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. 2024 Aug 22;20(8):e1012368.
doi: 10.1371/journal.ppat.1012368. eCollection 2024 Aug.

The assembly of neutrophil inflammasomes during COVID-19 is mediated by type I interferons

Luz E Cabrera  1 , Suvi T Jokiranta  2   3 , Sanna Mäki  1 , Simo Miettinen  1   4 , Ravi Kant  1   4   5 , Lauri Kareinen  1   4 , Tarja Sironen  1   4 , Jukka-Pekka Pietilä  6   7 , Anu Kantele  6   7 , Eliisa Kekäläinen  2   3   8 , Hanna Lindgren  9 , Pirkko Mattila  9 , Anja Kipar  4   10   11 , Olli Vapalahti  1   4   8 , Tomas Strandin  1
Affiliations

The assembly of neutrophil inflammasomes during COVID-19 is mediated by type I interferons

Luz E Cabrera et al. PLoS Pathog. .

Abstract

The severity of COVID-19 is linked to excessive inflammation. Neutrophils represent a critical arm of the innate immune response and are major mediators of inflammation, but their role in COVID-19 pathophysiology remains poorly understood. We conducted transcriptomic profiling of neutrophils obtained from patients with mild and severe COVID-19, as well as from SARS-CoV-2 infected mice, in comparison to non-infected healthy controls. In addition, we investigated the inflammasome formation potential in neutrophils from patients and mice upon SARS-CoV-2 infection. Transcriptomic analysis of polymorphonuclear cells (PMNs), consisting mainly of mature neutrophils, revealed a striking type I interferon (IFN-I) gene signature in severe COVID-19 patients, contrasting with mild COVID-19 and healthy controls. Notably, low-density granulocytes (LDGs) from severe COVID-19 patients exhibited an immature neutrophil phenotype and lacked this IFN-I signature. Moreover, PMNs from severe COVID-19 patients showed heightened nigericin-induced caspase1 activation, but reduced responsiveness to exogenous inflammasome priming. Furthermore, IFN-I emerged as a priming stimulus for neutrophil inflammasomes. These findings suggest a potential role for neutrophil inflammasomes in driving inflammation during severe COVID-19. Altogether, these findings open promising avenues for targeted therapeutic interventions to mitigate the pathological processes associated with the disease.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Increased IFN-I related gene expression in mature COVID-19 neutrophils.
The analysis was reduced to include only the samples with the highest purity (cell fraction over 0.65 of neutrophils), as identified by CIBERSORTx. (A) Principal component analysis (PCA) of the RNA-seq samples (n = 7 PMNs from HC, n = 10 PMNs from severe COVID-19, n = 8 PMNs from mild COVID-19, and n = 6 LDGs from severe COVID-19). (B) Ridgeline diagrams depicting the top 20 enriched signal pathways from the genes differentially expressed by PMNs versus LDGs during severe COVID-19: overrepresentation analysis (ORA) using KEGG database and gene-set enrichment analysis (GSEA) according to Reactome database. Both enrichment analyses were made using ExpressAnalyst and are sorted by P-value, obtained from Welch’s t-test. (C) Heatmap of differentially expressed IFN-related genes in COVID-19 PMNs and LDGs as compared to HC PMNs. RNA sequencing was performed on purified PMNs from healthy controls, mild COVID-19 and severe COVID-19, as well as LDGs from severe COVID-19. The heatmap was clustered by complete linkage and ordered by Spearman’s rank. FC = fold change.
Fig 2
Fig 2. Inflammasome related gene expression.
(A) Heatmap depicting selected differentially expressed inflammasome related genes from RNA sequencing performed in PMNs from HC, mild and severe COVID-19, as well as severe COVID-19 LDGs. Only the samples with the highest purity, determined by a cell fraction over 0.65 of neutrophils (identified by CIBERSORTx) are included. The heatmap was clustered by complete linkage and ordered by Spearman’s rank. (B-C) Ridgeline diagrams of overrepresentation analyses (ORA) according to KEGG database, depicting the top 10 enriched signaling pathways in PMNs during severe COVID-19 compared to (B) healthy controls and (C) mild COVID-19. (D) UMAP analysis of the COVID-19 Immune Atlas, which integrates 5 public COVID-19 PBMC single-cell transcriptomics datasets, created using CELLxGENE. (Top) UMAP showing the clustering of CD16+ cells (mature, FCGR3B expressing cells) and CD66b+ cells (immature, CEACAM8 expressing cells). Each dot represents a single cell colored according to the expression level of a selected gene. The color scale ranges from green (low expression) to purple (high expression). (Bottom) Pie chart summarizing the percentage of mature (black) and immature (blue) cells in the data. (E) The fraction of mature and immature neutrophils cells expressing inflammasome related genes identified in (D) are shown in bar graphs. For each gene, the proportion of expressing cells is shown in light blue, while the proportion of negative or not-expressing cells is shown in gray. Zoomed-in bar graph depicts the proportion of mature and immature cells expressing each gene.
Fig 3
Fig 3. Comparative neutrophil transcriptomics of COVID-19 and non-COVID-19 patients.
Bar graphs represent the activation levels of selected pathways and processes as identified by neutrophil transcriptomics. The analysis includes interpheron alpha (IFN-α) responses, interleukin (IL)-1β production, Toll-like receptor (TLR) signaling, NLRP3 inflammasome, and pyroptosis, as determined through the Gene Ontology (GO) database. The NOD-like receptor signaling pathway was investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the inflammasome pathway was explored via the REACTOME database (more information in S1 Table). The graphs compare the activation levels of these pathways in healthy controls (HC), non-COVID patients with similar symptoms (COVID-19 negative), and COVID-19 positive individuals. Statistical significance is denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. P values were calculated with Kruskall-Wallis test.
Fig 4
Fig 4. Inflammasome activation in PMNs during severe COVID-19.
PMNs were freshly isolated from blood and cultured at 2 million cells/ml (A) IL-1β and (B) IL-18 levels in 24 h cell culture supernatants from COVID-19 (n = 11 for IL-1β and 9 for IL-18) and HC PMNs (n = 6 for both). (C) IL-1β and (D) IL-18 levels in 24 h cell culture supernatant from PMNs exposed or non-exposed to purified SARS-CoV-2 viral particles (10 virus particles / PMN) (n = 3). (E) Caspase1 activity in PMNs following a 2 h stimulation with nigericin or purified SARS-CoV-2 viral particles (10 virus particles / PMN). For HC PMNs, n = 9 for mock and nigericin and n = 6 for SARS-CoV-2 exposure. For COVID-19 PMNs, n = 12 for mock and nigericin and n = 9 for SARS-CoV-2 exposure. *p < 0.05 and **p < 0.01. Data presented as mean ± SD. Tukey’s multiple comparisons test for mixed-effect analysis was applied for (E), meanwhile P values for (A-D) were calculated with the Mann-Whitney U-test.
Fig 5
Fig 5. IFN-I primes inflammasome activation while COVID-19 PMNs show defective inflammasome responses ex vivo.
Isolated HC or COVID-19 PMNs were non-stimulated or stimulated 4h with IFN-I (combination of 2.7*104 IU/ml IFN-α and IFN-β) or 20 ng/ml LPS (1st signal), followed by 4h with 2.5 μM nigericin or purified SARS-CoV-2 (10,1 virus/PMNs) (2nd signal). Then, (A) western blot of pro-IL-1β (31 kD) and active IL-1β (17 kD) was performed from HC PMNs supernatant and cell lysates, (B) IL-1β (n = 5 HC PMN and 9 COVID-19 PMN) and (C) MPO (n = 5 HC PMN and 9 COVID-19 PMN) were measured from supernatants by ELISA. (D-E) Effect of inflammasome inhibitor MCC950 (2 μg/ml, added simultaneously with nigericin) on IL-1β secretion in (D) HC and (E) severe COVID-19 PMN supernatant (n = 3). (F) LDH and (G) IL-8 in HC and severe COVID-19 PMN supernatants (n = 3). (H-K) RT-qPCR of selected mRNAs in IFN-I or LPS-primed HC and COVID-19 PMNs (n = 6–8 HC PMN and 7–10 COVID-19 PMN). (L-M) HC PMNs were stimulated with high dose IFN-I (2.7*105 IU/ml), normal dose IFN-I (2.7*104 IU/ml) and 20 ng/ml LPS. After 4 hr stimulation caspase1 activity was measured using median fluorescence intensity (MFI) of FAM-FLICA by flow cytometry (L, n = 5, representative histogram of one donor shown) and after 24 hr stimulation IL-1β release was measured by ELISA (M, n = 5). P values calculated with Kruskall-Wallis test for the comparison between treatments by group (HC or COVID-19 PMNs), and Mann-Whitney test for the comparison between HC and COVID-19 PMNs by individual treatment for (B-G), and Two-way ANOVA Tukey’s multiple comparisons test for (B, H-K). The treatments in L-M were compared to mock by one-way ANOVA for repeated measures, corrected for multiple comparisons with the two-stage step-up method of Benjamini, Krieger and Yekutieli. *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001. Data presented as mean ± SD.
Fig 6
Fig 6. Correlation analysis between clinical parameters and ex vivo PMN inflammasome activation.
(A) Spearman’s correlation matrix depicting the relationships among clinical parameters and results of ex vivo experimentation. For the WHO ordinal scale, the baseline parameters were used. (B-C) Linear regression analysis demonstrating the associations between: (B) Positive association between PMN Caspase1 activity, measured after ex vivo nigericin stimulation, and the levels of Calprotectin in the matched patient’s peripheral blood; (C) Negative association between ex vivo stimulated PMN IL-1β levels (IFN+Nig) and the blood neutrophil count in matched patients at the time of sampling (n = 12). LOS = length of stay. WHO = World Health Organization. Min = minimum. Casp1 = caspase1. LPS or IFN + nig = lipopolysaccharide or type I interferon + nigericin ex vivo stimulation.
Fig 7
Fig 7. Immature neutrophils express IL-18 in response to inflammasome activation.
(A-D) Isolated COVID-19 LDGs or HL-60 cells (differentiated for 5 days with 1% DMSO) were non-stimulated or stimulated 4h with IFN-I or LPS (1st signal), followed by 4h with nigericin (2nd signal) in the presence or absence of inflammasome inhibitors MCC950 or YVAD as previously. Secretion of (A, C) IL-1β and (B, D) IL-18 were measured from the supernatants by ELISA (n = 2 for LDGs and 3–5 for HL-60). *p < 0.05 and **p < 0.01. P values calculated with Kruskal-Wallis test. Data presented as mean ± SD. (E) Volcano plot of differentiated vs undifferentiated HL-60 cells gene expression from GSE93996, with inflammasome related genes marked in blue. Only significant DE genes are shown (adjusted p value < 0.05).
Fig 8
Fig 8. Neutrophil accumulation in the lungs correlates with viral loads in SARS-CoV-2 infected mice.
Female BALB/c mice were intranasally inoculated with 5*105 TCID50 SARS-CoV-2 MaVie strain or PBS as control and euthanized at 2 dpi or 4 dpi. (A) RNA was isolated from lungs and subjected to RT-qPCR targeting viral subE and GAPDH as housekeeping gene. The relative expression of subE was measured using the comparative Ct method as compared to mock-infected control (in which subE was undetectable but set to 40 Ct) **** p < 0.0001. P values calculated with Welch’s t-test. (B) Infectious virus was calculated from supernatants of lung single cell suspensions of infected mice as fluorescence focus forming units (FFU) in Vero E6 cells. * p < 0.05. P values calculated with Mann-Whitney test. (C) Quantification based on morphometric analysis that determines the area of immunolabelling for SARS-CoV-2 nucleoprotein in relation to total tissue area. (D) Quantification of Ly6G neutrophil/lymphocyte ratio in lung single cell suspensions by flow cytometry. (E) Quantification of total Ly6G neutrophil counts in lung single cell suspensions by flow cytometry. (F) Quantification of Ly6G based on morphometric analysis that determines the area of immunolabelling for Ly6G in relation to total tissue area in mock-infected controls. (G) Quantification of median fluorescence intensity (MFI) of CD11b expression in ly6G neutrophils by flow cytometry. Representative histograms of CD11b expression in Ly6G+ neutrophils is shown. P values for C-G were calculated with ordinary one-way ANOVA using Tukey’s multiple comparison correction. Black line represents the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (H) Fluorescent nuclear staining of representative magnetic-bead isolated Ly6G neutrophils by Hoechst33342. Panels A, C and F are representative of two independent experiments.
Fig 9
Fig 9. Neutrophils of SARS-CoV-2 infected mice display increased caspase1 activation ability that is dependent on IFN-I.
Female BALB/c mice were intranasally inoculated with 5* 105 TCID50 SARS-CoV-2 MaVie strain or PBS as control. (A) Lungs were harvested at 2 and 4 dpi and single cell suspensions stained with FAM-FLICA and Ly6G antibody followed by flow cytometric analysis. FAM-FLICA median fluorescence intensity (MFI) was recorded in Ly6G+ neutrophils (n = 4, representative histogram image shown). * p < 0.05, ** p < 0.01. P values calculated with one-way ANOVA using Tukey’s multiple comparisons. (B-E) Ly-6G+ neutrophils isolated from lung single cell suspensions based on positive selection with magnetic beads. (B-D) RNA was isolated and subjected to transcriptomic analysis by RNA-seq. (B) Principal component analysis (PCA) of the PBS-inoculated control and SARS-CoV-2 infected mice lung neutrophil RNA-seq samples. (C) Heatmap of the top differentially expressed genes (DEGs). (D) Volcano plots of DEGs between neutrophils isolated from SARS-CoV-2 infected mice versus uninfected PBS-inoculated mice. Blue points represent significant terms (adjusted p-value < 0.05), while smaller gray points represent non-significant terms. Relevant inflammasome and interferon related genes are shown with larger and darker blue points. (E) Caspase1 activity in isolated mice neutrophils following a 2 h stimulation with nigericin was assessed by a bioluminescence method (Caspase-Glo 1 Inflammasome Assay). (F-I) Mice were intraperitoneally inoculated with 250 μg anti-IFNAR or IgG1 isotype control directly after infection with SARS-CoV-2 and lung neutrophils isolated at 2 dpi (including also intranasally PBS-inoculated control mice without intraperitoneal injection). (F) Caspase1 activity was assessed following a 2 h stimulation with nigericin by bioluminescence method. (G-I) RNA was isolated from isolated neutrophils and fold change mRNA expressions of (G) Oasl2, (H) Caspase1 (Casp1) and (I) IL-1β (Il1b) was assessed by RT-qPCR in isotype control and anti-IFNAR treated infected mice as compared to mock-infected control mice. *p < 0.05, **p < 0.01 and ***p < 0.001. P values for A, E and F panels were calculated with ordinary one-way ANOVA using Tukey’s multiple comparisons correction, while Welch’s t-test was used for panels G-I. Data presented as mean ± SD.
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Grants and funding

This work was financed by grants by the Academy of Finland to T.S. (321809), A.K. (336439 and 335527); grants by the Helsinki University Hospital funds to O.V. (TYH 2021343); EU Horizon 2020 programme VEO (874735) to O.V.; Finnish governmental subsidy for Health Science Research (TYH 2021315) to A.K.; Paulon Säätiö to L.E.C.; Suomen Lääketieteen Säätiö to L.E.C.; Jane and Aatos Erkko foundation to O.V. The funders had no role in study design, data collection and analysis, nor decision to publish, or preparation of the manuscript.