Perceptions and engagement of patients with chronic conditions on the use of medical cannabis: a scoping review.

CONTEXT: Studies generally focus on one type of chronic condition and the effect of medical cannabis (MC) on symptoms; little is known about the perceptions and engagement of patients living with chronic conditions regarding the use of MC. OBJECTIVES: This scoping review aims to explore: (1) what are the dimensions addressed in studies on MC that deal with patients' perceptions of MC? and (2) how have patients been engaged in developing these studies and their methodologies? Through these objectives, we have identified areas for improving future research. METHODS: We searched five databases and applied exclusion criteria to select relevant articles. A thematic analysis approach was used to identify the main themes: (1) reasons to use, to stop using or not to use MC, (2) effects of MC on patients themselves and empowerment, (3) perspective and knowledge about MC, and (4) discussion with relatives and healthcare professionals. RESULTS: Of 53 articles, the main interest when assessing the perceptions of MC is to identify the reasons to use MC (n = 39), while few articles focused on the reasons leading to stop using MC (n = 13). The majority (85%) appraise the effects of MC as perceived by patients. Less than one third assessed patients' sense of empowerment. Articles determining the beliefs surrounding and knowledge of MC (n = 41) generally addressed the concerns about or the comfort level with respect to using MC. Only six articles assessed patients' stereotypes regarding cannabis. Concerns about stigma constituted the main topic while assessing relationships with relatives. Some articles included patients in the research, but none of them had co-created the data collection tool with patients. CONCLUSIONS: Our review outlined that few studies considered chronic diseases as a whole and that few patients are involved in the co-construction of data collection tools as well. There is an evidence gap concerning the results in terms of methodological quality when engaging patients in their design. Future research should evaluate why cannabis' effectiveness varies between patients, and how access affects the decision to use or not to use MC, particularly regarding the relationship between patients and healthcare providers. Future research should consider age and gender while assessing perceptions and should take into consideration the legislation status of cannabis as these factors could in fact shape perception. To reduce stigma and stereotypes about MC users, better quality and accessible information on MC should be disseminated.

Vagus Nerve Stimulation Modulates Inflammation in Treatment-Resistant Depression Patients: A Pilot Study.

Vagal neurostimulation (VNS) is used for the treatment of epilepsy and major medical-refractory depression. VNS has neuropsychiatric functions and systemic anti-inflammatory activity. The objective of this study is to measure the clinical efficacy and impact of VNS modulation in depressive patients. Six patients with refractory depression were enrolled. Depression symptoms were assessed with the Montgomery-Asberg Depression Rating, and anxiety symptoms with the Hamilton Anxiety Rating Scale. Plasmas were harvested prospectively before the implantation of VNS (baseline) and up to 4 years or more after continuous therapy. Forty soluble molecules were measured in the plasma by multiplex assays. Following VNS, the reduction in the mean depression severity score was 59.9% and the response rate was 87%. Anxiety levels were also greatly reduced. IL-7, CXCL8, CCL2, CCL13, CCL17, CCL22, Flt-1 and VEGFc levels were significantly lowered, whereas bFGF levels were increased (p values ranging from 0.004 to 0.02). This exploratory study is the first to focus on the long-term efficacy of VNS and its consequences on inflammatory biomarkers. VNS may modulate inflammation via an increase in blood-brain barrier integrity and a reduction in inflammatory cell recruitment. This opens the door to new pathways involved in the treatment of refractory depression.

ALCAM on human oligodendrocytes mediates CD4 T cell adhesion.

Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the CNS. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 Th17 cells can interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we used single cell RNA sequencing, flow cytometry and immunofluorescence studies on CNS tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule (MCAM) and activated leukocyte cell adhesion molecule (ALCAM) in multiple sclerosis, in experimental autoimmune encephalomyelitis and in controls, although their regulation differs between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of MCAM but not of ALCAM at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live imaging, immunofluorescence and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking ALCAM but not MCAM limited prolonged interactions between human primary oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferring significant protection of oligodendrocytic processes. In conclusion, we showed that human oligodendrocytes express MCAM and ALCAM, which are differently modulated by inflammation and T cell contact. We found that ALCAM is a ligand for Th17-polarized cells, contributing to their capacity to adhere and induce damage to human oligodendrocytes, and therefore could represent a relevant target for neuroprotection in multiple sclerosis.

Central activation of the fatty acid sensor GPR120 suppresses microglia reactivity and alleviates sickness- and anxiety-like behaviors.

G protein-coupled receptor 120 (GPR120, Ffar4) is a sensor for long-chain fatty acids including omega-3 polyunsaturated fatty acids (n-3 PUFAs) known for beneficial effects on inflammation, metabolism, and mood. GPR120 mediates the anti-inflammatory and insulin-sensitizing effects of n-3 PUFAs in peripheral tissues. The aim of this study was to determine the impact of GPR120 stimulation on microglial reactivity, neuroinflammation and sickness- and anxiety-like behaviors by acute proinflammatory insults. We found GPR120 mRNA to be enriched in  both murine and human microglia, and in situ hybridization revealed GPR120 expression in microglia of the nucleus accumbens (NAc) in mice. In a manner similar to or exceeding n-3 PUFAs, GPR120 agonism (Compound A, CpdA) strongly attenuated lipopolysaccharide (LPS)-induced proinflammatory marker expression in primary mouse microglia, including tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), and inhibited nuclear factor-ĸB translocation to the nucleus. Central administration of CpdA to adult mice blunted LPS-induced hypolocomotion and anxiety-like behavior and reduced TNF-α, IL-1ß and IBA-1 (microglia marker) mRNA in the NAc, a brain region modulating anxiety and motivation and implicated in neuroinflammation-induced mood deficits. GPR120 agonist pre-treatment attenuated NAc microglia reactivity and alleviated sickness-like behaviors elicited by central injection TNF-α and IL-1ß. These findings suggest that microglial GPR120 contributes to neuroimmune regulation and behavioral changes in response to acute infection and elevated brain cytokines. GPR120 may participate in the protective action of n-3 PUFAs at the neural and behavioral level and offers potential as treatment target for neuroinflammatory conditions.

Classification of T lymphocyte motility behaviors using a machine learning approach.

T lymphocytes migrate into organs and interact with local cells to perform their functions. How human T lymphocytes communicate with organ-specific cells and participate in pathobiological processes remains unresolved. Brain infiltration of T lymphocytes is associated with multiple neurological disorders. Thus, to characterize the behavior of human T lymphocytes reaching the human brain, we performed time-lapse microscopy on human CD8+ T lymphocytes co-cultured with either primary human astrocytes or neurons. Using traditional manual and visual assessment of microscopy data, we identified distinct CD8+ T lymphocyte motility behaviors. However, such characterization is time and labor-intensive. In this work, we trained and validated a machine-learning model for the automated classification of behaviors of CD8+ T lymphocytes interacting with astrocytes and neurons. A balanced random forest was trained for the binary classification of established classes of cell behaviors (synapse vs. kinapse) as well as visually identified behaviors (scanning, dancing, and poking). Feature selection was performed during 3-fold cross-validation using the minimum redundancy maximum relevance algorithm. Results show promising performances when tested on a held-out dataset of CD8+ T lymphocytes interacting with astrocytes with a new experimenter and a held-out independent dataset of CD8+ T lymphocytes interacting with neurons. When tested on the independent CD8+ T cell-neuron dataset, the final model achieved a binary classification accuracy of 0.82 and a 3-class accuracy of 0.79. This novel automated classification approach could significantly reduce the time required to label cell motility behaviors while facilitating the identification of interactions of T lymphocytes with multiple cell types.

GM-CSF distinctly impacts human monocytes and macrophages via ERK1/2-dependent pathways.

Human monocytes and macrophages are two major myeloid cell subsets with similar and distinct functions in tissue homeostasis and immune responses. GM-CSF plays a fundamental role in myeloid cell differentiation and activation. Hence, we compared the effects of GM-CSF on the expression of several immune mediators by human monocytes and monocyte-derived macrophages obtained from healthy donors. We report that GM-CSF similarly elevated the expression of CD80 and ICAM-1 and reduced HLA-DR levels on both myeloid cell subsets. However, GM-CSF increased the percentage of macrophages expressing surface IL-15 but reduced the proportion of monocytes carrying surface IL-15. Moreover, GM-CSF significantly increased the secretion of IL-4, IL-6, TNF, CXCL10, and IL-27 by macrophages while reducing the secretion of IL-4 and CXCL10 by monocytes. We show that GM-CSF triggered ERK1/2, STAT3, STAT5, and SAPK/JNK pathways in both myeloid subsets. Using a pharmacological inhibitor (U0126) preventing ERK phosphorylation, we demonstrated that this pathway was involved in both the GM-CSF-induced increase and decrease of the percentage of IL-15+ macrophages and monocytes, respectively. Moreover, ERK1/2 contributed to GM-CSF-triggered secretion of IL-4, IL-6, TNF, IL-27 and CXCL10 by macrophages. However, the ERK1/2 pathway exhibited different roles in monocytes and macrophages for the GM-CSF-mediated impact on surface makers (CD80, HLA-DR, and ICAM-1). Our data demonstrate that GM-CSF stimulation induces differential responses by human monocytes and monocyte-derived macrophages and that some but not all of these effects are ERK-dependent.

Impact of aging on treatment considerations for multiple sclerosis patients.

With a rapidly aging global population and improvement of outcomes with newer multiple sclerosis (MS)-specific disease-modifying therapies (DMTs), the epidemiology of MS has shifted to an older than previously described population, with a peak prevalence of the disease seen in the 55-65 years age group. Changes in the pathophysiology of MS appear to be age-dependent. Several studies have identified a consistent phase of disability worsening around the fifth decade of life. The latter appears to be independent of prior disease duration and inflammatory activity and concomitant to pathological changes from acute focal active demyelination to chronic smoldering plaques, slow-expanding lesions, and compartmentalized inflammation within the central nervous system (CNS). On the other hand, decreased CNS tissue reserve and poorer remyelinating capacity with aging lead to loss of relapse recovery potential. Aging with MS may imply longer exposure to DMTs, although treatment efficacy in patients >55 years has not been evaluated in pivotal randomized controlled trials and appears to decrease with age. Older individuals are more prone to adverse effects of DMTs, an important aspect of treatment individualization. Aging with MS also implies a higher global burden of comorbid illnesses that contribute to overall impairments and represent a crucial confounder in interpreting clinical worsening. Discontinuation of DMTs after age 55, when no evidence of clinical or radiological activity is detected, is currently under the spotlight. In this review, we will discuss the impact of aging on MS pathobiology, the effect of comorbidities and other confounders on clinical worsening, and focus on current therapeutic considerations in this age group.

Analysis of the microglia transcriptome across the human lifespan using single cell RNA sequencing.

BACKGROUND: Microglia are tissue resident macrophages with a wide range of critically important functions in central nervous system development and homeostasis. METHOD: In this study, we aimed to characterize the transcriptional landscape of ex vivo human microglia across different developmental ages using cells derived from pre-natal, pediatric, adolescent, and adult brain samples. We further confirmed our transcriptional observations using ELISA and RNAscope. RESULTS: We showed that pre-natal microglia have a distinct transcriptional and regulatory signature relative to their post-natal counterparts that includes an upregulation of phagocytic pathways. We confirmed upregulation of CD36, a positive regulator of phagocytosis, in pre-natal samples compared to adult samples in situ. Moreover, we showed adult microglia have more pro-inflammatory signature compared to microglia from other developmental ages. We indicated that adult microglia are more immune responsive by secreting increased levels of pro-inflammatory cytokines in response to LPS treatment compared to the pre-natal microglia. We further validated in situ up-regulation of IL18 and CXCR4 in human adult brain section compared to the pre-natal brain section. Finally, trajectory analysis indicated that the transcriptional signatures adopted by microglia throughout development are in response to a changing brain microenvironment and do not reflect predetermined developmental states. CONCLUSION: In all, this study provides unique insight into the development of human microglia and a useful reference for understanding microglial contribution to developmental and age-related human disease.

Investigating anti-inflammatory and immunomodulatory properties of brivaracetam and lacosamide in experimental autoimmune encephalomyelitis (EAE).

PURPOSE: Inflammation plays a role in drug-resistant epilepsy (DRE). We have previously reported an increased proportion of CD4 T cells displaying a pro-inflammatory profile in the peripheral blood of adults with DRE. Specific anti-epileptic drugs (AEDs) exhibit immunomodulatory properties that could increase the risk of infections but also contribute to their beneficial impact on DRE and other neurological diseases. The impact of novel generation AEDs on the profile of immune cells and on neuroinflammatory processes remains unclear. METHODS: We compared the influence of brivaracetam and lacosamide on the activation of human and murine peripheral immune cells in vitro and in vivo in active experimental autoimmune encephalomyelitis (EAE), a common mouse model of central nervous system inflammation. RESULTS: We found that brivaracetam and lacosamide at 2.5 µg/ml did not impair the survival and activation of human immune cells, but a higher dose of 25 µg/ml decreased mitogen-induced proliferation of CD8 T cells in vitro. Exposure to high doses of brivaracetam, and to a lesser extent lacosamide, reduced the proportion of CD25+ and CD107a+ CD8+ human T cells in vitro, and the frequency of CNS-infiltrating CD8+ T cells at EAE onset and CD11b+ myeloid cells at peak in vivo. Prophylactic administration of brivaracetam or lacosamide did not delay EAE onset but significantly improved the clinical course in the chronic phase of EAE compared to control. CONCLUSION: Novel generation AEDs do not impair the response to immunization with MOG peptide but improve the course of EAE, possibly through a reduction of neuroaxonal damage.

Specific alterations in NKG2D+ T lymphocytes in relapsing-remitting and progressive multiple sclerosis patients.

BACKGROUND: T lymphocytes exhibit numerous alterations in relapsing-remitting (RRMS), secondary progressive (SPMS), and primary progressive multiple sclerosis (PPMS). The NKG2D pathway has been involved in MS pathology. NKG2D is a co-activating receptor on subsets of CD4+ and most CD8+ T lymphocytes. The ligands of NKG2D are expressed at low levels in normal tissues but are elevated in MS postmortem brain tissues compared with controls. Whether the NKG2D pathway shows specific changes in different forms of MS remains unclear. METHODS: We performed unsupervised and supervised flow cytometry analysis to characterize peripheral blood T lymphocytes from RRMS, SPMS, and PPMS patients and healthy controls (HC). We used an in vitro microscopy approach to assess the role of NKG2D in the interactions between human CD8+T lymphocytes and human astrocytes. RESULTS: Specific CD8+, CD4+, and CD4-CD8- T cell populations exhibited altered frequency in MS patients' subgroups. The proportion of NKG2D+ T lymphocytes declined with age in PPMS patients but not in RRMS and HC. This reduced percentage of NKG2D+ cells was due to lower abundance of γδ and αß CD4-CD8- T lymphocytes in PPMS patients. NKG2D+ T lymphocytes were significantly less abundant in RRMS than in HC; this was caused by a decreased frequency of CD4-CD8- and CD8+ T lymphocytes and was not linked to age. Blocking NKG2D increased the motility of CD8+ T lymphocytes co-cultured with astrocytes expressing NKG2D ligand. Moreover, preventing NKG2D from interacting with its ligands increased the proportion of CD8+ T lymphocytes exhibiting a kinapse-like behavior characterized by short-term interaction while reducing those displaying a long-lasting synapse-like behavior. These results support that NKG2D participates in the establishment of long-term interactions between activated CD8+ T lymphocytes and astrocytes. CONCLUSION: Our data demonstrate specific alterations in NKG2D+ T lymphocytes in MS patients' subgroups and suggest that NKG2D contributes to the interactions between human CD8+ T lymphocytes and human astrocytes.

Single-Cell Transcriptomics Identifies Brain Endothelium Inflammatory Networks in Experimental Autoimmune Encephalomyelitis.

BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by infiltration of immune cells in multifocal areas of the CNS. The specific molecular processes allowing autoreactive immune cells to enter the CNS compartment through the blood-brain barrier remain elusive. METHODS: Using endothelial cell (EC) enrichment and single-cell RNA sequencing, we characterized the cells implicated in the neuroinflammatory processes in experimental autoimmune encephalomyelitis, an animal model of MS. Validations on human MS brain sections of the most differentially expressed genes in venous ECs were performed using immunohistochemistry and confocal microscopy. RESULTS: We found an upregulation of genes associated with antigen presentation and interferon in most populations of CNS-resident cells, including ECs. Interestingly, instead of transcriptionally distinct profiles, a continuous gradient of gene expression separated the arteriovenous zonation of the brain vasculature. However, differential gene expression analysis presented more transcriptomic alterations on the venous side of the axis, suggesting a prominent role of venous ECs in neuroinflammation. Furthermore, analysis of ligand-receptor interactions identified important potential molecular communications between venous ECs and infiltrated immune populations. To confirm the relevance of our observation in the context of human disease, we validated the protein expression of the most upregulated genes (Ackr1 and Lcn2) in MS lesions. DISCUSSION: In this study, we provide a landscape of the cellular heterogeneity associated with neuroinflammation. We also present important molecular insights for further exploration of specific cell processes that promote infiltration of immune cells inside the brain of experimental autoimmune encephalomyelitis mice.

Granulocyte-macrophage colony-stimulating factor-stimulated human macrophages demonstrate enhanced functions contributing to T-cell activation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated in numerous chronic inflammatory diseases, including multiple sclerosis (MS). GM-CSF impacts multiple properties and functions of myeloid cells via species-specific mechanisms. Therefore, we assessed the effect of GM-CSF on different human myeloid cell populations found in MS lesions: monocyte-derived macrophages (MDMs) and microglia. We previously reported a greater number of interleukin (IL)-15+ myeloid cells in the brain of patients with MS than in controls. Therefore, we investigated whether GM-CSF exerts its deleterious effects in MS by increasing IL-15 expression on myeloid cells. We found that GM-CSF increased the proportion of IL-15+ cells and/or IL-15 levels on nonpolarized, M1-polarized and M2-polarized MDMs from healthy donors and patients with MS. GM-CSF also increased IL-15 levels on human adult microglia. When cocultured with GM-CSF-stimulated MDMs, activated autologous CD8+ T lymphocytes secreted and expressed significantly higher levels of effector molecules (e.g. interferon-γ and GM-CSF) compared with cocultures with unstimulated MDMs. However, neutralizing IL-15 did not attenuate enhanced effector molecule expression on CD8+ T lymphocytes triggered by GM-CSF-stimulated MDMs. We showed that GM-CSF stimulation of MDMs increased their expression of CD80 and ICAM-1 and their secretion of IL-6, IL-27 and tumor necrosis factor. These molecules could participate in boosting the effector properties of CD8+ T lymphocytes independently of IL-15. By contrast, GM-CSF did not alter CD80, IL-27, tumor necrosis factor and chemokine (C-X-C motif) ligand 10 expression/secretion by human microglia. Therefore, our results underline the distinct impact of GM-CSF on human myeloid cells abundantly present in MS lesions.

MCAM+ brain endothelial cells contribute to neuroinflammation by recruiting pathogenic CD4+ T lymphocytes.

The trafficking of autoreactive leucocytes across the blood-brain barrier endothelium is a hallmark of multiple sclerosis pathogenesis. Although the blood-brain barrier endothelium represents one of the main CNS borders to interact with the infiltrating leucocytes, its exact contribution to neuroinflammation remains understudied. Here, we show that Mcam identifies inflammatory brain endothelial cells with pro-migratory transcriptomic signature during experimental autoimmune encephalomyelitis. In addition, MCAM was preferentially upregulated on blood-brain barrier endothelial cells in multiple sclerosis lesions in situ and at experimental autoimmune encephalomyelitis disease onset by molecular MRI. In vitro and in vivo, we demonstrate that MCAM on blood-brain barrier endothelial cells contributes to experimental autoimmune encephalomyelitis development by promoting the cellular trafficking of TH1 and TH17 lymphocytes across the blood-brain barrier. Last, we showcase ST14 as an immune ligand to brain endothelial MCAM, enriched on CD4+ T lymphocytes that cross the blood-brain barrier in vitro, in vivo and in multiple sclerosis lesions as detected by flow cytometry on rapid autopsy derived brain tissue from multiple sclerosis patients. Collectively, our findings reveal that MCAM is at the centre of a pathological pathway used by brain endothelial cells to recruit pathogenic CD4+ T lymphocyte from circulation early during neuroinflammation. The therapeutic targeting of this mechanism is a promising avenue to treat multiple sclerosis.

CLMP Promotes Leukocyte Migration Across Brain Barriers in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: In multiple sclerosis (MS), peripheral immune cells use various cell trafficking molecules to infiltrate the CNS where they cause damage.The objective of this study was to investigate the involvement of coxsackie and adenovirus receptor-like membrane protein (CLMP) in the migration of immune cells into the CNS of patients with MS. METHODS: Expression of CLMP was measured in primary cultures of human brain endothelial cells (HBECs) and human meningeal endothelial cells (HMECs), postmortem brain samples, and peripheral blood mononuclear cells (PBMCs) from patients with MS and controls by RNA sequencing, quantitative PCR, immunohistochemistry, and flow cytometry. In vitro migration assays using HBECs and HMECs were performed to evaluate the function of CLMP. RESULTS: Using bulk RNA sequencing of primary cultures of human brain and meningeal endothelial cells (ECs), we have identified CLMP as a new potential cell trafficking molecule upregulated in inflammatory conditions. We first confirmed the upregulation of CLMP at the protein level on TNFα-activated and IFNγ-activated primary cultures of human brain and meningeal ECs. In autopsy brain specimens from patients with MS, we demonstrated an overexpression of endothelial CLMP in active MS lesions when compared with normal control brain tissue. Flow cytometry of human PBMCs demonstrated an increased frequency of CLMP+ B lymphocytes and monocytes in patients with MS, when compared with that in healthy controls. The use of a blocking antibody against CLMP reduced the migration of immune cells across the human brain and meningeal ECs in vitro. Finally, we found CLMP+ immune cell infiltrates in the perivascular area of parenchymal lesions and in the meninges of patients with MS. DISCUSSION: Collectively, our data demonstrate that CLMP is an adhesion molecule used by immune cells to access the CNS during neuroinflammatory disorders such as MS. CLMP could represent a target for a new treatment of neuroinflammatory conditions.

IL-27 shapes the immune properties of human astrocytes and their impact on encountered human T lymphocytes.

BACKGROUND: Interleukin-27 (IL-27) can trigger both pro- and anti-inflammatory responses. This cytokine is elevated in the central nervous system (CNS) of multiple sclerosis (MS) patients, but how it influences neuroinflammatory processes remains unclear. As astrocytes express the receptor for IL-27, we sought to determine how these glial cells respond to this cytokine and whether such exposure alters their interactions with infiltrating activated T lymphocytes. To determine whether inflammation shapes the impact of IL-27, we compared the effects of this cytokine in non-inflamed and inflamed conditions induced by an IL-1ß exposure. MAIN BODY: Transcriptomic analysis of IL-27-exposed human astrocytes showed an upregulation of multiple immune genes. Human astrocytes increased the secretion of chemokines (CXCL9, CXCL10, and CXCL11) and the surface expression of proteins (PD-L1, HLA-E, and ICAM-1) following IL-27 exposure. To assess whether exposure of astrocytes to IL-27 influences the profile of activated T lymphocytes infiltrating the CNS, we used an astrocyte/T lymphocyte co-culture model. Activated human CD4+ or CD8+ T lymphocytes were co-cultured with astrocytes that have been either untreated or pre-exposed to IL­27 or IL-1ß. After 24 h, we analyzed T lymphocytes by flow cytometry for transcription factors and immune molecules. The contact with IL-27-exposed astrocytes increased the percentages of T-bet, Eomes, CD95, IL-18Rα, ICAM-1, and PD-L1 expressing CD4+ and CD8+ T lymphocytes and reduced the proportion of CXCR3-positive CD8+ T lymphocytes. Human CD8+ T lymphocytes co-cultured with human IL-27-treated astrocytes exhibited higher motility than when in contact with untreated astrocytes. These results suggested a preponderance of kinapse-like over synapse-like interactions between CD8+ T lymphocytes and IL-27-treated astrocytes. Finally, CD8+ T lymphocytes from MS patients showed higher motility in contact with IL-27-exposed astrocytes compared to healthy donors' cells. CONCLUSION: Our results establish that IL-27 alters the immune functions of human astrocytes and shapes the profile and motility of encountered T lymphocytes, especially CD8+ T lymphocytes from MS patients.

Contact-Dependent Granzyme B-Mediated Cytotoxicity of Th17-Polarized Cells Toward Human Oligodendrocytes.

Multiple sclerosis (MS) is characterized by the loss of myelin and of myelin-producing oligodendrocytes (OLs) in the central nervous system (CNS). Pro-inflammatory CD4+ Th17 cells are considered pathogenic in MS and are harmful to OLs. We investigated the mechanisms driving human CD4+ T cell-mediated OL cell death. Using fluorescent and brightfield in vitro live imaging, we found that compared to Th2-polarized cells, Th17-polarized cells show greater interactions with primary human OLs and human oligodendrocytic cell line MO3.13, displaying longer duration of contact, lower mean speed, and higher rate of vesicle-like structure formation at the sites of contact. Using single-cell RNA sequencing, we assessed the transcriptomic profile of primary human OLs and Th17-polarized cells in direct contact or separated by an insert. We showed that upon close interaction, OLs upregulate the expression of mRNA coding for chemokines and antioxidant/anti-apoptotic molecules, while Th17-polarized cells upregulate the expression of mRNA coding for chemokines and pro-inflammatory cytokines such as IL-17A, IFN-γ, and granzyme B. We found that secretion of CCL3, CXCL10, IFN-γ, TNFα, and granzyme B is induced upon direct contact in cocultures of human Th17-polarized cells with human OLs. In addition, we validated by flow cytometry and immunofluorescence that granzyme B levels are upregulated in Th17-polarized compared to Th2-polarized cells and are even higher in Th17-polarized cells upon direct contact with OLs or MO3.13 cells compared to Th17-polarized cells separated from OLs by an insert. Moreover, granzyme B is detected in OLs and MO3.13 cells following direct contact with Th17-polarized cells, suggesting the release of granzyme B from Th17-polarized cells into OLs/MO3.13 cells. To confirm granzyme B-mediated cytotoxicity toward OLs, we showed that recombinant human granzyme B can induce OLs and MO3.13 cell death. Furthermore, pretreatment of Th17-polarized cells with a reversible granzyme B blocker (Ac-IEPD-CHO) or a natural granzyme B blocker (serpina3N) improved survival of MO3.13 cells upon coculture with Th17 cells. In conclusion, we showed that human Th17-polarized cells form biologically significant contacts with human OLs and exert direct toxicity by releasing granzyme B.

Bacillus Calmette-Guerin vaccination and multiple sclerosis: A population-based birth cohort study in Quebec, Canada.

BACKGROUND AND PURPOSE: The bacillus Calmette-Guerin (BCG) vaccine could reduce the incidence of multiple sclerosis (MS) through immunomodulation. Previous studies, presenting some limitations, reported no association. We re-examined this association in a large cohort focusing on relapsing-remitting MS (RRMS). METHODS: The cohort included 400,563 individuals, and was linked with the Quebec provincial BCG vaccination registry and administrative health data. Individuals were followed up from 1983 to 2014 and then within Period 1 (1983-1996) and Period 2 (1997-2014), for the occurrence of MS. Incident MS cases were defined as those with ≥3 hospital or physician claims for MS. Subjects with ≥1 drug reimbursement for MS disease-modifying therapies were classified as RRMS. Cox proportional hazards regression was used to estimate hazard ratios (HRs) over the follow-ups, adjusting for potential confounders. Possible effect modification due to sex was assessed. RESULTS: A total of 178,335 (46%) individuals were BCG vaccinated. There were 274 (0.06%) incident MS cases identified in 1983-1996, and 1433 (0.4%) in 1997-2014. No association was found with RRMS, either in Period 1 (adjusted HR [HRadj ] = 0.96, 95% confidence interval [CI] = 0.63-1.45; 96 cases) or in Period 2 (HRadj  = 1.02, 95% CI = 0.85-1.23; 480 cases). The remaining MS cases, for whom the phenotype was unknown, were positively associated with BCG over the entire follow-up (HRadj  = 1.25, 95% CI = 1.10-1.41; 1131 cases) and in Period 2 (HRadj  = 1.33, 95% CI = 1.17-1.52; 953 cases). No interaction with sex was found. CONCLUSIONS: Findings suggest that BCG vaccination does not decrease the risk of RRMS, and that future studies should consider phenotypes of MS.

DICAM promotes TH17 lymphocyte trafficking across the blood-brain barrier during autoimmune neuroinflammation.

The migration of circulating leukocytes into the central nervous system (CNS) is a key driver of multiple sclerosis (MS) pathogenesis. The monoclonal antibody natalizumab proved that pharmaceutically obstructing this process is an effective therapeutic approach for treating relapsing-remitting MS (RRMS). Unfortunately, the clinical efficacy of natalizumab is somewhat offset by its incapacity to control the progressive forms of MS (PMS) and by life-threatening side effects in RRMS rising from the expression of its molecular target, very late antigen 4 (VLA4), on most immune cells and consequent impairment of CNS immunosurveillance. Here, we identified dual immunoglobulin domain containing cell adhesion molecule (DICAM) as a cell trafficking molecule preferentially expressed by T helper 17 (TH17)­polarized CD4+ T lymphocytes. We found that DICAM expression on circulating CD4+ T cells was increased in patients with active RRMS and PMS disease courses, and expression of DICAM ligands was increased on the blood-brain barrier endothelium upon inflammation and in MS lesions. Last, we demonstrated that pharmaceutically neutralizing DICAM reduced murine and human TH17 cell trafficking across the blood-brain barrier in vitro and in vivo, and alleviated disease symptoms in four distinct murine autoimmune encephalomyelitis models, including relapsing-remitting and progressive disease models. Collectively, our data highlight DICAM as a candidate therapeutic target to impede the migration of disease-inducing leukocytes into the CNS in both RRMS and PMS and suggest that blocking DICAM with a monoclonal antibody may be a promising therapeutic approach.

Stress Signal ULBP4, an NKG2D Ligand, Is Upregulated in Multiple Sclerosis and Shapes CD8+ T-Cell Behaviors.

BACKGROUND AND OBJECTIVES: We posit the involvement of the natural killer group 2D (NKG2D) pathway in multiple sclerosis (MS) pathology via the presence of specific NKG2D ligands (NKG2DLs). We aim to evaluate the expression of NKG2DLs in the CNS and CSF of patients with MS and to identify cellular stressors inducing the expression of UL16-binding protein 4 (ULBP4), the only detectable NKG2DL. Finally, we evaluate the impact of ULBP4 on functions such as cytokine production and motility by CD8+ T lymphocytes, a subset largely expressing NKG2D, the cognate receptor. METHODS: Human postmortem brain samples and CSF from patients with MS and controls were used to evaluate NKG2DL expression. In vitro assays using primary cultures of human astrocytes and neurons were performed to identify stressors inducing ULBP4 expression. Human CD8+ T lymphocytes from MS donors and age/sex-matched healthy controls were isolated to evaluate the functional impact of soluble ULBP4. RESULTS: We detected mRNA coding for the 8 identified human NKG2DLs in brain samples from patients with MS and controls, but only ULBP4 protein expression was detectable by Western blot. ULBP4 levels were greater in patients with MS, particularly in active and chronic active lesions and normal-appearing white matter, compared with normal-appearing gray matter from MS donors and white and gray matter from controls. Soluble ULBP4 was also detected in CSF of patients with MS and controls, but a smaller shed/soluble form of 25 kDa was significantly elevated in CSF from female patients with MS compared with controls and male patients with MS. Our data indicate that soluble ULBP4 affects various functions of CD8+ T lymphocytes. First, it enhanced the production of the proinflammatory cytokines GM-CSF and interferon-γ (IFNγ). Second, it increased CD8+ T lymphocyte motility and favored a kinapse-like behavior when cultured in the presence of human astrocytes. CD8+ T lymphocytes from patients with MS were especially altered by the presence of soluble ULBP4 compared with healthy controls. DISCUSSION: Our study provides new evidence for the involvement of NKG2D and its ligand ULBP4 in MS pathology. Our results point to ULBP4 as a viable target to specifically block 1 component of the NKG2D pathway without altering immune surveillance involving other NKG2DL.