The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5=-triphosphate (5=ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5=ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5=pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5=pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5=pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCEThe development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5=pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.H uman-pathogenic RNA viral infections, including influenza, dengue, and chikungunya, pose significant threats to human health and safety. For this reason, the development of prophylactic and therapeutic antivirals to treat and limit spread of infection remains a growing unmet medical need. Currently, there are no therapeutics for the prevention or treatment of dengue or chikungunya infections, and approved antiviral compounds to treat influenza have si...

Extracellular nucleotides are emerging as important inflammatory mediators. Here, we demonstrate that these molecules mediate LPS-induced neutrophil migration in vitro and in vivo. Apyrase, a nucleotide scavenger, reduced the ability of LPS-stimulated monocytes to recruit neutrophils, as assayed using a modified Boyden chamber. This effect resulted from the inhibition of IL-8 release from monocytes. Furthermore, LPS-induced IL-8 release by monocytes was attenuated significantly by P2Y 6 receptor antagonists, RB-2 and MRS2578. Reciprocally, UDP, the selective P2Y 6 agonist, induced IL-8 release by monocytes. As for LPS, the media of UDPstimulated monocytes were chemotactic for neutrophils; IL-8 accounted for ~50% of neutrophil migration induced by the media of LPS-or UDP-treated monocytes in transendothelial migration assays. It is important that in the murine air-pouch model, extracellular nucleotides were instrumental in LPS-induced neutrophil migration. Altogether, these data imply that LPS induces the release of nucleotides from monocytes and that by autocrine stimulation, the latter molecules regulate neutrophil migration caused by Gram-negative bacteria, suggesting a proinflammatory role of extracellular nucleotides in innate immunity.

Extracellular nucleotides regulate a variety of cellular responses involved in inflammation via the activation of P2 receptors. Here, we show that nucleotides regulate TLR2-induced neutrophil migration both in vivo and in vitro. The nucleotide scavenger apyrase inhibited neutrophil recruitment in murine air pouches injected with the TLR2 agonist Pam 3 CSK 4 . In agreement, the supernatants of either human primary monocytes or monocytic cells (THP-1 and U937) treated with Pam 3 CSK 4 recruited significantly fewer neutrophils when the former cells were treated in the presence of apyrase. As demonstrated with inhibitory Ab, these supernatants induced neutrophil migration due to IL-8 secretion. In addition, IL-8 secretion was markedly diminished by the non-selective P2 receptor antagonists reactive blue 2 and suramin, and by a selective P2Y 6 antagonist, MRS2578. Selective antagonists of P2Y 1 (MRS2500) and P2Y 11 (NF157) did not affect IL-8 release. The knockdown of either P2Y 2 or P2Y 6 with specific shRNA diminished IL-8 secretion from Pam 3 CSK 4 -treated THP-1 cells. Altogether, these results show that extracellular nucleotides, via P2Y 2 and P2Y 6 receptors, regulate neutrophil migration by controlling TLR2-induced IL-8 release from human monocytes. In line with our previous work on TLR4, this study further supports the importance of nucleotides in bacterial-induced neutrophil migration.Key words: Cell trafficking . Human monocytes . Inflammation . Neutrophils . Pam 3 CSK 4 IntroductionMonocytes play an important role in immune defences against invading bacteria and viruses via TLR activation [1]. TLR recognize highly conserved structural motifs expressed by microbes called PAMP. Monocytes express various TLR including TLR4 (that is activated by LPS, a cell wall component of Gramnegative bacteria), TLR2 (activated by peptidoglycan and lipopeptide, components of Gram-positive bacteria), TLR5 (activated by the bacterial flagellin) and TLR7/8 (activated by single-stranded viral RNA) [2][3][4][5]. In response to TLR activation by PAMP, monocytes release various cytokines and chemokines that orchestrate the innate immune responses [6][7][8]. For example, these cells secrete large amounts of the chemokine IL-8 that plays a major role in neutrophil recruitment at sites of infection [9].In addition to TLR, monocytes abundantly express P2 receptors that are activated by extracellular nucleotides. P2 receptors are divided into two subfamilies: the G-protein-coupled receptors (P2Y 1,2,4,6,[11][12][13][14] ) and the ligand-gated ion channels (P2X 1-7 ). The P2Y subtypes differ in their selectivity toward adenine (ATP, ADP) and uracil nucleotides (UTP, UDP) while all P2X receptors are activated by ATP [10,11]. Human primary monocytes and monocytic cell lines (THP-1 and U937) express the mRNA of various P2 receptors of which P2X 1,7 and P2Y 2,6,11 are common to all these cells [12,13]. There is growing evidence indicating that Results Extracellular nucleotides are involved in Pam 3 CSK 4 -induced neutrophil migration in vi...