SARS-CoV-2–associated ssRNAs activate inflammation and immunity via TLR7/8
Valentina Salvi, … , Silvano Sozzani, Daniela Bosisio
Valentina Salvi, … , Silvano Sozzani, Daniela Bosisio
Published August 6, 2021
Citation Information: JCI Insight. 2021;6(18):e150542. https://doi.org/10.1172/jci.insight.150542.
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Research Article Immunology

SARS-CoV-2–associated ssRNAs activate inflammation and immunity via TLR7/8

Abstract

The inflammatory and IFN pathways of innate immunity play a key role in the resistance and pathogenesis of coronavirus disease 2019 (COVID-19). Innate sensors and SARS-CoV-2–associated molecular patterns (SAMPs) remain to be completely defined. Here, we identified single-stranded RNA (ssRNA) fragments from the SARS-CoV-2 genome as direct activators of endosomal TLR7/8 and MyD88 pathway. The same sequences induced human DC activation in terms of phenotype and function, such as IFN and cytokine production and Th1 polarization. A bioinformatic scan of the viral genome identified several hundreds of fragments potentially activating TLR7/8, suggesting that products of virus endosomal processing potently activate the IFN and inflammatory responses downstream of these receptors. In vivo, SAMPs induced MyD88-dependent lung inflammation characterized by accumulation of proinflammatory and cytotoxic mediators and immune cell infiltration, as well as splenic DC phenotypical maturation. These results identified TLR7/8 as a crucial cellular sensor of ssRNAs encoded by SARS-CoV-2 involved in host resistance and the disease pathogenesis of COVID-19.

Authors

Valentina Salvi, Hoang Oanh Nguyen, Francesca Sozio, Tiziana Schioppa, Carolina Gaudenzi, Mattia Laffranchi, Patrizia Scapini, Mauro Passari, Ilaria Barbazza, Laura Tiberio, Nicola Tamassia, Cecilia Garlanda, Annalisa Del Prete, Marco A. Cassatella, Alberto Mantovani, Silvano Sozzani, Daniela Bosisio

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Figure 6

SAMPs activate murine cells in vitro and in vivo.

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SAMPs activate murine cells in vitro and in vivo. (A) Expression of TLR ...
(A) Expression of TLR mRNAs in RAW264.7 cells. Data are expressed as 2–ΔCt relative to RPL32 of 1 representative experiment out of 3. (B) RAW264.7 (1 × 106/mL) were pretreated for 1 hour with CQ (12.5 μM), then stimulated with 5 μg/mL SCV2-RNA or vehicle (-) for 24 hours. Secreted TNF-α was evaluated by ELISA. Data are expressed as mean ± SEM (n = 3); *P < 0.05 versus (-); §P<0.05 versus “(-) SCV2-RNA” by paired Student’s t test. (C) Expression of TLR mRNAs in splenocytes from WT (white circle) or MyD88–/– mice (black circle). Data are expressed as mean ± SEM (n = 3) of 2–ΔCt relative to RPL32 of 1 representative experiment out of 3. (D) Splenocytes (3 × 106/mL) from WT (white circle) or MyD88–/– mice (black circle) were stimulated with 5 μg/mL SCV2-RNA or vehicle (-) for 24 hours. Secreted TNF-α was evaluated by ELISA. Data are expressed as mean ± SEM (n = 3); *P < 0.05 versus (-) or #P < 0.05 versus “SCV2-RNA MyD88–/–” by paired Student’s t test . (E) Circulating IFN-α in WT (white circle) or MyD88–/– mice (black circle) treated with SCV2-RNA or vehicle (-) for 6 hours. Data are expressed as mean ± SEM [(-) n = 4, SCV2-RNA n = 7]; *P < 0.05 versus (-) or #P < 0.05 versus “SCV2-RNA MyD88–/–” by unpaired Student’s t test. of 1 representative experiment out of 3. (F–H) Activation of splenic pDCs (CD11cintMHC-II+B220+SiglecH+) (F), cDC1s (CD11c+MHC-II+CD8α+CD11b–) (G), or cDC2s (CD11c+MHC-II+CD8α–CD11b+) (H) from WT (white circle) or MyD88–/– mice (black circle), treated with SCV2-RNA or vehicle (-) for 6 hours evaluated in terms of CD40 and CD86 expression. Data are expressed as mean ± SEM of the MFI [(-) n = 4, SCV2-RNA n = 7]; *P < 0.05 versus (-) or #P < 0.05 versus “SCV2-RNA MyD88–/–” by unpaired Student’s t test.