388 HIV Inhibition of IL-12 Synthesis is Mediated by Altering MAPK Activity and Nuclear Factor Binding to the IL-12p40 Promoter F. M. Frappier*, K. A. Chambers, J. B. Angel Ottawa Hlth Res Inst Univ of Ottawa, Canada
Background: IL-12 is critical for the generation of cellular immune responses and its production is impaired in HIV infection. LPS and other agents can stimulate IL-12 synthesis, but the signaling pathways that lead to its production are not well characterized. We have previously shown that HIV directly inhibits IL-12p40 gene transcription in monocytic cells. Further delineating the mechanisms by which HIV inhibits IL-12 synthesis will provide important insight into the immunopathogenesis of HIV infection.
Methods: Potential HIV-induced alterations in nuclear factor binding to IL-12p40 promoter were assessed with electromobility shift assays (EMSA). Western analyses were then conducted to determine total and phosphorylated levels of the relevant upstream molecules (e.g., p38, JNK, ERK and IkappaBalpha) in HIV-infected and uninfected monocytic (THP-1) cells. To further investigate the role of these MAP kinases (MAPK) in IL-12p40 synthesis, MAPK inhibitors were used (SB203580 for p38, PD98059 for MEK/ERK1/2 ,and SP600125 for JNK). ELISA were used to quantitate IL-12p40 protein production from LPS stimulated THP-1 cells in the presence or absence of inhibitors. EMSA were performed to determine whether MAPK inhibition influenced binding of transcription factors to those sites of the IL-12p40 promoter that were altered with HIV infection.
Results: EMSA demonstrated decreased binding to NFkappaB, Sp-1 in HIV-infected THP-1 cells. In vitro HIV infection also resulted in decreased expression and phosphorylation of JNK, as well as decreased phosphorylation of p38, ERK1/2 and IkappaB alpha. In uninfected THP-1 cells, inhibition of p38 and JNK decreased IL-12p40 protein production, whereas ERK inhibition had no significant effect. Both the p38 and the JNK inhibitors decreased AP-1 binding activity while only the JNK inhibitor reduced NFkappaB binding. Consistent with its lack of effect on IL-12p40 protein, the ERK inhibitor had no effect on nuclear factor binding to any site studied. None of the inhibitors altered binding to Sp-1.
Conclusion: HIV infection of monocytic cells interferes with normal MAPK activity which appears to lead to downstream changes in binding of nuclear factors to the IL-12p40 promoter and hence impaired IL-12p40 transcription. Understanding precisely how HIV interferes with this critical signaling pathway has the potential to lead to the development of novel immune based therapies.
