Multiple Sclerosis Journal 2015; 21: (7) 963–965
ACTRIMS Symposium FOCIS Annual Meeting June 24, 2015 San Diego, California
CNS autoimmune disease and the innate immune system: Novel roles of pro-inflammatory and regulatory myeloid cells Session Chair: Benjamin M. Segal, M.D., University of Michigan (Ann Arbor, MI, USA) Polarization properties of human myeloid cell populations Jack Antel, Samuel Ludwin, Luke Healy, Bryce Durafourt, Amit Bar-Or, Craig S. Moore McGill University, Montreal Neurological InstituteNeurobiology, Montreal, QC, Canada Microglia are the long-lived endogenous myeloid cell population of the central nervous system (CNS). In contrast, blood-derived myeloid cell populations occupy the perivascular spaces under physiologic conditions and access the parenchyma under select pathologic conditions. The functional properties of these cell types as related to their participation in tissue injury and repair responses are modulated by signals derived from their microenvironment. Such dynamic properties can be referred to as “state of polarization.” The capacity to isolate microglia from the human adult CNS and maintain them in long-term dissociated culture allows analysis of their response to polarization inducing conditions; these responses can be compared with properties of blood-derived macrophages in vitro and in situ under select pathologic conditions. Both microglia and macrophages respond to M1 polarization conditions (GM-CSF/ IFNγ/LPS) by up-regulating CCR7 and CD80 and producing IL-12. Human microglia are less responsive to M2a polarizing conditions (M-CSF/IL-4/IL-13) compared with blood-derived macrophages as measured by relative expression of CD23, CD163, and CD206. The M2 phenotype of microglia is further expanded by exposure to IL-10 (M2c) and transforming growth factor beta (TGFβ). M2 microglia show enhanced myelin phagocytosis compared with both M1 microglia and similarly polarized macrophages. Soluble products released from M1 microglia, but not M2 microglia, mediate cytotoxicity of human oligodendrocyte progenitor cells (OPCs) in vitro; an effect mainly attributable to TNFα. P2Y12 is a cell surface purinergic receptor that can discriminate between brain-resident microglia and blood-derived macrophages. Under M2a conditions, P2Y12 is elevated in human microglia and plays a significant role in mediating migratory and inflammatory function upon stimulation with ADP, its endogenous ligand and a secreted molecule that is releases from neurons following injury. Analysis of parasite-infected human brain tissue, a Th2 microenvironment, confirms the presence of P2Y12+ M2 cells in situ. Targeting surface receptors whose expression is linked to
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functional properties of specific myeloid cell types under select microenvironmental conditions provides a potential therapeutic means to modulate the balance of injury and repair responses in human myeloid cells.
Role of neutrophils to the atypical EAE phenotype Yudong Liu, Hongwei Qin, Etty (Tika) Benveniste University of Alabama at Birmingham, Birmingham, AL, USA The JAK/STAT pathway is critical for development, regulation, and termination of immune responses, and dysregulation of the JAK/STAT pathway, i.e. hyper-activation, has pathological implications in autoimmune and neuroinflammatory diseases. Suppressor Of Cytokine Signaling 3 (SOCS3) regulates STAT3 activation in response to cytokines that play important roles in the pathogenesis of neuroinflammatory diseases, including IL-6 and IL-23. We previously demonstrated that myeloid-lineage specific deletion of SOCS3 (macrophages, neutrophils, dendritic cells, microglia) resulted in a severe, non-resolving atypical form of experimental autoimmune encephalomyelitis (EAE), characterized by lesions and inflammatory infiltrates in the cerebellum, elevated STAT activation in the cerebellum and elevated cytokine and chemokine expression. Clinically, these mice exhibit ataxia and tremors. In this study, we provide a detailed analysis of this model, demonstrating that the atypical EAE observed in LysMCre-SOCS3fl/fl mice is characterized by extensive neutrophil infiltration into the cerebellum and brainstem, increased iNOS levels in the cerebellum and brainstem, and prominent axonal damage. Importantly, infiltrating SOCS3deficient neutrophils produce high levels of CXCL2, CCL2, CXCL10, nitric oxide (NO), TNF-α and IL-1β. Kinetic studies demonstrate that neutrophil infiltration into the cerebellum and brainstem of LysMCre-SOCS3fl/fl mice closely correlates with atypical EAE clinical symptoms. Antibody-mediated depletion of neutrophils converts the atypical phenotype to the classical EAE phenotype, and in some cases, a mixed atypical/classical phenotype. Blocking CXCR2 signaling ameliorates atypical EAE development by reducing neutrophil infiltration into the cerebellum/brainstem. Thus, neutrophils lacking SOCS3 display elevated STAT3 activation, expression of pro-inflammatory
964 mediators, and play a critical role in the development of atypical EAE.
Mast cell–T cell interactions in the meninges amplify T-cell encephalitogenicity Melissa A. Brown, Margaret E. Walker-Caulfield, Abigail E. Russi Northwestern University Feinberg School of Medicine, Chicago, IL, USA There is much evidence that the meninges, tissues that surround the brain and spinal cord and enclose the cerebrospinal fluid, are an immune cell gateway to the central nervous system (CNS) and regulate the severity of autoimmune demyelinating disease. In experimental autoimmune encephalomyelitis (EAE), the murine model of multiple sclerosis (MS), T cells and other immune cells accumulate in the meninges and are activated in preclinical disease. Meningeal inflammation is also prominent in MS patients where it precedes the appearance of classic white matter lesions in many patients and is thought to drive clinical relapses. Importantly, inflammatory lesions in the meninges are often directly proximal to grey matter demyelination in the brain and correlate with a more severe clinical disease course. Our work in EAE has demonstrated that mast cells, normal residents of the meninges, play a pivotal role in orchestrating these immune events. Activated within a day of disease induction, meningeal mast cells release several mediators that regulate neutrophil trafficking and destabilize blood–brain barrier integrity. Mast cells also exert profound effects on the trafficking and re-activation of encephalitogenic T cells. Adoptively transferred MOG35-55-specific T cells are detected in the meninges as early as 3 days post transfer, and this T-cell transit is impaired in mast cell-deficient c-kit mutant (KitW/Wv) mice. Furthermore, T cells acquire the ability to express GM-CSF in the meninges, an event also dependent on mast cells. GM-CSF, a cytokine that promotes the recruitment and survival of myeloid cells in inflamed CNS tissues, is implicated in T cell encephalitogenicity. We provide in vitro and in vivo evidence that that activated T cells directly elicit mast cell IL-1β production, which in turn acts on T cells to promote GM-CSF production. Mast cell expression of caspase-1, an enzyme that functions in the inflammasome to cleave pro-IL-1β to its biologically active form is required for these events. Co-culture of wild type but not caspase-1-/- or IL-1β-/- mast cells with MOG35-55specific T cells elicits robust GM-CSF production by the T cells. In vivo, reconstitution of meningeal mast cell populations to KitW/Wv mice with wild type but not caspase-1-/- bone marrow derived mast cells restores the ability of transferred T cells to express GM-CSF, which results in pathologic myeloid cell infiltration of the CNS and disease susceptibility. Taken together, these data show that mast cell–T-cell cross-talk in the meninges causes the activation of both cell types and is essential for promoting the disease-inducing activity of T cells. Although the actions of mast cells are often considered to be restricted to allergic inflammation, these studies provide further support that mast cells can exert profound effects on CNS inflammatory processes as well.
Multiple Sclerosis Journal 2015; 21: (7) 963–965
ACTRIMS Symposium 21(7) Microglia and macrophage activation in remyelination Veronique E. Miron MRC Centre for Reproductive Health & MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, The University of Edinburgh, Edinburgh, UK Remyelination fails with progression of multiple sclerosis, highlighting the need to understand the cellular mechanisms underpinning successful remyelination in order to develop novel therapeutic approaches. Our previous work showed that temporal regulation of macrophage activation during remyelination controls specific phases of the repair process, with pro-inflammatory phenotypes driving oligodendrocyte progenitor cell proliferation early on and anti-inflammatory macrophages required for subsequent oligodendrocyte differentiation. Lineage tracing showed the contribution of microglia and monocyte-derived macrophages to both activation phenotypes. However, the requirement for the latter for remyelination is controversial and their differential contribution to oligodendrocyte progenitor responses is unknown. We first identified a role for peripherally derived macrophages in regulating oligodendrocyte progenitor behaviour during remyelination by using CCR2-/- mice in which circulation-derived monocytes are unable to extravasate into lesions, and observed that focal remyelinating lesions had altered progenitor responses. To investigate potential differences between microglia and peripherally derived macrophages in regulating progenitor responses relevant for remyelination, we activated primary microglia and bone marrow derived macrophages (BMDMs) to pro- and anti-inflammatory phenotypes and applied conditioned media to oligodendrocyte progenitor cells in vitro. Conditioned media from all sources applied to co-cultures of rat oligodendrocytes and dorsal root ganglion neurons similarly promoted oligodendrocyte process extension and axonal contact, but did not promote myelination. This was supported by observations of low numbers of all macrophage phenotypes in the final stages of remyelination in vivo. However, we observed a differential capacity of conditioned media generated from activated microglia vs. BMDMs in driving oligodendrocyte progenitor proliferation, differentiation and survival. Together, these findings highlight the requirement, yet differential contribution, of microglia and peripherally derived macrophage activation phenotypes to oligodendrocyte progenitor cell responses in early stages of remyelination.
Neutrophil-attracting CXC chemokines and G-CSF in EAE and MS pathogenesis Julie Rumble University of Michigan-Neurology, Ann Arbor, MI, USA A major function of T helper (Th)17 cells is to induce the production of factors that activate and mobilize neutrophils. We have previously shown that neutrophils are critical for blood-brain-barrier breakdown and clinical disease in actively induced experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). In the current study, we investigated the mechanisms by which neutrophil movement is regulated during both actively induced and passively transferred EAE. We found that neutrophils expanded in the bone marrow and the bloodstream before onset of clinical symptoms. Additionally, we measured an early spike in systemic levels of G-CSF, a neutrophil growth and mobilization factor, and CXCL1, a
ACTRIMS Symposium 21(7) neutrophil chemoattractant. This accumulation of neutrophils was not dependent on adjuvant, as it was observed at clinical onset in a spontaneous model of EAE as well as after passive transfer of either myelin-specific Th1 or Th17 CD4 T cells. Loss of G-CSF signaling abrogated disease in actively immunized mice and reduced disease in Th17 recipients, which was further ameliorated by blockade of CXCR2, a receptor for CXCL1. In relapsing MS patients, plasma levels of CXCL5, another neutrophil-related chemokine, were elevated during acute lesion formation. Systemic expression of CXCL1, CXCL5, and neutrophil elastase correlated with measures of MS lesion burden and clinical disability. Based on these results, we advocate that neutrophil-related molecules be further investigated as novel biomarkers and therapeutic targets in MS.
Neutrophil interactions at the blood–brain interface in neuroinflammation Luc Vallières Centre Hospitalier de l’Université Laval (CHUL), Quebec, QC, Canada
965 Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) that can be induced in animals to model immunological processes involved in human diseases such as multiple sclerosis and neuromyelitis optica. EAE is initiated by CD4+ T helper lymphocytes that recognize myelin peptides through their T-cell receptors. These cells do not act alone, but in concert with different myeloid cells, including monocyte-derived dendritic cells and macrophages, which, respectively, activate them by presenting myelin peptides and execute effector functions leading to demyelination. In addition, recent studies have established that neutrophils importantly contribute to EAE, although their precise role is still unclear. In this presentation, I will discuss the involvement of different molecules (e.g. IL-1β, TNF, IL-6, Angpt2, CXCL1, Icam1) in the recruitment of neutrophils at the blood–brain interface. I will also report the existence of two subsets of neutrophils in the CNS of EAE mice and the identification of IL-36γ as a neutrophil-specific cytokine highly expressed in EAE. Our ongoing studies should help to clarify the role of neutrophils in EAE and to identify new potential therapeutic targets for neuroinflammatory diseases.
Multiple Sclerosis Journal 2015; 21: (7) 963–965
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