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BACKGROUND: Acute exacerbations of chronic inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD), are frequently associated with rhinovirus (RV) infections. Despite these associations, the pathogenesis of virus-induced exacerbations is incompletely understood. We aimed to investigate effects of cigarette smoke (CS), a primary risk factor for COPD, on RV infection in airway epithelium and identify novel mechanisms related to these effects. METHODS: Primary bronchial epithelial cells (PBEC) from COPD patients and controls were differentiated by culture at the air-liquid interface (ALI) and exposed to CS and RV-A16. Bulk RNA sequencing was performed using samples collected at 6 and 24 h post infection (hpi), and viral load, mediator and L-lactate levels were measured at 6, 24 and 48hpi. To further delineate the effect of CS on RV-A16 infection, we performed growth differentiation factor 15 (GDF15) knockdown, L-lactate and interferon pre-treatment in ALI-PBEC. We performed deconvolution analysis to predict changes in the cell composition of ALI-PBEC after the various exposures. Finally, we compared transcriptional responses of ALI-PBEC to those in nasal epithelium after human RV-A16 challenge. RESULTS: CS exposure impaired antiviral responses at 6hpi and increased viral replication at 24 and 48hpi in ALI-PBEC. At 24hpi, CS exposure enhanced expression of RV-A16-induced epithelial interferons, inflammation-related genes and CXCL8. CS exposure increased expression of oxidative stress-related genes, of GDF15, and decreased mitochondrial membrane potential. GDF15 knockdown experiments suggested involvement of this pathway in the CS-induced increase in viral replication. Expression of glycolysis-related genes and L-lactate production were increased by CS exposure, and was demonstrated to contribute to higher viral replication. No major differences were demonstrated between COPD and non-COPD-derived cultures. However, cellular deconvolution analysis predicted higher secretory cells in COPD-derived cultures at baseline. CONCLUSION: Altogether, our findings demonstrate that CS exposure leads to higher viral infection in human bronchial epithelium by altering not only interferon responses, but likely also through a switch to glycolysis, and via GDF15-related pathways.
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Fumar Cigarrillos , Enfermedad Pulmonar Obstructiva Crónica , Virosis , Humanos , Interferones , Factor 15 de Diferenciación de Crecimiento , Fumar Cigarrillos/efectos adversos , LactatosRESUMEN
BACKGROUND AND OBJECTIVE: Smoking disturbs the bronchial-mucus-barrier. This study assesses the cellular composition and gene expression shifts of the bronchial-mucus-barrier with smoking to understand the mechanism of mucosal damage by cigarette smoke exposure. We explore whether single-cell-RNA-sequencing (scRNA-seq) based cellular deconvolution (CD) can predict cell-type composition in RNA-seq data. METHODS: RNA-seq data of bronchial biopsies from three cohorts were analysed using CD. The cohorts included 56 participants with chronic obstructive pulmonary disease [COPD] (38 smokers; 18 ex-smokers), 77 participants without COPD (40 never-smokers; 37 smokers) and 16 participants who stopped smoking for 1 year (11 COPD and 5 non-COPD-smokers). Differential gene expression was used to investigate gene expression shifts. The CD-derived goblet cell ratios were validated by correlating with staining-derived goblet cell ratios from the COPD cohort. Statistics were done in the R software (false discovery rate p-value < 0.05). RESULTS: Both CD methods indicate a shift in bronchial-mucus-barrier cell composition towards goblet cells in COPD and non-COPD-smokers compared to ex- and never-smokers. It shows that the effect was reversible within a year of smoking cessation. A reduction of ciliated and basal cells was observed with current smoking, which resolved following smoking cessation. The expression of mucin and sodium channel (ENaC) genes, but not chloride channel genes, were altered in COPD and current smokers compared to never smokers or ex-smokers. The goblet cell-derived staining scores correlate with CD-derived goblet cell ratios. CONCLUSION: Smoking alters bronchial-mucus-barrier cell composition, transcriptome and increases mucus production. This effect is partly reversible within a year of smoking cessation. CD methodology can predict goblet-cell percentages from RNA-seq.
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Enfermedad Pulmonar Obstructiva Crónica , Transcriptoma , Humanos , Transcriptoma/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Moco/metabolismo , Biopsia , Fumar/efectos adversos , Fumar/genéticaRESUMEN
Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type 2 cells (AEC2s) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1 Organ-Chip that has a flexible polydimethylsiloxane (PDMS) membrane enabling the application of dynamic stretch. AEC2s cultured for 7 days on inserts or the chip maintained expression of HTII-280, prosurfactant protein C (SP-C), SP-A and SP-B, and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2s cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared with static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2s and their application in microfluidic chip cultures supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.
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Células Epiteliales Alveolares , Organoides , Células Epiteliales Alveolares/metabolismo , Células Cultivadas , Células Epiteliales , Humanos , Pulmón , Organoides/metabolismo , Alveolos PulmonaresRESUMEN
BACKGROUND: Alveolar epithelial cell dysfunction plays an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), but remains incompletely understood. Some monogenic forms of pulmonary fibrosis are associated with expression of mutant surfactant protein C (SFTPC). The commonest pathogenic mutant, I73T, mislocalises to the alveolar epithelial cell plasma membrane and displays a toxic gain of function. Because the mechanisms explaining the link between this mutant and IPF are incompletely understood, we sought to interrogate SFTPC trafficking in health and disease to understand the functional significance of SFTPC-I73T relocalisation. METHODS: We performed mechanistic analysis of SFTPC trafficking in a cell model that reproduces the in vivo phenotype and validated findings in human primary alveolar organoids. RESULTS: We show that wild-type SFTPC takes an unexpected indirect trafficking route via the plasma membrane and undergoes the first of multiple cleavage events before reaching the multivesicular body (MVB) for further processing. SFTPC-I73T takes this same route, but its progress is retarded both at the cell surface and due to failure of trafficking into the MVB. Unable to undergo onward trafficking, it is recycled to the plasma membrane as a partially cleaved intermediate. CONCLUSION: These data show for the first time that all SFTPC transits the cell surface during normal trafficking, and the I73T mutation accumulates at the cell surface through both retarded trafficking and active recycling. This understanding of normal SFTPC trafficking and how the I73T mutant disturbs it provides novel insight into SFTPC biology in health and disease, and in the contribution of the SFTPC mutant to IPF development.
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Fibrosis Pulmonar Idiopática , Proteína C Asociada a Surfactante Pulmonar/metabolismo , Células Epiteliales Alveolares , Humanos , Fibrosis Pulmonar Idiopática/genética , Mutación , Proteína C Asociada a Surfactante Pulmonar/genética , TensoactivosRESUMEN
BACKGROUND: An inverse relation between Helicobacter pylori infection and asthma has been shown in epidemiological studies. Infection with H. pylori, or application of an extract of it before or after sensitization, inhibits allergic airway disease in mice. OBJECTIVES: The aim of this study was to investigate the effect of an extract of H. pylori on allergic airway disease induced by repeated allergen exposure in mice that were sensitized and challenged prior to extract application. METHOD: C57BL/6 mice were intranasally (i.n.) sensitized and challenged with house dust mite (HDM). After a minimum of 4 weeks, mice received the H. pylori extract intraperitoneally and were rechallenged i.n. with HDM. Allergen-specific antibodies were measured by ELISA. Cells present in the bronchoalveolar lavage fluid and dendritic cell (DC) subsets in the lung tissue were analyzed by flow cytometry. Tissue inflammation and goblet cell hyperplasia were assessed by histology. Cells of the mediastinal lymph node (mLN) were isolated and in vitro restimulated with HDM or H. pylori extract. RESULTS: Treatment with H. pylori extract before rechallenge reduced allergen-specific IgE, the DC numbers in the tissue, and goblet cell hyperplasia. Cells isolated from mLN of mice treated with the extract produced significantly more IL-10 and IL-17 after in vitro restimulation with HDM. mLN cells of H. pylori-treated mice that were re-exposed to the H. pylori extract produced significantly more interferon gamma. CONCLUSIONS: An extract of H. pylori is effective in reducing mucus production and various features of inflammation in HDM rechallenged mice.
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Alérgenos/inmunología , Antígenos Bacterianos/inmunología , Células Caliciformes/patología , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/inmunología , Helicobacter pylori/inmunología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/patología , Animales , Biomarcadores , Biopsia , Citocinas/metabolismo , Exposición a Riesgos Ambientales , Femenino , Infecciones por Helicobacter/microbiología , Hiperplasia , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Inmunofenotipificación , Ratones , Pyroglyphidae/inmunologíaRESUMEN
BACKGROUND: We have previously shown that 8 weeks' treatment with phenylbutyrate (PBA) (500mgx2/day) with or without vitamin D3 (vitD3) (5000 IU/day) as host-directed therapy (HDT) accelerated clinical recovery, sputum culture conversion and increased expression of cathelicidin LL-37 by immune cells in a randomized, placebo-controlled trial in adults with pulmonary tuberculosis (TB). In this study we further aimed to examine whether HDT with PBA and vitD3 promoted clinically beneficial immunomodulation to improve treatment outcomes in TB patients. METHODS: Cytokine concentration was measured in supernatants of peripheral blood mononuclear cells (PBMC) from patients (n = 31/group). Endoplasmic reticulum stress-related genes (GADD34 and XBP1spl) and human beta-defensin-1 (HBD1) gene expression were studied in monocyte-derived-macrophages (MDM) (n = 18/group) from PBMC of patients. Autophagy in MDM (n = 6/group) was evaluated using LC3 expression by confocal microscopy. RESULTS: A significant decline in the concentration of cytokines/chemokines was noted from week 0 to 8 in the PBA-group [TNF-α (ß = - 0.34, 95% CI = - 0.68, - 0.003; p = 0.04), CCL11 (ß = - 0.19, 95% CI = - 0.36, - 0.03; p = 0.02) and CCL5 (ß = - 0.08, 95% CI = - 0.16, 0.002; p = 0.05)] and vitD3-group [(CCL11 (ß = - 0.17, 95% CI = - 0.34, - 0.001; p = 0.04), CXCL10 (ß = - 0.38, 95% CI = - 0.77, 0.003; p = 0.05) and PDGF-ß (ß = - 0.16, 95% CI = - 0.31, 0.002; p = 0.05)] compared to placebo. Both PBA- and vitD3-groups showed a decline in XBP1spl mRNA on week 8 (p < 0.03). All treatment groups demonstrated increased LC3 expression in MDM compared to placebo over time (p < 0.037). CONCLUSION: The use of PBA and vitD3 as adjunct therapy to standard TB treatment promoted favorable immunomodulation to improve treatment outcomes. TRIALS REGISTRATION: This trial was retrospectively registered in clinicaltrials.gov, under identifier NCT01580007 .
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Tuberculosis Pulmonar/inmunología , Vitamina D/uso terapéutico , Vitaminas/uso terapéutico , Adulto , Péptidos Catiónicos Antimicrobianos/metabolismo , Colecalciferol , Citocinas/sangre , Estrés del Retículo Endoplásmico , Femenino , Humanos , Leucocitos Mononucleares , Macrófagos/inmunología , Masculino , Persona de Mediana Edad , Fenilbutiratos , ARN Mensajero , Estudios Retrospectivos , Resultado del Tratamiento , Tuberculosis Pulmonar/tratamiento farmacológico , Adulto Joven , beta-Defensinas , CatelicidinasRESUMEN
Long-term treatment with inhaled corticosteroids (ICS) might attenuate lung function decline and decrease airway inflammation in a subset of patients with chronic obstructive pulmonary disease (COPD), and discontinuing ICS treatment could result in further lung function decline. We hypothesised that airway inflammation increases after ICS withdrawal following long-term ICS treatment in COPD.In the GLUCOLD-1 study (GL1), 114 patients with moderate-severe COPD were randomised to 6-month or 30-month treatment with fluticasone propionate (500â µg twice daily), 30-month treatment with fluticasone/salmeterol (500/50â µg twice daily) or placebo. During the 5-year follow-up study (GL2), patients were followed prospectively while being treated by their physician. Bronchial biopsies and induced sputum were collected at baseline, at 30 months (end of GL1) and at 7.5â years (end of GL2) to assess inflammatory cell counts. Data were analysed using linear mixed-effects models.In patients using ICS during GL1 and using ICS 0-50% of the time during GL2 (n=61/85), there were significant increases in GL2 bronchial CD3+ (fold change per year calculated as GL2 minus GL1 2.68, 95% CI 1.87-3.84), CD4+ (1.91, 95% CI 1.33-2.75) and CD8+ cells (1.71, 95% CI 1.15-2.53), and mast cells (1.91, 95% CI 1.36-2.68). The sputum total cell counts increased significantly in GL2 (1.90, 95% CI 1.42-2.54), as did counts of macrophages (2.10, 95% CI 1.55-2.86), neutrophils (1.92, 95% CI 1.39-2.65) and lymphocytes (2.01, 95% CI 1.46-2.78).ICS discontinuation increases airway inflammation in patients with moderate-severe COPD, suggesting that the anti-inflammatory effects of ICS in COPD are not maintained after ICS discontinuation.
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Corticoesteroides/administración & dosificación , Combinación Fluticasona-Salmeterol/administración & dosificación , Fluticasona/administración & dosificación , Inflamación/tratamiento farmacológico , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Privación de Tratamiento , Administración por Inhalación , Anciano , Bronquios/patología , Broncodilatadores/uso terapéutico , Femenino , Estudios de Seguimiento , Volumen Espiratorio Forzado/efectos de los fármacos , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Países Bajos , Neutrófilos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/microbiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Esputo/citologíaRESUMEN
Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.
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Células Epiteliales/metabolismo , Proteína Fosfatasa 1/metabolismo , Infecciones por Pseudomonas/metabolismo , Respuesta de Proteína Desplegada/fisiología , Factores de Virulencia/metabolismo , Western Blotting , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/fisiología , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/microbiología , Células Epiteliales/patología , Humanos , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Exacerbations constitute a major cause of morbidity and mortality in patients suffering from chronic obstructive pulmonary disease (COPD). Both bacterial infections, such as those with non-typeable Haemophilus influenzae (NTHi), and exposures to diesel engine emissions are known to contribute to exacerbations in COPD patients. However, the effect of diesel exhaust (DE) exposure on the epithelial response to microbial stimulation is incompletely understood, and possible differences in the response to DE of epithelial cells from COPD patients and controls have not been studied. METHODS: Primary bronchial epithelial cells (PBEC) were obtained from age-matched COPD patients (n = 7) and controls (n = 5). PBEC were cultured at the air-liquid interface (ALI) to achieve mucociliary differentiation. ALI-PBECs were apically exposed for 1 h to a stream of freshly generated whole DE or air. Exposure was followed by 3 h incubation in presence or absence of UV-inactivated NTHi before analysis of epithelial gene expression. RESULTS: DE alone induced an increase in markers of oxidative stress (HMOX1, 50-100-fold) and of the integrated stress response (CHOP, 1.5-2-fold and GADD34, 1.5-fold) in cells from both COPD patients and controls. Exposure of COPD cultures to DE followed by NTHi caused an additive increase in GADD34 expression (up to 3-fold). Importantly, DE caused an inhibition of the NTHi-induced expression of the antimicrobial peptide S100A7, and of the chaperone protein HSP5A/BiP. CONCLUSIONS: Our findings show that DE exposure of differentiated primary airway epithelial cells causes activation of the gene expression of HMOX1 and markers of integrated stress response to a similar extent in cells from COPD donors and controls. Furthermore, DE further increased the NTHi-induced expression of GADD34, indicating a possible enhancement of the integrated stress response. DE reduced the NTHi-induced expression of S100A7. These data suggest that DE exposure may cause adverse health effects in part by decreasing host defense against infection and by modulating stress responses.
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Infecciones por Haemophilus/inmunología , Haemophilus influenzae/inmunología , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/microbiología , Emisiones de Vehículos/envenenamiento , Anciano , Bronquios/efectos de los fármacos , Bronquios/inmunología , Bronquios/microbiología , Células Cultivadas , Femenino , Haemophilus influenzae/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Especies Reactivas de Oxígeno/inmunología , Mucosa Respiratoria/efectos de los fármacosRESUMEN
Diesel emissions are the main source of air pollution in urban areas, and diesel exposure is linked with substantial adverse health effects. In vitro diesel exposure models are considered a suitable tool for understanding these effects. Here we aimed to use a controlled in vitro exposure system to whole diesel exhaust to study the effect of whole diesel exhaust concentration and exposure duration on mucociliary differentiated human primary bronchial epithelial cells (PBEC). PBEC cultured at the air-liquid interface were exposed for 60 to 375 min to three different dilutions of diesel exhaust (DE). The DE mixture was generated by an engine at 47% load, and characterized for particulate matter size and distribution and chemical and gas composition. Cytotoxicity and epithelial barrier function was assessed, as well as mRNA expression and protein release analysis. DE caused a significant dose-dependent increase in expression of oxidative stress markers (HMOX1 and NQO1; n = 4) at 6 h after 150 min exposure. Furthermore, DE significantly increased the expression of the markers of the integrated stress response CHOP and GADD34 and of the proinflammatory chemokine CXCL8, as well as release of CXCL8 protein. Cytotoxic effects or effects on epithelial barrier function were observed only after prolonged exposures to the highest DE dose. These results demonstrate the suitability of our model and that exposure dose and duration and time of analysis postexposure are main determinants for the effects of DE on differentiated primary human airway epithelial cells.
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Contaminantes Atmosféricos/toxicidad , Células Epiteliales/metabolismo , Material Particulado/toxicidad , Emisiones de Vehículos/toxicidad , Diferenciación Celular , Células Cultivadas , Estrés del Retículo Endoplásmico , Células Epiteliales/efectos de los fármacos , Expresión Génica , Humanos , Interleucina-8/metabolismo , Estrés Oxidativo , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patologíaRESUMEN
Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.
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Células Epiteliales/metabolismo , Sistema de Señalización de MAP Quinasas , Mutación Missense , Transducción de Señal , alfa 1-Antitripsina/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Línea Celular Tumoral , Citocinas/metabolismo , Estrés del Retículo Endoplásmico , Receptores ErbB/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Pulmón/patología , FN-kappa B/metabolismo , Cultivo Primario de Células , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , alfa 1-Antitripsina/biosíntesisRESUMEN
Which inflammatory markers in the bronchial mucosa of asthma patients are associated with decline of lung function during 14â years of prospective follow-up?To address this question, 19 mild-to-moderate, atopic asthmatic patients underwent spirometry and bronchoscopy at baseline and after 14â years of follow-up (t=14). Baseline bronchial biopsies were analysed for reticular layer thickness, eosinophil cationic protein (EG2), mast cell tryptase (AA1), CD3, CD4 and CD8. Follow-up biopsies were stained for EG2, AA1, neutrophil elastase, CD3, CD4, CD8, CD20, granzyme B, CD68, DC-SIGN, Ki67 and mucins.Decline in forced expiratory volume in 1â s (FEV1) % predicted was highest in patients with high CD8 (p=0.01, both pre- and post-bronchodilator) or high CD4 counts at baseline (p=0.04 pre-bronchodilator, p=0.03 post-bronchodilator). Patients with high CD8, CD3 or granzyme B counts at t=14 also exhibited faster decline in FEV1 (p=0.00 CD8 pre-bronchodilator, p=0.04 CD8 post-bronchodilator, p=0.01 granzyme B pre-bronchodilator, and p<0.01 CD3 pre-bronchodilator).Long-term lung function decline in asthma is associated with elevation of bronchial CD8 and CD4 at baseline, and CD8, CD3 and granzyme B at follow-up. This suggests that high-risk groups can be identified on the basis of inflammatory phenotypes.
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Asma/fisiopatología , Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Granzimas/metabolismo , Pruebas de Función Respiratoria , Adulto , Asma/terapia , Biopsia , Bronquios/fisiopatología , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial , Broncodilatadores/farmacología , Estudios de Cohortes , Estudios Transversales , Femenino , Estudios de Seguimiento , Volumen Espiratorio Forzado , Humanos , Inflamación , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Fenotipo , Estudios Prospectivos , EspirometríaRESUMEN
α1-antitrypsin deficiency is the most widely recognised genetic disorder causing chronic obstructive pulmonary disease (COPD). Mutant Z α1-antitrypsin expression has previously been linked to intracellular accumulation and polymerisation of this proteinase inhibitor. Subsequently, this has been described to underlie an exaggerated endoplasmic reticulum stress response and enhanced nuclear factor-κB signalling. However, whether monocyte-derived macrophages display the same features remains unknown. Monocytes from homozygous PiZZ α1-antitrypsin deficiency patients and PiMM controls were cultured for 6 days in the presence of granulocyte-macrophage or macrophage colony-stimulating factor to obtain pro- and anti-inflammatory macrophages (mφ-1 and mφ-2, respectively). We first showed that, in contrast to monocytes, pre-stressed mφ-1 and mφ-2 from healthy blood donors display an enhanced endoplasmic reticulum stress response upon a lipopolysaccharide trigger (XBP1 splicing, CHOP, GADD34 and GRP78 mRNA). However, this endoplasmic reticulum stress response did not differ between monocyte-derived macrophages and monocytes from ZZ patients compared to MM controls. Furthermore, these ZZ cells do not secrete higher cytokine levels, and α1-antitrypsin polymers were not detectable by ELISA. These data suggest that monocyte-derived macrophages are not the local source of Z α1-antitrypsin polymers found in the lung and that endoplasmic reticulum stress and pro-inflammatory cytokine release is not altered.
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Macrófagos/metabolismo , Monocitos/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Deficiencia de alfa 1-Antitripsina/sangre , Adulto , Citocinas/metabolismo , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Ensayo de Inmunoadsorción Enzimática , Femenino , Volumen Espiratorio Forzado , Homocigoto , Humanos , Inflamación/metabolismo , Pulmón/metabolismo , Macrófagos/citología , Masculino , Persona de Mediana Edad , Monocitos/citología , Mutación , FN-kappa B/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Transducción de Señal , Respuesta de Proteína Desplegada , alfa 1-Antitripsina/genética , Deficiencia de alfa 1-Antitripsina/complicacionesRESUMEN
Our study focuses on the intricate connection between tissue-level organization and ciliated organ function in humans, particularly in understanding the morphological organization of airways and their role in mucociliary clearance. Mucociliary clearance is a key mechanical defense mechanism of human airways, and clearance failure is associated with many respiratory diseases, including chronic obstructive pulmonary disease (COPD) and asthma. While single-cell transcriptomics have unveiled the cellular complexity of the human airway epithelium, our understanding of the mechanics that link epithelial structure to clearance function mainly stem from animal models. This reliance on animal data limits crucial insights into human airway barrier function and hampers the human-relevant in vitro modeling of airway diseases. This study, for the first time, maps the distribution of ciliated and secretory cell types along the airway tree in both rats and humans, noting species-specific differences in ciliary function and elucidates structural parameters of airway epithelia that predict clearance function in both native and in vitro tissues alike. By uncovering how tissue organization influences ciliary function, we can better understand disruptions in mucociliary clearance, which could have implications for various ciliated organs beyond the airways.
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Mucociliary clearance is a key mechanical defense mechanism of human airways, and clearance failure is linked to major respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. While single-cell transcriptomics have unveiled the cellular complexity of the human airway epithelium, our understanding of the mechanics that link epithelial structure to clearance function mainly stem from animal models. This reliance on animal data limits crucial insights into human airway barrier function and hampers the human-relevant in vitro modeling of airway diseases. Our study fills this crucial knowledge gap and for the first time (1) maps the distribution of ciliated and secretory cell types on the mucosal surface along the proximo-distal axis of the rat and human airway tree, (2) identifies species-specific differences in ciliary beat and clearance function, and (3) elucidates structural parameters of airway epithelia that predict clearance function in both native and in vitro tissues alike. Our broad range of experimental approaches and physics-based modeling translate into generalizable parameters to quantitatively benchmark the human-relevancy of mucociliary clearance in experimental models, and to characterize distinct disease states.
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Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying the effects of dynamic medium and airflow as well as stretch on human primary airway epithelial cell differentiation and maturation, including mucociliary clearance, using an organ-on-chip device. Perfused epithelial cell cultures displayed accelerated maturation and polarization of mucociliary clearance, and changes in specific cell-types when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in an increase in polarization of mucociliary clearance towards the applied flow, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and maturation and that their fine-tuned application could generate models of specific epithelial pathologies, including mucociliary (dys)function.
RESUMEN
α(1)-Antitrypsin (AAT) acts as an important neutrophil elastase inhibitor in the lung. Although the hepatocyte is considered to be the primary source of AAT, local production by monocytes, macrophages, and epithelial cells may contribute to the formation of an antielastase screen. Because monocytes can differentiate into a heterogeneous population of macrophages with subpopulations ranging from proinflammatory properties (MΦ-1) to antiinflammatory properties (ΜΦ-2) and into dendritic cells (DCs), we studied whether LPS, TNF-α, and oncostatin M (OSM) enhance AAT production differentially in cultured ΜΦ-1, ΜΦ-2, and DCs. Monocytes from healthy blood donors were cultured for 7 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor, or GM-CSF with IL-4 to obtain ΜΦ-1, ΜΦ-2, and immature (i)DCs, respectively. Cells were stimulated with LPS, TNF-α, or OSM, and AAT synthesis was assessed by quantitative RT-PCR, immunocytochemistry, and ELISA. Spontaneous release of AAT was higher in ΜΦ-1 than in ΜΦ-2 and iDCs, and only LPS significantly increased AAT production in ΜΦ-1, ΜΦ-2, and DC. TNF-α and OSM did not affect AAT secretion. The secretion levels of the related protease inhibitors α-1 antichymotrypsin and secretory leukocyte proteinase inhibitor were below the limits of detection by ELISA. In contrast to the protein data, analysis by quantitative RT-PCR showed that 24-hour LPS exposure caused a maximal 2.1-fold AAT mRNA increase in ΜΦ-1, a 21-fold increase in ΜΦ-2, and an 11-fold increase in DCs. These data suggest that cellular differentiation is a regulator of local AAT production.