key: cord-0884068-kllu9s0b authors: Salvi, Valentina; Nguyen, Hoang Oanh; Sozio, Francesca; Schioppa, Tiziana; Laffranchi, Mattia; Scapini, Patrizia; Passari, Mauro; Barbazza, Ilaria; Tiberio, Laura; Tamassia, Nicola; Garlanda, Cecilia; Del Prete, Annalisa; Cassatella, Marco A.; Mantovani, Alberto; Sozzani, Silvano; Bosisio, Daniela title: SARS-CoV-2-associated ssRNAs activate inflammation and immunity via TLR7/8 date: 2021-04-15 journal: bioRxiv DOI: 10.1101/2021.04.15.439839 sha: 3c4a934a0c05dacaf7b6d814ddee594d03054bba doc_id: 884068 cord_uid: kllu9s0b The inflammatory and IFN pathways of innate immunity play a key role in both 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 identify single-stranded RNA (ssRNA) fragments from 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 functions, 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 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 identify TLR7/8 as crucial cellular sensors of ssRNAs encoded by SARS-CoV-2 involved in host resistance and disease pathogenesis of COVID-19. DCs act as crucial messengers linking innate and adaptative immunity against viral infections 53 (7, 8) . Within DC heterogeneity, plasmacytoid DCs (pDCs) play an important role as the major source 54 of type I IFN in response to viral infection, while conventional DCs (cDCs) respond to a vast variety 55 of pathogens by producing pro-inflammatory cytokines and are the main responsible for T cell 56 activation (9) (10) (11) . pDCs sense ssRNA viruses through TLR7 (12) , an endosomal receptor activated by 57 genomic fragments rich in guanine (G) and uracil (U), derived by endosomal processing of the virus 58 independently of infection (13). By contrast, cDCs express the closely related TLR8 (14). Despite the 59 fact that TLR7 and TLR8 display high structural and functional homology, similar ligand specificity 60 (15) and recruit the same signaling intracellular adaptor molecule, MyD88 (16), the signaling 61 pathways of these two TLRs diverge in the functional significance, with TLR7 more involved in the 62 antiviral immune response and TLR8 mastering the production of pro-inflammatory cytokines. Both 63 cDCs and pDCs were shown to be reduced in the blood of severe acute COVID-19 patients (17, 18) as 64 a possible result of cell activation (19) , but the mechanisms of SARS-CoV-2 recognition and activation SARS-CoV-2-specific PAMPs activate TLR7 and TLR8 Salvi et al., We hypothesized that these sequences may represent so far unidentified SAMPs responsible for 77 viral recognition and immune activation via endosomal TLR triggering. The elevated number of 78 sequences detected suggests that, upon endosomal engulfment, the fragmentation of the SARS-CoV-2 79 genome may generate many TLR7/8-triggering sequences, thus displaying high chances to contact and 80 activate the IFN and inflammatory responses downstream these receptors. To validate the stimulatory potential on innate immune cells, two representative sequences, SCV2- RNA2A were used as negative controls (see materials and methods). Figure 1A shows that both 90 fragments efficiently activated cytokine secretion by moDCs. In particular, we observed potent 91 induction of pro-inflammatory cytokines (TNF-α, IL-6), of the Th1-polarizing cytokine IL-12 and RNA2 were more efficient than HIV-1-derived RNA40. In all experimental conditions, U/A alternated 95 SCV2-RNA1A and SCV2-RNA2A did not induce cytokine secretion. SCV2-RNA1 and SCV2-RNA2 also 96 induced moDC phenotypical maturation in terms of CD83, CD86 and CCR7 expression ( Figure 1B ). Similarly to cytokine secretion, upregulation of maturation markers by RNA40 was less effective. These These experiments demonstrated that phenotypical DC maturation induced by SAMPs ( Figure 1B The ability of SCV2-RNAs to activate DCs was further investigated using primary circulating cDCs 119 (comprising CD141 + cDC1 and CD1c + cDC2) and BDCA2 + pDCs. SCV2-RNA efficiently induced the 120 secretion of TNF-α and IL-6 ( Figure 3A ) and the expression of maturation markers, such as CD86 and 121 CCR7 ( Figure 3B DCs. Indeed, cDCs express TLR8 as their unique endosomal ssRNA receptor, while pDCs express TLR7 143 (14). Consistent with this, CU-CPT9a blocked the production of pro-inflammatory cytokines in cDCs 144 ( Figure 5A ). Our effort to block TLR7 signaling using commercially available receptor antagonists was 145 unsuccessful since none of these inhibitors blocked TLR7 activation in pDCs stimulated with R848 or 146 Imiquimod (data not shown). As an alternative strategy to demonstrate the involvement of TLR7 in 147 SCV2-RNA sensing we performed TLR desensitization (21). pDCs were stimulated with SCV2-RNA or 148 R848 or left untreated, washed, and then re-stimulated with R848. Figure 5B shows that, upon re- ( Figure 6F ). Activation of splenic cDC1s (CD11c + MHC-II + CD8a + CD11b -) and cDC2s (CD11c + MHC-II + 169 CD8a -CD11b + ) was also detected ( Figure 6G and H). Figure 7A shows that SAMP treatment induced In conclusion, this work describes that SARS-CoV-2 is as a potential powerful source of 237 immunostimulatory nucleic acid fragments and identifies the first SARS-CoV-2-specific PAMPs 238 endowed with the ability to promote inflammation and immunity triggering TLR7 and TLR8. Based The reference SARS-CoV-2 genome (NC_045512, positive strand) was scanned for GU-rich ssRNA 247 fragments with the SequenceSearcher tool in the Fuzzy mode (44). We defined "GU-enriched 248 sequences" short strings with a maximal length of 20 bp, that were composed for more than 40% of the 249 length by "GU" and/or "UG" pairs. The identified 491 GU-rich sequences were further selected based 250 on the content of at least one "UGUGU" Interferon Induction Motif (IIM)(21) (see Suppl. Table 1 ). TNF-a, IL-6, IL-12p70, CXCL8, CXCL9, CCL3 and mouse TNF-a were measured by ELISA assay 293 (R&D Systems). Human IFN-a was detected using specific Module Set ELISA kit (eBioscience). Mouse IFN-a was measured by a bioluminescence kit (InvivoGen). All assays were performed on cell 295 free supernatants according to the manufacturer's protocol. ) in cell transfected with control siRNA (mean + SEM n=4); *P< 0.05 versus respective "ctr siRNA" by one-way ANOVA with Dunnett's post-hoc test. (B, right panel) moDCs transfected with MyD88-specific siRNAs or with control siRNA were stimulated with 5 µg/ml SCV2-RNA or 25 µg/ml Poly I:C for 24 hours. The production of IL-6 was evaluated by ELISA. Data are expressed as percentage of production for each individual stimulation (n=4); *P< 0.05 versus respective "ctr siRNA" by one-way ANOVA with Dunnett's posthoc test. (C) moDCs were pre-treated with increasing concentration of CU-CPT9a for 1 hour and then stimulated with SCV2-RNA (5 µg/ml) or R848 (1 µg/ml) or LPS (100 ng/ml) for 24 hours. IL-6 production was evaluated by ELISA. Data are expressed as percentage of production for each individual stimulation (n=3); *P< 0.05 versus respective "0" by one-way ANOVA with Dunnett's post-hoc test. Data are expressed as 2 -∆ Ct relative to RPL32 of one representative experiment out of three. (B) RAW264.7 (1x10 6 /ml) were pre-treated 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 -∆ Ct relative to RPL32 of one representative experiment out of three. (D) Splenocytes (3x10 6 /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 one representative experiment out of three. (F-H) Activation of splenic pDCs (CD11c int MHC-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 median fluorescence intensity (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. 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