Faculty Bibliography

Objective. Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating beta2-glycoprotein I (beta2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. Methods. Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. Results. uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 +/- 0.1 vs 1.9 +/- 0.05 ng/ml (P < 0.0001), 6.6 +/- 0.3 vs 2.1 +/- 0.2 ng/ml (P < 0.0001) and 435 +/- 34 vs 220 +/- 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 +/- 0.1 vs 1.9 +/- 0.05 ng/ml; P = 0.0086, 6.2 +/- 1.4 vs 2.2 +/- 0.7 ng/ml; P = 0.147 and 412 +/- 61 vs 260 +/- 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. Conclusion. Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.

Cardiac neonatal lupus (NL) is presumed to arise from maternal autoantibody targeting an intracellular ribonucleoprotein, Ro60, which binds noncoding Y RNA and only becomes accessible to autoantibodies during apoptosis. Despite the importance of Ro60 trafficking in the development of cardiac NL, the mechanism underlying cell surface exposure is unknown. To evaluate the influence of Y RNA on the subcellular location of Ro60 during apoptosis and activation of macrophages, stable Ro60 knockout murine fibroblasts expressing wild-type or mutated FLAG-Ro60 were assessed. FLAG3-Ro60(K170A R174A) binds Y RNA, whereas FLAG3-Ro60(H187S) does not bind Y RNA; fibroblasts expressing these constructs showed equivalent intracellular expression of Ro60. In contrast, apoptotic fibroblasts containing FLAG3-Ro60(K170A R174A) were bound by anti-Ro60, whereas FLAG3-Ro60(H187S) was not surface expressed. RNA interference of mY3 RNA in wild-type fibroblasts inhibited surface translocation of Ro60 during apoptosis, whereas depletion of mY1 RNA did not affect Ro60 exposure. Furthermore, Ro60 was not exposed following overexpression of mY1 in the mY3-depleted fibroblasts. In an in vitro model of anti-Ro60-mediated injury, Y RNA was shown to be an obligate factor for TLR-dependent activation of macrophages challenged with anti-Ro60-opsonized apoptotic fibroblasts. Murine Y3 RNA is a necessary factor to support the surface translocation of Ro60, which is pivotal to the formation of immune complexes on apoptotic cells and a TLR-dependent proinflammatory cascade. Accordingly, the Y3 RNA moiety of the Ro60 ribonucleoprotein imparts a critical role in the pathogenicity of maternal anti-Ro60 autoantibodies.

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFalpha in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFalpha secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFalpha release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.

Introduction: This study explored the role of a novel CR3 agonist to attenuate pro-inflammatory signaling in subjects with common and variant ITGAM polymorphism, rs1143679. The mechanism underlying initiation and perpetuation of inflammation - with eventual end organ injury - in Systemic Lupus Erythemasosus (SLE) is not yet defined. Equally unclear are mechanisms to attenuate such inflammation. An important candidate for initiation and perpetuation of the inflammatory response may be the chronic stimulation of resident leukocytes through toll like receptors (TLR). Complement Receptor 3 (CR3), a heterodimeric receptor on the surface of various types of leucocytes, is known to decrease proinflammatory signals by dendritic cells when ligated to iC3b. The goal of this study was to evaluate whether a TLR-mediated pro-inflammatory stimulus is attenuated by a novel iC3b mimetic specific for CR3 - known as LA1 - on macrophages expressing CR3. ITGAM polymorphism rs1143679, encoding for a non-conserved R77H substitution CD11b alpha chain of CR3 is known to be associated with SLE across various ethnic groups. While the polymorphism is theorized to affect ligand binding to CR3 the functional significance of the polymorphism is unknown. A sub-goal was to evaluate if rs1143679 carrier status attenuated the effect of LA1. Patients and Methods: The effect of LA1 on basal and stimulated responses by macrophages of human subjects (twenty healthy donors) was evaluated. Rs1143679 carrier status of subjects was determined by allelic discrimination. Macrophages derived from CD14+ monocytes of healthy human donors were treated with R848 (a specific TLR7 ligand, 1 uM) and hY3 (2.5 ug), with and without LA1 (a recently described CR3 agonist, 15 uM). Quantification of TNFalpha secretion, the readout of TLR7 activation, was assessed by ELISA. Results: Treatment of macrophages with R848 significantly stimulated TNFalpha release compared with macrophages alone (1265 +/- 297 pg/ml versus 26 +/- 30 pg/ml, respectively, p = 0!

OBJECTIVE: The proposed pathogenesis of the cardiac manifestations of neonatal lupus (cardiac-NL) involves maternal autoantibodies to the RNPs SSA/Ro and SSB/La, enhanced by as-yet-unknown factors that likely involve dysregulation of both inflammatory and fibrotic fetal responses. This study was designed to improve the power to detect specific associations in genes with candidate biologic functions. METHODS: Using data from our genome-wide association study of 116 Caucasian children with cardiac-NL and 3,351 Caucasian controls, we tested for enrichment of single-nucleotide polymorphism (SNP) associations in genes with candidate biologic functions related to fibrosis, immune function, apoptosis, T cell function, cell infiltration, innate immune cell function, interferon, Toll-like receptors, and calcium channels. After linkage disequilibrium pruning and exclusion of the extended HLA region, a total of 15,103 SNPs in 3,068 genes remained. RESULTS: A highly significant enrichment of P values was observed for genes related to fibrosis (P = 2.27 x 10(-9) ), apoptosis (P = 7.67 x 10(-7) ), and innate immune cell (P = 2.53 x 10(-6) ), immune (P = 5.01 x 10(-4) ), T cell (P = 2.23 x 10(-4) ), and interferon functions (P = 1.64 x 10(-3) ). The most significant non-HLA associations included the sialyltransferase gene ST8SIA2 (rs1487982; odds ratio 2.20 [95% confidence interval 1.52-3.19], P = 3.37 x 10(-5) ), the integrin gene ITGA1 (rs2432143; odds ratio 2.31 [95% confidence interval 1.54-3.45], P = 4.54 x 10(-5) ), and the complement regulator gene CSMD1 (rs7002001; odds ratio 2.41 [95% confidence interval 1.57-3.72], P = 6.33 x 10(-5) ). CONCLUSION: This study identified novel candidate genes associated with cardiac-NL and highlights the value of studying this cohort for advancing knowledge regarding the genetic etiology of this syndrome. Identification of causal alleles is expected to provide critical insight into the molecular mechanisms responsible for linking maternal autoantibodies to cardiac scarring in these fetuses/neonates.

OBJECTIVE: Variation in the interferon regulatory factor 5 (IRF5) gene has been associated with risk of developing systemic lupus erythematosus (SLE), and this association is largely dependent upon anti-Ro autoantibodies. This study was undertaken to determine if the IRF5 genotype is associated with maternal diagnosis or progression of autoimmunity. METHODS: Genotyping of haplotype-tagging polymorphisms in IRF5 was performed in 93 subjects of European ancestry who were recruited to the Research Registry for Neonatal Lupus. All subjects had high-titer anti-Ro autoantibodies and had a child with neonatal lupus (NL); allele frequencies were compared to those in nonautoimmune controls. The mothers had SLE, Sjogren's syndrome (SS), or undifferentiated autoimmune syndrome (UAS), or were asymptomatic. RESULTS: The SLE risk haplotype of IRF5 was enriched in all anti-Ro-positive subjects except in those with SS (odds ratio [OR] 2.55, P = 8.8 x 10(-4) ). The SLE risk haplotype was even enriched in asymptomatic individuals with anti-Ro antibodies (OR 2.69, P = 0.019). The same haplotype was more prevalent in subjects who were initially asymptomatic but developed symptomatic SLE during followup (OR 5.83, P = 0.0024). Interestingly, SS was associated with 2 minor IRF5 haplotypes, and these same haplotypes were decreased in frequency in mothers with SLE and those with UAS. CONCLUSION: The IRF5 SLE risk haplotype was associated with anti-Ro antibody positivity in asymptomatic individuals, as well as with progression to SLE in asymptomatic anti-Ro-positive individuals. SS in mothers of children with NL was associated with different IRF5 haplotypes. These data suggest that IRF5 polymorphisms play a role in serologic autoimmunity in humans and may promote the progression to clinical autoimmunity.