Humoral Dysregulation Associated with Increased Systemic Inflammation among Injection Heroin Users

Injection drug use is a growing major public health concern. Injection drug users (IDUs) have a higher incidence of co-morbidities including HIV, Hepatitis, and other infections. An effective humoral response is critical for optimal homeostasis and protection from infection; however, the impact of injection heroin use on humoral immunity is poorly understood. We hypothesized that IDUs have altered B cell and antibody profiles.A comprehensive systems biology-based cross-sectional assessment of 130 peripheral blood B cell flow cytometry- and plasma- based features was performed on HIV-/Hepatitis C-, active heroin IDUs who participated in a syringe exchange program (n = 19) and healthy control subjects (n = 19). The IDU group had substantial polydrug use, with 89% reporting cocaine injection within the preceding month. IDUs exhibited a significant, 2-fold increase in total B cells compared to healthy subjects, which was associated with increased activated B cell subsets. Although plasma total IgG titers were similar between groups, IDUs had significantly higher IgG3 and IgG4, suggestive of chronic B cell activation. Total IgM was also increased in IDUs, as well as HIV Envelope-specific IgM, suggestive of increased HIV exposure. IDUs exhibited numerous features suggestive of systemic inflammation, including significantly increased plasma sCD40L, TNF-α, TGF-α, IL-8, and ceramide metabolites. Machine learning multivariate analysis distilled a set of 10 features that classified samples based on group with absolute accuracy.These results demonstrate broad alterations in the steady-state humoral profile of IDUs that are associated with increased systemic inflammation. Such dysregulation may impact the ability of IDUs to generate optimal responses to vaccination and infection, or lead to increased risk for inflammation-related co-morbidities, and should be considered when developing immune-based interventions for this growing population

Ibudilast, a Pharmacologic Phosphodiesterase Inhibitor, Prevents Human Immunodeficiency Virus-1 Tat-Mediated Activation of Microglial Cells

Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorders (HAND) occur, in part, due to the inflammatory response to viral proteins, such as the HIV-1 transactivator of transcription (Tat), in the central nervous system (CNS). Given the need for novel adjunctive therapies for HAND, we hypothesized that ibudilast would inhibit Tat-induced excess production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFα) in microglial cells. Ibudilast is a non-selective cyclic AMP phosphodiesterase inhibitor that has recently shown promise as a treatment for neuropathic pain via its ability to attenuate glial cell activation. Accordingly, here we demonstrate that pre-treatment of both human and mouse microglial cells with increasing doses of ibudilast inhibited Tat-induced synthesis of TNFα by microglial cells in a manner dependent on serine/threonine protein phosphatase activity. Ibudilast had no effect on Tat-induced p38 MAP kinase activation, and blockade of adenosine A2A receptor activation did not reverse ibudilast's inhibition of Tat-induced TNFα production. Interestingly, ibudilast reduced Tat-mediated transcription of TNFα, via modulation of nuclear factor-kappa B (NF-κB) signaling, as shown by transcriptional activity of NF-κB and analysis of inhibitor of kappa B alpha (IκBα) stability. Together, our findings shed light on the mechanism of ibudilast's inhibition of Tat-induced TNFα production in microglial cells and may implicate ibudilast as a potential novel adjunctive therapy for the management of HAND

Nuclear Factor-Kappa B Family Member RelB Inhibits Human Immunodeficiency Virus-1 Tat-Induced Tumor Necrosis Factor-Alpha Production

Human Immunodeficiency Virus-1 (HIV-1)-associated neurocognitive disorder (HAND) is likely neuroinflammatory in origin, believed to be triggered by inflammatory and oxidative stress responses to cytokines and HIV protein gene products such as the HIV transactivator of transcription (Tat). Here we demonstrate increased messenger RNA for nuclear factor-kappa B (NF-κB) family member, transcription factor RelB, in the brain of doxycycline-induced Tat transgenic mice, and increased RelB synthesis in Tat-exposed microglial cells. Since genetic ablation of RelB in mice leads to multi-organ inflammation, we hypothesized that Tat-induced, newly synthesized RelB inhibits cytokine production by microglial cells, possibly through the formation of transcriptionally inactive RelB/RelA complexes. Indeed, tumor necrosis factor-alpha (TNFα) production in monocytes isolated from RelB deficient mice was significantly higher than in monocytes isolated from RelB expressing controls. Moreover, RelB overexpression in microglial cells inhibited Tat-induced TNFα synthesis in a manner that involved transcriptional repression of the TNFα promoter, and increased phosphorylation of RelA at serine 276, a prerequisite for increased RelB/RelA protein interactions. The Rel-homology-domain within RelB was necessary for this interaction. Overexpression of RelA itself, in turn, significantly increased TNFα promoter activity, an effect that was completely blocked by RelB overexpression. We conclude that RelB regulates TNFα cytokine synthesis by competitive interference binding with RelA, which leads to downregulation of TNFα production. Moreover, because Tat activates both RelB and TNFα in microglia, and because Tat induces inflammatory TNFα synthesis via NF-κB, we posit that RelB serves as a cryoprotective, anti-inflammatory, counter-regulatory mechanism for pathogenic NF-κB activation. These findings identify a novel regulatory pathway for controlling HIV-induced microglial activation and cytokine production that may have important therapeutic implications for the management of HAND

Targeting HIV-1 Associated Neurocognitive Disorder (HAND): Endogenous and Exogenous Anti-Inflammatory Mechanisms

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2010.Neurological complications of Human Immunodeficiency Virus-1 (HIV-1) infection of the central nervous system (CNS) persist despite effective control of viral replication with combined antiretroviral therapy (cART). Collectively, these neurological complications are termed HIV-1-Associated Neurocognitive Disorder (HAND). HIV-1-induced neuroinflammation is caused by both viral products and host-derived pro-inflammatory mediators released in the CNS, neither of which are inhibited by cART. Considering this, the discovery of new anti-inflammatory mechanisms, both endogenous and exogenous, remains an important endeavor. In order to examine a potential anti-inflammatory role of nuclear factor-kappa B (NF-B) family member, transcription factor RelB, in the context of HIV-1-induced neuroinflammation, we measured RelB transcript levels in the brain of HIV-1 Tat transgenic mice as well as RelB protein levels in Tat exposed microglial cells. Our results show that there is increased RelB messenger RNA and protein in response to Tat. Additionally, RelB appears to inhibit tumor necrosis factor-alpha (TNF) production in a manner involving transcriptional repression of the TNF promoter via an inhibitory interaction between RelB and another NF-B protein, RelA. The Rel-homology domain within RelB is necessary for this interaction. Our data suggests that RelB regulates TNF cytokine synthesis by competitive interference binding with RelA, which leads to the downregulation of TNF production. The results shown here identify RelB as part of a novel regulatory pathway for controlling HIV-1-induced microglial activation and cytokine production. In an alternate approach, we examined the anti-inflammatory potential of ibudilast, a pharmacological phosphodiesterase inhibitor, in the context of HIV-1-induced microglial activation. Ibudilast is a non-selective cyclic AMP phosphodiesterase inhibitor that has recently shown promise as a treatment for other neuroinflammatory disorders through its ability to attenuate glial cell activation. We demonstrated that ibudilast inhibits Tat-induced pro-inflammatory cytokine and chemokine production in primary human microglial cells. Our results indicate that ibudilast’s abrogation of Tat-induced TNF production involves serine/threonine protein phosphatase activity. Additionally, ibudilast inhibits Tat-mediated transcription of TNF via modulation of NF-B signaling, as shown by analysis of the transcriptional activity of NF-B and of inhibitor of kappa B-alpha (IB) stability. Our results suggest that ibudilast may have potential as a novel adjunctive therapy for the management of HIV-1-induced neuroinflammation. The results presented in this thesis provide insight into potential therapies for the treatment of HAND, through the elucidation of new therapeutic targets involving NF-B signal transduction. Overall, our data further emphasizes the multiple complex functions of the NF-B transcription factor family, and identifies novel inhibitory mechanisms against HIV-1-mediated microglial activation and pro-inflammatory cytokine production, both of which significantly contribute to HIV-1-induced neuroinflammation

Inhibition of Tat-induced TNFα production by ibudilast is not reversed by blockade of adenosine A_2A receptor.

<p><i>A</i>, BV-2 cells (1.5×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with either anti-Tat or control IgG antibodies (8 µg/mL) or adenosine A_2A receptor agonist, CGS21680 (CGS, 1 µM) or antagonist, ZM241385 (ZM, 100 nM), as indicated. TNFα release was measured by ELISA. The Tat-treated samples were set to 100% and all other samples were compared to this value (the TNFα concentration for this sample was 3475 pg/µg total protein content). Results are shown as mean ± SEM of values derived from four replicates from a single representative experiment; two total experiments were performed. Statistical significance (p<0.001) is indicated, as compared to Tat-treated cells (***) or as compared to Tat+CGS-treated cells (###). <i>B</i>, BV-2 cells (1.2×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with ibudilast (Ib, 50 µM) or vehicle (Veh) alone or together with increasing concentrations of the adenosine A_2A receptor antagonist, ZM241385, as indicated. TNFα release was measured by ELISA. The Tat+Veh-treated samples were set to 100% and all other samples were compared to this value (the TNFα concentration for this sample was 696.2 pg/mL). Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. Statistical significance is indicated, as compared to Tat+Veh-treated cells (p<0.05, *) or as compared to Tat+ZM241385-treated cells (p<0.001, ###).</p

Ibudilast inhibits Tat-induced TNFα production via modulation of NF-κB signaling.

<p><i>A</i>, BV-2 cells (10×10⁶) were transiently transfected using Nucleofector (Amaxa/Lonza) with an NF-κB-dependent luciferase reporter plasmid. 16 h post-transfection, cells were left untreated or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with 50 µM ibudilast (Ib) or vehicle (Veh). Luciferase activity in whole cell lysates was determined. Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. Statistical significance is denoted (***, p<0.001) as compared to NT samples or (###, p<0.001) as compared to Tat-treated samples. A parallel transfection with a luciferase reporter containing responsive elements of the OCT-1 transcription factor upstream of a firefly luciferase gene was included as a control for the specificity of ibudilast's inhibition of NF-κB transcriptional activity. <i>B</i>, BV-2 cells (10×10⁶) were transiently transfected using Nucleofector (Amaxa/Lonza) with an NF-κB-dependent luciferase reporter plasmid alone or together with either a RelA-encoding plasmid or a vector control plasmid. 16 h post-transfection, cells were left untreated or were treated with 50 µM ibudilast (Ib) or vehicle (Veh) for 8 h. Luciferase activity in whole cell lysates was determined. Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. Co-transfection with a <i>Renilla</i> luciferase reporter plasmid was included as a control for transfection efficiency. For each sample the firefly luciferase reading was divided by the <i>Renilla</i> luciferase reading. C, BV-2 cells (10×10⁶) were transiently transfected using Nucleofector (Amaxa/Lonza) with a plasmid containing a luciferase reporter gene under transcriptional control of the mouse TNFα promoter region, alone or together with a RelA-encoding plasmid. 16 h post-transfection, cells were left untreated or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with 50 µM ibudilast (Ib) or vehicle (Veh). Luciferase activity in whole cell lysates was determined. Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. A parallel transfection with the OCT-1 luciferase reporter was included as a control for the specificity of ibudilast's inhibition of TNFα promoter activation. <i>D</i>, BV-2 cells (1.2×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 15 min. with or without pre-treatment for 30 min. with increasing concentrations of ibudilast or MG-132, as indicated. Whole cell lysates were subjected to immunoblot analysis using either IκBα-specific <i>(upper panel)</i> or α-Tubulin-specific <i>(lower panel)</i> antibodies. The results of a single representative experiment are shown; three total experiments were performed. <i>E</i>, BV-2 cells (1.2×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for the indicated periods of time with or without pre-treatment for 30 min. with increasing concentrations of ibudilast, as indicated. Whole cell lysates were subjected to immunoblot analysis using RelA phospho-serine 276-specific <i>(first panel)</i>, RelA phospho-serine 536-specific <i>(second panel)</i>, total RelA-specific <i>(third panel)</i>, or α-Tubulin-specific <i>(fourth panel)</i> antibodies. The results of a single representative experiment are shown; two total experiments were performed.</p

Ibudilast dose-dependently inhibits Tat-induced pro-inflammatory cytokine production in microglial cells.

<p><i>A</i>, Human microglial cells (1×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast or vehicle (Veh), as indicated. TNFα, IL-1β, IL-6, and MCP-1 levels in culture supernatants were analyzed by Multi-Plex cytokine array, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018633#s2" target="_blank">Methods</a>. The Tat+Veh-treated samples were set to 100% and all other samples were compared to this value. Results are shown as mean ± SD of values derived from two replicates from a single representative experiment. Statistical significance (***, p<0.001; *, p<0.05) is indicated, as compared to Tat+Veh-treated cells. The average cytokine/chemokine concentration of the Tat+Veh-treated sample was as follows; TNFα: 8166 pg/mL, IL-1β: 3.3 pg/mL, IL-6: 6027 pg/mL, and MCP-1: 1480 pg/mL. <i>B</i>, Similarly, murine microglial cells (BV-2; 1.2×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast or vehicle (Veh), as indicated. TNFα release was measured by ELISA. Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; three total experiments were performed. Statistical significance (p<0.001) is indicated, as compared to Tat+Veh-treated cells (***). The TNFα concentration of the Tat+Veh-treated sample was 1861 pg/mL. <i>C</i>, BV-2 cells (1.2×10⁵) were left untreated (NT) or were treated with increasing concentrations of ibudilast or vehicle (Veh), as indicated, for 8 h. The MTT assay was used as a measure of cell viability. Percent survival was calculated as compared to the untreated sample. Results are shown as mean ± SEM of values derived from four replicates from a single representative experiment; two total experiments were performed.</p

Ibudilast inhibits Tat-induced TNFα transcript levels.

<p>BV-2 cells (2.5×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 4 h with or without pre-treatment for 30 min. with increasing concentrations of ibudilast (Ib) or vehicle (Veh). Total RNA was collected, reverse transcribed using oligo-dT primers, and subjected to Real-Time SYBR Green RT-PCR amplification. Fold induction of TNFα mRNA species was normalized to those of GAPDH and presented as a function of the expression level in NT samples. Data represent mean ± SEM of values derived from three replicates from a single representative experiment. Statistical significance (***, p<0.001 or *, p<0.05) is denoted as compared to Tat-treated samples.</p

Ibudilast inhibits Tat-induced TNFα production in a protein phosphatase dependent manner.

<p><i>A</i>, BV-2 cells (1.2×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 8 h with or without pre-treatment for 30 min. with ibudilast (Ib, 50 µM) or vehicle (Veh) alone or together with the protein phosphatase inhibitor, okadaic acid (Oka, 50 nM). TNFα release was measured by ELISA. The Tat+Veh-treated samples were set to 100% and all other samples were compared to this value (the TNFα concentration for this sample was 1861 pg/mL). Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. Statistical significance is indicated, as compared to Tat+Veh-treated cells (p<0.001, ***) or as compared to Tat+Ib-treated cells (p<0.01, ##). <i>B</i>, Serine/Threonine phosphatase activity was measured in whole cell lysates from BV-2 cells (4.6×10⁵) treated with ibudilast (50 µM) for the indicated periods of time. Results are shown as mean ± SEM of values derived from three replicates from a single representative experiment; two total experiments were performed. Statistical significance (p<0.001) is indicated, ***. <i>C</i>, BV-2 cells (1.2×10⁵) were left untreated (NT) or were treated with ibudilast (50 µM) for 8 h or 24 h, as indicated. Whole cell lysates were subjected to immunoblot analysis using either PP2Ac-specific <i>(upper panel)</i> or α-Tubulin-specific <i>(lower panel)</i> antibodies. The results of a single representative experiment are shown; two total experiments were performed.</p

Ibudilast does not inhibit Tat-induced activation of p38 MAP kinase.

<p><i>A</i>, BV-2 cells (1.2×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 15 min. with or without pre-treatment for 5 min. with increasing concentrations of ibudilast, as indicated. Whole cell lysates were subjected to immunoblot analysis using either phospho-p38 MAPK-specific <i>(upper panel)</i> or p38 MAPK-specific <i>(lower panel)</i> antibodies. The results of a single representative experiment are shown; three total experiments were performed. <i>B</i>, BV-2 cells (2.4×10⁵) were left untreated (NT) or were treated with Tat (100 nM) for 20 min. with or without pre-treatment for 10 min. with increasing concentrations of ibudilast or the p38 MAP kinase inhibitor, SB203580, as indicated. Whole cell lysates were subjected to immunoprecipitation with an immobilized phospho-p38 MAP kinase antibody. Precipitated p38 was incubated with an ATF-2 fusion protein substrate. Phosphorylated ATF-2 levels were determined by immunoblot analysis with a phospho-ATF-2-specific antibody (<i>top panel</i>). Whole cell lysate supernatants collected after immunoprecipitation were subjected to immunoblot analysis using p38-specific (<i>middle panel</i>) or α-Tubulin-specific (<i>lower panel</i>) antibodies. Protein levels were quantified using ImageJ software <i>(bottom graph)</i>. Phospho-ATF-2 levels were normalized to total p38 MAPK levels and fold change compared to NT was calculated. The results of a single representative experiment are shown; two total experiments were performed.</p