Staphylococcus aureus stimulates neutrophil itaconate production that suppresses the oxidative burst

Abstract

Neutrophils are critical in the host defense against Staphylococcus aureus, a major human pathogen. However, even in the setting of a robust neutrophil response, S. aureus can evade immune clearance. Here, we demonstrate that S. aureus impairs neutrophil function by triggering the production of the anti-inflammatory metabolite itaconate. The enzyme that synthesizes itaconate, Irg1, is selectively expressed in neutrophils during S. aureus pneumonia. Itaconate inhibits neutrophil glycolysis and oxidative burst, which impairs survival and bacterial killing. In a murine pneumonia model, neutrophil Irg1 expression protects the lung from excessive inflammation but compromises bacterial clearance. S. aureus is thus able to evade the innate immune response by targeting neutrophil metabolism and inducing the production of the anti-inflammatory metabolite itaconate.

Keywords: CP: Immunology; CP: Microbiology; NADPH oxidase; Staphylococcus aureus; itaconate; neutrophils; oxidative burst; pathogenesis; pneumonia.

Figure 1.. Itaconate impairs bacterial clearance during S. aureus lung infection

(A) Relative abundance of metabolites in the bronchoalveolar lavage (BAL) fluid of mice infected with S. aureus (LAC) compared with mock-infected mice (PBS). (B) Itaconate in the BAL fluid of LAC-infected mice over the course of infection. (C–F) Bacterial colony-forming units (CFUs) (C), neutrophils (D), monocytes (E), and alveolar macrophages (F) in the BAL fluid and lungs of WT and itaconate-deficient (Irg1^−/−) mice 24 h after infection. Data are shown as mean ± SEM from n = (A and B) 3 mice or (C–F) 9–12 mice. Statistics are from Student’s t test; *p < 0.05.

Figure 2.. Neutrophils are the main source of itaconate during S. aureus lung infection

(A) Uniform manifold approximation and projection (UMAP) representation of all cell clusters in scRNA-seq data from the lungs of WT and Irg1^−/− mice 24 h after infection with S. aureus (LAC). (B) Relative abundance of different scRNA-seq cell populations in LAC-infected WT and Irg1^−/− mice. (C) UMAP representation of Irg1 expression in all cell clusters of LAC-infected WT mice. (D) Irg1 expression (qRT-PCR) in neutrophils after LAC infection. (E) Itaconate accumulation in the supernatant of neutrophils during LAC infection. (F and G) Neutrophils (F) and bacterial CFUs (G) in the BAL fluid and lung tissue of neutrophil-depleted (+αLy6G) and control mice 24 h after LAC infection. (H) Itaconate in the BAL fluid of neutrophil-depleted (+αLy6G) and control mice 24 h after lung infection. Data are shown as mean ± SEM from n = (D) 5 mice (3 replicates/mouse), (E) 3 mice (3 replicates/mouse), or (G–I) 6–7 mice. Statistics are from multiple t test with false discovery rate (FDR) correction; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3.. Itaconate impacts neutrophil survival and recruitment during infection

(A) Glycolysis, TCA cycle, and electron transport chain scores in the scRNA-seq neutrophil populations from WT and Irg1^−/− mouse lungs 24 h after LAC infection. (B) Neutrophil glycolytic activity (measured by extracellular acidification rate [ECAR]) during LAC infection in the presence of 0 or 5 mM itaconate; glucose was added at the first dotted line, oligomycin at the second, and 2-deoxyglucose at the third. (C) Neutrophil necroptosis and apoptosis scores in the scRNA-seq neutrophil populations. (D) Neutrophil cell death during LAC infection or mock-infection (PBS) in the presence of 0, 1, or 5 mM itaconate. (E) Enriched gene sets in the lung epithelial cell populations of LAC-infected WT vs. Irg1^−/− mice. (F) Neutrophil recruitment cytokines in the BAL fluid of WT or Irg1^−/− mice 24 h after LAC infection or mock infection. Data are shown as mean ± SEM from n = (B) 3 mice (3 replicates/mouse), (D) 4 mice (3 replicates/mouse), or (F) 14 mice. Statistics are from (A, C, and F) Student’s t test, (B) multiple t test with FDR correction, or (D) one-way ANOVA for each time point; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.. Itaconate inhibits bacterial killing by suppressing neutrophil oxidative burst

(A) Covalent modification of NADPH oxidase by itaconate. (B) Neutrophil oxygen consumption rate (OCR) during LAC infection in the presence of 0 or 5 mM itaconate; glucose added at the first dotted line, oligomycin at the second, and 2-deoxyglucose at the third. (C) Neutrophil superoxide production during LAC infection or mock infection (PBS) in the presence of 0, 1, or 5 mM itaconate or an NADPH oxidase inhibitor (DPI) compared with the positive control (PMA). (D) Bacterial killing by neutrophils in 0 or 5 mM itaconate. (E and F) Enriched gene sets in the scRNA-seq (E) epithelial and (F) endothelial cell populations of LAC-infected WT vs. Irg1^−/− mice. (G–I) H&E-stained lung sections (24 h post-infection) (G), inflammation scoring (H), and albumin (I) in the BAL fluid from PBS-treated and LAC-infected WT and Irg1^−/− mice. Data are shown as mean ± SEM from n = (B and C) 3 mice (3 replicates/mouse) or (D) 6 mice (3 replicates/mouse). Statistics are from (B) multiple t test with FDR correction, (C) one-way ANOVA, or (D) Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.