Secreted phospholipase A2 activity in experimental autoimmune encephalomyelitis and multiple sclerosis

BACKGROUND: There is increased interest in the contribution of the innate immune system to multiple sclerosis (MS), including the activity of acute inflammatory mediators. The purpose of this study was to test the involvement of systemic secreted phospholipase A2 (sPLA2) enzymes in experimental autoimmune encephalomyelitis (EAE), an MS model, and to determine if enzyme activity is elevated in MS patients. METHODS: A non-invasive urinary assay was developed in order to monitor enzymatically active sPLA2 levels in Dark Agouti rats after induction of EAE. Some Rats were treated with nonapeptide CHEC-9, an uncompetitive sPLA2 enzyme inhibitor, during the initial rise in urinary enzyme levels. Body weight and clinical EAE score were measured for 18 days post immunization (PI), after which the rats were sacrificed for H&E and myelin staining, and for ED-1 immunocytochemistry, the latter to quantify macrophages and activated microglia. The urinary sPLA2 assay was also applied to un-timed samples collected from a cross section of 44 MS patients and 14 healthy controls. RESULTS: Mean levels of enzymatically active sPLA2 in the urine increased following immunization and peaked between days 8–10 PI which was just prior to the onset of EAE symptoms. At this time, a transient attenuation of activity was detected in the urine of CHEC-9 treated rats consistent with the activity-dependent properties of the inhibitor. The peptide also reduced or abolished EAE symptoms compared to vehicle-injected controls. Histopathological changes in the spinal cords of the EAE rats correlated generally with clinical score including a significant reduction in ED-1+ cells after peptide treatment. Multiple Sclerosis patients also showed elevations in sPLA2 enzyme activity. Mean levels of sPLA2 were increased 6-fold in the urine of patients with active disease and 4-fold for patients in remission, regardless of immunomodulating therapy. CONCLUSION: The results suggest that sPLA2 enzymes, traditionally thought to be part the acute phase inflammatory response, are therapeutic targets for MS

The Iddm14 gene is Tcrbv-13S1A1: Prevention of Autoimmune Diabetes in the Rat with an Allele-Specific Depleting Antibody That Recognizes the Vβ13a T Cell Receptor Beta Chain

To identify new intervention strategies for autoimmune type 1 diabetes (T1D), we investigated several rat models of the disorder. We dissected the powerful Iddm14 diabetes susceptibility locus in eight T1D susceptible vs. resistant rat strains by single nucleotide polymorphism (SNP) haplotyping. We identified an allele of a T cell receptor (TCR) beta chain gene, Tcrb-V13S1A1 (encoding V13βa) as a candidate gene. In three separate trials, treating LEW.1WR1 rats, which are susceptible to T1D, with a depleting anti-Vβ13 monoclonal antibody reduced diabetes frequency from 75% (N=50) to 17% (N=30, p\u3c0.001. Anti-Vβ13 monoclonal antibody also prevented T1D in spontaneously diabetic BBDP rats. We then analyzed the phenotype of infiltrating T cells recovered from the cultured islets of LEW.1WR1 rats exposed to a diabetogenic trigger. Within 5 days, up to 22% of CD4+ T cells recovered from islets were V13β+, most of these CD25+FoxP3-. We also recovered Vβ13 transcripts from pre-diabetic islets and observed a limited number of Jβ variant transcripts, indicating an oligoclonal TCR response to pancreatic beta cells. These data indicate that, in susceptible rats, V13βa on diabetogenic T cells is required to recognize a critical T1D autoantigen. Interestingly, the diabetogenic and non-diabetogenic alleles of Vβ13 have non-conservative sequence differences in both CRR1 and CDR2. The data suggest that it is possible to prevent T1D in the rat with a very narrowly targeted deletional therapy. Preliminary data suggest that a specific alpha chain may preferentially pair with Vβ13a. We are currently generating rat T cell hybridoma clones with which to analyze the interaction of putative autoantigens with a diabetogenic TCR

The Missing Heritability in T1D and Potential New Targets for Prevention

Type 1 diabetes (T1D) is a T cell-mediated disease. It is strongly associated with susceptibility haplotypes within the major histocompatibility complex, but this association accounts for an estimated 50% of susceptibility. Other studies have identified as many as 50 additional susceptibility loci, but the effect of most is very modest (odds ratio (OR) 5) and that deletion of V beta 13+ T cells prevents diabetes. A role for the TCR is also suspected in NOD mice, but TCR regions have not been associated with human T1D. To investigate this disparity, we tested the hypothesis in silico that previous studies of human T1D genetics were underpowered to detect MHC-contingent TCR susceptibility. We show that stratifying by MHC markedly increases statistical power to detect potential TCR susceptibility alleles. We suggest that the TCR regions are viable candidates for T1D susceptibility genes, could account for missing heritability, and could be targets for prevention

A genetic locus complements resistance to Bordetella pertussis-induced histamine sensitization.

Histamine plays pivotal role in normal physiology and dysregulated production of histamine or signaling through histamine receptors (HRH) can promote pathology. Previously, we showed that Bordetella pertussis or pertussis toxin can induce histamine sensitization in laboratory inbred mice and is genetically controlled by Hrh1/HRH1. HRH1 allotypes differ at three amino acid residues with

eae36, a Locus on Mouse Chromosome 4, Controls Susceptibility to Experimental Allergic Encephalomyelitis in Older Mice and Mice Immunized in the Winter

Genetic factors are believed to contribute to multiple sclerosis (MS) susceptibility; however, strong evidence implicating intrinsic and environmental factors in the etiopathogenesis of MS also exists. Susceptibility to experimental allergic encephalomyelitis (EAE), the principal animal model of MS, is also influenced by nongenetic factors, including age and season at immunization. This suggests that age- and season-by-gene interactions exist and that different susceptibility loci may influence disease as a function of the two parameters. In this study, linkage analysis based on genome exclusion mapping was carried out using age and season at immunization restricted cohorts of (B10.S × SJL/J) F(2) intercross mice in an effort to identify such linkages. Significant linkage of EAE to eae4 and eae5 was detected with 6- to 12-week-old and summer cohorts. In contrast, significant linkage of EAE to eae4 and eae5 was not detected with the >12-week-old and winter/spring populations. Rather, significant linkage to D4Mit203 at 128.50 Mb on chromosome 4 was detected with animals that were >12 weeks old at the time of immunization or were immunized in the winter. This previously unidentified locus has been designated eae36. These results support the existence of age- and season-by-gene-specific interactions in the genetic control of susceptibility to autoimmune inflammatory disease of the central nervous system and suggest that late-onset MS may be immunogenetically distinct

T Cell Receptor Genotype and Ubash3a Determine Susceptibility to Rat Autoimmune Diabetes

Genetic analyses of human type 1 diabetes (T1D) have yet to reveal a complete pathophysiologic mechanism. Inbred rats with a high-risk class II major histocompatibility complex (MHC) haplotype (RT1B/Du) can illuminate such mechanisms. Using T1D-susceptible LEW.1WR1 rats that express RT1B/Du and a susceptible allele of the Ubd promoter, we demonstrate that germline knockout of Tcrb-V13S1A1, which encodes the Vβ13a T cell receptor β chain, completely prevents diabetes. Using the RT1B/Du-identical LEW.1W rat, which does not develop T1D despite also having the same Tcrb-V13S1A1 β chain gene but a different allele at the Ubd locus, we show that knockout of the Ubash3a regulatory gene renders these resistant rats relatively susceptible to diabetes. In silico structural modeling of the susceptible allele of the Vβ13a TCR and its class II RT1u ligand suggests a mechanism by which a germline TCR β chain gene could promote susceptibility to T1D in the absence of downstream immunoregulation like that provided by UBASH3A. Together these data demonstrate the critical contribution of the Vβ13a TCR to the autoimmune synapse in T1D and the regulation of the response by UBASH3A. These experiments dissect the mechanisms by which MHC class II heterodimers, TCR and regulatory element interact to induce autoimmunity

T Cell Receptor Genotype and Ubash3a Determine Susceptibility to Rat Autoimmune Diabetes

Genetic analyses of human type 1 diabetes (T1D) have yet to reveal a complete pathophysiologic mechanism. Inbred rats with a high-risk class II major histocompatibility complex (MHC) haplotype (RT1B/D(u)) can illuminate such mechanisms. Using T1D-susceptible LEW.1WR1 rats that express RT1B/D(u) and a susceptible allele of the Ubd promoter, we demonstrate that germline knockout of Tcrb-V13S1A1, which encodes the Vbeta13a T cell receptor beta chain, completely prevents diabetes. Using the RT1B/D(u)-identical LEW.1W rat, which does not develop T1D despite also having the same Tcrb-V13S1A1 beta chain gene but a different allele at the Ubd locus, we show that knockout of the Ubash3a regulatory gene renders these resistant rats relatively susceptible to diabetes. In silico structural modeling of the susceptible allele of the Vbeta13a TCR and its class II RT1(u) ligand suggests a mechanism by which a germline TCR beta chain gene could promote susceptibility to T1D in the absence of downstream immunoregulation like that provided by UBASH3A. Together these data demonstrate the critical contribution of the Vbeta13a TCR to the autoimmune synapse in T1D and the regulation of the response by UBASH3A. These experiments dissect the mechanisms by which MHC class II heterodimers, TCR and regulatory element interact to induce autoimmunity