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.

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.

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.

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.

Fish miR-146a promotes Singapore grouper iridovirus infection by regulating cell apoptosis and NF-κB activation.

miR-146a was reported to participate in various pathophysiological conditions in mammals, such as inflammation and immune responses, oncogenesis and cell damage. However, its function in low vertebrates has not been well elucidated. In this study, we characterized the expression profiles and functions of miR-146a in fish cells during iridovirus infection. We found that the reported fish miR-146a genes encoded an identical mature sequence, which shared high similarity with its mammalian orthologues, suggesting a putative functional conservation of miR-146a between fish and other vertebrates. Using a well-established infection model of Singapore grouper iridovirus (SGIV) in fathead minnow cells, we found that SGIV infection induced the expression of miR-146a to a dramatic extent. More importantly, we found that miR-146a promoted SGIV propagation, as demonstrated by higher expression of viral genes and increased virus titres in miR-146a-overexpressing cells. Mechanistically, we found that miR-146a overexpression suppressed, while miR-146a knockdown promoted, NF-κB activation and SGIV-induced cell apoptosis, two major cellular events involved in SGIV infection. Our study suggested that the induction of miR-146a by SGIV infection may function through a feed-forward mechanism to promote viral infection by restraining anti-viral cellular responses.

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.

Pyruvate dehydrogenase kinase 1 participates in macrophage polarization via regulating glucose metabolism.

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.

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.

Singapore grouper iridovirus-encoded semaphorin homologue (SGIV-sema) contributes to viral replication, cytoskeleton reorganization and inhibition of cellular immune responses.

Semaphorins are a large, phylogenetically conserved family of proteins that are involved in a wide range of biological processes including axonal steering, organogenesis, neoplastic transformation, as well as immune responses. In this study, a novel semaphorin homologue gene belonging to the Singapore grouper iridovirus (SGIV), ORF155R (termed SGIV-sema), was cloned and characterized. The coding region of SGIV-sema is 1728 bp in length, encoding a predicted protein with 575 aa. SGIV-sema contains a ~370 aa N-terminal Sema domain, a conserved plexin-semaphorin-integrin (PSI) domain, and an immunoglobulin (Ig)-like domain near the C terminus. SGIV-sema is an early gene product during viral infection and predominantly distributed in the cytoplasm with a speckled and clubbed pattern of appearance. Functionally, SGIV-sema could promote viral replication during SGIV infection in vitro, with no effect on the proliferation of host cells. Intriguingly, ectopically expressed SGIV-sema could alter the cytoskeletal structure of fish cells, characterized by a circumferential ring of microtubules near the nucleus and a disrupted microfilament organization. Furthermore, SGIV-sema was able to attenuate the cellular immune response, as demonstrated by decreased expression of inflammation/immune-related genes such as IL-8, IL-15, TNF-α and mediator of IRF3 activation (MITA), in SGIV-sema-expressing cells before and after SGIV infection. Ultimately, our study identified a novel, functional SGIV gene that could regulate cytoskeletal structure, immune responses and facilitate viral replication.

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.

MicroRNA let-7c regulates macrophage polarization.

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.

miR-145 regulates myofibroblast differentiation and lung fibrosis.

The expression of smooth muscle actin-α (SMA-α) by fibroblasts defines phenotypic transition to myofibroblasts and is a primary contributor to contractile force generation by these differentiated cells. Although the regulation of SMA-α expression has been the focus of many studies, there is presently only limited information concerning miRNA regulation of lung myofibroblast differentiation and the involvement of these miRNAs in pulmonary fibrosis. To determine the role of miR-145 in regulating lung myofibroblast differentiation and pulmonary fibrosis. Wild-type and miR-145(-/-) mice were studied. Lung fibrosis models and cell culture systems were employed. miR-145 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic and contractile activities of lung fibroblasts were determined. We found that miR-145 expression is upregulated in TGF-ß1-treated lung fibroblasts. miR-145 expression is also increased in the lungs of patients with idiopathic pulmonary fibrosis as compared to in normal human lungs. Overexpression of miR-145 in lung fibroblasts increased SMA-α expression, enhanced contractility, and promoted formation of focal and fibrillar adhesions. In contrast, miR-145 deficiency diminished TGF-ß1 induced SMA-α expression. miR-145 did not affect the activity of TGF-ß1, but promoted the activation of latent TGF-ß1. miR-145 targets KLF4, a known negative regulator of SMA-α expression. Finally, we found that miR-145(-/-) mice are protected from bleomycin-induced pulmonary fibrosis. miR-145 plays an important role in the differentiation of lung myofibroblasts. miR-145 deficiency is protective against bleomycin-induced lung fibrosis, suggesting that miR-145 may be a potential target in the development of novel therapies to treat pathological fibrotic disorders.

miR-31 is a negative regulator of fibrogenesis and pulmonary fibrosis.

Aberrant expression of miRNAs is closely associated with initiation and progression of pathological processes, including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in lung fibrosis is not well characterized. We sought to determine the role of miR-31 in regulating the fibrogenic, contractile, and migratory activities of lung fibroblasts and modulating of pulmonary fibrosis in vivo. In vivo lung fibrosis models and ex vivo cell culture systems were employed. Real-time PCR and Western blot analysis were used to determine gene expression levels. miR-31 mimics or inhibitors were transfected into pulmonary fibroblasts. Fibrogenic, contractile, and migratory activities of lung fibroblasts were determined. We found that miR-31 expression is reduced in the lungs of mice with experimental pulmonary fibrosis and in IPF fibroblasts. miR-31 inhibits the profibrotic activity of TGF-ß1 in normal lung fibroblasts and diminishes the fibrogenic, contractile, and migratory activities of IPF fibroblasts. In these experiments, miR-31 was shown to directly target integrin α(5) and RhoA, two proteins that have been shown to regulate activation of fibroblasts. We found that levels of integrin α(5) and RhoA are up-regulated in fibrotic mouse lungs. Knockdown of integrin α(5) and RhoA attenuated fibrogenic, contractile, and migratory activities of IPF fibroblasts, in a manner similar to that observed with miR-31. We also found that introduction of miR-31 ameliorated experimental lung fibrosis in mice. Our data suggest that miR-31 is an important regulator of the pathological activities of lung fibroblasts and may be a potential target in the development of novel therapies to treat pathological fibrotic disorders, including pulmonary fibrosis.

Identification of TLT2 as an engulfment receptor for apoptotic cells.

Clearance of apoptotic cells (efferocytosis) is critical to the homeostasis of the immune system by restraining inflammation and autoimmune response to intracellular Ags released from dying cells. TLRs-mediated innate immunity plays an important role in pathogen clearance and in regulation of the adaptive immune response. However, the regulation of efferocytosis by activation of TLRs has not been well characterized. In this study, we found that activation of TLR3 or TLR9, but not of TLR2, enhances engulfment of apoptotic cells by macrophages. We found that the activation of TLR3 upregulates the expression of triggering receptor expressed on myeloid cells (TREM)-like protein 2 (TLT2), a member of the TREM receptor family, on the surface of macrophages. Blocking TLT2 on the macrophage surface by either specific anti-TLT2 Ab or soluble TLT2 extracellular domain attenuated the enhanced ability of macrophages with TLR3 activation to engulf apoptotic cells. To the contrary, overexpression of TLT2 increased the phagocytosis of apoptotic cells. We found that TLT2 specifically binds to phosphatidylserine, a major "eat me" signal that is exposed on the surface of apoptotic cells. Furthermore, we found that TLT2 mediates phagocytosis of apoptotic cells in vivo. Thus, our studies identified TLT2 as an engulfment receptor for apoptotic cells. Our data also suggest a novel mechanism by which TREM receptors regulate inflammation and autoimmune response.

Extracellular histones inhibit efferocytosis.

The uptake and clearance of apoptotic cells by macrophages and other phagocytic cells, a process called efferocytosis, is a major component in the resolution of inflammation. Increased concentrations of extracellular histones are found during acute inflammatory states and appear to contribute to organ system dysfunction and mortality. In these studies, we examined the potential role of histones in modulating efferocytosis. We found that phagocytosis of apoptotic neutrophils or thymocytes by macrophages was significantly diminished in the presence of histones H3 or H4, but not histone H1. Histone H3 demonstrated direct binding to macrophages, an effect that was diminished by preincubation of macrophages with the opsonins growth arrest-specific gene 6 (Gas6) and milk fat globule-epidermal growth factor (EGF) 8 (MFG-E8). Incubation of histone H3 with soluble α(v)ß5 integrin and Mer, but not with α(v)ß3, diminished its binding to macrophages. Phagocytosis of apoptotic cells by alveolar macrophages in vivo was diminished in the presence of histone H3. Incubation of histone H3 with activated protein C, a treatment that degrades histones, abrogated its inhibitory effects on efferocytosis under both in vitro and in vivo conditions. The present studies demonstrate that histones have inhibitory effects on efferocytosis, suggesting a new mechanism by which extracellular histones contribute to acute inflammatory processes and tissue injury.