Human Nasal and Lung Tissues Infected Ex Vivo with SARS-CoV-2 Provide Insights into Differential Tissue-Specific and Virus-Specific Innate Immune Responses in the Upper and Lower Respiratory Tract

Abstract

The nasal mucosa constitutes the primary entry site for respiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the imbalanced innate immune response of end-stage coronavirus disease 2019 (COVID-19) has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here, we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with a rapid increase in tissue-associated viral subgenomic mRNA and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon-stimulated genes, cytokines, and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tracts that are specific to SARS-CoV-2. The studies shed light on the role of the nasal mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCE In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here, we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues infected in parallel with SARS-CoV-2 and influenza virus, we found distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal mucosal infection model can be employed to assess the impact of viral evolutionary changes and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

Keywords: COVID-19; SARS-CoV-2; human respiratory viruses; nasal mucosa; organ culture; tissue innate immune response.

FIG 1

Confocal microscopy analysis of SARS-CoV-2 receptors and cellular tropism in nasal turbinate and lung organ cultures. (A and B) Representative confocal micrographs of whole-mount turbinate (A) and lung (B) cultures, stained for the indicated SARS-CoV-2 receptor and the epithelial cell marker Ep-CAM. Yellow arrows point to cells exhibiting colocalization. DAPI-stained nuclei are shown in blue. Bar, 100 μm. (C and D) Representative confocal micrographs of whole-mount turbinate (C) and lung (D) cultures at 24 h after infection with SARS-CoV-2 or influenza virus A(H1N1) pdm09 as indicated, showing the respective viral nucleoprotein (NP) colocalization with the epithelial cell marker Ep-CAM. Yellow arrows point to cells exhibiting colocalization. DAPI-stained nuclei are shown in blue. Bar, 50 μm.

FIG 2

SARS-CoV-2 and influenza virus infection kinetics in nasal turbinate and lung organ cultures. Nasal turbinate and lung organ cultures were (each) infected in parallel with SARS-CoV-2 or influenza virus A(H1N1) pdm09 (2 × 10⁵ TCID₅₀/well). (A) Levels of tissue-associated SARS-CoV-2 N gene subgenomic RNA (sg-RNA) and influenza virus RNA were determined by RT-qPCR and normalized to β-actin (left panels); Infectious SARS-CoV-2 and influenza virus progeny titers in the supernatants of the same infected tissues were determined by a standard 50% tissue culture infectious dose (TCID₅₀) assay (right panels). The data shown are representative of at least 3 independent tissues, and each point represents the mean ± SEM for 5 biological replicates. (B) Percent of viral transcripts in the transcriptome of infected tissues. RNA was extracted from SARS-CoV-2 or influenza virus A(H1N1) pdm09-infected turbinate and lung tissues at 24 h postinfection and subjected to transcriptome analysis. Three independent donors’ turbinate tissues and five independent donors’ lung tissues (with two biological replicates for each experimental condition) were included in the analysis. Mean values for percentages of viral transcripts (out of all the sequence reads in the transcriptome) with SEM are shown. **, P < 0.01; ns, not significant.

FIG 3

Nasal turbinate tissue transcriptional response to SARS-CoV-2 and influenza virus infection. Nasal turbinate tissues were mock infected or infected in parallel with SARS-CoV-2 or influenza virus A(H1N1) pdm09 (2 × 10⁵ TCID₅₀/well). At 24 h postinfection, RNA was extracted and subjected to transcriptome analysis. Three independent donors’ tissues (with two biological replicates for each experimental condition) were included in the analysis. (A) Principal-component analysis (PCA) of the global transcriptional response of the nasal turbinate tissues to SARS-CoV-2 or influenza virus infection. The first two PCs are shown. (B) Venn diagram illustration of the number of unique and overlapping differentially expressed (DE) genes in SARS-CoV-2- and influenza virus-infected nasal turbinate tissues. (C) The 20 most profoundly upregulated genes in SARS-CoV-2-infected (versus mock-infected) nasal turbinate tissues. (D) Clustered heat map representation of all genes which exhibited a significant effect of infection on their expression in nasal turbinate tissues. Normalized expression values were scaled at gene level (scale is shown at top right) and then clustered by kmeans (with a k value of 4), as indicated. Representative pathways and molecular functions distinctively enriched in SARS-CoV-2-infected tissues (versus influenza virus-infected tissues), as reflected by the related upregulated genes in cluster 2 and downregulated genes in cluster 3, are indicated on the left. (E) Effect of SARS-CoV-2 and influenza virus infection on the expression of selected innate immunity genes in nasal turbinate cultures. RNA from SARS-CoV-2-, influenza virus A(H1N1) pdm09-, and mock-infected cultures was extracted at 24 h postinfection and analyzed for the indicated gene expression by RT-qPCR, normalized to the expression of the housekeeping β-actin gene.

FIG 4

Lung tissue transcriptional response to SARS-CoV-2 and influenza virus infection. Lung tissues were mock infected or infected in parallel with SARS-CoV-2 or influenza virus A(H1N1) pdm09 (2 × 10⁵ TCID₅₀/well). At 24 h postinfection, RNA was extracted and subjected to transcriptome analysis. Five independent donors’ tissues (with two biological replicates for each experimental condition) were included in the analysis. (A) Principal-component analysis (PCA) of the global transcriptional response of the lung tissues to SARS-CoV-2 or influenza virus infection. The first two PCs are shown. (B) Venn diagram illustration of the number of unique and overlapping differentially expressed (DE) genes in SARS-CoV-2- and influenza virus-infected lung tissues. (C) The 20 most-profoundly upregulated genes in SARS-CoV-2-infected (versus mock-infected) lung tissues. (D) Clustered heat map representation of all genes with a significant contribution of infection to their expression in lung tissues. Normalized expression values were scaled at gene level (scale is shown at top right) and then clustered by kmeans (with a k value of 2), as indicated. (E to G) IPA overlapping schemes of interferon signaling (E), inflammasome (F), and TNFR2 signaling (G) canonical pathways. Significantly differentially expressed genes between SARS-CoV-2-infected and mock-infected tissues are overlaid with those that were identified when influenza virus-infected tissues were compared to mock-infected tissues (pink outlines). Upregulated genes are colored in shades of red, from white (not significantly changed) to dark red (highly upregulated). Blank pink shapes stand for differentially expressed genes that were found in lung tissues infected with influenza virus but not with SARS-CoV-2. Images in panels E to G were generated by Qiagen Ingenuity Pathway Analysis (IPA). (H) Effect of SARS-CoV-2 and influenza virus infection on the expression of selected innate immunity genes in lung organ cultures. RNA from SARS-CoV-2-, influenza virus A(H1N1) pdm09-, and mock-infected cultures was extracted at 24 h postinfection and analyzed for the indicated gene expression by RT-qPCR, normalized to the expression of the housekeeping β-actin gene.

FIG 5

SARS-CoV-2- and influenza virus-modulated canonical pathways and interferon gene expression in nasal turbinate versus lung tissues. (A and B) Dot plots of selected IPA canonical pathways in nasal turbinate (A) and lung tissues (B). The size of the dot corresponds to the number of the significantly differentially expressed genes that participate in the pathway, and the color is according to –log(B-H p-value). (C) Interferon responses of nasal turbinate and lung tissues infected with SARS-CoV-2 or influenza virus. RNA from infected and mock-infected tissues was extracted at 24 h postinfection and analyzed for the indicated interferon mRNA expression by RT-qPCR, normalized to the housekeeping β-actin gene. The results are presented as fold change relative to mock infection. The results are representative of 7 independent nasal turbinate tissues and 7 independent lung tissues from different individuals. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

FIG 6

Schematic illustration of the early patterns of viral infection and local mucosal innate immune responses in human nasal turbinate and lung tissues. In the work presented here, we show that SARS-CoV-2 productively infects respiratory epithelial cells within the nasal turbinate tissues. By comparing the innate response patterns of nasal and lung tissues infected in parallel with SARS-CoV-2 and influenza virus, we revealed differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tracts. Our findings emphasize the role of the nasal mucosa in viral transmission and innate antiviral defense, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues shown here (contrasting with their robust response to influenza virus) could potentially (as indicated by the dotted arrow) underlie the unique late-phase lung damage of advanced COVID-19. ISGs, interferon-stimulated genes.