Chronic obstructive pulmonary disease (COPD) is a progressive lung disorder linked to oxidative stress, mitochondrial damage, and impaired mitophagy. Tanreqing (TRQ) inhalation solution is widely used for respiratory diseases but its mechanism against COPD remains poorly defined. Here we investigated the protective effects of TRQ and its underlying pathway in a COPD rat model and cigarette smoke extract (CSE)-stimulated A549 cells. Our results showed that TRQ treatment alleviated body weight loss, lung pathological injury, alveolar destruction, and airway remodeling in rats. TRQ reduced ROS and MDA levels, restored SOD activity, and improved mitochondrial function including ATP production, mitochondrial membrane potential, and mitochondrial permeability transition pore homeostasis. Moreover, TRQ enhanced autophagy and mitophagy by upregulating Bnip3, Nix, Pink1, and Parkin expression. In vitro, knockdown of Pink1 abolished TRQ-mediated mitophagy activation, confirming that the Pink1/Parkin pathway was essential for TRQ action. In conclusion, TRQ inhalation solution ameliorates COPD by suppressing oxidative stress and restoring mitochondrial homeostasis via activating Pink1/Parkin-mediated mitophagy. This study identifies TRQ as a promising candidate for COPD treatment.

Since humans spend the majority of their time indoors, exposure to indoor dust is a primary route for air pollutant inhalation. Although growing evidence links indoor dust to respiratory impairments, the mechanisms underlying its association with lung damage and immune homeostasis remain poorly understood. In this study, indoor dust samples collected from an industrial city in Northern China (Taiyuan, Shanxi Province) were used to establish a mouse inhalation model at environmentally relevant concentrations. Non-invasive pulmonary function tests using the whole-body plethysmography (WBP) system and histopathological examinations revealed that indoor dust exposure induced significant impairments in lung function, characterized by a 33.34% increase in Penh and a 19.06% reduction in Rpef in the 25 mg/kg group, alongside airflow limitation (reduced ventilatory capacity) and airway obstruction (inflammatory narrowing of the respiratory tract). Exposure also caused alveolar structural damage, interstitial inflammatory infiltration, and pulmonary fibrosis. Transcriptomic profiling (RNA-seq), flow cytometric immunophenotyping, and qRT-PCR further demonstrated disruption of immune homeostasis in the bone marrow (BM). These results suggest a potential interorgan crosstalk between the lung and BM mediated by inflammatory signaling, in which the lung-derived inflammatory signals might alter the hematopoietic function of the BM, while the BM could mobilize immune cells to sustain pulmonary inflammatory responses, further contributing to lung function impairment. This interorgan crosslink offers a new clue for understanding the mechanisms underlying indoor dust-related lung injury through lung-BM immune communication and for identifying candidate intervention targets for environmental respiratory diseases.

White matter repair relies on microglial clearance of cholesterol-rich myelin debris. Microglia have been reported to predominantly depend on de novo sterol synthesis to support this repair process. Lysosomal acid lipase (LAL) is the only known lysosomal enzyme capable of hydrolyzing cholesterol esters. In stark contrast to previous studies, we demonstrate here that LAL-mediated lysosomal lipolysis—not de novo sterol synthesis—serves as a central determinant of the microglial capacity to drive white matter repair. Using single-cell RNA sequencing, we identified a novel reparative microglial state characterized by simultaneously high expression of glycoprotein nonmetastatic melanoma protein B (GPNMB) and LAL. Following white matter injury, GPNMB+ microglia expanded and constituted the major microglial subset responsible for myelin debris engulfment. However, GPNMB+ microglia displayed context-dependent capacity to digest internalized myelin debris and mediate remyelination, with marked differences between reparable white matter injury and nonregenerative injury induced by white matter stroke (WMS). Transcriptomic profiling identified LAL as a key regulator of the reparative phenotype in GPNMB+ microglia. Independent of cytosolic lipases—widely regarded as synergistic mediators of LAL in cholesteryl ester hydrolysis, microglial LAL was both indispensable for myelin debris clearance and spontaneous remyelination in the reparable injury model, and sufficient to restore these processes following WMS-induced irreparable white matter injury. Mechanistically, LAL-mediated lysosomal lipolysis constituted the primary pathway for cholesteryl ester hydrolysis in microglia after white matter injury. This pathway converted cholesteryl esters into free cholesterol and activates liver X receptors (LXRs), both of which were required to reprogram microglial into the reparative state. Consistently, LXR activation alone was insufficient to rescue defective white matter repair caused by LAL deficiency. Hydroxypropyl-β-cyclodextrin (HβCD), an FDA-approved drug carrier, effectively lowers intracellular cholesterol levels through incompletely defined mechanisms. HβCD specifically upregulated LAL expression within white matter lesions and promoted remyelination via a LAL-dependent manner following WMS, supporting its potential as a therapeutic agent for WMS. Collectively, this study identifies lysosomal cholesterol ester hydrolysis as a novel therapeutic target for the treatment of irreversible white matter injury.

Pulmonary sarcoidosis has a heterogeneous clinical course, with a subset of patients progressing to chronic disease and fibrosis despite first-line corticosteroid therapy. The mechanisms driving disease progression are poorly understood, although limited evidence suggests a role for neutrophils. The present study aimed to characterise neutrophils and neutrophil effector molecules in progressive pulmonary sarcoidosis. We first performed in silico transcriptomic analyses to evaluate neutrophil gene signatures in granulomatous lung tissue from patients with progressive, fibrotic sarcoidosis compared with self-resolving disease. In parallel, we employed vimentin-induced mouse models and performed single-cell RNA-sequencing (scRNA-seq), flow cytometry, and immunostaining to examine neutrophils and assess their contribution to lung injury and fibrosis. Neutrophil gene signatures were significantly enriched in progressive sarcoidosis and positively associated with fibrosis-related pathways while inversely correlating with lung function. In the vimentin-induced model, scRNA-seq, flow cytometry, and bronchoalveolar lavage (BAL) differential analyses demonstrated neutrophil infiltration with altered transcriptomic profile and enhanced activation, which were amplified by repeated vimentin challenge. BAL myeloperoxidase activity and total protein, markers of neutrophil activation and lung injury, were significantly elevated and correlated with neutrophil content. Within granulomas, neutrophils were also abundant and neutrophil extracellular traps (NETs) were identified, along with the detection of collagen deposition and fibroblast activation. Together, these findings demonstrate that activation of neutrophil-driven inflammatory pathways is a prominent feature of progressive pulmonary sarcoidosis and can be triggered by extracellular vimentin. Its strong association with lung injury and fibrosis highlights neutrophils and NETosis as potential therapeutic targets for modifying disease trajectory in progressive sarcoidosis.

Background and ObjectiveAsthma-COPD overlap (ACO) is characterized by patients exhibiting features of both asthma and COPD. Currently, there is no specific treatment for ACO. This study aimed to investigate the therapeutic potential of targeting CD131, a shared receptor subunit for IL-3, IL-5 and GM-CSF, in ACO development and in preventing acute viral exacerbations.MethodsA two-hit mouse model of ACO was established by house dust mite (HDM) allergen sensitization to model asthma, and elastase treatment to model emphysema. In a separate model, human rhinovirus 1b (RV1b) was used to induce an acute asthma exacerbation. A neutralizing antibody against CD131 was used to block CD131 in vivo signalling.ResultsMice exposed to HDM and elastase developed cardinal features for asthma and COPD, including airway hyperreactivity (AHR) and emphysema. A mixed granulocytic inflammatory profile was identified in the lungs, including expansion of monocyte-derived macrophages, neutrophils and eosinophils. RT-qPCR analysis detected heightened gene expression of Mmp12, Il5 and Il13. Transcriptomic analysis further revealed pathway enrichment for type 2 inflammation and macrophage activation. Blockade of CD131 effectively reduced the lung inflammation and prevented the development of AHR, airway fibrosis and emphysema. Interestingly, pathway enrichment for Th1 response and interferon production detected in the model was not affected by the treatment. Consistently, CD131 antagonism prevented RV1b-induced asthma exacerbation without compromising RV1b clearance.ConclusionCD131 signalling coordinates multiple pathological pathways that drive airway inflammation and lung remodelling in ACO. Hence, CD131 antagonism represents a novel approach to combating the immunopathology in the complex ACO setting.

Background COPD is a heterogenous disease where chronic inflammation is implicated in airway remodelling and emphysema. The CD131 receptor is indispensable for signalling by the [3 common ([3c) family of cytokines encompassing granulocyte-macrophage colony-stimulating factor, interleukin (IL)-5 and IL-3, which instigate both type-2 and non-type-2 inflammatory responses. This study aims to determine whether antagonising CD131 signalling can prevent pulmonary inflammation, alveolar cell death and emphysema development. Methods We performed in-house and in silico transcriptomic analysis to investigate the gene expression of CD131 (CSF2RB) and pathway enrichment for [3c cytokine signalling in blood, sputum and lung biopsies of COPD patients. To model emphysema, transgenic mice expressing human CD131 were exposed to elastase or cigarette smoke (CS) and a fully human monoclonal antibody (CSL311) was employed to inhibit CD131 signalling. Results CD131 gene expression was significantly increased in COPD, along with an enrichment of gene set for [3c cytokine signalling. In transgenic mice subjected to emphysema models, CD131 antagonism effectively prevented lung injury, alveolar cell death and emphysema development. Mechanistically, RNA sequencing identified pathway enrichment for myeloid cell activation, type-2 immune response and macrophage alternative activation in elastase-induced emphysematous mice, mirroring human COPD. Blocking CD131 signalling almost completely reversed the global gene expression alterations associated with emphysema development. Conclusions CD131 signalling orchestrates pulmonary inflammation in COPD, resulting in immunopathology that underpins emphysema and lung function decline. Antagonising CD131 therefore represents a unique strategy to simultaneously target multiple pathogenic myeloid cell populations.

IntroductionSarcoidosis is a multisystem chronic inflammatory disorder of unknown etiology that primarily affects the lungs and currently has no cure. Macrophages are central to granuloma formation, and βc cytokines tightly regulate their activation and function. This study investigates the role of the βc receptor in granuloma development and evaluates βc antagonism as a potential therapeutic strategy for sarcoidosis.MethodsWe utilized an in vitro model of human granuloma formation using sarcoidosis patient human peripheral blood mononuclear cells (PBMCs) and an in vivo vimentin-induced pulmonary sarcoidosis model in unique humanized βc transgenic (hβcTg) mice to assess the efficacy of βc antagonism in reducing granuloma formation and evaluate the underlying mechanism of action.ResultsAnti-βc receptor antibody, CSL311, significantly reduced the formation of human granulomas from PBMCs exposed to purified protein derivative and decreased the pro-inflammatory cytokine production by the granulomas. Mechanistically, CSL311 inhibited hyperactivation of mTOR signaling and reduced lipid droplet formation in granuloma macrophages. In hβcTg mice challenged with vimentin, CSL311 effectively reduced both granuloma size and immune cell infiltration in the lung. RNA sequencing analysis of lung tissue further showed that CSL311 treatment suppressed the activation of vimentin-induced inflammatory, fibrotic, and lipid metabolic pathways.ConclusionWe identified that βc cytokines are critical regulators in driving inflammatory and metabolic processes that lead to granuloma formation in sarcoidosis. Precisely targeting the βc receptor effectively disrupts these pathogenic networks and offers a promising new strategy for mitigating sarcoidosis immunopathology.

Neutrophils infiltrate lung tumours and can exhibit an immunosuppressive phenotype that promotes tumour growth, yet their regulation in early-stage lung cancer remains unclear. This study investigates molecular regulators of neutrophils in early-stage lung adenocarcinoma (LUAD) using publicly available gene expression datasets. An early-stage LUAD dataset (GSE31210) was analysed using CIBERSORTx to estimate neutrophil abundance. Weighted gene co-expression network analysis (WGCNA) identified the hub gene most correlated with neutrophil scores. Single-cell RNA sequencing (scRNA-seq) was used to determine the cellular source and regulatory mechanisms of this gene. A high neutrophil score was a negative prognostic factor, validated in independent datasets. WGCNA identified C1QA as the key gene linked to neutrophil abundance. scRNA-seq and immunofluorescence staining confirmed macrophages as the primary source of C1QA. Cell–cell communication analysis suggested C1QA interacts with neutrophils via complement receptors, contributing to an immunosuppressive tumour microenvironment. RT-qPCR showed C1QA expression correlated with IL-10 and TGFB1, markers of immunosuppression. C1QA is a poor prognostic marker in LUAD, potentially driving immunosuppressive tumour-associated neutrophils. Targeting C1QA and its pathway may offer a novel therapeutic strategy in early-stage LUAD.

BACKGROUND:Longitudinal studies have identified childhood asthma as a risk factor for obstructive pulmonary disease (COPD) and asthma-COPD overlap (ACO) where persistent airflow limitation can develop more aggressively. However, a causal link between childhood asthma and COPD/ACO remains to be established. Our study aimed to model the natural history of childhood asthma and COPD and to investigate the cellular/molecular mechanisms that drive disease progression.METHODS:Allergic airways disease was established in three-week-old young C57BL/6 mice using house dust mite (HDM) extract. Mice were subsequently exposed to cigarette smoke (CS) and HDM for 8 weeks. Airspace enlargement (emphysema) was measured by the mean linear intercept method. Flow cytometry was utilised to phenotype lung immune cells. Bulk RNA-sequencing was performed on lung tissue. Volatile organic compounds (VOCs) in bronchoalveolar lavage-fluid were analysed to screen for disease-specific biomarkers.RESULTS:Chronic CS exposure induced emphysema that was significantly augmented by HDM challenge. Increased emphysematous changes were associated with more abundant immune cell lung infiltration consisting of neutrophils, interstitial macrophages, eosinophils and lymphocytes. Transcriptomic analyses identified a gene signature where disease-specific changes induced by HDM or CS alone were conserved in the HDM-CS group, and further revealed an enrichment of Mmp12, Il33 and Il13, and gene expression consistent with greater expansion of alternatively activated macrophages. VOC analysis also identified four compounds increased by CS exposure that were paradoxically reduced in the HDM-CS group.CONCLUSIONS:Early-life allergic airways disease worsened emphysematous lung pathology in CS-exposed mice and markedly alters the lung transcriptome.

Macrophages are unique immune cells attracting growing attention as a potential candidate for cell-based therapy for infectious diseases and cancer. Strategies that can reprogramme or gene-edit macrophages hold potential across a spectrum of acute and chronic conditions. Herein, lipid nanoparticles (LNPs) are developed containing the ionizable lipid SM-102, helper lipid monoolein which is known for self-assembly in aqueous solutions into the inverse cubic lyotropic liquid crystalline mesophase, and cholesterol as an mRNA nanocarrier. The immortalized alveolar macrophage cell line (MH-S cells) is utilized to investigate how cholesterol concentration impacts on mRNA delivery which is further validated using primary mouse alveolar macrophages isolated from the bronchoalveolar compartment and human monocyte derived macrophages. By using high-throughput synchrotron small angle X-ray scattering (SAXS), an acidification-induced non-ordered to ordered internal nanostructure transition of the formulated LNPs is observed, following the transition sequence of inverse micellar to hexagonal to cubic mesophase in the pH range from 7 to 4. Cholesterol is identified as another crucial component for superior mRNA transfection in macrophages, contributing to nanostructure transition and protein corona variation. Successful ex vivo mRNA transfection is also achieved in primary macrophages, highlighting the prospectivity of reprogramming macrophages as a cell therapy for lung diseases. This study highlights the critical roles of lipid nanostructure self-assembly, notably the inverse cubic and hexagonal phases, and cholesterol in successful intracellular delivery and transfection of mRNAs to lung macrophages, advancing RNA technology for cell-based therapy for lung diseases. image

Background: Patients with severe asthma can present with eosinophilic type 2 (T2), neutrophilic, or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common b (bc) receptor signals for 3 cytokines, GM-CSF, IL-5, and IL-3, which collectively mediate T2 and neutrophilic inflammation. Objective: To determine the pathogenesis of bc receptor- mediated inflammation and remodeling in severe asthma and to investigate bc antagonism as a therapeutic strategy for mixed granulocytic airway disease. Methods: bc gene expression was analyzed in bronchial biopsy specimens from patients with mild-to-moderate and severe asthma. House dust mite extract and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human bc transgenic mice. Lung tissue gene expression was analyzed by RNA sequencing. The mAb CSL311 targeting the shared cytokine binding site of bc was used to block bc signaling. Results: bc gene expression was increased in patients with severe asthma. CSL311 potently reduced lung neutrophils, eosinophils, and interstitial macrophages and improved airway pathology and lung function in the acute steroid-resistant house dust mite extract model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalized the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. Conclusions: bc cytokines drive steroid-resistant mixed myeloid CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens. (J Allergy Clin Immunol 2024;153:672-83.)

Severe lower respiratory tract disease following influenza A virus (IAV) infection is characterized by excessive inflammation and lung tissue damage, and this can impair lung function. The effect of toll-like receptor 7 (TLR7), which detects viral RNA to initiate antiviral and proinflammatory responses to IAV, on lung function during peak infection and in the resolution phase is not fully understood. Using wild-type (WT) C57BL/6 and TLR7 knockout (TLR7 KO) mice, we found that IAV infection induced airway dysfunction in both genotypes, although in TLR7 KO mice, this dysfunction manifested later, did not affect lung tissue elastance and damping, and was associated with a different immune phenotype. A positive correlation was found between lung dysfunction and the infiltration of neutrophils and Ly6Clo patrolling monocytes at day 7 post-infection. Conversely, in TLR7 KO mice, eosinophil and CD8+ cytotoxic T cells were associated with airway hyperactivity at day 14. IL-5 expression was higher in the airways of IAV-infected TLR7 KO mice, suggesting an enhanced Th2 response due to TLR7 deficiency. This study highlights an underappreciated duality of TLR7 in IAV disease: promoting inflammation-driven lung dysfunction during the acute infection but suppressing eosinophilic and CD8+ T cell-dependent hyperresponsiveness during disease resolution.

Chronic obstructive pulmonary disease (COPD) is a major, incurable respiratory condition that is primarily caused by cigarette smoking (CS). Neurocognitive disorders including cognitive dysfunction, anxiety and depression are highly prevalent in people with COPD. It is understood that increased lung inflammation and oxidative stress from CS exposure may ‘spill over’ into the systemic circulation to promote the onset of these extra-pulmonary comorbidities, and thus impacts the quality of life of people with COPD. The precise role of the ‘spill-over’ of inflammation and oxidative stress in the onset of COPD-related neurocognitive disorders are unclear. The present study investigated the impact of chronic CS exposure on anxiety-like behaviors and social recognition memory, with a particular focus on the role of the ‘spill-over’ of inflammation and oxidative stress from the lungs. Adult male BALB/c mice were exposed to either room air (sham) or CS (9 cigarettes per day, 5 days a week) for 24 weeks and were either daily co-administered with the NOX2 inhibitor, apocynin (5 mg/kg, in 0.01 % DMSO diluted in saline, i.p.) or vehicle (0.01 % DMSO in saline) one hour before the initial CS exposure of the day. After 23 weeks, mice underwent behavioral testing and physiological diurnal rhythms were assessed by monitoring diurnal regulation profiles. Lungs were collected and assessed for hallmark features of COPD. Consistent with its anti-inflammatory and oxidative stress properties, apocynin treatment partially lessened lung inflammation and lung function decline in CS mice. CS-exposed mice displayed marked anxiety-like behavior and impairments in social recognition memory compared to sham mice, which was prevented by apocynin treatment. Apocynin was unable to restore the decreased Bmal1-positive cells, key in cells in diurnal regulation, in the suprachiasmatic nucleus of the hypothalamus to that of sham levels. CS-exposed mice treated with apocynin was associated with a restoration of microglial area per cell and basal serum corticosterone. This data suggests that we were able to model the CS-induced social recognition memory impairments seen in humans with COPD. The preventative effects of apocynin on memory impairments may be via a microglial dependent mechanism.

Background and Purpose: Cardiovascular disease (CVD) affects up to half of the patients with chronic obstructive pulmonary disease (COPD), which exerts deleterious impact on health outcomes and survivability. Vascular endothelial dysfunction marks the onset of cardiovascular disease. The present study examined the effect of a potent NADPH Oxidase (NOX) inhibitor and free-radical scavenger, apocynin, on COPD-related CVD. Experimental Approach: Male BALB/c mice were exposed to either room air (Sham) or cigarette smoke (CS) generated from 9 cigarettes per day, 5 days a week for up to 24 weeks with or without apocynin treatment (5 mg·kg-1·day-1, intraperitoneal injection). Key Results: Eight-weeks of apocynin treatment reduced airway neutrophil infiltration (by 42%) and completely preserved endothelial function and endothelial nitric oxide synthase (eNOS) activity against the oxidative insults of CS exposure. These preservative effects were maintained up until the 24-week time point. 24-week of apocynin treatment exhibited marked benefits on airway inflammation (reduced infiltration of macrophage, neutrophil and lymphocyte) and lung function decline (hyperinflation), and prevented airway collagen deposition by CS exposure. Conclusion and Implications: Limiting NOX activity may slow COPD progression and lower CVD risk, particularly when signs of oxidative stress become evident.