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.

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.

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.

The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

Immune changes occur in experimental and clinical epilepsy. Here, we tested the hypothesis that during epileptogenesis and spontaneous recurrent seizures (SRS) an impairment of the endogenous anti-inflammatory pathway glucocorticoid receptor (GR)-annexin A1 (ANXA1) occurs. By administrating exogenous ANXA1, we studied whether pharmacological potentiation of the anti-inflammatory response modifies seizure activity and pathophysiology. We used an in vivo model of temporal lobe epilepsy based on intrahippocampal kainic acid (KA) injection. Video-electroencephalography, molecular biology analyses on brain and peripheral blood samples, and pharmacological investigations were performed in this model. Human epileptic cortices presenting type II focal cortical dysplasia (IIa and b), hippocampi with or without hippocampal sclerosis (HS), and available controls were used to study ANXA1 expression. A decrease of phosphorylated (phospho-) GR and phospho-GR/tot-GR protein expression occurred in the hippocampus during epileptogenesis. Downstream to GR, the anti-inflammatory protein ANXA1 remained at baseline levels while inflammation installed and endured. In peripheral blood, ANXA1 and corticosterone levels showed no significant modifications during disease progression except for an early and transient increase poststatus epilepticus. These results indicate inadequate ANXA1 engagement over time and in these experimental conditions. By analyzing human brain specimens, we found that where significant inflammation exists, the pattern of ANXA1 immunoreactivity was abnormal because the typical perivascular ANXA1 immunoreactivity was reduced. We next asked whether potentiation of the endogenous anti-inflammatory mechanism by ANXA1 administration modifies the disease pathophysiology. Although with varying efficacy, administration of exogenous ANXA1 somewhat reduced the time spent in seizure activity as compared to saline. These results indicate that the anti-inflammatory GR-ANXA1 pathway is defective during experimental seizure progression. The prospect of pharmacologically restoring or potentiating this endogenous anti-inflammatory mechanism as an add-on therapeutic strategy for specific forms of epilepsy is proposed.-Zub, E., Canet, G., Garbelli, R., Blaquiere, M., Rossini, L., Pastori, C., Sheikh, M., Reutelingsperger, C., Klement, W., de Bock, F., Audinat, E., Givalois, L., Solito, E., Marchi, N. The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

A pericyte-glia scarring develops at the leaky capillaries in the hippocampus during seizure activity.

OBJECTIVE: Inflammatory cerebrovascular damage occurs in epilepsy. Here, we tested the hypothesis that a pericyte-glia scar forms around the outer wall of hippocampal capillaries in a model of temporal lobe epilepsy associated with hippocampal sclerosis. We studied the participation of stromal cells expressing platelet-derived growth factor receptor beta (PDGFRß) and extracellular matrix modifications to the perivascular scar during epileptogenesis. METHODS: We used NG2DsRed/C57BL6 mice and induced status epilepticus (SE) followed by epileptogenesis and spontaneous recurrent seizures (SRS) by means of unilateral intrahippocampal injection of kainic acid (KA). For pharmacological assessment, we used organotypic hippocampal cultures (OHCs) where ictal electrographic activity was elicited by KA or bicuculline. RESULTS: NG2DsRed pericytes, GFAP astroglia, and IBA1 microglia are reactive and converge to form a pericapillary multicellular scar in the CA hippocampal regions during epileptogenesis and at SRS. The capillaries are leaky as indicated by fluorescein entering the parenchyma from the peripheral blood. Concomitantly, PDGFRß transcript and protein levels were significantly increased. Within the regional scar, a fibrotic-like PDGFRß mesh developed around the capillaries, peaking at 1 week post-SE and regressing, but not resolving, at SRS. Abnormal distribution or accumulation of extracellular matrix collagens III/IV occurred in the CA regions during seizure progression. PDGFRß/DAPI cells were in direct contact with or adjacent to the damaged NG2DsRed pericytes at the capillary interface, consistent with the notion of stromal cell reactivity or fibroblast formation. Inducing electrographic activity in OHCs was sufficient to augment PDGFRß reactivity around the capillaries. The latter effect was pharmacologically mimicked by treating OHCs with the PDGFRß agonist PDGF-BB and it was diminished by the PDGFRß inhibitor imatinib. SIGNIFICANCE: The reported multicellular activation and scar are traits of perivascular inflammation and hippocampal sclerosis in experimental epilepsy, with an implication for neurovascular dysfunction. Modulation of PDGFRß could be exploited to target inflammation in this chronic disease setting.

Seizure progression and inflammatory mediators promote pericytosis and pericyte-microglia clustering at the cerebrovasculature.

BACKGROUND: Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications. METHODS: In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1ß, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly. RESULTS: A disarray of NG2DsRed+ pericyte soma and ramifications was found 72 h post-SE and 1 week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72 h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1ß elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes. CONCLUSIONS: These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.

The pericyte-glia interface at the blood-brain barrier.

The cerebrovasculature is a multicellular structure with varying rheological and permeability properties. The outer wall of the brain capillary endothelium is enclosed by pericytes and astrocyte end feet, anatomically assembled to guarantee barrier functions. We, here, focus on the pericyte modifications occurring in disease conditions, reviewing evidence supporting the interplay amongst pericytes, the endothelium, and glial cells in health and pathology. Deconstruction and reactivity of pericytes and glial cells around the capillary endothelium occur in response to traumatic brain injury, epilepsy, and neurodegenerative disorders, impacting vascular permeability and participating in neuroinflammation. As this represents a growing field of research, addressing the multicellular reorganization occurring at the outer wall of the blood-brain barrier (BBB) in response to an acute insult or a chronic disease could disclose novel disease mechanisms and therapeutic targets.

Regulation of stress granule formation in human oligodendrocytes.

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.

Ferroptosis induces detrimental effects in chronic EAE and its implications for progressive MS.

Ferroptosis is a form of lipid peroxidation-mediated cell death and damage triggered by excess iron and insufficiency in the glutathione antioxidant pathway. Oxidative stress is thought to play a crucial role in progressive forms of multiple sclerosis (MS) in which iron deposition occurs. In this study we assessed if ferroptosis plays a role in a chronic form of experimental autoimmune encephalomyelitis (CH-EAE), a mouse model used to study MS. Changes were detected in the mRNA levels of several ferroptosis genes in CH-EAE but not in relapsing-remitting EAE. At the protein level, expression of iron importers is increased in the earlier stages of CH-EAE (onset and peak). While expression of hemoxygenase-1, which mobilizes iron from heme, likely from phagocytosed material, is increased in macrophages at the peak and progressive stages. Excess iron in cells is stored safely in ferritin, which increases with disease progression. Harmful, redox active iron is released from ferritin when shuttled to autophagosomes by 'nuclear receptor coactivator 4' (NCOA4). NCOA4 expression increases at the peak and progressive stages of CH-EAE and accompanied by increase in redox active ferrous iron. These changes occur in parallel with reduction in the antioxidant pathway (system xCT, glutathione peroxidase 4 and glutathione), and accompanied by increased lipid peroxidation. Mice treated with a ferroptosis inhibitor for 2 weeks starting at the peak of CH-EAE paralysis, show significant improvements in function and pathology. Autopsy samples of tissue sections of secondary progressive MS (SPMS) showed NCOA4 expression in macrophages and oligodendrocytes along the rim of mixed active/inactive lesions, where ferritin+ and iron containing cells are located. Cells expressing NCOA4 express less ferritin, suggesting ferritin degradation and release of redox active iron, as indicated by increased lipid peroxidation. These data suggest that ferroptosis is likely to contribute to pathogenesis in CH-EAE and SPMS.

CAR Protects Females from Diet-Induced Steatosis and Associated Metabolic Disorders.

Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common cause of chronic liver disease worldwide, affecting 70-90% of obese individuals. In humans, a lower NAFLD incidence is reported in pre-menopausal women, although the mechanisms affording this protection remain under-investigated. Here, we tested the hypothesis that the constitutive androstane nuclear receptor (CAR) plays a role in the pathogenesis of experimental NAFLD. Male and female wild-type (WT) and CAR knock-out (CAR-/-) mice were subjected to a high-fat diet (HFD) for 16 weeks. We examined the metabolic phenotype of mice through body weight follow-up, glucose tolerance tests, analysis of plasmatic metabolic markers, hepatic lipid accumulation, and hepatic transcriptome. Finally, we examined the potential impact of HFD and CAR deletion on specific brain regions, focusing on glial cells. HFD-induced weight gain and hepatic steatosis are more pronounced in WT males than females. CAR-/- females present a NASH-like hepatic transcriptomic signature suggesting a potential NAFLD to NASH transition. Transcriptomic correlation analysis highlighted a possible cross-talk between CAR and ERα receptors. The peripheral effects of CAR deletion in female mice were associated with astrogliosis in the hypothalamus. These findings prove that nuclear receptor CAR may be a potential mechanism entry-point and a therapeutic target for treating NAFLD/NASH.

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.

Mechanisms of metabolic stress induced cell death of human oligodendrocytes: relevance for progressive multiple sclerosis.

Oligodendrocyte (OL) injury and loss are central features of evolving lesions in multiple sclerosis. Potential causative mechanisms of OL loss include metabolic stress within the lesion microenvironment. Here we use the injury response of primary human OLs (hOLs) to metabolic stress (reduced glucose/nutrients) in vitro to help define the basis for the in situ features of OLs in cases of MS. Under metabolic stress in vitro, we detected reduction in ATP levels per cell that precede changes in survival. Autophagy was initially activated, although ATP levels were not altered by inhibitors (chloroquine) or activators (Torin-1). Prolonged stress resulted in autophagy failure, documented by non-fusion of autophagosomes and lysosomes. Consistent with our in vitro results, we detected higher expression of LC3, a marker of autophagosomes in OLs, in MS lesions compared to controls. Both in vitro and in situ, we observe a reduction in nuclear size of remaining OLs. Prolonged stress resulted in increased ROS and cleavage of spectrin, a target of Ca2+-dependent proteases. Cell death was however not prevented by inhibitors of ferroptosis or MPT-driven necrosis, the regulated cell death (RCD) pathways most likely to be activated by metabolic stress. hOLs have decreased expression of VDAC1, VDAC2, and of genes regulating iron accumulation and cyclophilin. RNA sequencing analyses did not identify activation of these RCD pathways in vitro or in MS cases. We conclude that this distinct response of hOLs, including resistance to RCD, reflects the combined impact of autophagy failure, increased ROS, and calcium influx, resulting in metabolic collapse and degeneration of cellular structural integrity. Defining the basis of OL injury and death provides guidance for development of neuro-protective strategies.

Obesity promotes fumonisin B1 hepatotoxicity.

Obesity, which is a worldwide public health issue, is associated with chronic inflammation that contribute to long-term complications, including insulin resistance, type 2 diabetes and non-alcoholic fatty liver disease. We hypothesized that obesity may also influence the sensitivity to food contaminants, such as fumonisin B1 (FB1), a mycotoxin produced mainly by the Fusarium verticillioides. FB1, a common contaminant of corn, is the most abundant and best characterized member of the fumonisins family. We investigated whether diet-induced obesity could modulate the sensitivity to oral FB1 exposure, with emphasis on gut health and hepatotoxicity. Thus, metabolic effects of FB1 were assessed in obese and non-obese male C57BL/6J mice. Mice received a high-fat diet (HFD) or normal chow diet (CHOW) for 15 weeks. Then, during the last three weeks, mice were exposed to these diets in combination or not with FB1 (10 mg/kg body weight/day) through drinking water. As expected, HFD feeding induced significant body weight gain, increased fasting glycemia, and hepatic steatosis. Combined exposure to HFD and FB1 resulted in body weight loss and a decrease in fasting blood glucose level. This co-exposition also induces gut dysbiosis, an increase in plasma FB1 level, a decrease in liver weight and hepatic steatosis. Moreover, plasma transaminase levels were significantly increased and associated with liver inflammation in HFD/FB1-treated mice. Liver gene expression analysis revealed that the combined exposure to HFD and FB1 was associated with reduced expression of genes involved in lipogenesis and increased expression of immune response and cell cycle-associated genes. These results suggest that, in the context of obesity, FB1 exposure promotes gut dysbiosis and severe liver inflammation. To our knowledge, this study provides the first example of obesity-induced hepatitis in response to a food contaminant.

Tissular Genomic Responses to Oral FB1 Exposure in Pigs.

Fumonisin B1 (FB1) is a widespread mycotoxin produced by fungal Fusarium species-mainly in maize, one of the plants most commonly used for food and feed. Pigs and horses are the animal species most susceptible to this mycotoxin. FB1 exposure can cause highly diverse clinical symptoms, including hepatotoxicity, immunotoxicity, and intestinal barrier function disturbance. Inhibition of ceramide synthetase is a well-understood ubiquitous molecular mechanism of FB1 toxicity, but other more tissue-specific effects remain to be elucidated. To investigate the effects of FB1 in different exposed tissues, we cross-analyzed the transcriptomes of fours organs: liver, jejunum, jejunal Peyer's patches, and spleen. During a four-week study period, pigs were fed a control diet or a FB1-contaminated diet (10 mg/kg feed). In response to oral FB1 exposure, we observed common biological processes in the four organs, including predominant and recurrent processes (extracellular matrix organization, integrin activation, granulocyte chemotaxis, neutrophil migration, and lipid and sterol homeostasis), as well as more tissue-specific processes that appeared to be related to lipid outcomes (cell cycle regulation in jejunum, and gluconeogenesis in liver).

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.

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.

Varying modalities of perinatal exposure to a pesticide cocktail elicit neurological adaptations in mice and zebrafish.

Epidemiological indications connect maternal and developmental presence or exposure to pesticides with an increased risk for a spectrum of neurological trajectories. To provide pre-clinical data in support of this hypothesis, we used two distinct experimental models. First, female and male mice were fed immediately prior to mating, and the resulting pregnant dams were continously fed during gestation and lactation periods using chow pellets containing a cocktail of six pesticides at tolerable daily intake levels. Male and female offspring were then tracked for behavioral and in vivo electrophysiological adaptations. Second, a zebrafish model allowed us to screen toxicity and motor-behavior outcomes specifically associated with the developmental exposure to a low-to-high concentration range of the cocktail and of each individual pesticide. Here, we report anxiety-like behavior in aging male mice maternally exposed to the cocktail, as compared to age and gender matched sham animals. In parallel, in vivo electrocorticography revealed a decrease in gamma (40-80 Hz) and an increase of theta (6-9 Hz) waves, delineating a long-term, age-dependent, neuronal slowing. Neurological changes were not accompanied by brain structural malformations. Next, by using zebrafish larvae, we showed an increase of all motor-behavioral parameters resulting from the developmental exposure to 10 µg/L of pesticide cocktail, an outcome that was not associated with midbrain structural or neurovascular modifications as assessed by in vivo 2-photon microscopy. When screening each pesticide, chlorpyrifos elicited modifications of swimming parameters at 0.1 µg/L, while other components provoked changes from 0.5 µg/L. Ziram was the single most toxic component inducing developmental malformations and mortality at 10 µg/L. Although we have employed non-equivalent modalities and timing of exposure in two dissimilar experimental models, these outcomes indicate that presence of a pesticide cocktail during perinatal periods represents an element promoting behavioral and neurophysiological modifications. The study limitations and the possible pertinence of our findings to ecotoxicology and public health are critically discussed.

Life-long Dietary Pesticide Cocktail Induces Astrogliosis Along with Behavioral Adaptations and Activates p450 Metabolic Pathways.

Exposure to environmental contaminants is a public health concern. However, pre-clinical studies that examine the impact of pesticides at low-dose and the long-term consequences are uncommon. Here, C57BL6/j male and female mice were daily fed from weaning and up to 12 months, corresponding to early-childhood into middle-age in humans, using chow pellets containing a cocktail of pesticides at tolerable daily intake levels. We found that 12 months of dietary exposure to pesticides was associated with a moderate perenchymal or perivascular astrogliosis in specific hippocampal sub-regions. The expression of platelet-derived growth factor receptor beta was modified at the perivascular level. Examination of Iba1+ microglial cells did not reveal sizeable changes. Concomitantly to astrogliosis, spontaneous spatial memory and sociability were modified in males at 12 months of dietary exposure to pesticides. Telemetry electrocorticograhic explorations ruled out the presence of epileptiform activity or theta-gamma wave modifications in these conditions. Long-term pesticides impacted the periphery where the hepatic P450 metabolic cytochromes Cyp4a14 and Cyp4a10 were significantly upregulated in male and female mice during the 12 months of exposure. The expression of ß-oxidation genes, such as Acox1, Cpt1a and Eci, was also significantly increased in male and female mice in response to pesticides. Collectively, our results indicate that a life-long exposure to a pesticide cocktail elicits sex-dependent, spatio-temporally restricted brain modifications and significant activation of P450 pathways in the periphery. These brain-peripheral adjustments are discussed as time or age-dependent vulnerability elements.

Perinatal exposure to a dietary pesticide cocktail does not increase susceptibility to high-fat diet-induced metabolic perturbations at adulthood but modifies urinary and fecal metabolic fingerprints in C57Bl6/J mice.

BACKGROUND: We recently demonstrated that chronic dietary exposure to a mixture of pesticides at low-doses induced sexually dimorphic obesogenic and diabetogenic effects in adult mice. Perinatal pesticide exposure may also be a factor in metabolic disease etiology. However, the long-term consequences of perinatal pesticide exposure remain controversial and largely unexplored. OBJECTIVES: Here we assessed how perinatal exposure to the same low-dose pesticide cocktail impacted metabolic homeostasis in adult mice. METHODS: Six pesticides (boscalid, captan, chlopyrifos, thiachloprid, thiophanate, and ziram) were incorporated in food pellets. During the gestation and lactation periods, female (F0) mice were fed either a pesticide-free or a pesticide-enriched diet at doses exposing them to the tolerable daily intake (TDI) level for each compound, using a 1:1 body weight scaling from humans to mice. All male and female offsprings (F1) were then fed the pesticide-free diet until 18 weeks of age, followed by challenge with a pesticide-free high-fat diet (HFD) for 6 weeks. Metabolic parameters, including body weight, food and water consumption, glucose tolerance, and urinary and fecal metabolomes, were assessed over time. At the end of the experiment, we evaluated energetic metabolism and microbiota activity using biochemical assays, gene expression profiling, and 1H NMR-based metabolomics in the liver, urine, and feces. RESULTS: Perinatal pesticide exposure did not affect body weight or energy homeostasis in 6- and 14-week-old mice. As expected, HFD increased body weight and induced metabolic disorders as compared to a low-fat diet. However, HFD-induced metabolic perturbations were similar between mice with and without perinatal pesticide exposure. Interestingly, perinatal pesticide exposure induced time-specific and sex-specific alterations in the urinary and fecal metabolomes of adult mice, suggesting long-lasting changes in gut microbiota. CONCLUSIONS: Perinatal pesticide exposure induced sustained sexually dimorphic perturbations of the urinary and fecal metabolic fingerprints, but did not significantly influence the development of HFD-induced metabolic diseases.