Regulation of interleukin-12p40 synthesis in HIV-infected myeloid cells

Abstract

Progressive immunodeficiency in HIV infection is paralleled by a decrease in IL-12 production, a cytokine crucial for cellular immune function. This thesis reports an examination of the requirement for productive viral infection, the role of other cytokines, and the intracellular molecular targets of HIV-mediated suppression of IL-12 p40 expression, with particular emphasis on regulation of the IL-12 p40 promoter, and LPS-induced activation of MAP kinases. The reduction in LPS-induced IL-12 p40 protein and mRNA following acute in vitro HIV infection of THP-1 cells and monocyte-derived macrophages (MDM) was not attributed to IL-10 or TGF-beta activity, and was not restored by priming with IL-4, IL-13, or IFN-gamma. Suppression of IL-12 was dependent upon productive viral infection, and was due, at least in part, to impaired transcription of IL-12 p40. HIV infection of THP-1 cells reduced nuclear factor binding to the NF-kappaB, AP-1, and Sp1 sites of the IL-12 p40 promoter. Nuclear factor binding to these sites, in addition to the Ets-2 element, was also altered by HIV infection of MDM. By site-directed mutagenesis, each of these transcription factor binding sites were determined to be necessary for IL-12 p40 promoter activation. Binding of NF-kappaB p50, p65, c-Rel, c-Fos, c-Jun, Sp1 and Sp3 proteins to the IL-12 p40 promoter was reduced by HIV infection of THP-1 cells, while p50, c-Rel, c-Fos, c-Jun, IRF-1, ICSBP, and Ets-2 binding was altered by HIV in MDM. HIV infection did not affect cellular levels of the nuclear factors, with exception of NF-kappaB p65 in THP-1 cells, and c-Fos in MDM. HIV infection of myeloid cells decreased levels of phosphorylated JNK MAPK, impaired phosphorylation of p38 MAPK, and suppressed phosphorylation and degradation of IkappaBalpha while ERK 1/2 MAPK was unaffected. Taken together, these results suggest that impaired IL-12 production in HIV-infected myeloid cells occurs, in part, via disruption of IL-12 p40 transcription, and is dependent on productive infection, and independent of the activity of other cytokines. Inhibition of IL-12 p40 transcription is associated with disruption of nuclear factor binding to the NF-kappaB, AP-1, Sp1, and Ets-2 elements of the human IL-12 p40 promoter, which may be a consequence of altered MAPK activation.