NRAP reduction rescues sarcomere defects in nebulin-related nemaline myopathy.

Nemaline myopathy (NM) is a rare neuromuscular disorder associated with congenital or childhood-onset of skeletal muscle weakness and hypotonia, which results in limited motor function. NM is a genetic disorder and mutations in 12 genes are known to contribute to autosomal dominant or recessive forms of the disease. Recessive mutations in nebulin (NEB) are the most common cause of NM affecting about 50% of patients. Because of the large size of the NEB gene and lack of mutational hot spots, developing therapies that can benefit a wide group of patients is challenging. Although there are several promising therapies under investigation, there is no cure for NM. Therefore, targeting disease modifiers that can stabilize or improve skeletal muscle function may represent alternative therapeutic strategies. Our studies have identified Nrap upregulation in nebulin deficiency that contributes to structural and functional deficits in NM. We show that genetic ablation of nrap in nebulin deficiency restored sarcomeric disorganization, reduced protein aggregates and improved skeletal muscle function in zebrafish. Our findings suggest that Nrap is a disease modifier that affects skeletal muscle structure and function in NM; thus, therapeutic targeting of Nrap in nebulin-related NM and related diseases may be beneficial for patients.

Macrophage Akt1 Kinase-Mediated Mitophagy Modulates Apoptosis Resistance and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disorder with increasing incidence. Mitochondrial oxidative stress in alveolar macrophages is directly linked to pulmonary fibrosis. Mitophagy, the selective engulfment of dysfunctional mitochondria by autophagasomes, is important for cellular homeostasis and can be induced by mitochondrial oxidative stress. Here, we show Akt1 induced macrophage mitochondrial reactive oxygen species (ROS) and mitophagy. Mice harboring a conditional deletion of Akt1 in macrophages (Akt1(-/-)Lyz2-cre) and Park2(-/-) mice had impaired mitophagy and reduced active transforming growth factor-ß1 (TGF-ß1). Although Akt1 increased TGF-ß1 expression, mitophagy inhibition in Akt1-overexpressing macrophages abrogated TGF-ß1 expression and fibroblast differentiation. Importantly, conditional Akt1(-/-)Lyz2-cre mice and Park2(-/-) mice had increased macrophage apoptosis and were protected from pulmonary fibrosis. Moreover, IPF alveolar macrophages had evidence of increased mitophagy and displayed apoptosis resistance. These observations suggest that Akt1-mediated mitophagy contributes to alveolar macrophage apoptosis resistance and is required for pulmonary fibrosis development.

Myeloid Heterogeneity Mediates Acute Exacerbations of Pulmonary Fibrosis.

Epidemiological evidence indicates that exposure to particulate matter is linked to the development of idiopathic pulmonary fibrosis (IPF) and increases the incidence of acute exacerbations of IPF. In addition to accelerating the rate of lung function decline, exposure to fine particulate matter (particulate matter smaller than 2.5 µm [PM2.5]) is a risk factor for increased mortality in subjects with IPF. In this article, we show that exposure to PM2.5 mediates monocyte recruitment and fibrotic progression in mice with established fibrosis. In mice with established fibrosis, bronchoalveolar lavage cells showed monocyte/macrophage heterogeneity after exposure to PM2.5. These cells had a significant inflammatory and anti-inflammatory signature. The mixed heterogeneity of cells contributed to the proinflammatory and anti-inflammatory response. Although monocyte-derived macrophages were recruited to the lung in bleomycin-injured mice treated with PM2.5, recruitment of monocytes expressing Ly6Chi to the lung promoted progression of fibrosis, reduced lung aeration on computed tomography, and impacted lung compliance. Ly6Chi monocytes isolated from PM2.5-exposed fibrotic mice showed enhanced expression of proinflammatory markers compared with fibrotic mice exposed to vehicle. Moreover, IPF bronchoalveolar lavage cells treated ex vivo with PM2.5 showed an exaggerated inflammatory response. Targeting Ly6Chi monocyte recruitment inhibited fibrotic progression in mice. Moreover, the adoptive transfer of Ly6Chi monocytes exacerbated established fibrosis. These observations suggest that enhanced recruitment of Ly6Chi monocytes with a proinflammatory phenotype mediates acute exacerbations of pulmonary fibrosis, and targeting these cells may provide a potential novel therapeutic target to protect against acute exacerbations of IPF.

NOX4-TIM23 interaction regulates NOX4 mitochondrial import and metabolic reprogramming.

Pulmonary fibrosis is a progressive lung disease characterized by macrophage activation. Asbestos-induced expression of nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4 (NOX4) in lung macrophages mediates fibrotic progression by the generation of mitochondrial reactive oxygen species (ROS), modulating mitochondrial biogenesis, and promoting apoptosis resistance; however, the mechanism(s) by which NOX4 localizes to mitochondria during fibrosis is not known. Here, we show that NOX4 localized to the mitochondrial matrix following asbestos exposure in lung macrophages via direct interaction with TIM23. TIM23 and NOX4 interaction was found in lung macrophages from human subjects with asbestosis, while it was absent in mice harboring a conditional deletion of NOX4 in lung macrophages. This interaction was localized to the proximal transmembrane region of NOX4. Mechanistically, TIM23 augmented NOX4-induced mitochondrial ROS and metabolic reprogramming to oxidative phosphorylation. Silencing TIM23 decreased mitochondrial ROS and oxidative phosphorylation. These observations highlight the important role of the mitochondrial translocase TIM23 interaction with NOX4. Moreover, this interaction is required for mitochondrial redox signaling and metabolic reprogramming in lung macrophages.

Financial Capability Interventions Used for Specific Diagnoses Related to Functional Impairment: A Scoping Review.

IMPORTANCE: Occupational therapists often address financial occupations of clients with acquired functional impairments who experience challenges with financial capability (FC). OBJECTIVE: To explore the intervention literature aimed at improving FC in five diagnostic adult populations. DATA SOURCES: MEDLINE, CINAHL, PsycInfo, EconLit, and EMBASE; researchers also completed backward and forward citation searching and contacted expert authors. STUDY SELECTION AND DATA COLLECTION: Two independent reviewers completed article screening, selection, and extraction using a scoping review approach; a priori inclusion criteria were peer-reviewed articles, written in English, involving adults with one of five diagnostic conditions, describing any intervention to improve FC. FINDINGS: Twenty-four articles met the inclusion criteria. Most articles were aimed at substance use or mental health populations (n = 20); fewer focused on brain injury (n = 2), multiple sclerosis (n = 1), or mixed-diagnosis (n = 1) populations. Only 4 were randomized controlled trials (RCTs). Interventions were heterogeneous and complex, including components of skills training (n = 21), individualized budgeting (n = 18), representative payeeship (n = 11), education (n = 10), structured goal setting (n = 7), savings building (n = 5), metacognitive strategies (n = 2), and assistive technology (n = 1). CONCLUSIONS AND RELEVANCE: Despite growth in the area, the literature regarding FC intervention is limited, with few RCTs and many populations unrepresented. The literature for a systematic review of FC intervention efficacy for these populations is insufficient, particularly because included studies used varied components, limiting comparison. Further research is imperative to guide evidence-based practice. Plain-Language Summary: This study is an overview of literature about interventions to address the financial occupations of clients with acquired functional impairments. The findings give occupational therapy researchers and clinicians the information they need to begin analyzing, using, and building the evidence to support the use of interventions to improve clients' financial capability and well-being.

Participation in a High-Structure General Chemistry Course Increases Student Sense of Belonging and Persistence to Organic Chemistry.

A parallel series of general chemistry courses for Life Science Majors was created in an effort to support students and improve general chemistry outcomes. We created a two-quarter enhanced general chemistry course series that is not remedial, but instead implements several evidence-based teaching practices including Process Oriented Guided Inquiry Learning (POGIL), Peer-Led Team Learning (PLTL), and the Learning Assistant (LA) model. We found that students who took enhanced general chemistry had higher persistence to the subsequent first organic chemistry course, and performed equally well in the organic course compared to their peers who took standard general chemistry. Students in the first enhanced general chemistry course also reported significantly higher belonging, although we were unable to determine if increased belonging was associated with the increased persistence to organic chemistry. Rather we found that the positive association between taking the enhanced general chemistry course and persistence to organic chemistry was mediated by higher grades received in the enhanced general chemistry course. Our findings highlight the responsibility we have as educators to carefully consider the pedagogical practices we use, in addition to how we assign student grades.

NOX4 regulates macrophage apoptosis resistance to induce fibrotic progression.

Pulmonary fibrosis is a progressive lung disease often occurring secondary to environmental exposure. Asbestos exposure is an important environmental mediator of lung fibrosis and remains a significant cause of disease despite strict regulations to limit exposure. Lung macrophages play an integral role in the pathogenesis of fibrosis induced by asbestos (asbestosis), in part by generating reactive oxygen species (ROS) and promoting resistance to apoptosis. However, the mechanism by which macrophages acquire apoptosis resistance is not known. Here, we confirm that macrophages isolated from asbestosis subjects are resistant to apoptosis and show they are associated with enhanced mitochondrial content of NADPH oxidase 4 (NOX4), which generates mitochondrial ROS generation. Similar results were seen in chrysotile-exposed WT mice, while macrophages from Nox4-/- mice showed increased apoptosis. NOX4 regulated apoptosis resistance by activating Akt1-mediated Bcl-2-associated death phosphorylation. Demonstrating the importance of NOX4-mediated apoptosis resistance in fibrotic remodeling, mice harboring a conditional deletion of Nox4 in monocyte-derived macrophages exhibited increased apoptosis and were protected from pulmonary fibrosis. Moreover, resolution occurred when Nox4 was deleted in monocyte-derived macrophages in mice with established fibrosis. These observations suggest that NOX4 regulates apoptosis resistance in monocyte-derived macrophages and contributes to the pathogenesis of pulmonary fibrosis. Targeting NOX4-mediated apoptosis resistance in monocyte-derived macrophages may provide a novel therapeutic target to protect against the development and/or progression of pulmonary fibrosis.

Post-translational regulation of PGC-1α modulates fibrotic repair.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease associated with mitochondrial oxidative stress. Mitochondrial reactive oxygen species (mtROS) are important for cell homeostasis by regulating mitochondrial dynamics. Here, we show that IPF BAL cells exhibited increased mitochondrial biogenesis that is, in part, due to increased nuclear expression of peroxisome proliferator-activated receptor-É£ (PPARÉ£) coactivator (PGC)-1α. Increased PPARGC1A mRNA expression directly correlated with reduced pulmonary function in IPF subjects. Oxidant-mediated activation of the p38 MAPK via Akt1 regulated PGC-1α activation to increase mitochondrial biogenesis in monocyte-derived macrophages. Demonstrating the importance of PGC-1α in fibrotic repair, mice harboring a conditional deletion of Ppargc1a in monocyte-derived macrophages or mice administered a chemical inhibitor of mitochondrial division had reduced biogenesis and increased apoptosis, and the mice were protected from pulmonary fibrosis. These observations suggest that Akt1-mediated regulation of PGC-1α maintains mitochondrial homeostasis in monocyte-derived macrophages to induce apoptosis resistance, which contributes to the pathogenesis of pulmonary fibrosis.

Cadmium-mediated lung injury is exacerbated by the persistence of classically activated macrophages.

Heavy metals released into the environment have a significant effect on respiratory health. Lung macrophages are important in mounting an inflammatory response to injury, but they are also involved in repair of injury. Macrophages develop mixed phenotypes in complex pathological conditions and polarize to a predominant phenotype depending on the duration and stage of injury and/or repair. Little is known about the reprogramming required for lung macrophages to switch between these divergent functions; therefore, understanding the mechanism(s) by which macrophages promote metabolic reprogramming to regulate lung injury is essential. Here, we show that lung macrophages polarize to a pro-inflammatory, classically activated phenotype after cadmium-mediated lung injury. Because metabolic adaptation provides energy for the diverse macrophage functions, these classically activated macrophages show metabolic reprogramming to glycolysis. RNA-Seq revealed up-regulation of glycolytic enzymes and transcription factors regulating glycolytic flux in lung macrophages from cadmium-exposed mice. Moreover, cadmium exposure promoted increased macrophage glycolytic function with enhanced extracellular acidification rate, glycolytic metabolites, and lactate excretion. These observations suggest that cadmium mediates the persistence of classically activated lung macrophages to exacerbate lung injury.

A novel tree shrew model of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapy. Animal models effectively reproducing IPF disease features are needed to study the underlying molecular mechanisms. Tree shrews are genetically, anatomically, and metabolically closer to humans than rodents or dogs; therefore, the tree shrew model presents a unique opportunity for translational research in lung fibrosis. Here we demonstrate that tree shrews have in vivo and in vitro fibrotic responses induced by bleomycin and pro-fibrotic mediators. Bleomycin exposure induced lung fibrosis evidenced by histological and biochemical fibrotic changes. In primary tree shrew lung fibroblasts, transforming growth factor beta-1 (TGF-ß1) induced myofibroblast differentiation, increased extracellular matrix (ECM) protein production, and focal adhesion kinase (FAK) activation. Tree shrew lung fibroblasts showed enhanced migration and increased matrix invasion in response to platelet derived growth factor BB (PDGF-BB). Inhibition of FAK significantly attenuated pro-fibrotic responses in lung fibroblasts. The data demonstrate that tree shrews have in vivo and in vitro fibrotic responses similar to that observed in IPF. The data, for the first time, support that the tree shrew model of lung fibrosis is a new and promising experimental animal model for studying the pathophysiology and therapeutics of lung fibrosis.

Macrophage Rac2 Is Required to Reduce the Severity of Cigarette Smoke-induced Pneumonia.

RATIONALE: Cigarette smoking is prevalent in the United States and is the leading cause of preventable diseases. A prominent complication of smoking is an increase in lower respiratory tract infections (LRTIs). Although LRTIs are known to be increased in subjects that smoke, the mechanism(s) by which this occurs is poorly understood. OBJECTIVES: Determine how cigarette smoke (CS) reduces reactive oxygen species (ROS) production by the phagocytic NOX2 (NADPH oxidase 2), which is essential for innate immunity in lung macrophages. METHODS: NOX2-derived ROS and Rac2 (Ras-related C3 botulinum toxin substrate 2) activity were determined in BAL cells from wild-type and Rac2-/- mice exposed to CS or cadmium and in BAL cells from subjects that smoke. Host defense to respiratory pathogens was analyzed in mice infected with Streptococcus pneumoniae. MEASUREMENTS AND MAIN RESULTS: NOX2-derived ROS in BAL cells was reduced in mice exposed to CS via inhibition of the small GTPase Rac2. These mice had greater bacterial burden and increased mortality compared with air-exposed mice. BAL fluid from CS-exposed mice had increased levels of cadmium, which mediated the effect on Rac2. Similar observations were seen in human subjects that smoke. To support the importance of Rac2 in the macrophage immune response, overexpression of constitutively active Rac2 by lentiviral administration increased NOX2-derived ROS, decreased bacterial burden in lung tissue, and increased survival compared with CS-exposed control mice. CONCLUSIONS: These observations suggest that therapies to maintain Rac2 activity in lung macrophages restore host defense against respiratory pathogens and diminish the prevalence of LRTIs in subjects that smoke.

Macrophages utilize the mitochondrial calcium uniporter for profibrotic polarization.

Fibrosis in multiple organs, including the liver, kidney, and lung, often occurs secondary to environmental exposure. Asbestos exposure is one important environmental cause of lung fibrosis. The mechanisms that mediate fibrosis is not fully understood, although mitochondrial oxidative stress in alveolar macrophages is critical for fibrosis development. Mitochondrial Ca2+ levels can be associated with production of reactive oxygen species. Here, we show that patients with asbestosis have higher levels of mitochondrial Ca2+ compared with normal patients. The mitochondrial calcium uniporter (MCU) is a highly selective ion channel that transports Ca2+ into the mitochondrial matrix to modulate metabolism. Asbestos exposure increased mitochondrial Ca2+ influx in alveolar macrophages from wild-type, but not MCU+/-, mice. MCU expression polarized macrophages to a profibrotic phenotype after exposure to asbestos, and the profibrotic polarization was regulated by MCU-mediated ATP production. Profibrotic polarization was abrogated when MCU was absent or its activity was blocked. Of more importance, mice that were deficient in MCU were protected from pulmonary fibrosis. Regulation of mitochondrial Ca2+ suggests that MCU may play a pivotal role in the development of fibrosis and could potentially be a therapeutic target for pulmonary fibrosis.-Gu, L., Larson-Casey, J. L., Carter, A. B. Macrophages utilize the mitochondrial calcium uniporter for profibrotic polarization.

Cu,Zn-Superoxide Dismutase-Mediated Redox Regulation of Jumonji Domain Containing 3 Modulates Macrophage Polarization and Pulmonary Fibrosis.

M2 macrophages are implicated in the development of pulmonary fibrosis as they generate profibrotic signals. The polarization process, at least in part, is regulated by epigenetic modulation. Because Cu,Zn-superoxide dismutase-induced H2O2 can polarize macrophages to a profibrotic M2 phenotype, we hypothesized that modulation of the redox state of the cell is involved in the epigenetic modulation of the macrophage phenotype. In this study, we show that signal transducer and activator of transcription 6 (STAT6) regulates Jumonji domain containing (Jmjd) 3, a histone H3 lysine 27 demethylase, and mutation of a redox-sensitive cysteine in STAT6 attenuates jmjd3 expression. Moreover, Jmjd3 deficiency abrogates profibrotic M2 gene expression. Treatment with leflunomide, which reduces mitochondrial reactive oxygen species production and tyrosine phosphorylation, inhibits jmjd3 expression and M2 polarization, as well as development of a fibrotic phenotype. Taken together, these observations provide evidence that the redox regulation of Jmjd3 is a unique regulatory mechanism for Cu,Zn-superoxide dismutase-mediated profibrotic M2 polarization. Furthermore, leflunomide, which reduces reactive oxygen species production and tyrosine phosphorylation, may prove to be therapeutic in the treatment of asbestos-induced pulmonary fibrosis.

Alternative activation of macrophages and pulmonary fibrosis are modulated by scavenger receptor, macrophage receptor with collagenous structure.

Alternative activation of alveolar macrophages is linked to fibrosis following exposure to asbestos. The scavenger receptor, macrophage receptor with collagenous structure (MARCO), provides innate immune defense against inhaled particles and pathogens; however, a receptor for asbestos has not been identified. We hypothesized that MARCO acts as an initial signaling receptor for asbestos, polarizes macrophages to a profibrotic M2 phenotype, and is required for the development of asbestos-induced fibrosis. Compared with normal subjects, alveolar macrophages isolated from patients with asbestosis express higher amounts of MARCO and have greater profibrotic polarization. Arginase 1 (40-fold) and IL-10 (265-fold) were higher in patients. In vivo, the genetic deletion of MARCO attenuated the profibrotic environment and pulmonary fibrosis in mice exposed to chrysotile. Moreover, alveolar macrophages from MARCO(-/-) mice polarize to an M1 phenotype, whereas wild-type mice have higher Ym1 (>3.0-fold) and nearly 7-fold more active TGF-ß1 in bronchoalveolar lavage (BAL) fluid (BALF). Arg(432) and Arg(434) in domain V of MARCO are required for the polarization of macrophages to a profibrotic phenotype as mutation of these residues reduced FIZZ1 expression (17-fold) compared with cells expressing MARCO. These observations demonstrate that a macrophage membrane protein regulates the fibrotic response to lung injury and suggest a novel target for therapeutic intervention.

Resonance Raman and temperature-dependent electronic absorption spectra of cavity and noncavity models of the hydrated electron.

Most of what is known about the structure of the hydrated electron comes from mixed quantum/classical simulations, which depend on the pseudopotential that couples the quantum electron to the classical water molecules. These potentials usually are highly repulsive, producing cavity-bound hydrated electrons that break the local water H-bonding structure. However, we recently developed a more attractive potential, which produces a hydrated electron that encompasses a region of enhanced water density. Both our noncavity and the various cavity models predict similar experimental observables. In this paper, we work to distinguish between these models by studying both the temperature dependence of the optical absorption spectrum, which provides insight into the balance of the attractive and repulsive terms in the potential, and the resonance Raman spectrum, which provides a direct measure of the local H-bonding environment near the electron. We find that only our noncavity model can capture the experimental red shift of the hydrated electron's absorption spectrum with increasing temperature at constant density. Cavity models of the hydrated electron predict a solvation structure similar to that of the larger aqueous halides, leading to a Raman O-H stretching band that is blue-shifted and narrower than that of bulk water. In contrast, experiments show the hydrated electron has a broader and red-shifted O-H stretching band compared with bulk water, a feature recovered by our noncavity model. We conclude that although our noncavity model does not provide perfect quantitative agreement with experiment, the hydrated electron must have a significant degree of noncavity character.

Modulation of the mevalonate pathway by akt regulates macrophage survival and development of pulmonary fibrosis.

Protein kinase B (Akt) is a key effector of multiple cellular processes, including cell survival. Akt, a serine/threonine kinase, is known to increase cell survival by regulation of the intrinsic pathway for apoptosis. In this study, we found that Akt modulated the mevalonate pathway, which is also linked to cell survival, by increasing Rho GTPase activation. Akt modulated the pathway by phosphorylating mevalonate diphosphate decarboxylase (MDD) at Ser(96). This phosphorylation in macrophages increased activation of Rac1, which enhanced macrophage survival because mutation of MDD (MDDS96A) induced apoptosis. Akt-mediated activation in macrophages was specific for Rac1 because Akt did not increase activity of other Rho GTP-binding proteins. The relationship between Akt and Rac1 was biologically relevant because Akt(+/-) mice had significantly less active Rac1 in alveolar macrophages, and macrophages from Akt(+/-) mice had an increase in active caspase-9 and -3. More importantly, Akt(+/-) mice were significantly protected from the development of pulmonary fibrosis, suggesting that macrophage survival is associated with the fibrotic phenotype. These observations for the first time suggest that Akt plays a critical role in the development and progression of pulmonary fibrosis by enhancing macrophage survival via modulation of the mevalonate pathway.

Asbestos-induced disruption of calcium homeostasis induces endoplasmic reticulum stress in macrophages.

Although the mechanisms for fibrosis development remain largely unknown, recent evidence indicates that endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) may act as an important fibrotic stimulus in diseased lungs. ER stress is observed in lungs of patients with idiopathic pulmonary fibrosis. In this study we evaluated if ER stress and the UPR was present in macrophages exposed to chrysotile asbestos and if ER stress in macrophages was associated with asbestos-induced pulmonary fibrosis. Macrophages exposed to chrysotile had elevated transcript levels of several ER stress genes. Macrophages loaded with the Ca(2+)-sensitive dye Fura2-AM showed that cytosolic Ca(2+) increased significantly within minutes after chrysotile exposure and remained elevated for a prolonged time. Chrysotile-induced increases in cytosolic Ca(2+) were partially inhibited by either anisomycin, an inhibitor of passive Ca(2+) leak from the ER, or 1,2-bis(2-aminophenoxyl)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular Ca(2+) chelator known to deplete ER Ca(2+) stores. Anisomycin inhibited X-box-binding protein 1 (XBP1) mRNA splicing and reduced immunoglobulin-binding protein (BiP) levels, whereas BAPTA-AM increased XBP1 splicing and BiP expression, suggesting that ER calcium depletion may be one factor contributing to ER stress in cells exposed to chrysotile. To evaluate ER stress in vivo, asbestos-exposed mice showed fibrosis development, and alveolar macrophages from fibrotic mice showed increased expression of BiP. Bronchoalveolar macrophages from asbestosis patients showed increased expression of several ER stress genes compared with normal subjects. These findings suggest that alveolar macrophages undergo ER stress, which is associated with fibrosis development.