Determination of PCB and PAH tissue levels in bottlenose dolphins that stranded in the Mississippi sound before and after the unusual mortality event in 2019.

Cetaceans are regarded as a sentinel species because their health and population changes can serve as indicators of effects on marine ecosystems. We characterized levels of 24 polycyclic aromatic hydrocarbons (PAHs) and 7 polychlorinated biphenyls (PCBs) in blubber, kidney, liver, and muscle of 138 bottlenose dolphins (Tursiops truncatus) that stranded in the Mississippi Sound (MSS) between 2010 and 2021. The samples were divided into four time periods: 2010-2018, 2019, 2020, and 2021, to assess whether there was a significant association between chemical levels in dolphin tissues and the 2019 unusual mortality event (UME) in the MSS resulting from the unprecedented freshwater incursion from 2 openings of the Bonnet Carré Spillway (BCS). We found 7 PCBs were readily detected in all tissues across time, and the major PAH was naphthalene, with detection of biphenyl and acenaphthene at lower levels and frequency. There was little change in tissue PCB levels over time; only naphthalene in the blubber was higher in tissues from dolphins that stranded before 2019. However, there were not significant changes in chemical levels in tissues from stranded bottlenose dolphins in the MSS before or after the UME in 2019.

Mild Disease Course of Experimental Autoimmune Encephalomyelitis without Pertussis Toxin: Brain Transcriptome Analysis Reveals Similar Signaling to Active Lesions in Multiple Sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is a powerful model to study multiple sclerosis (MS). One of the approaches for EAE is to actively immunize with myelin-derived peptides with immune adjuvants. One of the commonly used immune adjuvants is pertussis toxin (PTx), without which EAE disease is mild with relatively longer onset. However, pertussis toxin can also inhibit G protein-coupled receptor (GPCR) signaling so it can confound investigations into the role of GPCRs in EAE or therapies designed to target GPCRs. Since EAE via active immunization without PTx results in a relatively mild disease state, we wanted to confirm that appropriate signaling molecules for the disease were being induced in one target tissue (i.e., brain). RNA-Seq analysis of whole brain tissue demonstrated that the MS signaling pathway was strongly activated in symptomatic mice. In addition, there was activation of Th1 (IFN signaling), Th2 (IL-4 signaling), and Th17 (IL-17 signaling). In comparing canonical pathways from our mouse mild EAE brains with a human MS atlas, EAE shared the most pathways with active and inactive lesions. An advantage of this approach is that disease induction is slower to develop and results in modest clinical signs, which likely more closely mimic human disease onset.

Oxytocin and Vasopressin Gene Expression in the Brain as Potential Biomarkers for Cannabidiol Therapeutic Efficacy.

Over the last several years, there has been increased interest in cannabidiol (CBD) to treat various ailments such as pain, anxiety, insomnia, and inflammation. The potential for CBD as an anti-inflammatory therapy has come, in part, from its demonstrated ability to suppress neuroinflammation in autoimmune diseases, such as the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). The increased use of CBD strongly suggests that more research is necessary to elucidate its safety and efficacy and determine the mechanisms by which it acts. Thus, we conducted two separate studies. In the first, RNA sequencing (RNA-Seq) analysis of brains of female mice undergoing EAE in the presence and absence of CBD was conducted to identify potential genes that mediated its neuroprotective effects when efficacious. In the second, we assessed some of the same genes in male and female mice treated with CBD in the absence of an immune stimulus. Together, these data showed that CBD modestly increased oxytocin (Oxt) and arginine vasopressin (vasopressin, Avp) gene expression in the brains of mice, regardless of whether there was active inflammation. Overall, these data suggest that Oxt and Avp might act as biomarkers for CBD exposure.

Potential to use cannabinoids as adjunct therapy for dexamethasone: An in vitro study with canine peripheral blood mononuclear cells.

Dexamethasone (dex) is a potent glucocorticoid used to treat a variety of diseases. It is widely used in veterinary medicine in many species; for instance, in dogs, it can be used for emergent cases of anaphylaxis or trauma, management of immune-mediated hemolytic anemia or thrombocytopenia, certain cancers, allergic reactions, and topically for skin or eye inflammation. Dex is not without its side effects, especially when administered systemically, which might compromise compliance and effective treatment. Thus, adjunct therapies have been suggested to allow for decreased dex dosing and reduction in side effects while maintaining immunosuppressive efficacy. The goal of this study was to evaluate the potential for cannabinoids to serve as adjunct therapies for dex. Immune function was assessed in canine peripheral blood mononuclear cells (PBMCs) after treatment with dex with and without cannabidiol (CBD) and/or Δ9-tetrahydrocannabinol (THC). Dex suppressed IFN-γ protein secretion in a concentration-dependent manner and this suppression by low concentrations of dex was enhanced in the presence of CBD, THC, or the combination of CBD and THC. Similar effects were found with INFG and TNFA mRNA expression. These findings provide a rationale for using CBD or THC in vivo to reduce dex dosing and side effects.

Development of an in vitro peptide-stimulated T cell assay to evaluate immune effects by cannabinoid compounds.

Previous studies demonstrated that cannabinoids exhibit immunosuppressive effects in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). To ask questions about treatment timing and investigate mechanisms for immune suppression by the plant-derived cannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), an in vitro peptide stimulation of naive splenocytes (SPLC) was developed to mimic T cell activation in EAE. The peptide was derived from the myelin oligodendrocyte glycoprotein (MOG) protein, which is one component of the myelin sheath. MOG peptide is typically used with an immune adjuvant to trigger MOG-reactive T cells that attack MOG-containing tissues, causing demyelination and clinical disease in EAE. To develop the in vitro model, naïve SPLC were stimulated with MOG peptide on day 0 and restimulated on day 4. Cytokine analyses revealed that CBD and THC suppressed MOG peptide-stimulated cytokine production. Flow cytometric analysis showed that intracellular cytokines could be detected in CD4+ and CD8+ T cells. To determine if intracellular calcium was altered in the cultures, cells were stimulated for 4 days to assess the state of the cells at the time of MOG peptide restimulation. Both cannabinoid-treated cultures had a smaller population of the calcium-positive population as compared to vehicle-treated cells. These results demonstrate the establishment of an in vitro model that can be used to mimic MOG-reactive T cell stimulation in vivo.

Protecting Human and Animal Health: The Road from Animal Models to New Approach Methods.

Animals and animal models have been invaluable for our current understanding of human and animal biology, including physiology, pharmacology, biochemistry, and disease pathology. However, there are increasing concerns with continued use of animals in basic biomedical, pharmacological, and regulatory research to provide safety assessments for drugs and chemicals. There are concerns that animals do not provide sufficient information on toxicity and/or efficacy to protect the target population, so scientists are utilizing the principles of replacement, reduction, and refinement (the 3Rs) and increasing the development and application of new approach methods (NAMs). NAMs are any technology, methodology, approach, or assay used to understand the effects and mechanisms of drugs or chemicals, with specific focus on applying the 3Rs. Although progress has been made in several areas with NAMs, complete replacement of animal models with NAMs is not yet attainable. The road to NAMs requires additional development, increased use, and, for regulatory decision making, usually formal validation. Moreover, it is likely that replacement of animal models with NAMs will require multiple assays to ensure sufficient biologic coverage. The purpose of this manuscript is to provide a balanced view of the current state of the use of animal models and NAMs as approaches to development, safety, efficacy, and toxicity testing of drugs and chemicals. Animals do not provide all needed information nor do NAMs, but each can elucidate key pieces of the puzzle of human and animal biology and contribute to the goal of protecting human and animal health. SIGNIFICANCE STATEMENT: Data from traditional animal studies have predominantly been used to inform human health safety and efficacy. Although it is unlikely that all animal studies will be able to be replaced, with the continued advancement in new approach methods (NAMs), it is possible that sometime in the future, NAMs will likely be an important component by which the discovery, efficacy, and toxicity testing of drugs and chemicals is conducted and regulatory decisions are made.

The mechanism by which cannabidiol (CBD) suppresses TNF-α secretion involves inappropriate localization of TNF-α converting enzyme (TACE).

Cannabidiol (CBD) is a phytocannabinoid derived from Cannabis sativa that exerts anti-inflammatory mechanisms. CBD is being examined for its putative effects on the neuroinflammatory disease, multiple sclerosis (MS). One of the major immune mediators that propagates MS and its mouse model experimental autoimmune encephalomyelitis (EAE) are macrophages. Macrophages can polarize into an inflammatory phenotype (M1) or an anti-inflammatory phenotype (M2a). Therefore, elucidating the impact on macrophage polarization with CBD pre-treatment is necessary to understand its anti-inflammatory mechanisms. To study this effect, murine macrophages (RAW 264.7) were pre-treated with CBD (10 µM) or vehicle (ethanol 0.1 %) and were either left untreated (naive; cell media only), or stimulated under M1 (IFN-γ + lipopolysaccharide, LPS) or M2a (IL-4) conditions for 24 hr. Cells were analyzed for macrophage polarization markers, and supernatants were analyzed for cytokines and chemokines. Immunofluorescence staining was performed on M1-polarized cells for the metalloprotease, tumor necrosis factor-α-converting enzyme (TACE), as this enzyme is responsible for the secretion of TNF-α. Overall results showed that CBD decreased several markers associated with the M1 phenotype while exhibiting less effects on the M2a phenotype. Significantly, under M1 conditions, CBD increased the percentage of intracellular and surface TNF-α but decreased secreted TNF-α. This phenomenon might be mediated by TACE as staining showed that CBD sequestered TACE intracellularly. CBD also prevented RelA nuclear translocation. These results suggest that CBD may exert its anti-inflammatory effects by reducing M1 polarization and decreasing TNF-α secretion via inappropriate localization of TACE and RelA.

Canine immune cells express high levels of CB1 and CB2 cannabinoid receptors and cannabinoid-mediated alteration of canine cytokine production is vehicle-dependent.

With the increased popularity and societal acceptance of marijuana and cannabidiol (CBD) use in humans, there is an interest in using cannabinoids in veterinary medicine. There have been a few placebo-controlled clinical trials in dogs suggesting that cannabis-containing extracts are beneficial for dogs with inflammatory diseases such as osteoarthritis, and there is growing interest in their immunosuppressive potential for the treatment of immune-mediated diseases. Since cannabinoids exhibit anti-inflammatory and immunosuppressive effects in many species, the purpose of these studies was to examine whether the plant-derived cannabinoids, CBD and Δ9-tetrahydrocannabinol (THC), would also suppress immune function in canine peripheral blood mononuclear cells (PBMCs). Another goal was to characterize expression of the cannabinoid receptors, CB1 and CB2, in canine immune cells. We hypothesized that CBD and THC would suppress stimulated cytokine expression and that both cannabinoid receptors would be expressed in canine immune cells. Surprisingly, cannabinoid suppressive effects in canine PMBCs were quite modest, with the most robust effect occurring at early stimulation times and predominantly by THC. We further showed that cannabinoid-mediated suppression was dog- and vehicle-dependent with CBD and THC delivered in dimethyl sulfoxide (DMSO) producing more immune suppressive effects as compared to ethanol (ETOH). PCR, flow cytometry, and immunohistochemical staining demonstrated that both CB1 and CB2 are expressed in canine immune cells. Together these data show that canine immune cells are sensitive to suppression by cannabinoids, but more detailed studies are needed to further understand the mechanisms and broad effects of these compounds in the dog.

Age-Dependent Effects of Transgenic 2D2 Mice Used to Induce Passive Experimental Autoimmune Encephalomyelitis in C57BL/6 Mice.

INTRODUCTION: Multiple sclerosis (MS) is a neurodegenerative autoimmune disease that worsens with age. Here, we examined the influence of age on passive experimental autoimmune encephalomyelitis (P-EAE), a model to study MS, using young and mature adult 2D2 transgenic donor mice to induce pathology in WT C57BL6/J mice. METHODS: Lymphocytes from young adult (i.e., 10-week-old) or mature adult (i.e., 6-month-old) transgenic donor mice were characterized by flow cytometry prior to injection of cultured leukocytes into adult female WT recipient mice, with a special focus on transgenic T cell phenotypes. RESULTS: Our findings show age-dependent changes in memory T cell phenotypes correlated with more severe clinical and histological disease when donor cells originated from young as compared to mature adult mice. CONCLUSION: Not only do these results demonstrate that the age of the 2D2 transgenic donor mice is critical in establishing P-EAE, but the differential effects might also identify age-dependent factors that contribute to EAE and perhaps MS.

Cnr1-/- has minimal impact on chlorpyrifos-mediated effects in the mouse endocannabinoid system, but it does alter lipopolysaccharide-induced cytokine levels in splenocytes.

Chlorpyrifos (CPF) is an organophosphate pesticide that can inhibit endocannabinoid (eCB) metabolizing enzymes in animal models at levels that do not significantly alter acetylcholinesterase (AChE) in the central nervous system (CNS). Previous studies indicated that repeated low-level CPF exposure in developing rats increased the levels of eCBs in the brain. Because eCBs play a role in immune homeostasis through their engagement with cannabinoid receptors, we investigated the role of cannabinoid receptor 1 (CB1, encoded by the Cnr1 gene) on the CPF-mediated effects in the spleen and lung of neonatal and adult female mice. We treated neonatal and adult female Cnr1-/- mice with 2.5 mg/kg oral CPF or vehicle for 7 days. Tissues were harvested 4 h after the last CPF dose to evaluate eCB metabolic enzyme activity, levels of eCBs, and tissue immunophenotype. There were a small number of genotype-dependent alterations noted in the endpoints following CPF treatment that were specific to age and tissue type, and differences in eCB metabolism caused by CPF treatment did not correlate to changes in eCB levels. To explore the role of CB1 in CPF-mediated effects on immune endpoints, in vitro experiments were performed with WT murine splenocytes exposed to chlorpyrifos oxon (CPO; oxon metabolite of CPF) and challenged with lipopolysaccharide (LPS). While CPO did not alter LPS-induced pro-inflammatory cytokine levels, inactivation of CB1 by the antagonist SR141716A augmented LPS-induced IFN-γ levels. Additional experiments with WT and Cnr1-/- murine splenocytes confirmed a role for CB1 in altering the production of LPS-induced pro-inflammatory cytokine levels. We conclude that CPF-mediated effects on the eCB system are not strongly dependent on CB1, although abrogation of CB1 does alter LPS-induced cytokine levels in splenocytes.

Consensus on the Key Characteristics of Immunotoxic Agents as a Basis for Hazard Identification.

BACKGROUND: Key characteristics (KCs), properties of agents or exposures that confer potential hazard, have been developed for carcinogens and other toxicant classes. KCs have been used in the systematic assessment of hazards and to identify assay and data gaps that limit screening and risk assessment. Many of the mechanisms through which pharmaceuticals and occupational or environmental agents modulate immune function are well recognized. Thus KCs could be identified for immunoactive substances and applied to improve hazard assessment of immunodulatory agents. OBJECTIVES: The goal was to generate a consensus-based synthesis of scientific evidence describing the KCs of agents known to cause immunotoxicity and potential applications, such as assays to measure the KCs. METHODS: A committee of 18 experts with diverse specialties identified 10 KCs of immunotoxic agents, namely, 1) covalently binds to proteins to form novel antigens, 2) affects antigen processing and presentation, 3) alters immune cell signaling, 4) alters immune cell proliferation, 5) modifies cellular differentiation, 6) alters immune cell-cell communication, 7) alters effector function of specific cell types, 8) alters immune cell trafficking, 9) alters cell death processes, and 10) breaks down immune tolerance. The group considered how these KCs could influence immune processes and contribute to hypersensitivity, inappropriate enhancement, immunosuppression, or autoimmunity. DISCUSSION: KCs can be used to improve efforts to identify agents that cause immunotoxicity via one or more mechanisms, to develop better testing and biomarker approaches to evaluate immunotoxicity, and to enable a more comprehensive and mechanistic understanding of adverse effects of exposures on the immune system. https://doi.org/10.1289/EHP10800.

Carboxylesterase 1d Inactivation Augments Lung Inflammation in Mice.

Carboxylesterases are members of the serine hydrolase superfamily and metabolize drugs, pesticides, and lipids. Previous research showed that inhibition of carboxylesterase 1 (CES1) in human macrophages altered the immunomodulatory effects of lipid mediators called prostaglandin glyceryl esters, which are produced by cyclooxygenase-catalyzed oxygenation of the endocannabinoid 2-arachidonoylglycerol (2-AG). Ces1d - the mouse ortholog of human CES1 - is the most abundant Ces isoform in murine lung tissues and alveolar macrophages and a major target of organophosphate poisons. Monoacylglycerol lipase (Magl) is also expressed in murine lung and is the main enzyme responsible for 2-AG catabolism. Several metabolic benefits are observed in Ces1d-/- mice fed a high-fat diet; thus, we wondered whether pharmacological and genetic inactivation of Ces1d in vivo might also ameliorate the acute inflammatory response to lipopolysaccharide (LPS). C57BL/6 mice were treated with WWL229 (Ces1d inhibitor) or JZL184 (Magl inhibitor), followed 30 min later by either LPS or saline. Wild-type (WT) and Ces1d-/- mice were also administered LPS to determine the effect of Ces1d knockout. Mice were sacrificed at 6 and 24 h, and cytokines were assessed in serum, lung, liver, and adipose tissues. Lipid mediators were quantified in lung tissues, while activity-based protein profiling and enzyme assays determined the extent of lung serine hydrolase inactivation by the inhibitors. WWL229 was shown to augment LPS-induced lung inflammation in a female-specific manner, as measured by enhanced neutrophil infiltration and Il1b mRNA. The marked Ces inhibition in female lung by 4 h after drug treatment might explain this sex difference, although the degree of Ces inhibition in female and male lungs was similar at 6 h. In addition, induction of lung Il6 mRNA and prostaglandin E2 by LPS was more pronounced in Ces1d-/- mice than in WT mice. Thus, WWL229 inhibited lung Ces1d activity and augmented the female lung innate immune response, an effect observed in part in Ces1d-/- mice and Ces1d/CES1-deficient murine and human macrophages. In contrast, JZL184 attenuated LPS-induced Il1b and Il6 mRNA levels in female lung, suggesting that Ces1d and Magl have opposing effects. Mapping the immunomodulatory molecules/pathways that are regulated by Ces1d in the context of lung inflammation will require further research.

Potential for TCDD to induce regulatory functions in B cells as part of the mechanism for T cell suppression in EAE.

Part of the mechanism by which 2,3,7.8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses immune function involves induction of regulatory T cells and suppression of effector T cells. The goal of this project was to examine whether TCDD's suppression of effector T cells was due in part to inducing B regulatory cells (Bregs). TCDD's potential to increase the percentage and/or function of CD24+CD38+ B cells was assessed in response to lipopolysaccharide (LPS) + interleukin (IL)-4 in vitro and in a mild model of experimental autoimmune encephalomyelitis (EAE) in vivo. In vitro, TCDD did not consistently increase the percentage of CD19+CD24+CD38+ cells using splenocytes, purified B cells or bone marrow (BM) cells. However, TCDD increased IL-10 in all three culture preparations, and TCDD increased the percentage of CD5+CD24+CD38+ cells producing IL-10. In EAE, TCDD did not affect the percentage of the CD24+CD38+ cell population in CD19, B220 or CD5 B cells in splenocytes (SPLC), lymph nodes (LN) nor BM cells at end-stage disease. On the other hand, TCDD increased the CD19+CD24+CD38+ percentage in the spinal cord (SC) in EAE. Moreover, TCDD-treated B cells isolated from spleens or TCDD-treated BM cells in EAE mice modestly reduced the ability of naïve effector T cells to express interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Together these data show that TCDD can induce regulatory functions in B cells, although it was not obvious simply by examining the expression of regulatory markers but by assessing function by cytokine production or mixed lymphocyte responses.

Standardized Cannabis Smoke Extract Induces Inflammation in Human Lung Fibroblasts.

Cannabis (marijuana) is the most commonly used illicit product in the world and is the second most smoked plant after tobacco. There has been a rapid increase in the number of countries legalizing cannabis for both recreational and medicinal purposes. Smoking cannabis in the form of a joint is the most common mode of cannabis consumption. Combustion of cannabis smoke generates many of the same chemicals as tobacco smoke. Although the impact of tobacco smoke on respiratory health is well-known, the consequence of cannabis smoke on the respiratory system and, in particular, the inflammatory response is unclear. Besides the combustion products present in cannabis smoke, cannabis also contains cannabinoids including Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). These compounds are hydrophobic and not present in aqueous solutions. In order to understand the impact of cannabis smoke on pathological mechanisms associated with adverse respiratory outcomes, the development of in vitro surrogates of cannabis smoke exposure is needed. Therefore, we developed a standardized protocol for the generation of cannabis smoke extract (CaSE) to investigate its effect on cellular mechanisms in vitro. First, we determined the concentration of Δ9-THC, one of the major cannabinoids, by ELISA and found that addition of methanol to the cell culture media during generation of the aqueous smoke extract significantly increased the amount of Δ9-THC. We also observed by LC-MS/MS that CaSE preparation with methanol contains CBD. Using a functional assay in cells for CB1 receptors, the major target of cannabinoids, we found that this CaSE contains Δ9-THC which activates CB1 receptors. Finally, this standardized preparation of CaSE induces an inflammatory response in human lung fibroblasts. This study provides an optimized protocol for aqueous CaSE preparation containing biologically active cannabinoids that can be used for in vitro experimentation of cannabis smoke and its potential impact on various indices of pulmonary health.

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD's effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD's EAE disease attenuation.

Isolation of Transcriptomic-Quality Total RNA from Mouse Spinal Cords.

Assessing cells, proteins, and total RNA in the spinal cord is vital for advancing our understanding of neuroinflammation and neurodegenerative diseases. For instance, immune cells infiltrate the spinal cord in the experimental autoimmune encephalomyelitis (EAE) model, commonly used to study multiple sclerosis. Thus, it is valuable to assess total RNA to determine the neuronal and inflammatory profiles in the spinal cord. Further, RNA profiles are useful for deciphering the effects of drugs or chemicals on neuroinflammation and neurodegenerative diseases such as EAE. The purpose of this protocol and the online video illustrating it is to describe and demonstrate the expulsion of the spinal cord from the mouse spinal column and homogenization of the spinal cord using liquid nitrogen for optimal RNA isolation. Although we present this method with spinal cords from EAE mice, the technique is broadly applicable, including RNA isolation from the spinal cords of healthy mice. Proper performance of these steps is critical to achieving a sufficient yield of transcriptomic-quality spinal cord RNA when combined with final isolation using commercially available kits. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Isolation of the spinal cord from the spinal column Support Protocol: Preparation of blunt-end needle for spinal cord isolation Basic Protocol 2: Spinal cord homogenization using liquid nitrogen Basic Protocol 3: Assessment of RNA purity, quantification, and integrity.

Immunomodulation by cannabinoids: Current uses, mechanisms, and identification of data gaps to be addressed for additional therapeutic application.

The endocannabinoid system plays a critical role in immunity and therefore its components, including cannabinoid receptors 1 and 2 (CB1 and CB2), are putative druggable targets for immune-mediated diseases. Whether modulating endogenous cannabinoid levels or interacting with CB1 or CB2 receptors directly, cannabinoids or cannabinoid-based therapeutics (CBTs) show promise as anti-inflammatory or immune suppressive agents. Herein we provide an overview of cannabinoid effects in animals and humans that provide support for the use of CBTs in immune-mediated disease such as multiple sclerosis (MS), inflammatory bowel disease (IBD), asthma, arthritis, diabetes, human immunodeficiency virus (HIV), and HIV-associated neurocognitive disorder (HAND). This is not an exhaustive review of cannabinoid effects on immune responses, but rather provides: (1) key studies in which initial and/or novel observations were made in animal studies; (2) critical human studies including meta-analyses and randomized clinical trials (RCTs) in which CBTs have been assessed; and (3) evidence for the role of CB1 or CB2 receptors in immune-mediated diseases through genetic analyses of single nucleotide polymorphisms (SNPs) in the CNR1 and CNR2 genes that encode CB1 or CB2 receptors, respectively. Perhaps most importantly, we provide our view of data gaps that exist, which if addressed, would allow for more rigorous evaluation of the efficacy and risk to benefit ratio of the use of cannabinoids and/or CBTs for immune-mediated diseases.

The CB1 Receptor Differentially Regulates IFN-γ Production In Vitro and in Experimental Autoimmune Encephalomyelitis.

Introduction: Activation of the peripheral immune system and the infiltration of immune cells into the central nervous system are both key features of the experimental autoimmune encephalomyelitis (EAE) model. By exploring how the endocannabinoid system works to modulate this response, we can better understand how exogenous cannabinoids, such as THC, might be used to modulate the immune responses of multiple sclerosis patients. Materials and Methods: In this study, we examined the role of the CB1 receptor in IFN-γ and IL-17A production in the EAE model and in vitro stimulations of naive splenocytes using Cnr1-/- mice and wild-type (WT) littermates. We also introduce a novel method of scoring spinal cord histological sections to show the differences in disease severity between Cnr1-/- and WT mice with EAE. Results: Clinical scores of Cnr1-/-/EAE and WT/EAE mice showed more severe disease progression in Cnr1-/- mice, which was confirmed using our new histological scoring method. In the peripheral immune system, IFN-γ production by restimulated splenocytes from Cnr1-/-/EAE mice, compared with WT/EAE mice, was increased and the primary source of IFN-γ was a CD3- cell population; however, IFN-γ production by Cnr1-/- splenocytes was decreased compared with WT splenocytes when the primary source of IFN-γ was CD3+ T cells in cultures from naive mice stimulated by either anti-CD3/anti-CD28 antibodies or Staphylococcal superantigens. Conclusion: These findings suggest a duality to the CB1 receptor's effects on the peripheral immune response, which varies based on the specific cell types stimulated. Knowledge of the complex nature of a receptor is an important part of determining its potential usefulness as a therapeutic target, and these findings further define the role of CB1 in IFN-γ responses.