Abstract
The innate immune system, which senses invading pathogens, plays a critical role as the first line of host defense. After recognition of foreign RNA ligands (e.g., RNA viruses), host cells generate an innate immune or antiviral response via the interferon-mediated signaling pathway. Retinoic acid-inducible gene I (RIG-1) acts as a major sensor that recognizes a broad range of RNA ligands in mammals; however, chickens lack a RIG-1 homolog, meaning that RNA ligands should be recognized by other cellular sensors such as melanoma differentiation-associated protein 5 (MDA5) and toll-like receptors (TLRs). However, it is unclear which of these cellular sensors compensates for the loss of RIG-1 to act as the major sensor for RNA ligands. Here, we show that chicken MDA5 (cMDA5), rather than chicken TLRs (cTLRs), plays a pivotal role in the recognition of RNA ligands, including poly I:C and influenza virus. First, we used a knockdown approach to show that both cMDA5 and cTLR3 play roles in inducing interferon-mediated innate immune responses against RNA ligands in chicken DF-1 cells. Furthermore, targeted knockout of cMDA5 or cTLR3 in chicken DF-1 cells revealed that loss of cMDA5 impaired the innate immune responses against RNA ligands; however, the responses against RNA ligands were retained after loss of cTLR3. In addition, double knockout of cMDA5 and cTLR3 in chicken DF-1 cells abolished the innate immune responses against RNA ligands, suggesting that cMDA5 is the major sensor whereas cTLR3 is a secondary sensor. Taken together, these findings provide an understanding of the functional role of cMDA5 in the recognition of RNA ligands in chicken DF-1 cells and may facilitate the development of an innate immune-deficient cell line or chicken model.
Highlights
Host pattern recognition receptors (PRRs), which are part of the innate immune system, form the first line of defense against invading pathogens
To better understand the pivotal roles played by chicken MDA5 (cMDA5), cTLR3, and cTLR7 in the absence of RIG-1, we first examined the expressions of mRNAs encoding cMDA5, cTLR3, and cTLR7 in chicken DF-1 cells
We performed small interfering RNA (siRNA)-meditated gene silencing of cMDA5, cTLR3, and cTLR7 to determine their functional roles in IFN-mediated innate immunity
Summary
Host pattern recognition receptors (PRRs), which are part of the innate immune system, form the first line of defense against invading pathogens. RIG-1 recognizes a broad range of RNA viruses harboring 5 triphosphate (5 ppp) moieties, including influenza virus and Sendai virus [5, 12,13,14], whereas MDA5 recognizes double-stranded RNA (dsRNA) patterns, including poly I:C, picornaviruses, and encephalomyocarditis virus (EMCV) [15, 16]. Both RIG-1 and MDA5 contain N-terminal caspase activation and recruitment domains (CARDs), which activate downstream signaling pathways by interacting with the MAVS protein after virus recognition [17, 18]. It is notable that RIG-1 and MDA5 can be regulated negatively or positively by DHX58 receptors, which lack CARDs [19, 20]
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