Genetic analysis of the influence of neuroantigen-complete Freund's adjuvant emulsion structures on the sexual dimorphism and susceptibility to experimental allergic encephalomyelitis

Abstract

The induction of organ-specific autoimmune diseases, such as experimental allergic encephalomyelitis (EAE) the principal animal model of multiple sclerosis (MS), relies on the use of complete Freund's adjuvant (CFA) emulsions. In this study we report that the physical structure of the particles comprising neuroantigen-CFA emulsions significantly influences the genetic control of the incidence and sexual dimorphism seen in EAE. Immunization of (B10.S/SgMcdJ x SJL/J) F(2) mice segregating the quantitative trait loci (QTL) controlling EAE in susceptible SJL/J and resistant B10.S/SgMcdJ mice with emulsions consisting of particles where the Mycobacterium tuberculosis and neuroantigens are localized on the phase surfaces led to severe EAE in 98.8% of the mice, overriding all sex-specific and non-sex-specific genetic checkpoints. In contrast, F(2) mice immunized with emulsions where the bacterial products and encephalitogens are buried inside the water/oil vesicles exhibited a significant reduction in disease incidence (7.5%) and a sexual dimorphism (5% male versus 10% female). A genome scan identified QTL on chromosomes 7 and 11 controlling the sexual dimorphism as a function of the physical structure of the emulsion. The chromosome 11 QTL co-localizes with eae6b, and with Il12b and heptatitis A virus cellular receptor 2 (Havcr2, formerly known as Timd3), both of which are candidate genes for this QTL. Sequence analysis of the SJL/J and B10.S/SgMcdJ alleles indicates that both gene products are structurally monomorphic. Expression analysis also excluded both as candidates for this sex-specific QTL. These results reinforce the importance of gene-environment interactions in initiating and propagating autoimmune disease of the central nervous system, particularly in the context of susceptibility to MS and disease heterogeneity.

Figure 1.

Genetic linkage on chromosome 7. CIM results for linkage of susceptibility to EAE (disease index) to chromosome 7 using the combined male and female F₂ intercross mice immunized with int-ENC-CFA emulsion. Permutation-derived significance cut-offs were based on 1000 permutations at α = 0.05. Significance cutoffs for this trait are 14.8.

Figure 2.

Genetic linkage on chromosome 11. CIM results for linkage of susceptibility to EAE (disease index) to chromosome 11 using the combined male and female F₂ intercross mice immunized with int-ENC-CFA emulsion. Permutation-derived significance cut-offs were based on 1000 permutations at α = 0.05. Significance cutoffs for this trait are 14.8.

Figure 3.

cDNA sequences for Havcr2/Timd3 alleles. SJL/J and B10.S/SgMcdJ were obtained using total RNA isolated from adult spleen. cDNAs were PCR-amplified using Taq polymerase and specific primer pairs flanking the mRNA coding region of the gene. The amplified fragments were TA-cloned and PCR-screened for inserts using gene-specific primers. At least three clones for each PCR fragment were sequenced from both insert termini. Although there is one mutation between the nucleotide sequences of B10.S/SgMcdJ and SJL/J Havcr2/Timd3 alleles, at the third base position of codon 76 (Thr); this change, A->G, is silent.

Figure 4.

Havcr2/Timd3 expression by CD4⁺ T cells. CD4⁺ T cells were isolated from B10.S/SgMcdJ and SJL/J mice and Havcr2/Timd3 expression levels assessed by TaqMan PCR using cDNA generated by reverse transcription of mRNA isolated from naïve cells and CD4⁺ T cells stimulated with either anti-CD3 and anti-CD28 or anti-CD3 and anti-CD28 and IFNγ. Expression levels were normalized with respect to B10.S/SgMcdJ levels. Data are presented as the normalized means ± SD. No significant differences in Havcr2/Timd3 expression were observed between B10.S/SgMcdJ and SJL/J under any of the conditions studies (P > 0.05).

Figure 5.

Il12β expression by SJL/J and B10.S/SgMcdJ splenic cells. IL12β expression was assessed by TaqMan PCR using cDNA generated by reverse transcription of mRNA isolated from individual, freshly excised spleens. Expression levels were normalized with respect to B10.S/SgMcdJ levels. Data are presented as the normalized means ± SD. Statistical significance was assessed using Student’s t-test (P = 0.24).