Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5–7 d after initiation of pulmonary injury. TGF-β1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-β1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.

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.

Toll-like receptors (TLRs) are major receptors that enable inflammatory cells to recognize invading microbial pathogens. MicroRNAs are small non-coding RNAs that play important regulatory roles in a variety of biological processes. In this study, we found that a microRNA, miR-147, was induced upon stimulation of multiple TLRs and functioned as a negative regulator of TLR-associated signaling events in murine macrophages. We first demonstrated that the NMES1 transcript was a functional primary miR-147. miR-147 was induced in LPS-stimulated mouse macrophages and under in vivo conditions in the lungs of LPS-treated mice. Expression of miR-147 was greater after cellular activation by TLR4 than after engagement of either TLR2 or TLR3, suggesting that maximal induction of miR-147 required activation of both NF-κB and IRF3. TLR4-induced miR-147 expression was both MyD88- and TRIF-dependent. The miR-147 promoter was responsive to TLR4 stimulation and both NF-κB and STAT1α bound to the miR-147 promoter. miR-147 mimics or induced expression of miR-147 decreased, whereas miR-147 knockdown increased inflammatory cytokine expression in macrophages stimulated with ligands to TLR2, TLR3, and TLR4. These data demonstrate a negative-feedback loop in which TLR stimulation induces miR-147 to prevent excessive inflammatory responses.

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.

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.

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.