Journal: Science immunology

Article Title: Sensing of SARS-CoV-2 by pDCs and their subsequent production of IFN-I contribute to macrophage-induced cytokine storm during COVID-19

doi: 10.1126/sciimmunol.add4906

Figure Lengend Snippet: ( A ) pDCs purified from PBMCs of HDs were cultured in medium alone [unstimulated (Unst)] or with either the TLR9 ligand CpG C274 (0.5 μM) or the inactivated (Inact) SARS-CoV-2 (MOI of 0.25) for 3, 6, 10, and 18 hours ( n = 4). IFN-α2 expression levels were quantified by qPCR. ( B to D ) pDCs purified from PBMCs of HDs ( n = 4) were incubated for 6 and 18 hours either alone, with SARS-CoV-2 (MOI of 0.25), or with CpG C274 (0.5 μM). Expression of IL-6 and TNF (B) and IFN-I and IFN-III (C) was quantified by qPCR (rel Ct). (D) Heatmap was generated with the log of the mean of each gene. ( E and F ) Purified pDCs (E) and PBMCs (F) from HDs ( n = 6) were cultured for 24 hours with medium only or with live SARS-CoV-2 at an MOI of 0.25 alone or with the TLR7 inhibitor IRS661 (2 μM). Production and gene expression of IFN-α were quantified by ELISA and qPCR, respectively. ( G and H ) pDCs purified from PBMCs of HDs ( n = 4 to 8) were cultured for 24 hours with inactivated SARS-CoV-2 either alone or in the presence of clathrin inhibitor CPZ (30 μM) or dynamin inhibitor DH (100 μM). Production of IFN-α was quantified by ELISA (G), and SARS-CoV-2 ribonucleoprotein was quantified by qPCR (H). All results are represented as means ± SEM. Statistical significance was evaluated using a Friedman test with Dunn’s multiple comparisons posttest or a Mann-Whitney test. * P < 0.05 and ** P < 0.01.

Article Snippet: For blocking experiment, cells were preincubated for 1 hour with the ACE2 inhibitor (2 μM; Novus Biologicals), TLR7 inhibitor IRS661 (2 μM; TriLink BioTechnologies), PI3Kδ inhibitor CAL-101 (10 μM; Selleck Chemicals), clathrin inhibitor chlorpromazine (CPZ; 30 μM; Sigma-Aldrich), or dynamin inhibitor dynasore hydrate (DH; 100 μM; Sigma-Aldrich) followed by the addition of inactivated SARS-CoV-2 at an MOI of 0.25.

Techniques: Purification, Cell Culture, Expressing, Incubation, Generated, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Science immunology

Article Title: Sensing of SARS-CoV-2 by pDCs and their subsequent production of IFN-I contribute to macrophage-induced cytokine storm during COVID-19

doi: 10.1126/sciimmunol.add4906

Figure Lengend Snippet: ( A ) PCA of the differentiated genes in either unstimulated (Unst), IFN-α–stimulated macrophages, or SARS-pDC SN–stimulated macrophages, followed by the addition of LPS for 3 hours (2 ng/ml) when indicated. PC1 and PC2 capture percent variation associated with either individual or combination treatments. ( B ) K -means clustering ( K = 7) of DEGs induced by a greater than twofold change with FDR < 0.05 under the conditions shown in (A). ( C ) Macrophages purified from PBMCs of HDs ( n = 4) were incubated for 24 hours with either Unst-pDC SN, IFN-α, or SARS-pDC SN either alone or followed by the addition of LPS. The number of genes differentiated by IFN-α, SARS-pDC SN, or LPS was normalized to that of the unstimulated condition. ( D ) Heatmap showing the inflammatory genes related to COVID-19 in macrophages incubated under the same conditions as in (A). ( E ) Top activated pathways of the differentiated genes induced by more than twofold, with FDR < 0.05 in macrophages preincubated with SARS-pDC SN and then cultured for 3 hours with LPS versus LPS alone. ( F ) Macrophages purified from PBMCs of HDs ( n = 6) were cultured for 24 hours with either SARS-pDC SN alone or SARS-pDC SN with the TLR7 inhibitor IRS661 followed by the addition of LPS for 6 hours. Gene expression levels and production of TNF and IL-6 were quantified by qPCR and ELISA, respectively. ( G ) Macrophages purified from PBMCs of HDs ( n = 6) were incubated alone (Unst) or with IFN-α for 24 hours. LPS was then added (when indicated) for 3 hours, and the FAIRE assay was performed on the promoter regions of IL6, TNF, and IL12p40. Results are represented as means ± SEM. Statistical significance was evaluated using a Mann-Whitney test and one-way ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: For blocking experiment, cells were preincubated for 1 hour with the ACE2 inhibitor (2 μM; Novus Biologicals), TLR7 inhibitor IRS661 (2 μM; TriLink BioTechnologies), PI3Kδ inhibitor CAL-101 (10 μM; Selleck Chemicals), clathrin inhibitor chlorpromazine (CPZ; 30 μM; Sigma-Aldrich), or dynamin inhibitor dynasore hydrate (DH; 100 μM; Sigma-Aldrich) followed by the addition of inactivated SARS-CoV-2 at an MOI of 0.25.

Techniques: Purification, Incubation, Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY