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Presentation Number: 1973
Systemic lupus erythematosus (SLE) is the prototype of systemic autoimmune disease which has complex genetic interactions. Antinuclear antibody (ANA) is a hallmark of SLE. The B6.FcγRIIB-/- mice develops spontaneous anti-chromatin antibodies and glomerulonephritis resembling SLE in human. Addition of the Y-linked autoimmune accelerator (Yaa) in B6.FcγRIIB-/-, augmented the lupus severity and geared towards RNA-related antigens (nucleolar pattern). To determine whether the switch of ANA pattern in autoantibody RNA specificity in B6.FcγRIIB-/-.Yaa mice is B cell intrinsic, we created mixed bone marrow chimeras of congenic IgHa.B6. FcγRIIB-/- and IgHb.B6. FcγRIIB-/-Yaa and tested the reconstituted mice for the ANA using antibody specific for a or b allotype. Only Yaa derived antibodies were antinucleolar while non-Yaa B cells produced anti-chromatin antibodies. This result confirms that Yaa B cells are intrinsically biased towards RNA-related antigens. The Yaa gene expression in B cells suggests the defect in Yaa mice is likely to be involved in the excessive activation of B cells. We reasoned that a mutation that reduces the B cells activation should reverse the Yaa phenotypes. To test this hypothesis, we crossed B6.Yaa or B6.FcγRIIB-/-Yaa mice with B6.Btk-/- mice to generate Yaa Btk- mice and control Yaa Btk+ littermates. While B6.Yaa mice lack marginal zone B cells, addition of the Btk- in those mice can rescue the marginal zone B cells. Also, the Btk- can abolish the production of ANA and prevent lupus disease in B6.FcγRIIb-/-Yaa mice. These results suggested Yaa gene mediated through Btk-signaling pathway. To discover the underlying genetic abnormality in Yaa mice, microarray analyses of follicular B cells were performed. Only 26 genes were detected to be upregulated in B6.Yaa. Interestingly, 4 of these 26 genes (Msl31, TLR7, Tmsb4x and Rab9) are located consecutively on the X-chromosome. We hypothesized that in Yaa, the Y-chromosome might have a genomic translocation that results in duplication of X-chromosome linked genes. Quantitative RT-PCR of these genes tested in genomic DNA has shown double amount of genomic DNA in Yaa males compared to B6 males. Fluorescence in situ hybridization using BAC probes from the X chromosome confirmed that Yaa males have a 4 Mb expansion of the pseudoautosomal region (PAR) that includes the TLR7 gene. The duplication of TLR7 genes makes Yaa B cells increased expression of TLR7 protein, more responsive to TLR7 ligands and augments the Btk-dependent signaling pathway. These results reveal high divergence in PAR among mouse strains with significant functional consequences in disease susceptibility and also provide evidence support for the emerging concept that lupus arises through uncontrolled responses to host ligands for TLRs.
P. Pisitkun, None.