Cutting Edge: Influenza A Virus Activates TLR3-Dependent Inflammatory and RIG-I-Dependent Antiviral Responses in Human Lung Epithelial Cells

Abstract

Influenza A virus (IAV) triggers a contagious acute respiratory disease that causes considerable mortality annually. Recently, we established a role for the pattern-recognition TLR3 in the response of lung epithelial cells to IAV-derived dsRNA. However, additional nucleic acid-recognition proteins have lately been implicated as key viral sensors, including the RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene (MDA)-5. In this study, we investigated the respective role of TLR3 vs RIG-I/MDA-5 signaling in human respiratory epithelial cells infected by IAV using BEAS-2B cells transfected with vectors encoding either a dominant-negative form of TLR3 or of mitochondrial antiviral signaling protein (MAVS; a signaling intermediate of RIG-I and MDA-5), or with plasmids overexpressing functional RIG-I or MDA-5. We demonstrate that the sensing of IAV by TLR3 primarily regulates a proinflammatory response, whereas RIG-I (but not MDA-5) mediates both a type I IFN-dependent antiviral signaling and a proinflammatory response.

Role of human Toll‐like receptors in naturally occurring influenza A infections

“…Increased TLR (7, 8, and 9) expression was found to correlate significantly with the key intracellular signaling molecules (MAPKs, NF‐κB/IκB) and higher levels of pro‐inflammatory cytokines including IL‐6 and sTNFR‐1 3, 5, 7, 14, 20, 22, 27, 28, 32, 33. Consistent with earlier in vitro studies, associations with the “adaptive” cytokines (e.g., Th1‐related IFN‐γ, CXCL10/IP‐10, CXCL9/MIG) were also observed, particularly for TLR9 and TLR8, which signal through the MyD88 pathway 3, 4, 10, 34.…”

“…This reflects the less specific nature of innate immunity, which can be advantageous when considering TLR targeting as a means of prophylaxis in influenza (discussed below). There is evidence to show that the RLRs are concomitantly upregulated and possibly play a contributory role in mediating the pro‐inflammatory cytokine responses 3, 7, 10, 15, 33. Although both RIG‐1 and MDA‐5 often showed upregulation in influenza and other respiratory viral infections, innate responses against influenza viruses are likely dependent on RIG‐1 and suppressible by the viral NS‐1 protein 7, 13, 26, 33…”

“…These observations were unlikely the sole result of adaptive immunity, as patients were studied within 48 hours of their illnesses 3, 5, 20. The alternative explanation of a higher level of viral replication inducing a smaller increase in these viral‐sensing TLRs also appeared unlikely 3, 4, 5, 7, 8. Our results are in line with recent mice experiments which showed that TLR activation rapidly produces virus inhibitory responses (predominantly through type I IFNs and IFN‐stimulated mechanisms), conferring protection against influenza challenge 3, 4, 10, 14.…”

“…Broadly, viral nucleic acids (dsRNA, ssRNA, CpG oligodeoxynucleotides) are detected by the endosomal TLRs (3, 7, 8, 9), whereas components of bacteria (peptidoglycans/lipoproteins, lipopolysaccharides) are detected by the cell surface TLRs (2, 4) 3, 4. Recent in vitro and in vivo studies on influenza pathogenesis have shown that TLR activation induces expression of type I interferons and pro‐inflammatory cytokines (e.g., IL‐6, TNF‐α), limiting viral replication and dissemination, mediating tissue inflammation, and link to adaptive immunity development 3, 7, 8, 9, 10, 11, 123, 7, 10, 13.…”

“…Recent in vitro and in vivo studies on influenza pathogenesis have shown that TLR activation induces expression of type I interferons and pro‐inflammatory cytokines (e.g., IL‐6, TNF‐α), limiting viral replication and dissemination, mediating tissue inflammation, and link to adaptive immunity development 3, 7, 8, 9, 10, 11, 123, 7, 10, 13. Recently, data from animal studies have further shown that targeting the TLRs (with agonists) may rapidly upregulate the innate immunity to provide broad‐range, virus strain non‐specific protection against lethal influenza challenge 4, 11, 14, 15, 16, 17.…”