Involvement of chicken melanoma differentiation-associated gene 5 in innate and adaptive immune response to infectious bursal disease virus infection.

摘要

The present study was undertaken: 1) to determine the molecular identity and functional genomics of chicken melanoma differentiation-associated gene 5 (MDA5); 2) to determine if infectious bursal disease virus (IBDV) infection activated chicken MDA5 (chMDA5) signaling pathway to initiate and amplify innate immune responses; and 3) to determine if the innate immune response activated by chMDA5 following IBDV stimulation bridged to activate adaptive immune responses. For objective 1, chMDA5 was cloned and sequenced and had an open reading frame of 3309 base pairs, encoding an alternatively translated chMDA5 of 1102 amino acids. The domain architecture was conserved among MDA5 molecules from various vertebrates. The chMDA5 shared a high genetic identity with zebrafinch MDA5, but not fish or mammalian MDA5. ChMDA5 transcripts were detected in different chicken tissues. Transfection of chMDA5-expressing plasmid led to a dose-dependent upregulation of chicken interferon- β (IFN-β). DF-1 cells transfected with polyinosinic-polycytidylic acid (poly(I:C)) had significant upregulations (p<0.05) of chicken MDA5 and IFN-β mRNA compared to those in the controls. These results indicated that although chMDA5 was phylogenetically different from mammalian MDA5, the domain architecture remained conserved, and chMDA5 functioned to signal the activation of chicken IFN-β when overexpressing or being stimulated by poly(I:C). For objective 2, IBDV LP1 replicated in DF-1 cells and induced significantly upregulated (p<0.05) expression levels of chicken MDA5, IFN regulatory factor-3 (IRF-3), IFN-β, 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). Expressed chMDA5 and IBDVderived double-stranded RNA (dsRNA) were localized or colocalized in the cytoplasm of DF-1 cells. A significantly enhanced (p<0.05) IBDV replication was observed in chMDA5-knockdown DF-1 cells at 24 hr post infection (hpi). The upregulation of chicken MDA5, IPS-1, IRF-3, IFN-β, PKR, OAS, Mx and MHC class I in DF-1 cells upon IBDV infection was associated with the chMDA5 signaling pathway. DF-1 cells with chMDA5 overexpression had significantly reduced (p<0.05) replication of IBDV and induced significantly upregulated (p<0.05) chicken MDA5, IRF-3, IFN-β, PKR, OAS, Mx and MHC class I. The results indicated that IBDV infection activated the chMDA5 signaling pathway to initiate and amplify the innate immune response. For objective 3, IBDV titers and RNA loads increased up to 3.4x107 PFU/mL and 1114 ng/μL, respectively, in IBDV-infected HD11 cells. IBDV infection in HD11 cells induced significantly upregulated (p<0.05) expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, interleukin-1β (IL-1β, IL-6, IL-8, IL-10, inducible nitric oxide synthase (iNOS) and MHC class I up to 59, 693, 3.8, 286, 22, 5, 146, 4.3, 4.3, 15 and 4.4 folds, respectively. Nitric oxide productions in the culture supernatants increased significantly (p<0.05) up to 6.5 μM at 24 hpi. The expressed chMDA5 and IBDV-derived dsRNA were localized or co-localized in the cytoplasm of HD11 cells. ChMDA5-knockdown HD11 cells had a significantly higher (p<0.05) IBDV RNA load at 24 hpi. HD11 cells with chMDA5 knockdown had significantly lower (p<0.05) nitric oxide productions and expression levels of chicken MDA5, IFN-β PKR, OAS, Mx, IL-1β IL-6, IL-8, IL- 12(p40), IL-18, iNOS, MHC class I and CD86 at 24 hpi. In addition, chMDA5- overexpressed HD11 cells had significantly reduced (p<0.05) IBDV titers and RNA loads and significantly enhanced (p<0.05) nitric oxide productions during IBDV infection. They also had significantly higher (p<0.05) expression levels of chicken MDA5, IFN-β, PKR, OAS, Mx, IL-1β, IL-6, IL-8, IL-12(p40), IL-10 and iNOS at 2 hpi. The results indicated that chMDA5 in chicken macrophages recognized IBDV infection, initiated a chMDA5-related innate immune response and activated adaptive immunity modulating genes. In conclusion, chickens had MDA5 and chicken MDA5 functioned as a cytosolic dsRNA sensor. Chicken MDA5 could sense IBDV to induce the immune response related to the chMDA5 signaling pathway and the induced innate immunity bridged to activate adaptive immune response.