SARS-CoV-2-Associated ssRNAs Activate Human Neutrophils in a TLR8-Dependent Fashion.

COVID-19 disease is characterized by a dysregulation of the innate arm of the immune system. However, the mechanisms whereby innate immune cells, including neutrophils, become activated in patients are not completely understood. Recently, we showed that GU-rich RNA sequences from the SARS-CoV-2 genome (i.e., SCV2-RNA1 and SCV2-RNA2) activate dendritic cells. To clarify whether human neutrophils may also represent targets of SCV2-RNAs, neutrophils were treated with either SCV2-RNAs or, as a control, R848 (a TLR7/8 ligand), and were then analyzed for several functional assays and also subjected to RNA-seq experiments. Results highlight a remarkable response of neutrophils to SCV2-RNAs in terms of TNFα, IL-1ra, CXCL8 production, apoptosis delay, modulation of CD11b and CD62L expression, and release of neutrophil extracellular traps. By RNA-seq experiments, we observed that SCV2-RNA2 promotes a transcriptional reprogramming of neutrophils, characterized by the induction of thousands of proinflammatory genes, similar to that promoted by R848. Furthermore, by using CU-CPT9a, a TLR8-specific inhibitor, we found that SCV2-RNA2 stimulates neutrophils exclusively via TLR8-dependent pathways. In sum, our study proves that single-strand RNAs from the SARS-CoV-2 genome potently activate human neutrophils via TLR8, thus uncovering a potential mechanism whereby neutrophils may contribute to the pathogenesis of severe COVID-19 disease.

The PDE4 Inhibitor Tanimilast Blunts Proinflammatory Dendritic Cell Activation by SARS-CoV-2 ssRNAs.

Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases associated with chronic inflammatory conditions, such as COPD, psoriasis and atopic dermatitis. Tanimilast (international non-proprietary name of CHF6001) is a novel, potent and selective inhaled PDE4 inhibitor in advanced clinical development for the treatment of COPD. To begin testing its potential in limiting hyperinflammation and immune dysregulation associated to SARS-CoV-2 infection, we took advantage of an in vitro model of dendritic cell (DC) activation by SARS-CoV-2 genomic ssRNA (SCV2-RNA). In this context, Tanimilast decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CCL3, CXCL9, and CXCL10) and of Th1-polarizing cytokines (IL-12, type I IFNs). In contrast to ß-methasone, a reference steroid anti-inflammatory drug, Tanimilast did not impair the acquisition of the maturation markers CD83, CD86 and MHC-II, nor that of the lymph node homing receptor CCR7. Consistent with this, Tanimilast did not reduce the capability of SCV2-RNA-stimulated DCs to activate CD4+ T cells but skewed their polarization towards a Th2 phenotype. Both Tanimilast and ß-methasone blocked the increase of MHC-I molecules in SCV2-RNA-activated DCs and restrained the proliferation and activation of cytotoxic CD8+ T cells. Our results indicate that Tanimilast can modulate the SCV2-RNA-induced pro-inflammatory and Th1-polarizing potential of DCs, crucial regulators of both the inflammatory and immune response. Given also the remarkable safety demonstrated by Tanimilast, up to now, in clinical studies, we propose this inhaled PDE4 inhibitor as a promising immunomodulatory drug in the scenario of COVID-19.

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

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.