidation (FAO) via CPT–1 or cell death via ceramide accumulation. PPARα activation promotes the cellular uptake of long-chain fatty acids (LCFAs), which are converted to LCFA. These are either used for fatty acid oxidation (FAO), contributing to M2 macrophage polarization, or converted to ceramides, whose accumulations promotes cell death. Inhibition of CPT–1 blocks FAO, shifting the conversion of LCFAs toward ceramide production. Created with BioRender. (b–f) C57BL/6 hox-derived macrophages were treated with DMSO (vehicle control) or Etomoxir for 1 hr, then infected with STm at MOI 20 for 3hrs (e) or 90 mins, followed by gentamicin treatment in fresh media for 90 mins (b–c, f). Fluorescence microscopy analysis (b-c,e) and representative images (d) of internalized STm (b), lipid body dynamics (c) and cell death (e). (e) Microscopy analysis of cell death measured by the percentage of propidiumiodide-stained nuclei (dead) relative to Hoechst-stained nuclei (total). (f) RNA was extracted from macrophages and TaqMan Primer/Probe sets were used for q-PCR. Relative gene expression was normalized to housekeeping gene Eif1a. Bar graphs represent the mean ± SEM. Statistical significance was determined using 2-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p <.0001). Figures are representative of three independent experiments.

Figure 5.

Etomoxir treatment of STm-infected mice increases ceramide production, reduces inflammatory gene expression, and increases PC and PGE2 levels in cecal tissue. Male mice (n = 4-10 per group) were infected with GFP-expressing STm via oral gavage 24-hours following oral streptomycin treatment. Where applicable, mice were administered etomoxir in PBS with 10% DMSO via i.P. injection at the time of infection and 24-hrs post-infection. Cecum and mesenteric lymph nodes (MLNs) were collected and analyzed 48-hrs post-infection. Bar graphs represent mean and SEM. (a, b) Cecum pathology scoring and representative images for formalin-fixed, H&E-stained cecum sections. (c) RNA was extracted from cecal homogenate, and TaqMan Primer/Probe sets were used for q-PCR. Relative gene expression was normalized to housekeeping gene Eif1a. Bar graphs depicting the mean ± SEM. (d) Average relative intensity of ceramide in cecal tissue. (e, f) Average relative intensity of phosphatidylcholine (PC) and oxidized PC (OxPC) (e) and PGE2 (f) in cecal tissue. (g) Flow cytometry analysis of M1 and M2 macrophages from MLN single-cell suspensions. Bar graphs represent the mean ± SEM. Statistical significance was determined using one-way or 2-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < .0001). Figures are representative of three independent experiments.