Safety and efficacy of tolebrutinib, an oral brain-penetrant BTK inhibitor, in relapsing multiple sclerosis: a phase 2b, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Tolebrutinib is an oral, CNS-penetrant, irreversible inhibitor of Bruton's tyrosine kinase, an enzyme expressed in B lymphocytes and myeloid cells including microglia, which are major drivers of inflammation in multiple sclerosis. We aimed to determine the dose-response relationship between tolebrutinib and the reduction in new active brain MRI lesions in patients with relapsing multiple sclerosis. METHODS: We did a 16-week, phase 2b, randomised, double-blind, placebo-controlled, crossover, dose-finding trial at 40 centres (academic sites, specialty clinics, and general neurology centres) in ten countries in Europe and North America. Eligible participants were adults aged 18-55 years with diagnosed relapsing multiple sclerosis (either relapsing-remitting or relapsing secondary progressive multiple sclerosis), and one or more of the following criteria: at least one relapse within the previous year, at least two relapses within the previous 2 years, or at least one active gadolinium-enhancing brain lesion in the 6 months before screening. Exclusion criteria included a diagnosis of primary progressive multiple sclerosis or a diagnosis of secondary progressive multiple sclerosis without relapse. We used a two-step randomisation process to randomly assign eligible participants (1:1) to two cohorts, then further randomly assign participants in each cohort (1:1:1:1) to four tolebrutinib dose groups (5, 15, 30, and 60 mg administered once daily as an oral tablet). Cohort 1 received tolebrutinib for 12 weeks, then matched placebo (ie, identical looking tablets) for 4 weeks; cohort 2 received 4 weeks of placebo followed by 12 weeks of tolebrutinib. Participants and investigators were masked for dose and tolebrutinib-placebo administration sequence; investigators, study team members, and study participants did not have access to unmasked data. MRI scans were done at screening and every 4 weeks over 16 weeks. The primary efficacy endpoint was the number of new gadolinium-enhancing lesions detected on the scan done after 12 weeks of tolebrutinib treatment (assessed at week 12 for cohort 1 and week 16 for cohort 2), relative to the scan done 4 weeks previously, and compared with the lesions accumulated during 4 weeks of placebo run-in period in cohort 2. Efficacy data were analysed in a modified intention-to-treat population, using a two-step multiple comparison procedure with modelling analysis. Safety was assessed for all participants who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov (NCT03889639), EudraCT (2018-003927-12), and WHO (U1111-1220-0572), and has been completed. FINDINGS: Between May 14, 2019, and Jan 2, 2020, we enrolled and randomly assigned 130 participants to tolebrutinib: 33 to 5 mg, 32 to 15 mg, 33 to 30 mg, and 32 to 60 mg. 129 (99%) completed the treatment regimen and 126 were included in the primary analysis. At treatment week 12, there was a dose-dependent reduction in the number of new gadolinium-enhancing lesions (mean [SD] lesions per patient: placebo, 1·03 [2·50]; 5 mg, 1·39 [3·20]; 15 mg, 0·77 [1·48]; 30 mg, 0·76 [3·31]; 60 mg, 0·13 [0·43]; p=0·03). One serious adverse event was reported (one patient in the 60 mg group was admitted to hospital because of a multiple sclerosis relapse). The most common non-serious adverse event during tolebrutinib treatment was headache (in one [3%] of 33 in the 5 mg group; three [9%] of 32 in the 15 mg group; one [3%] of 33 in the 30 mg group; and four [13%] of 32 in the 60 mg group). No safety-related discontinuations or treatment-related deaths occurred. INTERPRETATION: 12 weeks of tolebrutinib treatment led to a dose-dependent reduction in new gadolinium-enhancing lesions, the 60 mg dose being the most efficacious, and the drug was well tolerated. Reduction of acute inflammation, combined with the potential to modulate the immune response within the CNS, provides a scientific rationale to pursue phase 3 clinical trials of tolebrutinib in patients with relapsing and progressive forms of multiple sclerosis. FUNDING: Sanofi.

Evolution of clinical trials in multiple sclerosis.

Clinical trials have advanced the treatment of multiple sclerosis (MS) by demonstrating the safety and efficacy of disease-modifying therapies (DMTs). This review discusses major changes to MS clinical trials in the era of DMTs. As treatment options for MS continue to increase, patients in modern MS trials present earlier and with milder disease compared with historic MS populations. While placebo-controlled trials for some questions may still be relevant, DMT trials in relapsing-remitting MS (RRMS) are no longer ethical. The replacement of the placebo arm by an active comparator arm in trials have raised the cost of trials by requiring larger sample sizes to detect on-study changes in treatment effects. Efforts to improve trial efficiency in RRMS have focused on exploring adaptive designs and relying on sensitive magnetic resonance imaging measures of disease activity. In trials for progressive forms of MS (PMS), the lack of sensitive outcome measures that can be used in shorter-term trials have delayed the development of effective treatments. Recent shifting of the focus to advancing trials in PMS has identified paraclinical outcome measurements with improved potential, and the testing of agents for neuroprotection and remyelination is in progress.

Siponimod versus placebo in secondary progressive multiple sclerosis (EXPAND): a double-blind, randomised, phase 3 study.

BACKGROUND: No treatment has consistently shown efficacy in slowing disability progression in patients with secondary progressive multiple sclerosis (SPMS). We assessed the effect of siponimod, a selective sphingosine 1-phosphate (S1P) receptor1,5 modulator, on disability progression in patients with SPMS. METHODS: This event-driven and exposure-driven, double-blind, phase 3 trial was done at 292 hospital clinics and specialised multiple sclerosis centres in 31 countries. Using interactive response technology to assign numbers linked to treatment arms, patients (age 18-60 years) with SPMS and an Expanded Disability Status Scale score of 3·0-6·5 were randomly assigned (2:1) to once daily oral siponimod 2 mg or placebo for up to 3 years or until the occurrence of a prespecified number of confirmed disability progression (CDP) events. The primary endpoint was time to 3-month CDP. Efficacy was assessed for the full analysis set (ie, all randomly assigned and treated patients); safety was assessed for the safety set. This trial is registered with ClinicalTrials.gov, number NCT01665144. FINDINGS: 1651 patients were randomly assigned between Feb 5, 2013, and June 2, 2015 (1105 to the siponimod group, and 546 to the placebo group). One patient did not sign the consent form, and five patients did not receive study drug, all of whom were in the siponimod group. 1645 patients were included in the analyses (1099 in the siponimod group and 546 in the placebo). At baseline, the mean time since first multiple sclerosis symptoms was 16·8 years (SD 8·3), and the mean time since conversion to SPMS was 3·8 years (SD 3·5); 1055 (64%) patients had not relapsed in the previous 2 years, and 918 (56%) of 1651 needed walking assistance. 903 (82%) patients receiving siponimod and 424 (78%) patients receiving placebo completed the study. 288 (26%) of 1096 patients receiving siponimod and 173 (32%) of 545 patients receiving placebo had 3-month CDP (hazard ratio 0·79, 95% CI 0·65-0·95; relative risk reduction 21%; p=0·013). Adverse events occurred in 975 (89%) of 1099 patients receiving siponimod versus 445 (82%) of 546 patients receiving placebo; serious adverse events were reported for 197 (18%) patients in the siponimod group versus 83 (15%) patients in the placebo group. Lymphopenia, increased liver transaminase concentration, bradycardia and bradyarrhythmia at treatment initiation, macular oedema, hypertension, varicella zoster reactivation, and convulsions occurred more frequently with siponimod than with placebo. Initial dose titration mitigated cardiac first-dose effects. Frequencies of infections, malignancies, and fatalities did not differ between groups. INTERPRETATION: Siponimod reduced the risk of disability progression with a safety profile similar to that of other S1P modulators and is likely to be a useful treatment for SPMS. FUNDING: Novartis Pharma AG.

Safety and Efficacy of Siponimod (BAF312) in Patients With Relapsing-Remitting Multiple Sclerosis: Dose-Blinded, Randomized Extension of the Phase 2 BOLD Study.

IMPORTANCE: This dose-blinded extension of the phase 2 BOLD (BAF312 on MRI Lesion Given Once Daily) Study in relapsing-remitting multiple sclerosis provides evidence on disease activity and safety of a range of siponimod doses for up to 24 months. OBJECTIVE: To assess the safety and efficacy of siponimod for up to 24 months during the dose-blinded extension of the BOLD Study. DESIGN, SETTING, AND PARTICIPANTS: At extension baseline in a randomized clinical trial, patients taking siponimod continued at the originally assigned dose and patients taking placebo were rerandomized to the 5 siponimod doses. Initial treatment was titrated over 10 days. A total of 252 eligible patients were treated at specialized multiple sclerosis centers for this study conducted from August 30, 2010, through June 3, 2013. INTERVENTIONS: Siponimod at 10-mg, 2-mg, 1.25-mg, 0.5-mg, and 0.25-mg doses. MAIN OUTCOMES AND MEASURES: Safety assessment included blood tests, documentation of adverse events at regular scheduled visits and Holter monitoring; key efficacy measures were annualized relapse rate and magnetic resonance imaging lesion activity. RESULTS: Among the 252 eligible patients, the mean (SD) ages were 37.2 (8.4) years, 35.2 (9.1) years, 34.0 (7.6) years, 35.1 (9.2) years, and 36.8 (9.1) years in the 0.25-mg, 0.5-mg, 1.25-mg, 2-mg, and 10-mg groups. Of the 252 patients, 184 (73%) entered the extension and received siponimod (10 mg: n = 33; 2 mg: n = 29; 1.25 mg: n = 43; 0.5 mg: n = 29; and 0.25 mg: n = 50); 159 (86.4%) completed the dose-blinded extension. The incidence of adverse events was similar across treatment groups (10 mg: 87.9%; 2 mg: 89.7%; 1.25 mg: 88.4%; 0.5 mg: 96.6%; and 0.25 mg: 84.0%). Nine patients reported serious adverse events (2 mg: 3/29 [10.3%], 1.25 mg: 1/43 [2.3%], 0.5 mg: 4/29 [13.8%], and 0.25 mg: 1/50 [2.0%]; no serious adverse event was reported for more than 1 patient and no new safety signals occurred compared with the BOLD Study. Dose titration mitigated symptomatic bradycardic events. Reductions in mean (95% CI) gadolinium-enhancing T1 lesion counts from the last BOLD assessment were sustained in the 10-mg, 2-mg, 1.25-mg, and 0.5-mg dose groups (0 [0-0], 0.1 [0-1.9], 0.1 [0-2.6], and 0.1 [0-2.8] at month 24, respectively). At the 3 highest vs 2 lowest doses, the estimated new/newly enlarging T2 lesion counts (95% CIs) were lower during months 6 to 12 (0.5 [0.2-1.3], 0.4 [0.2-1.1], and 0.2 [0.1-0.6] vs 1.3 [0.6-2.8] and 1.4 [0.7-2.7]), months 12 to 18 (0.4 [0.1-1.1], 0.4 [0.1-1.3], and 0.4 [0.2-1.0] vs 1.0 [0.4-2.6] and 3.6 [1.7-7.6]), and months 18 to 24 (0 [0-not estimable], 0.9 [0.1-7.6], and 0.1 [0-1.7] vs 1.6 [0.3-7.7] and 2.0 [0.4-9.5]). Annualized relapse rates (95% CIs) up to month 24 were similarly lower for the 3 highest doses: 0.22 (0.12-0.40) for 10 mg, 0.20 (0.10-0.38) for 2 mg, and 0.14 (0.08-0.26) for 1.25 mg vs 0.33 (0.19-0.56) for 0.5 mg and 0.33 (0.21-0.50) for 0.25 mg. CONCLUSIONS AND RELEVANCE: For up to 24 months of siponimod treatment, multiple sclerosis disease activity was low and there were no new safety signals; investigation in phase 3 trials is encouraged. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01185821.

Siponimod for patients with relapsing-remitting multiple sclerosis (BOLD): an adaptive, dose-ranging, randomised, phase 2 study.

BACKGROUND: Siponimod is an oral selective modulator of sphingosine 1-phosphate receptor types 1 and type 5, with an elimination half-life leading to washout in 7 days. We aimed to determine the dose-response relation of siponimod in terms of its effects on brain MRI lesion activity and characterise safety and tolerability in patients with relapsing-remitting multiple sclerosis. METHODS: In this double-blind, adaptive dose-ranging phase 2 study, we enrolled adults (aged 18-55 years) with relapsing-remitting multiple sclerosis at 73 medical centres in Europe and North America. We tested two patient cohorts sequentially, separated by an interim analysis at 3 months. We randomly allocated patients in cohort 1 (1:1:1:1) to receive once-daily siponimod 10 mg, 2 mg, or 0·5 mg, or placebo for 6 months. We randomly allocated patients in cohort 2 (4:4:1) to siponimod 1·25 mg, siponimod 0·25 mg, or placebo once-daily for 3 months. Randomisation was done with a central, automated system and patients and investigators were masked to treatment assignment. The primary endpoint was dose-response, assessed by percentage reduction in monthly number of combined unique active lesions at 3 months for siponimod versus placebo; this endpoint was analysed by a multiple comparison procedure with modelling techniques in all patients with at least one MRI scan up to 3 months. We assessed safety in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT00879658. FINDINGS: Between March 30, 2009, and Oct 22, 2010, we recruited 188 patients into cohort 1 and 109 patients into cohort 2. We showed a dose-response relation (p=0·0001) across the five doses of siponimod, with reductions in combined unique active lesions at 3 months compared with placebo of 35% (95% CI 17-57) for siponimod 0·25 mg (51 patients included in the primary endpoint analysis), 50% (29-69) for siponimod 0·5 mg (43 patients), 66% (48-80) for siponimod 1·25 mg (42 patients), 72% (57-84) for siponimod 2 mg (45 patients), and 82% (70-90) for siponimod 10 mg (44 patients). In patients treated for 6 months, 37 (86%) of 43 patients who received siponimod 0·5 mg had adverse events (eight serious), as did 48 (98%) of 49 patients who received siponimod 2 mg (four serious), 48 (96%) of 50 patients who received siponimod 10 mg (three serious), and 36 (80%) of 45 controls (none serious). For individuals treated to 3 months, 38 (74%) of 51 patients who received siponimod 0·25 mg had adverse events (none serious), as did 29 (69%) of 42 patients who received siponimod 1·25 mg (two serious) and 13 (81%) of 16 controls (none serious). INTERPRETATION: Therapeutic effects of siponimod on MRI lesion activity in model-based analyses and its tolerability in relapsing-remitting multiple sclerosis warrant investigation in a phase 3 trial. FUNDING: Novartis Pharma AG.

Gene expression profiling in multiple sclerosis: a disease of the central nervous system, but with relapses triggered in the periphery?

The aetiology of multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system (CNS), includes both genetic and environmental factors, but the pathogenesis is still incompletely known. We performed gene expression profiling on paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) samples from 26 MS patients without immunomodulatory treatment, sampled in relapse or remission, and 18 controls using Human Genome U133 plus 2.0 arrays (Affymetrix). In the CSF, 939 probe sets detected differential expression in MS patients compared to controls, but none in PBMCs, confirming that CSF cells might mirror the disease processes. The regulation of selected transcripts in CSF of MS patients was confirmed by quantitative PCR. Unexpectedly however, when comparing MS patients in relapse to those in remission, 266 probe sets detected differential expression in PBMCs, but not in CSF cells, indicating the importance of events outside of the CNS in the triggering of relapse.

Pivotal advance: HMGB1 expression in active lesions of human and experimental multiple sclerosis.

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS, most frequently starting with a series of bouts, each followed by complete remission and then a secondary, progressive phase during which the neurological deficit increases steadily. The underlying molecular mechanisms responsible for disease progression are still unclear. Herein, we demonstrate that high mobility group box chromosomal protein 1 (HMGB1), a DNA-binding protein with proinflammatory properties, is evident in active lesions of MS and experimental autoimmune encephalomyelitis (EAE) and that HMGB1 levels correlate with active inflammation. Furthermore, the expression of the innate HMGB1 receptors--receptor for advanced glycation end products, TLR2, and TLR4--was also highly increased in MS and rodent EAE. Additionally, in vitro activation of rodent CNS-derived microglia and bone marrow-derived macrophages demonstrated that microglia were equally as capable as macrophages of translocating HMGB1 following LPS/IFN-gamma stimulation. Significant expression of HMGB1 and its receptors on accumulating activated macrophages and resident microglia may thus provide a positive feedback loop that amplifies the inflammatory response during MS and EAE pathogenesis.

Vra4 congenic rats with allelic differences in the class II transactivator gene display altered susceptibility to experimental autoimmune encephalomyelitis.

Presentation of Ag bound to MHC class II (MHC II) molecules to CD4+ T cells is a key event in adaptive immune responses. Genetic differences in MHC II expression in the rat CNS were recently positioned to allelic variability in the CIITA gene (Mhc2ta), located within the Vra4 locus on rat chromosome 10. In this study, we have examined reciprocal Vra4-congenic strains on the DA and PVGav1 backgrounds, respectively. After experimental nerve injury the strain-specific MHC II expression on microglia was reversed in the congenic strains. Similar findings were obtained after intraparenchymal injection of IFN-gamma in the brain. Expression of MHC class II was also lower on B cells and dendritic cells from the DA.PVGav1-Vra4- congenic strain compared with DA rats after in vitro stimulation with IFN-gamma. We next explored whether Vra4 may affect the outcome of experimental autoimmune disease. In experimental autoimmune encephalomyelitis induced by immunization with myelin oligodendrocyte glycoprotein, DA.PVGav1-Vra4 rats displayed a lower disease incidence and milder disease course compared with DA, whereas both PVGav1 and PVGav1.DA-Vra4 rats were completely protected. These results demonstrate that naturally occurring allelic differences in Mhc2ta have profound effects on the quantity of MHC II expression in the CNS and on immune cells and that this genetic variability also modulates susceptibility to autoimmune neuroinflammation.

Variation in interleukin 7 receptor alpha chain (IL7R) influences risk of multiple sclerosis.

Multiple sclerosis is a chronic, often disabling, disease of the central nervous system affecting more than 1 in 1,000 people in most western countries. The inflammatory lesions typical of multiple sclerosis show autoimmune features and depend partly on genetic factors. Of these genetic factors, only the HLA gene complex has been repeatedly confirmed to be associated with multiple sclerosis, despite considerable efforts. Polymorphisms in a number of non-HLA genes have been reported to be associated with multiple sclerosis, but so far confirmation has been difficult. Here, we report compelling evidence that polymorphisms in IL7R, which encodes the interleukin 7 receptor alpha chain (IL7Ralpha), indeed contribute to the non-HLA genetic risk in multiple sclerosis, demonstrating a role for this pathway in the pathophysiology of this disease. In addition, we report altered expression of the genes encoding IL7Ralpha and its ligand, IL7, in the cerebrospinal fluid compartment of individuals with multiple sclerosis.

Temporal expression and cellular origin of CC chemokine receptors CCR1, CCR2 and CCR5 in the central nervous system: insight into mechanisms of MOG-induced EAE.

BACKGROUND: The CC chemokine receptors CCR1, CCR2 and CCR5 are critical for the recruitment of mononuclear phagocytes to the central nervous system (CNS) in multiple sclerosis (MS) and other neuroinflammatory diseases. Mononuclear phagocytes are effector cells capable of phagocytosing myelin and damaging axons. In this study, we characterize the regional, temporal and cellular expression of CCR1, CCR2 and CCR5 mRNA in the spinal cord of rats with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE). While resembling human MS, this animal model allows unique access to CNS-tissue from various time-points of relapsing neuroinflammation and from various lesional stages: early active, late active, and inactive completely demyelinated lesions. METHODS: The expression of CCR1, CCR2 and CCR5 mRNA was studied with in situ hybridization using radio labelled cRNA probes in combination with immunohistochemical staining for phenotypic cell markers. Spinal cord sections from healthy rats and rats with MOG-EAE (acute phase, remission phase, relapse phase) were analysed. In defined lesion stages, the number of cells expressing CCR1, CCR2 and CCR5 mRNA was determined. Data were statistically analysed by the nonparametric Mann-Whitney U test. RESULTS: In MOG-EAE rats, extensive up-regulation of CCR1 and CCR5 mRNA, and moderate up-regulation of CCR2 mRNA, was found in the spinal cord during episodes of active inflammation and demyelination. Double staining with phenotypic cell markers identified the chemokine receptor mRNA-expressing cells as macrophages/microglia. Expression of all three receptors was substantially reduced during clinical remission, coinciding with diminished inflammation and demyelination in the spinal cord. Healthy control rats did not show any detectable expression of CCR1, CCR2 or CCR5 mRNA in the spinal cord. CONCLUSION: Our results demonstrate that the acute and chronic-relapsing phases of MOG-EAE are associated with distinct expression of CCR1, CCR2, and CCR5 mRNA by cells of the macrophage/microglia lineage within the CNS lesions. These data support the notion that CCR1, CCR2 and CCR5 mediate recruitment of both infiltrating macrophages and resident microglia to sites of CNS inflammation. Detailed knowledge of expression patterns is crucial for the understanding of therapeutic modulation and the validation of CCR1, CCR2 and CCR5 as feasible targets for therapeutic intervention in MS.

Genetics of autoimmune neuroinflammation.

Detection of gene variants affecting the risk for multiple sclerosis provides insights into mechanisms central for autoaggressive neuroinflammation. Major histocompatibility complex (MHC) class II genes, and probably also MHC class I genes, regulate both human multiple sclerosis and rodent experimental autoimmune encephalomyelitis. However, the functional understanding of the MHC regulation requires further experimentation. Genome scans in human multiple sclerosis have failed to demonstrate significant non-MHC loci with genome-wide significance, but approximately 50 such loci have been described in different rodent experimental autoimmune encephalomyelitis models. Positional cloning of individual rodent genes is difficult, but genes or small genome regions now emerge. Association studies in large human cohorts are needed to confirm the human relevance of rodent genes and such cohorts will also be used for single nucleotide polymorphism-based whole-genome screening. It is realistic to assume that several non-MHC genes regulating autoimmune neuroinflammation, including target tissue responses, will be pinpointed in the next ten years. At the moment there are a few hot candidates, including MHC2TA, PRKCA and IL7R.

Advanced intercross line mapping of Eae5 reveals Ncf-1 and CLDN4 as candidate genes for experimental autoimmune encephalomyelitis.

Eae5 in rats was originally identified in two F(2) intercrosses, (DA x BN) and (E3 x DA), displaying linkage to CNS inflammation and disease severity in experimental autoimmune encephalomyelitis (EAE), respectively. This region overlaps with an arthritis locus, Pia4, which was also identified in the (E3 x DA) cross. Two congenic strains, BN.DA-Eae5 and BN.DA-Eae5.R1, encompassing the previously described Eae5 and Pia4, were established. DA alleles within the chromosome 12 fragment conferred an increase in disease susceptibility as well as increased inflammation and demyelination in the CNS as compared with BN alleles. To enable a more precise fine mapping of EAE regulatory genes, we used a rat advanced intercross line between the EAE-susceptible DA strain and the EAE-resistant PVG.1AV1 strain. Linkage analysis performed in the advanced intercross line considerably narrowed down the myelin oligodendrocyte glycoprotein-EAE regulatory locus (Eae5) to a approximately 1.3-megabase region with a defined number of candidate genes. In this study we demonstrate a regulatory effect of Eae5 on MOG-EAE by using both congenic strains as well as fine mapping these effects to a region containing Ncf-1, a gene associated with arthritis. In addition to structural polymorphisms in Ncf-1, both sequence polymorphisms and expression differences were identified in CLDN4. CLDN4 is a tight junction protein involved in blood-brain barrier integrity. In conclusion, our data strongly suggests Ncf-1 to be a gene shared between two organ-specific inflammatory diseases with a possible contribution by CLDN4 in encephalomyelitis.

CX3CL1 (fractalkine) and CX3CR1 expression in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis: kinetics and cellular origin.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). It is associated with local activation of microglia and astroglia, infiltration of activated macrophages and T cells, active degradation of myelin and damage to axons and neurons. The proposed role for CX3CL1 (fractalkine) in the control of microglia activation and leukocyte infiltration places this chemokine and its receptor CX3CR1 in a potentially strategic position to control key aspects in the pathological events that are associated with development of brain lesions in MS. In this study, we examine this hypothesis by analyzing the distribution, kinetics, regulation and cellular origin of CX3CL1 and CX3CR1 mRNA expression in the CNS of rats with an experimentally induced MS-like disease, myelin oligodendrocyte glycoprotein (MOG)-induced autoimmune encephalomyelitis (EAE). METHODS: The expression of CX3CL1 and its receptor CX3CR1 was studied with in situ hybridization histochemical detection of their mRNA with radio labeled cRNA probes in combination with immunohistochemical staining of phenotypic cell markers. Both healthy rat brains and brains from rats with MOG EAE were analyzed. In defined lesional stages of MOG EAE, the number of CX3CR1 mRNA-expressing cells and the intensity of the in situ hybridization signal were determined by image analysis. Data were statistically evaluated by ANOVA, followed by Tukey\primes multiple comparison test. RESULTS: Expression of CX3CL1 mRNA was present within neuronal-like cells located throughout the neuraxis of the healthy rat. Expression of CX3CL1 remained unaltered in the CNS of rats with MOG-induced EAE, with the exception of an induced expression in astrocytes within inflammatory lesions. Notably, the brain vasculature of healthy and encephalitic animals did not exhibit signs of CX3CL1 mRNA expression. The receptor, CX3CR1, was expressed by microglial cells in all regions of the healthy brain. Induction of MOG-induced EAE was associated with a distinct accumulation of CX3CR1 mRNA expressing cells within the inflammatory brain lesions, the great majority of which stained positive for markers of the microglia-macrophage lineage. Analysis in time-staged brain lesions revealed elevated levels of CX3CR1 mRNA in microglia in the periplaque zone, as well as a dramatically enhanced accumulation of CX3CR1 expressing cells within the early-active, late-active and inactive, demyelinated lesions. CONCLUSION: Our data demonstrate constitutive and regulated expression of the chemokine CX3CL1 and its receptor CX3CR1 by neurons/astrocytes and microglia, respectively, within the normal and inflamed rat brain. Our findings propose a mechanism by which neurons and reactive astrocytes may control migration and function of the surrounding microglia. In addition, the accumulation of CX3CR1 expressing cells other than microglia within the inflammatory brain lesions indicate a possible role for CX3CL1 in controlling invasion of peripheral leucocytes to the brain.

Disparate MHC class II haplotypes in myelin oligodendrocyte glycoprotein- and myelin basic protein-induced experimental autoimmune encephalomyelitis.

The major histocompatibility complex (MHC) regulates multiple sclerosis (MS) and its model experimental autoimmune encephalomyelitis (EAE). We created four new intra-MHC recombinant rat strains, between the MHC haplotypes RT1(n) (BN) and RT1(l) (LEW) on the LEW background, to define disease regulation and localization within the MHC. Immunization with recombinant myelin oligodendrocyte glycoprotein (a.a.1-125; MOG)/IFA induced EAE in strains expressing the MHC class II allele RT1.B(n), whereas strains expressing the RT1.B(l) were resistant. In myelin basic protein peptide (MBP(GP)63-88)/CFA-induced EAE, RT1.B(l) expressing strains were susceptible whereas strains expressing the RT1.B(n) were resistant. High levels of antigen-specific IFN-gamma secreting lymphoid cells and antigen-specific serum IgG antibodies were only recorded in rats with an MHC class II allele that permitted MOG- or MBP-EAE, respectively. Genetically, we localized the MHC regulation of the investigated EAE models to the central part of the MHC, containing the MHC class II (RT1.B/D) and the centromeric parts of the MHC class III. No influences were evident from the classical MHC class I (RT1.A), the telomeric parts of the MHC class III or the non-classical MHC class I (RT1.C/E/M) in contrast to previous reports. The MHC class II haplotype-specific regulation of EAE induced with two different CNS antigens demonstrates a strikingly specific MHC-association even within the same target organ.

Eae19, a new locus on rat chromosome 15 regulating experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) and its animal model, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE), share a complex genetic predisposition with contributions from the major histocompatibility complex class II genes and many other genes. Linkage mapping in F(2) crosses between the susceptible DA rat strain and the resistant ACI or BN rat strains in various models of autoimmune neuroinflammation have repeatedly displayed suggestive linkage to a region on rat chromosome 15. A direct study of this region was undertaken in congenic strains by transferring resistant ACI alleles to the susceptible DA background. Phenotypic analysis demonstrated lower maximal and cumulative EAE scores in the DA.ACI-D15Rat6-D15Rat71 (C15), DA.ACI-D15Rat6-D15Rat48, D15Rat126-D15Rat71 (C15R3b), and DA.ACI-D15Rat23-D15rat71 (C15R4) strains compared to the parental DA rat strain. Linkage analysis was then performed in a (DA x PVG.AV1)F(7) advanced intercross line, resulting in a LOD score of 4.7 for the maximal EAE score phenotype at the peak marker D15Rat71 and a confidence interval of 13 Mb, overlapping with the congenic fragment defined by the C15R3b and the C15R4 strains. Thus, a new MOG-EAE locus with the designation Eae19 is identified on rat chromosome 15. There are 32 confirmed or predicted genes in the confidence interval, including immune-responsive gene 1 and neuronal ceroid lipofuscinose gene 5. Definition of loci such as Eae19 enables the characterization of genetically regulated, evolutionary conserved disease pathways in complex neuroinflammatory diseases.

MHC and non-MHC gene regulation of disease susceptibility and disease course in experimental inflammatory peripheral neuropathy.

With a panel of rat strains, we demonstrate a strong impact of the MHC genotype on susceptibility and disease course in experimental autoimmune neuritis induced with peripheral nerve myelin or the P2 peptide 58-81 (KNTEISFKLGQEFEETTADNRKTK). Beside the MHC genotype, non-MHC genes determined disease susceptibility and resistance. The type of disease induced with P2 58-81 was strongly correlated to the strength of the MHC class II isotype interaction with P2 58-81. These findings suggest a link between susceptibility and acute versus chronic disease courses on one hand and the strength of the MHC class II molecule/peptide affinity on the other hand.

An advanced intercross line resolves Eae18 into two narrow quantitative trait loci syntenic to multiple sclerosis candidate loci.

Identification of polymorphic genes regulating inflammatory diseases may unravel crucial pathogenic mechanisms. Initial steps to map such genes using linkage analysis in F(2) intercross or backcross populations, however, result in broad quantitative trait loci (QTLs) containing hundreds of genes. In this study, an advanced intercross line in combination with congenic strains, was used to fine-map Eae18 on rat chromosome 10 in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE). Myelin oligodendrocyte glycoprotein-induced EAE is a chronic relapsing disease that closely mimics key features of multiple sclerosis. Congenic DA.ACI rat strains localized Eae18 to an approximately 30-Mb large region. Fine-mapping was then performed in an advanced intercross line consisting of a (DA x PVG.1AV1)F(7) intercross, resulting in two adjacent EAE-regulating QTLs designated Eae18a and Eae18b. The two QTLs span 5.5 and 3 Mb, respectively, and the 3-Mb Eae18b contains as few as 10 genes, including a cluster of chemokine genes (CCL1, CCL2, CCL7, and CCL11). Eae18a and Eae18b are syntenic to human chromosome 17p13 and 17q11, respectively, which both display linkage to multiple sclerosis. Thus, Eae18 consists of at least two EAE-regulating genes, providing additional evidence that clustering of disease-regulating genes in QTLs is an important phenomenon. The overlap between Eae18a and Eae18b with previously identified QTLs in humans and mice further supports the notion that susceptibility alleles in inflammatory disease are evolutionary conserved between species.

T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3) and TIM-1 molecules are differentially expressed on human Th1 and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis.

T cell Ig- and mucin-domain-containing molecules (TIMs) comprise a recently described family of molecules expressed on T cells. TIM-3 has been shown to be expressed on murine Th1 cell clones and has been implicated in the pathogenesis of Th1-driven experimental autoimmune encephalomyelitis. In contrast, association of TIM-1 polymorphisms to Th2-related airway hyperreactivity has been suggested in mice. The TIM molecules have not been investigated in human Th1- or Th2-mediated diseases. Using real-time (TaqMan) RT-PCR, we show that human Th1 lines expressed higher TIM-3 mRNA levels, while Th2 lines demonstrated a higher expression of TIM-1. Analysis of cerebrospinal fluid mononuclear cells obtained from patients with multiple sclerosis revealed significantly higher mRNA expression of TIM-1 compared with controls. Moreover, higher TIM-1 expression was associated with clinical remissions and low expression of IFN-gamma mRNA in cerebrospinal fluid mononuclear cells. In contrast, expression of TIM-3 correlated well with high expression of IFN-gamma and TNF-alpha. These data imply the differential expression of human TIM molecules by Th1 and Th2 cells and may suggest their differential involvement in different phases of a human autoimmune disease.

Prior poliomyelitis-IVIg treatment reduces proinflammatory cytokine production.

The postpolio syndrome (PPS) is characterized by progressive disabilities decades after recovery from the acute paralytic disease. There are reports on intrathecal inflammatory reactions in PPS, including increased expression of cytokines by cerebrospinal fluid (CSF) mononuclear cells (CSF-MC). This is potentially of relevance for the clinical condition. We here explored if cytokine expression in the CSF of PPS patients could be modulated by high-dose intravenous immunoglobulins (IvIg). The expression of TNF-alpha, IFN-gamma, IL-10 and IL-4 mRNAs was measured by real-time RT-PCR in CSF and peripheral blood mononuclear cells (PBMC) of 16 PPS patients before, and 6-8 weeks after IvIg treatment, and in 26 patients with noninflammatory other neurological diseases (OND). TNF-alpha, IFN-gamma and IL-10 CSF mRNA levels were elevated in samples from untreated persons with PPS compared to OND. Upon IvIg treatment, IFN-gamma and TNF-alpha mRNA levels were dramatically reduced, while IL-10 remained unchanged. Placebo-controlled studies are now warranted to evaluate if IvIg treatment also has any effects on the clinical manifestations of PPS.

Differential expression of the chemokine receptors CX3CR1 and CCR1 by microglia and macrophages in myelin-oligodendrocyte-glycoprotein-induced experimental autoimmune encephalomyelitis.

Chemokines are important for the recruitment of immune cells into sites of inflammation. To better understand their functional roles during inflammation we have here studied the in vivo expression of receptors for the chemokines CCL3/CCL5/CCL7 (MIP-1alpha/RANTES/MCP-3) and CX3CL1 (fractalkine), CCR1 and CX3CR1, respectively, in rat myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. Combined in situ hybridization and immunohistochemistry demonstrated intensely upregulated CCR1 mRNA expression in early, actively demyelinating plaques, whereas CX3CR1 displayed a more generalized expression pattern. CX3CR1 mRNA expressing cells were identified as microglia on the basis of their cellular morphology and positive GSA/B4 lectin staining. In contrast, CCR1 mRNA was preferentially expressed by ED1+ GSA/B4+ macrophages. The notion of differential chemokine receptor expression in microglia and monocyte-derived macrophages was corroborated at the protein level by extraction and flow cytometric sorting of cells infiltrating the spinal cord using gating for the surface markers CD45, ED-2 and CD11b. These observations suggest a differential receptor expression between microglia and monocyte-derived macrophages and that mainly the latter cell type is responsible for active demyelination. This has great relevance for the possibility of therapeutic intervention in demyelinating diseases such as multiple sclerosis, for example by targeting signaling events leading to monocyte recruitment.