Methods: Primary macrophages from SJL/J (TMEV susceptible), B10.S (TMEV resistant) were challenged with TMEV and IRF1 and IRF7 protein evaluated by western blot. Following challenge with TMEV both sets of macrophages expressed IRF1 and IRF7. To determine the ability of IRF1 and IRF7 to stimulate p19 promoter activity, the RAW264.7 macrophage cell line was transfected the p19 promoter luciferase-reporter vector with or without IRF1 or IRF7 expression vectors before challenge with TMEV. Both IRF1 and IRF7 overexpression stimulated p19 promoter activity. To confirm whether IRF1, IRF3, and IRF7 interact with the p19 promoter, fluorescent-tagged oligos corresponding to two IRF consensus sites (IRFE1 and IRFE2) of the p19 promoter were mixed with nuclear extracts of TMEV-challenged RAW264.7 cells and evaluated by EMSA supershift.
Results: The results confirm that IRF7 but not IRF1 or IRF3 bound to p19IRFE1 while IRF3 and IRF7 but not IRF1 bound to p19IRFE2.
Therefore, IRF1, IRF3, and IRF7 play a significant role in expression of pro-inflammatory IL-23 in macrophages challenged with viruses and thus could have a role in sustaining inflammation as a result of chronic virus infection.
Keywords: Gene expression, Immune response, Immunology, Inflammatory mediators and Periodontal disease