Fig 1.
SARS-CoV-2 stimulation induces a TLR7/8-dependent type I and III IFN response in PBMC.
Peripheral blood mononuclear cells (PBMC) were left untreated (not stimulated, ns) or Mock-treated as negative controls, or stimulated for 24 hours with the TLR7/8 agonist R848 (5μM) as positive control, with SARS-CoV-2 (CoV2) at different multiplicity of infection (MOI; 0.02, 0.04 and 0.1) in presence or absence of a 30 minute pre-treatment with a specific TLR7/8 inhibitor (I-TLR7/8, 1μM). (A) Relative expression of IFN-αs, IFN-β, IFN-λ1 genes was measured by quantitative real time PCR analysis. All quantification data were normalized to TBP level by using the equation 2-ΔCt. (B) Production of IFN-αs was tested by specific ELISA in 24 hour-collected cell culture supernatants. (C) Mx1 gene expression was quantified by real time PCR as described above. Shown results were mean relative values ± SEM of 5 independent experiments. P-values were depicted as follows: *p≤0.05; ** p≤0.001.
Fig 2.
SARS-CoV-2 stimulation induces a TLR7/8-dependent cytokine and chemokine production in PBMC.
Peripheral blood mononuclear cells (PBMC) were left untreated (not stimulated, ns) or Mock-treated as negative controls, or stimulated for 24 hours with the TLR7/8 agonist R848 (5μM) as positive control, with SARS-CoV-2 (CoV2) at different multiplicity of infection (MOI; 0.02, 0.04 and 0.1) in presence or absence of a 30 minute pre-treatment with a specific TLR7/8 inhibitor (I-TLR7/8, 1μM). Production of cytokines (IL-6, TNF-α, IL-1β, IL-10, IL-12p70) (A) and chemokines (CXCL-10, CXCL-9, CCL-2, CXCL-8, CCL-5) (B) was tested by multiparametric cytokine bead arrays in collected cell culture supernatants. Shown results were mean relative values ± SEM of 5 independent experiments. P-values were depicted as follows: *p≤0.05.
Fig 3.
Type I IFN-induced anti-viral state impacts on SARS-CoV-2 infected Calu-3 lung epithelial cell line.
(A) The human lung epithelial cell line Calu-3 was left untreated (not stimulated, ns), infected for 24 hours with SARS-CoV-2 at the back titration dose calculated in peripheral blood mononuclear cells (PBMC) (104 TCID50/ml for 0.02 MOI) or treated with 24 hour-supernatants collected from either ns PBMC cultures (sup PBMC_ns) or from SARS-CoV-2 (MOI = 0.02)-treated PBMC cultures (sup PBMC_CoV2). Relative expression of Mx1 and IL-6 genes was measured by quantitative real time PCR analysis. All quantification data were normalized to TBP level by using the equation 2-ΔCt. (B) Calu-3 cell line was infected with SARS-CoV-2 at the back titrated dose calculated in PBMC (104 TCID50/ml for 0.02 MOI) in presence of 24 hour-supernatants collected from either ns PBMC cultures (sup PBMC_ns) or from SARS-CoV-2 (MOI = 0.02)-treated PBMC cultures (sup PBMC_CoV2). Mx1 gene expression was quantified by real time PCR and quantification data normalized to TBP level by using the equation 2-ΔCt. (C) Virus titers were evaluated in supernatants from infected Calu-3 cultures described in (B) by Endpoint Dilution Assay by using the Reed-Muench formula and reported as TCID50/ml. Shown results were mean relative values ± SEM of 3 independent experiments. P-values were depicted as follows: *p≤0.05; ** p≤0.001.
Fig 4.
SARS-CoV-2-induced IFN-α production is dependent on TLR7 and Neuropilin 1 in pDC.
(A) Purified plasmacytoid dendritic cells (pDC) were left untreated (not stimulated, ns) or Mock-treated as negative controls, or stimulated for 24 hours with the TLR7/8 agonist R848 (5μM) as positive control, with SARS-CoV-2 (CoV2; MOI = 0.1) in presence or absence of a 30 minute pre-treatment with a specific TLR7/8 inhibitor (I-TLR7/8, 1μM) or with UV-I-SARS-CoV-2 (UV-I-CoV2, MOI = 0.1). IFN-α production was tested by ELISA in collected cell culture supernatants. (B) pDC and Calu-3 cell line were stimulated with 1000 U/mL of recombinant IFN-α or IFN-β. Gene expression of ACE-2 and TMPRSS2 was measured by quantitative RT-PCR. (C) pDC were treated with CoV2 (MOI = 0.1) in presence or absence of a 30 minute pre-treatment with increasing doses of anti-Neuropilin 1 monoclonal antibody (anti-NRP1, 1 or 2 μg/ml) or anti-human IgG control (IgG ctrl, 2 μg/ml). IFN-α production was tested by ELISA in culture supernatants. Shown results were mean relative values ± SEM of 3 independent experiments. P-values were depicted as follows: *p≤0.05; ** p≤0.001.
Fig 5.
SARS-CoV-2 stimulation drives TLR7-dependent phenotypical modification in pDC.
Purified plasmacytoid dendritic cells (pDC) were either left untreated (not stimulated, ns) or Mock-treated as negative controls, or stimulated for 24 hours with the TLR7/8 agonist R848 (5μM) as positive control, with SARS-CoV-2 (CoV2; MOI = 0.1) in presence or absence of a 30 minute pre-treatment with a specific TLR7/8 inhibitor (I-TLR7/8, 1μM). (A) pDC were then stained with anti-BDCA4, PD-L1, CD80 and CD86 antibodies. The percentage (%) of pDC sub-populations was evaluated by flow cytometry in live/single BDCA4+ pDC; in particular P1-pDC (PD-L1+CD80-, in blue), P2-pDC (PD-L1+CD80+, in red) and P3-pDC (PD-L1-CD80+, in green). Representative dot plot profile out of 3 different experiments independently conducted is shown. (B) Surface expression of CD86, PD-L1 and CD80 was determined as mean fluorescence intensity (MFI) by flow cytometer analysis. (C) Production of TNF-α and IL-6 was tested by cytometric bead assay in 24 hour-collected pDC culture supernatants. Shown results were mean relative values ± SEM of 3 independent experiments. P-values were depicted as follows: *p≤0.05; ** p≤0.001.
Fig 6.
pDC differently activate and express chemokine receptors in COVID-19 asymptomatic and hospitalized patients.
Freshly isolated peripheral blood mononuclear cells (PBMC) from asymptomatic (CP-AS, n = 8) and hospitalized COVID-19 patients (CP, n = 6) as well as matched healthy donors (HD, n = 5) were stained with a cocktails of antibodies to study by flow cytometry plasmacytoid dendritic cell (pDC) frequency and absolute number (A), diversification and activation status (Lineage, CD123, BDCA-4, HLA-DR, PD-L1, CD80 and CD86) (B, C, D), as well as expression of chemokine receptors (CXCR4, CXCR3, CCR7 and CD62-L) (E, F). The percentage (%) of shown pDC sub-populations (B, C, E) was evaluated in live/single Lineage-CD123+BDCA4+HLADR+ gated pDC and depicted for each studied patients or HD together with mean ± SEM values. Surface expression of PD-L1, CD80, CD86 (D) and CXCR4, CXCR3, CD62-L, CCR7 (F), was determined as mean fluorescence intensity (MFI) and shown results were mean relative values ± SEM of analyzed patients or HD. P-values were depicted as follows: *p≤0.05; ** p≤0.001; *** p≤0.0001.
Fig 7.
COVID-19 asymptomatic and hospitalized patients display a specular anti-viral and pro-inflammatory profile.
Peripheral blood mononuclear cells (PBMC) and sera were collected from asymptomatic (CP-AS, n = 8) and hospitalized COVID-19 patients (CP, n = 6) as well as matched healthy donors (HD, n = 5). (A) Relative expression of Mx1 gene was measured by quantitative real time PCR analysis and normalized to TBP level by using the equation 2-ΔCt in total RNA isolated from ex vivo PBMC. (B) Production of IFN-αs was tested in serum samples by a specific ELISA kit. Production of cytokines (IL-6, TNF-α, IL-1β, IL-10) (C) and chemokines (CXCL-10, CCL-2, CXCL-8) (D) was tested by multiparametric cytokine bead arrays in collected serum samples. Shown results were mean relative values ± SEM of analyzed patients or HD. P-values were depicted as follows: *p≤0.05; ** p≤0.001; *** p≤0.0001.