HIV-induced dysregulation of IFN-gamma signaling and programmed cell death in primary human monocytes
Creators: name: Alhetheel, Abdulkarim Fahad
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 2010
Subjects: subject: Biology, Microbiology.

description: Cytokine responsiveness in monocytes / macrophages (M/M) is critical in their phagocytic, antimicrobial and antigen presenting function. It has been suggested that HIV can impair cell function by disrupting signal transduction induced in response to antigen and cytokines in different immune cell types including CD4+ T cells, CD8+ T cells, and monocytes. A study by Kryworuchko et al. showed that Interleukin (IL)-2-induced Signal Transducer and Activator of Transcription (STAT) 5 activation was inhibited in CD8+ T cells from a subset of HIV-infected patient's naive to therapy, but was restored, at least in part, after antiretroviral therapy (ART). In view of the important biological role of cytokine signaling via the STAT pathway in the regulation of M/M phagocytosis, proliferation, and cell survival, I hypothesized that disruption of the JAK/STAT pathway may lead to dysregulation of M/M effector functions and programmed cell death (PCD) during the course of chronic HIV infection. Therefore, my objectives were first to evaluate cytokine-induced JAK/STAT signaling in monocytes obtained from HIV- individuals and compare it to that found in two groups of HIV+ patients (ART-treated patients for &gt;1 year and patients off therapy for &gt;6 months). Second, I sought to determine the biological impact and the molecular mechanisms responsible for the alterations observed. Analysis of patient monocytes showed no clear difference in responsiveness among the three groups in the case of Interferon (IFN)-alpha, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 stimulation. However, in the case of IFN-gamma stimulation, the STAT1 transcription factor was significantly upregulated in HIV+ patients off therapy compared to HIV- controls and HIV+ patients on ART. Upregulation of STAT1 activation was not due to alterations in IFN-gamma receptor expression but rather the result of increased total STAT1 expression levels. Treatment of monocytic cells with HIV proteins Gp120 and Vpr was able to induce STAT1 expression in these cells. Investigations into the significance of the STAT1 hyperactivation in patient monocytes showed that, surprisingly, among the STAT1 responsive genes (HLA-DR, IRF-1, CXCL9, and CXCL10) studied, only chemokine CXCL9 expression was elevated in HIV+ patients on ART compared to the other groups. Since IFN-gamma-induced STAT1 activation is associated with PCD and IL-10 has inhibitory effects on IFN-gamma-induced signaling and its downstream effects, I evaluated monocyte cell death in response to IFN-gamma and IL-10 in all groups. Interestingly, spontaneous and IFN-gammainduced monocyte PCD were elevated in HIV+ patients compared to HIV- controls. Spontaneous PCD was significantly correlated with increased total STAT1 expression but not plasma levels of TRAIL. Interestingly, pretreatment with IL-10 could rescue monocytes from this fate. Further investigation of how PCD occurs in normal monocytes and how it is regulated by IFN-gamma and IL-10, showed that IFN-gamma enhanced spontaneous TRAIL secretion and caspase 8 activation. In contrast, IL-10 inhibited spontaneous and IFN-gamma-induced TRAIL secretion and caspase 8 activation. Surprisingly and despite this, spontaneous and IFN-gamma-induced monocyte PCD appeared to be independent of caspase activation but rather depended on autophagy. LC3-II expression, an autophagy marker, was upregulated spontaneously in cultured monocytes, enhanced further upon IFN-gamma stimulation but surprisingly, was also upregulated by IL-10. At the level of the molecular mechanism, I observed that blocking the STAT or Phosphatidyl inositol-3 kinase (PI3K) signaling pathways inhibited PCD in response to IFN-gamma and could also thwart the cytoprotective effects of IL-10. Concordantly, blocking STAT or PI3K activation reduced LC3-II expression III response to IFN-gamma or IL-10 stimulation in monocytes. In conclusion, these results suggest that dysregulation of IFN-gamma signaling may contribute to monocyte dysfunction during HIV infection. Furthermore, IL-10 may have a role in enhancing monocyte survival during chronic HIV infection. Thus, understanding the regulation of monocyte PCD via autophagy pathway may have important implications concerning the elimination of this important cellular reservoir for HIV.