Genetics of experimental allergic encephalomyelitis supports the role of T helper cells in multiple sclerosis pathogenesis

Abstract

Objective: The major histocompatibility complex (MHC) is the primary genetic contributor to multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), but multiple additional interacting loci are required for genetic susceptibility. The identity of most of these non-MHC genes is unknown. In this report, we identify genes within evolutionarily conserved genetic pathways leading to MS and EAE.

Methods: To identify non-MHC binary and quantitative trait loci (BTL/QTL) important in the pathogenesis of EAE, we generated phenotype-selected congenic mice using EAE-resistant B10.S and EAE-susceptible SJL mice. We hypothesized that genes linked to EAE BTL/QTL and MS-GWAS can be identified if they belong to common evolutionarily conserved pathways, which can be identified with a bioinformatic approach using Ingenuity software.

Results: Many known BTL/QTL were retained and linked to susceptibility during phenotype selection, the most significant being a region on chromosome 17 distal to H2 (Eae5). We show in pathway analysis that T helper (T(H))-cell differentiation genes are critical for both diseases. Bioinformatic analyses predicted that Eae5 is important in CD4 T-effector and/or Foxp3(+) T-regulatory cells (Tregs), and we found that B10.S-Eae5(SJL) congenic mice have significantly greater numbers of lymph node CD4 and Tregs than B10.S mice.

Interpretation: These results support the polygenic model of MS/EAE, whereby MHC and multiple minor loci are required for full susceptibility, and confirm a critical genetic dependence on CD4 T(H)-cell differentiation and function in the pathogenesis of both diseases.

Figure 1

The risk of EAE susceptibility in B10.S-SJL-Eae5^S cross-intercross progeny bearing Eae5^S/S or Eae5^S/b is compared to progeny bearing Eae5^b/b after immunization with 2×PLP+CFA. The relative odds ratio in B10.S-SJL-Eae5^S mice that carried susceptible alleles at Eae5 over those that carried resistant Eae5^b/b alleles, classified by the year of the study. The year 2003 corresponds primarily to N6 mice, 2004 to N7 and N8 mice, etc. The Eae5^S/S-dependent susceptibility was lost after several generations of backcrossing to B10.S. One attempt to rescue the phenotype of these Eae5- bearing mice is shown for 2008, when they were inoculated with 1×MSCH+PTX. s/s, homozygous SJL at Eae5; s/b, heterozygous SJL/B10.S at Eae5; b/b, homozygous B10.S at Eae5; *, P<0.05; **, P<0.01. Percentage of affected mice (Eae5 ^s/− vs. Eae5^b/b): 2008 with PTX 97% vs.77%; 2008, 10% vs.15%; 2007, 32% vs.42%; 2006, 38% vs.55%; 2005, 49% vs.67%; 2004, 51% vs.34%; 2003, 39% vs.10%

Figure 2

In silico analysis to determine epistatic interactions in EAE and MS. Starting with known 44 MS-GWAS genes and their interactors (defined as protein-protein interactions or gene regulation relationships), a total of 1612 murine orthologues formed our initial analysis point in Stage 1. For Stage 2, we filtered these genes against positions of known EAE-BTL or against regions retained during phenotype selection. In Stage 3 we queried these genes against the mouse phenome database to determine polymorphisms (≥ 1 bp) in the coding and noncoding regions of these genes as well as 20MB up- and downstream of the boundaries. These refined lists were then combined and analyzed by Core pathway analysis in Ingenuity. A highly significant (p<0.001 based on permutation test) interaction map for Conserved EAE/MS Pathways was then generated to elucidate epistatic interactions important in EAE and MS.

Figure 3

IPA analysis of polymorphic genes retained in B10.S-SJL-Eae5^S phenotype selected congenic mice. Eae5 candidate genes on chr 17 are indicated in green, and the polymorphic genes retained in the B10.S-SJL-Eae5^S congenic mice that interact with the eae5 candidates are in blue (chr 2), purple (chr 3), yellow (chr 12), and brown (chr 13). The Eae5 candidate genes are present in pathways that function within CD4 T cells, and these 5 pathways are unified by IL2, VEGFA, VAV1, SRF, and RUNX2. The loss of any one or more of the connected non-chr 17 genes could lead to the loss of the EAE-phenotype associated with Eae5.

Figure 4

The Eae5 haplotype and location of candidate genes. Traditional microsatellite mapping and SNP genotyping by phototyping were used to identify breakpoints in chr 17 for key strains. SNPs used are labeled by their position in megabases (Mb) and were developed from genomic databases at MPD. C57Bl/10J and A.SW were the parental strains used to create the B10.S H2 congenic and B10.S and SJL were the parental strains used to create the B10.S-Eae5^SJL congenic strain. The distal border of Eae5 in the B10.S-Eae5^SJL congenics is indeterminate.

Figure 5

B10.S-Eae5^SJL mice have an increased number of lymph node CD4⁺ T cells and Foxp3⁺ Treg cells. Total number of cells (A), CD4⁺ T cells (B), and CD4⁺Foxp3⁺ Treg cells (C) in the lymph nodes of naïve B10.S (■, n = 10) and B10.S-Eae5^SJL (□, n = 6) mice. Statistical significance was determined using the Mann-Whitney test (**p<0.01; ***p<0.001). Data are representative of two independent experiments.