American Journal of Respiratory Cell and Molecular Biology最新文献

Pseudomonas aeruginosa (PA) is responsible for significant morbidity and mortality particularly in patients with chronic lung diseases such as chronic obstructive pulmonary disease (COPD), bronchiectasis, cystic fibrosis (CF) as well as hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP). The rise of antibiotic-resistant PA strains necessitates alternative treatment strategies. Among the different toxins secreted by PA, Exotoxin-A (Exo-A) becomes cytotoxic when cleaved by furin. This study investigates the therapeutic potential of furin inhibitor BOS-318 in mitigating acute lung injury induced by Exo-A and PA infection. Furin inhibition significantly improved survival rates and reduced lung injury in mouse pneumonia models using Exo-A and PA103. Additionally, BOS-318 accelerated bacterial clearance in vivo, and increased phagocytosis by alveolar macrophages. Bulk RNA-seq done on whole lung homogenate at 6 h revealed an immune profile with decreased natural killer (NK) cell signaling in the BOS-318-treated group, possibly due to a decrease in NK recruitment observed at 24 h, suggesting a role of furin in shaping the immune response. Moreover, administration of BOS-318 as a therapeutic strategy results in a protection of the lung epithelium. Overall, our findings demonstrate that furin inhibition protects against PA-induced acute lung injury and hastens bacterial clearance. These results are the first to characterize furin inhibition in animal models and supports its potential use as an adjunctive therapeutic strategy for treating PA infections.

Lung transplantation prolongs survival for many patients with end-stage lung diseases. Though long-term outcomes are limited due to allograft inflammation leading to chronic rejection. In this study, we aimed to identify the role of NK cell receptors in lung transplant recipient outcomes. We hypothesized that Cystic Fibrosis may be a model for systemic inflammation. Peripheral blood mononuclear cells were collected from recipients with CF (n = 6), COPD (n = 6), and healthy donors (n = 7) for NK cell immunophenotyping via spectral flow cytometry and functional killing assays. Plasma B7H6 was also measured in two independent lung transplant cohorts to test the association with rejection. We identified a CF-specific reduction in NKp30 receptor expression, validated functionally against cells expressing the B7H6 ligand. The NKp30 reduction was not NK cell subset-specific, suggesting a systemic influence. Further, we found B7H6 in vitro reduced NKp30-mediated killing of target cells in a dose-dependent fashion. Analysis of soluble B7H6 concentrations in plasma revealed higher soluble B7H6 in CF recipients relative to other groups, suggesting a potentially broader role of soluble B7H6 in lung transplant outcomes. Consequently, B7H6 was higher in recipients without acute graft dysfunction, and higher B7H6 plasma concentrations conferred reduced risk of chronic lung allograft dysfunction and mortality. Single cell RNA sequencing showed B7H6 transcripts were most prevalent on ciliated airway epithelial cells and bronchoalveolar lavage monocytes and that airway B7H6 transcripts were reduced in CLAD. Thus, our data reveal a new role of the NKp30-B7H6 axis in potentiating lung allograft outcomes.

Lactate is increased in the lungs of patients with Idiopathic Pulmonary Fibrosis (IPF) and in mice with experimental lung fibrosis, and lactate has been linked to the pathogenesis of lung fibrosis. IPF fibroblasts in hypoxic conditions generate increased lactate due to an imbalance of lactate dehydrogenase isoforms that induce pyruvate conversion to lactate. Monocarboxylate transporter 4 (MCT4) functions as a key lactate export protein, but its role has not been studied in lung fibrosis. We hypothesized that MCT4 would have a critical role in the ability of IPF fibroblasts to generate extracellular lactate and found that MCT4 was significantly upregulated in IPF fibroblasts under hypoxic conditions. In contrast, the lactate importer protein, monocarboxylate transporter 1 (MCT1) did not significantly change in control or IPF fibroblasts. Pharmacologic inhibition and silencing of MCT4 reduced extracellular lactate generation by IPF fibroblasts. Supporting its role in lung fibrogenesis, MCT4 was increased in bleomycin-injured mice and in lungs from IPF patients. Importantly, normal fibroblasts incubated with conditioned media from IPF fibroblasts exposed to hypoxic conditions had increased α-smooth muscle actin expression that was attenuated by inhibition of MCT4 in the donor IPF fibroblasts or by inhibition of the lactate receptor GPR-81 in the recipient normal fibroblasts. Together, these findings implicate MCT4 in the ability of IPF fibroblasts to increase extracellular lactate and highlight the role of lactate signaling via G-protein coupled receptor-81 in normal fibroblast differentiation. We propose a novel paradigm in which lactate export, driven by increased MCT4 expression, promotes fibrosis in oxygen-deficient microenvironments.

The vacuolar H+-ATPase (V-ATPase) is an enzymatic complex responsible for pumping H + into the cytosol, thereby maintaining intracellular pH; however, its role in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is unclear. In this study, the functional relevance of V-ATPase and hypoxia inducible factor (HIF)-1 were assessed using alveolar-specific ATP6V0C knockout mice (Atp6v0c  AT2-KO) and HIF1A knockout mice (Hif1a  AT2-KO), respectively. ATP6V0C expression levels were measured in serum and bronchoalveolar lavage fluid (BALF) of ARDS patients. ATP6V0C expression was increased in lung tissues from ALI murine models and BALF from severe ARDS patients. Genetic deficiency of ATP6V0C in alveoli attenuated the functional, histological, and inflammatory hallmarks of lipopolysaccharide (LPS)-induced ALI, but did not alter the host's susceptibility to bacterial pathogens. Mechanistically, transcriptomic analyses revealed that ATP6V0C-regulated genes are highly enriched in HIF-1 signaling pathway. HIF-1α was upregulated synchronously with ATP6V0C in injured lungs, while co-immunoprecipitation (Co-IP) confirmed their interaction. Following LPS instillation, the signs of ALI were further exacerbated in Hif1a  fl/fl mice pretreated with lung epithelial tropic adeno-associated virus (AAV) carrying ATP6V0C, yet not in Hif1a  AT2-KO mice. HIF-1α, as a transcriptional factor, in turn, regulated ATP6V0C expression, forming a positive feedback loop. ATP6V0C levels were increased in BALF, yet not serum in ARDS patients. ATP6V0C levels in BALF correlate with ARDS severity. In summary, our study identified an ATP6V0C-HIF-1α detrimental feedback loop that exacerbates epithelial apoptosis and inflammation, thereby driving the progression of ALI. Targeting the ATP6V0C-HIF-1α loop may hence present a promising therapeutic strategy against ALI/ARDS.

Repetitive injury is hypothesized to lead to progressive tissue fibrosis and end-stage organ failure. Whether tissue-resident mesenchymal cell populations retain epigenetic memory of prior injuries that contribute to this pathological process is unknown. Here we used a genetic lineage labeling approach to mark the lung mesenchyme prior to injury, then performed multi-modal analyses on isolated lung mesenchyme during the initiation, progression and resolution of the fibrotic response. Our results demonstrate the remarkable epigenetic and transcriptional plasticity of the lung mesenchyme during fibrotic activation and de-activation. Despite this plasticity, we also find that the lung mesenchyme exhibits an enhanced fibrotic program upon re-injury. We identify RUNX1 as a critical driver of both fibrotic activation and fibrotic memory. Comparison of fresh isolated and cultured lung mesenchyme demonstrates that RUNX1 is spontaneously activated in standard culture conditions, previously masking these roles of RUNX1. Targeted knockdown of RUNX1 dampens fibrotic mesenchymal cell activation immediately after cell isolation, but with reduced efficacy after only days of culture, confirming its functional importance to both early activation and long-term memory. Collectively, our findings implicate RUNX1 in the initiation and memory of fibrotic mesenchymal cell activation that together prime enhanced mesenchymal cell responses upon repeated injury.

Extracellular vesicles (EVs) are increasingly recognized as critical mediators of intercellular communication, transferring a diverse repertoire of proteins, nucleic acids, and bioactive lipids that modulate the functional phenotype of recipient cells in both paracrine and endocrine manner. While the roles of EV-transported microRNAs (miRs) and proteins in pulmonary diseases have been extensively studied, the contribution of EV-encapsulated bioactive lipid mediators to the pathophysiology of pulmonary disorders, including acute respiratory distress syndrome (ARDS), remains largely underexplored. Here, we review the biosynthesis of bioactive lipids, their incorporation into EVs, and their roles in regulating pulmonary inflammation, injury, and resolution. We first highlight upstream signaling pathways, such as toll-like receptor 4 (TLR4) and the nuclear factor of activated T-cells cytoplasmic member 3 (NFATc3), which regulate the expression of lipid biosynthetic enzymes. We then examine how EV-encapsulated pro-inflammatory and pro-resolving lipids contribute to ARDS pathogenesis and clinical outcomes. Evidence supporting the role of EV-transported pro-resolving lipid mediators as key regulators of inflammation resolution and restoration of pulmonary homeostasis is also reviewed, along with their therapeutic potential in mitigating ARDS. Finally, we identify critical gaps in our understanding of how EV lipids modulate ARDS pathophysiology and discuss the challenges and opportunities for therapeutic targeting.

The alternative reading frame (ARF) protein, encoded by the CDKN2A locus, is well-recognized for its role in tumor suppression. Emerging evidence has highlighted ARF as a critical regulator of innate immunity and inflammation, with links to increased susceptibility to cardio-metabolic diseases. This study investigates the role of ARF in lung homeostasis and reveals that its deficiency in mice affects lipid metabolism and leads to pulmonary abnormalities resembling pulmonary alveolar proteinosis (PAP). ARF-deficient mice exhibited abnormal surfactant clearance, characterized by lipid and protein accumulation in the alveoli, foamy alveolar macrophages (AMs) with enlarged and vacuolated morphology, and increased bronchoalveolar lavage fluid (BALF) turbidity. These changes were linked to disrupted surfactant homeostasis resulting from an imbalance between increased lipid uptake (via upregulation of scavenger receptors such as SR-A1 and CD36) and impaired lipid efflux, evidenced by reduced expression of the cholesterol transporter SR-BI. These mice also display reduced AMs numbers, increased eosinophil and neutrophil infiltration, consistent with secondary PAP. Additionally, a distinctive chemokine and cytokine profile (elevated Ccl12, Ccl2, Cxcl1, and IL10) was observed, which may be associated with type 2 immune responses and alternative AMs polarization. Interestingly, ARF deficiency also appears to compromise AMs maintenance through effects on self-renewal and survival. Pulmonary function tests revealed increased tissue elastance and damping, suggesting early-stage lung stiffness. Collectively, these findings highlight the essential role of ARF in lung homeostasis and lipid regulation, providing insights into its potential involvement in PAP pathogenesis.

Mannose receptor C type 2 (MRC2) is highly expressed in the lung and is the major endocytic receptor for the internalization and degradation of collagen in mesenchymal cells. Using Mrc2 knockout (KO) mice, we previously showed that MRC2 is required for efficient clearance of collagen in bleomycin-induced fibrosis. However, MRC2 also interacts with various cell-surface receptors and ligands beyond collagens, indicating that MRC2 may have additional, previously unrecognized functions in fibroblasts. To uncover novel pathways regulated by MRC2, we took an unbiased approach to compare the transcriptomic profile of MRC2-deficient lung fibroblasts to WT after in vitro culture. RNA-seq analysis revealed upregulation of the expression of several extracellular matrix genes but unexpectedly showed changes in expression of several cell cycle genes, including that encoding Forkhead box M1 (FOXM1), a key regulator of cell cycle progression, and enrichment of pathways involved in mitosis and cell division. Both in vitro and in vivo functional assays demonstrated that a greater proportion of MRC2-deficient lung stromal cells progress through the cell cycle more rapidly than WT cells, thereby accelerating overall proliferation. Inhibitor experiments showed that actively proliferating Mrc2 KO fibroblasts are more reliant on FOXM1 activity compared to WT cells, suggesting that FOXM1 is a critical mediator in fibroblast proliferation in the absence of MRC2. Our findings point to an unexpected role for this endocytic receptor in regulation of lung stromal cell proliferation.

Pneumonias remain a leading cause of death worldwide. Seeking novel strategies to protect susceptible patients, we have reported that inhaled delivery of a diacylated lipopeptide and a synthetic CpG oligodeoxynucleotide (ODN) protects animals against a broad range of infectious pneumonias by stimulating antimicrobial responses from the lung epithelium. Toll-like receptor 9 (TLR9) is well-established as the primary cellular receptor for CpG ODNs. However, we recently reported that ODNs also stimulate TLR9-independent generation of antimicrobial mitochondrial reactive oxygen species. By testing a variety of synthetic ODN molecules, we found that ODNs containing a phosphorothioate backbone, but not those with a phosphodiester backbone, induce TLR9-independent pathogen killing in lungs and improve mouse survival. Phosphorothioate-backboned ODN binds mitochondrial protein voltage-dependent anion channel 1 (VDAC1) at its N terminus, initiating pneumonia-protective metabolic reprogramming in lung epithelial cells that yield the protective antimicrobial effect. Thus, the phosphorothioate backbone of ODN is a critical structural pattern that activates TLR9-independent, metabolically-modulated innate immune protection that may be harnessed to protect vulnerable patients against pneumonia.