Regulation of human immunodeficiency virus type 1 infectivity by the ERK mitogen-activated protein kinase signaling pathway

ERK1 and ERK2 mitogen-activated protein kinases (MAPK) play a critical role in regulation of cell proliferation and differentiation in response to mitogens and other extracellular stimuli. Mitogens and cytokines that activate MAPK in T cells have been shown to activate human immunodeficiency virus type 1 (HIV-1) replication. Little is known about the signal transduction pathways that activate HIV-1 replication in T cells upon activation by extracellular stimulation. Here, we report that activation of MAPK through the Ras/Raf/MEK signaling pathway enhances the infectivity of HIV-1 virions. Virus infectivity was enhanced by treatment of cells with MAPK stimulators, such as serum and phorbol myristate acetate, as well as by coexpression of constitutively activated Ras, Raf, or MEK (MAPK kinase) in the absence of extracellular stimulation. Treatment of cells with PD 098059, a specific inhibitor of MAPK activation, or with a MAPK antisense oligonucleotide reduced the infectivity of HIV-1 virions without significantly affecting virus production or the levels of virion-associated Gag and Env proteins. MAPK has been shown to regulate HIV-1 infectivity by phosphorylating Vif (X. Yang and D. Gabuzda, J. Biol. Chem. 273:29879-29887, 1998). However, MAPK activation enhanced virus infectivity in some cells lines that do not require Vif function. The HIV-1 Rev, Tat, p17(Gag), and Nef proteins were directly phosphorylated by MAPK in vitro, suggesting that other HIV-1 proteins are potential substrates for MAPK phosphorylation. These results suggest that activation of the ERK MAPK pathway plays a role in HIV-1 replication by enhancing the infectivity of HIV-1 virions through Vif-dependent as well as Vif-independent mechanisms. MAPK activation in producer cells may contribute to the activation of HIV-1 replication when T cells are activated by mitogens and other extracellular stimuli.     

Modulation of macrophage subtypes by IRF5 determines osteoclastogenic potential

Bone-resorbing osteoclasts are differentiated from macrophages (MΦ) by M-CSF and RANKL. MΦ can be mainly classified into M1 and M2 MΦ, which are proinflammatory and anti-inflammatory, respectively, but little is known about their osteoclastogenic potential. Here, we investigated the osteoclastogenic potential of MΦ subtypes. When the two MΦ subtypes were differentiated into osteoclasts using M-CSF and RANKL, M2 MΦ more potently differentiated into osteoclasts than M1 MΦ. M2 MΦ generated with IL-4 or IL-10 also showed enhanced osteoclast differentiation compared with M1 MΦ induced by IFN-γ and lipopolysaccharide. In addition, robust bone-resorptive capacity and giant actin rings, which are features of mature osteoclasts, were observed in M2, but not M1 MΦ, under the osteoclast differentiation condition. Osteoclast differentiation was significantly increased in CD206⁺ M2 MΦ but not in CD86⁺ M1 MΦ. Compared with M1 MΦ, c-Fms and RANK were highly expressed in M2 MΦ. Enhanced osteoclastogenesis of M2 MΦ was mediated through sustained ERK activation, followed by efficient c-Fos and NFATc1 induction. Notably, the osteoclastogenic potential of M1 MΦ converted into M2 MΦ by exposure to M-CSF was higher than that of M2 MΦ converted into M1 MΦ by exposure to GM-CSF. Silencing IRF5, which is responsible for M1 MΦ polarization, increased osteoclast differentiation by enhancing c-Fms expression and activation of ERK, c-Fos, CREB, and NFATc1, which was inhibited by overexpression of IRF5. Collectively, M2 MΦ are suggested to be more efficient osteoclast precursors than M1 MΦ because of the attenuated expression of IRF5.     

Inhibition of mixed lineage kinase 3 prevents HIV-1 Tat-mediated neurotoxicity and monocyte activation

The HIV-1 gene products Tat and gp120 are toxic to neurons and can activate cells of myeloid origin, properties that are thought to contribute to the clinical manifestations of HIV-1-associated dementia (HAD). To investigate the intracellular signaling mechanisms involved in these events, the effect of Tat and gp120 on mixed lineage kinase (MLK) 3 activation was examined. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons; this was abolished by the addition of an inhibitor of MLK3 (CEP1347). CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, overexpression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat. MLK3-dependent downstream signaling events were implicated in the neuroprotective and monocyte-deactivating pathways triggered by CEP1347. Thus, the inhibition of p38 MAPK and JNK protected neurons from Tat-induced apoptosis, whereas the inhibition of p38 MAPK, but not of JNK, was sufficient to prevent Tat- and gp120-mediated activation of monocytes. These results suggest that the normal function of MLK3 is compromised by HIV-1 neurotoxins (Tat, gp120), resulting in the activation of downstream signaling events that result in neuronal death and monocyte activation (with release of inflammatory cytokines). In aggregate, our data define MLK3 as a promising therapeutic target for intervention in HAD.     

Macropinocytosis and TAK1 mediate anti-inflammatory to pro-inflammatory macrophage differentiation by HIV-1 Nef

Macrophages (MΦ) are functionally classified into two types, anti-inflammatory M2 and pro-inflammatory M1. Importantly, we recently revealed that soluble HIV-1 proteins, particularly the pathogenetic protein Nef, preferentially activate M2-MΦ and drive them towards an M1-like MΦ, which might explain the sustained immune activation seen in HIV-1-infected patients. Here, we show that the preferential effect of Nef on M2-MΦ is mediated by TAK1 (TGF-β-activated kinase 1) and macropinocytosis. As with MAP kinases and NF-κB pathway, Nef markedly activated TAK1 in M-CSF-derived M2-MΦ but not in GM-CSF-derived M1-MΦ. Two Nef mutants, which were unable to activate MAP kinases and NF-κB pathway, failed to activate TAK1. Indeed, the TAK1 inhibitor 5Z-7-oxozeaenol as well as the ectopic expression of a dominant-negative mutant of TAK1 or TRAF2, an upstream molecule of TAK1, inhibited Nef-induced signaling activation and M1-like phenotypic differentiation of M2-MΦ. Meanwhile, the preferential effect of Nef on M2-MΦ correlated with the fact the Nef entered M2-MΦ more efficiently than M1-MΦ. Importantly, the macropinosome formation inhibitor EIPA completely blocked the internalization of Nef into M2-MΦ. Because the macropinocytosis activity of M2-MΦ was higher than that of M1-MΦ, our findings indicate that Nef enters M2-MΦ efficiently by exploiting their higher macropinocytosis activity and drives them towards M1-like MΦ by activating TAK1.     

Selenium suppressed the LPS-induced inflammation of bovine endometrial epithelial cells through NF-κB and MAPK pathways under high cortisol background

The bovine uterus is susceptible to infection, and the elevated cortisol level due to stress are common in cows after delivery. The essential trace element selenium plays a pivotal role in the antioxidant and anti-inflammatory defence system of body. This study investigated whether selenium supplementation protected endometrial cells from inflammation in the presence of high-level cortisol. The primary bovine endometrial epithelial cells were subjected to Escherichia coli lipopolysaccharide to establish cellular inflammation model. The gene expression of inflammatory mediators and proinflammatory cytokines was measured by quantitative PCR. The key proteins of NF-κB and MAPK signalling pathways were detected by Western blot and immunofluorescence. The result showed that pre-treatment of Na₂SeO₃ (1, 2 and 4 μΜ) decreased the mRNA expression of proinflammatory genes, inhibited the activation of NF-κB and suppressed the phosphorylation of extracellular signal-regulated kinase, P38MAPK and c-Jun N-terminal kinase. This inhibition of inflammation was more apparent in the presence of high-level cortisol (30 ng/mL). These results indicated that selenium has an anti-inflammatory effect, which is mediated via NF-κB and MAPK signalling pathways and is augmented by cortisol in bovine endometrial epithelial cells.     

IL-17A and IL-17F repair HIV-1 gp140 damaged Caco-2 cell barriers by upregulating tight junction genes

It is widely accepted that impairment of the intestinal epithelial barrier from HIV/AIDS contributes significantly to microbial translocation and systemic immune activation. Such factors present potential targets for novel treatments aimed toward a functional cure. However, the extracellular mechanisms of intestinal barrier repair are poorly understood. In the current study, we investigated the abilities of IL-17A and IL-17F to repair the damaged barrier caused by HIV-1 gp140 using Caco-2 monolayers. It was found that HIV-1 gp140 downregulated the expression of tight junction-associated genes and disrupted the barrier integrity of Caco-2 monolayers. However, IL-17A and IL-17F treatment reversed the HIV-1 gp140-induced barrier dysfunction by upregulating the expression of tight junction-associated genes, the combination of which resulted in a stronger induction of barrier repair. Furthermore, the effects of IL-17A and IL-17F were reduced by downregulation of Act1 with siRNA and inhibition of NF-κB and MAPK pathways with BAY11-7082 and U0126, respectively. These data indicated that the NF-κB and MAPK pathways are involved in the repair of barrier integrity mediated by IL-17A and IL-17F, and IL-17 pathways are potential targets for gut barrier restoration therapies during HIV/AIDS.     

HIV-1 Tat increases BAG3 via NF-κB signaling to induce autophagy during HIV-associated neurocognitive disorder

The human immunodeficiency virus-1 (HIV-1) regulatory protein Tat plays an important role during HIV-1-associated neurocognitive disorders (HAND) by inducing neuronal autophagy. In this study, we used immunohistochemistry, immunofluorescence, western blot, qRT-PCR, and RNA interference to elucidate the involvement of Bcl-2-associated athanogene 3 (BAG3) in the pathogenesis of HIV-1 Tat-induced autophagy during HAND. We found that BAG3 expression is elevated in astrocytes in frontal cortex of macaques infected with simian immunodeficiency virus-human immunodeficiency chimeric virus (SHIV). In addition, in human primary glioblastoma cells (U87), HIV-1 Tat upregulated BAG3 in an NF-κB-dependent manner to induce autophagy. Importantly, suppression of BAG3 or inhibition of NF-κB activity reversed the HIV-1 Tat-induced autophagy. These results indicate that HIV-1 Tat induces autophagy by upregulating BAG3 via NF-κB signaling, which suggests BAG3 and NF-κB could potentially serve as novel targets for HAND therapies.     

RNA-binding protein AUF1 regulates lipopolysaccharide-induced IL10 expression by activating IkappaB kinase complex in monocytes

Exposure of monocytes and macrophages to endotoxin/lipopolysaccharide (LPS) from Gram-negative bacteria activates the NF-κB signaling pathway. At early times, this leads to their production of proinflammatory cytokines, but subsequently, they produce anti-inflammatory interleukin-10 (IL-10) to quell the immune response. LPS-mediated induction of IL10 gene expression requires the p40 isoform of the RNA-binding protein AUF1. As LPS exerts modest effects upon IL10 mRNA stability, we hypothesized that AUF1 controls the expression of signaling proteins. Indeed, knockdown of AUF1 impairs LPS-mediated p38 mitogen-activated protein kinase (MAPK) and NF-κB signaling, and the expression of an RNA interference-refractory p40(AUF1) cDNA restores both signaling pathways. To define the molecular mechanisms by which p40(AUF1) controls IL10 expression, we focused on the NF-κB pathway in search of AUF1-regulated targets. Here, we show that p40(AUF1) serves to maintain proper levels of the kinase TAK1 (transforming growth factor-β-activated kinase), which phosphorylates the IKKβ subunit within the IκB kinase complex to activate NF-κB-regulated genes. However, p40(AUF1) does not control the TAK1 mRNA levels but instead promotes the translation of the mRNA. Thus, p40(AUF1) regulates a critical node within the NF-κB signaling pathway to permit IL10 induction for the anti-inflammatory arm of an innate immune response.     

Unfractionated heparin inhibits lipopolysaccharide-induced inflammatory response through blocking p38 MAPK and NF-κB activation on endothelial cell

Heparins, including unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH), are glycosaminoglycans that are largely used as anti-thrombotic drugs. While the mechanisms of their anticoagulant actions in blood have been extensively studied, their effects on the inflammation of the endothelium are still under investigation since the endothelium plays a central role in sepsis. Furthermore, UFH is much cheaper than LMWH. The aim of this study was to determine how UFH regulates lipopolysaccharide (LPS)-induced inflammatory response on endothelial cells in vitro, and define the role of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) in mediating this effect. Human pulmonary microvascular endothelial cells (HPMECs) were pretreated with UFH (0.01U/ml-10U/ml), prior to stimulation with LPS (10μg/ml). Markers of systemic inflammation and endothelial activation were assessed. Interleukin (IL)-1β, IL-6, E-selectin, intercellular adhesion molecule (ICAM)-1 release were subsequently measured at 2h, 6h and 12h. Phosphorylation of p38 MAPK at 2h, 6h and nuclear translocation of the proinflammatory NF-κB at 2h were assessed. In HPMEC, UFH significantly attenuated LPS-induced production of IL-1β, IL-6, E-selectin and ICAM-1, as well as phosphorylation of p38 MAPK and NF-κB translocation, especially in 10U/ml. In conclusion, UFH at high dose significantly protects against endothelial-cell-mediated immune response. The inhibition of p38 MAPK and NF-κB activation certainly represents one of the mechanisms by which UFH exerts its anti-inflammatory effect.     

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke

Multi-protein complexes, termed “inflammasomes,” are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.     

Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.     

Inhibition of active HIV-1 replication by NF-κB inhibitor DHMEQ

Previous reports indicate that nuclear factor (NF)-κB regulates induction of human immunodeficiency virus type 1 (HIV-1) gene expression in latently infected cells. However, the role of NF-κB in cells with active HIV-1 replication is not well understood. In this study, we examined the effect of a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on HIV-1 replication in a human T cell line and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PHA-PBMCs). We further explored the mechanism of DHMEQ-mediated inhibition of HIV-1 replication. DHMEQ inhibited HIV-1 replication in HIV-1-infected Molt-4 and PHA-PBMCs. DHMEQ inhibited constitutive NF-κB activity in HIV-1-infected PHA-PBMCs and HIV long terminal repeat promoter activity driven by tumor necrosis factor (TNF)-α and the trans-activator Tat. The single-round assay using vesicular stomatitis virus-pseudotyped virus in the human T cell line M8166 indicated that DHMEQ treatment resulted in decreased integration of HIV-1 provirus into the host genome and decreased HIV-1 expression. These results indicate that NF-κB regulates early events as well as the initial and accelerated expression of HIV-1 in its life cycle. Therefore, we conclude that NF-κB is a molecular target for controlling active HIV-1 replication.     

Immunomodulatory beta-glucan from Lentinus edodes activates mitogen-activated protein kinases and nuclear factor-kappaB in murine RAW 264.7 macrophages

Lentinan, a cell wall β-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.     

Heterotrimeric Gi/Go proteins modulate endothelial TLR signaling independent of the MyD88-dependent pathway

The innate immune recognition of bacterial lipopolysaccharide (LPS) is mediated by Toll-like receptor 4 (TLR4) and results in activation of proinflammatory signaling including NF-κB and MAPK pathways. Heterotrimeric G proteins have been previously implicated in LPS signaling in macrophages and monocytes. In the present study, we show that pertussis toxin sensitive heterotrimeric G proteins (Gα(i/o)) are involved in the activation of MAPK and Akt downstream of TLR2, TLR3, and TLR4 in endothelial cells. Gα(i/o) are also required for full activation of interferon signaling downstream of TLR3 and TLR4 but are not required for the activation of NF-κB. We find that Gα(i/o)-mediated activation of the MAPK is independent of the canonical MyD88, interleukin-1 receptor-associated kinase, and tumor necrosis factor receptor-associated factor 6 signaling cascade in LPS-stimulated cells. Taken together, the data presented here suggest that heterotrimeric G proteins are widely involved in TLR pathways along a signaling cascade that is distinct from MyD88-TRAF6.