Liat Hayardeny

Modulation of Autoimmune Demyelination by Laquinimod via Induction of Brain-Derived Neurotrophic Factor

Laquinimod is a promising, orally available compound that has been successfully evaluated in placebo-controlled phase II/III studies of relapsing-remitting multiple sclerosis (MS). Studies are ongoing to further define laquinimods modulatory mechanisms. Analyses in the animal model of experimental autoimmune encephalomyelitis (EAE) demonstrate that laquinimod reduces infiltration of leukocytes into the central nervous system, induces a Th1 to Th2/3 shift, and suppresses Th17 responses. To evaluate the potential neuroprotective capacity of laquinimod via modulation of brain-derived neurotrophic factor (BDNF), we analyzed the expression of BDNF in blood samples from 203 MS patients treated with laquinimod. Furthermore, we investigated the effect of laquinimod in EAE using a conditional BDNF knockout strain lacking BDNF expression in myeloid cells and T cells (LLF mice). Treatment with laquinimod resulted in a significant and persistent increase in BDNF serum levels of MS patients when compared to baseline and placebo-treated patients. LLF mice treated with laquinimod display a more severe EAE disease course in comparison to wild-type mice. Furthermore, laquinimod-treated wild-type monocytes secreted an anti-inflammatory cytokine pattern in comparison to untreated wild-type monocytes and treated LLF monocytes. Adoptive transfer of laquinimod stimulated monocytes into mice with EAE ameliorated the disease course. Consistent with immunomodulatory properties, laquinimod skewed monocytes toward a regulatory phenotype and also acted via modulation of BDNF, which may contribute to neuroprotection in MS patients.

Laquinimod interferes with migratory capacity of T cells and reduces IL-17 levels, inflammatory demyelination and acute axonal damage in mice with experimental autoimmune encephalomyelitis

We investigated the effect of laquinimod on inflammatory demyelination, axonal damage, cytokine profiles and migratory capacities of lymphocytes in C57BL/6 mice with active EAE induced with MOG(35-55) peptide. The mice were treated at disease induction and after disease onset. Spinal cords were assessed histologically. Cytokines and adhesive properties were analyzed in splenocytes. Preventive and therapeutic laquinimod treatment reduced clinical signs, inflammation, and demyelination. VLA-4-mediated adhesiveness and pro-inflammatory cytokines such as IL-17 were down-regulated in treated animals. Within lesions, treated mice showed similar axonal densities, but less acute axonal damage than controls. Laquinimod might thus protect myelin and axons by decreasing pro-inflammatory cytokines and impairing the migratory capacity of lymphocytes.

Reduced astrocytic NF-κB activation by laquinimod protects from cuprizone-induced demyelination

Laquinimod (LAQ) is a new oral immunomodulatory compound that reduces relapse rate, brain atrophy and disability progression in multiple sclerosis (MS). LAQ has well-documented effects on inflammation in the periphery, but little is known about its direct activity within the central nervous system (CNS). To elucidate the impact of LAQ on CNS-intrinsic inflammation, we investigated the effects of LAQ on cuprizone-induced demyelination in mice in vivo and on primary CNS cells in vitro. Demyelination, inflammation, axonal damage and glial pathology were evaluated in LAQ-treated wild type and Rag-1-deficient mice after cuprizone challenge. Using primary cells we tested for effects of LAQ on oligodendroglial survival as well as on cytokine secretion and NF-κB activation in astrocytes and microglia. LAQ prevented cuprizone-induced demyelination, microglial activation, axonal transections, reactive gliosis and oligodendroglial apoptoses in wild type and Rag-1-deficient mice. LAQ significantly decreased pro-inflammatory factors in stimulated astrocytes, but not in microglia. Oligodendroglial survival was not affected by LAQ in vitro. Astrocytic, but not microglial, NF-κB activation was markedly reduced by LAQ as evidenced by NF-κB reporter assay. LAQ also significantly decreased astrocytic NF-κB activation in cuprizone-treated mice. Our data indicate that LAQ prevents cuprizone-induced demyelination by attenuating astrocytic NF-κB activation. These effects are CNS-intrinsic and not mediated by peripheral immune cells. Therefore, LAQ downregulation of the astrocytic pro-inflammatory response may be an important mechanism underlying its protective effects on myelin, oligodendrocytes and axons. Modulation of astrocyte activation may be an attractive therapeutic target to prevent tissue damage in MS.

Pharmacogenetics of glatiramer acetate therapy for multiple sclerosis reveals drug-response markers.

Genetic-based optimization of treatment prescription is becoming a central research focus in the management of chronic diseases, such as multiple sclerosis, which incur a prolonged drug-regimen adjustment. This study was aimed to identify genetic markers that can predict response to glatiramer acetate (Copaxone) immunotherapy for relapsing multiple sclerosis. For this purpose, we genotyped fractional cohorts of two glatiramer acetate clinical trials for HLA-DRB1*1501 and 61 single nucleotide polymorphisms within a total of 27 candidate genes. Statistical analyses included single nucleotide polymorphism-by-single nucleotide polymorphism and haplotype tests of drug-by-genotype effects in drug-treated versus placebo-treated groups. We report the detection of a statistically significant association between glatiramer acetate response and a single nucleotide polymorphism in a T-cell receptor &bgr; (TRB@) variant replicated in the two independent cohorts (odds ratio=6.85). Findings in the Cathepsin S (CTSS) gene (P=0.049 corrected for all single nucleotide polymorphisms and definitions tested, odds ratio=11.59) in one of the cohorts indicate a possible association that needs to be further investigated. Additionally, we recorded nominally significant associations of response with five other genes, MBP, CD86, FAS, IL1R1 and IL12RB2, which are likely to be involved in glatiramer acetates mode-of-action, both directly and indirectly. Each of these association signals in and of itself is consistent with the no-association null-hypothesis, but the number of detected associations is surprising vis-à-vis chance expectation. Moreover, the restriction of these associations to the glatiramer acetate-treated group, rather than the placebo group, clearly demonstrates drug-specific genetic effects. These findings provide additional progress toward development of pharmacogenetics-based personalized treatment for multiple sclerosis.

Oral treatment with laquinimod augments regulatory T-cells and brain-derived neurotrophic factor expression and reduces injury in the CNS of mice with experimental autoimmune encephalomyelitis

Laquinimod is an orally active molecule that showed efficacy in clinical trials in multiple sclerosis. We studied its effects in the CNS, when administered by therapeutic regimen to mice inflicted with experimental autoimmune encephalomyelitis (EAE). Laquinimod reduced clinical and inflammatory manifestations and elevated the prevalence of T-regulatory cells in the brain. In untreated mice, in the chronic disease stage, brain derived neurotrophic factor (BDNF) expression was impaired. Laquinimod treatment restored BDNF expression to its level in healthy controls. Furthermore, CNS injury, manifested by astrogliosis, demyelination and axonal damages, was significantly reduced following laquinimod treatment, indicating its immunomodulatory and neuroprotective activity.

Laquinimod modulates B cells and their regulatory effects on T cells in multiple sclerosis.

Laquinimod is an orally administered drug under development for the treatment of Multiple Sclerosis (MS), lacking a fully elucidated mode of action. We assessed the immunomodulatory effects of laquinimod in vitro on human B cells from healthy or MS patients, cultured alone or with CD4(+) T cells. Laquinimod modulated B cell markers, mainly by increasing the regulatory ones CD25, IL10 and CD86, and decreased IL4, while increasing IL10 and TGFβ in both B and T cells, in a B cell-mediated manner. These findings shed additional light on the mechanisms underlying the effects of laquinimod in MS and potentially other immune-mediated diseases.

Laquinimod reduces neuroaxonal injury through inhibiting microglial activation

Laquinimod is an emerging oral medication for multiple sclerosis (MS) that reduces brain atrophy and progression of disability in two Phase III clinical trials. The mechanism of these effects is unclear. Persistent activation of microglia occurs in MS and contributes to injury. Thus, we investigated whether laquinimod alters properties of microglia in culture and in experimental autoimmune encephalomyelitis (EAE), and whether this reduces neurodegeneration.

Laquinimod prevents inflammation-induced synaptic alterations occurring in experimental autoimmune encephalomyelitis

Background There are two generally accepted strategies for treating multiple sclerosis (MS), preventing central nervous system (CNS) damage indirectly through immunomodulatory interventions and/or repairing CNS damage by promoting remyelination. Both approaches also provide neuroprotection since they can prevent, indirectly or directly, axonal damage. Objective Recent experimental and clinical evidence indicates that the novel immunomodulatory drug laquinimod can exert a neuroprotective role in MS. Whether laquinimod-mediated neuroprotection is exerted directly on neuronal cells or indirectly via peripheral immunomodulation is still unclear. Methods C57Bl/6 experimental autoimmune encephalomyelitis (EAE) mice, immunised with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, were treated for 26 days with subcutaneous daily injections of laquinimod (from 1 to 25 mg/kg). Patch clamp electrophysiology was performed on acute brain striatal slices from EAE mice treated with daily (25 mg/kg) laquinimod and on acute brain striatal slices from control mice bathed with laquinimod (1–30 µM). Results Both preventive and therapeutic laquinimod treatment fully prevented the alterations of GABAergic synapses induced by EAE, the first limiting also glutamatergic synaptic alterations. This dual effect might, in turn, have limited glutamatergic excitotoxicity, a phenomenon previously observed early during EAE and possibly correlated with later axonal damage. Furthermore, laquinimod treatment also preserved cannabinoid CB1 receptor sensitivity, normally lost during EAE. Finally, laquinimod per se was able to regulate synaptic transmission by increasing inhibitory post-synaptic currents and, at the same time, reducing excitatory post-synaptic currents. Conclusions Our data suggest a novel neuroprotective mechanism by which laquinimod might in vivo protect from neuronal damage occurring as a consequence of inflammatory immune-mediated demyelination.

Immune surveillance of the central nervous system in multiple sclerosis--relevance for therapy and experimental models.

Treatment of central nervous system (CNS) autoimmune disorders frequently involves the reduction, or depletion of immune-competent cells. Alternatively, immune cells are being sequestered away from the target organ by interfering with their movement from secondary lymphoid organs, or their migration into tissues. These therapeutic strategies have been successful in multiple sclerosis (MS), the most prevalent autoimmune inflammatory disorder of the CNS. However, many of the agents that are currently approved or in clinical development also have severe potential adverse effects that stem from the very mechanisms that mediate their beneficial effects by interfering with CNS immune surveillance. This review will outline the main cellular components of the innate and adaptive immune system that participate in host defense and maintain immune surveillance of the CNS. Their pathogenic role in MS and its animal model experimental autoimmune encephalomyelitis (EAE) is also discussed. Furthermore, an experimental model is introduced that may assist in evaluating the effect of therapeutic interventions on leukocyte homeostasis and function within the CNS. This model or similar models may become a useful tool in the repertoire of pre-clinical tests of pharmacological agents to better explore their potential for adverse events.

Laquinimod rescues striatal, cortical and white matter pathology and results in modest behavioural improvements in the YAC128 model of Huntington disease

Increasing evidence supports a role for abnormal immune activation and inflammatory responses in Huntington disease (HD). In this study, we evaluated the therapeutic potential of laquinimod (1 and 10 mg/kg), a novel immunomodulatory agent shown to be protective in a number of neuroinflammatory conditions, in the YAC128 mouse model of HD. Treatment with laquinimod for 6 months rescued atrophy in the striatum, in certain cortical regions, and in the corpus callosum of YAC128 HD mice. Diffusion tensor imaging showed that white matter microstructural abnormalities in the posterior corpus callosum were improved following treatment with low dose (1 mg/kg) laquinimod, and were paralleled by reduced levels of interleukin-6 in the periphery of YAC128 HD mice. Functionally, treatment with laquinimod (1 and 10 mg/kg) led to modest improvements in motor function and in depressive-like behaviour. Taken together, these results suggest that laquinimod may improve some features of pathology in HD, and provides support for the role of immune activation in the pathogenesis of HD.