Chronic obstructive pulmonary disease (COPD) is characterized by a deficiency within the lungs of regulatory T cells (Tregs) and an excess of Th17 cells, termed T17/Treg imbalance. Conventional dendritic cells type-1 (cDC1) and type-2 (cDC2) are known to drive Treg and Th17 polarization respectively, but their roles in COPD are incompletely understood. Using a murine cigarette smoke (CS)-exposure model, we found that after eight weeks of CS exposure the percentage of lung cDC1, but not cDC2, was significantly decreased ( < 0.05) relative to air-exposed controls. Following intranasal adoptive transfer into naïve recipients, cDC1 were less likely to be retained in the lungs and instead were enriched in mediastinal lymph nodes. Co-culture with DCs from cDC1-deficient BATF3-/- mice induced greater expression of intracellular IL-17 protein by naïve lung CD4+ T cells compared to DCs from BATF3+/+ mice ( < 0.001), which instead more greatly induced intracellular IFN-gamma. LPS-stimulated lung DCs from CS-exposed wild-type mice produced significantly higher amounts of the Th17-polarizing cytokines IL-6 and IL-23. Congruently, whole lung homogenates of CS-exposed mice had increased IL-17 protein expression compared to air-exposed mice ( < 0.05). Naïve CD4+ T cells co-cultured with lung DCs from CS-exposed mice produced more IL-17 ( < 0.01) than co-culture with lung DC from air-exposed mice. Thus, loss of cDC1 and predominance of cDC2 in the lungs of CS-exposed mice drive naïve CD4+ T cells towards IL-17 production, and could play a role in the Th17/Treg imbalance seen in COPD.

Despite recent advances in preventative options, respiratory syncytial virus (RSV) infection is still a major cause of hospitalizations of young children and older adults, with no specific treatment available. The aryl hydrocarbon receptor (AHR) is a transcription factor originally identified as the mediator of the toxic effects of environmental pollutants but later shown to be also activated by dietary and endogenous ligands. AHR is involved in various physiological and pathophysiological processes, including host response to infections. Many clinically relevant viruses have been shown to induce AHR activation as a strategy to evade antiviral immunity and promote replication, including the severe acute respiratory syndrome coronavirus 2. It is currently not known whether RSV infection affects the AHR pathway. In this study, we investigated the effects of RSV infection on the AHR signaling pathway by using in vitro and in vivo experimental models. We found that RSV infection led to inhibition of the AHR-dependent gene transcription in human airway epithelial cells and in the lungs of mice. Human lung epithelial cells lacking AHR showed upregulation of genes related to inflammatory response and airway remodeling, as well as increased production of proinflammatory mediators in response to RSV infection. In contrast, administration of the dietary AHR ligand indole-3-carbinol to mice led to beneficial effects on RSV-associated disease, including anti-inflammatory and antiviral activity. Collectively, our results suggest that the AHR has a protective role during RSV infection, and therefore its modulation can be explored as a novel therapeutic target for RSV-induced disease. Our study reveals that respiratory syncytial virus (RSV) downregulates the aryl hydrocarbon receptor (AHR) pathway in human airway epithelial cells and mouse lungs. Loss of the AHR in lung cells led to an exacerbated inflammatory response, and the AHR ligand indole-3-carbinol (I3C) showed in vivo anti-inflammatory and antiviral activity during RSV infection. Our data suggest that AHR plays a protective role during RSV infection and can be explored as a novel therapeutic target.

Newborn infants especially those born preterm often require supplemental oxygen therapy, however, exposure to supraphysiological oxygen (hyperoxia) can disrupt normal lung development and contribute to neonatal lung injury, including bronchopulmonary dysplasia (BPD). Mitochondrial dysfunction is increasingly recognized as a contributor to oxidative lung diseases including BPD. However, the effects of hyperoxia on mitochondrial function and mucociliary differentiation in the developing airway epithelium remain poorly understood. This study tested the hypothesis that hyperoxia impairs neonatal airway mucociliary differentiation by disrupting mitochondrial bioenergetic function. Neonatal tracheal airway epithelial cells (nTAECs) from term infants (n=5) were cultured in a 3D air-liquid interface (ALI) model and exposed to 60% O₂ during the mid-phase of differentiation (ALI day 7-14). Cellular phenotype was assessed using immunofluorescence staining and gene expression analyses. Mitochondrial function was evaluated through Seahorse metabolic flux analysis, and global protein changes were characterized by quantitative proteomics. Hyperoxia significantly impaired terminal differentiation with reduced ciliated and goblet cells. Seahorse assay revealed a decrease in baseline oxygen consumption and mitochondrial ATP production, accompanied by a compensatory increase in glycolytic ATP production. Quantitative proteomics identified disruption of mitochondrial Complex I as a central feature of the hyperoxic response. Downstream proteomic pathway analyses further confirmed the metabolic shift from mitochondrial to glycolytic ATP production and demonstrated altered epithelial differentiation pathways, including NOTCH and TGF-β signaling. These findings reveal that hyperoxia impairs mitochondrial bioenergetics and alters metabolic programming, leading to disrupted mucociliary differentiation. Future studies should evaluate mitochondrial oxidative fitness as a therapeutic target in neonatal lung disease.

The potential for early life air pollutant exposure to result in later onset respiratory disease in children and adults is an emerging public health concern. Fetal growth restriction (FGR) and childhood respiratory infections are associated with impaired lung function in adulthood, and later in life, death from chronic obstructive pulmonary disease (COPD). We previously showed that early gestational exposure of rats to the oxidant air pollutant, ozone, resulted in asymmetrical FGR and lung developmental delays. Herein, we investigate effects of early gestational, periadolescent, and combined ozone exposure on offspring health, lung injury, antioxidant reserve, and innate immune responses. Results revealed similar ozone effects in all offspring irrespective of exposure timing in terms of minor weight loss, reduced body temperature (1.5-2.0°C), and moderate lung injury. Lung injury was inversely correlated with lung antioxidant capacity. Progeny of ozone-exposed dams (i.e., FGR-prone offspring) showed greater variability in ventilatory responses (EF, Penh) and increased Penh correlated with greater lung injury. FGR-prone offspring had more variable, often blunted immunoinflammatory responses to subsequent ozone exposure. Enhanced expression for antioxidant (Nrf2-related or ARE) genes were observed in FGR-prone males, whereas decreased expression for hypoxia (Hif-related or HRE) and RAAS genes (, , and ) was observed in FGR-prone females, potentially suggesting that cross talk between redox transcription factors, Hif/RAAS, NF-κB, and Nrf2 led to differential responses. Collectively, these findings indicate that early life oxidant air pollutant exposure and resultant redox and RAAS dysregulation may impact both lung development and innate immune responses in a sex-dependent manner, effects that may increase vulnerability to respiratory infections. This research investigates exposure factors and potential mechanisms contributing both to FGR and altered innate immune responses, effects that may impair lung function, limit immunity to respiratory pathogens, and perpetuate lung disease risk across the life span. Results suggest that oxidative stress and resultant redox and RAAS imbalance occurring at critical developmental intervals could be a central mechanism by which exposure to oxidant air pollutants negatively affect fetal growth, lung growth, and innate immune responsiveness.

Bronchopulmonary dysplasia (BPD) associated pulmonary hypertension (PH) or BPD-PH is a lung disease of infants with significant morbidity. Adrenomedullin (Adm) is an angiogenic peptide that signals through calcitonin receptor-like receptor (Calcrl) and receptor activity modifying protein 2 (RAMP2). deficiency potentiates hyperoxia-induced experimental BPD-PH in mice; however, whether overexpression can mitigate this lung disease is unclear. Thus, we tested the hypothesis that overexpression attenuates hyperoxia (HO)-induced murine experimental BPD-PH by using a novel transgenic mouse that overexpresses globally ( mice). One-day-old mice or their wild-type littermates ( mice) were exposed to HO ([Formula: see text] 70%) for 14 days and allowed to recover in normoxia (NO, [Formula: see text] 21%) for an additional 14 days. Controls were maintained in NO for 28 days. On postnatal day (P) 14, we harvested the lungs to determine the extent of expression and apoptosis. On P28, we quantified alveolarization, lung vascularization, and PH. HO-exposed mice demonstrated increased lung apoptosis, decreased alveolarization and lung vascularization, and indices of PH, indicating that neonatal HO exposure causes BPD-PH. However, overexpression attenuated experimental BPD-PH, as evident by the decreased extent of hyperoxia-induced lung apoptosis and inflammation, alveolar and vascular simplification, pulmonary vascular remodeling, and PH in mice than in mice. Collectively, our results demonstrate that overexpression attenuates HO-induced murine experimental BPD-PH, emphasizing the therapeutic potential of Adm for BPD-PH in preterm infants. The deficiency of the proangiogenic peptide, adrenomedullin (Adm), exacerbates the severe infantile lung disorder, bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH), in mice. However, whether Adm therapy can mitigate this disease is unclear. Our study, conducted with a rigorous methodology, suggests a potential solution. Using a novel mouse that overexpresses Adm to overcome the pharmacological limitations of the peptide, we demonstrate that Adm can mitigate this disorder, highlighting the therapeutic potential of Adm for human BPD-PH.

Lung surfactant (LS) plays an essential role in preventing lung collapse due to physical forces by forming surface-active lipid-protein membranous films at the respiratory air-liquid interface. Throughout its biological cycle, LS exists in a variety of metabolically related, conspicuous morphological forms. Epithelial alveolar type II cells store LS as intracellular, tightly packed, multilayered organelles known as lamellar bodies. These are secreted as still-condensed material in the form of lamellar body-like particles, which, upon adsorption, give rise to the interfacial film and surface-associated structures. Surfactant material purified from bronchoalveolar lavage fluids has been extensively examined by conventional transmission electron microscopy (TEM), providing important information about LS ultrastructure. However, potential artifacts associated with classical TEM preparation methods-such as staining, dehydration, resin embedding, and sectioning-hinder the observation of surfactant biological samples in their truly native state. In this work, we have taken advantage of cutting-edge cryo-microscopy techniques to visualize the structural complexity present in LS preparations without fixation, in a frozen-hydrated state, and thus closer to physiological conditions. The implementation of cryo-preservation approaches has allowed us to unveil unprecedented ultrastructural details of the diverse morphological states in which LS is present in the alveolar spaces, such as the presence of a protein-based pore connecting the lumen of the LBPs with the external milieu, and an onion-like structure that suggests a mechanism that uses the energy accumulated upon LB assembly in the pneumocytes for a rapid release of the membranous complexes to the exterior. These morphological features shed light on the dynamic processes by which LS is unpacked from secreted condensed states to the more disorganized, interconnected membranous networks that sustain breathing mechanics.

A sustained contraction of the airway smooth muscle increases its contractile capacity through a time-dependent process called force adaptation and concordantly increases the response to methacholine in healthy subjects. Whether this occurs in asthma remains to be investigated. The present study aimed at evaluating force adaptation on the methacholine response in asthmatic patients. Thirty-four very mild to mild asthmatic patients underwent a methacholine challenge on two separate visits. Although the same cumulative concentration was administered on both visits, one challenge was preceded by force adaptation induced by inhaling low concentrations of methacholine at 5-min intervals. On each visit, respiratory mechanics were monitored before and throughout the methacholine challenge by oscillometry, and the degree of inflammation was assessed by measuring the fraction of exhaled nitric oxide. The results demonstrate that the final response to methacholine was greater in the challenge with than without force adaptation. For example, whereas the average change in respiratory system reactance caused by the methacholine challenge without force adaptation was 55.4 ± 67.9%, it amounted to 118.1 ± 150.7% with force adaptation ( = 0.0069). Interestingly, force adaptation on the methacholine response was weakly but negatively correlated with the degree of inflammation. In fact, when patients were split into two groups, one with the least inflammation and one with the most inflammation, force adaptation potentiated the methacholine response in the former but not in the latter. We conclude that although force adaptation potentiates the response to methacholine in asthmatic patients, this effect is mainly driven by patients with very little inflammation. A sustained contraction of the airway smooth muscle (ASM) increases its contractility through force adaptation, but whether this alters the methacholine response in asthma is unknown. Asthmatic patients underwent two methacholine challenges with identical cumulative concentration but one including a period of ASM preactivation. The preactivation enhanced the response in patients with little inflammation but not in more inflamed patients. This suggests that force adaptation increases the methacholine response only in patients with low inflammation.

The pulmonary alveolar-capillary niche is a highly specialized interface that balances gas exchange with maintenance functions and repair. Advances in single cell transcriptomics have uncovered endothelial heterogeneity, which underlies developmental angiogenesis and plastic responses to injury. Emerging evidence from a neonatal hyperoxia model highlights CAP1 to CAP2 transitions and the role of p53 in maintaining lineage fidelity. Beyond intrinsic lineage plasticity, circulating mediators such as cell-free hemoglobin drive endothelial barrier disruption through oxidative injury and lipid modification. As new signaling pathways and therapeutics targets emerge, complementary strategies are being developed at the cellular level, including adoptive transfer of mesenchymal stromal and immune cells, although mechanisms of endothelial adhesion and homing remain incompletely defined. Finally, biomechanical forces such as shear stress have become critical contextual cues for endothelial signaling, yet remain underrepresented in some experimental models. Together, these insights underscore the central role of endothelial heterogeneity, injury responses, and environmental cues in shaping pulmonary vascular health and repair, with implications for designing targeted therapies in both pediatric and adult lung disease.

Intravenous bolus (ivb) injection of fentanyl triggers a vagal-mediated immediate apnea and subsequent respiratory depression in anesthetized rats. This study compared the gender-dependence of these responses in conscious rats and roles of peripheral and central opioid receptors (ORs), especially µ- and mu1 opioid receptor (µ-ORs) in the genesis of these responses. Cardiorespiratory responses to ivb injection of fentanyl (50 µg·kg) were recorded in male and female conscious rats (). The same protocols were performed after: naloxone (NLX) and naloxone methiodide (NLM) to systemically and peripherally antagonize ORs, respectively (); D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and methylnaltrexone (MNTX) to systemically and peripherally block opioid mu-receptors (); and naloxonazine (NLZ) to systemically block µ-ORs (). ivb injection of fentanyl induced an immediate life-threatening apnea (∼1.5 min) and severe bradycardia, which was followed by cardiorespiratory depression lasting for ∼55 min with little difference between genders. NLX fully eliminated and CTAP substantially blunted all cardiorespiratory responses to fentanyl, whereas NLM and MNTX substantially minimized the immediate apnea and reduced bradycardia by ∼50% with limited impact on the subsequent cardiorespiratory depression. NLZ nearly abolished the fentanyl-evoked responses. Our results indicate that peripherally restricted OR (particularly µ-OR) antagonism prevents the fentanyl-induced immediate apnea, but fails to change the subsequent respiratory depression. The cardiorespiratory responses to rapid intravenous injection of fentanyl have not been fully investigated. We demonstrate in this study that intravenous bolus injection of fentanyl triggers an immediate sustained apnea and subsequent respiratory depression without remarkable gender-difference in conscious rats. The immediate apnea is triggered by activating peripheral opioid receptors and the subsequent respiratory depression is mediated by activating central opioid receptors, in which µ1-opioid receptors play a key role.

Session III of the inaugural biennial Research Symposium on Pulmonary Injury and Repair of the Endothelium (ReSPIRE) highlighted key advancements in endothelial-inflammatory cell interactions. The work presented illustrates a growing recognition that pulmonary endothelial cell interactions and direct cross talk with inflammatory cells are integral in both health and disease in the developing and aging lung. Data presented detail the impact of targeting neutrophil- and macrophage-endothelial interactions in acute lung injury, and the role of fibroblast-endothelial inflammatory communication in interstitial pulmonary fibrosis of the aging lung. In the developing lung, the paradoxical responses of the pulmonary circulation to inflammatory cell interactions and mediators illustrate the complexities in cross talk. State-of-the-art advances in intravital microscopy have recently revealed our ability to visualize and measure the mechanotransduction involved in neutrophil migration. This review highlights these recent advances and suggests future directions for understanding pulmonary endothelial-inflammatory cell cross talk.

Idiopathic pulmonary fibrosis (IPF) is a serious respiratory disease with a poor prognosis and limited treatments. IPF is characterised by accumulation of extracellular matrix, destruction of gas exchange units and decline in lung function. The pathogenesis and underlying mechanisms are poorly understood although a combination of environmental and genetic factors is implicated. Epigenetic modifications represent a facet of gene regulation likely important to key pathways including transforming growth factor (TGF-β1) signalling, fibroblast-to-myofibroblast differentiation, epithelial-to-mesenchymal transition, cellular aging and apoptosis. Methylation, acetylation, non-coding RNAs, telomere length and G-quadruplexes have all been investigated with varying levels of progress but are certainly potential targets for drug development. This review discusses the underlying epigenetic biology of IPF, associated clinical applications and potential novel treatments.

The co-development and interactions between structural and immune cells in the human fetal lung can be studied using transcriptomic and proteomic approaches. Here, we utilized imaging mass cytometry with a 31-antibody panel to visualize immune and structural cell development in human fetal lung tissue from elective abortions across the pseudoglandular and canalicular stages, spanning post-conception weeks (pcw) 6 (n=1), 8 (n=1), 10 (n=1), 13 (n=2) and 18 (n=3). This approach allows us to map the developing structural components of the human fetal lung. During the early pseudoglandular stage, keratin-8 (KRT8) epithelial structures appeared, gradually developing into KRT8EpCAM budding tips and elongated luminal structures. By the late pseudoglandular and canalicular stages, these luminal structures were lined by KRT8D2-40 cells, with D2-40 (Podoplanin) known to be expressed in alveolar epithelial and lymphatic endothelial cells. Surrounding these structures were layers of α-smooth muscle actin cells. The immune compartment was predominantly myeloid in origin. CD206CD68 macrophages were present as early as the pseudoglandular stage, whereas HLA-DR myeloid cells appeared later around 13 pcw. Cellular interaction analysis revealed an accumulation of HLA-DR cells near the KRT8EpCAM structural regions, suggestive of interactions between these cells during lung development. Our findings illustrate the dynamic development of structural and immune cell components in the human fetal lung throughout the pseudoglandular and canalicular stages. Furthermore, the observed interactions between structural and HLA-DR immune cells support the notion that immune-structural cross talk plays a role in human fetal lung development.

Lung fibroblasts generate and respond to mechanical, biochemical, and matrix cues present in their microenvironment. With the advent of next-generation sequencing technologies, multiple studies describe transcriptionally unique fibroblast subpopulations in the human lung. However, limited published data suggest a loss of fibroblast native phenotypes and functions after culture ex vivo. In this study, we characterized changes in transcriptional programs of human lung mesenchyme isolated from freshly procured tissue and maintained in traditional cell culture conditions. Our results demonstrate that fibroblasts isolated and cultured in this manner adopt transcriptional programs largely distinct from those observed . To recapitulate distinct native fibroblast states , we sought to develop a screening approach to identify cues promoting native fibroblast identities. From published single-cell data, we identified unique transcriptional markers of alveolar and adventitial fibroblast subpopulations and validated the sensitivity of ELISAs for detecting changes in secreted markers of these fibroblast subpopulations. We then stimulated primary human lung fibroblasts with soluble cues known to act on fibroblasts, quantifying changes in secreted and transcriptional markers by ELISA and qPCR. While our small pilot screen did not identify single cues capable of fully recapitulating fibroblast states, it established a system that can be expanded to broadly screen additional cues and pointed toward factors likely to be critical in developing better culture models for studying human lung fibroblast function and plasticity.

The respiratory system is integrated to optimize efficiency. Dysfunction of one element often impacts others. For example, in COPD, both OSA and small airways dysfunction are associated with worse emphysema. In BPD, cystic lung disease and tracheobronchomalacia are often comorbid. Further, childhood asthma predisposes to COPD. While mouse models have elucidated key mechanisms in respiratory disease, to date no models have accounted for how conducting airway dysfunction impacts alveolar structure and function. We report a novel murine partial tracheal occlusion (PTO) model and a complementary esophageal pressure monitoring technique to begin answering these questions. A fifty percent reduction in trachea diameter was achieved using a 19-gauge needle to prevent complete closure of a microsurgical clip on the anterior trachea. Esophageal pressure was measured by advancing a 3.5 French pressure transducing catheter 3 cm into the esophagus. In 8-10-week-old C57BL/6 mice, PTO did not cause appreciable alteration of distal lung structure despite a 10 mmHg increase in transpulmonary pressure gradient. However, PTO after tracheal aspiration of 0.5 units of porcine pancreatic elastase (PPE) resulted in 20 μm greater (p<0.001) mean linear intercept (MLI) than PPE+sham. This model can be leveraged in mouse models of asthma, BPD, and COPD to understand how conducting airway dysfunction and increased transpulmonary pressure impacts distal lung structure. The PTO model is a relatively simple, well tolerated model of conducting airway dysfunction that potentiates distal lung injury and expands our understanding of how mechanical forces influence pathological remodeling processes in the distal lung.

Survivors of COVID-19 can experience long-term lung complications (pulmonary sequelae), but the underlying mechanisms remain unclear. While most patients with COVID-19 lung injury eventually recover essentially completely, some experience significant residual damage. To investigate the underlying differences, we analyzed, using bronchoalveolar lavage fluid (BALF), the alveolar immune cell compartments of a group of patients with post-COVID-19 interstitial lung disease (ILD) six months after acute COVID-19. Patients were categorized into two groups, based on High-Resolution Computed Tomography (HRCT) evaluation a year later: those with persistent HRCT abnormalities compatible with fibrosis (persistent post-COVID-19 ILD, n=6) and those with resolved lung lesions (resolved post-COVID-19 ILD, n=13). Additionally, 6 patients with pre-existing ILD were included in the study, after recovery from COVID-19. Bulk RNA transcriptomics analyses of BALF cells revealed innate immunity and inflammation pathways of neutrophil and monocyte chemotaxis to be enriched in patients with persistent HRCT abnormalities post-COVID-19, consistent with an increase in monocyte-like cell recruitment in the lungs. Pro-fibrotic SPP1 gene expression was significantly upregulated similarly to other fibrotic lung diseases. Conversely, patients with resolved post-COVID-19 ILD showed enhanced BALF cell gene expression signatures indicative of adaptive immune response activation. BALF gene expression patterns of low T-cell activation, high pro-fibrotic macrophage activation and neutrophil chemotaxis were similarly observed in patients with pre-existing fibrotic ILD following COVID-19. These findings suggest that immune response imbalance leading to prolonged activation of innate immunity and subdued adaptive immune responses may be associated with persistent post-COVID-19 ILD and the development of pulmonary fibrosis.

Hyperoxia (HO) and mechanical ventilation (MV) are the mainstay of treatment for patients with acute respiratory failure, but both interventions can also accelerate further lung injury, highlighting the need for better therapeutic approaches. We previously found that HO decreases epithelial TREK-1 expression and promotes epithelial inflammation, but the consequences of TREK-1 deficiency in a clinically relevant system of combined HO+ST (Stretch) exposure remain unknown. We found that in both mouse lung tissue and primary human alveolar epithelial cells HO+ST downregulates TREK-1 protein levels. The injurious consequences of TREK-1 downregulation are evidenced in alveolar epithelial cells following pharmacological and genetic TREK-1 inhibition and in lungs of TREK-1 KO mice by potentiation of HO+ST-induced cytosolic ROS production, caspase-8 and caspase-1 activation, IL-1beta production, and MIP-1alpha and CXCL-10/IP-10 secretion. In addition, HO+MV exposed TREK-1 KO mice show increased histological lung injury scores, total cell, macrophage and neutrophil counts in the BALF. Mechanistically, HO+ST depolarized the epithelial electrical membrane potential (Em) and raised iCa levels, which was potentiated after pharmacological and genetic TREK-1 inhibition. Both Ca influx through voltage-gated Ca channels and Ca release from intracellular stores increased iCa levels following TREK-1 inhibition. Intratracheal administration of two structurally different pharmacological TREK-1 activators (ML335, BL1249) improved HO+ST -induced BALF total and differential cell counts, total protein levels, ROS production, caspase -8 and capase-1 production, and cytokine concentrations. Therefore, these findings highlight TREK-1 as new potential target for intervention against HO+ST/MV -induced lung and epithelial injury and lay the groundwork for future rational drug development.

Cadmium (Cd), a toxic heavy metal found in air pollution, poses serious risks to lung health due to its efficient pulmonary absorption and prolonged biological half-life. This study examines how ad libitum (AL), time-restricted feeding (TRF), and intermittent fasting (IF) influence Cd-induced lung injury and immune responses in mice. Adult male C57BL/6 mice were preacclimated to AL, TRF, or IF regimens for 3 wk, followed by intratracheal exposure to cadmium chloride (CdCl; 0.5 mg/kg). Lung mechanics were assessed using flexiVent, bronchoalveolar lavage (BAL) fluid was analyzed for inflammation, and immune profiling was performed on spleens and mediastinal lymph nodes (MLNs) 14 days postexposure. Cd exposure increased immune cell infiltration in BAL fluid. IF mice showed significantly elevated inflammatory cytokines, while TRF mice had a modest increase. Histological analysis revealed greater lung inflammation in TRF mice, whereas lung mechanics were more impaired in IF mice, suggesting distinct injury profiles. Immune profiling showed that IF reduced activated and effector T-cell populations in the spleen but increased them in MLNs, indicating a shift in immune localization. Furthermore, compared to the AL, Cd-exposed IF mice had minimal changes in T-cell distribution but reduced effector CD4 and CD8 T-cells in the spleen and an increase in MLNs. In contrast, TRF mice exhibited minimal changes in T-cell distribution. These findings suggest that dietary regimens modulate immune responses and lung injury following Cd exposure. Feeding patterns play a critical role in shaping susceptibility to environmental toxicants and should be considered in future toxicological and immunological studies. This study reveals that time-restricted feeding (TRF) and intermittent fasting (IF) distinctly modulate cadmium-induced lung injury and immune responses in mice. TRF worsened lung inflammation, while IF impaired lung function and altered immune cell distribution, indicating divergent mechanisms. These findings highlight how feeding patterns influence pulmonary responses to environmental toxicants and suggest that metabolic rhythms may shape airway immunity, offering new insight into dietary modulation as a potential strategy in lung injury management.

Sex appears to modulate interactions between neural mechanisms involved in regulating pulmonary ventilation during mild hypoxic exercise. Therefore, we compared pulmonary ventilation responses elicited by isolated and combined stimulation of the carotid chemoreflex and muscle mechanoreflex between males and females. Twenty-eight healthy adults (14 females) underwent four experimental manipulations: ) normoxic rest (no stimulation), ) hypoxic rest (carotid chemoreflex stimulation), ) normoxic passive movement (muscle mechanoreflex stimulation), and ) hypoxic passive movement (reflexes costimulation). Isocapnia was maintained using a rebreathing system, and hypoxia was induced by breathing 12% oxygen. Passive movement involved 30-s bouts of unilateral knee manipulation at 300°/s, with surface electromyography confirming absence of voluntary muscle contractions. In males, the pulmonary ventilation response to passive limb movement (last 10 s change vs. rest) was greater under hypoxia than normoxia (means ± SD: hypoxia = 3.6 ± 2.0 vs. normoxia = 1.6 ± 2.4 L/min; = 0.003), whereas no difference was observed in females (hypoxia = 1.9 ± 2.4 vs. normoxia = 2.2 ± 1.5 L/min; = 1.000). Moreover, pulmonary ventilation remained elevated in males (hypoxia = 2.7 ± 2.4 vs. normoxia = -0.1 ± 2.2; < 0.001) but not in females (hypoxia = 0.4 ± 3.3 vs. normoxia = 0.5 ± 1.5; = 1.000), 30 s following passive limb movement under hypoxia. These findings support a synergistic carotid chemoreflex-muscle mechanoreflex interaction in males but not in females. The persistent ventilatory elevation poststimulation indicates that short-term potentiation contributes to this synergistic interaction in males. Pulmonary ventilation response to passive limb movement is greater under hypoxia than normoxia in males but not in females. These results support a synergistic interaction between the carotid chemoreflex and muscle mechanoreflex in males but not in females. In addition, pulmonary ventilation remains elevated in males but not in females after the cessation of passive limb movement under hypoxia, suggesting that short-term potentiation may be a mechanism mediating this synergistic reflex interaction in males.

Particulate matter less than 2.5 μm (PM) contributes to many chronic respiratory disorders, but the mechanisms for this are not fully understood. The actions of PM on lung epithelial cells have been well studied, but their effect on lung fibroblasts has not been as extensively reported. Bone morphogenetic protein (BMP) 2, part of the transforming growth factor cytokine family, plays crucial roles in development, morphogenesis, and repair, and functions as a critical mediator in the pathogenesis of lung diseases such as pulmonary fibrosis and chronic obstructive lung disease. Here, we investigate the impact of PM on fibroblast BMP2 production and the role of BMP2 in mediating fibroblast-to-myofibroblast differentiation and matrix generation. Treatment of fibroblasts to PM resulted in a dose-dependent rise in BMP2 mRNA and protein secretion, which was specific to BMP2 and not observed with other BMP family members. In normal quiescent fibroblasts, BMP2 induced an increase in collagen and α-smooth muscle actin expression. Interestingly, BMP2 exerted an opposite effect in TGF-β1-differentiated myofibroblasts, whereby BMP2 downregulated collagen levels. These differential responses aligned with variations in p38 and ERK1/2 phosphorylation. Fibroblasts treated with high concentrations of PM demonstrated reduced collagen and α-smooth muscle actin expression, an effect reversed by BMP2 silencing or gremlin, a BMP2 antagonist. Overall, PM was observed to induce BMP2 production in fibroblasts and this was associated with suppression of fibroblast activation and matrix production by PM. These findings highlight a potential mechanism whereby PM contributes to lung disease via impairment of fibroblast regenerative and repair capabilities.

Defining pre-clinical models is of utmost importance for pleural mesothelioma (PM) to improve prognosis and predict therapeutic response. Using cells isolated from pleural fluid (PF) and diagnostic pleural biopsy (PB), we generated PM patient-derived organoids (PM-PDOs) and reactive-mesothelial (RM)-patient derived organoids (RM-PDO) aiming at assessing the proportion of successful cultures both from PF and PB. We also compared the architectural and immune-histochemical features of PM-PDOs to those of parental tissues and evaluated the PM-PDOs response to chemo-immunotherapy. We obtained 11 successful PM-PDOs from 15 PF/PB (73.3%). The rate of success was higher in epithelioid PM (88.8%) compared to biphasic PM (40.0%) (p=0.175), and when using PF (60.0%) compared to PB (20.0%) (p=0.001). We also obtained 3 RM effective cultures from 6 asbestos-exposed patients (50%) with non-specific pleuritis. Transcriptome analysis identified gene expression profile in PM-PDOs, which differentiate from RM-PDOs. PM-PDOs successfully maintained the histological architecture and molecular markers of their parental tumour tissues. The macrophagic component (CD68 and CD163) was an important component in RM-PDOs and was present in all three PM histotypes. Epithelioid PM-PDOs showed resistance to both Cis/PeMtx and pembrolizumab plus peripheral blood mononuclear cells (PBMCs), while both biphasic and sarcomatoid subtypes were sensitive to immunotherapy. Notably, immunotherapy induced an upregulation of PD-L1 expression and activated the STAT3/NF-κB signaling pathway, suggesting a mechanism of immune evasion. PF offers a valuable source of cancer and stromal cells to generate PDO, reinforcing its clinical utility for patients who cannot undergo invasive procedures.