Genetic and immunological regulation of multiple sclerosis and experimental autoimmune encephalomyelitis

Abstract

Multiple Sclerosis (MS) is the most common chronic inflammatory disorders of the central nervous system (CNS) caused by a combination of environmental factors and multiple genes. Animal models offer possibilities to keep environmental factors and selected genes constant and very large families may be created. Therefore, an animal model known as experimental autoimmune encephalomyelitis (EAE), which shows functional and pathological similarities to human MS is here utilized to characterize underlying genes and pathogenic mechanisms. We established congenic strains, PVG.LEW-Eae30-31, DA.PVG-Eae20-22, DA.BN-Eae4 & LEW.BN-Eae4 and DA.PVG-Eae19, encompassing previously identified quantitative trait loci (QTL) on rat chromosomes (RNO) 1, 4, 9 and 15 respectively. The DA and LEW strains are EAE susceptible both in the myelin oligodendrocyte glycoprotein (MOG) - and spinal cord (SC) -induced models while the PVG and BN strains are EAE-resistant.

With a combination of the tenth (G10) generation of an advanced intercross line (AIL) (DAxPVG.AV1) and congenic breeding (DAc15ACI), Eae19 on RNO15 was narrowed down to confidence interval of 13Mb. This region harbors 32 predicted and confirmed genes with some of them with an immunological role. Among the QTLs overlapping with Eae19, adjuvant-induced arthritis QTL 4 (Aia4) might be the most interesting one since EAE regulatory effects were demonstrated in rat strains congenic for arthritis-regulating QTL. We identified two closely located QTLs on RNO4, Eae20 and Eae21, in addition to a third epistatic QTL to Eae20, Eae22. The three QTLs were mapped on a region spanning 16.8Mb co-localized with several QTLs regulating experimental autoimmune diseases such as Oia2, Pia1 and Cia13. We demonstrated an influence of Eae20 on incidence and severity of EAE while Eae21 was found to regulate EAE once a certain threshold of the disease is exceeded.

By breeding interval-specific congenic lines we were able to narrow down the susceptibility region of Eae4 to ~1 cM conferring a milder clinical EAE course and lower expression and protein secretion of TNF, IFNgamma and IL-2 in ConA-stimulated splenocytes compared to the susceptible background strains. We here positionally cloned Eae4 to be Vav1 and correlated the disease-predisposing variant to higher pro-inflammatory cytokine regulation and low proportion of Foxp3+CD4+ regulatory T cells (Tregs). Association studies in seven human cohorts demonstrated association to one disease-predisposing CA haplotype (OR=1.18) that provides higher VAV1 expression and two protecting haplotypes (CG; OR=0.86 and TG, OR=0.90) associated to lower VAV1 expression. A higher expression of VAV1 was even noticed in peripheral blood (PBMCs) and cerebrospinal fluid (CSF) of MS patients compared to controls and correlated with TNF and IFN-gamma expression.

Linkage analysis in G10 and G12 of AIL resolved the region on RNO1 in two narrow QTLs, Eae30 and Eae31. Eae30 showed linkage to MOG-EAE, anti-MOG antibodies and levels of IL-6. Eae31 was linked to both EAE and PIA, anti-MOG antibodies and levels of TNF and IL-6. Association studies in four Nordic cohorts correlated a disease-predisposing variants of RGMA from Eae30 to MS (OR=1.33) to higher expression of IFN-gamma in CSF of MS patients. In Eae31, one positively and one negatively associated haplotype were detected in IL-21R (rs8060368-rs961914 CT, OR=0.87 and CC, OR= 1.16). The CT haplotype of IL21R also correlated to lower IFN-gamma expression in PBMCs of MS patients.

In conclusion, thanks to a combination of high-resolution mapping in AIL we were able to fine-map several EAE-regulating QTLs. Applying the strategy of shared QTLs between autoimmune diseases and performing association studies in MS cohorts, we were able to point to three MS-associated genes.