Ependyma: a new target for autoantibodies in neuromyelitis optica?

Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system characterized by the presence of autoantibodies (called NMO-IgG) targeting aquaporin-4. Aquaporin-4 is expressed at the perivascular foot processes of astrocytes, in the glia limitans, but also at the ependyma. Most studies have focused on studying the pathogenicity of NMO-IgG on astrocytes, and NMO is now considered an astrocytopathy. However, periependymal lesions are observed in NMO suggesting that ependymal cells could also be targeted by NMO-IgG. Ependymal cells regulate CSF-parenchyma molecular exchanges and CSF flow, and are a niche for sub-ventricular neural stem cells. Our aim was to examine the effect of antibodies from NMO patients on ependymal cells. We exposed two models, i.e. primary cultures of rat ependymal cells and explant cultures of rat lateral ventricular wall whole mounts, to purified IgG of NMO patients (NMO-IgG) for 24 hours. We then evaluated the treatment effect using immunolabelling, functional assays, ependymal flow analysis and bulk RNA sequencing. For each experiment, the effects were compared with those of purified IgG from a healthy donors and non-treated cells. We found that: (i) NMO-IgG induced aquaporin-4 agglomeration at the surface of ependymal cells and induced cell enlargement in comparison to controls. In parallel, it induced an increase in gap junction connexin-43 plaque size; (ii) NMO-IgG altered the orientation of ciliary basal bodies and functionally impaired cilia motility; (iii) NMO-IgG activated the proliferation of sub-ventricular neural stem cells; (iv) treatment with NMO-IgG up-regulated the expression of pro-inflammatory cytokines and chemokines in the transcriptomic analysis. Our study showed that NMO-IgG can trigger an early and specific reactive phenotype in ependymal cells, with functional alterations of intercellular communication and cilia, activation of sub-ventricular stem cell proliferation and the secretion of pro-inflammatory cytokines. These findings suggest a key role for ependymal cells in the early phase of NMO lesion formation.

Connexins in neuromyelitis optica: a link between astrocytopathy and demyelination.

Neuromyelitis optica, a rare neuroinflammatory demyelinating disease of the CNS, is characterized by the presence of specific pathogenic autoantibodies directed against the astrocytic water channel aquaporin 4 (AQP4) and is now considered as an astrocytopathy associated either with complement-dependent astrocyte death or with astrocyte dysfunction. However, the link between astrocyte dysfunction and demyelination remains unclear. We propose glial intercellular communication, supported by connexin hemichannels and gap junctions, to be involved in demyelination process in neuromyelitis optica. Using mature myelinated cultures, we demonstrate that a treatment of 1 h to 48 h with immunoglobulins purified from patients with neuromyelitis optica (NMO-IgG) is responsible for a complement independent demyelination, compared to healthy donors' immunoglobulins (P < 0.001). In parallel, patients' immunoglobulins induce an alteration of connexin expression characterized by a rapid loss of astrocytic connexins at the membrane followed by an increased size of gap junction plaques (+60%; P < 0.01). This was co-observed with connexin dysfunction with gap junction disruption (-57%; P < 0.001) and increased hemichannel opening (+17%; P < 0.001), associated with glutamate release. Blocking connexin 43 hemichannels with a specific peptide was able to prevent demyelination in co-treatment with patients compared to healthy donors' immunoglobulins. By contrast, the blockade of connexin 43 gap junctions with another peptide was detrimental for myelin (myelin density -48%; P < 0.001). Overall, our results suggest that dysregulation of connexins would play a pathogenetic role in neuromyelitis optica. The further identification of mechanisms leading to connexin dysfunction and soluble factors implicated, would provide interesting therapeutic strategies for demyelinating disorders.

Purified IgG from aquaporin-4 neuromyelitis optica spectrum disorder patients alters blood-brain barrier permeability.

BACKGROUND: Neuromyelitis optica spectrum disorders (NMOSD) is a primary astrocytopathy driven by antibodies directed against the aquaporin-4 water channel located at the end-feet of the astrocyte. Although blood-brain barrier (BBB) breakdown is considered one of the key steps for the development and lesion formation, little is known about the molecular mechanisms involved. The aim of the study was to evaluate the effect of human immunoglobulins from NMOSD patients (NMO-IgG) on BBB properties. METHODS: Freshly isolated brain microvessels (IBMs) from rat brains were used as a study model. At first, analysis of the secretome profile from IBMs exposed to purified NMO-IgG, to healthy donor IgG (Control-IgG), or non-treated, was performed. Second, tight junction (TJ) proteins expression in fresh IBMs and primary cultures of brain microvascular endothelial cells (BMEC) was analysed by Western blotting (Wb) after exposition to NMO-IgG and Control-IgG. Finally, functional BBB properties were investigated evaluating the presence of rat-IgG in tissue lysate from brain using Wb in the rat-model, and the passage of NMO-IgG and sucrose in a bicameral model. RESULTS: We found that NMO-IgG induces functional and morphological BBB changes, including: 1) increase of pro-inflammatory cytokines production (CXCL-10 [IP-10], IL-6, IL-1RA, IL-1ß and CXCL-3) in IBMs when exposed to NMO-IgG; 2) decrease of Claudin-5 levels by 25.6% after treatment of fresh IBMs by NMO-IgG compared to Control-IgG (p = 0.002), and similarly, decrease of Claudin-5 by at least 20% when BMEC were cultured with NMO-IgG from five different patients; 3) a higher level of rat-IgG accumulated in periventricular regions of NMO-rats compared to Control-rats and an increase in the permeability of BBB after NMO-IgG treatment in the bicameral model. CONCLUSION: Human NMO-IgG induces both structural and functional alterations of BBB properties, suggesting a direct role of NMO-IgG on modulation of BBB permeability in NMOSD.

The Balance in T Follicular Helper Cell Subsets Is Altered in Neuromyelitis Optica Spectrum Disorder Patients and Restored by Rituximab.

Neuromyelitis optica spectrum disorder (NMOSD) is a rare and severe auto-immune disease of the central nervous system driven by pathogenic antibodies mainly directed against aquaporin-4 (AQP4-Ab). Treatment of NMOSD currently relies on immunosuppressants (mycophenolate mofetil, azathioprine) or B-cell-depleting therapy (rituximab). B-cell differentiation into antibody-producing cells requires T follicular helper cells (Tfh). There are several Tfh subsets that differentially affect B-cell differentiation; Tfh2 and Tfh17 subsets strongly support B-cell differentiation. By contrast, Tfh1 lack this capacity and T follicular regulatory cells (Tfr), inhibit B-cell differentiation into antibody-producing cells. We performed a broad characterization of circulating Tfh subsets in 25 NMOSD patients and analyzed the impact of different treatments on these subsets. Untreated NMOSD patients presented a Tfh polarization toward excessive B-helper Tfh subsets with an increase of Tfh17 and (Tfh2+Tfh17)/Tfh1 ratio and a decrease of Tfr and Tfh1. Rituximab restored the Tfh polarization to that of healthy controls. There was a trend toward a similar result for azathioprine and mycophenolate mofetil. Our results suggest that NMOSD patients present an impaired balance in Tfh subsets favoring B-cell differentiation which may explain the sustained antibody production. These findings provide new insights into the pathophysiology of NMOSD, and further suggest that Tfh and Tfr subsets could be considered as potential therapeutic target in NMOSD because of their upstream role in antibody production.

Tissue remodeling in periplaque regions of multiple sclerosis spinal cord lesions.

Our knowledge of multiple sclerosis (MS) neuropathology has benefited from a number of studies that provided an in-depth description of plaques and, more recently, diffuse alterations of the normal-appearing white or grey matter. However, there have been few studies focusing on the periplaque regions surrounding demyelinated plaques, notably in MS spinal cords. In this context, the present study aimed to analyze the molecular immunopathology of periplaque demyelinated lesions (PDLs) in the spinal cord of patients with a progressive form of MS. To achieve this goal, the neuropathological features of PDLs were analyzed in postmortem tissues derived from the cervical spinal cord of 21 patients with primary or secondary progressive MS. We found that PDLs covered unexpectedly large areas of incomplete demyelination and were characterized by the superimposition of pro- and anti-inflammatory molecular signatures. Accordingly, macrophages/microglia accumulated in PDLs but exhibited a poor phagocytic activity toward myelin debris. Interestingly, while genes of the oligodendrocyte lineage were consistently down-regulated in PDLs, astrocyte-related molecules such as aquaporin 4, connexin 43 and the glutamate transporter EAAT1, were significantly upregulated in PDLs at the mRNA and protein levels. Overall, our work indicates that in the spinal cord of patients with a progressive form of MS, a tissue remodeling process that is temporally remote from plaque development takes place in PDLs. We propose that in spinal cord PDLs, this process is supported by subtle alterations of astrocyte functions and by low-grade inflammatory events that drive a slowly progressive loss of myelin and a failure of remyelination.

Aquaporin-4 antibody-negative neuromyelitis optica: distinct assay sensitivity-dependent entity.

OBJECTIVE: To optimize aquaporin-4 (AQP4) antibody (Ab) detection and to assess the influence of the increased sensitivity of the assay on the demographic and disease-related characteristics of a group of AQP4-Ab-negative patients. METHODS: Serum samples were obtained from patients included in the French NOMADMUS database with a definite diagnosis of neuromyelitis optica (NMO) (n = 87) and were compared with controls (n = 54). They were tested by indirect immunofluorescence and cell-based assays (CBAs) in various conditions and with several plasmids. RESULTS: We identified the CBA on live cells transfected with the untagged AQP4-M23 isoform as the best method, with a sensitivity of 74.4% and a specificity of 100%. We demonstrated a direct relationship between improvement of the sensitivity of the detection method and the distinctiveness and characteristics of the AQP4-Ab-negative NMO group. Whereas with the classic indirect immunofluorescence or current AQP4-M1 CBA we found only slight differences between the 2 populations, using the AQP4-M23 CBA, we demonstrated that patients with AQP4-Ab-negative NMO expressed specific demographic and disease-related features. They were characterized by an equal male/female ratio (p < 0.001), a Caucasian ethnicity (p = 0.029), and an overrepresentation of simultaneous optic neuritis and transverse myelitis at first episode (p = 0.015). In terms of disability, they experienced a better visual acuity at last follow-up compared with seropositive NMO (p = 0.007). CONCLUSION: This raises the question of a distinct physiopathology for patients with AQP4-Ab-negative NMO and of their place in the spectrum of the disease.

Insight into the role of CRMP2 (collapsin response mediator protein 2) in T lymphocyte migration: the particular context of virus infection.

Lymphocyte migration into the central nervous system is a critical step in the physiopathology of a variety of neurological diseases, including multiple sclerosis and virus-induced neuroinflammation. To better understand the molecular mechanisms involved in cells migration, we focused our studies on collapsin response mediator proteins (CRMPs), a group of phosphoproteins that mediate neural cell motility. There is now evidence that collapsin response mediator protein 2 (CRMP2) plays critical roles in the polarization (uropod formation) of T lymphocytes and their subsequent migration. CRMP2 was known to respond to semaphorin, ephrin and neurotrophin signaling in neurons. The link between the chemokine CXCL12, CRMP2 activity and cell migration has been demonstrated in T lymphocytes. These observations and comparisons of the activity of CRMPs in immune and non-immmune cells are summarized here. The ability of a human retrovirus to enhance lymphocyte migration through the modulation of CRMP2 activity is also discussed. In conclusion, viruses have the ability to manipulate the lymphocyte motility machinery, intensifying neural tissue invasion in infected patients.

Human T lymphotropic virus type 1 increases T lymphocyte migration by recruiting the cytoskeleton organizer CRMP2.

Recruitment of virus-infected T lymphocytes into the CNS is an essential step in the development of virus-associated neuroinflammatory diseases, notably myelopathy induced by retrovirus human T leukemia virus-1 (HTLV-1). We have recently shown the key role of collapsin response mediator protein 2 (CRMP2), a phosphoprotein involved in cytoskeleton rearrangement, in the control of human lymphocyte migration and in brain targeting in animal models of virus-induced neuroinflammation. Using lymphocytes cloned from infected patients and chronically infected T cells, we found that HTLV-1 affects CRMP2 activity, resulting in an increased migratory potential. Elevated CRMP2 expression accompanies a higher phosphorylation level of CRMP2 and its more pronounced adhesion to tubulin and actin. CRMP2 forms, a full length and a shorter, cleaved one, are also affected. Tax transfection and extinction strategies show the involvement of this viral protein in enhanced full-length and active CRMP2, resulting in prominent migratory rate. A role for other viral proteins in CRMP2 phosphorylation is suspected. Full-length CRMP2 confers a migratory advantage possibly by preempting the negative effect of short CRMP2 we observe on T lymphocyte migration. In addition, HTLV-1-induced migration seems, in part, supported by the ability of infected cell to increase the proteosomal degradation of short CRMP2. Finally, gene expression in CD69(+) cells selected from patients suggests that HTLV-1 has the capacity to influence the CRMP2/PI3K/Akt axis thus to positively control cytoskeleton organization and lymphocyte migration. Our data provide an additional clue to understanding the infiltration of HTLV-1-infected lymphocytes into various tissues and suggest that the regulation of CRMP2 activity by virus infection is a novel aspect of neuroinflammation.

Anti-aquaporin-4 antibodies in Devic's neuromyelitis optica: therapeutic implications.

Devic's neuromyelitis optica (DNMO) is a demyelinating and inflammatory disease of the central nervous system (CNS) essentially restricted to the spinal cord and the optic nerves. It is a rare disorder with a prevalence estimated at less than 1/100,000 in Western countries. Since the first description by Eugène Devic in 1894, the relationship between DNMO and multiple sclerosis (MS) has been controversial. Recent clinical, epidemiological, pathological and immunological data demonstrate that MS and DNMO are distinct entities. This distinction between DNMO and MS is crucial, as prognosis and treatment are indeed different. DNMO is now considered to be an autoimmune, antibody-mediated disease especially since the identification of a specific serum autoantibody, named NMO-IgG and directed against the main water channel of the CNS, aquaporin-4 (AQP4). The assessment of AQP4 antibodies (Abs) has initially been proposed to differentiate DNMO and MS. It has also enlarged the clinical spectrum of DNMO and proved to be helpful in predicting relapses and conversion to DNMO after a first episode of longitudinally extensive transverse myelitis or isolated optic neuritis. Lastly, the discovery of the pathogenic role of AQP4 Abs in DNMO leads to a better understanding of detailed DNMO immunopathology and the elaboration of relevant novel treatment strategies specific to DNMO. In this review, we summarize the present and future therapeutic implications generated by the discovery of the various pathogenic mechanisms of AQP4 Abs in DNMO pathophysiology.

Oligodendrocytes are damaged by neuromyelitis optica immunoglobulin G via astrocyte injury.

Devic's neuromyelitis optica is an inflammatory demyelinating disorder normally restricted to the optic nerves and spinal cord. Since the identification of a specific autoantibody directed against aquaporin 4, neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody, neuromyelitis optica has been considered an entity distinct from multiple sclerosis. Recent findings indicate that the neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody has a pathogenic role through complement-dependent astrocyte toxicity. However, the link with demyelination remains elusive. Autoantibodies can act as receptor agonists/antagonists or alter antigen density in their target cells. We hypothesized that the neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody impairs astrocytic function and secondarily leads to demyelination. Rat astrocytes and oligodendrocytes from primary cultures and rat optic nerves were exposed long-term (24 h) to immunoglobulin G in the absence of complement. Immunoglobulin G was purified from the serum of patients with neuromyelitis optica who were either neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody positive or negative, as well as from healthy controls. Flow cytometry analysis showed a reduction of membrane aquaporin 4 and glutamate transporter type 1 on astrocytes following contact with immunoglobulin G purified from neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody positive serum only. The activity of glutamine synthetase, an astrocyte enzyme converting glutamate into glutamine, decreased in parallel, indicating astrocyte dysfunction. Treatment also reduced oligodendrocytic cell processes and approximately 30% oligodendrocytes died. This deleterious effect was confirmed ex vivo; exposed optic nerves showed reduction of myelin basic protein. Immunoglobulin G from neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody seronegative patients and from healthy controls had no similar effect. Neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody did not directly injure oligodendrocytes cultured without astrocytes. A toxic bystander effect of astrocytes damaged by neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody on oligodendrocytes was identified. Progressive accumulation of glutamate in the culture medium of neuromyelitis optica-immunoglobulin G/aquaporin 4-antibody-treated glial cells supported the hypothesis of a glutamate-mediated excitotoxic death of oligodendrocytes in our models. Moreover, co-treatment of glial cultures with neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody and d+2-amino-5-phosphonopentanoic acid, a competitive antagonist at the N-methyl-d-aspartate/glutamate receptor, partially protected oligodendrocytes. Co-immunolabelling of oligodendrocyte markers and neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody showed that astrocytic positive processes were in close contact with oligodendrocytes and myelin in rat optic nerves and spinal cord, but far less so in other parts of the central nervous system. This suggests a bystander effect of neuromyelitis optica-immunoglobulin G-damaged astrocytes on oligodendrocytes in the nervous tissues affected by neuromyelitis optica. In conclusion, in these cell culture models we found a direct, complement-independent effect of neuromyelitis optica-immunoglobulin G/aquaporin 4 antibody on astrocytes, with secondary damage to oligodendrocytes possibly resulting from glutamate-mediated excitotoxicity. These mechanisms could add to the complement-induced damage, particularly the demyelination, seen in vivo.

Inflammation in neuroviral diseases.

During any viral infection of the central nervous system (CNS), the extent and nature of neural cell alterations are dictated by the localization of virus replication and, possibly, persistence. However, one additional source of CNS damage comes from the immune response that develops following CNS viral infection. Indeed, despite of its major role in controlling virus spread in the infected CNS, the immune system is equipped with numerous molecular effectors shared with the nervous system that may greatly alter the homeostasis and function of neural cells. Proinflammatory cytokines and metalloproteases belong to this inflammatory cascade. Besides neurovirulence, the crosstalk engaged between neural and immune cells is a major factor determining the outcome of neuroviral infections.

Altered expression of CRMPs in the brain of bovine spongiform encephalopathy-infected mice during disease progression.

While recent studies suggest that synaptic alterations are first events in the mechanisms of prion-mediated neurodegeneration, little is known on the identity of the neuronal plasticity-related genes potentially concerned. Here the expression of 4 Collapsin Response Mediator Proteins (CRMPs), a family of signal transduction proteins involved in brain development and altered in Alzheimer's disease, was studied in the brain of C57Bl/6 mice infected with the BSE strain of prion agent, using RT-PCR and Western-blot methods. At the terminal stage of the disease, gene expression of each CRMP had decreased, while at the mid-stage of the disease only CRMP4 (mRNA and protein) expression had increased, concomitant to the start of PrP(Sc) accumulation in the brainstem. Altogether our findings picked out originally CRMPs, and especially CRMP4, as potential contributors to prion pathogenesis.

Phosphorylation of collapsin response mediator protein 2 on Tyr-479 regulates CXCL12-induced T lymphocyte migration.

In the central nervous system, collapsin response mediator protein 2 (CRMP2) is a transducer protein that supports the semaphorin-induced guidance of axons toward their cognate target. However, we previously showed that CRMP2 is also expressed in immune cells and plays a crucial role in T lymphocyte migration. Here we further investigated the molecular mechanisms underlying CRMP2 function in chemokine-directed T-cell motility. Examining Jurkat T-cells treated with the chemokine CXCL12, we found that 1) CXCL12 induces a dynamic re-localization of CRMP2 to uropod, the flexible structure of migrating lymphocyte, and increases its binding to the cytoskeletal protein vimentin; 2) CXCL12 decreases phosphorylation of the glycogen synthase kinase-3beta-targeted residues CRMP2-Thr-509/514; and 3) tyrosine Tyr-479 is a new phosphorylation CRMP2 residue and a target for the Src-family kinase Yes. Moreover, phospho-Tyr-479 increased under CXCL12 signaling while phospho-Thr-509/514 decreased. The functional importance of this tyrosine phosphorylation was demonstrated by Y479F mutation that strongly reduced CXCL12-mediated T-cell polarization and motility as tested in a transmigration model and on neural tissue. We propose that differential phosphorylation by glycogen synthase kinase-3beta and Yes modulates the contribution of CRMP2 to cytoskeletal reorganization during chemokine-directed T-cell migration. In addition to providing a novel mechanism for T lymphocyte motility, our findings reveal CRMP2 as a transducer of chemokine signaling.

Processing and nuclear localization of CRMP2 during brain development induce neurite outgrowth inhibition.

Collapsin response mediator proteins (CRMPs) are believed to play a crucial role in neuronal differentiation and axonal outgrowth. Among them, CRMP2 mediates axonal guidance by collapsing growth cones during development. This activity is correlated with the reorganization of cytoskeletal proteins. CRMP2 is implicated in the regulation of several intracellular signaling pathways. Two subtypes, A and B, and multiple cytosolic isoforms of CRMP2B with apparent masses between 62 and 66 kDa have previously been reported. Here, we show a new short isoform of 58 kDa, expressed during brain development, derived from C-terminal processing of the CRMP2B subtype. Although full-length CRMP2 is restricted to the cytoplasm, using transfection experiments, we demonstrate that a part of the short isoform is found in the nucleus. Interestingly, at the tissue level, this short CRMP2 is also found in a nuclear fraction of brain extract. By mutational analysis, we demonstrate, for the first time, that nuclear translocation occurs via nuclear localization signal (NLS) within residues Arg(471)-Lys(472) in CRMP2 sequence. The NLS may be unmasked after C-terminal processing; thereby, this motif may be surface-exposed. This short CRMP2 induces neurite outgrowth inhibition in neuroblastoma cells and suppressed axonal growth in cultured cortical neurons, whereas full-length CRMP2 promotes neurite elongation. The NLS-mutated short isoform, restricted to the cytoplasm, abrogates both neurite outgrowth and axon growth inhibition, indicating that short nuclear CRMP2 acts as a dominant signal. Therefore, post-transcriptional processing of CRMP2 together with its nuclear localization may be an important key in the regulation of neurite outgrowth in brain development.

A role for the neuronal protein collapsin response mediator protein 2 in T lymphocyte polarization and migration.

The semaphorin-signaling transducer collapsin response mediator protein 2 (CRMP2) has been identified in the nervous system where it mediates Sema3A-induced growth cone navigation. In the present study, we provide first evidence that CRMP2 is present in the immune system and plays a critical role in T lymphocyte function. CRMP2 redistribution at the uropod in polarized T cells, a structural support of lymphocyte motility, suggests that it may regulate T cell migration. This was evidenced in primary T cells by small-interfering RNA-mediated CRMP2 gene silencing and blocking Ab, as well as CRMP2 overexpression in Jurkat T cells tested in a chemokine- and semaphorin-mediated transmigration assay. Expression analysis in PBMC from healthy donors showed that CRMP2 is enhanced in cell subsets bearing the activation markers CD69+ and HLA-DR+. Heightened expression in T lymphocytes of patients suffering from neuroinflammatory disease with enhanced T cell-transmigrating activity points to a role for CRMP2 in pathogenesis. The elucidation of the signals and mechanisms that control this pathway will lead to a better understanding of T cell trafficking in physiological and pathological situations.

T-cells in neuronal injury and repair: semaphorins and related T-cell signals.

There are many parallels between the hematopoietic and the nervous systems in terms of mechanisms regulating their development and functions. In neuroinflammatory diseases, interaction between the immune and nervous systems through shared molecules is suspected to trigger an inappropriate crosstalk and lead to demyelination and axonal loss. Here we focus on semaphorins and their functions in the nervous and immune systems and point out the deleterious effect of an immune semaphorin, semaphorin 4D (Sema4D)/CD100, on oligodendrocyte integrity and survival. We propose immune semaphorins as new candidates involved in the pathogenic mechanisms of neuroinflammatory diseases, promoting demyelination, and impairing neuroregeneration.

Impairment of blood-cerebrospinal fluid barrier properties by retrovirus-activated T lymphocytes: reduction in cerebrospinal fluid-to-blood efflux of prostaglandin E2.

The choroid plexus epithelium forms the interface between the blood and the CSF. In conjunction with the tight junctions restricting the paracellular pathway, polarized specific transport systems in the choroidal epithelium allow a fine regulation of CSF-borne biologically active mediators. The highly vascularized stroma delimited by the choroidal epithelium can be a reservoir for retrovirus-infected or activated immune cells. In this work, new insight in the implication of the blood-CSF barrier in neuroinfectious and inflammatory diseases is provided by using a differentiated cellular model of the choroidal epithelium, exposed to infected T lymphocytes. We demonstrate that T cells activated by a retroviral infection, but not non-infected cells, reduce the transporter-mediated CSF-to-blood efflux of organic anions, in particular that of the potent pro-inflammatory prostaglandin PGE2, via the release of soluble factors. A moderate alteration of the paracellular permeability also occurs. We identified the viral protein Tax, oxygenated free radicals, matrix-metalloproteinases and pro-inflammatory cytokines as active molecules released during the exposure of the epithelium to infected T cells. Among them, tumour necrosis factor and interleukin 1 are directly involved in the mechanism underlying the decrease in some choroidal organic anion efflux. Given the strong involvement of CSF-borne PGE2 in sickness behaviour syndrome, these data suggest that the blood-CSF barrier plays an important role in the pathophysiology of neuroinflammation and neuroinfection, via changes in the transport processes controlling the CSF biodisposition of PGE2.

Cumulative mutations in the genome of Echovirus 6 during establishment of a chronic infection in precursors of glial cells.

Although Enteroviruses are mainly described as responsible for acute diseases, their role in severe chronic pathology has been also established. Echovirus 6-like sequences have been detected by PCR analysis in central nervous system specimens from patients presenting with Amyotrophic Lateral Sclerosis. These findings suggested a persistent infection with viruses that underwent, genetic changes precluding viral particle release. To support this hypothesis, we developed a model system of Echovirus 6 chronic infection in precursors of glial cells. The nucleotide sequences of the 5'non-translated region (5'NTR), 2A and 3C regions of the virus developing persistent genome were analysed during establishment of the chronic phenotype. This study revealed that at day 160 of chronic infection, several mutations were observed: one mutation at nucleotide 108 upstream the domain II of the internal ribosome entry site (IRES) structure, one mutation at nucleotide 30 in the cloverleaf, and two mutations in the 2A region (translated in His48 to Tyr, and Ile 123 to Met). No mutations were detected in the 3C region. The impact of these mutations on viral replication have been analysed in a rabbit reticulocyte lysate (RRL) translation assay supplemented with HeLa cell lysate, and by plaque assay. Viruses with these mutations displayed a phenotype with a significant reduction of replication, while in vitro translation was not affected by the nucleotide 108 mutation. This model allowed the description of molecular changes observed in the genome of Echovirus 6 during the establishment of a chronic infection phenotype, and may be helpful for the understanding of the mechanisms leading Enteroviruses to develop chronic infections in man.

Semaphorin CD100 from activated T lymphocytes induces process extension collapse in oligodendrocytes and death of immature neural cells.

An inappropriate cross talk between activated T lymphocytes infiltrating the CNS and neural cells can sustain the onset and progression of demyelination and axonal degeneration in neuroinflammatory diseases. To mimic this deleterious cross talk, we designed an experimental paradigm consisting of transient cocultures of T lymphocytes chronically activated by retrovirus infection (not virus productive) with human multipotent neural precursors or primary oligodendrocytes from rat brain. We showed that activated T lymphocytes induced apoptotic death of multipotent neural progenitors and immature oligodendrocytes after a progressive collapse of their process extensions. These effects were reminiscent of those induced by brain semaphorin on neural cells. Blockade by specific Abs of soluble CD100 (sCD100)/semaphorin 4D released by activated T cells, or treatment with rsCD100, demonstrated that this immune semaphorin has the ability to collapse oligodendrocyte process extensions and to trigger neural cell apoptosis, most likely through receptors of the plexin family. The specific presence of sCD100 in the cerebrospinal fluid and of CD100-expressing T lymphocytes in the spinal cord of patients suffering with neuroinflammatory demyelination pointed to the potential pathological effect of sCD100 in the CNS. Thus, our results show that CD100 is a new important element in the deleterious T cell-neural cell cross talk during neuroinflammation and suggest its role in demyelination or absence of remyelination in neuroinflammatory diseases including multiple sclerosis and human T lymphotropic virus type 1-associated myelopathy.

Pro-inflammatory cytokines modulate matrix metalloproteinase secretion and organic anion transport at the blood-cerebrospinal fluid barrier.

Neuroinflammation and neuroinfection trigger cytokine-mediated responses that include an increase in the cerebrospinal fluid (CSF) levels of pro-inflammatory matrix metalloproteinases (MMPs) and organic anions such as leukotrienes and prostaglandins. The choroid plexus (CP) epithelium forming the interface between the blood and the CSF regulates the CSF concentration of bioactive organic anions and is involved in neuro-immune regulation. We demonstrated that both fourth and lateral ventricle CPs are a source of pro- and active MMP-2 and MMP-9 in the brain. Using a cellular model of the blood-CSF barrier, we showed that a pro-inflammatory cytokine treatment leads to an increase in the choroidal MMP secretion at either the apical or the basolateral membrane, depending on the ventricular origin of the choroidal cells. This effect was not concomitant with an alteration in the structural blood-CSF barrier. Neither was the pool of antioxidant sulfhydryls in the choroidal cells challenged. In contrast, the efficiency of the choroidal epithelium to clear the CSF from organic anions was highly reduced. Thus, during inflammation, the CPs could be one source of MMPs found in the CSF facilitate leucocyte migration by secreting MMPs into the choroidal stroma, and promote the inflammatory process by failing in its ability to clear deleterious compounds from the brain.