Both genetic and environmental interactions affect systemic lupus erythematosus (SLE) development and pathogenesis. One known genetic factor associated with lupus is a haplotype of the interferon regulatory factor 5 (IRF5) gene. Analysis of global gene expression microarray data using gene set enrichment analysis identified multiple interferon- and inflammation-related gene sets significantly overrepresented in cells with the risk haplotype. Pathway analysis using expressed genes from the significant gene sets impacted by the IRF5 risk haplotype confirmed significant correlation with the interferon pathway, Toll-like receptor pathway, and the B-cell receptor pathway. SLE patients with the IRF5 risk haplotype have a heightened interferon signature, even in an unstimulated state (P = 0.011), while patients with the IRF5 protective haplotype have a B cell interferon signature similar to that of controls. These results identify multiple genes in functionally significant pathways which are affected by IRF5 genotype. They also establish the IRF5 risk haplotype as a key determinant of not only the interferon response, but also other B-cell pathways involved in SLE.

Epstein-Barr virus has been implicated in the etiology of systemic lupus erythematosus (SLE) through serologic and immunologic studies. A potential mechanism for this influence is through molecular mimicry. The EBV nuclear antigen EBNA-1 contains a region, PPPGRRP, with considerable homology to the initial sequence targeted by antibodies in Sm B’ autoimmunity, PPPGMRPP. This study examined whether immunization of rabbits and mice with peptides containing the PPPGRRP sequence from EBNA-1 constructed on a poly-lysine backbone was able to drive the development of autoantibodies against the Smith antigen (Sm) and the related antigenic complex, the U1 nuclear ribonucleoproteins (nRNP). PPPGRRP immunization, and immunization with an EBNA-1 fragment containing PPPGRRP, led to autoantibodies in both rabbits and mice at high frequency (83% of rabbits and 43% of mice). Five out of six immunized rabbits developed either leucopenia or lymphopenia or both. The fine specificity of antibody binding against the lupus-associated autoantigens Sm B’, nRNP A, and nRNP C after immunization with the EBNA-1-derived peptides was very similar to the early antibody binding patterns against these proteins in human SLE. This similarity, as well as the prevalence of autoimmunity after immunization with these peptides, identifies PPPGRRP as a strong candidate for molecular mimicry in SLE etiology.

Objective:

The U1 small nuclear ribonucleoproteins (nRNPs) are common targets of autoantibodies in lupus and other autoimmune diseases. However, the etiology and progression of autoimmune responses directed against these antigens are not well understood. Using a unique collection of serial human samples from before and after nRNP antibody development, we investigated early humoral events in the development of anti-nRNP autoimmunity.

Methods:

Lupus patients with sera available from both before and after nRNP antibody precipitin development were identified from the Oklahoma Clinical Immunology Serum Repository. Antibodies in the serial samples were analyzed by ELISA, Western blotting, solid-phase epitope mapping and competition assays.

Results:

The first detected nRNP antibodies targeted 6 common initial epitopes in nRNP A, 2 in nRNP C and 9 in nRNP 70K. The initial epitopes of nRNP A and nRNP C were significantly enriched for proline (p=0.0004, p=0.048) and shared up to 95% sequence homology. The initial nRNP 70K humoral epitopes differed from nRNP A and C. The initial antibodies to nRNP A and nRNP C were cross-reactive with the Sm B′-derived peptide PPPGMRPP. Antibody binding against all three nRNP subunits diversified significantly over time.

Conclusions:

nRNP A and nRNP C autoantibodies initially targeted restricted, proline-rich motifs. Antibody binding subsequently spread to other epitopes. The similarity and cross-reactivity between the initial targets of nRNP and Sm autoantibodies identifies a likely commonality in etiology and a focal point for intermolecular epitope spreading.

Serologic association, cross-reactivity of select EBV-specific antibodies with SLE autoantigens, SLE-like autoimmunity after immunization with EBV peptides, increased EB viral load in SLE patients, and SLE-specific alterations in EBV humoral and cellular immunity implicate Epstein–Barr virus (EBV) in the development of systemic lupus erythematosus (SLE). To investigate SLE-specific differences in EBV gene expression, levels of eight EBV genes were compared between SLE patients and controls by using both ex vivo-infected and un-manipulated peripheral blood mononuclear cells (PBMCs). Expression levels of mRNA were significantly greater by Wilcoxen signed rank test in the ex vivo-infected SLE patient-derived cells for 4 of 8 EBV genes, including BLLF1, 3.2-fold (p<0.004); LMP-2, 1.7-fold (p<0.008); EBNA-1, 1.7-fold (p<0.01); and BcRF1, a proposed DNA binding protein, 1.7-fold (p<0.02). The frequency of LMP-1 gene expression was significantly greater by Chi square analysis in the peripheral blood from SLE patients than controls (44% of patients, 10% of controls p<0.05). PBMCs from SLE patients had greater expression of latent genes as well as increased expression of both latent and lytic genes after infection, suggesting that EBV may participate in SLE etiology through several mechanisms. Such altered infection patterns may contribute to the increased levels of EBV and the molecular mimicry seen in sera from SLE patients.