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 À/À or RNASET2 À/À but not RNASE2 À/À RNASET2 À/À 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.
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 , boosted anti-melanoma NK- and CD8 T-cell attack, and augmented 3pRNA antitumor efficacy 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..
The SARS-CoV-2 pandemic has underscored the need for rapidly employable prophylactic and antiviral treatments against emerging viruses. Targeted stimulation of antiviral innate immune receptors can trigger a broad antiviral response that also acts against new, unknown viruses. Here, we utilized 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, one to seven days before viral challenge, improved survival of mice by up to 50 %. Survival was also improved with therapeutic 3pRNA treatment starting one day after viral challenge. This improved outcome was associated with lower viral load in oropharyngeal swabs and in the lungs and brain 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, as well as its potential as a broad-spectrum approach to constrain the spread of newly emerging viruses until virus-specific therapies and vaccines become available.
The SARS-CoV-2 pandemic has underscored the need for rapidly employable prophylactic and antiviral treatments against emerging viruses. Nucleic acid agonists of the innate immune system can be administered to activate an effective antiviral program for prophylaxis in exposed populations, a measure of particular relevance for SARS-CoV-2 infection due to its efficient evasion of the host antiviral response. In this study, we utilized the K18-hACE2 mouse model of COVID-19 to examine whether prophylactic activation of the antiviral receptor RIG-I protects mice from SARS-CoV-2 infection. Systemic treatment of mice with a specific RIG-I ligand one to seven days prior to infection with a lethal dose of SARS-CoV-2 improved their survival of by up to 50 %. Improved survival was associated with lower viral load in oropharyngeal swabs and in the lungs and brain of RIG-I-treated mice. Moreover, despite antiviral protection, the surviving mice that were treated with RIG-I ligand developed adaptive SARS-CoV-2-specific immunity. These results reveal that prophylactic RIG-I activation by synthetic RNA oligonucleotides is a promising strategy to convey short-term, unspecific antiviral protection against SARS-CoV-2 infection and may be a suitable broad-spectrum approach to constraining the spread of newly emerging viruses until virus-specific therapies and vaccines become available.
Adaptive processes of the innate immune system, known as trained immunity (TI), are critical to human health and disease, yet they have not been systematically investigated downstream of antiviral sensing. Here, we elucidate the potential of the antiviral cytosolic RNA receptor retinoic acid-inducible gene I (RIG-I) to train, prime and tolerize the innate immune system. Using a specific RIG-I agonist, we observed that repetitive stimulation enhanced interferon-stimulated gene (ISG) and pro-inflammatory cytokine induction in human primary monocytes, epithelial cells and fibroblasts and afforded non-specific antiviral protection. RNA sequencing revealed broad, cell type-specific transcriptional changes, indicative of priming of ISGs and training of the NFκB pathway, without measurable tolerization, while ATAC sequencing in monocytes demonstrated chromatin remodeling and enhanced accessibility of key transcription factor-binding motifs such as STAT1. Moreover, while STAT1 signaling was critically required, it was not sufficient to recapitulate RIG-I induced TI. Altogether, our data demonstrate that RIG-I-mediated TI promotes an immunologically alert state with important implications for host defense and the application of RIG-I ligands in anti-infective and anti-tumoral therapies.
21 ] i may in turn activate TRPA 1 leading to a greater rise in [Ca 21 ] i . We also recognize that these studies were performed in vitro, which may not fully recapitulate what occurs in vivo. Finally, studies using primary bronchial epithelial cells from deceased donors are intrinsically limited by the availability of donor cells, especially in the pediatric age group. Additionally, history of atopic status and lung function is frequently not available, as well as comorbidities and cause of death. Thus, it is possible that the age effect could be explained by other factors and future studies will need to be done using cells from identified samples from patients with documented asthma status and severity.In conclusion, the results of this study show for the first time that lower airway epithelium from asthmatic children displays elevated basal TRPV 1 activity when compared with that of nonasthmatic controls. We have also shown that RSV infection of epithelial cells from asthmatic children-but not from adults-leads to an increase in overall TRPV 1 activation. Pharmacological inhibition of TRPV 1 may lead in the future to strategies that might reduce the impact of RSV infections in both asthmatic and nonasthmatic children.
Human TLR8 is an essential sensor of bacterial RNA which induces proinflammatory and Th1 cytokines. Crystallography revealed that TLR8 binds both uridine and short single-stranded RNA, but the RNases that process the RNA are still unknown. Herein, we demonstrate that two endosomal endoribonucleases, RNaseT2 and RNase2, can process RNA for TLR8 recognition. In the endosome, RNase2 and -T2 act synergistically to release uridine from oligoribonucleotides, with RNaseT2 cleaving preferentially before and RNase2 after uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic ligands all required RNase processing for TLR8 activation, and uridine supplementation restored RNA recognition in RNASE2−/− or RNASET2−/− but not double knockout cells. Strikingly, peripheral blood mononuclear cells from RNaseT2 hypomorphic patients did not respond to bacterial RNA but did to small-molecule TLR8 agonists. Our data provide a novel insight into TLR8 activation and its differences between cell types and species.
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