Mitochondrial arginase-2 is essential in preventing excessive inflammation
RCSI researchers discover new way to halt IL-10 metabolic reprogramming of inflammatory macrophages.
Macrophages are a subtype of immune cell that play key roles in the regulation of infection in our bodies.
Macrophages clear an infection through the induction of phagocytosis, the release of pro-inflammatory cytokines and toxic mediators. In this inflammatory state, they are classified as ‘M1’ macrophages. If inflammation persists however, they can also become destructive and lead to the severe tissue damage observed in variety of autoimmune diseases such as Multiple Sclerosis.
On the other hand, macrophages can also adopt a ‘M2’ phenotype when they produce anti-inflammatory mediators and potentiate cell proliferation, tissue repair and the healing process. Therefore, it is desirable to reprogram inflammatory macrophages from an M1 state into an anti-inflammatory M2 state as a therapeutic avenue to control inflammatory and autoimmune diseases.
Interestingly, it is becoming increasingly clear that macrophages are also governed by metabolic pathways.
M1-like macrophages have pro-inflammatory metabolic processes, with increased aerobic glycolysis, downregulation of mitochondrial oxidative phosphorylation and accumulation of TCA cycle metabolites. On the other hand, M2 macrophages can enhance oxidative phosphorylation to support their anti-inflammatory properties.
The metabolic switches that govern an M1-M2 switch are relatively unknown, but an understanding of this process may yield novel insights for therapeutic intervention.
Arginine2 (Arg2) has been newly identified as an important gene that is enhanced in M2 macrophages and which acts to resolve inflammation. Dr Claire McCoy of the School of Pharmacy and Biomolecular Sciences at RCSI University of Medicine and Health Sciences led this multi-centre international collaborative study, published in Nature Communications here.
Their discovery highlights how Arg2 alters the oxidative machinery in macrophages in response to the M2 agonist, IL-10 and offers a novel therapeutic target for the treatment of inflammatory and autoimmune diseases.
The study commenced by examining macrophage gene expression using micro-arrays.
The micro-array compared wild-type murine bone marrow-derived macrophages stimulated with a potent ‘M1 agonist’ LPS versus wild-type macrophages stimulated with LPS+IL-10 ‘M2 agonist’. The genetic changes were then compared to the same stimulations carried out in miR155 knockout (miR-155-/-) macrophages.
Arg2 was identified as a novel gene regulated by both IL-10 and miR-155, an indicator that this gene was important in M2 polarisation.
The catalytic activity of arginase was demonstrated by measuring urea production in the conversion of L-arginine to L-ornithine. Treatment of macrophages with LPS increased urea production. Co-stimulation of macrophages with LPS+IL-10 significantly increased urea production, an effect not seen in IL-10 knockout cells. A significant drop in urea production was seen in Arg2 knockout (Arg2-/-) macrophages treated with LPS and IL-10 confirming a role for Arg2 in arginine metabolism in macrophages.
Using a mitochondrial import assay, the authors identified that Arg2 protein localises to the mitochondria in macrophages and remains in the mitochondria following mitochondrial membrane depolarisation. Mitochondrial localisation of Arg2 following LPS and IL-10 stimulation of macrophages was confirmed by immunoblotting of mitochondrial fractions. Likewise, Arg2 was visualised in co-localisation with the mitochondrial protein Tom20, with increased mitochondrial levels in LPS+IL-10 stimulated macrophages.
Mitochondrial dynamics were examined in macrophages stimulated with LPS+IL-10. When examined with confocal microscopy, LPS-only stimulated cells showed significantly increased mitochondrial fission as indicated by average mitochondrion size <1 μm. In contrast, the mitochondria of cells treated with LPS+IL10 showed significantly reduced numbers in fission and mitochondria were largely in a state of fusion indicated by size >3 μm.
This effect was lost in Arg2-/- macrophages indicating that Arg2 is critical for mitochondrial dynamics and suggests that Arg2 may also play an important role in mitochondrial metabolism.
Researchers next assessed oxidative phosphorylation by measurement of basal oxygen consumption rate, maximal respiratory capacity and oxidative phosphorylation induced ATP production. LPS stimulated macrophages are known to decrease oxidative phosphorylation and exogenous IL-10 can restore oxidative phosphorylation in IL-10-/- macrophages treated with LPS.
Oxidative phosphorylation was measured in Arg2-/- macrophages and showed reduced restoration of oxidative phosphorylation when IL-10 was added to LPS treatment. The restoration of oxidative phosphorylation appeared to be confined to the Arg2 isoform with little contribution from the Arg1 isoform.
Moreover, mutation of the catalytic site in Arg2 demonstrated that its enzymatic activity is essential for restoration of oxidative phosphorylation
Authors next wished to explore how Arg2 promoted oxidative metabolism within the macrophage mitochondria, focusing on metabolic processes including the TCA cycle and the electron transport chain.
Metabolites were measured using tandem gas chromatography-mass spectrometry. Macrophages stimulated with LPS+IL-10 generated increased levels of fumarate and malate compared to controls. These metabolites are generated downstream of the succinate dehydrogenase enzyme — succinate dehydrogenase is a key protein in the TCA cycle and forms complex II in the electron transport chain.
To examine activity of complexes in electron transport chain, the authors measured the oxygen consumption rate of cells provided with complex substrates.
The consumption of oxygen by ETC complex I and II was upregulated in macrophages stimulated with LPS+IL-10 compared to unstimulated or LPS-only stimulated cells. Arg2-/- macrophages showed significantly reduced oxygen consumption at complex II but no difference was noted in oxygen consumption at complex I.
An MTT assay to measure succinate dehydrogenase activity confirmed Arg2-/- macrophages had lower levels of succinate dehydrogenase activity compared to untreated Arg2+/+ macrophages or Arg2+/+ macrophages stimulated with LPS+IL-10.
The effects of LPS on cell metabolism, including TCA cycle disruption and reduced oxidative phosphorylation, are known to result in an inflammatory phenotype with direct generation of mitochondrial reactive oxygen species. This is associated with stabilisation of HIF-1α, increased IL-1β and reduced anti-oxidant response.
The effects of Arg2 on mitochondrial reactive oxygen species was investigated using MitoSox stained cells detected by flow cytometry.
A significant increase in reactive oxygen species was observed in Arg2-/- macrophages stimulated with LPS+IL-10 compared to Arg2+/+ cells. This was accompanied by increased HIF-1α protein levels in Arg2-/- macrophages as detected by immunoblotting. ELISA-detected IL-1β levels were suppressed by IL-10 in Arg2+/+ macrophages but not in Arg2-/- cells.
This study has demonstrated that Arg2 has a key role in producing an anti-inflammatory state in macrophages downstream of the IL-10/miR-55 axis.
The research provides valuable insight into the mechanisms for IL-10 anti-inflammatory activity with a specific focus on upregulation of Arg2 as a novel target of IL-10. Arg2 has a critical role in altering mitochondrial bioenergetics and restoring oxidative phosphorylation. The mechanism for facilitating oxidative respiration has been shown by authors to involve complex II activity in the electron transport chain.
Importantly, Arg2 is critical for limiting the pro-inflammatory cytokine, IL-1, a cytokine that if left unchecked has a damaging effect in inflammatory and autoimmune conditions.
It is anticipated that enhancing the expression of Arg2 in macrophages may lead to the development of novel therapeutic strategies for these conditions.
Journal Article Information:
Mitochondrial arginase-2 is essential for IL-10 metabolic reprogramming of inflammatory macrophages
Nature Communications. 12, 1460 (2021)
https://doi.org/10.1038/s41467-021-21617-2
