Session Title: Systemic Lupus Erythematosus - Animal Models
Session Type: Abstract Submissions (ACR)
Background/Purpose: We have previously shown that B6 mice with an introgressed homozygous NZB chromosome (c) 1 interval (70 to 100 cM) develop high titres of antinuclear antibodies and severe glomerulonephritis (GN), with approximately 40% of the mice dying by 8 months age. Using subcongenic mice with shorter intervals in this region we found that T cell and dendritic cell (DC) defects, derived from several genetic loci, synergize to convert the preclinical disease in c1(96-100) mice to fatal GN in c1(70-100) mice through expansion of pro-inflammatory T cell subsets. EAT-2, an adapter molecule in the SLAM signaling pathway that is located in the 70-96 cM region, has a promoter polymorphism in NZB mice that is predicted to lead to decreased expression. In this study we examine whether altered expression of this molecule leads to the abnormal DC function observed in these mice.
Methods: Expression levels of EAT-2 were evaluated in bone marrow derived DC from c1 congenic and B6 mice using qRT-PCR and Western blots. siRNAs targeting EAT-2 gene were introduced into B6 and c1 congenic DC. Subsequently, naïve OVA-specific TCR transgenic (OTII) T cells from B6 and c1 congenic mice were isolated and co-cultured with EAT-2 silenced or scrambled control treated DC in the presence of OVA peptide. In parallel DC were stimulated with anti-CD40 before and after knock-down of EAT-2. Production of cytokines (IL-12, IL-6, IFN-g) by DC and T cells were analyzed by flow cytometry.
Results: Expression of EAT-2 was reduced by ~70% in DC from c1(70-100) mice as compared to c1(96-100) and B6 mice. Silencing of the EAT-2 gene in DC from B6 and congenic mice resulted in increased production of IL-12 as compared to scrambled control and was associated with increased differentiation of OVA-specific T cells from both B6 and c1 congenic mice to a Th1 phenotype. Knock-down of EAT-2 in DC from all strains of mice did not affect IL-6 production when co-cultured with B6.OTII T cells, however augmented IL-6 production was seen for c1(96-100) and c1(70-100) DC when co-cultured with naïve T cells from c1 congenic mice. This was accompanied by somewhat enhanced production of IL-21, but not IL-17, by c1 congenic OTII T cells. SLAM/SLAM homotypic interactions inhibit production of IL-12 and IL-6 by CD40L-activated DCs. Consistent with a role for EAT-2 in this inhibition, knock-down of EAT-2 resulted in increased production of IL-12 by CD40-stimulated B6 and c1(96-100) DC. This was recapitulated in c1(70-100) DC, which demonstrated increased production of IL-12, and a trend to increased production of IL-6, as compared to B6 or c1(96-100) DC following CD40 stimulation.
Conclusion: EAT-2 negatively regulates cytokine production in DC downstream of the SLAM molecules and a genetic polymorphism leading to low levels of EAT-2 in c1(70-100) mice may contribute to the increased production of IL-12 we have previously observed for their DC.
J. E. Wither,
« Back to 2014 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-eat-2-as-a-lupus-susceptibility-gene-in-new-zealand-black-nzb-mice-that-regulates-dendritic-cell-function/