Methamphetamine alters T cell cycle entry and progression: role in immune dysfunction.

We and others have demonstrated that stimulants such as methamphetamine (METH) exerts immunosuppressive effects on the host's innate and adaptive immune systems and has profound immunological implications. Evaluation of the mechanisms responsible for T-cell immune dysregulation may lead to ways of regulating immune homeostasis during stimulant use. Here we evaluated the effects of METH on T cell cycle entry and progression following activation. Kinetic analyses of cell cycle progression of T-cell subsets exposed to METH demonstrated protracted G1/S phase transition and differentially regulated genes responsible for cell cycle regulation. This result was supported by in vivo studies where mice exposed to METH had altered G1 cell cycle phase and impaired T-cell proliferation. In addition, T cells subsets exposed to METH had significant decreased expression of cyclin E, CDK2 and transcription factor E2F1 expression. Overall, our results indicate that METH exposure results in altered T cell cycle entry and progression. Our findings suggest that disruption of cell cycle machinery due to METH may limit T-cell proliferation essential for mounting an effective adaptive immune response and thus may strongly contribute to deleterious effect on immune system.

Impaired Subset Progression and Polyfunctionality of T Cells in Mice Exposed to Methamphetamine during Chronic LCMV Infection.

Methamphetamine (METH) is a widely used psychostimulant that severely impacts the host's innate and adaptive immune systems and has profound immunological implications. T cells play a critical role in orchestrating immune responses. We have shown recently how chronic exposure to METH affects T cell activation using a murine model of lymphocytic choriomeningitis virus (LCMV) infection. Using the TriCOM (trinary state combinations) feature of GemStone™ to study the polyfunctionality of T cells, we have analyzed how METH affected the cytokine production pattern over the course of chronic LCMV infection. Furthermore, we have studied in detail the effects of METH on splenic T cell functions, such as cytokine production and degranulation, and how they regulate each other. We used the Probability State Modeling (PSM) program to visualize the differentiation of effector/memory T cell subsets during LCMV infection and analyze the effects of METH on T cell subset progression. We recently demonstrated that METH increased PD-1 expression on T cells during viral infection. In this study, we further analyzed the impact of PD-1 expression on T cell functional markers as well as its expression in the effector/memory subsets. Overall, our study indicates that analyzing polyfunctionality of T cells can provide additional insight into T cell effector functions. Analysis of T cell heterogeneity is important to highlight changes in the evolution of memory/effector functions during chronic viral infections. Our study also highlights the impact of METH on PD-1 expression and its consequences on T cell responses.

Methamphetamine alters microglial immune function through P2X7R signaling.

BACKGROUND: Purinoceptors have emerged as mediators of chronic inflammation and neurodegenerative processes. The ionotropic purinoceptor P2X7 (P2X7R) is known to modulate proinflammatory signaling and integrate neuronal-glial circuits. Evidence of P2X7R involvement in neurodegeneration, chronic pain, and chronic inflammation suggests that purinergic signaling plays a major role in microglial activation during neuroinflammation. In this study, we investigated the effects of methamphetamine (METH) on microglial P2X7R. METHODS: ESdMs were used to evaluate changes in METH-induced P2X7R gene expression via Taqman PCR and protein expression via western blot analysis. Migration and phagocytosis assays were used to evaluate functional changes in ESdMs in response to METH treatment. METH-induced proinflammatory cytokine production following siRNA silencing of P2X7R in ESdMs measured P2X7R-dependent functional changes. In vivo expression of P2X7R and tyrosine hydroxylase (TH) was visualized in an escalating METH dose mouse model via immunohistochemical analysis. RESULTS: Stimulation of ESdMs with METH for 48 h significantly increased P2X7R mRNA (*p < 0.0336) and protein expression (*p < 0.022). Further analysis of P2X7R protein in cellular fractionations revealed increases in membrane P2X7R (*p < 0.05) but decreased cytoplasmic expression after 48 h METH treatment, suggesting protein mobilization from the cytoplasm to the membrane which occurs upon microglial stimulation with METH. Forty-eight hour METH treatment increased microglial migration towards Fractalkine (CX3CL1) compared to control (****p < 0.0001). Migration toward CX3CL1 was confirmed to be P2X7R-dependent through the use of A 438079, a P2X7R-competitive antagonist, which reversed the METH effects (****p < 0.0001). Similarly, 48 h METH treatment increased microglial phagocytosis compared to control (****p < 0.0001), and pretreatment of P2X7R antagonist reduced METH-induced phagocytosis (****p < 0.0001). Silencing the microglial P2X7R decreased TNF-α (*p < 0.0363) and IL-10 production after 48 h of METH treatment. Additionally, our studies demonstrate increased P2X7R and decreased TH expression in the striata of escalating dose METH animal model compared to controls. CONCLUSIONS: This study sheds new light on the functional role of P2X7R in the regulation of microglial effector functions during substance abuse. Our findings suggest that P2X7R plays an important role in METH-induced microglial activation responses. P2X7R antagonists may thus constitute a novel target of therapeutic utility in neuroinflammatory conditions by regulating pathologically activated glial cells in stimulant abuse.

Methamphetamine induces trace amine-associated receptor 1 (TAAR1) expression in human T lymphocytes: role in immunomodulation.

The novel transmembrane G protein-coupled receptor, trace amine-associated receptor 1 (TAAR1), represents a potential, direct target for drugs of abuse and monoaminergic compounds, including amphetamines. For the first time, our studies have illustrated that there is an induction of TAAR1 mRNA expression in resting T lymphocytes in response to methamphetamine. Methamphetamine treatment for 6 h significantly increased TAAR1 mRNA expression (P < 0.001) and protein expression (P < 0.01) at 24 h. With the use of TAAR1 gene silencing, we demonstrate that methamphetamine-induced cAMP, a classic response to methamphetamine stimulation, is regulated via TAAR1. We also show by TAAR1 knockdown that the down-regulation of IL-2 in T cells by methamphetamine, which we reported earlier, is indeed regulated by TAAR1. Our results also show the presence of TAAR1 in human lymph nodes from HIV-1-infected patients, with or without a history of methamphetamine abuse. TAAR1 expression on lymphocytes was largely in the paracortical lymphoid area of the lymph nodes with enhanced expression in lymph nodes of HIV-1-infected methamphetamine abusers rather than infected-only subjects. In vitro analysis of HIV-1 infection of human PBMCs revealed increased TAAR1 expression in the presence of methamphetamine. In summary, the ability of methamphetamine to activate trace TAAR1 in vitro and to regulate important T cell functions, such as cAMP activation and IL-2 production; the expression of TAAR1 in T lymphocytes in peripheral lymphoid organs, such as lymph nodes; and our in vitro HIV-1 infection model in PBMCs suggests that TAAR1 may play an important role in methamphetamine -mediated immune-modulatory responses.

Methamphetamine mediates immune dysregulation in a murine model of chronic viral infection.

Methamphetamine (METH) is a highly addictive psychostimulant that not only affects the brain and cognitive functions but also greatly impacts the host immune system, rendering the body susceptible to infections and exacerbating the severity of disease. Although there is gathering evidence about METH abuse and increased incidence of HIV and other viral infections, not much is known about the effects on the immune system in a chronic viral infection setting. We have used the lymphocytic choriomeningitis virus (LCMV) chronic mouse model of viral infection in a chronic METH environment and demonstrate that METH significantly increases CD3 marker on splenocytes and programmed death-1 (PD-1) expression on T cells, a cell surface signaling molecule known to inhibit T cell function and cause exhaustion in a lymphoid organ. Many of these METH effects were more pronounced during early stage of infection, which are gradually attenuated during later stages of infection. An essential cytokine for T-lymphocyte homeostasis, Interleukin-2 (IL-2) in serum was prominently reduced in METH-exposed infected mice. In addition, the serum pro-inflammatory (TNF, IL12 p70, IL1ß, IL-6, and KC-GRO) and Th2 (IL-2, IL-10, and IL-4) cytokine profiles were also altered in the presence of METH. Interestingly CXCR3, an inflammatory chemokine receptor, showed significant increase in the METH treated LCMV infected mice. Similarly, compared to only infected mice, epidermal growth factor receptor (EGFR) in METH exposed LCMV infected mice were up regulated. Collectively, our data suggest that METH alters systemic, peripheral immune responses and modulates key markers on T cells involved in pathogenesis of chronic viral infection.

Enhanced aggressiveness of benzopyrene-induced squamous carcinomas in transgenic mice overexpressing the proprotein convertase PACE4 (PCSK6).

PACE4 (PCSK6) is a proprotein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco-associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5-PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF-1R autophosphorylation. Squamous cell carcinomas, obtained from K5-PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50% increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5-PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression.

P2X4 receptor regulates alcohol-induced responses in microglia.

Mounting evidence indicates that alcohol-induced neuropathology may result from multicellular responses in which microglia cells play a prominent role. Purinergic receptor signaling plays a key role in regulating microglial function and, more importantly, mediates alcohol-induced effects. Our findings demonstrate that alcohol increases expression of P2X4 receptor (P2X4R), which alters the function of microglia, including calcium mobilization, migration and phagocytosis. Our results show a significant up-regulation of P2X4 gene expression as analyzed by real-time qPCR (***p < 0.002) and protein expression as analyzed by flow cytometry (**p < 0.004) in embryonic stem cell-derived microglial cells (ESdM) after 48 hours of alcohol treatment, as compared to untreated controls. Calcium mobilization in ethanol treated ESdM cells was found to be P2X4R dependent using 5-BDBD, a P2X4R selective antagonist. Alcohol decreased migration of microglia towards fractalkine (CX3CL1) by 75 % following 48 h of treatment compared to control (***p < 0.001). CX3CL1-dependent migration was confirmed to be P2X4 receptor-dependent using the antagonist 5-BDBD, which reversed the effects as compared to alcohol alone (***p < 0.001). Similarly, 48 h of alcohol treatment significantly decreased phagocytosis of microglia by 15 % compared to control (*p < 0.05). 5-BDBD pre-treatment prior to alcohol treatment significantly increased microglial phagocytosis (***p < 0.001). Blocking P2X4R signaling with 5-BDBD decreased the level of calcium mobilization compared to ethanol treatment alone. These findings demonstrate that P2X4 receptor may play a role in modulating microglial function in the context of alcohol abuse.

Proprotein convertase inhibition results in decreased skin cell proliferation, tumorigenesis, and metastasis.

PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine kinase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.

Methamphetamine causes mitrochondrial oxidative damage in human T lymphocytes leading to functional impairment.

Methamphetamine (METH) abuse is known to be associated with an inordinate rate of infections. Although many studies have described the association of METH exposure and immunosuppression, so far the underlying mechanism still remains elusive. In this study, we present evidence that METH exposure resulted in mitochondrial oxidative damage and caused dysfunction of primary human T cells. METH treatment of T lymphocytes led to a rise in intracellular calcium levels that enhanced the generation of reactive oxygen species. TCR-CD28 linked calcium mobilization and subsequent uptake by mitochondria in METH-treated T cells correlated with an increase in mitochondrion-derived superoxide. Exposure to METH-induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, increased mitochondrial mass, enhanced protein nitrosylation and diminished protein levels of complexes I, III, and IV of the electron transport chain. These changes paralleled reduced IL-2 secretion and T cell proliferative responses after TCR-CD28 stimulation indicating impaired T cell function. Furthermore, antioxidants attenuated METH-induced mitochondrial damage by preserving the protein levels of mitochondrial complexes I, III, and IV. Altogether, our data indicate that METH can cause T cell dysfunction via induction of oxidative stress and mitochondrial injury as underlying mechanism of immune impairment secondary to METH abuse.

Replication-deficient rabies virus-based vaccines are safe and immunogenic in mice and nonhuman primates.

Although current postexposure prophylaxis rabies virus (RV) vaccines are effective, approximately 40,000-70,000 rabies-related deaths are reported annually worldwide. The development of effective formulations requiring only 1-2 applications would significantly reduce mortality. We assessed in mice and nonhuman primates the efficacy of replication-deficient RV vaccine vectors that lack either the matrix (M) or phosphoprotein (P) gene. A single dose of M gene-deficient RV induced a more rapid and efficient anti-RV response than did P gene-deficient RV immunization. Furthermore, the M gene-deleted RV vaccine induced 4-fold higher virus-neutralizing antibody (VNA) levels in rhesus macaques than did a commercial vaccine within 10 days after inoculation, and at 180 days after immunization rhesus macaques remained healthy and had higher-avidity antibodies, higher VNA titers, and a more potent antibody response typical of a type 1 T helper response than did animals immunized with a commercial vaccine. The data presented in this article suggest that the M gene-deleted RV vaccine is safe and effective and holds the potential of replacing current pre- and postexposure RV vaccines.

Immune modulating effect by a phosphoprotein-deleted rabies virus vaccine vector expressing two copies of the rabies virus glycoprotein gene.

The type of immune response induced by a vaccine is a critical factor that determines its effectiveness in preventing infection or disease. Inactivated and live rabies virus (RV) vaccine strains elicit an IgG1-biased and IgG1/IgG2a-balanced antibody response, respectively. However, IgG2a antibodies are potent inducers of anti-viral effector functions, and therefore, a viral vaccine vector that can elicit an IgG2a-biased antibody response may be more effective against RV infection. Here we describe the humoral immune response of a live replication-deficient phosphoprotein (P)-deleted RV vector (SPBN-DeltaP), or a recombinant P-deleted virus that expresses two copies of the RV glycoprotein (G) gene (SPBN-DeltaP-RVG), and compare it to a UV-inactivated RV. Mice inoculated with UV-inactivated RV induced predominantly an IgG1-specific antibody response, while live recombinant SPBN-DeltaP exhibited a mixed IgG1/IgG2a antibody response, which is consistent with the isotype profiles from the replication-competent parental viruses. Survivorship in mice after pathogenic RV challenge indicates a 10-fold higher efficiency of live SPBN-DeltaP compared to UV-inactivated SPBN-DeltaP. In addition, SPBN-DeltaP-RVG induced a more rapid and robust IgG2a response that protected mice more effectively than SPBN-DeltaP. Of note, 10(3)ffu of SPBN-DeltaP-RVG-induced anti-RV antibodies that were 100% protective in mice against pathogenic RV challenge. The increased immune response was directed not only against RV G but also against the ribonucleoprotein (RNP), indicating that the expression of two RV G genes from SPBN-DeltaP-RVG enhances the immune response to other RV antigens as well. In addition, Rag2 mice inoculated intramuscularly with 10(5)ffu/mouse of SPBN-DeltaP showed no clinical signs of rabies, and no viral RNA was detected in the spinal cord or brain of inoculated mice. Therefore, the safety of the P-deleted vectors along with the onset and magnitude of the IgG2a-induced immune response by SPBN-DeltaP-RVG indicate that this vector holds great promise as either a therapeutic or preventative vaccine against RV or other infectious diseases.

PACE4 expression in mouse basal keratinocytes results in basement membrane disruption and acceleration of tumor progression.

Collagen type IV degradation results in disruption and breakdown of the normal basement membrane architecture, a key process in the initiation of tumor microinvasion into the connective tissue. PACE4, a proprotein convertase, activates membrane type matrix metalloproteinases (MT-MMPs) that in turn process collagenase type IV. Because PACE4 is overexpressed in skin carcinomas and in vitro overexpression of PACE4 resulted in enhanced invasiveness, we investigated whether or not in vivo PACE4 expression leads to the acquisition of invasiveness and increased tumorigenesis. Two transgenic mouse lines were designed by targeting PACE4 to the epidermal basal keratinocytes. Transgenic keratinocytes showed increased processing of MT1-MMP and MT2-MMP resulting in collagenase IV activation and collagen type IV degradation. Higher collagenolytic activity partially disrupted normal basement membrane architecture favoring epithelial endophytic growth into the dermis and accelerating invasion and metastasis after chemical carcinogenesis. PACE4 overexpression resulted in enhanced susceptibility to carcinogenesis and tumor progression pointing to a new target for blocking tumor cell invasiveness.

A murine model of ex vivo angiogenesis using aortic disks grown in fibrin clot.

The rat aortic ring model is well utilized for evaluation of angiogenesis. We report here an alternative assay employing an ex vivo mouse aorta angiogenesis model that can be extensively manipulated and serially evaluated using digital-assisted image analysis. Mouse aortas were harvested, cut into 2-mm disks, and cultured in fibrin matrix with growth media. Radial vascular outgrowths arose from the cut edge of the aortic disk and were digitally photographed and serially quantified. A variety of culture conditions were evaluated to determine their ability to alter angiogenesis in this model. Vessel outgrowth became apparent on day 3 and continued through day 10 with linear growth occurring between days 3 and 6. Increasing concentrations of serum from 0% to 40% resulted in stimulation of angiogenesis after day 3. Suramin and endostatin dramatically inhibited angiogenesis, which was more profound when applied at day 0 than when linear growth could be identified (day 3). Cells isolated from vessel outgrowths were predominantly endothelial in origin by immunocytochemistry and FACS analysis. We demonstrate that angiogenesis in an ex vivo murine model can be easily quantified using digital image analysis, responds appropriately to stimulation and inhibition, and exhibits differential results based on time of inhibitor administration. Antiangiogenic agents may be most effective if administered before development of accelerated vessel growth.