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In COVID-19, cytokine release syndrome can cause severe lung tissue damage leading to acute respiratory distress syndrome (ARDS). Here, we address the effects of IFNγ, TNFα, IL-1β and IL-6 on the growth arrest of alveolar A549 cells, focusing on the role of the IFN regulatory factor 1 (IRF1) transcription factor. The efficacy of JAK1/2 inhibitor baricitinib has also been tested. A549 WT and IRF1 KO cells were exposed to cytokines for up to 72 h. Cell proliferation and death were evaluated with the resazurin assay, analysis of cell cycle and cycle-regulator proteins, LDH release and Annexin-V positivity; the induction of senescence and senescence-associated secretory phenotype (SASP) was evaluated through β-galactosidase staining and the quantitation of secreted inflammatory mediators. While IL-1 and IL-6 proved ineffective, IFNγ plus TNFα caused a proliferative arrest in A549 WT cells with alterations in cell morphology, along with the acquisition of a secretory phenotype. These effects were STAT and IRF1-dependent since they were prevented by baricitinib and much less evident in IRF1 KO than in WT cells. In alveolar cells, STATs/IRF1 axis is required for cytokine-induced proliferative arrest and the induction of a secretory phenotype. Hence, baricitininb is a promising therapeutic strategy for the attenuation of senescence-associated inflammation. [ABSTRACT FROM AUTHOR]

Background. In COVID-19, an uncontrolled inflammatory response might worsen lung damage, leading to acute respiratory distress syndrome (ARDS). Recent evidence points to the induction of inducible nitric oxide synthase (NOS2/iNOS) as a component of inflammatory response since NOS2 is upregulated in critical COVID-19 patients. Here, we explore the mechanisms underlying the modulation of iNOS expression in human alveolar cells. Methods. A549 WT and IRF1 KO cells were exposed to a conditioned medium of macrophages treated with SARS-CoV-2 spike S1. Additionally, the effect of IFNγ, IL-1β, IL-6, and TNFα, either alone or combined, was addressed. iNOS expression was assessed with RT-qPCR and Western blot. The effect of baricitinib and CAPE, inhibitors of JAK/STAT and NF-kB, respectively, was also investigated. Results. Treatment with a conditioned medium caused a marked induction of iNOS in A549 WT and a weak stimulation in IRF1 KO. IFNγ induced NOS2 and synergistically cooperated with IL-1β and TNFα. The inhibitory pattern of baricitinb and CAPE indicates that cytokines activate both IRF1 and NF-κB through the JAK/STAT1 pathway. Conclusions. Cytokines secreted by S1-activated macrophages markedly induce iNOS, whose expression is suppressed by baricitinib. Our findings sustain the therapeutic efficacy of this drug in COVID-19 since, besides limiting the cytokine storm, it also prevents NOS2 induction. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxemia and high-permeability pulmonary edema. A hallmark of the disease is the presence of lung inflammation with features of diffuse alveolar damage. The molecular pathogenetic mechanisms of COVID-19-associated ARDS (CARDS), secondary to SARS-CoV-2 infection, are still not fully understood. Here, we investigate the effects of a cytokine-enriched conditioned medium from Spike S1-activated macrophage on alveolar epithelial A549 cells in terms of cell proliferation, induction of autophagy, and expression of genes related to protein degradation. The protective effect of baricitinib, employed as an inhibitor of JAK-STAT, has been also tested. The results obtained indicate that A549 exhibits profound changes in cell morphology associated to a proliferative arrest in the G0/G1 phase. Other alterations occur, such as a blockade of protein synthesis and the activation of autophagy, along with an increase of the intracellular amino acids content, which is likely ascribable to the activation of protein degradation. These changes correlate to the induction of IFN-regulatory factor 1 (IRF-1) due to an increased secretion of IFN-γ in the conditioned medium from S1-activated macrophages. The addition of baricitinib prevents the observed effects. In conclusion, our findings suggest that the IFN-γ-IRF-1 signaling pathway may play a role in the alveolar epithelial damage observed in COVID-19-related ARDS. [ABSTRACT FROM AUTHOR]

Background. Clinical and experimental evidence point to a dysregulated immune response caused by SARS-CoV-2 as the primary mechanism of lung disease in COVID-19. However, the pathogenic mechanisms underlying COVID-19-associated ARDS (Acute Respiratory Distress Syndrome) remain incompletely understood. This study aims to explore the inflammatory responses of alveolar epithelial cells to either the spike S1 protein or to a mixture of cytokines secreted by S1-activated macrophages. Methods and Results. The exposure of alveolar A549 cells to supernatants from spike-activated macrophages caused a further release of inflammatory mediators, with IL-8 reaching massive concentrations. The investigation of the molecular pathways indicated that NF-kB is involved in the transcription of IP-10 and RANTES, while STATs drive the expression of all the cytokines/chemokines tested, with the exception of IL-8 which is regulated by AP-1. Cytokines/chemokines produced by spike-activated macrophages are also likely responsible for the observed dysfunction of barrier integrity in Human Alveolar Epithelial Lentivirus-immortalized cells (hAELVi), as demonstrated by an increased permeability of the monolayers to mannitol, a marked decrease of TEER and a disorganization of claudin-7 distribution. Conclusion. Upon exposure to supernatants from S1-activated macrophages, A549 cells act both as targets and sources of cytokines/chemokines, suggesting that alveolar epithelium along with activated macrophages may orchestrate lung inflammation and contribute to alveolar injury, a hallmark of ARDS. [ABSTRACT FROM AUTHOR]

Alveolar epithelium, besides exerting a key role in gas exchange and surfactant production, plays important functions in host defense and inflammation. Pathological conditions associated to alveolar dysfunction include Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The use of predictive in vitro models of human alveolar epithelium is nowadays required for the study of disease mechanisms, as well as of pharmacokinetic parameters of pulmonary drugs delivery. Here, we employed a novel 3D model of human alveoli, namely EpiAlveolar™, consisting of primary alveolar epithelial cells, pulmonary endothelial cells and fibroblasts, that reflects properly the in vivo -like conditions. In EpiAlveolar™ we performed a characterization of Organic Cation Transporters (OCTs and OCTNs) expression and activity and we found that OCTN2, OCT1 and OCT3 are expressed on the basolateral membrane; instead, ATB0,+ transporter for cationic and neutral amino acids, which shares with OCTN2 the affinity for carnitine as substrate, is readily detectable and functional at the apical side. We also show that these transporters differentially interact with anticholinergic drugs. Overall, our findings reveal close similarities of EpiAlveolar™ with the tracheal/bronchial epithelium (EpiAirway™ model) and entrust this alveolar tissue as a potential tool for the screening of biopharmaceuticals molecules. • SLC6A14/ATB0,+ is strongly expressed at the apical side of alveolar epithelium. • OCT1, OCT3, and OCTN2 are active at the basolateral side. • All these transporters are involved in the interaction with respiratory drugs. [ABSTRACT FROM AUTHOR]