An epithelial-immune circuit amplifies inflammasome and IL-6 responses to SARS-CoV-2

Abstract

Elevated levels of cytokines IL-1β and IL-6 are associated with severe COVID-19. Investigating the underlying mechanisms, we find that while primary human airway epithelia (HAE) have functional inflammasomes and support SARS-CoV-2 replication, they are not the source of IL-1β released upon infection. In leukocytes, the SARS-CoV-2 E protein upregulates inflammasome gene transcription via TLR2 to prime, but not activate, inflammasomes. SARS-CoV-2-infected HAE supply a second signal, which includes genomic and mitochondrial DNA, to stimulate leukocyte IL-1β release. Nuclease treatment, STING, and caspase-1 inhibition but not NLRP3 inhibition blocked leukocyte IL-1β release. After release, IL-1β stimulates IL-6 secretion from HAE. Therefore, infection alone does not increase IL-1β secretion by either cell type. Rather, bi-directional interactions between the SARS-CoV-2-infected epithelium and immune bystanders stimulates both IL-1β and IL-6, creating a pro-inflammatory cytokine circuit. Consistent with these observations, patient autopsy lungs show elevated myeloid inflammasome gene signatures in severe COVID-19.

Keywords: AIM2; IL-1β; IL-6; NLRP1; NLRP3; SARS-CoV-2; STING; TLR2; inflammasome.

Graphical abstract

Figure 1

Primary, well-differentiated HAE form inflammasomes and secrete IL-1β but not in response to SARS-CoV-2 infection (A) IL-1β in pooled HAE supernatants following treatment with indicated stimuli ± transfection (TFX). (B) IL-1β and IL-18 in HAE supernatants following stimulation. (C) IL-1β in HAE supernatants following SARS-CoV-2 infection. (D) Viral titer from HAE apical wash following SARS-CoV-2 infection. (E) Viral N RNA expression relative to ACTB. (F) IL1B and IL18 expression relative to ACTB. (G) Apical IL-1β secretion from HAE following SARS-CoV-2 infection or stimulation. (H) Viral titer from HAE apical wash following infection. (I) LDH in HAE apical wash following SARS-CoV-2 infection. Data points represent individual HAE donors with bars shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^∗∗∗∗p < 0.0001 calculated by ordinary one-way (A and I) or two-way (B and D) ANOVA followed by Dunnett’s (A and I) or Tukey’s (B and D) multiple comparison test. See also Figure S1.

Figure 2

SARS-CoV-2 primes the inflammasome in PBMCs, while co-culture of PBMCs with infected epithelial cells fully activates inflammasome-mediated IL-1β secretion (A) Viral titer from PBMC supernatants. (B) Viral N RNA expression in PBMC relative to ACTB. (C) IL-1β release from PBMC supernatants following SARS-CoV-2 infection or stimulation. (D) IL1B transcript expression relative to ACTB. (E) Indicated transcript expression relative to ACTB after 4-h SARS-CoV-2 exposure. (F) IL-1β in PBMC supernatants following SARS-CoV-2 exposure alone or in combination with indicated signal I or II stimuli. (G) IL1B transcript expression relative to ACTB in PBMC at 4 HPI with SARS-CoV-2 ± Bay11-7082 (10 μM), chloroquine (10 μM), and C29 (25 μM). (H) IL1B expression relative to ACTB following 3-h treatment with 1 μg/mL S, E, or Pam3-CSK4. (I) IL-1β secretion from PBMCs following 3-h treatment described in (H) and subsequent ATP treatment (5 mM; 1 h). (J) PCR-amplified detection of dsDNA in HAE supernatants ± SARS-CoV-2 (MOI 0.5) at 24 HPI. (K and L) IL-1β in supernatants after infection in PBMCs, Vero-E6 cells, or co-culture of PBMCs with infected Vero-E6 cells (K) ± benzonase (20 units/mL) or H151 (10 μM) and (L) ± VX-765 (20 μg/mL) or MCC950 (5 μM). Data point color indicates specific PBMC donors and matched Vero-E6 values across conditions. Data points on graphs represent individual PBMC donors with bars or lines shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^∗∗∗∗p < 0.0001 as calculated by ordinary one-way (E, F, H, and I) or two-way (A, D, G, K, and L) ANOVA followed by Tukey’s (A, D, F, K, and L), Dunnet’s (E, H, and I), or Sidak’s (G) multiple comparison test. See also Figure S2.

Figure 3

Myeloid cells in COVID-19 autopsy lungs show hallmarks of inflammasome priming and activation primarily in cells lacking SARS-CoV-2 antigen (A) Representative images (left) and quantification (right) of RNA-ISH in control and COVID-19 autopsy lungs. (B) Representative images (left) and quantification (right) of fluorescent dual RNA-ISH labeling indicated inflammasome genes with CD68 or MPO. (C) Representative maximum Z projection (left) and quantification (right) of ASC immunostaining in control or COVID-19 autopsy lungs. (D) Representative images of ASC specks with cell-type markers in COVID-19 autopsy lungs (left) and quantifications (right). (E) Representative images of COVID-19 autopsy lungs of ASC specks in combination with SARS-CoV-2 antigen and quantification (right). All scale bars represent 20 μm. Data points on graphs represent individual donor lungs with bars shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^∗∗∗∗p < 0.0001 calculated by Student’s two-tailed, unpaired t test. See also Figure S3.

Figure 4

Meta-analysis of COVID-19 patient BALF scRNA-seq reveals differential expression of inflammasome pathway by disease state (A) UMAP of healthy and COVID-19 patient BALF scRNA-seq from pooled studies (healthy: n = 3; mild/moderate COVID-19: n = 5; severe COVID-19: n = 26). Each dot represents a cell, and each dot is colored according to cluster identity. Clusters are annotated by cell type. ‡ denotes the HLA-DR^low myeloid population. (B) UMAP shown in (A) but colored by patient disease state (healthy, mild/moderate, or severe). (C) Inflammasome scores for each cluster of the UMAP shown in (A). General cluster identity indicated below cluster number: M, myeloid cells; T, T cells; B, B cells, and E, epithelial cells. (D) Inflammasome scores for all myeloid cells in COVID-19 disease states and healthy patients. Each dot represents a cell. (E) Inflammasome scores for individual cells (individual points) within each myeloid cluster across COVID-19 disease states. (F) Inflammasome score variance within each myeloid cluster by disease state. (C)–(F) include projected datasets. Statistical calculations are described in the STAR Methods and text, except for (F) which was determined by paired two-sided t test ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^∗∗∗∗p < 0.0001. See also Figure S4.

Figure 5

Enriched expression of inflammasome genes and cytokines in specific myeloid subpopulations in severe COVID-19 Violin plots of normalized gene expression in each myeloid cluster by disease status. Includes projected data. Each cell shown by an individual dot. Each panel shows a different gene: (A) IL1B, (B) NLRP3, (C) AIM2, and (D) MEFV. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^∗∗∗∗p < 0.0001. Statistical calculations described in STAR Methods and text. See also Figure S5.

Figure 6

IL-1β stimulates IL-6 secretion from PBMCs and HAE (A–C) PBMCs or HAE treated with 100 pg/mL or 1 ng/mL IL-1β. (A) IL6 transcripts relative to ACTB in PBMCs (B) IL6 expression relative to ACTB in HAE. (C) IL-6 secretion from pooled HAE supernatants or PBMCs. (D) Schematic of HAE-PBMC co-culture experiment. Created with BioRender.com. (E) IL-1β in supernatants from experiment in (D). (F) IL-6 in supernatants from experiment in (D). (G) Representative images of RNA-ISH in COVID-19 autopsy lungs. Scale bar represents 5 μm. Data points on graphs represent individual donors (HAE or PBMC) or donor pairs (HAE + PBMC) with bars shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, or ^∗∗∗∗p < 0.0001 as calculated by two-way ANOVA followed by Dunnett’s (A and B) or Tukey’s multiple comparison test (C, E, and F). See also Figure S6.