Metabolic Alterations Contribute to Enhanced Inflammatory Cytokine Production in Irgm1-deficient Macrophages

Abstract

The immunity-related GTPases (IRGs) are a family of proteins that are induced by interferon (IFN)-γ and play pivotal roles in immune and inflammatory responses. IRGs ostensibly function as dynamin-like proteins that bind to intracellular membranes and promote remodeling and trafficking of those membranes. Prior studies have shown that loss of Irgm1 in mice leads to increased lethality to bacterial infections as well as enhanced inflammation to non-infectious stimuli; however, the mechanisms underlying these phenotypes are unclear. In the studies reported here, we found that uninfected Irgm1-deficient mice displayed high levels of serum cytokines typifying profound autoinflammation. Similar increases in cytokine production were also seen in cultured, IFN-γ-primed macrophages that lacked Irgm1. A series of metabolic studies indicated that the enhanced cytokine production was associated with marked metabolic changes in the Irgm1-deficient macrophages, including increased glycolysis and an accumulation of long chain acylcarnitines. Cells were exposed to the glycolytic inhibitor, 2-deoxyglucose, or fatty acid synthase inhibitors to perturb the metabolic alterations, which resulted in dampening of the excessive cytokine production. These results suggest that Irgm1 deficiency drives metabolic dysfunction in macrophages in a manner that is cell-autonomous and independent of infectious triggers. This may be a significant contributor to excessive inflammation seen in Irgm1-deficient mice in different contexts.

Keywords: Irgm1; cytokine induction; fatty acid metabolism; glycolysis; immunity-related GTPase; interferon gamma; macrophage; respiration.

FIGURE 1.

Irgm1 deficiency leads to enhanced cytokine production in uninfected mice. Sera collected from three WT and three Irgm1-deficient mice were used for cytokine measurements using a multiplex assay. Shown are average levels of the indicated cytokines in Irgm1-deficient mice relative to levels in WT mice. Error bars, S.E. *, p < 0.05.

FIGURE 2.

IFN-γ-primed macrophages show enhanced secretion of inflammatory cytokines in the absence of Irgm1. A, BMM from WT and Irgm1-deficient mice were primed with IFN-γ for 24 h. Cytokine levels were measured in conditioned media using a cytokine array dot blot. B and C, BMM from WT and Irgm1-deficient mice were primed with IFN-γ or were maintained under control conditions for 24 h. Levels of RANTES (B) and MCP-1 (C) were measured in conditioned media using ELISA. Shown are average relative levels measured in triplicate samples for the indicated cytokines. Error bars, S.E. *, p < 0.05. **, p < 0.001.

FIGURE 3.

Irgm1-deficient macrophages show an increase in glycolytic activity. BMM from WT and Irgm1-deficient mice were primed with IFN-γ or were maintained under control conditions for 24 h. The cells were then subjected to a glycolytic stress test and measurement of the ECAR as a proxy for glycolysis. The experiment was repeated four times, using cells isolated from different mice each time. Shown are representative ECAR measurements from a stress test (A) and a representative ECAR measurement (B). The cells were also used for metabolomic measurement of lactate levels (C), analyzing cells isolated from three separate mice per genotype. Cells were subjected to a mitochondrial stress test and measurement of the OCR as a proxy for oxidative phosphorylation. The experiment was repeated four times, using cells isolated from different mice each time. D, average OCR from a representative experiment. E, average ECAR/OCR ratios over four experiments. In other experiments, BMM from WT and Irgm1-deficient mice were maintained under control conditions, were primed with IFN-γ, or were activated with IFN-γ for 24 h and LPS for the final 16 h. Protein lysates were isolated and used for immunoblotting with an antibody mixture of electron transport chain (ETC) components. Shown are a representative blot (F) and sum intensities of the bands normalized to actin and averaged over three experiments (G). Error bars, S.E. *, p < 0.05.

FIGURE 4.

Increased levels of long chain acylcarnitines in IFN-γ-primed macrophages lacking Irgm1. Groups of three WT and three Irgm1-deficient BMM isolated from separate mice were maintained under control conditions, primed with IFN-γ for 24 h, or activated with IFN-γ for 24 h and LPS for the final 16 h. Lysates were prepared from the cells and were used for measurement of acylcarnitine levels using LC-MS. Shown is a heat map of the relative -fold changes in acylcarnitine concentration in IFN-γ-primed and IFN-γ/LPS-activated BMM, relative to concentrations in BMM of that genotype under control conditions. Raw acylcarnitine levels can be seen in supplemental Table S2.

FIGURE 5.

Inhibition of autophagy does not affect RANTES production in IFN-γ-primed macrophages. BMM of the indicated genotypes were plated and maintained under control conditions or primed with IFN-γ for 24 h. Conditioned media were collected and used for ELISA to measure RANTES (A) or IL-1β (B). Shown are averages of at least three experiments. Error bars, S.E. *, p < 0.05.

FIGURE 6.

The punctate mitochondrial morphology is increased in IFN-γ-primed macrophages lacking Irgm1 and is dependent on fatty acid synthesis and reactive oxygen species. A–D, BMM of the indicated genotype were plated on coverslips. Following various treatments, the cells were immunostained with antibodies to the mitochondrial marker TOM20. The mitochondrial morphology of cells was assessed in a blinded fashion in 50 cells/genotype/experiment. Shown is the average percentage of cells with an overall punctate mitochondrial morphology. In A, the cells were maintained under control conditions, were primed with IFN-γ for 24 h, or were activated with IFN-γ and LPS for 16 h. In B, the cells were maintained under control conditions or were primed with IFN-γ for 24 h. In C, the cells were maintained under control conditions or were primed with IFN-γ for 24 h with or without the fatty acid synthesis inhibitors, cerulenin or C75. In D, the cells were maintained under control conditions or primed with IFN-γ for 24 h with or without the reactive oxygen species quencher NAC. E, BMM of the indicated genotypes were maintained under control conditions, were primed with IFN-γ for 24 h, or were activated with IFN-γ and LPS for 16 h with or without the reactive oxygen species quencher, NAC. ROS levels were measured with the fluorescent probe 2′,7′-dichlorofluorescindiacetate (DCFDA). F and G, BMM of the indicated genotypes were maintained under control conditions or primed with IFN-γ for 24 h with or without the reactive oxygen species quencher NAC. MCP-1 or RANTES was measured in conditioned media from the cells using ELISA. For each figure, shown are average values from experiments performed at least three times. Error bars, S.E. *, p < 0.05; ***, p < 0.001.

FIGURE 7.

Blocking glycolysis or fatty acid synthesis mitigates the increased pro-inflammatory cytokine secretion in IFN-γ-primed macrophages lacking Irgm1. BMM of the indicated genotypes were plated in triplicate and maintained under control conditions, primed with IFN-γ, primed with IFN-γ, and simultaneously exposed to glycolytic inhibitor 2-DG (A and B) or primed with IFN-γ and simultaneously exposed to the fatty acid synthase inhibitors the fatty acid synthase inhibitor C75 or cerulenin (C and D) for 24 h. Conditioned media were isolated and used to perform ELISA to measure either RANTES (A and C) or MCP-1(B and D). Shown are average values over at least three separate experiments using BMM cultures from separate mice. Error bars, S.E. *, p < 0.05; ***, p < 0.001.