Wang, Feilong

Abstract Metabolic reprogramming is a fundamental determinant of immune cell fate and effector function during inflammation, yet how distinct metabolic pathways regulate pathogenic lung macrophage activity and contribute to fibrotic remodeling remains incompletely understood. Here, we integrated single-cell RNA sequencing, lineage-specific genetic mouse models, and fate-mapping approaches to define the metabolic pathways that regulate the pro-fibrotic activity of lung macrophages. We found that such cells undergo pronounced aerobic glycolysis in the bleomycin-induced mouse model of pulmonary fibrosis. Myeloid cell-specific depletion of lactate dehydrogenase A (Ldha), but not of mitochondrial pyruvate carrier (Mpc), markedly attenuated lung fibrosis and improved survival. Mechanistically, enhanced glycolytic flux increased lactate production, which promoted histone lysine lactylation and facilitated Arginase 1 (Arg1) expression in lung macrophages, thereby driving fibrotic progression. Using monocyte fate-mapping, we further demonstrated that Arg1 expression is largely restricted to recruited monocyte-derived macrophages rather than lung-resident macrophages. Notably, selective deletion of Ldha in granulocyte-monocyte progenitors and their progeny was sufficient to suppress Arg1 expression and reduce fibrosis severity. In human lung samples, we observed significantly elevated expression of ARG1 and key glycolytic enzymes in patients with idiopathic pulmonary fibrosis, despite species-specific differences in the immune cell types expressing ARG1. Together, these findings identify an aerobic glycolysis–lactate–histone lactylation axis that regulates pro-fibrotic myeloid cell function and represents a potential therapeutic target in pulmonary fibrosis.

AbstractTargeting NLRP3 inflammasome has been recognized as a promising therapeutic strategy for the treatment of numerous common diseases. UK5099, a long‐established inhibitor of mitochondrial pyruvate carrier (MPC), is previously found to inhibit macrophage inflammatory responses independent of MPC expression. However, the mechanisms by which UK5099 inhibit inflammatory responses remain unclear. Here, it is shown that UK5099 is a potent inhibitor of the NLRP3 inflammasome in both mouse and human primary macrophages. UK5099 selectively suppresses the activation of the NLRP3 but not the NLRC4 or AIM2 inflammasomes. Of note, UK5099 retains activities on NLRP3 in macrophages devoid of MPC expression, indicating this inhibitory effect is MPC‐independent. Mechanistically, UK5099 abrogates mitochondria‐NLRP3 interaction and in turn inhibits the assembly of the NLRP3 inflammasome. Further, a single dose of UK5099 persistently reduces IL‐1β production in an endotoxemia mouse model. Importantly, structure modification reveals that the inhibitory activities of UK5099 on NLRP3 are unrelated to the existence of the activated double bond within the UK5099 molecule. Thus, this study uncovers a previously unknown molecular target for UK5099, which not only offers a new candidate for the treatment of NLRP3‐driven diseases but also confounds its use as an MPC inhibitor in immunometabolism studies.

Background: Atherosclerosis is an inflammatory disease with T cells and macrophages as the leading plaque cell populations. The immunoproteasome (IP) influences T cell survival and differentiation but its impact on macrophage survival and differentiation remains unknown, which was the objective of this study. Methods: Murine monocyte/macrophage Raw264.7 cells were stimulated by IFN-gamma (20 ng/mL) plus LPS (10pg/mL) or IL-4 (20 ng/mL) for 18 hours to induce M1 and M2 macrophages, and these differentiated macrophages were then re-exposed to IFN- gamma and IL-4 for another 24 hours, without and with IP subunit beta 5i inhibition by siRNA or Onx0914. Nitric oxide synthase and arginase activity served as differentiation markers (analyzed by enzymatic assays) as did the cytokine/chemokine profile (analyzed by antibody arrays). Cell viability was tested by an XTT assay and apoptosis by TUNEL FACS. Results: IP beta5i knockout/inhibition did not influence M1 and M2 macrophage differentiation. Also, no significant differences in apoptosis were observed between RAW264.7 cells treated with and without beta 5i inhibitor during macrophage differentiation. With re-exposure to IFN- gamma and IL-4, however, there was more apoptosis and less survival of differentiated M1 macrophages with beta5i inhibition. Conversely, beta 5i inhibition achieved less apoptosis and more survival of differentiated M2 macrophages when switching IL-4- to IFN-gamma-stimulating conditions. Conclusions: IP inhibition does not influence macrophage differentiation but survival of differentiated macrophages of potential significance for the atherosclerotic disease process.

Abstract Glycolysis is essential for the classical activation of macrophages (M1), but how glycolytic pathway metabolites engage in this process remains to be elucidated. Glycolysis culminates in the production of pyruvate, which can be transported into the mitochondria by the mitochondrial pyruvate carrier (MPC) followed by conversion to citrate and utilization in the TCA cycle. Alternatively, pyruvate can be metabolized to lactate under aerobic conditions, which had been considered to be the dominant route in the setting of classical macrophage activation. However, based on studies that used UK5099 as a MPC inhibitor and showed reduction in key inflammatory cytokines, the mitochondrial route has been considered to be of significance for M1 activation as well. Herein, using a genetic depletion model, we found that MPC is dispensable for metabolic reprogramming and the activation of M1. In addition, MPC depletion in myeloid cells has no impact on inflammatory responses and macrophage polarization toward M1 phenotype in the endotoxemia mice model. While UK5099 reaches maximal MPC inhibitory capacity at approximately 2–5µM, higher concentrations are required to inhibit inflammatory cytokine production in M1 and this is independent of MPC expression. Taken together, this study found MPC-mediated metabolism is dispensable for the classical activation of macrophages, and UK5099 inhibits inflammatory responses in M1 macrophages due to effects other than MPC inhibition.

Abstract Glycolysis is essential for the classical activation of macrophages (M1), but how glycolytic pathway metabolites engage in this process remains to be elucidated. Glycolysis culminates in the production of pyruvate, which can be transported into the mitochondria by the mitochondrial pyruvate carrier (MPC) followed by conversion to citrate and utilization in the TCA cycle. Alternatively, pyruvate can be metabolized to lactate under aerobic conditions, which had been considered to be the dominant route in the setting of classical macrophage activation. However, based on studies that used UK5099 as a MPC inhibitor and showed reduction in key inflammatory cytokines, the mitochondrial route has been considered to be of significance for M1 activation as well. Herein, using a genetic depletion model, we found that MPC is dispensable for metabolic reprogramming and the activation of M1. While UK5099 reaches maximal MPC inhibitory capacity at approximately 2–5µM, higher concentrations are required to inhibit inflammatory cytokine production in M1 and this is independent of MPC expression. Apart from MPC inhibition, UK5099 at high doses impairs glutamate oxidation, mitochondrial membrane potential and HIF-1α stabilization. Taken together, UK5099 inhibits inflammatory responses in M1 macrophages due to effects other than MPC inhibition.