Table 1.
Baseline clinical characteristics of the study population.
Fig 1.
NLRP3 inflammasome pathway is upregulated during SARS-CoV-2 infection and is associated with disease severity.
(A) Gene Set Enrichment Analysis (GSEA) on nasopharyngeal swab dataset (GSE152075) showing the enrichment of the NLRP3 inflammasome pathway (REACTOME_THE_NLRP3_INFLAMMASOME). The genes shown in the transcriptomic analysis in Fig 1B–1I are the significantly upregulated genes out of 6 screened genes involved in the NLRP3 inflammasome pathway. (B) RNAseq profiles of the nasopharyngeal swabs (GSE152075) of SARS-CoV-2 infected individuals (430) and healthy controls (54) showed a log2 fold change in NLRP3 (1.24), CASP-1 (1.82) and IL-1β (1.07). (C) Increased expression of important players in the NLRP3 inflammasome pathway (NLRP3 (1.25), CASP-1 (1.21) and IL-1β (1.38)) in whole blood of severe transcriptome of COVID-19 patients (20) as compared to control subjects (10) using RNAseq data EGAS00001004503. (D) RNAseq performed on leukocyte dataset (GEO: GSE157103) with 37 severe and 51 non-severe COVID-19 patients with severity based on ICU admission. (E) Single-cell RNA sequencing dataset (GSE149689) showing log2 fold change in the expression of NLRP3 (0.25), CASP-1 (0.3) and IL-1β (1.2) in monocytes of PBMCs from severe and mildly infected COVID-19 patients. (F) Log2 fold changed of NLRP3 (0.4), CASP-1 (0.5) and IL-1β (0.55) using single-cell RNA sequence in M1-like and M2-like macrophages in the BALF obtained from 6 severe and 3 moderate COVID-19 patients (GSE145926), as well as 3 healthy controls. (G) GSE182264 data of murine peritoneal macrophages transfected with the Spike protein (n = 5) in comparison to control samples (n = 5). (H) GSE208320 compared data between two samples of THP-1-derived macrophages transfected with the SARS-CoV-2 spike protein and two control samples. (I) GSE169241 datasets examined SARS-CoV-2 infected human embryonic stem cell (ES) derived macrophages in three SARS-CoV-2 infected and three control samples. Results are presented as log2 fold change ± SE of gene expression between cases and controls. (J) The protein levels of NLRP3 in whole blood of severe COVID-19 patients as compared to healthy controls. (K) The plasma levels of IL-1β in severe COVID-19 patients as compared to healthy controls and mild cases. Results are presented as mean (± SEM) and relative to the control group. Statistic test: comparison was done using unpaired t-test or one-way ANOVA *P<0.05, **P<0.01, ***P<0.001).
Fig 2.
VitD3 attenuates NLRP3 inflammasome pathway in the blood of severe COVID-19 patients and in PBMCs treated with SARS-CoV-2 spike protein.
(A) The mRNA levels of NLRP3, ASC, CASP-1, IL-1β and IL-18 in whole blood of VitD3 treated and untreated COVID-19 patients.18S was used as a house keeping gene for qRT-PCR. (B) The protein levels of NLRP3, ASC and cleaved CASP-1 in whole blood of VitD3 treated and untreated COVID-19 patients. (C) The plasma levels of IL-1β in VitD3 treated (n = 21; 19 males and 2 females) and untreated severe COVID-19 patients (n = 24; 20 males and 4 females). Results are presented as mean (± SEM) and relative to the untreated group. The protein level of CASP-1 and IL-1β in VitD3 (50nM of calcitriol) treated PBMCs. (D) Protein levels of CASP-1 and IL-1β in VitD3 and/or LPS primed, and SARS-CoV-2 spike (50nM of calcitriol and/or 100ng/ml LPS and10nM Spike for 16 h) treated PBMCs. (E) The level of IL-1β in culture supernatant in VitD3 and/or spike treated PBMCs. Results are presented as mean (± SEM) and relative to the control PBMCs. Statistic test: comparison was done using unpaired t-test or one-way ANOVA *P<0.05, **P<0.01, ***P<0.001).
Fig 3.
Downregulating the NLRP3 inflammasome pathway is associated with an enhanced Type 1 IFNs signaling.
Lower levels of NLRP3 proteins in VitD3 treated COVID-19 patients were associated with enhanced mRNA expression of RIG-1/MDA-5 signaling pathway, interferon regulatory protein IRF-9 and interferon stimulating genes MX-1 and ISG-15 as compared with untreated COVID-19 patients (A-D). Lower levels of plasma IL-1β in VitD3 COVID-19 treated patients were associated with enhanced RIG-1/MDA-5 signaling pathway, interferon regulatory protein IRF-9 and interferon stimulating genes MX-1 and ISG-15 as compared with untreated COVID-19 patients (E-H). Statistical tests: Pearson or Spearman correlation with P value of <0.05 considered significant.
Fig 4.
VitD3 regulation of the NLRP3 inflammasome pathway is associated with a reduction in disease severity markers.
Lower levels of NLRP3 proteins in VitD3 treated COVID-19 patients were associated with lower levels of hyperinflammatory markers such D-dimer, IL-6 and IL-17 detected by ELISA as compared with untreated COVID-19 patients (A-C). Lower levels of plasma IL-1β in VitD3 COVID-19 treated patients were associated with lower levels of hyperinflammatory markers such D-dimer, IL-6 and IL-17 as compared with untreated COVID-19 patients (D-F). Statistical tests: Pearson or Spearman correlation with P value of <0.05 considered significant.
Fig 5.
Graphical illustration of main findings of the study.
The NLRP3 inflammasome pathway and associated hyperinflammation markers as determined by IL-1β, IL-6, IL-17 and D-Dimer are attenuated upon VitD3 treatment in severe COVID-19 patients. This is associated with an enhancement of the anti-viral immune response as detected by the ability of VitD3 to enhance RIG-1/MDA-5 and JAK-STAT signaling pathways along with the production of ISGs like MX-1 and ISG-15.