Immune Sensing of Synthetic, Bacterial, and Protozoan RNA by Toll-like Receptor 8 Requires Coordinated Processing by RNase T2 and RNase 2.

Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

RIG-I-induced innate antiviral immunity protects mice from lethal SARS-CoV-2 infection.

The SARS-CoV-2 pandemic has underscored the need for rapidly usable prophylactic and antiviral treatments against emerging viruses. The targeted stimulation of antiviral innate immune receptors can trigger a broad antiviral response that also acts against new, unknown viruses. Here, we used the K18-hACE2 mouse model of COVID-19 to examine whether activation of the antiviral RNA receptor RIG-I protects mice from lethal SARS-CoV-2 infection and reduces disease severity. We found that prophylactic, systemic treatment of mice with the specific RIG-I ligand 3pRNA, but not type I interferon, 1-7 days before viral challenge, improved survival of mice by up to 50%. Survival was also improved with therapeutic 3pRNA treatment starting 1 day after viral challenge. This improved outcome was associated with lower viral load in oropharyngeal swabs and in the lungs and brains of 3pRNA-treated mice. Moreover, 3pRNA-treated mice exhibited reduced lung inflammation and developed a SARS-CoV-2-specific neutralizing antibody response. These results demonstrate that systemic RIG-I activation by therapeutic RNA oligonucleotide agonists is a promising strategy to convey effective, short-term antiviral protection against SARS-CoV-2 infection, and it has great potential as a broad-spectrum approach to constrain the spread of newly emerging viruses until virus-specific therapies and vaccines become available.

RIG-I Resists Hypoxia-Induced Immunosuppression and Dedifferentiation.

A hypoxic tumor microenvironment is linked to poor prognosis. It promotes tumor cell dedifferentiation and metastasis and desensitizes tumor cells to type-I IFN, chemotherapy, and irradiation. The cytoplasmic immunoreceptor retinoic acid-inducible gene-I (RIG-I) is ubiquitously expressed in tumor cells and upon activation by 5'-triphosphate RNA (3pRNA) drives the induction of type I IFN and immunogenic cell death. Here, we analyzed the impact of hypoxia on the expression of RIG-I in various human and murine tumor and nonmalignant cell types and further investigated its function in hypoxic murine melanoma. 3pRNA-inducible RIG-I-expression was reduced in hypoxic melanoma cells compared with normoxic controls, a phenomenon that depended on the hypoxia-associated transcription factor HIF1α. Still, RIG-I functionality was conserved in hypoxic melanoma cells, whereas responsiveness to recombinant type-I IFN was abolished, due to hypoxia-induced loss of type I IFN receptor expression. Likewise, RIG-I activation in hypoxic melanoma cells, but not exposure to recombinant IFNα, provoked melanocyte antigen-specific CD8+ T-cell and NK-cell attack. Scavenging of hypoxia-induced reactive oxygen species by vitamin C restored the inducible expression of RIG-I under hypoxia in vitro, boosted in vitro anti-melanoma NK- and CD8+ T-cell attack, and augmented 3pRNA antitumor efficacy in vivo These results demonstrate that RIG-I remains operational under hypoxia and that RIG-I function is largely insensitive to lower cell surface expression of the IFNα receptor. RIG-I function could be fortified under hypoxia by the combined use of 3pRNA with antioxidants. Cancer Immunol Res; 5(6); 455-67. ©2017 AACR.