HIV-1 Nef activates STAT1 in human monocytes/macrophages through the release of soluble factors

Abstract

IntroductionThe pathology of acquired immunodeficiency syndrome (AIDS) should be considered as the sum of effects generated by human immunodeficiency virus (HIV) replication, with direct T-cell destruction, and by a deep alteration in the pattern of soluble factors. The latter largely depends on the extent of viral replication, because it is hindered by effective antiretroviral therapy. HIV/ simian immunodeficiency virus (SIV) Nef, a multifunctional 27-to 34-kd protein expressed early in viral replication, was demonstrated to be a strong candidate for many of the pathogenic effects of HIV/SIV. This was first highlighted by the evidence that monkeys failed to develop the disease on infection with nef-deleted SIV, 1 and later confirmed by the observation that nef transgenic mice developed a syndrome strictly related to AIDS. 2 Furthermore, Nef perturbs the pattern of secreted factors in different cell types. As an example, the engagement of Nef with the chain of T-cell receptor leads to release of Fas-ligand from T cells in an antigenindependent manner. 3 In addition, the expression of Nef in monocytes/macrophages leads to a massive release of macrophage inflammatory protein 1␣/␤ (MIP-1␣/␤) chemokines and of still unknown factor-activating lymphocytes. 4 These soluble factors possibly promote recruitment and activation of T lymphocytes, which become susceptible to HIV replication. Nef is also able to alter some cellular functions, that is, CD4 internalization/ recycling, 5-7 major histocompatibility complex class I surface expression, 8 and src tyrosine kinases signaling (reviewed in Herna Remkema 9 ) through intracellular mechanisms.We were interested in investigating whether the expression of Nef influences the activation of signal tranducers and activators of transcription (STAT) molecules (reviewed in Schindler and Darnell, 10 Leaman et al, 11 Darnell,12 Stark et al, 13 and Bromberg and Darnell 14 ). STAT-governed pathways were first described by Darnell and colleagues by studying the interferon (IFN)-induced intracellular signal transduction. 15 Seven different STATs have been characterized so far. Activation of STATs is involved in the response of a wide number of cytokines, growth factors, and hormones. Typically, binding of cytokines with specific receptors lacking intrinsic kinase activity in its cytoplasmic tail induces receptor aggregation and recruitment of members of Janus kinases. These become activated by phosphorylating themselves and tyrosine residues of the receptor cytoplasmic tails. The receptor phosphotyrosines serve as docking sites for the binding of inactive STAT through the Src-homology 2 (SH2) domains. STAT monomers become phosphorylated at a constant tyrosine residue and dimerize. The activated dimers translocate to the nucleus thereby binding to specific DNA response elements, ultimately influencing gene expression programs. Hence, alterations in the STAT pathways could substantially influence cellular homeostasis. Few reports regarding the effects of HIV-1 infection on STAT activation/...

HIV-1 Proteins Preferentially Activate Anti-Inflammatory M2-Type Macrophages

“…Using the widely used method to prepare M2-MF (M-CSF derived) and M1-MF (GM-CSF derived) (13-16), we examined whether soluble Tat, gp120, or Nef differentially activated these MF by comparing the activation of MAPK, such as p38, JNK, and ERK. As reported (18)(19)(20)(21)(22)(23)(24)(25)(26)(27), all soluble HIV-1 proteins clearly activated p38 (Supplemental Fig. 1B).…”

“…5B). It was shown that Nef activates Stat family proteins in MF through the release of proinflammatory cytokines and chemokines (24,27). Indeed, we found the activation of Stat1 and Stat3 in Nef-treated M2-MF (Fig.…”

Vav exchange factor counteracts the HIV‐1 Nef‐mediated decrease of plasma membrane GM1 and NF‐AT activity in T cells

“…VSV-G pseudotyped HIV-1 NL4-3 ¿ env and VSV-G pseudotyped HIV-1 NL4-3 ¿ env/nef [56] (10 ng/10 6 cells) were used to infect Jurkat and Vav(CL9) cells. After 24 h from infection, cells were stained with FITC-labeled cholera toxin B subunit (CT-B, Sigma) to measure GM1 expression.…”

DC contact with HIV‐1‐infected cells leads to high levels of Env‐mediated virion endocytosis coupled with enhanced HIV‐1 Ag presentation

“…Both human embryonic kidney epithelial 293T and 293/RGP HIV-1 inducible packaging cells [43] were grown in DMEM plus 10% FBS. Both human primary monocytes and human primary CD4 1 lymphocytes were isolated from PBMC and cultured as reported previously [44]. iDC were differentiated from purified monocytes by culturing for 5-8 days in RPMI medium supplemented with 20% FBS, 500 U/mL of GM-CSF (Serotec, Oxford, UK), and 30 ng/mL of IL-4 (R&D Systems, Oxford, UK).…”

“…Preparations of different (VSV-G) HIV-1 strains were obtained from the supernatants of 293T cells co-transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, CA)-based method as described previously [45], and titrated by measuring both the CAp24 contents by quantitative ELISA (Innogenetics, Gent, Belgium), and the reverse-transcriptase activity, as described previously [44]. Infections were carried out by spinoculation performed at 400g for 30 min at room temperature using 50-500 ng CAp24 equivalent of VSV-G pseudotyped HIV-1/10 5 cells.…”