Dataset Information

Targeted Knockout of MDA5 and TLR3 in the DF-1 Chicken Fibroblast Cell Line Impairs Innate Immune Response Against RNA Ligands.

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.

SUBMITTER: Lee SB

PROVIDER: S-EPMC7204606 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Publications

Targeted Knockout of MDA5 and TLR3 in the DF-1 Chicken Fibroblast Cell Line Impairs Innate Immune Response Against RNA Ligands.

Lee Su Bin SB Park Young Hyun YH Chungu Kelly K Woo Seung Je SJ Han Soo Taek ST Choi Hee Jung HJ Rengaraj Deivendran D Han Jae Yong JY

Frontiers in immunology 20200430

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 <i>via</i> 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 o ...[more]

PMID: 32425931

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Project description:The objective of the present study was to determine if chicken melanoma differentiation-associated gene 5 (MDA5) senses infectious bursal disease virus (IBDV) infection to initiate and amplify an innate immune response in the chicken MDA5 (chMDA5) signaling pathway. Chicken embryo fibroblast DF-1 cells were infected with IBDV LP1 at a multiplicity of infection (MOI) of 0.5 or 10. In addition, knockdown and overexpression of chMDA5 were performed by transfecting DF-1 cells with chMDA5-targeting small interfering RNA (siRNA) or chMDA5-expressing DNA. The transfected cells were infected with IBDV LP1 at an MOI of 10. Cell culture supernatants and lysates were collected at 2, 8, 16 and 24 hours postinfection (hpi) for IBDV titer determination and RNA extraction, respectively. IBDV RNA loads and mRNA expression levels of chicken MDA5, interferon-? (IFN-?) promoter stimulator 1 (IPS-1), interferon regulatory factor-3 (IRF-3), IFN-?, double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase (OAS), myxovirus resistance gene (Mx), and major histocompatibility complex class I (MHC class I) were determined by real-time RT-PCR. The IBDV titer increased up to 1.4 × 10(7) plaque-forming units (PFU)/mL at 24 hpi, and the IBDV RNA load reached 464 ng/?L at 24 hpi. The mRNA expression levels of chicken MDA5, IRF-3, IFN-?, PKR, OAS, Mx and MHC class I in IBDV-infected DF-1 cells exhibited significant (p < 0.05) upregulation up to 906-, 199-, 26,310-, 12-, 66,144-, 64,039- and 33-fold, respectively. Expressed chMDA5 from transfection and double-stranded RNA from IBDV infection were localized or colocalized in the cytoplasm of DF-1 cells at 16 hpi. When chMDA5 was knocked down in DF-1 cells, IBDV titers and RNA loads were significantly higher (p < 0.05) than those in DF-1 cells without chMDA5 knockdown at 24 hpi. The expression levels of chicken MDA5, IRF-3, IFN-? and MHC class I in chMDA5-knockdown DF-1 cells were significantly lower (p < 0.05) at 16 and 24 hpi. DF-1 cells overexpressing chMDA5 by transfection with chMDA5 expressing DNA had significantly lower (p < 0.05) IBDV titers and RNA loads at 16 and 24 hpi and showed significantly higher (p < 0.05) expression of chicken MDA5, IRF-3, IFN-?, PKR, OAS, Mx and MHC class I at 2 hpi. The results indicated that chicken MDA5 recognized IBDV infection and that this interaction resulted in the activation of chMDA5-related innate immune genes and upregulation of chicken MHC class I.

Dataset Information

Targeted Knockout of MDA5 and TLR3 in the DF-1 Chicken Fibroblast Cell Line Impairs Innate Immune Response Against RNA Ligands.

Publications

Targeted Knockout of MDA5 and TLR3 in the DF-1 Chicken Fibroblast Cell Line Impairs Innate Immune Response Against RNA Ligands.

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