Venkatesh L. Hegde

Resveratrol (trans-3,5,4′-Trihydroxystilbene) Ameliorates Experimental Allergic Encephalomyelitis, Primarily via Induction of Apoptosis in T Cells Involving Activation of Aryl Hydrocarbon Receptor and Estrogen Receptor

Resveratrol (trans-3,5,4′-trihydroxystilbene), a polyphenolic compound found in plant products, including red grapes, exhibits anticancer, antioxidant, and anti-inflammatory properties. Using an animal model of multiple sclerosis (MS), we investigated the use of resveratrol for the treatment of autoimmune diseases. We observed that resveratrol treatment decreased the clinical symptoms and inflammatory responses in experimental allergic encephalomyelitis (EAE)-induced mice. Furthermore, we observed significant apoptosis in inflammatory cells in spinal cord of EAE-induced mice treated with resveratrol compared with the control mice. Resveratrol administration also led to significant down-regulation of certain cytokines and chemokines in EAE-induced mice including tumor necrosis factor-α, interferon-γ, interleukin (IL)-2, IL-9, IL-12, IL-17, macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T-cell expressed and secreted (RANTES), and Eotaxin. In vitro studies on the mechanism of action revealed that resveratrol triggered high levels of apoptosis in activated T cells and to a lesser extent in unactivated T cells. Moreover, resveratrol-induced apoptosis was mediated through activation of aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) and correlated with up-regulation of AhR, Fas, and FasL expression. In addition, resveratrol-induced apoptosis in primary T cells correlated with cleavage of caspase-8, caspase-9, caspase-3, poly(ADP-ribose) polymerase, and release of cytochrome c. Data from the present study demonstrate, for the first time, the ability of resveratrol to trigger apoptosis in activated T cells and its potential use in the treatment of inflammatory and autoimmune diseases including, MS.

Cannabinoids as novel anti-inflammatory drugs

Cannabinoids are a group of compounds that mediate their effects through cannabinoid receptors. The discovery of Δ9-tetrahydrocannabinol (THC) as the major psychoactive principle in marijuana, as well as the identification of cannabinoid receptors and their endogenous ligands, has led to a significant growth in research aimed at understanding the physiological functions of cannabinoids. Cannabinoid receptors include CB1, which is predominantly expressed in the brain, and CB2, which is primarily found on the cells of the immune system. The fact that both CB1 and CB2 receptors have been found on immune cells suggests that cannabinoids play an important role in the regulation of the immune system. Recent studies demonstrated that administration of THC into mice triggered marked apoptosis in T cells and dendritic cells, resulting in immunosuppression. In addition, several studies showed that cannabinoids downregulate cytokine and chemokine production and, in some models, upregulate T-regulatory cells (Tregs) as a mechanism to suppress inflammatory responses. The endocannabinoid system is also involved in immunoregulation. For example, administration of endocannabinoids or use of inhibitors of enzymes that break down the endocannabinoids, led to immunosuppression and recovery from immune-mediated injury to organs such as the liver. Manipulation of endocannabinoids and/or use of exogenous cannabinoids in vivo can constitute a potent treatment modality against inflammatory disorders. This review will focus on the potential use of cannabinoids as a new class of anti-inflammatory agents against a number of inflammatory and autoimmune diseases that are primarily triggered by activated T cells or other cellular immune components.

Attenuation of Experimental Autoimmune Hepatitis by Exogenous and Endogenous Cannabinoids: Involvement of Regulatory T Cells

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. Natural cannabinoids such as Δ9-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases. We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury. Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3+ T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC. The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels. In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation.

Treatment of Mice with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Leads to Aryl Hydrocarbon Receptor-Dependent Nuclear Translocation of NF-! B and Expression of Fas Ligand in Thymic Stromal Cells and Consequent Apoptosis in T Cells 1

We investigated the role of aryl hydrocarbon receptor (AhR) in the regulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced apoptosis in thymic T cells. AhR knockout (KO) mice were resistant to TCDD-induced thymic atrophy and apoptosis when compared with the AhR wild-type mice. TCDD triggered the expression of several apoptotic genes, including FasL in AhR wild-type but not AhRKO mice. TCDD-induced increase in FasL was seen only in thymic stromal but not thymic T cells. When TCDD-exposed stromal cells were mixed with untreated thymic T cells, increased apoptosis was detected in T cells that involved Fas-FasL interactions. Thus, apoptosis in T cells was not detected when TCDD-treated stromal cells from FasL-defective or AhRKO mice were mixed with wild-type T cells or when TCDD-exposed wild-type stromal cells were mixed with Fas-deficient T cells. TCDD treatment, in vivo and in vitro, led to colocalization and translocation of NF-κB subunits (p50, p65) to the nucleus in stromal but not T cells from AhR wild-type mice. NF-κB activation was not observed in stromal cells isolated from TCDD-treated AhRKO mice. Mutations in NF-κB-binding sites on the FasL promoter showed that TCDD regulates FasL promoter activity through NF-κB. TCDD treatment in vivo caused activation of the death receptor and mitochondrial pathways of apoptosis. Cross-talk between the two pathways was not necessary for apoptosis inasmuch as TCDD-treated Bid KO mice showed thymic atrophy and increased apoptosis, similar to the wild-type mice. These findings demonstrate that AhR regulates FasL and NF-κB in stromal cells, which in turn plays a critical role in initiating apoptosis in thymic T cells.

Δ9-Tetrahydrocannabinol-Induced Apoptosis in Jurkat Leukemia T Cells Is Regulated by Translocation of Bad to Mitochondria

Plant-derived cannabinoids, including Δ9-tetrahydrocannabinol (THC), induce apoptosis in leukemic cells, although the precise mechanism remains unclear. In the current study, we investigated the effect of THC on the upstream and downstream events that modulate the extracellular signal-regulated kinase (ERK) module of mitogen-activated protein kinase pathways primarily in human Jurkat leukemia T cells. The data showed that THC down-regulated Raf-1/mitogen-activated protein kinase/ERK kinase (MEK)/ERK/RSK pathway leading to translocation of Bad to mitochondria. THC also decreased the phosphorylation of Akt. However, no significant association of Bad translocation with phosphatidylinositol 3-kinase/Akt and protein kinase A signaling pathways was noted when treated cells were examined in relation to phosphorylation status of Bad by Western blot and localization of Bad to mitochondria by confocal analysis. Furthermore, THC treatment decreased the Bad phosphorylation at Ser112 but failed to alter the level of phospho-Bad on site Ser136 that has been reported to be associated with phosphatidylinositol 3-kinase/Akt signal pathway. Jurkat cells expressing a constitutively active MEK construct were found to be resistant to THC-mediated apoptosis and failed to exhibit decreased phospho-Bad on Ser112 as well as Bad translocation to mitochondria. Finally, use of Bad small interfering RNA reduced the expression of Bad in Jurkat cells leading to increased resistance to THC-mediated apoptosis. Together, these data suggested that Raf-1/MEK/ERK/RSK-mediated Bad translocation played a critical role in THC-induced apoptosis in Jurkat cells. (Mol Cancer Res 2006;4(8):549–62)

Perinatal Exposure to Δ9-Tetrahydrocannabinol Triggers Profound Defects in T Cell Differentiation and Function in Fetal and Postnatal Stages of Life, Including Decreased Responsiveness to HIV Antigens

Marijuana abuse is very prominent among pregnant women. Although marijuana cannabinoids have been shown to exert immunosuppression in adults, virtually nothing is known about the effects of marijuana use during pregnancy on the developing immune system of the fetus and during postnatal life. We noted that murine fetal thymus expressed high levels of the cannabinoid receptors CB1 and CB2. Moreover, perinatal exposure to Δ9-tetrahydrocannabinol (THC) had a profound effect on the fetus as evidenced by a decrease in thymic cellularity on gestational days 16, 17, and 18 and postgestational day 1 and marked alterations in T cell subpopulations. These outcomes were reversed by CB1/CB2 antagonists, suggesting that THC-mediated these effects through cannabinoid receptors. Thymic atrophy induced in the fetus correlated with caspase-dependent apoptosis in thymocytes. Thymic atrophy was the result of direct action of THC and not based on maternal factors inasmuch as THC was able to induce T cell apoptosis in vitro in fetal thymic organ cultures. It is noteworthy that perinatal exposure to THC also had a profound effect on the immune response during postnatal life. Peripheral T cells from such mice showed decreased proliferative response to T cell mitogen as well as both T cell and antibody response to HIV-1 p17/p24/gp120 antigens. Together, our data demonstrate for the first time that perinatal exposure to THC triggers profound T cell dysfunction, thereby suggesting that the offspring of marijuana abusers who have been exposed to THC in utero may be at a higher risk of exhibiting immune dysfunction and contracting infectious diseases including HIV.

Bryostatin-1 Enhances the Maturation and Antigen-Presenting Ability of Murine and Human Dendritic Cells

In this study, we investigated the effect of bryostatin-1 (Bryo-1), an antineoplastic agent, on dendritic cell (DC) maturation, activation, and functions. Murine bone marrow-derived DCs on culture with Bryo-1 alone, Bryo-1 + calcium ionophore (CI), but not CI alone exhibited morphologic changes characteristic of mature DCs and expressed increased levels of CD40, CD80, and CD86. Moreover, Bryo-1 + CI–treated DCs exhibited enhanced antigen-presenting ability to naive and antigen-specific T cells and alloreactive T cells. Bryo-1 + CI–mediated activation of DCs involved protein kinase C (PKC), especially PKC-α, -δ, and -ι, and addition of PKC inhibitors impaired their ability to activate T cells. Bryo-1 + CI treatment of DCs did not activate mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, p38 MAPK, or stress-activated protein kinase/c-Jun NH2-terminal kinase pathways. Finally, treatment of DCs with Bryo-1 alone and Bryo-1 + CI, but not CI alone, induced nuclear translocation of nuclear factor κB as studied by confocal microscopy. DCs generated from human peripheral blood monocytes or from human cord blood CD34+ hematopoietic stem cells, when cultured with Bryo-1 + CI, also showed maturation and increased T-cell stimulatory activity. Bryo-1 + CI was more potent in inducing maturation and activation of DCs when compared with other agents such as tumor necrosis factor α, lipopolysaccharide, or phorbol 12-myristate 13acetate + CI. Collectively, the current study shows for the first time that Bryo-1 alone or in combination with CI may promote the maturation of DCs and therefore may be useful in development of DC-based cancer immunotherapy.

Histone Modifications Are Associated with Δ9-Tetrahydrocannabinol-mediated Alterations in Antigen-specific T Cell Responses

Background: Marijuana has been shown to have an immunomodulatory activity. Results: ChIP-Seq results show genome-wide changes in histone methylation in immune cells treated with THC. Conclusion: Histone modifications are associated with THC-mediated alterations in antigen-specific T cell response. Significance: This study provides insights into the potential role of epigenetic changes induced by THC in gene regulation. Marijuana is one of the most abused drugs due to its psychotropic effects. Interestingly, it is also used for medicinal purposes. The main psychotropic component in marijuana, Δ9-tetrahydrocannabinol (THC), has also been shown to mediate potent anti-inflammatory properties. Whether the immunomodulatory activity of THC is mediated by epigenetic regulation has not been investigated previously. In this study, we employed ChIP-Seq technology to examine the in vivo effect of THC on global histone methylation in lymph node cells of mice immunized with a superantigen, staphylococcal enterotoxin B. We compared genome-wide histone H3 Lys-4, Lys-27, Lys-9, and Lys-36 trimethylation and histone H3 Lys-9 acetylation patterns in such cells exposed to THC or vehicle. Our results showed that THC treatment leads to the association of active histone modification signals to Th2 cytokine genes and suppressive modification signals to Th1 cytokine genes, indicating that such a mechanism may play a critical role in the THC-mediated switch from Th1 to Th2. At the global level, a significant portion of histone methylation and acetylation regions were altered by THC. However, the overall distribution of these histone methylation signals among the genomic features was not altered significantly by THC, suggesting that THC activates the expression of a subset of genes while suppressing the expression of another subset of genes through histone modification. Functional classification of these histone marker-associated genes showed that these differentially associated genes were involved in various cellular functions, from cell cycle regulation to metabolism, suggesting that THC had a pleiotropic effect on gene expression in immune cells. Altogether, the current study demonstrates for the first time that THC may modulate immune response through epigenetic regulation involving histone modifications.

Targeting Cannabinoid Receptors as a Novel Approach in the Treatment of Graft-versus-Host Disease: Evidence from an Experimental Murine Model

Allogeneic hematopoietic cell transplantation (HCT) is widely used to treat patients with life-threatening malignant and nonmalignant hematological diseases. However, allogeneic HCT often is accompanied by severe and lethal complications from graft-versus-host disease (GVHD), in which activated donor T cells recognize histocompatibility antigenic mismatches and cause significant toxicity in the recipient. In the current study, we tested the hypothesis that activation of cannabinoid receptors on donor-derived T cells may prevent GVHD. We tested the effect of Δ9-tetrahydrocannabinol (THC) in an acute model of GVHD that was induced by transferring parental C57BL/6 (B6) spleen cells into (C57BL/6 × DBA/2) F1(BDF1) mice. Transfer of B6 cells into BDF1 mice produced severe acute GVHD in the recipient, characterized by lymphoid hyperplasia, weight loss, T helper l cytokine production and mortality. THC administration led to early recovery from body weight loss, reduced tissue injury in the liver and intestine, as well as complete survival. THC treatment reduced the expansion of donor-derived effector T cells and blocked the killing of host-derived immune cells while promoting Foxp3+ regulatory T cells. Impaired hematopoiesis seen during GVHD was rescued by treatment with THC. The ability of THC to reduce the clinical GVHD was reversed, at least in part, by administration of cannabinoid receptor (CB) 1 and CB2 antagonists, thereby demonstrating that THC-mediated amelioration of GVHD was cannabinoid receptor-dependent. Our results demonstrate for the first time that targeting cannabinoid receptors may constitute a novel treatment modality against acute GVHD.

MDSCs drive the process of endometriosis by enhancing angiogenesis and are a new potential therapeutic target

Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid‐derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T‐cell proliferation. Depletion of MDSCs by antiGr‐1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR‐2. These CXC‐chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2‐deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2‐KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC‐chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.