Faculty Bibliography

Objectives: Isolated congenital heart block (CHB) is highly associated with maternal anti-Ro/SSA antibodies. CHB carries a substantial mortality, approaching 30%, and morbidity, with over 60% of surviving affected children requiring lifelong pacemakers. To date, complete atrioventricular (AV) block is irreversible. This is a rare disease and factors beyond requisite maternal autoantibody are unknown. Data from twin studies and the 10-fold increased recurrence rate of cardiac neonatal lupus (NL) in subsequent pregnancies indicate a strong genetic contribution to risk. We posit that fetal genes influence the response to maternal autoantibodies. Methods: Children of European ancestry (n=116) with cardiac NL were identified from the U.S. Research Registry for Neonatal Lupus. Cases were genotyped using the Illumina 370K SNP platform and merged with 3351 controls from the International Consortium on Systemic Lupus Erythematosus Genetics (SLEGEN). Standard quality control and admixture-adjusted tests of association were computed. Results: Outside the HLA region, a strong association was detected at 21q22, upstream from the transcriptional regulator ERG (rs743446, p=5.45E-06, OR = 2.40). Within the HLA, associated regions include PSORS1C1 (rs3130544, p = 1.94E-07, OR = 2.77) and a missense mutation (proline to serine) at TCF19 (rs7750641, p = 1.58E-07, OR = 2.79), at Class I; several variants in the MICB-NFKBIL1- LTA-TNF-LTB-AIF1 region at Class III (rs2230365, p= 1.00E-03, OR=0.46; rs2857595, p= 1.96E-09, OR = 2.37; rs3128982, p= 6.40E-06, OR=1.86; and rs3099844, p= 4.52E-10, OR= 3.34; and the C6orf10 locus at class II (rs3115553, p=2.69E-05, OR=1.81; rs6457536, pa=1.74E-05, OR=1.84; and rs7775397 (p = 1.35E-09, OR=3.30). These are consistent with our previous results (Clancy 2002). With the exception of the HLA region, no loci previously implicated in autoimmune diseases achieved genome-wide significance in the CHB children. Conclusion: These results suggest that genetic variation near ERG, PSORS1C1, LTA/TNF/LTB and C6orf10 in the fetus may promote an abnormal tissue response initiated by exposure to maternal autoantibodies. Identification of risk loci is an incremental step towards discovery of a fetal genetic component that contributes to the anti-SSA/Ro associated development of life-long cardiac damage

Passively acquired anti-Ro associated congenital heart block (CHB) likely results from pathologic crosstalk between inflammatory and fibrosing pathways eventuating in scarring. The target, Ro60, complexed with uridine rich ssRNA induces TLR7-dependent macrophage production of supernatants capable of trans-differentiating human fetal cardiac fibroblasts. This study addressed the molecular components responsible for the TLR-dependent profibrosing effects. In seeking the link between an inflammatory response of macrophages and the profibrotic effect on fibroblasts, a microarray analysis comparing the mRNA expression profile of macrophages stimulated with hY3 (ssRNA associated with Ro60) or immune complexes consisting of Ro60-hY3-IgG fraction containing anti-Ro60 antibodies (IC) in the presence or absence of IRS661 (antagonist of TLR7) was performed. Gene expression of the vasoconstrictor endothelin-1 (ET-1), recently shown to promote dermal fibrosis, was significantly upregulated by ~ 4 fold and confirmed by RT-PCR. Incubation of macrophages with either hY3 or IC increased secretion of ET-1 from 2.64 pg/mL (baseline) to 9.67 pg/mL (p <.0001) and 7.52 pg/mL (p =.0003) respectively. Pre-treatment with IRS661 decreased hY3-induced secretion to 3.83 pg/mL and IC to 3.98 pg/mL. The direct effect of ET-1 (100 nM) on these fibroblasts (shown to express both ETa and ETb receptors) resulted in a profibrosing phenotype as demonstrated by a) TGFbeta secretion (13 pg/mL vs. 772 pg/mL, p =.03), b) increased collagen release (18 ng/mL baseline vs. 772 ng/mL, p =.05, and c) expression of a-smooth muscle actin (alpha-smac). ET-1-induced TGFbeta secretion was significantly decreased (p =.03) by each of the following: BQ-123, an ETa antagonist (119 pg/mL), BQ-788, an ETb antagonist (145 pg/mL), and anti-ET-1 antibody (211 pg/mL), but not isotype control antibody (691 pg/mL). Similarly, ET-1-induced collagen secretion was significantly decreased (p =.05) by BQ-123 (147 ng/mL collagen), BQ-788 (247 ng/mL), anti-ET-1 antibody (253 ng/mL), and anti-TGFbeta antibody (239 ng/ml), but not isotype control (815 ng/mL). Predictably, pretreatment of fibroblasts with either BQ-123, BQ-788 or incubation of supernatants with anti-ET-1 antibody, but not isotype control, significantly inhibited the profibrosing readouts (increased TGFbeta, collagen and alpha-smac) induced by supernatants from macrophages stimulated with either hY3 or IC. Additionally, fibroblasts transfected with either ETa or ETb siRNA, but not scrambled siRNA, also significantly inhibited the profibrosing readouts induced stimulated macrophages. Immuno-histochemistry of a heart from a fetus dying with CHB revealed ET-1/2/3 antibody staining but not isotype IgG control in the septal region in areas showing calcification, fibrosis, and mononuclear cell infiltrates (not seen in an age-matched fetal heart). In conclusion, these data suggest that macrophage secretion of ET-1 may be one mechanism linking TLR inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB

Purpose: Organ injury induced by antibodies characteristic of Sjogren Syndrome and Systemic Lupus Erythematosus, while varied in the adult and fetus, may share in common a link between apoptosis and ultimate fibrosis. In congenital heart block (CHB), binding of maternal anti-Ro/La antibodies to apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases uPA/uPAR-dependent plasmin activation. Immunological staining of CHB hearts reveals AV node TGFb staining and TGFb activation promotes fibroblast trans-differentiation, a scarring phenotype. Since the uPA/uPAR system plays a role in TGFb activation, this study evaluated whether anti-Ro/La binding to apoptotic cardiocytes via plasmin activation stimulates TGFb and promotes a profibrosing phenotype. Methods and Results: Initial analysis showed increased TGFb in supernatants from co-cultures of healthy cards and apoptotic cards incubated with IgG fractions from mothers whose sera contain anti-Ro/La antibodies and who had a child with CHB (apo-CHB-IgG) compared to co-cultures of healthy cards and apoptotic cards incubated with control IgG (apo-nl-IgG). Using a luciferase bioassay of active TGFb, supernatants from co-cultures of healthy cards and apo-CHB-IgG cards exhibited increased levels of active TGFb compared to those cocultured with apo-nl-IgG cards (511pg/ml CHB-IgG RLU vs 217 nl-IgG RLU; p=0.007; n=7). Abrogation of RLU via anti-TGFb antibody or TGFb inhibitor (SB431542) confirmed TGFb activation was solely due to TGFb. Significantly increased uPA levels (903 pg/ml vs 508 pg/ml; p =0.01; n=5) and uPA activity (0.8 units vs 0.04 units; p=0.005; n=3) were demonstrated in supernatants generated from coculture of healthy cards and apo CHB-IgG cards compared to healthy cards and apo nl-IgG, respectively. To determine whether uPA activity was responsible for TGFb activation, coculture experiments were conducted in which the apo-CHB-IgG cards were treated with anti-uPAR or anti-uPA antibodies or the plasmin inhibitor aprotinin prior to coculturing with healthy cards. In all instances treatments attenuated TGFb activation and uPA activity. To evaluate the profibrotic role of the observed TGFb activation, supernatants from either apo-CHB-IgG or apo-nl-IgG cocultures with healthy cards were applied to serum deprived cardiac fibroblasts. Only supernatants derived from cocultures of healthy cards and apo-CHB-IgG cardiocytes promoted transdifferentiation as evidenced by increased SMAc staining, an effect that was decreased when fibroblasts were treated with supernatants where cocultures were pretreated with uPAR antibodies. Supporting the hypothesis that increased uPA activity is causally related to the pathogenesis of CHB, cord blood samples from 12 of 18 children with CHB exhibited increased uPA activity and uPAR cleavage compared to 4 of 14 non CHB children exposed to maternal anti-Ro antibodies. Conclusions: These data suggest that binding of anti-Ro/La antibodies to apoptotic cardiocytes by virtue of increased uPAR-dependent uPA activity trigger TGFb activation thus initiating and amplifying a cascade of events that promote myofibroblast transdifferentiation and scar

Purpose: Coagulation is one of the first pathways to be elicited by vascular injury, and its activation is followed by proinflammatory phenomena, in part due to loss of the anti-inflammatory activity of both the Protein C pathway and membrane Endothelial Protein C receptor (mEPCR). It has been recently demonstrated that mEPCR is highly expressed in the cortical peritubular capillaries of kidneys from patients (pts) with active lupus nephritis compared to normal human kidney. Profound upregulation of mEPCR was observed even in areas absent tubulointerstitial damage. This study addressed the hypothesis that changes in the microvasculature extend beyond the clinically targeted organ and that dysregulation is a fundamental characteristic of SLE. Methods: The study included SLE pts in whom renal disease was considered active as assessed by proteinuria and urinary sediment. Renal biopsies were performed in all pts. Thirty skin biopsies from non-lesional nonsunexposed skin (buttocks) were obtained in 26 pts (23 females, 3 males) and five healthy controls (4 females, 1 male). The paraffin skin sections were individually stained with specific antibodies against mEPCR and adiponectin (protective markers), ICAM-1 (proinflammatory) and CD31 (pan endothelial marker). Immunohistochemistry (IHC) was scored by counting peroxidase-brown labeled blood vessels (10-20 microns in diameter) without knowledge of the clinical information associated with the biopsy. The number of blood vessels with an intensity of at least 1+ were quantitatively scored with ranges 1-12. To account for the number of blood vessels per slide, the CD31 count had to be 12 to be included in the analysis. Results: The 28 renal biopsies comprised the following ISN/RPS classifications: 4 Class III, 7 Class IV, 8 Class V, I Class VI, 3 Class III/V, 3 Class IV/V. Nineteen percent of the pts had a GFR <60 (mean GFR, 82 ml/min). Abnormal laboratory values for complement and anti-dsDNA antibodies were reported in 72% and 75% of pts, respectively. Nephrotic range proteinuria was present in 37%. For IHC skin assessments of the controls, the mean score for mEPCR was 1 (highest 2), ICAM-1 was 4 (highest 7) and adiponectin was 1 (highest 2). In 17/25 (68%) of the SLE non-lesional non-sun exposed skin sections, mEPCR was expressed above the highest control. In 16/30 (53%) ICAM-1 staining exceeded 7. In contrast, only 6/25 (19%) expressed adiponectin above 2. For each specific stain there were no apparent differences between biopsy class, degree of proteinuria, presence of anti dsDNA or low complement levels. However, pts with mEPCR staining above 2 had higher GFR measurements than those with staining < 2 (88 ml/min +/- 31 versus 53 +/- 32, p= 0.0168). In contrast, GFR was unrelated to ICAM-1 and adiponectin expression. Conclusion: These data are consistent with the notion that there is widespread activation of the microvasculature. The capacity of endothelial cells to utilize anticoagulation pathways is not restricted to the kidney and expression of mEPCR in the microcirculation likely represents an attempt to limit microvascular inflammation in kidney and skin

Objective: In patients with systemic lupus erythematosus (SLE), IRF5 genotype is associated with anti-Ro autoantibodies. It is not clear whether this association with autoantibody profile is specific to SLE disease status. To examine this question, we assessed IRF5 genotypes in a large cohort of individuals who all had high titer anti-Ro autoantibodies and carried a variety of diagnoses including healthy/asymptomatic, Sjogren's syndrome (SS), and SLE. Methods: We studied 85 European-ancestry individuals recruited to the Research Registry for Neonatal Lupus who all had high titer anti-Ro autoantibodies and a child with neonatal lupus. The diagnoses of these subjects were as follows: 15 healthy/asymptomatic, 10 SLE, 19 SLE/SS, 20 SS, and 21 undifferentiated autoimmune disease (UAS). IRF5 SNPs were genotyped using Taqman primer-probe sets to define previously reported European-derived SLE risk and protective haplotypes. Results: The IRF5 SLE-risk haplotype defined by the rs10488631 C allele was enriched in subjects of all diagnoses except SS when compared to large published data sets of European-American controls [OR=2.30 (1.50-3.32), p=8.4x10^-5]. Each of the asymptomatic, SLE, SLE/SS, and UAS mothers showed a very similar increase in rs10488631 allele frequency when examined individually (ORs range from 2.00 to 3.15, no significant differences between the asymptomatic, SLE, SLE/SS and UAS groups). The rs10488631 C allele frequency in the subjects diagnosed with SS was essentially the same as controls (OR=1.05). Interestingly, the rs3807306 C allele was significantly increased in the SS patients as compared to subjects from the other 4 diagnostic categories [OR=2.34 (1.14-4.79), p=0.026]. Conclusions: There was a significant increase in the frequency of the IRF5 SLE-risk haplotype in subjects with anti-Ro antibodies with varying diagnoses which was of comparable magnitude to the anti-Ro/IRF5 association observed in SLE (OR<2). Interestingly, the SS cohort was distinct from the other neonatal lupus mothers with respect to IRF5 genotype, and a different SNP was associated with SS status. These data suggest that the genetic association of IRF5 with autoimmunity is strongly influenced by serologic profile, and that the anti-Ro association with IRF5 genotype extends beyond the SLE disease state to some degree

Purpose: Membrane endothelial protein C receptor (mECPR), a cofactor for the anti-inflammatory activity and anti-coagulant activity of the Protein C pathway, is a prominent inducer of anti-apoptotic pathways in endothelial cells, and thus maintains vascular tone and normal blood flow in the microvasculature. We previously found that mEPCR is highly expressed in the cortical peritubular capillaries of kidneys from patients with active lupus nephritis, as compared with normal human kidneys. This profound upregulation of mEPCR was observed even in areas absent tubulointerstitial damage; we therefore hypothesized that mEPCR may be an important anti-injury molecule in the cascade(s) leading to renal damage in Systemic Lupus Erythematosus. Method: We used the autoimmune murine strain NZB/W F1 mice, and a nonautoimmune strain of mice with a podocyte-specific microRNA knockout of dicer (dicer k/o), which both display proteinuria. Healthy control mice were also utilized. Tissue was harvested from spleen, liver and kidney. Paraffin sections were stained using an anti-mEPCR antibody via avidin-biotin method. The isotope for mEPCR was used as a negative control. Intensity of immunostaining was scored from 0 to 3+, and staining was considered positive if >1+ and negative when 0 or trace (0.5+). The percent of tissue with positive staining for mEPCR was scored as focal or diffuse. Results: At four months of age, anti-ds DNA and anti-ss DNA were evident in all NZB/W F1 mice. At early onset of proteinuria, the mEPCR immunostain of cortical peritubular capillaries (PTCs) was, in four cases, 2+ and diffuse, with an isotype control that stained appropriately negative. mEPCR was also negative in the cortical PTCs of 6 healthy control mice. Mice with dicer k/o develop proteinuria at 6 weeks; however, expression of mEPCR was negative in the cortical PTCs. In order to evaluate whether the changes in the microvasculature extend beyond the clinically targeted organ, paraffin sections of liver and spleen were evaluated for mEPCR. The liver of each NZB/W F1 mouse displayed, at the sinusoids, a 2+, diffuse profile of mEPCR expression. In addition, in the spleen of NZB/W F1 we observed a 3+ focal mEPCR staining at the vasculature which supports the white pulp. The expression of mEPCR was negative for liver and spleen in healthy mice and in nonautoimmune nephritic mice. Conclusion: Consistent with the human studies, evaluation of the cortical peritubular capillaries from NZMB/W F1 mice, a classic murine model of lupus nephritis but not a murine model with nephritis secondary to a genetic podocytopathy, revealed an increase in mEPCR. These data are consistent with the notion that there is widespread activation of the microvasculature. The capacity of endothelial cells to utilize anticoagulation pathways is not restricted to the kidney, and expression of mEPCR in the microcirculation likely represents an attempt to limit microvascular inflammation in spleen, liver and kidney

OBJECTIVE: Identifying the frequency of recurrent cardiac manifestations of neonatal lupus (NL) in a second child is critical to understanding the pathogenesis of anti-SSA/Ro-mediated injury and would improve counseling strategies regarding future pregnancies and power the design of clinical prevention trials. Accordingly, this study was undertaken to address the recurrence rates of cardiac NL and associated risk factors in a large US-based cohort. METHODS: Families enrolled in the Research Registry for Neonatal Lupus were evaluated for rates of recurrence of cardiac NL and potential risk factors, with a focus on pregnancies immediately following the birth of an affected child. RESULTS: The overall rate of recurrence of cardiac NL in 161 pregnancies of 129 mothers with anti-SSA/Ro antibodies was 17.4% (95% confidence interval 11.1-23.6%). Analysis of the potential risk factors among 129 mothers with a pregnancy immediately following the birth of a child with cardiac NL showed that the maternal diagnosis was not associated with the outcome in a subsequent pregnancy. In this group, 23% of mothers who were either asymptomatic or had an undifferentiated autoimmune syndrome, compared with 14% of mothers with systemic lupus erythematosus or Sjogren's syndrome, had a second child with cardiac NL (P = 0.25). The recurrence rate was not statistically significantly different in mothers who had taken steroids compared with those who had not taken steroids (16% versus 21%; P = 0.78). The antibody status of the mother was not predictive of outcome in subsequent pregnancies. Moreover, death of the first child with cardiac NL was not predictive of recurrence of cardiac NL in a subsequent pregnancy (P = 0.31). The risk of cardiac NL was similar between male and female children (17.2% versus 18.3%; P = 1.0). CONCLUSION: In this cohort, the overall recurrence rate for cardiac NL was 17%. The recurrence rate appeared to be unaffected by maternal health, use of steroids, antibody status, severity of cardiac disease in the first affected child, or sex of the subsequent child

OBJECTIVE: /B> To evaluate autoimmune disease progression in asymptomatic and pauci-symptomatic mothers of children with neonatal lupus (NL). METHODS: Clinical information on mothers enrolled in the Research Registry (RRNL) was obtained from medical records. Genotyping was performed for -308A/G TNFalpha, 869T/C TGFbeta, and -889C/T IL1alpha. RESULTS: Of the 321-mothers enrolled, 229 had at least six months of follow-up. Twenty-six of the fifty-one mothers who were asymptomatic at the NL-child's birth progressed: 12 developed pauci-undifferentiated autoimmune syndrome (UAS), two poly-UAS, seven SS, four SLE and one SLE/SS. The median time to develop any symptom was 3.15 years. Sixteen of the 37 mothers classified as pauci-UAS at the NL-child's birth progressed: five developed poly-UAS, six SS, four SLE, and one SLE/SS. Of the pauci-UAS mothers enrolled within one year, the median time to progression was 6.7 years. Four mothers developed lupus nephritis (two asymptomatic, two pauci-UAS). The probability of an asymptomatic mother developing SLE by 10 years was 18.6%, and developing probable/definite SS was 27.9%. NL-manifestations did not predict disease progression in an asymptomatic mother. Mothers with anti-SSA/Ro and anti-SSB/La were nearly twice as likely to develop an autoimmune disease as mothers with anti-SSA/Ro only. Only TGFbetaT/T was significantly higher in SLE-mothers compared to asymptomatic-mothers (p=0.03). CONCLUSION: Continued follow-up of asymptomatic NL-mothers is warranted since nearly half progress, albeit few develop SLE. While the anti-SSB/La antibodies may be a risk factor for progression, further work is needed to determine reliable biomarkers in otherwise healthy women with anti-SSA/Ro antibodies identified solely because of an NL-child