MafB regulates NLRP3 inflammasome activation by sustaining p62 expression in macrophages.

Activation of the NLRP3 inflammasome is a two-step process: the priming and the activating. The priming step involves the induction of NLRP3 and pro-IL-1ß, while the activating step leads to the full inflammasome activation triggered by a NLRP3 activator. Although mechanisms underlying the NLRP3 inflammasome activation have been increasingly clear, the regulation of this process remains incompletely understood. In this study, we find that LPS and Pseudomonas aeruginosa cause a rapid downregulation in MafB transcription in macrophages, which leads to a quick decline in the level of MafB protein because MafB is short-lived and constantly degraded by the ubiquitin/proteasome system. We find that MafB knockdown or knockout markedly enhances the NLRP3, but not the NLRP1, NLRC4, or AIM2, inflammasome activation in macrophages. Conversely, pharmacological induction of MafB diminishes the NLRP3 inflammasome activation. Mechanistically, we find that MafB sustains the expression of p62, a key mediator of autophagy/mitophagy. We find that MafB inhibits mitochondrial damage, and mitochondrial ROS production and DNA cytoplasmic release. Furthermore, we find that myeloid MafB deficient mice demonstrate increased systemic and lung IL-1ß production in response to LPS treatment and P. aeruginosa infection and deficient lung P. aeruginosa clearance in vivo. In conclusion, our study demonstrates that MafB is an important negative regulator of the NLRP3 inflammasome. Our findings suggest that strategies elevating MafB may be effective to treat immune disorders due to excessive activation of the NLRP3 inflammasome.

PAI-1 Regulation of p53 Expression and Senescence in Type II Alveolar Epithelial Cells.

Cellular senescence contributes importantly to aging and aging-related diseases, including idiopathic pulmonary fibrosis (IPF). Alveolar epithelial type II (ATII) cells are progenitors of alveolar epithelium, and ATII cell senescence is evident in IPF. Previous studies from this lab have shown that increased expression of plasminogen activator inhibitor 1 (PAI-1), a serine protease inhibitor, promotes ATII cell senescence through inducing p53, a master cell cycle repressor, and activating p53-p21-pRb cell cycle repression pathway. In this study, we further show that PAI-1 binds to proteasome components and inhibits proteasome activity and p53 degradation in human lung epithelial A549 cells and primary mouse ATII cells. This is associated with a senescence phenotype of these cells, manifested as increased p53 and p21 expression, decreased phosphorylated retinoblastoma protein (pRb), and increased senescence-associated beta-galactose (SA-ß-gal) activity. Moreover, we find that, although overexpression of wild-type PAI-1 (wtPAI-1) or a secretion-deficient, mature form of PAI-1 (sdPAI-1) alone induces ATII cell senescence (increases SA-ß-gal activity), only wtPAI-1 induces p53, suggesting that the premature form of PAI-1 is required for the interaction with the proteasome. In summary, our data indicate that PAI-1 can bind to proteasome components and thus inhibit proteasome activity and p53 degradation in ATII cells. As p53 is a master cell cycle repressor and PAI-1 expression is increased in many senescent cells, the results from this study will have a significant impact not only on ATII cell senescence/lung fibrosis but also on the senescence of other types of cells in different diseases.

CD38 Mediates Lung Fibrosis by Promoting Alveolar Epithelial Cell Aging.

Rationale: A prevailing paradigm recognizes idiopathic pulmonary fibrosis (IPF) originating from various alveolar epithelial cell (AEC) injuries, and there is a growing appreciation of AEC aging as a key driver of the pathogenesis. Despite this progress, it is incompletely understood what main factor(s) contribute to the worsened alveolar epithelial aging in lung fibrosis. It remains a challenge how to dampen AEC aging and thereby mitigate the disease progression. Objectives: To determine the role of AEC CD38 (cluster of differentiation 38) in promoting cellular aging and lung fibrosis. Methods: We used single-cell RNA sequencing, real-time PCR, flow cytometry, and Western blotting. Measurements and Main Results: We discovered a pivotal role of CD38, a cardinal nicotinamide adenine dinucleotide (NAD) hydrolase, in AEC aging and its promotion of lung fibrosis. We found increased CD38 expression in IPF lungs that inversely correlated with the lung functions of patients. CD38 was primarily located in the AECs of human lung parenchyma and was markedly induced in IPF AECs. Similarly, CD38 expression was elevated in the AECs of fibrotic lungs of young mice and further augmented in those of old mice, which was in accordance with a worsened AEC aging phenotype and an aggravated lung fibrosis in the old animals. Mechanistically, we found that CD38 elevation downregulated intracellular NAD, which likely led to the aging promoting impairment of the NAD-dependent cellular and molecular activities. Furthermore, we demonstrated that genetic and pharmacological inactivation of CD38 improved these NAD dependent events and ameliorated bleomycin-induced lung fibrosis. Conclusions: Our study suggests targeting alveolar CD38 as a novel and effective therapeutic strategy to treat this pathology.

Lung Myofibroblasts Promote Macrophage Profibrotic Activity through Lactate-induced Histone Lactylation.

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-ß1 (transforming growth factor-ß1)-induced lung myofibroblasts and in the BAL fluids (BALFs) from mice with TGF-ß1- 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.

ATF4 Mediates Mitochondrial Unfolded Protein Response in Alveolar Epithelial Cells.

Although endoplasmic reticulum (ER) unfolded protein response (UPRER) is well known, mitochondrial unfolded protein response (UPRmt) has not been recognized in alveolar epithelial cells. Furthermore, ER stress and mitochondrial dysfunction are frequently encountered in alveolar epithelial cells from an array of lung disorders. However, these two scenarios have been often regarded as separate mechanisms contributing to the pathogeneses. It is unclear whether there is interplay between these two phenomena or an integrator that couples these two signaling cascades in the stressed alveolar epithelial cells from those pathologies. In this study, we defined UPRmt in alveolar epithelial cells and identified ATF4 (activating transcription factor 4), but not ATF5, as the key regulator of UPRmt. We found that UPRER led to UPRmt and mitochondrial dysfunction in an ATF4-dependent manner. In contrast, mitochondrial stresses did not activate UPRER. We found that alveolar epithelial ATF4 and UPRmt were induced in aged mice with experimental pulmonary fibrosis as well as in patients with idiopathic pulmonary fibrosis. Finally, we found that the inducible expression of ATF4 in mouse alveolar epithelial cells aggravated pulmonary UPRmt, lung inflammation, body weight loss, and death upon bleomycin-induced lung injury. In conclusion, ER stress induces ATF4-dependent UPRmt and mitochondrial dysfunction, indicating a novel mechanism by which ER stress contributes to the pathogeneses of a variety of pulmonary disorders.

Monocyte-derived alveolar macrophage apolipoprotein E participates in pulmonary fibrosis resolution.

Recent studies have presented compelling evidence that it is not tissue-resident, but rather monocyte-derived alveolar macrophages (TR-AMs and Mo-AMs, respectively) that are essential to development of experimental lung fibrosis. However, whether apolipoprotein E (ApoE), which is produced abundantly by Mo-AMs in the lung, plays a role in the pathogenesis is unclear. In this study, we found that pulmonary ApoE was almost exclusively produced by Mo-AMs in mice with bleomycin-induced lung fibrosis. We showed that, although ApoE was not necessary for developing maximal fibrosis in bleomycin-injured lung, it was required for the resolution of this pathology. We found that ApoE directly bound to Collagen I and mediated Collagen I phagocytosis in vitro and in vivo, and this process was dependent on low-density lipoprotein receptor-related protein 1 (LPR1). Furthermore, interference of ApoE/LRP1 interaction impaired the resolution of lung fibrosis in bleomycin-treated WT mice. In contrast, supplementation of ApoE promoted this process in ApoE-/- animals. In conclusion, Mo-AM-derived ApoE is beneficial to the resolution of lung fibrosis, supporting the notion that Mo-AMs may have distinct functions in different phases of lung fibrogenesis. The findings also suggest a potentially novel therapeutic target for treating lung fibrosis, to which effective remedies remain scarce.

Inhibition of Glutaminase 1 Attenuates Experimental Pulmonary Fibrosis.

It has been increasingly recognized lately that aberrant cellular metabolism plays an important role in the pathogenesis of pulmonary fibrosis. In our previous systemic studies, we found that human lung myofibroblasts undergo glutaminolytic reprogramming, which is mediated by an increased expression of glutaminase (Gls) 1. We showed that augmented glutaminolysis critically regulates collagen production by promoting its stabilization in human lung myofibroblasts. Our study indicates that lung fibroblast Gls1 is a promising therapeutic target for this disease. In this investigation, we primarily focused on delineating the in vivo role of fibroblast Gls1 in mouse models of pulmonary fibrosis and determining the efficacy of Gls1 inhibition in treating this pathology. We now show that fibroblast Gls1 is upregulated in fibrotic mouse lungs. We present evidence that mice with ablation of fibroblast Gls1 are protected from bleomycin-induced lung fibrosis. We show that the Gls1 inhibitor, CB-839, is therapeutically efficacious in treating both bleomycin- and transforming growth factor-ß1-induced pulmonary fibrosis. Our study has thus established a solid rationale for advancing Gls1 inhibitors, particularly CB-839, to the next stage of testing in the treatment of this disease.

Long noncoding RNA Malat1 regulates differential activation of macrophages and response to lung injury.

Macrophage activation, i.e., classical M1 and the alternative M2, plays a critical role in many pathophysiological processes, such as inflammation and tissue injury and repair. Although the regulation of macrophage activation has been under extensive investigation, there is little knowledge about the role of long noncoding RNAs (lncRNAs) in this event. In this study, we found that lncRNA Malat1 expression is distinctly regulated in differentially activated macrophages in that it is upregulated in LPS-treated and downregulated in IL-4-treated cells. Malat1 knockdown attenuates LPS-induced M1 macrophage activation. In contrast, Malat1 knockdown enhanced IL-4-activated M2 differentiation as well as a macrophage profibrotic phenotype. Mechanistically, Malat1 knockdown led to decreased expression of Clec16a, silencing of which phenocopied the regulatory effect of Malat1 on M1 activation. Interestingly, Malat1 knockdown promoted IL-4 induction of mitochondrial pyruvate carriers (MPCs) and their mediation of glucose-derived oxidative phosphorylation (OxPhos), which was crucial to the Malat1 regulation of M2 differentiation and profibrotic phenotype. Furthermore, mice with either global or conditional myeloid knockout of Malat1 demonstrated diminished LPS-induced systemic and pulmonary inflammation and injury. In contrast, these mice developed more severe bleomycin-induced lung fibrosis, accompanied by alveolar macrophages displaying augmented M2 and profibrotic phenotypes. In summary, we have identified what we believe is a previously unrecognized role of Malat1 in the regulation of macrophage polarization. Our data demonstrate that Malat1 is involved in pulmonary pathogeneses in association with aberrant macrophage activation.

Impairment of Fatty Acid Oxidation in Alveolar Epithelial Cells Mediates Acute Lung Injury.

Profound impairment in cellular oxygen consumption, referred to as cytopathic dysoxia, is one of the pathological hallmarks in the lungs of patients with pathogen-induced acute lung injury (ALI). However, the underlying mechanism for this functional defect remains largely unexplored. In this study, we found that primary mouse alveolar epithelial cells (AECs) conducted robust fatty acid oxidation (FAO). More importantly, FAO was strikingly impaired in AECs of mice with LPS-induced ALI. The metabolic deficiency in these cells was likely due to decreased expression of key mediators involved in FAO and mitochondrial bioenergenesis, such as peroxisome proliferator-activated receptor γ coactivator (PGC)-1α, carnitine palmitoyltransferase 1A, and medium-chain acyl-CoA dehydrogenase (CAD). We found that treatment of alveolar epithelial line MLE-12 cells with BAL fluids from mice with ALI decreased FAO, and this effect was largely replicated in MLE-12 cells treated with the proinflammatory cytokine TNF-α, which was consistent with downregulations of PGC-1α, carnitine palmitoyltransferase 1A, long-chain CAD, and medium-chain CAD in the same treated cells. Furthermore, we found that the BAL fluids from ALI mice and TNF-α inhibited MLE-12 bioenergenesis and promoted cell apoptosis. In delineation of the role of FAO in ALI in vivo, we found that conditional ablation of AEC PGC-1α aggravated LPS-induced ALI. In contrast, fenofibrate, an activator of the PPAR-α/PGC-1α cascade, protected mice from this pathology. In summary, these data suggest that FAO is essential to AEC bioenergenesis and functional homeostasis. This study also indicates that FAO impairment-induced AEC dysfunction is an important contributing factor to the pathogenesis of ALI.

IFN Regulatory Factor 2 Inhibits Expression of Glycolytic Genes and Lipopolysaccharide-Induced Proinflammatory Responses in Macrophages.

Rapid initiation and timely resolution of inflammatory response in macrophages are synergistic events that are known to be equally critical to optimal host defense against pathogen infections. However, the regulation of these processes, in particular by a specific cellular metabolic program, has not been well understood. In this study, we found that IFN regulatory factor 2 (IRF2) underwent an early degradation in a proteasome-mediated pathway in LPS-treated mouse macrophages, followed by a later recovery of the expression via transactivation. We showed that IRF2 was anti-inflammatory in that knockdown of this protein promoted the production of LPS-induced proinflammatory mediators. Mechanistically, although IRF2 apparently did not target the proximal cytoplasmic signaling events upon LPS engagements, it inhibited HIF-1α-dependent expression of glycolytic genes and thereby cellular glycolysis, sequential events necessary for the IRF2 anti-inflammatory activity. We found that macrophages in endotoxin tolerant state demonstrated deficiency in LPS-augmented glycolysis, which was likely caused by failed downregulation of IRF2 and the ensuing upregulation of the glycolytic genes in these cells. In contrast to observations with LPS, knockdown of IRF2 decreased IL-4-induced macrophage alternative activation. The pro-IL-4 activity of IRF2 was dependent on KLF4, a key mediator of the alternative activation, which was transcriptionally induced by IRF2. In conclusion, our data suggest that IRF2 is an important regulator of the proinflammatory response in macrophages by controlling HIF-1α-dependent glycolytic gene expression and glycolysis. This study also indicates IRF2 as a novel therapeutic target to treat inflammatory disorders associated with dysregulations of macrophage activations.

Glutaminolysis Promotes Collagen Translation and Stability via α-Ketoglutarate-mediated mTOR Activation and Proline Hydroxylation.

Glutaminolysis is the metabolic process of glutamine, aberration of which has been implicated in several pathogeneses. Although we and others recently found a diversity of metabolic dysregulation in organ fibrosis, it is unknown if glutaminolysis regulates the profibrotic activities of myofibroblasts, the primary effector in this pathology. In this study, we found that lung myofibroblasts demonstrated significantly augmented glutaminolysis that was mediated by elevated glutaminase 1 (Gls1). Inhibition of glutaminolysis by specific Gls1 inhibitors CB-839 and BPTES as well as Gls1 siRNA blunted the expression of collagens but not that of fibronectin, elastin, or myofibroblastic marker smooth muscle actin-α. We found that glutaminolysis enhanced collagen translation and stability, which were mediated by glutaminolysis-dependent mTOR complex 1 activation and collagen proline hydroxylation, respectively. Furthermore, we found that the amount of the glutaminolytic end product α-ketoglutarate (α-KG) was increased in myofibroblasts. Similar to glutaminolysis, α-KG activated mTOR complex 1 and promoted the expression of collagens but not of fibronectin, elastin, or smooth muscle actin-α. α-KG also remarkably inhibited collagen degradation in fibroblasts. Taken together, our studies identified a previously unrecognized mechanism by which a major metabolic program regulates the exuberant production of collagens in myofibroblasts and suggest that glutaminolysis is a novel therapeutic target for treating organ fibrosis, including idiopathic pulmonary fibrosis.

Metabolic characterization and RNA profiling reveal glycolytic dependence of profibrotic phenotype of alveolar macrophages in lung fibrosis.

Metabolic reprogramming has been intrinsically linked to macrophage activation. Alveolar macrophages are known to play an important role in the pathogenesis of pulmonary fibrosis. However, systematic characterization of expression profile in these cells is still lacking. Furthermore, main metabolic programs and their regulation of cellular phenotype are completely unknown. In this study, we comprehensively analyzed the expression profile and main metabolic programs in alveolar macrophages from mice with or without experimental pulmonary fibrosis. We found that alveolar macrophages from both bleomycin and active TGF-ß1-induced fibrotic mouse lungs demonstrated a primarily profibrotic M2-like profile that was distinct from the well-defined M1 or any of the M2 subtypes. More importantly, we found that fibrotic lung alveolar macrophages assumed augmented glycolysis, which was likely attributed to enhanced expression of multiple key glycolytic mediators. We also found that fatty acid oxidation was upregulated in these cells. However, the profibrotic M2-like profile of fibrotic lung alveolar macrophages was not dependent on fatty acid oxidation and synthesis or lipolysis, but instead on glycolysis, in contrast to the typical IL-4-induced macrophages M(IL-4). Additionally, glutaminolysis, a key metabolic program that has been implicated in numerous pathologies, was not required for the profibrotic M2-like phenotype of these macrophages. In summary, our study identifies a unique expression and metabolic profile in alveolar macrophages from fibrotic lungs and suggests glycolytic inhibition as an effective antifibrotic strategy in treating lung fibrosis.

miR-34a promotes fibrosis in aged lungs by inducing alveolarepithelial dysfunctions.

Idiopathic pulmonary fibrosis is a well-known age-related disease. However, much less recognized has been the aging associated pathogenesis of this disorder. As we and others previously showed that dysregulation of micro-RNAs (miRNAs) was an important mechanism involved in pulmonary fibrosis, the role of these molecules in this pathology in the aged population has not been investigated (Cushing L, Kuang PP, Qian J, Shao F, Wu J, Little F, Thannickal VJ, Cardoso WV, Lü J. Am J Respir Cell Mol Biol 45: 287-294, 2011; Liu G, Friggeri A, Yang Y, Milosevic J, Ding Q, Thannickal VJ, Kaminski N, Abraham E. J Exp Med 207: 1589-1597, 2010; Pandit KV, Corcoran D, Yousef H, Yarlagadda M, Tzouvelekis A, Gibson KF, Konishi K, Yousem SA, Singh M, Handley D, Richards T, Selman M, Watkins SC, Pardo A, Ben-Yehudah A, Bouros D, Eickelberg O, Ray P, Benos PV, Kaminski N. Am J Respir Crit Care Med 182: 220-229, 2010). In this study, by using a lung fibrosis model established in old mice, we found that ablation of miR-34a protected aged animals from developing experimental lung fibrosis. miR-34a was upregulated in lung epithelial cells, but not in lung fibroblasts of aged mice, and miR-34a expression was further increased in epithelial cells of the fibrotic lungs of these old animals. We found that miR-34a induced dysfunctions in alveolar epithelial cells (AECs), as evidenced by increased cellular senescence and apoptosis and mitochondrial aberrations. More importantly, these abnormalities were attenuated in AECs of the fibrotic lungs of aged miR-34a-/- mice. We found that miR-34a targeted Sirt1, a master anti-aging regulator, and two key cell cycle modulators, E2F3 and cyclin E2, in lung epithelial cells, and the repression of these targets was relieved in miR-34a-deficient AECs. In summary, our data suggest that elevated AEC miR-34a plays a critical role in the pathogenesis of pulmonary fibrosis in the aged population. Our study also indicates miR-34a to be a more precise miRNA target for treating this disease that overwhelmingly affects people of advanced age.

miR-34a Inhibits Lung Fibrosis by Inducing Lung Fibroblast Senescence.

Cellular senescence has been implicated in diverse pathologies. However, there is conflicting evidence regarding the role of this process in tissue fibrosis. Although dysregulation of microRNAs is a key mechanism in the pathogenesis of lung fibrosis, it is unclear whether microRNAs function by regulating cellular senescence in the disease. In this study, we found that miR-34a demonstrated greater expression in the lungs of patients with idiopathic pulmonary fibrosis and in mice with experimental pulmonary fibrosis, with its primary localization in lung fibroblasts. More importantly, miR-34a was up-regulated significantly in both human and mouse lung myofibroblasts. We found that mice with miR-34a ablation developed more severe pulmonary fibrosis than did wild-type animals after fibrotic lung injury. Mechanistically, we found that miR-34a induced a senescent phenotype in lung fibroblasts because this microRNA increased senescence-associated ß-galactosidase activity, enhanced expression of senescence markers, and decreased cell proliferative capacities. Consistently, we found that primary lung fibroblasts from fibrotic lungs of miR-34a-deficient mice had a diminished senescent phenotype and enhanced resistance to apoptosis as compared with those from wild-type animals. We also identified multiple miR-34a targets that likely mediated its activities in inducing senescence in lung fibroblasts. In conclusion, our data suggest that miR-34a functions through a negative feedback mechanism to restrain fibrotic response in the lungs by promoting senescence of pulmonary fibroblasts.

MicroRNA-27a-3p Is a Negative Regulator of Lung Fibrosis by Targeting Myofibroblast Differentiation.

Although microRNAs (miRs) have been well recognized to play an important role in the pathogenesis of organ fibrosis, there is a lack of evidence as to whether miRs directly regulate the differentiation of myofibroblasts, the putative effector cells during pathological fibrogenesis. In this study, we found that levels of miR-27a-3p were up-regulated in transforming growth factor-ß1-treated human lung fibroblasts in a Smad2/3-dependent manner and in fibroblasts isolated from lungs of mice with experimental pulmonary fibrosis. However, both basal and transforming growth factor-ß1-induced expression of miR-27a-3p were reduced in lung fibroblasts from patients with idiopathic pulmonary fibrosis compared with that from normal control subjects. Overexpression of miR-27a-3p inhibited, whereas knockdown of miR-27a-3p enhanced, the differentiation of lung fibroblasts into myofibroblasts. We found that miR-27a-3p directly targeted the phenotypic marker of myofibroblasts, α-smooth muscle actin, and two key Smad transcription factors, Smad2 and Smad4. More importantly, we found that therapeutic expression of miR-27a-3p in mouse lungs through lentiviral delivery diminished bleomycin-induced lung fibrosis. In conclusion, our data suggest that miR-27a-3p functions via a negative-feedback mechanism in inhibiting lung fibrosis. This study also indicates that targeting miR-27a-3p is a novel therapeutic approach to treat fibrotic organ disorders, including lung fibrosis.

Glycolytic Reprogramming in Myofibroblast Differentiation and Lung Fibrosis.

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-ß1-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.

The monocarboxylate transporter 4 is required for glycolytic reprogramming and inflammatory response in macrophages.

There has been fast growing evidence showing that glycolysis plays a critical role in the activation of immune cells. Enhanced glycolysis leads to increased formation of intracellular lactate that is exported to the extracellular environment by monocarboxylate transporter 4 (MCT4). Although the biological activities of extracellular lactate have been well studied, it is less understood how the lactate export is regulated or whether lactate export affects glycolysis during inflammatory activation. In this study, we found that MCT4 is up-regulated by TLR2 and TLR4, but not TLR3 agonists in a variety of macrophages. The increased expression of MCT4 was mediated by MYD88 in a NF-κB-dependent manner. Furthermore, we found that MCT4 is required for macrophage activation upon TLR2 and TLR4 stimulations, as evidenced by attenuated expression of proinflammatory mediators in macrophages with MCT4 knockdown. Mechanistically, we found that MCT4 knockdown leads to enhanced intracellular accumulation of lactate and decreased glycolysis in LPS-treated macrophages. We found that LPS-induced expression of key glycolytic enzymes hexokinase 2 and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 is diminished in macrophages with MCT4 knockdown. Our data suggest that MCT4 up-regulation represents a positive feedback mechanism in macrophages to maintain a high glycolytic rate that is essential to a fully activated inflammatory response.

miR-27a regulates inflammatory response of macrophages by targeting IL-10.

Although microRNAs were shown to participate in innate immune responses, it is not completely understood how they regulate negative immunomodulatory events. IL-10 is an important anti-inflammatory mediator that prevents excessive inflammation and associated immunological pathologies. Although the regulation of IL-10 expression has been well studied at both the transcriptional and translational levels, it is less clear how microRNAs control IL-10 expression during inflammation. In this study, we found that miR-27a is downregulated in macrophages following stimulation through TLR2 and TLR4, but not TLR3. Upregulation of miR-27a enhanced the expression of proinflammatory cytokines in TLR2/4-activated macrophages. Conversely, knockdown of miR-27a diminished cytokine expression. Mechanistically, we found that miR-27a negatively regulates IL-10 expression; upregulation of miR-27a decreases, whereas downregulation of miR-27a increases, IL-10 expression in activated macrophages. Likely due to the decreased expression of IL-10, upregulation of miR-27a diminished IL-10-dependent STAT3 phosphorylation in TLR4-activated macrophages. Consistent with IL-10 being a potential mediator for the role of miR-27a in the immune response, blocking IL-10 abolished the enhancing effect of miR-27a on TLR4-activated inflammation. In conclusion, our study identified miR-27a downregulation as a negative-regulatory mechanism that prevents overly exuberant TLR2- and TLR4-driven inflammatory responses.

The human long noncoding RNA lnc-IL7R regulates the inflammatory response.

Long noncoding RNAs (lncRNAs), once thought to be transcriptional noise, have been recently shown to regulate a variety of biological processes. However, there is not much knowledge regarding their roles in the inflammatory response. In this study, we performed human lncRNA microarray assays and identified a number of lncRNAs that demonstrated altered expression in response to LPS stimulation. Of these lncRNAs, lnc-IL7R, which overlaps with the 3'untranslated region (3'UTR) of the human interleukin-7 receptor α-subunit gene (IL7R) gene, was significantly upregulated in LPS-treated cells. Functionally, lnc-IL7R was capable of diminishing the LPS-induced inflammatory response, demonstrated by elevated expression of LPS-induced E-selectin, VCAM-1, IL-6, and IL-8 in lnc-IL7R knockdown cells. Mechanistically, we found that lnc-IL7R knockdown diminished trimethylation of histone H3 at lysine 27 (H3K27me3), a hallmark of silent transcription, at the proximal promoters of the inflammatory mediators. Our data suggest that lnc-IL7R contributes another layer of complexity in regulation of the inflammatory response.

miR-125a-5p regulates differential activation of macrophages and inflammation.

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.