Expression and Function of RIG-I in Oral Keratinocytes and Fibroblasts

Akiko Fukui , Department of Oral and Maxillofacial Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Kouji Ohta DDS, PhD, Department of Oral and Maxillofacial Surgery, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Hideo Shigeishi , Department of Oral and Maxillofacial Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Yoko Ishida , Department of Oral and Maxillofacial Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Hiromi Nishi , Hiroshima University, Hiroshima, Japan
Masaaki Takechi DDS, PhD, Department of Oral and Maxillofacial Surgery, Applied Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
Statement of the problem:The innate immune system constitutes the first line of host defense against invading viruses. Oral mucosa is thought to be constantly exposed to various pathogens, and serves as a barrier against microorganism invasion. Innate immune response by oral mucosal cells like oral keratinocytes and fibroblasts may be the first line of host defense against viral infection.  Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic pattern recognition receptors including DExH box family RNA helicase proteins.RIG-I recognizes viral dsRNA in the cytoplasm, and RIG-I-mediated signaling regulates antiviral type I IFN, and inflammatory chemokine production. Here, we tested the hypothesis that oral mucosal cell participation in host defense against viral infection via RIG-I.

Materials and Methods: RIG-I expression was examined in oral keratinocytes (RT7) and oral fibroblasts (GT1) and buccal mucosal cells derived swabs using and RT-PCR and immunohistochemistry. RT7 and GT1 were exposed to dsRNA virus mimic Poly I:C-LMW/LyoVec (PLV; Invivogen, San Diego, CA, USA ).  Expression of IFN-β and CXCL10 via RIG-I was examined by Real-time RT-PCR and ELISA. Phosphorylation of IRF3 and STAT1 were detected by western blotting.

Methods of data analysis: Data were analyzed using Student’s t-test or one-way analysis of variance (ANOVA), and the results are presented as the mean ± standard deviation.

Results: RT-PCR results showed that RT7, GT1 and buccal mucosal cells derived from swabs constitutively expressed RIG-I mRNA. RIG-I was detected in the cytoplasm of both RT7 and GT1 when analyzed by immunocytochemistry. When RT7 and GT1 exposed to PLV, mRNA levels of IFN-β showed  significant increases in both RT7 and GT1, and CXCL10 was dramatically induced by PLV in a dose-dependent manner.The production levels of both IFN- β and CXCL10 induced by PLV were specifically  attenuated by RIG-I specific small interfering RNA(siRNAs).  PLV increased IFN- β and  CXCL10 productions in both RT7 and GT1 via RIG-I concurrent with phosphorylation of IRF3 and STAT1. PLV-induced CXCL10 production was attenuated by neutralization of IFN- β and blocking of IFN-α/β receptor (IFNAR).Indicating that primal IFN-β production via the RIG-I-IRF3 axis, which eventually induces CXCL10 production via the IFNAR -STAT1 axis.

Conclusion: We propose that RIG-I in oral keratinocytes and fibroblasts may cumulatively develop host-defense mechanisms against viral infection in oral mucosa.

Reference

Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K,

Akira S, Fujita T: The RNA helicase RIG-I has an essential function in double-stranded

RNA-induced innate antiviral responses. Nat Immunol 2004;5: 730-737.

 

Fukui A, Ohta K, Nishi H , Shigeishi H, Tobiume K, Takechi M, Kamata N:

Interleukin-8 and CXCL10 expression in oral keratinocytes and fibroblasts via Toll-like receptors. Microbiol Immunol 2013;57:198-206

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