SARS-CoV-2 infection of human-induced pluripotent stem cells-derived lung lineage cells evokes inflammatory and chemosensory responses by targeting mitochondrial pathways

Abstract

The COVID-19 disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily affects the lung, particularly the proximal airway and distal alveolar cells. NKX2.1+ primordial lung progenitors of the foregut (anterior) endoderm are the developmental precursors to all adult lung epithelial lineages and are postulated to play an important role in viral tropism. Here, we show that SARS-CoV-2 readily infected and replicated in human-induced pluripotent stem cell-derived proximal airway cells, distal alveolar cells, and lung progenitors. In addition to the upregulation of antiviral defense and immune responses, transcriptomics data uncovered a robust epithelial cell-specific response, including perturbation of metabolic processes and disruption in the alveolar maturation program. We also identified spatiotemporal dysregulation of mitochondrial heme oxygenase 1 (HMOX1), which is associated with defense against antioxidant-induced lung injury. Cytokines, such as TNF-α, INF-γ, IL-6, and IL-13, were upregulated in infected cells sparking mitochondrial ROS production and change in electron transport chain complexes. Increased mitochondrial ROS then activated additional proinflammatory cytokines leading to an aberrant cell cycle resulting in apoptosis. Notably, we are the first to report a chemosensory response resulting from SARS-CoV-2 infection similar to that seen in COVID-19 patients. Some of our key findings were validated using COVID-19-affected postmortem lung tissue sections. These results suggest that our in vitro system could serve as a suitable model to investigate the pathogenetic mechanisms of SARS-CoV-2 infection and to discover and test therapeutic drugs against COVID-19 or its consequences.

Keywords: SARS-CoV-2; chemosensory response; induced pluripotent stem cells; inflammatory response; lung epithelial cells; mitochondrial damage.

Figure 1

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection and replication inside induced pluripotent stem cells (iPSC)‐derived lung epithelial lineages. (a) Differentiation snapshot capturing various stages involved in the generation of lung cells from iPSC along with distinct markers expressed along different lung cell types (b) Lung progenitor cells—transcription factor NKX2.1, alveolar progenitor—SOX9, developing lung bud marker‐ FOXP2, and distal alveolar type II cells expressing SP‐C (c) Proximal airway cells expressing goblet cell—FOXJ1 costained with SOX2, ciliated cell—ARL13B costained with tight junction protein ‐ZO1, basal cell‐ P63, Clara cell secretory protein—CC10 (d) Lung cell types showing ACE2 and TMPRSS2 at protein levels (e) Immunofluorescence analysis of SARS‐CoV‐2 spike protein (green) in uninfected lung cells. Nuclei were counterstained with DAPI (blue) (f, g) Real‐time PCR levels of viral N gene and ORF gene in the cell after 24 and 72 h of SARS‐CoV‐2 infection (h) Representative plaque assay plate picture of SARS‐CoV‐2 infection (i) Plaque assay plates quantified and levels of viral infection virus in culture supernatant represented as titer values. Scale bars represent 100 µm

Figure 2

Severe acute respiratory syndrome coronavirus 2 infection results in a massive inflammatory response. (a) Number of significantly up/downregulated genes identified by comparing with respective uninfected control represented in a table. (b, c) Gene ontology fold enrichment for infection and inflammatory response. Heat map color, white to red. (d−f) Real‐time PCR validation of selected genes (d) Inflammation—TNF‐α, IL‐6, IL‐13 (e) Apoptosis—BCL2, CDK1, FOS (f) Tissue repair—TGFβ1, VEGF (g) Heat map of genes from key signaling pathway involved in inflammation—IFNγ, NF‐κΒ; heat map color, red to green through black. NF‐kB, nuclear factor kappa B; TGF, tumor growth factor; NF, tumor necrosis factor; VEGF, vascular endothelial growth factor

Figure 3

Severe acute respiratory syndrome coronavirus 2 infection negatively impacts alveolar maturation, induces fibrosis, and triggers chemosensory changes. (a, b) messanger RNA (mRNA) level changes in lung developmental program represented as FPKM values for (a) proximal airway genes and (b) distal alveolar genes (c, d) Real‐time polymerase chain reaction (PCR) validation of selected genes (c) Pulmonary secretion—Muc5ac, AQP5 (d) Fibrosis—α‐collagen, MMP2, MMP9 (e) Gene ontology showing changes in sensory perception functional modules (f) Genes responsible for bitter taste upregulated in infected samples and sweet taste downregulated (g) qPCR validation of bitter taste gene—TAS2R5, TAS2R38 (h) Heat map showing expression of PNEC genes. (i) Proximal airway cells expressing PNEC marker TUJ1 costained with basal cell marker P63. Heat map color, blue to yellow through white. Scale bars represent 100 µm

Figure 4

Mitochondrial damage is associated with inflammation and apoptosis. (a) mRNA level changes in ACE2 and NOX4 are represented as FPKM values (b, c) MRPS and NDUF family genes are represented as heat maps. (d) Genes related to reactive oxygen species are represented as heat maps (e) Key mitochondrial genes involved in the electron transport chain are represented as heat maps (f) Real‐time qPCR validation of mitochondrial electron transport chain complex genes—MT‐CO1, MT‐CYB, MT‐ND1, and SDHA across all cell types upon infection (g) Protein network analysis showing a direct correlation between mitochondrial HMOX with inflammatory and apoptotic genes. (h) Airway and alveolar lung cells expressing heme oxygenase 1 (HMOX1) protein (i) messanger RNA level changes in HMOX1 and HMOX2 represented as FPKM value (j) Real‐time qPCR analysis showing the dysregulation of HMOX1 during the course of infection. Heat map color, red to green through black. Scale bars represent 100 µm

Figure 5

Disruption of alveolar spaces and inflammation in COVID‐19 infected lung tissues support in vitro findings. (a, b) Hematoxylin and eosin‐stained COVID lung section showing (a) ARDS like pathology with edema and hyaline membrane (b) features of pneumonia (c−f) Lung surfactant—SPC, secretory protein CC10 highlights the alveolar lining cells in normal lung and disrupted staining along the alveolar walls with no proper air spaces (g−j) Inflammatory cytokines—IL‐6, IL‐13 stained macrophages in normal lung and massive cellular infiltration in COVID lung. Scale bars represent 100 µm

Figure 6

A schematic representing the working model of our study, starting from entry of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) inside the lung epithelial cell types, triggering an inflammatory response, chemosensory changes, and impaired proximal‐distal lung patterning. These alterations eventually resulted in mitochondrial dysfunction and pulmonary fibrosis via specific cellular and molecular events. HMOX1, heme oxygenase 1; iPSC, induced pluripotent stem cells.