Immune-Mediated Inflammatory Responses of Alveolar Epithelial Cells: Implications for COVID-19 Lung Pathology

Abstract

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.

Keywords: COVID-19; IL-8; chemokines; cytokines; epithelial barrier dysfunction; human alveolar epithelial cells; human macrophages.

Figure 1

Alveolar epithelial A549 cells were incubated for 4 h with the indicated concentrations of spike S1 protein (Panel A) or with conditioned medium (CM) obtained by incubating monocyte-derived macrophages (MDM) for 16 h in the absence (CM_cont) or in the presence of 10 nM spike S1 (CM_S1) (Panel B). The expression of the indicated genes was measured by means of RT-qPCR and calculated relative to untreated cells (=1; dotted line) upon normalization for the housekeeping gene RPL15, as described in Methods. Data are means ± SEM of four determinations, each performed in duplicate. * p < 0.05, *** p < 0.001 vs. untreated cells.

Figure 2

A549 were incubated for 24 h with conditioned medium from monocyte-derived macrophages (MDM) maintained for 16 h in the presence of 10 nM S1 (CM_S1). The amount of the indicated cytokines and chemokines in CM_S1 was measured with an ELISA assay before (pre-treatment) and after (post-treatment) treatment of A549 cells, as described in Methods. Data are means ± SEM of four independent experiment, each performed in quadruplicate. * p < 0.05, ** p < 0.01 vs. CM_S1 pre-treatment.

Figure 3

A549 were incubated for the indicated times with conditioned medium (CM) from untreated (CM_cont) or S1-treated (CM_S1) monocyte-derived macrophages (MDM). The expression of the indicated proteins was assessed by means of Western Blot analysis, as detailed in Methods; representative blots are shown and the densitometric analysis of three different experiments is shown.

Figure 4

A549 were incubated for 4 h with conditioned medium (CM) from untreated (CM_cont) or S1-treated (CM_S1) monocyte-derived macrophages (MDM), either in the absence or in the presence of the indicated inhibitors, added 30 min before the treatment. The expression of the indicated genes was measured by means of RT-qPCR and calculated relative to CM_cont (=1, dotted line) upon normalization for the housekeeping gene RPL15, as described in Methods. Data are means ± SEM of four determinations, each performed in duplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CM_S1.

Figure 5

hAELVi, cultured under air–liquid interface (ALI) conditions for 20 d, were incubated with conditioned medium (CM) from untreated (CM_cont) or S1-treated (CM_S1) monocyte-derived macrophages (MDM). Transepithelial electrical resistance (TEER) values were measured at the indicated times (Panel A) and mannitol fluxes were monitored after 24 h (Panel B), as described in Methods. Data represent the mean ± SEM of three independent determinations. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. CM_cont (Panel C). After 24 h cells were fixed and immunostained for ZO-1 (green) and claudin-7 (red); for each condition, a single horizontal confocal section of a representative field is shown, with orthogonal projections. Bottom images show the merged signals of ZO-1, claudin-7 and Hoecst33342 (blue) employed for nuclear staining (see Methods). The experiment was performed twice with similar results. Bar = 20 μm.