Immunoresponsive gene 1 facilitates TLR4 agonist-induced augmentation of innate antimicrobial immunity

Abstract

Treatment with the toll-like receptor 4 agonist monophosphoryl lipid A conditions innate immunocytes to respond robustly to subsequent infection, a phenotype termed innate immune memory. Our published studies show that metabolic reprogramming of macrophages is a prominent feature of the memory phenotype. We undertook studies to define the functional contributions of tricarboxylic acid cycle reprogramming to innate immune memory. We observed that priming of wild-type mice with monophosphoryl lipid A potently facilitated accumulation of the tricarboxylic acid cycle metabolite itaconate at sites of infection and enhanced microbial clearance. Augmentation of itaconate accumulation and microbial clearance was ablated in Irg1-deficient mice. We further observed that monophosphoryl lipid A potently induces expression of Irg1 and accumulation of itaconate in macrophages. Compared to wild-type macrophages, the ability of Irg1-deficient macrophages to kill Pseudomonas aeruginosa was impaired. We further observed that itaconate is directly antimicrobial against P. aeruginosa at pH 5, which is characteristic of the phagolysosome, and is facilitated by reactive oxygen species. Monophosphoryl lipid A-induced augmentation of glycolysis, oxidative phosphorylation, and accumulation of the tricarboxylic acid cycle metabolites succinate and malate was decreased in Irg1 knockout macrophages compared to wild-type controls. RNA sequencing revealed suppressed transcription of genes associated with phagolysosome function and increased expression of genes associated with cytokine production and chemotaxis in Irg1-deficient macrophages. This study identifies a contribution of itaconate to monophosphoryl lipid A-induced augmentation of innate antimicrobial immunity via facilitation of microbial killing as well as impact on metabolic and transcriptional adaptations.

Keywords: immune-responsive gene 1; innate immune memory; itaconate; macrophages; monophosphoryl lipid A.

Fig. 1.

MPLA-induced itaconate in the peritoneal fluid is associated with enhanced P. aeruginosa clearance, with no effect on peritoneal leukocytes or plasma cytokines. (A) Experimental protocol. (B) Itaconate level in peritoneal fluid. (C) Bacterial burden in peritoneal fluid. (D) Abundances of innate leukocytes at the site of infection. (E) Plasma IL-6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represent medians, and each dot represents biological replicates.

Fig. 2.

Irg1 ablation does not affect phagocytosis and respiratory burst in innate leukocytes. (A) Phagocytic capacity of indicated cells types in vivo. (B) Phagocytic capacity of BMDMs. (C) Respiratory burst activity of indicated cell types in vivo. (D) Respiratory burst activity of BMDMs (left) and ROS production of BMDMs with and without fMLP stimulation (right). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

MPLA induces Irg1 expression and itaconate production in murine bone marrow–derived macrophages. (A) BMDM treatment strategies. (B) Irg1 expression after MPLA treatment. (C) Irg1 protein expression by Western blot. (D) Intracellular itaconate in MPLA-stimulated BMDMs. (E) Intracellular itaconate in LPS-stimulated BMDMs previously stimulated with vehicle or MPLA. (F) Extracellular itaconate in media of BMDMs in panel F. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represent means, and each dot represents a biological replicate. ND, not detected.

Fig. 4.

Effect of Irg1 on metabolism of MPLA-treated BMDMs. (A) Glycolytic capacity and maximal respiration of BMDMs by Seahorse extracellular flux analysis. (B) Relative metabolite abundances in BMDMs normalized to norvaline internal standard peak area (n = 3). (C) ¹³C isotopologues of metabolites in BMDMs incubated with [U-¹³C₆]glucose for 24 h prior to metabolite harvest (n = 3). (D) Itaconate formation pathway. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represent means, and each dot represents a biological replicate. ND, not detected.

Fig. 5.

Transcriptional response of WT and Irg1KO BMDMs to MPLA. (A) Principal component analysis of KEGG-catalogued gene expression in BMDMs treated with MPLA. (B) Differentially expressed genes in MPLA-treated BMDMs. (C, D) Up- and downregulated GO pathways in between Irg1KO and WT BMDMs at multiple time points post-MPLA. The top 10 most significant pathways at every time point are listed in the table. Numbers of differentially regulated genes in each pathway are denoted by circle size.

Fig. 6.

Transcriptional response of MPLA-treated BMDMs to LPS. (A) Principal component analysis of KEGG-catalogued gene expression in BMDMs treated with MPLA, 4 h LPS. (B) DEGs in BMDMs. (C, D) Up- and downregulated GO pathways in BMDMs. The top 10 most significant pathways of every treatment group are listed in the table. Numbers of differentially regulated genes in each pathway are denoted by circle size.

Fig. 7.

Irg1, but not BLOC3, contributes to acute control of P. aeruginosa infection in BMDMs. (A) In vitro killing experimental protocol. (B) Initial bacterial load of BMDMs measuring phagocytosis after vehicle or 24-h MPLA treatment. (C) Bacterial load 3 h after initial lysis time point, measuring the bacteria not killed by the BMDMs during the incubation period. (D) Fraction of phagocytosed bacteria that are killed in 3 h ((B-C)/B) in BMDMs treated with MPLA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represent means, and each dot represents a biological replicate (D) and all technical replicates of 5 independent experiments (B, C). Statistical analyses were performed using biological replicate average values as inputs even when technical replicates are displayed.

Fig. 8.

Itaconate inhibits P. aeruginosa growth at pH 5 and in cooperation with H₂O₂. (A) Representative P. aeruginosa growth curve with multiple concentrations of itaconate at either pH 5 or pH 7 (left). Time to reach OD of 1.0 of all wells from 2 independent experiments (right). (B) Representative P. aeruginosa growth curves at pH 5 with multiple concentration of itaconate alone or combined with hydrogen peroxide (left). Time to reach OD of 1.0 of all wells from 2 independent experiments (right).