J Toxicol Environ Health A

Exposure to organic dusts is associated with increased respiratory morbidity and mortality in agricultural workers. Organic dusts in dairy farm environments are complex, polydisperse mixtures of toxic and immunogenic compounds. Previous toxicological studies focused primarily on exposures to the respirable size fraction; however, organic dusts in dairy farm environments are known to contain larger particles. Given the size distribution of dusts from dairy farm environments, the nasal and bronchial epithelia represent targets of agricultural dust exposures. In this study, well-differentiated normal human bronchial epithelial cells and human nasal epithelial cells were exposed to two different size fractions (PM10 and PM>10) of dairy parlor dust using a novel aerosol-to-cell exposure system. Levels of proinflammatory transcripts (interleukin [IL]-8, IL-6, and tumor necrosis factor [TNF]-\uce\ub1) were measured 2 h after exposure. Lactate dehydrogenase (LDH) release was also measured as an indicator of cytotoxicity. Cell exposure to dust was measured in each size fraction as a function of mass, endotoxin, and muramic acid levels. To our knowledge, this is the first study to evaluate the effects of distinct size fractions of agricultural dust on human airway epithelial cells. Our results suggest that both PM10 and PM>10 size fractions elicit a proinflammatory response in airway epithelial cells and that the entire inhalable size fraction needs to be considered when assessing potential risks from exposure to agricultural dusts. Further, data suggest that human bronchial cells respond differently to these dusts than human nasal cells, and therefore that the two cell types need to be considered separately in airway cell models of agricultural dust toxicity.R01 ES019325/ES/NIEHS NIH HHS/United StatesR01ES019325/ES/NIEHS NIH HHS/United StatesU54OH008085/OH/NIOSH CDC HHS/United States2016-01-01T00:00:00Z25965193PMC443033

Secretory to multi-ciliated cell imbalance by altered cellular progeny in end-stage COPD facilitates resilience to environmental pollutants

Background: Air pollution is a major risk factor for patients suffering from chronic res-piratory conditions including chronic obstructive pulmonary disease (COPD), as it drives episodes of exacerbation and subsequently disease progression. Analysis of the differentiation process of the primary human bronchial epithelial cells (pHBECs) from non-chronic lung disease (non-CLD) and CLD/ COPD-diseased tissue samples is of critical importance to understand the underlying pathophysiological mech-anisms that characterize the disease specific response to air pollutant exposure at the first line of defense, i.e. the human bronchial epithelium. In addition, pHBECs culture con-tributes to potential identification of preventive and therapeutic strategies in CLD. Materials and Methods: We established 3D air-liquid interface (ALI) cultures in pHBECs isolated from large airway resections of diseased (n=3 COPD-II and n=6 COPD-IV) and healthy (non-CLD, n=4) patients. To mimic air pollution, pHBECs were exposed to relevant aerosolized nanoparticles (NPs, i.e. carbon black soot surrogate NP (CNP) and Zinc oxide (ZnO)) using the pre-clinical, highly standardized VITROCELL® CLOUD 12 nebulization system (Waldkirch, Germany). ALI cultures, validated for their disease specific, biomimetic cellular composition using trans-mural bronchial punches (BP), were analyzed for functional consequences of NP exposure via transepi-thelial electrical resistance (TEER), WST-1, LDH, 3D confocal immunofluorescence (IF), transcriptome, secretome as well as ciliary beating frequencies (CBF) of multi-ciliated cells (MCC). To highlight the cell differentiation trajectory that explains the outlined cell composition and functional changes postexposure, single cell RNA-seq drop-seq analysis and immunofluorescence (IF) stainings of native bronchial tissue sam-ples were performed. Results: ZnO exposure induced effects on the amount of MCC and function exceeded the effects observed by CNP or LPS exposure. Exposure to moderate ZnO doses in-duced a decrease in the number of MCC in COPD-II (20.35±14.07%) and COPD-IV pHBECs (18.51±11.86%) when compared to non-CLD cells (47.01±2.80%), as well as an elevated number of MUC5AC+ cells in COPD-IV cultures (12.75±2.90%) when compared to non-CLD cultures (5.17±2.43%). These findings were accompanied by a concentration dependent reduction in epithelial barrier integrity (TEER), metabolic cell viability (WST-1) and membrane integrity (LDH release) in non-CLD and COPD-II pHBECs when compared to COPD-IV pHBECs. Following ZnO and CNP exposure, COPD-IV cultures were characterized by transcriptional regulation of genes involved in secretory cells (SC)-MCC differentiation axis (cilium assembly and organization), TLR-mediated innate immunity and regulation of extracellular matrix remodeling (ECM). Also at baseline level, transcriptome analysis revealed an overrepresentation of ECM gene clusters in COPD-IV cultures. Cellular composition of pHBECs ALI cultures resembled the ex vivo picture achieved by culturing BPs at ALI. These findings highlight an oligo-ciliated hypersecretory phe-notype in COPD-IV cultures, with a skewed basal cells (BC)-induced cell trajectory towards SC at the expense of the more vulnerable MCC. Terminal differentiation into MCC resulted from progenitor SC (MUC5AC+, CC10+). The outlined phenotype was in line with an aberrant expression of MCC genes together with a pathologic CBF spectrum in COPD-IV cultures. Drop-seq single cell RNA-seq analysis in both non-CLD and COPD-IV cultures on ALI day 0 and ALI day 28 revealed two distinct BC populations (basal_1, basal_2) as pro-genitor cells for the SC-MCC differentiation axis. BC present a strong shift between non-CLD and COPD-IV patients on both ALI day 0 and day 28. Specifically, basal_1 cells characterized the COPD-IV cultures, being predominantly detected on ALI day 0 and exclusively on ALI day 28 in COPD-IV derived pHBECs. Conversely, basal_2 cells characterized the non-CLD derived pHBECs on both ALI day 0 and 28. These signatures were validated by IF stainings of native bronchial tissue samples. Conclusion. In summary, our results identify the predominance of SC in the large air-ways of patients suffering from COPD-IV resulting in a greater functional resilience of the pHBECs to environmental small particle exposure underlined by the unsuccessful drive to induce trans-differentiation of the SC cells into MCC.Hintergrund: Luftverschmutzung ist der wichtigste Risikofaktor für Patienten mit chronischen obstruktiven Lungenerkrankung (COPD). Die chronische Nanopartikel (NP) Exposition führt zur wiederholten Exazerbationen und zu einem unvermeidbaren Fortschreiten der COPD Erkrankung. Die Analyse des Differenzierungsprozesses der primären humanen Bronchialepithelzel-len (pHBECs) von Patienten ohne chronische Lungenerkrankungen (non-chronic lung disease/ non-CLD) und mit chronischen Lungenerkrankungen (CLD/ e.g. COPD) ist von entscheidender Bedeutung, die pathophysiologischen Mechanismen des Bronchial-epithels nach Luftverschmutzung zu charakterisieren sowie präventive und therapeuti-sche Strategien für CLD zu etablieren. Materialen und Methoden: Wir haben eine 3D Zellkultur an der Luft-Flüssigkeit-Grenzschicht (air liquid interface/ ALI) mit pHBECs aus den proximalen Hauptbron-chien von COPD (n = 3 COPD-II und n = 6 COPD-IV) und gesunden (n = 4 non-CLD) Patienten etabliert. Um die Umweltverschmutzung in vitro zu simulieren, wurde eine Exposition der pHBECs mit relevanten aerosolierten NP (z.B. Carbon soot surrogate NP (CNP) und Zinkoxid (ZnO)) mit Hilfe eines standardisierten Expositionssystems (VIT-ROCELL® CLOUD 12, Waldkirch, Germany) durchgeführt. Die Validierung von ALI-Zellkulturen und deren krankheitsspezifischen und biomimetischen Zellzusammenset-zung erfolgte mit Hilfe eines neuen 3D Kulturmodells unter Verwendung von frischen nativen humanen Bronchialwand-Präparaten (bronchial punches/ BPs), die die intakte Struktur der gesamten Bronchialwand aufrechterhalten. Für die Validierung der 3D ALI pHBECs Kulturen erfolgte die Analyse des transepithelialen elektrischen Widerstands (TEER), der Zellviabilität (WST-1) und der Membranintegrität (LDH), sowie die 3D konfokale Immunfluoreszenz (IF), Transkriptom- , Sekretomanalyse, und die Analyse der Zilienschlagfrequenz (ciliary beating frequency/ CBF). Um die Änderungen der Zellzusammensetzung und der Epithelfunktion nach NP Exposition genauer zu analysie-ren, wurden eine single cell RNA-seq drop-seq Analyse und IF von nativen Bronchus-gewebeproben ergänzend durchgeführt. Ergebnisse: Die ZnO-induzierte Effekte auf die Zilien-tragenden Zellen (multi-ciliated cells/ MCC) übertrafen die bei CNP- oder LPS-Exposition beobachteten Effekte. Die Exposition mit moderaten ZnO NP Dosierungen führte zu einer leichten Zahlabnahme der MCC in COPD-II (20,35 ± 14,07%) und COPD-IV pHBECs (18,51 ± 11,86%) im Vergleich zu non-CLD Zellen (47,01 ± 2,80%), sowie zu einer erhöhten Zahl von MUC5AC+ Zellen in COPD-IV Kulturen (12,75 ± 2,90%) im Vergleich zu non-CLD Zellen (5,17 ± 2,43%). Diese Befunde korrelieren mit einem konzentrationsabhängigen Verlust des transepithelialen elektrischen Widerstands (TEER), der Zellviabilität (WST-1) und der Membranintegrität (LDH-Freisetzung) in non-CLD und COPD-II im Vergleich zu COPD-IV pHBECs. Die ZnO und CNP Exposition führte zu einer aberranten Überexpression von Zilien spe-zifischen Genen in den COPD-IV Kulturen. Die Transkriptomanalyse der COPD-IV Kulturen nach ZnO und CNP Exposition zeigte eine Aktivierung der Mukuszellen-MCC Differenzierungsachse, der TLR-Immunität und der extrazellulären Matrix Biosyntese (extracellular matrix remodeling/ ECM). Des Weiteren bestätigen die unbehandelten COPD-IV Kulturen eine signifikante ECM Genexpression. Darüber hinaus ähnelte die Zellzusammensetzung der pHBEC-ALI-Kulturen dem ex vivo Bild der BPs. Diese Ergebnisse demonstrieren einen „oligo-ciliated“ hypersekretori-schen Phänotyp der COPD-IV ALI Kulturen im Rahmen einer verzerrten, Basalzellen-induzierten Zelldifferenzierung in Richtung sekretorischen Zellen, auf Kosten der anfäl-ligen MCC. Die sekretorischen Zellen (MUC5AC+, CC10+) sind Vorläuferzellen für MCC. Dieser Phänotyp stimmte mit einer aberranten MCC Genexpression sowie einem pathologischen CBF Spektrum in COPD-IV Kulturen überein. Die drop-seq single cell Analyse bei non-CLD und COPD-IV Patienten zeigt zwei un-terschiedliche Basalzellpopulationen (basal_1, basal_2) am ALI Tag 0 und 28. Die ba-sal_1 Zellen sind überwiegend am Tag 0 und ausschließlich am Tag 28 in COPD-IV Kulturen identifizierbar. Die basal_2 Zellen charakterisieren die non-CLD-Kulturen so-wohl am Tag 0 als auch am Tag 28. Die single cell Signaturen wurden durch IF Färbun-gen validiert. Schlussfolgerung: Der hypersekretorische Phänotyp der COPD-IV ALI Kulturen führt zu einer größeren funktionellen Widerstandsfähigkeit des Bronchialepithels nach NP Exposition und resultiert aus einer erfolglosen Transdifferenzierung der sekretorischen Zellen in MCC

Differential Response of Human Nasal and Bronchial Epithelial Cells Upon Exposure to Size-Fractionated Dairy Dust

<div><p>Exposure to organic dusts is associated with increased respiratory morbidity and mortality in agricultural workers. Organic dusts in dairy farm environments are complex, polydisperse mixtures of toxic and immunogenic compounds. Previous toxicological studies focused primarily on exposures to the respirable size fraction; however, organic dusts in dairy farm environments are known to contain larger particles. Given the size distribution of dusts from dairy farm environments, the nasal and bronchial epithelia represent targets of agricultural dust exposures. In this study, well-differentiated normal human bronchial epithelial cells and human nasal epithelial cells were exposed to two different size fractions (PM₁₀ and PM_>10) of dairy parlor dust using a novel aerosol-to-cell exposure system. Levels of proinflammatory transcripts (interleukin [IL]-8, IL-6, and tumor necrosis factor [TNF]-α) were measured 2 h after exposure. Lactate dehydrogenase (LDH) release was also measured as an indicator of cytotoxicity. Cell exposure to dust was measured in each size fraction as a function of mass, endotoxin, and muramic acid levels. To our knowledge, this is the first study to evaluate the effects of distinct size fractions of agricultural dust on human airway epithelial cells. Our results suggest that both PM₁₀ and PM_>10 size fractions elicit a proinflammatory response in airway epithelial cells and that the entire inhalable size fraction needs to be considered when assessing potential risks from exposure to agricultural dusts. Further, data suggest that human bronchial cells respond differently to these dusts than human nasal cells, and therefore that the two cell types need to be considered separately in airway cell models of agricultural dust toxicity.</p></div