Spontaneous NLRP3 inflammasome-driven IL-1-β secretion is induced in severe COVID-19 patients and responds to anakinra treatment

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained.

Objectives: To dissect the mechanisms underlying SARS-CoV-2-associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1β, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes.

Methods: We analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line.

Results: We found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein-containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient's treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation.

Conclusions: These results provide further evidence that IL-1β targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Keywords: IL-1β; NLRP3 inflammasome; SARS-CoV-2; inflammation.

Graphical abstract

Fig 1

NLRP3 and caspase-1 activation, and IL-1β and IL-18 spontaneous secretion, by monocytes of patients with COVID-19. A, Above: ASC speck detection in monocytes from patients with severe COVID-19 by FACS. Representative dot plots of CD14⁺ monocytes from HDs (left), patients with COVID-19 (center), and HDs treated with LPS + ATP, used as a positive control. ASC specks are detected gating as ASC pulse width vs ASC pulse area. Percentages of ASC speck + cells are shown. Below: left, quantification of ASC speck⁺ CD14⁺ monocytes as identified by FACS; right, percentage of cells positive for active caspase-1 quantified by FACS, using FLICA-Caspase-1. B, Representative confocal images of NLRP3 and ASC speck formation in monocytes from HDs and patients with COVID-19. Images are a result of a 3-dimensional reconstruction of Z stack. Cells were stained with anti-ASC antibody (yellow), NLRP3 (green), and DAPI (blue). Scale bar is 5 μm. C,In vitro inflammatory cytokine secretion by monocytes as measured in culture supernatant after 18 hours with or without stimulation with the indicated stimuli. BF, Brightfield; DAPI, 4'-6-diamidino-2-phenylindole, dihydrochloride; FACS, fluorescence-activated cell sorting; FLICA, fluorochrome-labeled inhibitors of caspases; HD, healthy donor; IQR, interquartile range; MCC, MCC950 NLRP3 inhibitor. Data are expressed as median ± IQR. ∗P < .05, ∗∗P < .01 as assessed by Mann-Whitney t test.

Fig 2

Patients’ treatment with anakinra inhibits NLRP3-dependent cytokine secretion by monocytes. A,In vitro inflammatory cytokine secretion by monocytes as measured in culture supernatant after 18 hours with or without stimulation with the indicated stimuli before (day 0) and after 3 and 7 days of treatment in patients affected by severe COVID-19. B, Dot plot visualizes significantly enriched gene sets of 4 patients with COVID-19. GSEA was used to perform gene set enrichment. The analysis was based on a custom collection. Gene set names and patient identifiers are reported on the left and bottom sides, respectively. Log-fold change was used as a score for running GSEA on prerank. The gene expression profiles of each patient at day 7 and day 0 were instrumental to estimate the log-fold change of all genes. NES is a measure of gene set enrichment that accounts for the size of the gene set. The NES value of each gene set in the collection was encoded in the dot color. Colors ranged from red (positive NES) to blue (negative NES). The probability of false-positive enrichment increases with the number of gene sets tested. To account for multiple hypothesis testing, GSEA computes a q value for each gene set. The −log₁₀ of the q value was encoded in the dot size. Legend is shown in the middle right. A gene set with nominal P value lower than .05 and FDR q value lower than 0.15 was considered significantly enriched. IQR, Interquartile range; MCC, MCC950 NLRP3 inhibitor. Data are expressed as median ± IQR. ∗P < .05, ∗∗P < .01 as assessed by Mann-Whitney t test.

Fig 3

Inflammatory activation in patients with COVID-19. A, Concentration of the inflammatory cytokine IL-6, IL-18, and IL-1β, and chemokines CXCL9 and IP-10, measured by BD CBA assays and ELISA kit for Human Soluble Protein and peripheral blood type I interferon score measured by quantitative PCR in patients with COVID-19 before and after treatment with anakinra. Dashed lines represent the mean + 2 SD of HD control values. B, Serum secretion of IL-18 and IL-1β at day 0 in patients affected by severe COVID-19. CXCL9, CXC motif chemokine ligand 9; HD, healthy control; IP-10, CXC motif chemokine ligand 10. ∗P < .05, ∗∗P < .01 as assessed by Mann-Whitney t test.

Fig 4

ORF3a-dependent ASC speck formation in THP1-cell–derived macrophages. A, Representative confocal images of ASC speck formation (in yellow) in the different experimental conditions (empty plasmid, ORF3a, LPS/ATP). Images are a result of a 3-D reconstruction of Z stack. Cells were stained with anti-ASC antibody (yellow), anti-NLRP3 (green), and DAPI (blue). Transfected cells PCMV6-AC-rfp-ORF3a and PCMV6-AC-rfp are in red. Scale bar is 5 μm. B, Representative confocal images of ASC specks (yellow) that colocalized with ORF3a protein (red). Images are a result of a 3-D reconstruction of Z stack. Cells were stained with anti-ASC antibody (yellow) and DAPI (blue). Transfected cells PCMV6-AC-rfp-ORF3a are in red. Scale bar is 5 μm. C, Percentage of THP1-macrophage cells forming ASC specks quantified from n = 100 cells per condition in quadruplicate (n = 4). THP1 macrophage cells were stimulated with LPS (100 ng/mL) for 3 hours followed by ATP (5 mM) for 30 minutes or transfected with PCMV6-AC-rfp-ORF3a and PCMV6-AC-rfp. 3-D, Three-dimensional; DAPI, 4'-6-diamidino-2-phenylindole, dihydrochloride; IQR, interquartile range. Data are expressed as median ± IQR (Fig 4, B and C). ∗P < .05 and ∗∗P < .01 as assessed by Mann-Whitney test.