Primary Human Airway Epithelial Cells Research Articles

Proof of stability of an RSV Controlled Human Infection Model challenge agent

In 2018, SGS Belgium NV developed RSV-NICA (Respiratory Syncytial Virus-Nasobronchial Infective Challenge Agent), an RSV type A challenge agent for use in RSV Controlled Human Infection Model (CHIM) studies.It is widely recognized that the stability of RSV can be influenced by a variety of environmental parameters, such as temperature and pH. Consequently, our objective was to evaluate the stability of the viral titer of RSV-NICA following five years of controlled storage and to determine the uniformity of the viral titers across different vials of a GMP-qualified batch of RSV-NICA. In addition, we examined the capacity of RSV-NICA to infect human primary airway epithelial cells (MucilAir™), the principal target cells of RSV, and evaluated the influence of single and recurrent freeze–thaw cycles on the infectious viral titer of the challenge agent.The aliquoted RSV-NICA virus stock was subjected to standard virological and molecular methods to gather data on the titer and consistency of the viral titer contained within 24 representative vials of the stock. Our findings illustrate that over a span of five years of cryo-storage, the infectious viral titer in 75% of the tested vials exhibited a comparable average infectious viral titer (4.75 ± 0.06 vs 4.99 ± 0.11; p-value = 0.14). A considerable reduction down to an undetectable level of infectious virus was observed in the remaining vials. RSV-NICA demonstrated its capacity to effectively infect differentiated human airway epithelial cells, with active virus replication detected in these cells through increasing RSV genome copy number over time. Virus tropism for ciliated cells was suggested by the inhibition of cilia beating coupled with an increase in viral RNA titers. No discernable impact on membrane barrier function of the epithelial lung tissues nor cytotoxicity was detected. Pooling of vials with infectious titers > 4.0 log10 TCID50/ml and freeze-thawing of these combined vials showed no deterioration of the infectious titer. Furthermore, pooling and re-aliquoting of vials spanning the entire range of viral titers (including vials with undetectable infectious virus) along with subjecting the vials to three repeated freeze–thaw cycles did not result in a decrease of the infectious titers in the tested vials.Taken together, our findings indicate that long-term cryo-storage of vials containing RSV-NICA challenge agent may influence the infectious viral titer of the virus, leading to a decrease in the homogeneity of this titer throughout the challenge stock. However, our study also demonstrates that when heterogeneity of the infectious titer of an RSV stock is observed, rounds of pooling, re-aliquoting and subsequent re-titration serve as an effective method not only to restore the homogeneity of the infectious titer of an RSV-A stock, but also to optimize patient-safety, scientific and operational aspects of viral inoculation of study participants during at least the period of one RSV CHIM trial. RSV-NICA is a stable, suitable CHIM challenge agent that can be utilized in efficacy trials for RSV vaccines and antiviral entities.

The FAM13A Long Isoform Regulates Cilia Movement and Co-ordination in Airway Mucociliary Transport.

SNPs in the FAM13A locus are amongst the most commonly reported risk alleles associated with chronic obstructive pulmonary disease (COPD) and other respiratory diseases, however the physiological role of FAM13A is unclear. In humans, two major protein isoforms are expressed at the FAM13A locus: 'long' and 'short', but their functions remain unknown, partly due to a lack of isoform conservation in mice. We performed in-depth characterisation of organotypic primary human airway epithelial cell subsets and show that multiciliated cells predominantly express the FAM13A long isoform containing a putative N-terminal Rho GTPase activating protein (RhoGAP) domain. Using purified proteins, we directly demonstrate RhoGAP activity of this domain. In Xenopus laevis, which conserve the long isoform, Fam13a-deficiency impaired cilia-dependent embryo motility. In human primary epithelial cells, long isoform deficiency did not affect multiciliogenesis but reduced cilia co-ordination in mucociliary transport assays. This is the first demonstration that FAM13A isoforms are differentially expressed within the airway epithelium, with implications for the assessment and interpretation of SNP effects on FAM13A expression levels. We also show that the long FAM13A isoform co-ordinates cilia-driven movement, suggesting that FAM13A risk alleles may affect susceptibility to respiratory diseases through deficiencies in mucociliary clearance. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

Polycyclic aromatic hydrocarbon (PAH) activation of AhR inhibits glutathione S-transferase (GST) activity in primary human small airway epithelial cells (hSAECs)

Background: PAH represents the organic phase of particulate matter (PM) in air pollution emitted via the incomplete combustion of various compounds (i.e. diesel fuel, wood, and plastics). It is widely regarded that PAH activation of aryl hydrocarbon receptor (AhR) plays an important role in pro-inflammatory signaling, however the mechanisms remain unknown. In contrast- endogenous AhR agonists, such as 6-Formylindolo[3,2-b]carbazole (FICZ), exert pleiotropic protective effects against inflammation, fibrosis, and oxidative stress. The objective of our research program is to delineate the differences between activation of AhR by PAH (with adverse effects) compared to its beneficial homeostatic activation by endogenous FICZ. PM may induce oxidative stress in part through inhibition of antioxidant defense mechanisms. GSTs are a superfamily of enzymes that plays an important role in protecting cells from oxidative damage (by conjugating reduced glutathione to substrates under pro-oxidizing conditions). We hypothesize that PAH activation of AhR contributes to oxidative stress via inhibition of GST activity in primary hSAECs. Methods: Primary hSAECs were subjected to varying dosages of PAH [0, 250, 500, 1000, 5000 ng/mL] to model airborne PM exposure levels, and to increasing doses of FICZ [0, 0.50, 10, 100, 1000 nM] to model normal and excessively high endogenous ligand activation of AhR. We determined the relative potencies of PAH and FICZ by measuring CYP1A1 and CYP1B1 gene expression. Oxidative stress was measured using DHE-labeling and GST activity was measured with enzymatic kits (Sigma Aldrich). AhR activity was pharmacologically inhibited using 10μM CH223191. Statistical analysis was conducted using SigmaPlot 14.0 (Systat Software). Results: PAH and FICZ maximally stimulate hSAEC CYP1A1 and CYP1B1 expression at 1000ng/mL and 100nM, respectively, and are therefore defined as equipotent concentrations used in our studies. In a representative experiment in hSAECs, PAH (1000ng/mL) significantly increased hSAEC oxidative stress vs FICZ (100nM) n=6, p<0.01), while significantly decreasing GST specific activity vs equipotent FICZ activation of AhR in primary hSAECs from a 66-year-old female without lung disease (26.6±0.874 vs 28.3±.611 nmol/mL/min); n=6 from 2 independent assays; p=0.032. Similar differential effects of PAH vs FICZ activation of AhR on GST were seen in additional experiments with male hSAECs and Calu-3 cells (male). Importantly, CH223191 inhibition of GST enzymatic activity suggests that AhR plays an important role in protecting cells from oxidative stress. Conclusions: Our studies indicate that airborne PAH exposure can have detrimental health effects on the lung via aberrant AhR signaling. Significance: Understanding activity of AhR signaling can leads to novel therapeutic options for redox disorders in the lung. Funding: VA 5I01BX001777-05 (RP) and R01HL137033 (MH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A viral assembly inhibitor blocks SARS-CoV-2 replication in airway epithelial cells

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for innovative therapies with high genetic barriers to resistance. Therefore, there is pronounced interest in identifying new pharmacological targets in the SARS-CoV-2 viral life cycle. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. In this study, we investigate the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). We show that PAV-104 inhibits >99% of infection with diverse SARS-CoV-2 variants in immortalized AECs, and in primary human AECs cultured at the air-liquid interface (ALI) to represent the lung microenvironment in vivo. Our data demonstrate that PAV-104 inhibits SARS-CoV-2 production without affecting viral entry, mRNA transcription, or protein synthesis. PAV-104 interacts with SARS-CoV-2 nucleocapsid (N) and interferes with its oligomerization, blocking particle assembly. Transcriptomic analysis reveals that PAV-104 reverses SARS-CoV-2 induction of the type-I interferon response and the maturation of nucleoprotein signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19 with a mechanism of action that is distinct from existing clinical management approaches.

Altered circadian gene expression in primary human airway epithelial cells in asthma

Altered circadian gene expression in primary human airway epithelial cells in asthma

Klebsiella pneumoniae peptide hijacks a Streptococcus pneumoniae permease to subvert pneumococcal growth and colonization.

Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.

Akt-driven TGF-β and DKK1 Secretion Impairs F508del CF Airway Epithelium Polarity.

Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked-down (KD) for CFTR (CFTR KD) were compared to control counterparts, grown at an air-liquid interface (ALI). We show that CFTR mutation in primary HAECs and CFTR KD cells promote the overexpression and over-secretion of TGF-β1 and DKK1 when cultured at ALI. These dynamic changes result in hyperactivation of the TGF-β pathway and inhibition of the Wnt pathway through degradation of β-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-β and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-β, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF.

Dysfunctional neutrophil type 1 interferon responses in preschool children with recurrent wheezing and IL-4–mediated aeroallergen sensitization

BackgroundThe innate mechanisms associated with viral exacerbations in preschool children with recurrent wheezing are not understood. ObjectiveWe assessed differential gene expression in blood neutrophils from preschool children with recurrent wheezing, stratified by aeroallergen sensitization, at baseline and after exposure to polyinosinic-polycytidylic acid (poly(I:C)). We also examined whether poly(I:C)-stimulated blood neutrophils influenced airway epithelial gene expression. MethodsBlood neutrophils were purified and cultured overnight with poly(I:C) and underwent next-generation sequencing with Reactome pathway analysis. Primary human small airway epithelial cells were treated with poly(I:C)-treated neutrophil culture supernatants and were analyzed for Type 1 interferon gene expression with a targeted array. Symptoms and exacerbations were assessed in participants over 12 months. Results436 genes were differently expressed in neutrophils from children with versus without aeroallergen sensitization at baseline, with significant downregulation of Type 1 interferons. These Type 1 interferons were significantly upregulated in sensitized children after poly(I:C) stimulation. Confirmatory experiments demonstrated similar upregulation of Type 1 interferons in IL-4 treated neutrophils stimulated with poly(I:C). Poly(I:C)-treated neutrophil supernatants from children with aeroallergen sensitization also induced a Type 1 interferon response in epithelial cells. Children with aeroallergen sensitization also had higher symptom scores during exacerbations and these symptom differences persisted for three days after prednisolone treatment. ConclusionsType 1 interferon responses are dysregulated in preschool children with aeroallergen sensitization, which is in turn associated with exacerbation severity. Given the importance of Type 1 interferon signaling in viral resolution, additional studies of neutrophil Type 1 interferon responses are needed in this population.

Sphingosine kinase 1-specific inhibitor PF543 reduces goblet cell metaplasia of bronchial epithelium in an acute asthma model.

Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.

Liquid plug propagation in computer-controlled microfluidic airway-on-a-chip with semi-circular microchannels.

This paper introduces a two-inlet, one-outlet lung-on-a-chip device with semi-circular cross-section microchannels and computer-controlled fluidic switching that enables a broader systematic investigation of liquid plug dynamics in a manner relevant to the distal airways. A leak-proof bonding protocol for micro-milled devices facilitates channel bonding and culture of confluent primary small airway epithelial cells. Production of liquid plugs with computer-controlled inlet channel valving and just one outlet allows more stable long-term plug generation and propagation compared to previous designs. The system also captures both plug speed and length as well as pressure drop concurrently. In one demonstration, the system reproducibly generates surfactant-containing liquid plugs, a challenging process due to lower surface tension that makes the plug formation less stable. The addition of surfactant decreases the pressure required to initiate plug propagation, a potentially significant effect in diseases where surfactant in the airways is absent or dysfunctional. Next, the device recapitulates the effect of increasing fluid viscosity, a challenging analysis due to higher resistance of viscous fluids that makes plug formation and propagation more difficult particularly in airway-relevant length scales. Experimental results show that increased fluid viscosity decreases plug propagation speed for a given air flow rate. These findings are supplemented by computational modeling of viscous plug propagation that demonstrates increased plug propagation time, increased maximum wall shear stress, and greater pressure differentials in more viscous conditions of plug propagation. These results match physiology as mucus viscosity is increased in various obstructive lung diseases where it is known that respiratory mechanics can be compromised due to mucus plugging of the distal airways. Finally, experiments evaluate the effect of channel geometry on primary human small airway epithelial cell injury in this lung-on-a-chip. There is more injury in the middle of the channel relative to the edges highlighting the role of channel shape, a physiologically relevant parameter as airway cross-sectional geometry can also be non-circular. In sum, this paper describes a system that pushes the device limits with regards to the types of liquid plugs that can be stably generated for studies of distal airway fluid mechanical injury.

Single-cell resolution of human airway epithelial cells exposed to bronchiolitis obliterans-associated chemicals.

Bronchiolitis obliterans (BO) is a fibrotic lung disease characterized by progressive luminal narrowing and obliteration of the small airways. In the nontransplant population, inhalation exposure to certain chemicals is associated with BO; however, the mechanisms contributing to disease induction remain poorly understood. This study's objective was to use single-cell RNA sequencing for the identification of transcriptomic signatures common to primary human airway epithelial cells after chemical exposure to BO-associated chemicals-diacetyl or nitrogen mustard-to help explain BO induction. Primary airway epithelial cells were cultured at air-liquid interface and exposed to diacetyl, nitrogen mustard, or control vapors. Cultures were dissociated and sequenced for single-cell RNA. Differential gene expression and functional pathway analyses were compared across exposures. In total, 75,663 single cells were captured and sequenced from all exposure conditions. Unbiased clustering identified 11 discrete phenotypes, including 5 basal, 2 ciliated, and 2 secretory cell clusters. With chemical exposure, the proportion of cells assigned to keratin 5+ basal cells decreased, whereas the proportion of cells aligned to secretory cell clusters increased compared with control exposures. Functional pathway analysis identified interferon signaling and antigen processing/presentation as pathways commonly upregulated after diacetyl or nitrogen mustard exposure in a ciliated cell cluster. Conversely, the response of airway basal cells differed significantly with upregulation of the unfolded protein response in diacetyl-exposed basal cells, not seen in nitrogen mustard-exposed cultures. These new insights provide early identification of airway epithelial signatures common to BO-associated chemical exposures.NEW & NOTEWORTHY Bronchiolitis obliterans (BO) is a devastating fibrotic lung disease of the small airways, or bronchioles. This original manuscript uses single-cell RNA sequencing for identifying common signatures of chemically exposed airway epithelial cells in BO induction. Chemical exposure reduced the proportion of keratin 5+ basal cells while increasing the proportion of keratin 4+ suprabasal cells. Functional pathways contributory to these shifts differed significantly across exposures. These new results highlight similarities and differences in BO induction across exposures.

Parkin Promotes Airway Inflammatory Response to Interferon Gamma.

Increased type 2 interferon (i.e., IFN-γ) signaling has been shown to be involved in airway inflammation in a subset of asthma patients who often show high levels of airway neutrophilic inflammation and poor response to corticosteroid treatment. How IFN-γ mediates airway inflammation in a mitochondrial dysfunction setting (e.g., Parkin up-regulation) remains poorly understood. The goal of this study was to determine the role of Parkin, an E3 ubiquitin ligase, in IFN-γ-mediated airway inflammation and the regulation of Parkin by IFN-γ. A mouse model of IFN-γ treatment in wild-type and Parkin knockout mice, and cultured human primary airway epithelial cells with or without Parkin gene deficiency were used. Parkin was found to be necessary for the production of neutrophil chemokines (i.e., LIX and IL-8) and airway neutrophilic inflammation following IFN-γ treatment. Mechanistically, Parkin was induced by IFN-γ treatment both in vivo and in vitro, which was associated with less expression of a Parkin transcriptional repressor Thap11. Overexpression of Thap11 inhibited Parkin expression in IFN-γ-stimulated airway epithelial cells. Our data suggest a novel mechanism by which IFN-γ induces airway neutrophilic inflammation through the Thap11/Parkin axis. Inhibition of Parkin expression or activity may provide a new therapeutic target for the treatment of excessive neutrophilic inflammation in an IFN-γ-high environment.

Mechanisms of antiviral activity of the new hDHODH inhibitor MEDS433 against respiratory syncytial virus replication

Human respiratory syncytial virus (RSV) is an important cause of acute lower respiratory infections, for which no effective drugs are currently available. The development of new effective anti-RSV agents is therefore an urgent priority, and Host-Targeting Antivirals (HTAs) can be considered to target RSV infections. As a contribution to this antiviral avenue, we have characterized the molecular mechanisms of the anti-RSV activity of MEDS433, a new inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of de novo pyrimidine biosynthesis. MEDS433 was found to exert a potent antiviral activity against RSV-A and RSV-B in the one-digit nanomolar range. Analysis of the RSV replication cycle in MEDS433-treated cells, revealed that the hDHODH inhibitor suppressed the synthesis of viral genome, consistently with its ability to specifically target hDHODH enzymatic activity. Then, the capability of MEDS433 to induce the expression of antiviral proteins encoded by Interferon-Stimulated Genes (ISGs) was identified as a second mechanism of its antiviral activity against RSV. Indeed, MEDS433 stimulated secretion of IFN-β and IFN-λ1 that, in turn, induced the expression of some ISG antiviral proteins, such as IFI6, IFITM1 and IRF7. Singly expression of these ISG proteins reduced RSV-A replication, thus likely contributing to the overall anti-RSV activity of MEDS433. Lastly, MEDS433 proved to be effective against RSV-A replication even in a primary human small airway epithelial cell model. Taken as a whole, these observations provide new insights for further development of MEDS433, as a promising candidate to develop new strategies for treatment of RSV infections.

Anti-SARS-CoV-2 activity of cyanopeptolins produced by Nostoc edaphicum CCNP1411

Despite the advances in contemporary medicine and availability of numerous innovative therapies, effective treatment and prevention of SARS-CoV-2 infections pose a challenge. In the search for new anti-SARS-CoV-2 drug candidates, natural products are frequently explored. Here, fifteen cyanopeptolins (CPs) were isolated from the Baltic cyanobacterium Nostoc edaphicum and tested against SARS-CoV-2. Of these depsipeptides, the Arg-containing structural variants showed the strongest inhibition of the Delta SARS-CoV-2 infection in A549ACE2/TMPRSS2 cells. The functional assays indicated a direct interaction of the Arg-containing CP978 with the virions. CP978 also induced a significant decline in virus replication in the primary human airway epithelial cells (HAE). Of the four tested SARS-CoV-2 variants, Wuhan, Alpha, Omicron and Delta, only Wuhan was not affected by CP978. Finally, the analyses with application of confocal microscopy and with the SARS-CoV-2 pseudoviruses showed that CP978-mediated inhibition of viral infection results from the direct binding of the cyanopeptolin with the coronaviral S protein. Considering the potency of viral inhibition and the mode of action of CP978, the significance of the peptide as antiviral drug candidate should be further explored.

Nepalese indoor cookstove smoke extracts alter human airway epithelial gene expression, DNA methylation and hydroxymethylation

Household air pollution caused by inefficient cooking practices causes 4 million deaths a year worldwide. In Nepal, 86% of the rural population use solid fuels for cooking. Over 25% of premature deaths associated with air pollution are respiratory in nature. Here we aimed to identify molecular signatures of different cookstove and fuel type exposures in human airway epithelial cells, to understand the mechanisms mediating cook stove smoke induced lung disease. Primary human airway epithelial cells in submerged culture were exposed to traditional cook stove (TCS), improved cook stove (ICS) and liquefied petroleum gas (LPG) stove smoke extracts. Changes to gene expression, DNA methylation and hydroxymethylation were measured by bulk RNA sequencing and HumanMethylationEPIC BeadChip following oxidative bisulphite conversion, respectively. TCS smoke extract alone reproducibly caused changes in the expression of 52 genes enriched for oxidative stress pathways. TCS, ICS and LPG smoke extract exposures were associated with distinct changes to DNA methylation and hydroxymethylation. A subset of TCS induced genes were associated with differentially methylated and/or hydroxymethylated CpGs sites, and enriched for the ferroptosis pathway and the upstream regulator NFE2L2. DNA methylation and hydroxymethylation changes not associated with a concurrent change in gene expression, were linked to biological processes and molecular pathways important to airway health, including neutrophil function, transforming growth factor beta signalling, GTPase activity, and cell junction organisation. Our data identified differential impacts of TCS, ICS and LPG cook stove smoke on the human airway epithelium transcriptome, DNA methylome and hydroxymethylome and provide further insight into the association between indoor air pollution exposure and chronic lung disease mechanisms.

Representative measles virus infection requires appropriate airway epithelia culture conditions.

For many years, measles virus (MeV) was assumed to first enter the host via the apical surface of airway epithelial cells and subsequently spread systemically. We and others reported that MeV has an overwhelming preference for entry at the basolateral surface of airway epithelial cells, which led to a fundamental new understanding of how MeV enters a human host. This unexpected observation using well-differentiated primary cultures of airway epithelia from human donors contradicted previous studies using immortalized cultured cells. Here, we show that appropriate differentiation and cell morphology of primary human airway epithelial cells are critical to recapitulate MeV infection patterns and pathogenesis of the in vivo airways. By simply culturing primary cells in media containing serum or passaging primary cultures, erroneous results quickly emerge. These results have broad implications for data interpretation related to respiratory virus infection, spread, and release from human airway epithelial cells.

Interferon-independent processes constrain measles virus cell-to-cell spread in primary human airway epithelial cells.

Amplification of measles virus (MeV) in human airway epithelia may contribute to its extremely high contagious nature. We use well-differentiated primary cultures of human airway epithelial cells (HAE) to model ex vivo how MeV spreads in human airways. In HAE, MeV spreads cell-to-cell for 3-5 days, but then, infectious center growth is arrested. What stops MeV spread in HAE is not understood, but interferon (IFN) is known to slow MeV spread in other in vitro and in vivo models. Here, we assessed the role of type I and type III IFN in arresting MeV spread in HAE. The addition of IFN-β or IFN-λ1 to the medium of infected HAE slowed MeV infectious center growth, but when IFN receptor signaling was blocked, infectious center size was not affected. In contrast, blocking type-I IFN receptor signaling enhanced respiratory syncytial virus spread. HAE were also infected with MeV mutants defective for the V protein. The V protein has been demonstrated to interact with both MDA5 and STAT2 to inhibit activation of innate immunity; however, innate immune reactions were unexpectedly muted against the V-defective MeV in HAE. Minimal innate immunity activation was confirmed by deep sequencing, quantitative RT-PCR, and single-cell RNA-seq analyses of the transcription of IFN and IFN-stimulated genes. We conclude that in HAE, IFN-signaling can contribute to slowing infectious center growth; however, IFN-independent processes are most important for limiting cell-to-cell spread. IMPORTANCE Fundamental biological questions remain about the highly contagious measles virus (MeV). MeV amplifies within airway epithelial cells before spreading to the next host. This final step likely contributes to the ability of MeV to spread host-to-host. Over the course of 3-5 days post-infection of airway epithelial cells, MeV spreads directly cell-to-cell and forms infectious centers. Infectious center formation is unique to MeV. In this study, we show that interferon (IFN) signaling does not explain why MeV cell-to-cell spread is ultimately impeded within the cell layer. The ability of MeV to spread cell-to-cell in airway cells without appreciable IFN induction may contribute to its highly contagious nature. This study contributes to the understanding of a significant global health concern by demonstrating that infectious center formation occurs independent of the simplest explanation for limiting viral transmission within a host.

Human umbilical cord mesenchymal stromal cell small extracellular vesicle transfer of microRNA-223-3p to lung epithelial cells attenuates inflammation in acute lung injury in mice.

Acute lung injury (ALI), manifested as strong pulmonary inflammation and alveolar epithelial damage, is a life-threatening disease with high morbidity and mortality. Small extracellular vesicles (sEVs), secreted by multiple types of cells, are critical cellular communication mediators and can inhibit inflammation by transferring bioactive molecules, such as microRNAs (miRNAs). Thus, we hypothesized that sEVs derived from mesenchymal stromal cells (MSC sEVs) could transfer miRNAs to attenuate inflammation of lung epithelial cells during ALI. C57BL/6 male mice were intratracheally administered LPS (10mg/kg). Six hours later, the mice were randomly administered with MSC sEVs (40µg per mouse in 150µl of saline), which were collected by ultracentrifugation. Control group received saline administration. After 48h, the mice were sacrificed to evaluate pulmonary microvascular permeability and inflammatory responses. In vitro, A549 cells and primary human small airway epithelial cells (SAECs) were stimulated with LPS with or without MSC sEVs treatment. In vitro, MSC sEVs could also inhibit the inflammation induced by LPS in A549 cells and SAECs (reducing TNF-α, IL-1β, IL-6 and MCP-1). Moreover, MSC sEV treatment improved the survival rate, alleviated pulmonary microvascular permeability, and inhibited proinflammatory responses (reducing TNF-α, IL-1β, IL-6 and JE-1) in ALI mice. Notably, miR-223-3p was found to be served as a critical mediator in MSC sEV-induced regulatory effects through inhibition of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) in lung epithelial cells. Overall, these findings suggest that MSC sEVs may offer a novel promising strategy for ALI.

A WNT4- and DKK3-driven canonical to noncanonical Wnt signaling switch controls multiciliogenesis.

Multiciliated cells contain hundreds of cilia whose directional movement powers the mucociliary clearance of the airways, a vital host defense mechanism. Multiciliated cell specification requires canonical Wnt signaling, which then must be turned off. Next, ciliogenesis and polarized ciliary orientation are regulated by noncanonical Wnt/planar cell polarity (Wnt/PCP) signaling. The mechanistic relationship between the Wnt pathways is unknown. We show that DKK3, a secreted canonical Wnt regulator and WNT4, a noncanonical Wnt ligand act together to facilitate a canonical to noncanonical Wnt signaling switch during multiciliated cell formation. In primary human airway epithelial cells, DKK3 and WNT4 CRISPR knockout blocks, whereas ectopic expression promotes, multiciliated cell formation by inhibiting canonical Wnt signaling. Wnt4 and Dkk3 single-knockout mice also display defective ciliated cells. DKK3 and WNT4 are co-secreted from basal stem cells and act directly on multiciliated cells via KREMEN1 and FZD6, respectively. We provide a novel mechanism that links specification to cilium biogenesis and polarization for proper multiciliated cell formation.

Myeloid-associated differentiation marker is associated with type 2 asthma and is upregulated by human rhinovirus infection.

Human rhinoviruses are known to predispose infants to asthma development during childhood and are often associated with exacerbations in asthma patients. MYADM epithelial expression has been shown to associate with asthma severity. The goal of this study was to determine if MYADM expression patterns were altered in asthma and/or rhinovirus infection and if increased MYADM expression is associated with increased asthma-associated factors. Utilizing H1HeLa cells and differentiated primary human airway epithelial cells (AECs), we measured the expression of MYADM and inflammatory genes by qRT-PCR in the presence or absence of RV-1B infection or poly I:C treatment and with siRNA knockdown of MYADM. Expression of MYADM in the asthmatic lung was determined in the ovalbumin (ova)-challenged murine model. MYADM expression was upregulated in the lungs from ova-treated mice and in particular on the subsurface vesicle membrane in airway epithelial cells. Upon infection with RV-1B, human AECs grown at an air-liquid interface had increased the MYADM expression predominantly detected in ciliated cells. We found that the presence of MYADM was required for expression of several inflammatory genes both in a resting state and after RV-1B or poly I:C treatments. Our studies show that in a mouse model of asthma and during RV-1B infection of primary human AECs, increased MYADM expression is observed. In the mouse model of asthma, MYADM expression was predominantly on the luminal side of airway epithelial cells. Additionally, MYADM expression was strongly associated with increases in inflammatory genes, which may contribute to more severe asthma and RV-linked asthma exacerbations.