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[Preprint]. 2021 Jan 8:2020.07.10.197343.
doi: 10.1101/2020.07.10.197343.

SARS-CoV-2 induces human plasmacytoid pre-dendritic cell diversification via UNC93B and IRAK4

Fanny Onodi  1 , Lucie Bonnet-Madin  2 , Laurent Meertens  2 , Léa Karpf  1 , Justine Poirot  1 , Shen-Ying Zhang  3   4   5 , Capucine Picard  4   6   7 , Anne Puel  3   4   5 , Emmanuelle Jouanguy  3   4   5 , Qian Zhang  5 , Jérôme Le Goff  1   8 , Jean-Michel Molina  2   8 , Constance Delaugerre  2   8 , Jean-Laurent Casanova  3   4   5   7   9 , Ali Amara  2 , Vassili Soumelis  1   10
Affiliations

SARS-CoV-2 induces human plasmacytoid pre-dendritic cell diversification via UNC93B and IRAK4

Fanny Onodi et al. bioRxiv. .

Update in

Abstract

Several studies have analyzed antiviral immune pathways in late-stage severe COVID-19. However, the initial steps of SARS-CoV-2 antiviral immunity are poorly understood. Here, we have isolated primary SARS-CoV-2 viral strains, and studied their interaction with human plasmacytoid pre-dendritic cells (pDC), a key player in antiviral immunity. We show that pDC are not productively infected by SARS-CoV-2. However, they efficiently diversified into activated P1-, P2-, and P3-pDC effector subsets in response to viral stimulation. They expressed CD80, CD86, CCR7, and OX40 ligand at levels similar to influenza virus-induced activation. They rapidly produced high levels of interferon-α, interferon-λ1, IL-6, IP-10, and IL-8. All major aspects of SARS-CoV-2-induced pDC activation were inhibited by hydroxychloroquine. Mechanistically, SARS-CoV-2-induced pDC activation critically depended on IRAK4 and UNC93B1, as established using pDC from genetically deficient patients. Overall, our data indicate that human pDC are efficiently activated by SARS-CoV-2 particles and may thus contribute to type I IFN-dependent immunity against SARS-CoV-2 infection.

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Conflict of interest statement

Competing interest

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. SARS-CoV-2 induces activation and diversification of primary human pDC.
Sorted blood pDC from healthy donors were cultured for 24h with either Medium, SARS-CoV-2, or Influenza virus A (Flu). (A) Dotplot showing pDC activation and diversification through the expression of PD-L1 and CD80 into P1-, P2-, and P3-subpopulations. Results from one healthy donor representative of n=8. (B) Quantification of the three populations. Bars represent medians of n=8 healthy donors from 6 independent experiments. **P < 0.01; ***P < 0.005 Mann Whitney test. (C) Dotplot showing pDC activation from different strains of SARS-CoV-2 isolated from two patients. Results from one healthy donor representative of n=3. (D) Percentage of live pDC after 24h of culture with either Medium, SARS-CoV-2, or Influenza virus A (Flu). n=8 healthy donors from 6 independent experiments. ***P < 0.005 Mann Whitney test. (E) Histogram of ACE2-expression on pDC, Vero E6 and 293T-ACE2 (black) compared to the isotype (light grey). Results from one experiment representative of n=3. (F) Intracellular production of SARS-CoV-2 Ribonucleoprotein in Vero E6 and pDC at 2, 24 or 48h post infection (hpi) with SARS-CoV-2. Results from one experiment representative of n=3. (G) Infectious viral titers in the supernatants of SARS-CoV-2-infected Vero E6 and pDC at 2, 24, 48 or 72h post infection (hpi). Results from one experiment representative of n=3.
Figure 2.
Figure 2.. SARS-CoV-2 induces pDC activation in a dose dependent manner.
Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI of 0.04, 0.2, or 1. (A) Dotplot showing pDC activation through the expression of PD-L1 and CD80. Results from one healthy donor representative of n=3. (B) Quantification of the three populations. Bars represent medians of n=3 healthy donors from 3 independent experiments. *P < 0.05; ns: not significant; Mann Whitney test. (C) PDC geometric mean (MFI) of activation markers after 24h of culture with either Medium, Influenza virus A, or SARS-CoV-2 at a MOI of 1. Histograms represent medians and bars interquartile of n=5 healthy donors from 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; Kruskal-Wallis with Dunn’s multiple comparison’s post test.
Figure 3.
Figure 3.. SARS-CoV-2-activated pDC produce pro-inflammatory cytokines.
Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2. (A) Quantification of secreted pro-inflammatory cytokines after 24h of culture. Bars represent medians of n=5 healthy donors from 3 independent experiments. **P < 0.01; ns: not significant; Mann Whitney test. (B) Dotplot showing pDC activation through the expression of PD-L1 and CD80 (upper plots), and intracellular IFN-α and TNF-α in P1, P2 or P3 populations (lower plots). Results from one healthy donor representative of n=4. (C) Percentages of IFN-α single positive, IFN-α+ TNF-α+ double positive, and TNF-α single positive cells in P0, P1, P2 or P3 populations. Histograms represent medians and bars interquartile of n=4 healthy donors from 3 independent experiments. *P < 0.05; Mann Whitney test.
Figure 4.
Figure 4.. SARS-CoV-2-induced pDC activation is inhibited by hydroxychloroquine.
Sorted blood pDC from healthy donors were cultured for 24h with either Medium, Influenza virus A (Flu), or SARS-CoV-2 at a MOI 1 with or without the presence of hydroxychloroquine (HCQ). (A) Dotplot showing pDC diversification in P1-, P2-, and P3-subpopulations in the presence of HCQ. Results from one healthy donor representative of n=3. (B) Quantification of the three populations. Bars represent medians of n=3 healthy donors from 3 independent experiments. *P < 0.05; ns: not significant; Mann Whitney test. (C) Histograms of pDC’s activation markers. Results from one healthy donor representative of n=3. (D) Geometric mean (MFI) of activation markers. Histograms represent medians and bars interquartile of n=3 healthy donors from 3 independent experiments. (E) Quantification of pro-inflammatory cytokines production. Bars represent medians of n=3 healthy donors from 3 independent experiments. *P < 0.05; ns: not significant; Mann Whitney test.
Figure 5.
Figure 5.. SARS-CoV-2-induced pDC activation requires IRAK4 and UNC93B1.
Sorted blood pDC from mutated patients and healthy donors were cultured for 24h with either Medium or SARS-CoV-2. (A) Dotplot showing pDC diversification in P1-, P2-, and P3-subpopulations from magnetically sorted blood pDC from homozygous IRAK4−/− (n = 1), UNC93B1−/− (n = 2), TLR3−/− (n = 1) donors, and gender aged-matched healthy donors (n = 2) were cultured for 24h with either Medium, or SARS-CoV-2 at a MOI 1. (B) Quantification of pro-inflammatory cytokines production by in the supernatant of activated pDC for 24h in response to SARS-CoV-2 challenge. Bars represent medians; ND: Not Detectable.
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