Background The nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) inflammasome is a signalling hub associated with the pathogenesis of neuroinflammatory conditions such as multiple sclerosis (MS). NLRP3 inflammasome activation requires interplay between pathogen-/damage-associated molecular patterns and other soluble factors, which initiates inflammation to promote the secretion of the cytokine, interleukin (IL)-1β. Objective To determine if the expression of NLRP3 inflammasome signalling components is altered in the brain and in immune cells in MS. Methods Using post-mortem brain tissue from 21 cases, including 8 non-MS control, 7 primary progressive (PP) MS and 6 secondary progressive (SP) MS cases, alongside peripheral blood mononuclear cells (PBMCs) isolated from 45 subjects including healthy controls (n = 23), and people with (pw) a relapsing remitting (RR) (n = 15), SP (n = 5) or PP (n = 2) form of MS, we profiled the expression of NLRP3 inflammasome components, both centrally in CNS white matter, and peripherally in immune cells. Results The expression of NLRP3, IL1B, IL18, CASP1 and PYCARD transcripts were elevated in chronic active lesions (CALs) in PPMS cases, with no significant alterations determined in SPMS CNS tissue. Furthermore, NLRP3-dependent IL-1β release, alongside NLRP3, IL1B and GSDMD expression, were significantly elevated in immune cells isolated from pwMS (primarily RRMS), when compared to PBMCs from healthy controls. Conclusion The expression of NLRP3 inflammasome components is dysregulated in MS, both in the brain and in PBMCs. This suggests that uncontrolled NLRP3 inflammasome activity takes place at certain stages of MS.

Dual-specificity phosphatase 1 (DUSP1) is a negative regulator of mitogen-activated protein kinase (MAPK) activity, and a player in the control of glial function. Several disease models have identified that the expression of DUSP1 is altered in tissue samples, which may contribute to disease pathogenesis. Using post-mortem brain tissue from a cohort of progressive multiple sclerosis (MS) cases, this study set out to determine if the expression of DUSP1 is altered in the brain in MS. We employed the use of a combination of PCR, western immunoblotting and immunohistochemical analysis to profile the expression of DUSP1. Data presented herein indicate that DUSP1 is significantly downregulated at gene and protein level in MS white matter tissue, when compared to samples from a non-MS control cohort. We also identify that DUSP1 is expressed on Iba1+ cells in the brain and that ITGAM (CD11b) expression is elevated in white matter in progressive MS. These findings provide evidence that DUSP1 is dysregulated centrally in CNS white matter in progressive MS.

BACKGROUND:Advancements continue to be made in the development of medicines containing components of the Cannabis sativa L. plant. Consultants can prescribe specific cannabis-based products for a restricted set of indications in Ireland, with neurologists being at the forefront of therapy. Much debate on the therapeutic potential/efficacy of such cannabis-based products exists. AIM:The objective of this study was to conduct a national survey to determine the perspectives/views of Irish neurologists regarding the use of cannabis-based medicines. METHODS:An online anonymous survey was conducted to capture the perspectives and experiences of neurologists in Ireland regarding cannabis-based therapeutics. RESULTS:Thirty-four neurologists completed the survey in full, with study participants rating their knowledge of cannabis-based medicines as average. Data presented herein indicate that there is a need for educational programmes on the cannabinoid system and cannabinoid-based medicines, and the findings indicate that neurologists are interested in the use of cannabinoid-based medicines in their practice. Study participants were more divided with regard to the clarity of the process for accessing cannabis-based medicines, and the consensus is that the application process is unclear. Approximately one-third of participants have made an application to access medicinal cannabis-based products on behalf of a patient. CONCLUSIONS:Data presented herein indicates that the majority of neurologists surveyed are aware of the current systems in place to access cannabis-based products for medicinal use in Ireland and that some engagement with these systems has taken place. A key finding is that educational programmes on the cannabinoid system and cannabis-based medicines are required.

Cannabinoid regulation of endosomal signalling via innate immune toll-like receptors (TLRs) is understudied. Endosomal cell signalling via TLR7 and TLR8 governs cellular responses to infection with viral and bacterial single-stranded RNA. TLR7/8 activation is associated with neuroinflammation, with inappropriate activation of TLR7/8 linked to the propagation of autoimmune disease. Following activation, TLR7 and TLR8 control the cellular production of cytokines, chemokines and type I interferons (IFNs). In this study we focused on two clinically relevant plant-derived (phyto) cannabinoids, cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), given that cannabinoid-based therapeutics containing these compounds are currently available in the form of sativex® (nabiximols) and epidiolex®. The study aim was to determine the anti-inflammatory effects of CBD and THC, when delivered in isolation and in a sativex-like combination (1:1), on TLR7/8-induced inflammation in immune cells. We employed the use of CL075 (3M-002), a thiazoloquinolone derivative that acts as an agonist of both TLR7 and TLR8. Using THP-1-derived macrophages and primary peripheral blood mononuclear cells (PBMCs) from healthy control subjects, we demonstrate that TLR7/8 activation promoted the time- and concentration-dependent production of the chemokine CXCL10, cytokine TNFα and type I IFNs in both macrophages and PBMCs. TLR7/8 activation promoted nuclear factor (NF)-κB activation, p38 MAPK phosphorylation and the transcription of interferon regulator factor 7 (IRF7). We assessed the anti-inflammatory effects of CBD and THC, when delivered alone and in a 1:1 combination, on CL075-stimulated inflammatory mediator production in macrophages/PBMCs. Data presented herein indicate that CBD and THC, particularly when delivered in a 1:1 combination, can act as TLR7/8 immunomodulatory drugs to dampen inflammation in macrophages and PBMCs. This study provides evidence that phytocannabinoids target TLR7/8-induced viral signalling on endosomal compartments to control inflammation in immune cells.

Toll-like receptors (TLRs) have become a focus in biomedicine and biomedical research given the roles of this unique family of innate immune proteins in immune activation, infection, and autoimmunity. It is evident that TLR dysregulation, and subsequent alterations in TLR-mediated inflammatory signalling, can contribute to disease pathogenesis, and TLR targeted therapies are in development. This review highlights evidence that cannabinoids are key regulators of TLR signalling. Cannabinoids include component of the plant Cannabis sativa L. (C. sativa), synthetic and endogenous ligands, and overall represent a class of compounds whose therapeutic potential and mechanism of action continues to be elucidated. Cannabinoid-based medicines are in the clinic, and are furthermore under intense investigation for broad clinical development to manage symptoms of a range of disorders. In this review, we present an overview of research evidence that signalling linked to a range of TLRs is targeted by cannabinoids, and such cannabinoid mediated effects represent therapeutic avenues for further investigation. First, we provide an overview of TLRs, adaptors and key signalling events, alongside a summary of evidence that TLRs are linked to disease pathologies. Next, we discuss the cannabinoids system and the development of cannabinoid-based therapeutics. Finally, for the bulk of this review, we systematically outline the evidence that cannabinoids (plant-derived cannabinoids, synthetic cannabinoids, and endogenous cannabinoid ligands) can cross-talk with innate immune signalling governed by TLRs, focusing specifically on each member of the TLR family. Cannabinoids should be considered as key regulators of signalling controlled by TLRs, and such regulation should be a major focus in terms of the anti-inflammatory propensity of the cannabinoid system.

Cannflavin A (CFL-A), a flavonoid present in the hemp plant Cannabis sativa L. (C. sativa), has anti-inflammatory and neuroprotective capacity. Research continues to elucidate the anti-inflammatory effects of components of C. sativa, with evidence that plant-derived cannabinoids and terpenes can mediate anti-inflammatory activity by targeting toll-like receptor (TLR) signalling, the sensors of pathogen-associated molecules. This study set out to determine if TLR-mediated inflammatory signalling is a CFL-A target using the endotoxin lipopolysaccharide (LPS) to induce TLR4 signalling in human THP-1-derived macrophages. TLR4 activation promoted the production of the chemokine CXCL10 and cytokines IL-1β and TNFα. Treatment with CFL-A dose-dependently attenuated TLR4-induced CXCL10 and IL-1β secretion, with our findings also indicating that the inhibitory effects of CFL-A on chemokine/cytokine secretion are in line with an NF-κB inhibitor. This study highlights TLR4 signalling as a cannflavin target in macrophages.

Multiple sclerosis (MS) is a neurological autoimmune condition associated with many symptoms including spasticity, pain, limb numbness and weakness. It is characterised by inflammatory demyelination and axonal degeneration of the brain and spinal cord. A range of disease-modifying therapies (DMTs) are available to suppress inflammatory disease activity in MS, however, there is a pressing need for new therapeutic avenues as DMTs have a limited ability to suppress confirmed disability progression. A body of literature indicates that innate immune inflammation is linked to MS progression. The nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing protein 3 (NLRP3) inflammasome has a well-established function in innate immunity which is closely associated with the pathogenesis of neuroinflammatory conditions. Evidence suggests that the inflammasome may be a therapeutic target in disorders such as MS and at present, inhibitors of the NLRP3 inflammasome are in pre-clinical development. Therefore, this review systematically highlights the pathogenic role of inflammasomes in MS, presenting an overview of research evidence linking inflammasome-related polymorphisms to MS susceptibility, and gathering evidence investigating NLRP3 biomarkers in MS. The role of the NLRP3 inflammasome in murine models of MS is furthermore discussed. Finally, a significant component of this review focuses on evidence that NLRP3 signalling components are novel drug targets in MS. Overall this review defines the role of the inflammasome in MS pathogenesis and identifies inflammasome inhibitor targets that warrant full investigation in MS and related disorders.

Human embryology is a complex topic that brings together core components of anatomy and physiology to describe the developmental process from fertilisation to birth. Embryonic development is a challenging topic of study that is core to the curricula for health science students. There are challenges ingrained in teaching and learning embryology, due to the three-dimensional dynamic processes that occur as the embryo develops. This study aimed to develop and assess two newly developed animations depicting key processes in embryology, namely gastrulation and neurulation, as supplemental learning aids for students. Indeed, animated teaching tools to enhance the learning of gastrulation and neurulation are not widely available. A multi-disciplinary team of physiologists, biochemists, anatomists, and a computer scientist developed the animation sets. A student cohort of 81 first-year health science students were enrolled in this study over a period of three academic years. Both animations are in line with the course content of the first-year health science students undertaking the Human Health and Disease BSc at Trinity College Dublin, who were the study participants. Participants were randomly assigned to a non-animation control group and an animation group. Each set of animated teaching aids was broken down into individual clips which were given identifiable headings to allow the user to interchange between clips to facilitate a more personal learning experience. The animation group had open access to the animations for a three-week period. Questionnaires were designed to assess participants’ attitude to the animations and their knowledge of embryology, both at the start of the study and three weeks later following access to the animations. Data presented herein indicate that students incorporated the animated teaching aids into digital home study and that the use of the animations acted as a supplemental tool that increased student knowledge in key areas of human embryology. From a qualitative point of view, students described the animations as enjoyable and helpful in visualising complex processes. This study indicates that the development of gastrulation and neurulation animated learning tools allow for a more engaging learning experience, facilitating student’s engagement with academically challenging concepts in human embryology.

Background Neuroblastoma (NB) is a childhood cancer of the sympathetic nervous system, and its prognosis is poor. NB undergoes transcription changes to use aerobic glycolysis as its primary metabolic pathway. Aerobic glycolysis provides an immediate source of ATP in metastasis proliferation, and therefore may be exploited in dietary therapies, such as the ketone diet. Procedure In this study, the expression of glycolytic and ketolytic genes in the context of MYCN oncogene amplification, tumor staging, and Kaplan-Meier survivability was investigated using the R2: Genomics analysis and visualisation platform (http://r2.amc.nl). The R2 database is a platform for gene expression and prognostic data for primary tumour samples. Six NB genomics datasets were accessed and further analysed in GraphPad Prism to investigate the relationships between glycolytic and ketolytic gene expression and prognosis. Results Glycolytic gene expression is increased in MYCN amplified, metastatic tumours, and is associated with worse event free survival. Ketolytic gene expression is lower in metastatic tumours and is associated with better event free survivability. The glycolytic gene expression profile of NB suggests that elevated levels correlate with tumorigenicity and low probability of survival. Ketolytic gene expression patterns suggest a decreased reliance for ketolytic energy, which may be exploited to slow tumorigenic growth. Conclusions This study validates the glycolytic gene expression profiles in metastatic and MYCN-amplified NB tumours and suggest the potential use of these genes in prognosis prediction. Furthermore, the study highlights the reliability and utility of the R2 database as an oncogenomic tool for NB research.

This Special Issue brings an update on some of the advances in research in the cannabinoid field, with focus on the impact of cannabinoids on inflammation [...]

Burning mouth syndrome (BMS) is a neuropathic pain disorder associated with a burning sensation on oral mucosal surfaces with frequently reported xerostomia, dysgeusia and tingling or paraesthetic sensations. However, patients present no clinically evident causative lesions. The poor classification of the disorder has resulted in a diagnostic challenge, particularly for the clinician/dentist evaluating these individuals. Major research developments have been made in the BMS field in recent years to address this concern, principally in terms of the pathophysiological mechanisms underlying the disorder, in addition to therapeutic advancements. For the purpose of this review, an update on the pathophysiological mechanisms will be discussed from a neuropathic, immunological, hormonal and psychological perspective. This review will also focus on the many therapeutic strategies that have been explored for BMS, including antidepressants/antipsychotics, non-steroidal anti-inflammatories, hormone replacement therapies, phytotherapeutic compounds and non-pharmacological interventions, overall highlighting the lack of controlled clinical studies to support the effectiveness of such therapeutic avenues. Particular focus is given to the cannabinoid system and the potential of cannabis-based therapeutics in managing BMS patients.

The innate immune response to bacterial and viral molecules involves the coordinated production of cytokines, chemokines, and type I interferons (IFNs), which is orchestrated by toll-like receptors (TLRs). TLRs, and their intracellular signalling intermediates, are closely associated with multiple sclerosis (MS) pathogenesis. Recent data from our laboratory reported that the plant-derived cannabinoids, Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), regulate viral and bacterial inflammatory signalling pathways controlled by TLR3 and TLR4 in macrophages. The aim of this study was to assess the impact of THC and CBD, when delivered in isolation and in combination (1:1), on TLR3- and TLR4-dependent signalling in peripheral blood mononuclear cells (PBMCs) from people with MS (pwMS; n = 21) and healthy controls (HCs; n = 26). We employed the use of poly(I:C) and lipopolysaccharide (LPS) to induce viral TLR3 and bacterial TLR4 signalling, and PBMCs were pre-exposed to plant-derived highly purified THC (10 μM), CBD (10 μM), or a combination of both phytocannabinoids (1:1 ratio, 10:10 μM), prior to LPS/poly(I:C) exposure. TLR3 stimulation promoted the protein expression of the chemokine CXCL10 and the type I IFN-β in PBMCs from both cohorts. THC and CBD (delivered in 1:1 combination at 10 μM) attenuated TLR3-induced CXCL10 and IFN-β protein expression in PBMCs from pwMS and HCs, and this effect was not seen consistently when THC and CBD were delivered alone. In terms of LPS, TLR4 activation promoted TNF-α expression in PBMCs from both cohorts, and, interestingly, CBD when delivered alone at 10 μM, and in combination with THC (in 1:1 combination at 10 μM), exacerbated TLR4-induced TNF-α protein expression in PBMCs from pwMS and HCs. THC and CBD displayed no evidence of toxicity in primary PBMCs. No significant alteration in the relative expression of TLR3 and TLR4 mRNA, or components of the endocannabinoid system, including the cannabinoid receptor CB1 (encoded by CNR1 gene) and CB2 (encoded by CNR2 gene), and endocannabinoid metabolising enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGLL), was determined in PBMCs from pwMS versus HCs. Given their role in inflammation, TLRs are clinical targets, and data herein identify CBD and THC as TLR3 and TLR4 modulating drugs in primary immune cells in vitro. This offers insight on the cellular target(s) of phytocannabinoids in targeting inflammation in the context of MS.

Background and Purpose Cannabidiol (CBD) has been shown to differentially regulate the mechanistic target of rapamycin complex 1 (mTORC1) in preclinical models of disease, where it reduces activity in models of epilepsies and cancer and increases it in models of multiple sclerosis (MS) and psychosis. Here, we investigate the effects of phytocannabinoids on mTORC1 and define a molecular mechanism. Experimental Approach A novel mechanism for phytocannabinoids was identified using the tractable model system, Dictyostelium discoideum. Using mouse embryonic fibroblasts, we further validate this new mechanism of action. We demonstrate clinical relevance using cells derived from healthy individuals and from people with MS (pwMS). Key Results Both CBD and the more abundant cannabigerol (CBG) enhance mTORC1 activity in D. discoideum. We identify a mechanism for this effect involving inositol polyphosphate multikinase (IPMK), where elevated IPMK expression reverses the response to phytocannabinoids, decreasing mTORC1 activity upon treatment, providing new insight on phytocannabinoids' actions. We further validated this mechanism using mouse embryonic fibroblasts. Clinical relevance of this effect was shown in primary human peripheral blood mononuclear cells, where CBD and CBG treatment increased mTORC1 activity in cells derived from healthy individuals and decreased mTORC1 activity in cells derived from pwMS. Conclusion and Implications Our findings suggest that both CBD and the abundant CBG differentially regulate mTORC1 signalling through a mechanism dependent on the activity of the upstream IPMK signalling pathway, with potential relevance to the treatment of mTOR-related disorders, including MS.

Cannabidiol (CBD), one of the primary non-euphoric components in the Cannabis sativa L. plant, has undergone clinical development over the last number of years as a therapeutic for patients with Lennox-Gastaut syndrome and Dravet syndromes. This phytocannabinoid demonstrates functional and pharmacological diversity, and research data indicate that CBD is a comparable antioxidant to common antioxidants. This review gathers the latest knowledge regarding the impact of CBD on oxidative signalling, with focus on the proclivity of CBD to regulate antioxidants and control the production of reactive oxygen species. CBD is considered an attractive therapeutic agent for neuroimmune disorders, and a body of literature indicates that CBD can regulate redox function at multiple levels, with a range of downstream effects on cells and tissues. However, pro-oxidant capacity of CBD has also been reported, and hence caution must be applied when considering CBD from a therapeutic standpoint. Such pro- and antioxidant functions of CBD may be cell- and model-dependent and may also be influenced by CBD dose, the duration of CBD treatment and the underlying pathology.

Toll-like receptors (TLRs) are sensors of pathogen-associated molecules that trigger inflammatory signalling in innate immune cells including macrophages. All TLRs, with the exception of TLR3, promote intracellular signalling via recruitment of the myeloid differentiation factor 88 (MyD88) adaptor, while TLR3 signals via TollInterleukin-1 Receptor (TIR)-domain-containing adaptor-inducing interferon (IFN)beta (TRIF) adaptor to induce MyD88-independent signalling. Furthermore, TLR4 can activate both MyD88-dependent and -independent signalling (via TRIF). The study aim was to decipher the impact of the highly purified plant-derived (phyto) cannabinoids Delta(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD), when delivered in isolation and in combination (1:1), on MyD88-dependent and -independent signalling in macrophages. We employed the use of the viral dsRNA mimetic poly(I:C) and endotoxin lipopolysaccharide (LPS), to induce viral TLR3 and bacterial TLR4 signalling in human Tamm-Horsfall protein-1 (THP-1)-derived macrophages, respectively. TLR3/TLR4 stimulation promoted the activation of interferon (IFN) regulatory factor 3 (IRF3) and TLR4 promoted the activation of nuclear factor (NF)-kappa B signalling, with downstream production of the type I IFN-beta, the chemokines CXCL10 and CXCL8, and cytokine TNF-alpha. THC and CBD (both at 10 mu M) attenuated TLR3/4-induced IRF3 activation and induction of CXCL10/IFN-beta, while both phytocannabinoids failed to impact TLR4-induced IKB-alpha degradation and TNF-alpha/CXCL8 expression. The role of CB1, CB2 and PPAR gamma receptors in mediating the effect of THC and CBD on MyD88-independent signalling was investigated. TLRs are attractive therapeutic targets given their role in inflammation and initiation of adaptive immunity, and data herein indicate that both CBD and THC preferentially modulate TLR3 and TLR4 signalling via MyD88-independent mechanisms in macrophages. This offers mechanistic insight into the role of phytocannabinoids in modulating cellular inflammation.