It has been recently reported that the endogenous expression of HIV-1 Nef in human monocyte/macrophages induces the release of chemokines and other as yet unidentified soluble factors leading to multiple effects of pathogenic significance, such as the recruitment and activation of quiescent lymphocytes. However, the description of underlying molecular mechanisms remained elusive. We recently demonstrated that human monocyte-derived macrophages (MDM) efficiently internalize soluble rNef, thereby inducing effects largely resembling those observed in cells endogenously expressing Nef. By exploiting the rNef/MDM model, we sought to gain more insights on the molecular mechanisms underlying the response of MDM to Nef. Array analysis for the detection of transcripts from a large number of monokines, chemokines, cytokines, and receptors thereof showed that MDM promptly responded to rNef treatment by increasing the transcription of genes for several inflammatory factors. Analysis of supernatants revealed that rNef treatment induced the release of macrophage inflammatory proteins 1α and 1β, IL-1β, IL-6, and TNF-α. Conversely, rNefs mutated in domains critical for the interaction with the endocytotic machinery (i.e., EE155-156QQ, and DD174-175AA) were ineffective. Interestingly, we found that the Nef-dependent release of inflammatory factors correlated with the activation of the NF-κB transcription factor, mainly in its p50/p50 homodimeric form, and in a de novo protein synthesis-independent manner. Our data add new hints supporting the idea that the presence of Nef is per se heavily detrimental for monocyte/macrophages and relative cross-talking cell types.

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/...

Numerous evidence has demonstrated the involvement in growth control of interferon (IFN) regulatory factor-1 (IRF-1), which shows tumor suppressor activity. IRF-1 is a well-studied member of the IRF transcription factors that reveals functional diversity in the regulation of cellular response by activating expression of a diverse set of target genes, depending on the cell type and on the specific stimuli. IRF-1 gene rearrangements may be a crucial point in the pathogenesis of some cancer types. Furthermore, different aspects of the tumor suppressor function of IRF-1 may be explained, at least in part, by the observations that IRF-1 is a regulator of cell cycle and apoptosis and that its inactivation accelerates cell transformation. Studies on gene knockout mice contributed greatly to the clarification of these multiple IRF-1 functions. We summarize our current knowledge of the antigrowth effect of IRF-1, focusing also on a more general involvement of IRF-1 in mediating negative regulation of cell growth induced by numerous cytokines and other biologic response modifiers.

The viral protein Nef is a virulence factor that plays multiple roles during the early and late phases of human immunodeficiency virus (HIV) replication. Nef regulates the cell surface expression of critical proteins (including down-regulation of CD4 and major histocompatibility complex class I), T-cell receptor signaling, and apoptosis, inducing proapoptotic effects in uninfected bystander cells and antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 ligand signaling pathway in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T lymphocytes, rendering them susceptible to HIV infection. There is also increasing evidence that in vitro cell treatment with Nef induces signaling effects. Exogenous Nef treatment is able to induce apoptosis in uninfected T cells, maturation in dendritic cells, and suppression of CD40-dependent immunoglobulin class switching in B cells. Previously, we reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a cycloheximide-independent activation of NF-B and the synthesis and secretion of a set of chemokines/cytokines that activate STAT1 and STAT3. Here, we show that Nef treatment is capable of hijacking cellular signaling pathways, inducing a very rapid regulatory response in MDMs that is characterized by the rapid and transient phosphorylation of the ␣ and ␤ subunits of the IB kinase complex and of JNK, ERK1/2, and p38 mitogenactivated protein kinase family members. In addition, we have observed the activation of interferon regulatory factor 3, leading to the synthesis of beta interferon mRNA and protein, which in turn induces STAT2 phosphorylation. All of these effects require Nef myristoylation.The 27-to 34-kDa Nef protein is an important virulence factor of primate lentiviruses; it is the regulatory protein expressed earliest and most abundantly in the infection cycle. Studies of animal models and seropositive patients showed that Nef-defective viruses led to an attenuated clinical phenotype with a reduced viral load (29,30,50,59,60). In addition, nef transgenic mice develop an AIDS-like disease (51) characterized by failure to thrive/weight loss, diarrhea, wasting, premature death, thymus atrophy, loss of CD4 ϩ T cells, interstitial pneumonitis, and tubulointerstitial nephritis. Inside the cell, Nef induces effects that are genetically distinguishable yet highly conserved and that appear to be mediated via specific protein-protein interaction domains (7,33,44). Nef is cotranslationally modified by an N-terminal myristoylation site whose lipidation is required for membrane association. However, cellular-fractionation assays from transient transfections showed that less than 50% of the protein was localized at membranes, while the remaining portion was found to be cytosolic (25,34,58,68,98). The protein adopts a two-domain structure that is characterized by a flexible N-terminal arm of about 60 amino acids, followed by a well-conserved and folded core...

The BRAF inhibitors vemurafenib, dabrafenib and encorafenib are used in the treatment of patients with BRAF-mutant melanoma. They selectively target BRAF kinase and thus interfere with the mitogen-activated protein kinase (MAPK) signalling pathway that regulates the proliferation and survival of melanoma cells. In addition to their molecularly targeted activity, BRAF inhibitors have immunomodulatory effects. The MAPK pathway is involved in T-cell receptor signalling, and interference in the pathway by BRAF inhibitors has beneficial effects on the tumour microenvironment and anti-tumour immune response in BRAF-mutant melanoma, including increased immune-stimulatory cytokine levels, decreased immunosuppressive cytokine levels, enhanced melanoma differentiation antigen expression and presentation of tumour antigens by HLA 1, and increased intra-tumoral T-cell infiltration and activity. These effects promote recognition of the tumour by the immune system and enhance anti-tumour T-cell responses. Combining BRAF inhibitors with MEK inhibitors provides more complete blockade of the MAPK pathway. The immunomodulatory effects of BRAF inhibition alone or in combination with MEK inhibition provide a rationale for combining these targeted therapies with immune checkpoint inhibitors. Available data support the synergy between these treatment approaches, indicating such combinations provide an additional beneficial effect on the tumour microenvironment and immune response in BRAF-mutant melanoma.