Histamine and neuroinflammation: insights from murine experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a chronic inflammatory, neurodegenerative disease of the CNS whose pathogenesis remains largely unknown, and available therapies are rarely successful in reversing neurological deficits or stopping disease progression. Ongoing studies on MS and the widely used murine model of experimental autoimmune encephalomyelitis (EAE) are focused on the many components of this complex and heterogeneous neurodegenerative disease in the hope of providing a mechanism-based characterization of MS that will afford successful strategies to limit and repair the neuronal damage. Recently, histamine has been postulated to have a key regulatory role in EAE and MS pathogenesis. Histamine is a mediator of inflammation and immune responses, exerting its many actions through four G protein-coupled receptors (H1,2,3,4R) that signal through distinct intracellular pathways and have different therapeutic potentials as they vary in expression, isoform distribution, signaling properties, and function. Immune cells involved in MS/EAE, including dendritic cells (DCs) and T lymphocytes, express H1R, H2R and H4R, and histamine may have varying and counteracting effects on a particular cell type, depending on the receptor subtypes being activated. Here, we review evidence of the complex and controversial role of histamine in the pathogenesis of MS and EAE and evaluate the therapeutic potential of histaminergic ligands in the treatment of autoimmune diseases

Immunosenescence and Autoimmunity: Exploiting the T-Cell Receptor Repertoire to Investigate the Impact of Aging on Multiple Sclerosis

T-cell receptor (TCR) repertoire diversity is a determining factor for the immune system capability in fighting infections and preventing autoimmunity. During life, the TCR repertoire diversity progressively declines as a physiological aging progress. The investigation of TCR repertoire dynamics over life represents a powerful tool unraveling the impact of immunosenescence in health and disease. Multiple Sclerosis (MS) is a demyelinating, inflammatory, T-cell mediated autoimmune disease of the Central Nervous System in which age is crucial: it is the most widespread neurological disease among young adults and, furthermore, patients age may impact on MS progression and treatments outcome. Crossing knowledge on the TCR repertoire dynamics over MS patients’ life is fundamental to investigate disease mechanisms, and the advent of high- throughput sequencing (HTS) has significantly increased our knowledge on the topic. Here we report an overview of current literature about the impact of immunosenescence and age-related TCR dynamics variation in autoimmunity, including MS

Diverse inflammatory threats modulate astrocytes Ca2+ signaling via connexin43 hemichannels in organotypic spinal slices

Neuroinflammation is an escalation factor shared by a vast range of central nervous system (CNS) pathologies, from neurodegenerative diseases to neuropsychiatric disorders. CNS immune status emerges by the integration of the responses of resident and not resident cells, leading to alterations in neural circuits functions. To explore spinal cord astrocyte reactivity to inflammatory threats we focused our study on the effects of local inflammation in a controlled micro-environment, the organotypic spinal slices, developed from the spinal cord of mouse embryos. These organ cultures represent a complex in vitro model where sensory-motor cytoarchitecture, synaptic properties and spinal cord resident cells, are retained in a 3D fashion and we recently exploit these cultures to model two diverse immune conditions in the CNS, involving different inflammatory networks and products. Here, we specifically focus on the tuning of calcium signaling in astrocytes by these diverse types of inflammation and we investigate the mechanisms which modulate intracellular calcium release and its spreading among astrocytes in the inflamed environment. Organotypic spinal cord slices are cultured for two or three weeks in vitro (WIV) and exposed for 6 h to a cocktail of cytokines (CKs), composed by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1 β) and granulocyte macrophage-colony stimulating factor (GM-CSF), or to lipopolysaccharide (LPS). By live calcium imaging of the ven- tral horn, we document an increase in active astrocytes and in the occurrence of spontaneous calcium oscillations displayed by these cells when exposed to each inflammatory threat. Through several pharmacological treatments, we demonstrate that intracellular calcium sources and the activation of connexin 43 (Cx43) hemichannels have a pivotal role in increasing calcium intercellular communication in both CKs and LPS conditions, while the Cx43 gap junction communication is apparently reduced by the inflammatory treatments

Cytokine inflammatory threat, but not LPS one, shortens GABAergic synaptic currents in the mouse spinal cord organotypic cultures

Background: Synaptic dysfunction, named synaptopathy, due to inflammatory status of the central nervous system (CNS) is a recognized factor potentially underlying both motor and cognitive dysfunctions in neurodegenerative diseases. To gain knowledge on the mechanistic interplay between local inflammation and synapse changes, we compared two diverse inflammatory paradigms, a cytokine cocktail (CKs; IL-1\u3b2, TNF-\u3b1, and GM-CSF) and LPS, and their ability to tune GABAergic current duration in spinal cord cultured circuits. Methods: We exploit spinal organotypic cultures, single-cell electrophysiology, immunocytochemistry, and confocal microscopy to explore synaptic currents and resident neuroglia reactivity upon CK or LPS incubation. Results: Local inflammation in slice cultures induced by CK or LPS stimulations boosts network activity; however, only CKs specifically reduced GABAergic current duration. We pharmacologically investigated the contribution of GABAAR \u3b1-subunits and suggested that a switch of GABAAR \u3b11-subunit might have induced faster GABAAR decay time, weakening the inhibitory transmission. Conclusions: Lower GABAergic current duration could contribute to providing an aberrant excitatory transmission critical for pre-motor circuit tasks and represent a specific feature of a CK cocktail able to mimic an inflammatory reaction that spreads in the CNS. Our results describe a selective mechanism that could be triggered during specific inflammatory stress

3D Organotypic Spinal Cultures: Exploring Neuron and Neuroglia Responses Upon Prolonged Exposure to Graphene Oxide

Graphene-based nanomaterials are increasingly engineered as components of biosensors, interfaces or drug delivery platforms in neuro-repair strategies. In these developments, the mostly used derivative of graphene is graphene oxide (GO). To tailor the safe development of GO nanosheets, we need to model in vitro tissue responses, and in particular the reactivity of microglia, a sub-population of neuroglia that acts as the first active immune response, when challenged by GO. Here, we investigated central nervous system (CNS) tissue reactivity upon long-term exposure to GO nanosheets in 3D culture models. We used the mouse organotypic spinal cord cultures, ideally suited for studying long-term interference with cues delivered at controlled times and concentrations. In cultured spinal segments, the normal presence, distribution and maturation of anatomically distinct classes of neurons and resident neuroglial cells are preserved. Organotypic explants were developed for 2 weeks embedded in fibrin glue alone or presenting GO nanosheets at 10, 25 and 50 \u3bcg/mL. We addressed the impact of such treatments on premotor synaptic activity monitored by patch clamp recordings of ventral interneurons. We investigated by immunofluorescence and confocal microscopy the accompanying glial responses to GO exposure, focusing on resident microglia, tested in organotypic spinal slices and in isolated neuroglia cultures. Our results suggest that microglia reactivity to accumulation of GO flakes, maybe due to active phagocytosis, may trim down synaptic activity, although in the absence of an effective activation of inflammatory response and in the absence of neuronal cell death

Mouse vaccination with dendritic cells loaded with prion protein peptides overcomes tolerance and delays scrapie.

Prion diseases are presumed to be caused by the accumulation in the brain of a pathological protein called prion protein (PrP) scrapie which results from the transconformation of cellular PrP, a ubiquitous glycoprotein expressed in all mammals. Since all isoforms of PrP are perceived as self by the host immune system, a major problem in designing efficient immunoprophylaxis or immunotherapy is to overcome tolerance. The present study was aimed at investigating whether bone-marrow-derived dendritic cells (DCs) loaded with peptides previously shown to be immunogenic in PrP-deficient mice, can overcome tolerance in PrP-proficient wild-type mice and protect them against scrapie. Results show that, in such mice, peptide-loaded DCs elicit both lymphokine release by T cells and antibody secretion against native cellular PrP. Repeated recalls with peptide-loaded DCs reduces the attack rate of 139A scrapie inoculated intraperitoneally and retards disease duration by 40 days. Most interestingly, survival time in individual mice appears to be correlated with the level of circulating antibody against native cellular PrP

Histamine regulates actin cytoskeleton in human toll-like receptor 4-activated monocyte-derived dendritic cells tuning CD4+ T lymphocyte response

Histamine, a major mediator in allergic diseases, differentially regulates the polarizing ability of dendritic cells after Toll-like receptor (TLR) stimulation, by not completely explained mechanisms. In this study we investigated the effects of histamine on innate immune reaction during the response of human monocyte-derived DCs (mDCs) to different TLR stimuli: LPS, specific for TLR4, and Pam3Cys, specific for heterodimer molecule TLR1/TLR2. We investigated actin remodeling induced by histamine together with mDCs phenotype, cytokine production, and the stimulatory and polarizing ability of Th0. By confocal microscopy and RT-PCR expression of Rac1/CdC42 Rho GTPases, responsible for actin remodeling, we show that histamine selectively modifies actin cytoskeleton organization induced by TLR4, but not TLR2 and this correlates with increased IL4 production and decreased IFNγ by primed T cells. We also demonstrate that histamine-induced cytoskeleton organization is at least in part mediated by down-regulation of small Rho GTPase CdC42 and the protein target PAK1, but not by down-regulation of Rac1. The presence and relative expression of histamine receptors HR1–4 and TLRs were determined as well. Independently of actin remodeling, histamine down-regulates IL12p70 and CXCL10 production in mDCs after TLR2 and TLR4 stimulation. We also observed a trend of IL10 up-regulation that, despite previous reports, did not reach statistical significance