The Cytotoxicity of PM2.5 and its Effect on the Secretome of Normal Human Bronchial Epithelial Cells

Exposure to airborne fine particulate matter (PM2.5) induced various adverse health effects, such as metabolic syndrome, systemic inflammation and respiratory infection. Many works have studied the influence of PM2.5 exposure to intracellular proteome and the underlying mechanism. But the extracellular proteome changes under PM2.5 exposure, and the correlation between secretome changes and PM2.5-induced cytotoxicity remains confusing. Herein, the cytotoxicity of PM2.5 on normal human bronchial epithelia cells (BEAS-2B) was evaluated and the secretome profile of BEAS-2B cells before and after PM2.5 exposure was investigated. The secretion of 83 proteins (58 up-regulated and 25 down-regulated) was differentially expressed upon PM2.5 treatment. In addition to apoptosis, extracellular matrix (ECM) organization, complement activation and RNA splicing were also found to be involved in PM2.5 mediated cytotoxicity. These results provide an insight into the underlying mechanism of respiratory injury caused by PM2.5.

β2-Adrenoceptor Activation Stimulates IL-6 Production via PKA, ERK1/2, Src, and Beta-Arrestin2 Signaling Pathways in Human Bronchial Epithelia

Objective β2-Adrenoceptor agonists are widely used to treat asthma because of their bronchial-dilation effects. We previously reported that isoprenaline, via the apical and basolateral β2-adrenoceptor, induced Cl− secretion by activating cyclic AMP (cAMP)-dependent pathways in human bronchial epithelia. Despite these results, whether and how the β2-adrenoceptor-mediated cAMP-dependent pathway contributes to pro-inflammatory cytokine release in human bronchial epithelia remains poorly understood. Methods We investigated β2-adrenoceptor-mediated signaling pathways involved in the production of two pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, in 16HBE14o- human bronchial epithelia. The effects of isoprenaline or formoterol were assessed in the presence of protein kinase A (PKA), exchange protein directly activated by cAMP (EPAC), Src, and extracellular signal-regulated protein kinase (ERK)1/2 inhibitors. The involvement of β-arrestin2 was examined using siRNA knockdown. Results Isoprenaline and formoterol (both β2 agonists) induced IL-6, but not IL-8, release, which could be inhibited by ICI 118,551 (β2 antagonist). The PKA-specific inhibitor, H89, partially inhibited IL-6 release. Another intracellular cAMP receptor, EPAC, was not involved in IL-6 release. Isoprenaline-mediated IL-6 secretion was attenuated by dasatinib, a Src inhibitor, and PD98059, an ERK1/2 inhibitor. Isoprenaline treatment also led to ERK1/2 phosphorylation. In addition, knockdown of β-arrestin2 by siRNA specifically suppressed cytokine release when a high concentration of isoprenaline (1 mM) was used. Conclusion Our results suggest that activation of the β2-adrenoceptor in 16HBE14o- cells stimulated the PKA/Src/ERK1/2 and/or β-arrestin2 signaling pathways, leading to IL-6 release. Therefore, our data reveal that β2-adrenoceptor signaling plays a role in the immune regulation of human airway epithelia.

Separating the contributions of SLC26A9 and CFTR to anion secretion in primary human bronchial epithelia (HBE)

Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. While the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel which has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies which suggest it is a significant modifier of CF disease severity. In spite of this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remains poorly understood, in part because its activity is difficult to separate from the activity of CFTR. Here we present results using primary, human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE, and that its constitutive channel activity is unaffected by knock down of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single nucleotide polymorphisms in SLC26A9 modulate airway disease.

β2-adrenoceptor Activation Stimulates IL-6 Production via PKA, ERK1/2, Src, and Beta-arrestin2 Signaling Pathways in Human Bronchial Epithelia

Background: β 2 -adrenoceptor agonists are widely used to treat asthma because of their bronchial-dilation effects. However, a recent study describing a side effect of aggravating eosinophilic inflammation in the mouse airway epithelia by β 2 -adrenoceptor agonists could impact the future clinical use of these bronchodilators. We previously reported that isoprenaline, via the apical and basolateral β 2 -adrenoceptor, induced Cl - secretion by activating cyclic AMP (cAMP)-dependent pathways in human bronchial epithelia. Despite these results, whether and how the β 2 -adrenoceptor-mediated cAMP-dependent pathway contributes to pro-inflammatory cytokine release in human bronchial epithelia remains poorly understood.Methods: We investigated β 2 -adrenoceptor-mediated signaling pathways involved in the production of two pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, in 16HBE14o- human bronchial epithelia. The effects of isoprenaline or formoterol were assessed in the presence of protein kinase A (PKA), exchange protein directly activated by cAMP (EPAC), Src, and extracellular signal-regulated protein kinase (ERK)1/2 inhibitors. The involvement of b-arrestin2 was examined using siRNA knockdown. Results: Both isoprenaline and formoterol (both β 2 agonists) induced IL-6, but not IL-8, release, which could be inhibited by ICI 118551 (β 2 antagonist). The PKA-specific inhibitor, H89, partially inhibited IL-6 release. Another intracellular cAMP receptor, EPAC, was not involved in IL-6 release. Isoprenaline-mediated IL-6 secretion was attenuated by dasatinib, a Src inhibitor, and PD98059, an ERK1/2 inhibitor. Isoprenaline treatment also led to ERK1/2 phosphorylation. In addition, knockdown of β-arrestin2 by siRNA specifically suppressed cytokine release when a high concentration of isoprenaline (1 mM) was used. Conclusion: Our results suggest that activation of the β 2 -adrenoceptor in 16HBE14o- cells stimulated the PKA/Src/ERK1/2 and/or β-arrestin2 signaling pathways, leading to IL-6 release. Therefore, our data reveal that β 2 -adrenoceptor signaling plays a role in the immune regulation of human airway epithelia.

SARS-CoV-2 transmission via apical syncytia release from primary bronchial epithelia and infectivity restriction in children epithelia

The beta-coronavirus SARS-CoV-2 is at the origin of a persistent worldwide pandemic. SARS-CoV-2 infections initiate in the bronchi of the upper respiratory tract and are able to disseminate to the lower respiratory tract eventually causing acute severe respiratory syndrome with a high degree of mortality in the elderly. Here we use reconstituted primary bronchial epithelia from adult and children donors to follow the infection dynamic following infection with SARS-CoV-2. We show that in bronchial epithelia derived from adult donors, infections initiate in multi-ciliated cells. Then, infection rapidly spread within 24-48h throughout the whole epithelia. Within 3-4 days, large apical syncytia form between multi-ciliated cells and basal cells, which dissipate into the apical lumen. We show that these syncytia are a significant source of the released infectious dose. In stark contrast to these findings, bronchial epithelia reconstituted from children donors are intrinsically more resistant to virus infection and show active restriction of virus spread. This restriction is paired with accelerated release of IFN compared to adult donors. Taken together our findings reveal apical syncytia formation as an underappreciated source of infectious virus for either local dissemination or release into the environment. Furthermore, we provide direct evidence that children bronchial epithelia are more resistant to infection with SARS-CoV-2 providing experimental support for epidemiological observations that SARS-CoV-2 cases fatality is linked to age.

Antiviral Properties of the NSAID Drug Naproxen Targeting the Nucleoprotein of SARS-CoV-2 Coronavirus

There is an urgent need for specific antiviral treatments directed against SARS-CoV-2 to prevent the most severe forms of COVID-19. By drug repurposing, affordable therapeutics could be supplied worldwide in the present pandemic context. Targeting the nucleoprotein N of the SARS-CoV-2 coronavirus could be a strategy to impede viral replication and possibly other essential functions associated with viral N. The antiviral properties of naproxen, a non-steroidal anti-inflammatory drug (NSAID) that was previously demonstrated to be active against Influenza A virus, were evaluated against SARS-CoV-2. Intrinsic fluorescence spectroscopy, fluorescence anisotropy, and dynamic light scattering assays demonstrated naproxen binding to the nucleoprotein of SARS-Cov-2 as predicted by molecular modeling. Naproxen impeded recombinant N oligomerization and inhibited viral replication in infected cells. In VeroE6 cells and reconstituted human primary respiratory epithelium models of SARS-CoV-2 infection, naproxen specifically inhibited viral replication and protected the bronchial epithelia against SARS-CoV-2-induced damage. No inhibition of viral replication was observed with paracetamol or the COX-2 inhibitor celecoxib. Thus, among the NSAID tested, only naproxen combined antiviral and anti-inflammatory properties. Naproxen addition to the standard of care could be beneficial in a clinical setting, as tested in an ongoing clinical study.