Rôle différentiel des cellules épithéliales intestinales et pulmonaires dans le recrutement des cellules Th17 vers les sites de réplication du virus de l'immunodéficience humaine de type 1

L’infection à VIH-1 est associée à une forte déplétion des lymphocytes T CD4+ à polarisation Th17 au niveau des tissus lymphoïdes associés aux muqueuses intestinales (GALT, gut-associated lymphoid tissues). Ceci conduit à la translocation microbienne, qui est une cause d’activation immunitaire chronique et de progression de la maladie. Les cellules épithéliales (CE) jouent un rôle critique dans le maintien de l’intégrité et de l’homéostasie au niveau des muqueuses intestinales via le recrutement des cellules de l’immunité innée (e.g., neutrophiles) et adaptative (e.g., cellules Th17). Les neutrophiles produisent des molécules antivirales (e.g., défensines-) et ont la capacité de limiter la réplication virale au niveau des muqueuses. Les cellules Th17 jouent un double rôle lors de l’infection à VIH. Elles contribuent d’une part à la défense contre différents pathogènes opportunistes en augmentant, via la production d’IL-17, la capacité des CE à attirer les cellules Th17 et les neutrophiles. D’autre part, les cellules Th17 jouent un rôle délétère en tant que cibles de réplication virale et sources de cytokines pro-inflammatoires. La fréquence des cellules Th17 est diminuée dans les GALT mais pas dans les poumons des patients infectés par le VIH, suggérant qu’il existe des mécanismes différents par lesquels les cellules Th17 sont recrutées vers ces sites anatomiques. Nous avons testé l’hypothèse selon laquelle le VIH interfère avec la capacité des CE intestinales et non pas pulmonaires à produire des chimiokines (CK) responsables de l’attraction des cellules Th17 et des neutrophiles. Nous avons démontré que les CE intestinales et pulmonaires produisent des CK spécifiques pour les cellules Th17 (CCL20) et les neutrophiles (CXCL8) en réponse à des stimuli pro-inflammatoires tels que l’IL-1 et le TNF-. Le TNF- agit en synergie avec l’IL-17, un « signal de danger » récemment identifié, et augmente la capacité des CE intestinales mais pas pulmonaires à produire la chimiokine CCL20. Cette synergie s’explique par l’augmentation préférentielle de l’expression du récepteur à l’IL-17 à la surface des CE intestinales suite à la stimulation par le TNF-. L’exposition au VIH n’affecte pas la production de CCL20 et de CXCL8 par les CE intestinales, mais altère la capacité des CE alvéolaires à produire ces chimiokines en accord avec la permissivité sélective de ces dernières à l’infection par le VIH. En conclusion, nos résultats démontrent que (i) le VIH n’interfère pas directement avec la capacité des CE intestinales à recruter des cellules Th17 et des neutrophils et que (ii) la production de CCL20 par ces cellules est dépendantes de la synergie entre le TNF- et l’IL-17. Ainsi, la déplétion des cellules Th17 et la pénurie en IL-17 dans les GALT des sujets infectés pourrait causer de façon préférentielle des altérations fonctionnelles au niveau des CE intestinales, se traduisant par l’altération du recrutement des cellules Th17 en réponse au CCL20.The HIV-1 infection is associated with a severe loss of CD4+ T-cells with Th17 polarization from the gut-associated lymphoid tissues (GALT). These alterations lead to microbial translocation, which is a cause of chronic immune activation and disease progression in HIV-infected subjects. Epithelial cells (EC) play a critical role in maintaining mucosal integrity and homeostasis in the GALT by mechanisms including recruitment of innate (e.g., neutrophils) and adaptive immunity cells (e.g., Th17 cells). Neutrophils produce antiviral molecules (e.g., -defensins) that may limit HIV replication at mucosal sites. Th17 cells play a dual role in HIV pathogenesis. Th17 cells contribute to the defence against different opportunistic pathogens by increasing the ability of epithelial cells to attract neutrophils in an IL-17-dependent manner. On the other hand, Th17 cells play a deleterious role in HIV pathogenesis as they are sites of productive viral replication and a source of pro-inflammatory cytokines. The frequency of Th17 cells is decreased in the GALT but not in the lungs of HIV-infected individuals, suggesting distinct mechanisms of Th17 recruitment in these anatomic sites in the context of HIV pathogenesis. In this manuscript we tested the hypothesis that HIV differentially interfere with the ability of intestinal but not pulmonary EC to produce chemokines that attract Th17 cells and neutrophils. We demonstrated that both intestinal and pulmonary EC produce chemokines that specifically attract Th17 cells (CCL20) and neutrophils (CXCL8) upon stimulation with the pro-inflammatory cytokines IL-1 and TNF- . TNF-α acted in synergy with IL-17, a recently identified « danger signal », and increases the capacity of intestinal but not pulmonary EC to produce CCL20. This synergistic effect can be explained by the preferential upregulation of IL-17 receptor expression on intestinal EC upon TNF- stimulation. The exposure of intestinal EC to HIV did not affect their ability to produce CCL20 and CXCL8; however, exposure to HIV altered the production of these chemokines by alveolar EC, consistent with their selective permissiveness to infection. In conclusion, our results demonstrate that (i) HIV does not interfere directly with the ability of intestinal EC to attract Th17 cells and neutrophils and that (ii) the ability of intestinal EC to recruit the Th17 cells via CCL20 production is selectively dependent on the synergy between TNF- and IL-17. Thus, the depletion of Th17 cells and the shortage in IL-17 in the GALT of HIV-infected subjects may preferentially lead to functional alterations of the intestinal barrier resulting by the alteration of Th17 recruitment in response to CCL20

Differential impact of environmental factors on systemic and localized autoimmunity

The influence of environmental factors on the development of autoimmune disease is being broadly investigated to better understand the multifactorial nature of autoimmune pathogenesis and to identify potential areas of intervention. Areas of particular interest include the influence of lifestyle, nutrition, and vitamin deficiencies on autoimmunity and chronic inflammation. In this review, we discuss how particular lifestyles and dietary patterns may contribute to or modulate autoimmunity. We explored this concept through a spectrum of several autoimmune diseases including Multiple Sclerosis (MS), Systemic Lupus Erythematosus (SLE) and Alopecia Areata (AA) affecting the central nervous system, whole body, and the hair follicles, respectively. A clear commonality between the autoimmune conditions of interest here is low Vitamin D, a well-researched hormone in the context of autoimmunity with pleiotropic immunomodulatory and anti-inflammatory effects. While low levels are often correlated with disease activity and progression in MS and AA, the relationship is less clear in SLE. Despite strong associations with autoimmunity, we lack conclusive evidence which elucidates its role in contributing to pathogenesis or simply as a result of chronic inflammation. In a similar vein, other vitamins impacting the development and course of these diseases are explored in this review, and overall diet and lifestyle. Recent work exploring the effects of dietary interventions on MS showed that a balanced diet was linked to improvement in clinical parameters, comorbid conditions, and overall quality of life for patients. In patients with MS, SLE and AA, certain diets and supplements are linked to lower incidence and improved symptoms. Conversely, obesity during adolescence was linked with higher incidence of MS while in SLE it was associated with organ damage. Autoimmunity is thought to emerge from the complex interplay between environmental factors and genetic background. Although the scope of this review focuses on environmental factors, it is imperative to elaborate the interaction between genetic susceptibility and environment due to the multifactorial origin of these disease. Here, we offer a comprehensive review about the influence of recent environmental and lifestyle factors on these autoimmune diseases and potential translation into therapeutic interventions

Accumulation of meningeal lymphocytes correlates with white matter lesion activity in progressive multiple sclerosis

Subpial cortical demyelination is an important component of multiple sclerosis (MS) pathology contributing to disease progression, yet mechanism(s) underlying its development remain unclear. Compartmentalized inflammation involving the meninges may drive this type of injury. Given recent findings identifying substantial white matter (WM) lesion activity in patients with progressive MS, elucidating whether and how WM lesional activity relates to meningeal inflammation and subpial cortical injury is of interest. Using postmortem FFPE tissue blocks (range, 5-72 blocks; median, 30 blocks) for each of 27 patients with progressive MS, we assessed the relationship between meningeal inflammation, the extent of subpial cortical demyelination, and the state of subcortical WM lesional activity. Meningeal accumulations of T cells and B cells, but not myeloid cells, were spatially adjacent to subpial cortical lesions, and greater immune cell accumulation was associated with larger subpial lesion areas. Patients with a higher extent of meningeal inflammation harbored a greater proportion of active and mixed active/inactive WM lesions and an overall lower proportion of inactive and remyelinated WM lesions. Our findings support the involvement of meningeal lymphocytes in subpial cortical injury and point to a potential link between inflammatory subpial cortical demyelination and pathological mechanisms occurring in the subcortical WM

B cell:Glial cell interactions and contribution to CNS- compartmentalized inflammation in multiple sclerosis

Growing evidence suggests that B cells play important roles during disease relapses and possibly also during the progressive stages of multiple sclerosis (MS). A number of studies have implicated antibody-independent roles of B cells in the periphery of patients, including presence of abnormally higher proportions of pro-inflammatory B cells (Beff) compared to anti-inflammatory B cells (Breg), which is thought to induce pro-inflammatory T cell responses. In the inflamed central nervous system (CNS) of MS patients, B cells are abnormally fostered and found in different compartments, including the meningeal immune-cell collections closely linked with subpial cortical injury and progressive disease. However, there remain major gaps in our knowledge of B cell persistence and their potential contributions to CNS-compartmentalized inflammation. The aim of my doctoral thesis is to shed light on cellular mechanisms involving B cell:glial cell cross-talk and how these interactions may propagate local inflammation and injury as well as contribute to progressive disease in MS. In the first part of my thesis (Chapter 2), I demonstrate that human astrocytes support the survival of B cells through soluble factors. These soluble factors derived from pre-stimulated astrocytes not only supported B cell survival but also their activation (increased co-stimulatory molecule expression), which further induced T cell proliferation. I further demonstrate that astrocyte-secreted factors supported survival and activation of MS-relevant B cell subsets derived from relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients. In chapter 3, I investigated the bi-directional interaction between B cells and myeloid cells (microglia and macrophages) to determine how these interactions may impact the propagation of CNS-compartmentalized inflammation. First, I show that soluble factors derived from M1-activated microglia supported B cell activation, whereas M2c microglia induced B apoptotic death of MS-relevant B cell subsets. I further demonstrate that soluble factors derived from pro-inflammatory (Beff) B cells (abnormally implicated in the periphery of MS) substantially increased the secretion of the pro-inflammatory cytokines (IL-12, IL-6 & TNF) and diminished IL-10 production by microglia and macrophage. Interestingly, this secretion of pro-inflammatory cytokines by macrophages was dependent on B cell-derived GM-CSF. Lastly, I demonstrate that supernatants derived from distinct B cell subsets (Beff & Breg) were capable of modulating microglia and macrophage phenotype (expression of CD80 & TREM-2), while reciprocally modulating myeloid phagocytosis of myelin.My overall doctoral investigations enhances our understanding of cellular mechanisms that may be associated with progressive disease. My findings indicate that B cells and glial cells have the capacity to interact and that they may contribute to cascades of pro-inflammatory events. In related work to which I have contributed, we demonstrated a selective cytotoxic effect to oligodendrocytes and neurons in response to soluble factors of B cells derived from MS patients but B cells from matching controls. If these interactions occur in vivo they may propagate CNS-compartmentalized inflammation and subpial injury. Taken together, these findings constitute a conceptual advance pointing to novel cellular mechanisms that may contribute to subpial cortical pathology and progressive MS. Future work will aim to elucidate the molecular mechanisms underlying these B cell:glial cell interactions. I am hopeful that my results will eventually help the development of more targeted therapies that can limit or modulate these interaction in a way that is beneficial for progressive MS – a major unmet clinical need.Les études réalisées dans le domaine de la sclérose en plaques (SEP) ont montré le rôle clé des cellules B au cours des poussées, et probablement lors de la phase progressive de la maladie. Plusieurs travaux montrent le rôle des cellules B, dans la pathophysiologie de la maladie indépendamment de la production d'anticorps. Les patients atteints de SEP présenteraient une proportion plus élevée de cellules B pro-inflammatoires (Beff), en comparaison aux cellules B anti-inflammatoires (Breg). De plus, les cellules Beff sont à même d'induire des réponses T pro-inflammatoires. Dans le système nerveux central (SNC) inflammé, les cellules B persistent anormalement au sein du compartiments des agrégats de cellules immunitaires associées aux méninges. Ces derniers sont étroitement liées aux lésions superficielles du cortex, et à la progression de la maladie. Toutefois, nos connaissances restent limitées quant à la persistance des cellules B et leur potentielle contribution à une inflammation compartimentalisée dans le SNC. Le but de ma thèse est d'apporter quelques éclairages sur les mécanismes cellulaires mis en jeu lors des interactions bidirectionnelles entre les cellules B et gliales, et comment ce type d'interactions propagerait l'inflammation locale, induirait la dégénérescence cérébrale et la progression.Dans la première partie de ma thèse (Chapitre 2), j'ai pu démontré que les astrocytes humains permettraient la survie des cellules B via des produits solubles. Les produits solubles dérivés d'astrocytes pré-stimulés ont amélioré la survie des B, et augmenté l'activation des cellules B (expression de molécules de co-stimulation). Ces derniers, ont permis d'induire une meilleure réponse proliférative des cellules T. Dans le même sens, j'ai pu démontrer que ces facteurs solubles augmentaient la survie et l'activation de sous-populations de cellules B caractéristiques de la SEP, de patients exprimant la forme récurrente rémittente et progressive secondaire. Dans le troisième volet de cette thèse, j'ai investigué l'interaction bidirectionnelle entre les cellules B et les microglies/macrophages, et comment ces interactions impacteraient la propagation de l'inflammation compartimentalisée. D'abord, j'ai démontré que les produits solubles des microglies-M1 augmentent l'activation des cellules B, alors que les microglies-M2c induisent la mort cellulaire des différentes sous-populations de B. Par ailleurs, j'ai démontré que les facteurs solubles dérivés des Beff (élevées dans la périphérie de la SEP) ont augmentée de manière considérable la sécrétion de cytokines pro-inflammatoires (IL-12, IL-6 &TNF) par les microglies et les macrophages, de manière dépendante du GM-CSF produit par les Beff. Ces mêmes facteurs solubles ont induit la diminution de la production d'IL-10 par les cellules myéloïdes. Finalement, j'ai démontré que les surnageants de cellules (Beff & Breg) ont le pouvoir de moduler le phénotype (CD80 & TREM-2) des cellules myéloïdes, et de moduler de manière réciproque la phagocytose de la myéline.Globalement, mes travaux ont permis l'élucidation de mécanismes cellulaires qui pourraient être associés à la SEP progressive. Mes résultats indiquent que les interactions entre cellules B et gliales pourraient contribuer à la cascade d'évènements pro-inflammatoires. J'ai ainsi démontré que les facteurs solubles de cellules B dérivées de SEP induisaient la cytotoxicité des oligodendrocytes et des neurones. Si ces interactions sont initiées in vivo, elles pourraient contribuer à la propagation d'inflammation compartimentalisée dans le SNC, et à la dégénérescence qui mènent aux lésions superficielles corticales. Mes résultats ont contribués a des avancées conceptuelles indiquant l'engagement d'un nouveau mécanisme cellulaire contribuant la progression de la maladie. D'autres travaux axés sur les mécanismes moléculaires apparaissent nécessaires pour générer des thérapies ciblées pour la prise en charge des formes progressive

Chronic clomipramine treatment reverses depressogenic-like effects of a chronic treatment with dexamethasone in rats

Corticosteroids are widely used in medicine, for their anti-inflammatory and immunosuppressive actions, but can lead to troubling psychiatric side-effects. In fact, corticosteroids can induce many symptoms and syndromes, for example, mood disorders, anxiety and panic disorder, suicidal thinking and behavior. Furthermore, chronic stress and the administration of exogenous glucocorticoids are reported to induce affective changes in humans and rodents that relate to depressive state. Animal models are highly useful tools for studying the depression etiology. Face validity, construct validity, and predictive validity are the main criteria to evaluate animal depression models. The present study aimed to investigate the behavioral, cognitive, and biochemical effects of a chronic administration of DEX on Wistar rats. Wistar rats were administered daily with DEX (1.5 mg/kg, i.p., 21 days) or saline, the clomipramine treatment (2 mg/kg, i.p.) was realized just after the DEX injections for 21 days. DEX induced changes were evaluated by: forced swimming, novelty suppressed feeding, saccharin preference, open field, Morris water maze, and oxidative stress state in the brain. Results showed that chronic DEX administration conduct to a range of depression-related behavioral traits, including anhedonia, despair, weight loss, anxiety-like behavior, and cognitive impairments, which fill the face validity criterion. The DEX induced behavioral changes may result from the massive production of oxidative stress agents. This sustains the etiological hypothesis claiming that hyper-circulating glucocorticoid resulting from HPA dysfunction induces damage in certain neural structures related to depressive disorder, essentially the hippocampus. The antidepressant treatment has restored the behavioral state of rats which fills the predictive validity criterion

Fisetin disposition and metabolism in mice: Identification of geraldol as an active metabolite.: Fisetin disposition and metabolism in mice

International audienceAlthough the natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been recently identified as an anticancer agent with antiangiogenic properties in mice, its in vivo pharmacokinetics and metabolism are presently not characterized. Our purpose was to determine the pharmacokinetics and metabolism of fisetin in mice and determine the biological activity of a detected fisetin metabolite. After fisetin administration of an efficacious dose of 223 mg/kg i.p. in mice, the maximum fisetin concentration reached 2.5 μg/ml at 15 min and the plasma concentration declined biphasically with a rapid half-life of 0.09 h and a terminal half-life of 3.1h. Three metabolites were detected, one of which was a glucuronide of fisetin (M1), whereas another glucuronide (M2) was a glucuronide of a previously unknown fisetin metabolite (M3). HPLC-MS/MS analysis indicated that M3 was a methoxylated metabolite of fisetin (MW=300 Da). The UV spectrum of M3 was identical to that of fisetin and standard 3,4',7-trihydroxy-3'-methoxyflavone (geraldol). In addition, because M3 co-eluted with standard geraldol in 4 different chromatographic ternary gradient conditions, M3 was therefore assigned to geraldol. Of interest, this metabolite was shown to achieve higher concentrations than fisetin in Lewis lung tumors. We also compared the cytotoxic and antiangiogenic activities of fisetin and geraldol in vitro and it was found that the latter was more cytotoxic than the parent compound toward tumor cells, and that it could also inhibit endothelial cells migration and proliferation. In conclusion, these results suggest that fisetin metabolism plays an important role in its in vivo anticancer activities

B Cells in the Multiple Sclerosis Central Nervous System: Trafficking and Contribution to CNS-Compartmentalized Inflammation

Clinical trial results of peripheral B cell depletion indicate abnormal proinflammatory B cell properties, and particularly antibody-independent functions, contribute to relapsing MS disease activity. However, potential roles of B cells in progressive forms of disease continue to be debated. Prior work indicates that presence of B cells is fostered within the inflamed MS central nervous system (CNS) environment, and that B cell-rich immune cell collections may be present within the meninges of patients. A potential association is reported between such meningeal immune cell collections and the subpial pattern of cortical injury that is now considered important in progressive disease. Elucidating the characteristics of B cells that populate the MS CNS, how they traffic into the CNS and how they may contribute to progressive forms of the disease has become of considerable interest. Here, we will review characteristics of human B cells identified within distinct CNS subcompartments of patients with MS, including the cerebrospinal fluid, parenchymal lesions, and meninges, as well as the relationship between B cell populations identified in these subcompartments and the periphery. We will further describe the different barriers of the CNS and the possible mechanisms of migration of B cells across these barriers. Finally, we will consider the range of human B cell responses (including potential for antibody production, cytokine secretion, and antigen presentation) that may contribute to propagating inflammation and injury cascades thought to underlie MS progression

Cross-talk between B cells, microglia and macrophages, and implications to central nervous system compartmentalized inflammation and progressive multiple sclerosisResearch in context

Summary: Background: B cells can be enriched within meningeal immune-cell aggregates of multiple sclerosis (MS) patients, adjacent to subpial cortical demyelinating lesions now recognized as important contributors to progressive disease. This subpial demyelination is notable for a ‘surface-in’ gradient of neuronal loss and microglial activation, potentially reflecting the effects of soluble factors secreted into the CSF. We previously demonstrated that MS B-cell secreted products are toxic to oligodendrocytes and neurons. The potential for B-cell–myeloid cell interactions to propagate progressive MS is of considerable interest. Methods: Secreted products of MS-implicated pro-inflammatory effector B cells or IL-10-expressing B cells with regulatory potential were applied to human brain-derived microglia or monocyte-derived macrophages, with subsequent assessment of myeloid phenotype and function through measurement of their expression of pro-inflammatory, anti-inflammatory and homeostatic/quiescent molecules, and phagocytosis (using flow cytometry, ELISA and fluorescently-labeled myelin). Effects of secreted products of differentially activated microglia on B-cell survival and activation were further studied. Findings: Secreted products of MS-implicated pro-inflammatory B cells (but not IL-10 expressing B cells) substantially induce pro-inflammatory cytokine (IL-12, IL-6, TNFα) expression by both human microglia and macrophage (in a GM-CSF dependent manner), while down-regulating their expression of IL-10 and of quiescence-associated molecules, and suppressing their myelin phagocytosis. In contrast, secreted products of IL-10 expressing B cells upregulate both human microglia and macrophage expression of quiescence-associated molecules and enhance their myelin phagocytosis. Secreted factors from pro-inflammatory microglia enhance B-cell activation. Interpretation: Potential cross-talk between disease-relevant human B-cell subsets and both resident CNS microglia and infiltrating macrophages may propagate CNS-compartmentalized inflammation and injury associated with MS disease progression. These interaction represents an attractive therapeutic target for agents such as Bruton's tyrosine kinase inhibitors (BTKi) that modulate responses of both B cells and myeloid cells. Funding: Stated in Acknowledgments section of manuscript