Huachun Cui

RATIONALE: Dysregulation of cellular metabolism has been shown to participate in several pathologic processes. However, the role of metabolic reprogramming is not well appreciated in the pathogenesis of organ fibrosis. OBJECTIVES: To determine if glycolytic reprogramming participates in the pathogenesis of lung fibrosis and assess the therapeutic potential of glycolytic inhibition in treating lung fibrosis. METHODS: A cell metabolism assay was performed to determine glycolytic flux and mitochondrial respiration. Lactate levels were measured to assess glycolysis in fibroblasts and lungs. Glycolytic inhibition by genetic and pharmacologic approaches was used to demonstrate the critical role of glycolysis in lung fibrosis. MEASUREMENTS AND MAIN RESULTS: Augmentation of glycolysis is an early and sustained event during myofibroblast differentiation, which is dependent on the increased expression of critical glycolytic enzymes, in particular, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3). Augmented glycolysis contributes to the stabilization of hypoxia-inducible factor 1-α, a master regulator of glycolytic enzymes implicated in organ fibrosis, by increasing cellular levels of tricarboxylic acid cycle intermediate succinate in lung myofibroblasts. Inhibition of glycolysis by the PFKFB3 inhibitor 3PO or genomic disruption of the PFKFB3 gene blunted the differentiation of lung fibroblasts into myofibroblasts, and attenuated profibrotic phenotypes in myofibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis. Inhibition of glycolysis by 3PO demonstrates therapeutic benefit in bleomycin-induced and transforming growth factor-β1-induced lung fibrosis in mice. CONCLUSIONS: Our data support the novel concept of glycolytic reprogramming in the pathogenesis of lung fibrosis and provide proof-of-concept that targeting this pathway may be efficacious in treating fibrotic disorders, such as idiopathic pulmonary fibrosis.

Augmented glycolysis due to metabolic reprogramming in lung myofibroblasts is critical to their profibrotic phenotype. The primary glycolysis byproduct, lactate, is also secreted into the extracellular milieu, together with which myofibroblasts and macrophages form a spatially restricted site usually described as fibrotic niche. Therefore, we hypothesized that myofibroblast glycolysis might have a non-cell autonomous effect through lactate regulating the pathogenic phenotype of alveolar macrophages. Here, we demonstrated that there was a markedly increased lactate in the conditioned media of TGF-β1 (transforming growth factor-β1)-induced lung myofibroblasts and in the BAL fluids (BALFs) from mice with TGF-β1- or bleomycin-induced lung fibrosis. Importantly, the media and BALFs promoted profibrotic mediator expression in macrophages. Mechanistically, lactate induced histone lactylation in the promoters of the profibrotic genes in macrophages, consistent with the upregulation of this epigenetic modification in these cells in the fibrotic lungs. The lactate inductions of the histone lactylation and profibrotic gene expression were mediated by p300, as evidenced by their diminished concentrations in p300-knockdown macrophages. Collectively, our study establishes that in addition to protein, lipid, and nucleic acid molecules, a metabolite can also mediate intercellular regulations in the setting of lung fibrosis. Our findings shed new light on the mechanism underlying the key contribution of myofibroblast glycolysis to the pathogenesis of lung fibrosis.

Macrophages demonstrate a high level of plasticity, with the ability to undergo dynamic transition between M1 and M2 polarized phenotypes. The role of microRNAs (miRNAs) in regulating macrophage polarization has been largely undefined. In this study, we found that miRNA let-7c is expressed at a higher level in M-BMM (M2 macrophages) than in GM-BMM (M1 macrophages). let-7c levels are also greater in alveolar macrophages from fibrotic lungs as compared with those from normal lungs. let-7c expression was decreased when M-BMM converted to GM-BMM, whereas it increased when GM-BMM converted to M-BMM. LPS stimulation reduced let-7c expression in M-BMM. We found that overexpression of let-7c in GM-BMM diminished M1 phenotype expression while promoting polarization to the M2 phenotype. In contrast, knockdown of let-7c in M-BMM promoted M1 polarization and diminished M2 phenotype expression. We found that let-7c targets C/EBP-δ, a transcriptional factor that plays an important role in inflammatory response. Furthermore, we found that let-7c regulates bactericidal and phagocytic activities of macrophages, two functional phenotypes implicated in macrophage polarization. Our data suggest that the miRNA let-7c plays an important role in regulating macrophage polarization.

The M1 and M2 polarized phenotypes dictate distinctive roles for macrophages as they participate in inflammatory disorders. There has been growing interest in the role of cellular metabolism in macrophage polarization. However, it is currently unclear whether different aspects of a specific metabolic program coordinately regulate this cellular process. In this study, we found that pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, plays an important role in the differential activation of macrophages. Knockdown of PDK1 diminished M1, whereas it enhanced M2 activation of macrophages. Mechanistically, PDK1 knockdown led to diminished aerobic glycolysis in M1 macrophages, which likely accounts for the attenuated inflammatory response in these cells. Furthermore, we found that mitochondrial respiration is enhanced during and required by the early activation of M2 macrophages. Suppression of glucose oxidation, but not that of fatty acids, inhibits this process. Consistent with its inhibitory role in early M2 activation, knockdown of PDK1 enhanced mitochondrial respiration in macrophages. Our data suggest that two arms of the glucose metabolism synergistically regulate the differential activation of macrophages. Our findings also highlight the central role of PDK1 in this event via controlling glycolysis and glucose oxidation.

Macrophage activation is a central event in immune responses. Macrophages undergoing classical activation (M1 macrophages) are proinflammatory, whereas alternatively activated macrophages (M2 macrophages) are generally anti-inflammatory. miRNAs play important regulatory roles in inflammatory response. However, the manner in which miRNAs regulate macrophage activation in response to different environmental cues has not been well defined. In this study, we found that M-BMM macrophages (M2) express greater levels of miR-125a-5p than do GM-BMM macrophages (M1). Stimulation of macrophages through TLR2 and TLR4 but not through TLR3 enhanced miR-125a-5p expression. Up-regulation of miR-125a-5p after TLR2/4 activation requires the adaptor MYD88 but not TRIF. Overexpression of miR-125a-5p diminished M1 phenotype expression induced by LPS but promoted M2 marker expression induced by IL-4. In contrast, knockdown of miR-125a-5p promoted M1 polarization and diminished IL-4-induced M2 marker expression. We found that miR-125a-5p targets KLF13, a transcriptional factor that has an important role in T lymphocyte activation and inflammation. KLF13 knockdown had similar effects on M1 activation as did miR-125a-5p overexpression. In addition, miR-125a-5p regulates phagocytic and bactericidal activities of macrophages. Our data suggest that miR-125a-5p has an important role in suppressing classical activation of macrophages while promoting alternative activation. Macrophage activation is a central event in immune responses. Macrophages undergoing classical activation (M1 macrophages) are proinflammatory, whereas alternatively activated macrophages (M2 macrophages) are generally anti-inflammatory. miRNAs play important regulatory roles in inflammatory response. However, the manner in which miRNAs regulate macrophage activation in response to different environmental cues has not been well defined. In this study, we found that M-BMM macrophages (M2) express greater levels of miR-125a-5p than do GM-BMM macrophages (M1). Stimulation of macrophages through TLR2 and TLR4 but not through TLR3 enhanced miR-125a-5p expression. Up-regulation of miR-125a-5p after TLR2/4 activation requires the adaptor MYD88 but not TRIF. Overexpression of miR-125a-5p diminished M1 phenotype expression induced by LPS but promoted M2 marker expression induced by IL-4. In contrast, knockdown of miR-125a-5p promoted M1 polarization and diminished IL-4-induced M2 marker expression. We found that miR-125a-5p targets KLF13, a transcriptional factor that has an important role in T lymphocyte activation and inflammation. KLF13 knockdown had similar effects on M1 activation as did miR-125a-5p overexpression. In addition, miR-125a-5p regulates phagocytic and bactericidal activities of macrophages. Our data suggest that miR-125a-5p has an important role in suppressing classical activation of macrophages while promoting alternative activation.