Effects of patient pleural effusion fluids on the BBSome components expression of human benign mesothelial cellsJagirdar, Rajesh M.; Rouka, Erasmia; Pitaraki, Eleanna; Sarrigeorgiou, Ioannis; Kotsiou, Ourania S.; Sinis, Sotiris I.; Papazoglou, Eleftherios D.; Marnas, Periklis; Malami, Zoi; Lymberi, Peggy; Giannou, Anastasios D.; Hatzoglou, Chrissi; Gourgoulianis, Konstantinos I.; Zarogiannis, Sotirios G.
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00373.2023pmid: 39470611
Background: Malignant pleural mesothelial cells are affected by the extracellular milieu while such data on benign cells are scarce. Benign cells sense the extracellular environment with the Primary Cilium (PC) and its molecular complex, the BBSome, is critical for this process. Here we aimed at assessing the changes in BBSome genes expression in ordinary 2D and spheroid 3D cell cultures after incubation with pleural effusion fluids (PF) of several etiologies. Methods: Benign human mesothelial cells MeT-5A were incubated with PF from patients with mesothelioma (Meso-PF), breast cancer (BrCa-PF), hemothorax (Hemo-PF) and congestive heart failure (CHF-PF). Gene expression of BBS1, 2, 4, 5, 7, 9, 18 was assessed by quantitative real-time PCR (qRT-PCR) to monitor PF-induced gene expression changes. MeT-5A cell migration using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) during PF incubation was also determined. Results: BBSome gene expression upon influence of BrCa-PF and Hemo-PF was more pronounced in 2D compared to 3D, inducing global changes in 2D. CHF-PF and Meso-PF also induced changes in 2D but not as many, while in all cases MeT-5A grown in 3D were more resistant to the effects of the PF. Meso-PF decreased 2D cell migration, while the disturbance of PC in all PF cases resulted in decreased cell migration. Conclusions: These data suggest distinct BBSome molecular profile changes in benign mesothelial cells exposed to malignant and benign PF, in each case, in both 2D and 3D. Cell migration is sensitive to drug disturbance with PC modulators in PF-exposed cells.
Ultrastructural analysis of lamellar bodies in type II alveolar epithelial cells in the human lungVanhecke, Dimitri; Nyengaard, Jens Randel; Haenni, Beat; Schipke, Julia; Ochs, Matthias
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00284.2024pmid: 39437759
Pulmonary surfactant is produced by type II alveolar epithelial cells (AEC2) and stored in lamellar bodies (LBs) prior to secretion. Here, we characterize AEC2 and their LBs in the human lung ultrastructurally and quantitatively. Five human lungs were analyzed by transmission electron microscopy, serial section electron tomography and stereology. A human lung contained about 24 billion AEC2 with a mean size of about 650 µm³. The number of AEC2 as well as the total volume of LBs per lung, about 1.9 mL, strongly correlated with total lung volume. A single AEC2 contained an LB volume of about 74 µm³. This amount was packed in about 324 LBs with a mean size of 0.24 µm³. Three morphologically distinct subpopulations of LBs were identified: 1.) isolated LBs which make up the majority (average 300 per AEC2), 2.) LBs connected to each other via pores (average 23 per AEC2), and 3.) LBs connected to the plasma membrane via a fusion pore (average 1 per AEC2). Along this sequence of subpopulations, the mean size of LBs increased. LBs that are connected either with each other or to the plasma membrane contained about 14% of an AEC2´s LB volume. This is in line with the concept of an intermediate surfactant pool, stored in LBs either directly or indirectly connected to the plasma membrane. In summary, this study provides quantitative reference data on surfactant-storing LBs in AEC2 as well as morphological evidence for an intermediate surfactant pool in the human lung.
Antenatal steroids enhance long-term neonatal lung outcomes and are associated with placental alterations in experimental chorioamnionitisNetsanet, Adom; Seedorf, Gregory J.; Abman, Steven H.; Taglauer, Elizabeth S.
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00204.2024pmid: 39701125
Intrauterine inflammation from chorioamnionitis (CA) is associated with placental dysfunction and increased risk of bronchopulmonary dysplasia (BPD), the chronic lung disease of prematurity. Antenatal steroid (ANS) treatment improves early respiratory outcomes for premature infants. However, it remains unclear whether ANS improve long-term respiratory outcomes, and whether these effects are mediated through improvement of placental dysfunction and/or direct impact on the fetal lung. We hypothesized that maternal ANS therapy preserves long-term lung development and impacts placental structural changes and gene expression in experimental CA with features of BPD. Pregnant rat dams were administered either saline (CTL), intraamniotic (IA) endotoxin (ETX), ETX plus intramuscular (IM) betamethasone (ETX+BETA) or IM BM alone (BETA) on embryonic day 20 (e20). We collected placental tissue at delivery (e22) and infant lung tissue on day of life (DOL)14. In comparison with controls, IA ETX had impaired infant lung growth and function. Maternal BM treatment of ETX-exposed pregnant dams reduced infant total lung resistance by 15.3% (p<0.05), improved infant lung compliance by 9.5% (p<0.05), preserved alveolar and vascular growth (p<0.05), and improved RVH by 42.4% (p<0.05). ETX+BETA pregnancies were also associated with normalization of placental spiral artery modification and altered placental gene expression. These included upregulation of placental prolactin which has regulatory effects on pregnancy homeostasis and has been clincially associated with decreased BPD risk. The current study identifies parallel lung and placental changes associated with ANS treatment, providing a foundation for future studies to identify alternate antenatal therapies with more specific efficacy for BPD prevention
Surfactant Protein-A and its immunomodulatory roles in infant respiratory syncytial virus infection: a potential for therapeutic intervention?Mapindra, Muhammad Pradhika; Castillo-Hernandez, Tania; Clark, Howard; Madsen, Jens
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00199.2024pmid: 39662519
The vast majority of early-life hospital admissions globally highlight Respiratory Syncytial Virus (RSV), the leading cause of neonatal lower respiratory tract infections, as the major culprit behind the poor neonatal outcomes following respiratory infections. Unlike those of older children and adults, the immune system of neonates looks rather unique, therefore mostly counting on the innate immune system and antibodies of maternal origins. The collaborations between cells and immune compartments during infancy inclines to bias toward a T-helper 2 (Th2) immune profile and thereby away from a T-helper 1 (Th1) immune response. What makes it more problematic is that RSV infection also tends to elicit a stronger Th2-biased immune response and drive an aberrant allergy-like inflammation. It is thus evident how RSV infections potentially pave the way for wheezing recurrences and childhood asthma later in life. Surfactant, the essential lung substance for normal breathing processes in mammals, has immunomodulatory properties including lung collectins such as Surfactant Protein-A (SP-A), which is the most abundant protein component of surfactant. Deficiency of SP-A has been found to be associated with impaired pathogen clearance and exacerbated immune responses during infections. We therefore conducted a review of the literature to describe pathomechanisms of RSV infections during blunted neonatal immunity ultimately enabling allergy-like inflammatory events within the developing lungs, and where the protective humoral collectin SP-A potentially participates in the pulmonary immune homeostasis in the meantime.
Impaired antiviral immunity in frequent exacerbators of chronic obstructive pulmonary diseaseFinney, Lydia J.; Fenwick, Peter; Kemp, Samuel V.; Singanayagam, Aran; Edwards, Michael R.; Belchamber, Kylie B. R.; Kebadze, Tatiana; Regis, Eteri; Donaldson, Gavin D.; Mallia, Patrick; Donnelly, Louise E.; Johnston, Sebastian L.; Wedzicha, Jadwiga A.
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00118.2024pmid: 39560620
Background Respiratory viruses cause chronic obstructive pulmonary disease (COPD) exacerbations. Rhinoviruses (RVs) are the most frequently detected. Some COPD patients experience frequent exacerbations ({greater than or equal to}2 exacerbations/ year). The relationship between exacerbation frequency and anti-viral immunity remains poorly understood.Objectives To investigate the relationship between exacerbation frequency and anti-viral immunity in COPDMethods Alveolar macrophages and bronchial epithelial cells (BECs) were obtained from COPD patients and healthy participants. Alveolar macrophages were infected with RV-A16 multiplicity of infection 5 (MOI 5) and BECs infected with RV-A16 MOI 1 for 24 hours. Interferons (IFN) and pro-inflammatory cytokines IL-1β, IL-6, CXCL8 and TNF were measured in cell supernatants using mesoscale discovery platform. Viral load and interferon stimulated genes were measured in cell lysates using qPCR.Results Spontaneous and RV induced IFN-β, IFN-γ and CXCL-11 release were significantly reduced in alveolar macrophages from COPD patients compared to healthy subjects. IFN-β was further impaired in uninfected alveolar macrophages from COPD patients with frequent exacerbations 82.0 pg/mL vs infrequent exacerbators 234.7 pg/mL P=0.008 and RV-infected alveolar macrophages from frequent exacerbators 158.1 pg/mL vs infrequent exacerbators 279.5 pg/mL P=0.022. Release of proinflammatory cytokines CXCL8, IL-6, TNF and IL-1β was higher in uninfected BECs from COPD patients compared to healthy subjects but there was no difference in pro-inflammatory response to RV between groups.Conclusions IFN responses to RV was impaired in alveolar macrophages from COPD patients and further reduced in patients with frequent exacerbations.
Deep phenotyping of pulmonary edema and pulmonary vascular permeability in COVID-19 ARDSSchippers, Job R.; Atmowihardjo, Leila N.; Duijvelaar, Erik; Knaap, Lars G.; Netea, Mihai G.; Meijboom, Lilian J.; Bos, Lieuwe D. J.; Bogaard, Harm Jan; Aman, Jurjan
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00196.2024pmid: 39437755
Background Clinical monitoring of pulmonary edema due to vascular hyperpermeability in ARDS poses significant clinical challenges. Presently, no biological or radiological markers are available for quantifying pulmonary edema. Our aim was to phenotype pulmonary edema and pulmonary vascular permeability in patients with COVID-19 ARDS. Methods Transpulmonary thermodilution measurements were conducted in 65 COVID-19 ARDS patients on the day of intubation to determine extravascular lung water index (EVLWi) and pulmonary vascular permeability index (PVPi). In parallel, ventilatory parameters, clinical outcomes, the volume of lung opacity measured by chest CT, and plasma proteomics (358 unique proteins) were compared between tertiles based on the EVLWi and PVPi. Regression models were used to associate EVLWi and PVPi with plasma, radiological, and clinical parameters. Computational pathway analysis was performed on significant plasma proteins in the regression models. Results Patients with the highest EVLWi values at intubation exhibited poorer oxygenation parameters and more days on the ventilator. Extravascular lung water strongly correlated with the total volume of opacity observed on CT(r=0.72), while the PVPi had weaker associations with clinical and radiological parameters. Plasma protein concentrations demonstrated a stronger correlation with PVPi than with EVLWi. The highest tertile of PVPi was associated with proteins linked to the acute phase response (cytokine and chemokine signaling) and extracellular matrix turnover. Conclusions In the clinical setting of COVID-19 ARDS, pulmonary edema (EVLWi) can be accurately quantified through chest CT and parallels deterioration in ventilatory parameters and clinical outcomes. Vascular permeability (PVPi) is strongly reflected by inflammatory plasma proteins.
Neutrophil elastase activates macrophage calpain as a mechanism for phagocytic failureMa, Jonathan; Kummarapurugu, Apparao B.; Zheng, Shuo; Ghio, Andrew J.; Deshpande, Laxmikant S.; Voynow, Judith A.
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00132.2024pmid: 39499256
Neutrophil elastase (NE), elevated in the cystic fibrosis (CF) airway, causes macrophage phagocytic failure. We previously reported that NE increases the release of protease Calpain-2 in macrophages. We hypothesized that NE mediates macrophage failure through activation of Calpains. We demonstrate that Calpain inhibition rescued NE induced macrophage phagocytic failure in murine alveolar macrophages in both cftr-null and wild type genotypes. We then sought to determine how NE regulates Calpain-2. Human monocyte derived macrophages (hMDM) from persons with CF (PwCF) and non-CF subjects, were treated with NE or control vehicle and cell lysates prepared to evaluate Calpain-2 protein abundance by Western, and Calpain activity by a specific activity kit. Calpain is activated by intracellular calcium and inactivated by an endogenous inhibitor, Calpastatin. Human MDM were thus treated with NE or control vehicle and cell lysates were analyzed for increased intracellular calcium by Fluo-4 assay and for Calpastatin protein abundance by Western. NE increased Calpain-2 protein and activity, degraded Calpastatin, and increased intracellular calcium in macrophages. At baseline there are no differences in Calpain activity, Calpain-2 and Calpastatin expression, and intracellular calcium between CF and non-CF macrophages. NE increased macrophage Calpain-2 protein and Calpain activity by two potential mechanisms: degradation of Calpastatin, and/or increased intracellular calcium. In summary, Calpain inhibition restored NE-induced macrophage phagocytic failure suggesting a potential CFTR-independent target for phagocytic failure in the CF airway.
Leukocyte kinetics and bacterial clearance during Streptococcus pneumoniae pneumonia and contributions of ICAM-1McPeek, Matthew K.; Gomez, John C.; Martin, Jessica R.; Iannone, Marie Anne; Dang, Hong; Doerschuk, Claire M.
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00039.2024pmid: 39437756
Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule that is highly expressed on the pulmonary capillary endothelium, alveolar epithelium and other cell types within the lung. ICAM-1 plays important roles in leukocyte adhesion, migration, and motility. To determine the contributions of ICAM-1 to bacterial clearance and leukocyte kinetics during pneumonia, mice were inoculated with S. pneumoniae and evaluated 1, 4 and 7 days later. Our results show that Icam1-/- mice have a greater number of viable bacteria within the lung at each time point. The impaired clearance observed in Icam1-/- mice was not due to an impediment in leukocyte recruitment. In fact, Icam1-/- mice had a greater number of neutrophils and recruited inflammatory macrophages in the lung tissue and the alveoli/airways on day 7. In contrast, fewer alveolar macrophages were present in the BAL of Icam1-/- mice. The loss of body weight and the concentrations of inflammatory mediators in the BAL were also significantly greater in Icam1-/- mice. Mechanistic studies to understand the defect in clearance show that neutrophils and macrophage subpopulations had no defect in phagocytosis or acidification of phagosomes. RNA sequencing reveals many differences in gene expression, but no suggestion of a defect in phagocytosis or killing. Thus, that ICAM-1 is necessary for the clearance of S. pneumoniae and for the resolution of pneumonia, but is not required for the recruitment of neutrophils or inflammatory macrophages into the pneumonic lung parenchyma or the alveoli/airways during S. pneumoniae-induced pneumonia.
Dynamic driving pressure predicts ventilator-induced lung injury in mice with and without endotoxin-induced acute lung injuryWallbank, Alison; Sosa, Alexander; Colson, Andrew; Farooqi, Huda; Kaye, Elizabeth; Warner, Katharine; Albers, David J.; Sottile, Peter D.; Smith, Bradford J.
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00176.2024pmid: 39601347
Mechanical ventilation is a necessary lifesaving intervention for patients with Acute Respiratory Distress Syndrome (ARDS) but it can cause ventilator induced lung injury (VILI), which contributes to the high ARDS mortality rate (≈40%). Bedside determination of optimally lung-protective ventilation settings is challenging because the evolution of VILI is not immediately reflected in clinically available, patient-level, data. The goal of this work was therefore to test ventilation waveform-derived parameters that represent the degree of ongoing VILI and can serve as targets for ventilator adjustments. VILI was generated at three different positive end expiratory pressures in a murine inflammation-mediated (lipopolysaccharide, LPS) acute lung injury model and in initially healthy controls. LPS injury increased expression of proinflammatory cytokines and caused widespread atelectasis, predisposing the lungs to VILI as measured in structure, mechanical function, and inflammation. Changes in lung function were used as response variables in an elastic net regression model that predicted VILI severity from tidal volume, dynamic driving pressure (PDDyn), mechanical power calculated by integration during inspiration or the entire respiratory cycle, and power calculated according to Gattinoni’ s equation. Of these, PDDyn best predicted functional outcomes of injury using either data from the entire dataset or from 5-minute time windows. The windowed data shows higher predictive accuracy after a ≈1-hour ‘run in’ period and worse accuracy immediately following recruitment maneuvers. This analysis shows that low driving pressure is a computational biomarker associated with better experimental VILI outcomes and supports the use of driving pressure to guide ventilator adjustments to prevent VILI.
PDGFRα-positive cell-derived TIMP-1 modulates adaptive immune responses to influenza A viral infectionDutta, Saugata; Zhu, Yin; Almuntashiri, Sultan; Peh, Hong Yong; Zuñiga, Joaquin; Zhang, Duo; Somanath, Payaningal R.; Ramírez, Gustavo; Irineo-Moreno, Valeria; Jiménez-Juárez, Fabiola; López-Salinas, Karen; Regino, Nora; Campero, Paloma; Crocker, Stephen J.; Owen, Caroline A.; Wang, Xiaoyun
2025 AJP - Lung Cellular and Molecular Physiology
doi: 10.1152/ajplung.00104.2024pmid: 39585242
TIMP-1 (tissue inhibitor of metalloproteinases-1) is a physiologic inhibitor of the matrix metalloproteinases (MMPs), but little is known about the role of TIMP-1 in regulating the pathogenesis of influenza A virus (IAV) infection. Here, we performed both in vivo and in vitro experiments to investigate the regulation and function of TIMP-1 during IAV infection. Specifically, plasma levels of TIMP-1 are significantly increased in human subjects and wild-type (WT) mice infected with 2009 H1N1 IAV compared with levels in uninfected controls. Also, TIMP-1 is strikingly upregulated in PDGFRα positive (PDGFRα+) cells in IAV-infected murine lungs as demonstrated using conditional KO (cKO) mice with a specific deletion of Timp-1 in PDGFRα+ cells. Our in vitro data indicated that TIMP-1 is induced by TGF-β during lipofibroblast (lipoFBs)-to-myofibroblast (myoFB) transdifferentiation. Timp-1 deficiency protects mice from H1N1 IAV-induced weight loss, mortality, and lung injury. IAV-infected Timp-1 deficient mice showed increased macrophages, and B and T cell counts in bronchoalveolar lavage (BAL) on day 7 post-infection (p.i.), but reduced BAL neutrophil counts. Increased Cxcl12 levels were detected in both BAL cells and lungs from Timp-1 deficient mice on day 3 p.i. Taken together, our data strongly link TIMP-1 to IAV pathogenesis. We identified that PDGFRα-lineage cells are the main cellular source of elevated TIMP-1 during IAV infection. Loss of Timp-1 attenuates IAV-induced mortality and promotes T and B cell recruitment. Thus, TIMP-1 may be a novel therapeutic target for IAV infection.