Faculty Bibliography

Background/Purpose: Two Apolipoprotein L1 (APOL1) risk variants (RV), G1 and G2, are enriched in ancestrally African populations due to a conferred superior resistance to Trypanosoma brucei. This improved infectious evolutionary fitness comes at the cost of propensity toward progressive renal disease by multiple causes including SLE. In Ghana's Ashanti people, Allelic frequencies for G0, G1, and G2 have been reported at 0.46, 0.49, and 0.13 respectively. Despite their high frequencies, and the established role in SLE nephritis, no study has examined outcomes in a Ghanaian SLE cohort. Accordingly, this prospective study evaluated APOL1 risk traits including renal, damage accrual, and mortality in 101 Ghanaian patients followed at Korle bu Teaching Hospital in Accra, Ghana.
Method(s): From 05/2015-04/2018, 101 Ghanaian patients meeting at least 4 ACR criteria for SLE were followed prospectively with data evaluated every six months. DNA was extracted from saliva and patients were stratified by APOL1 genotype as follows: reference allele (G0/G0), RV heterozygote (RV/G0), and RV homozygotes (RV/RV). Sera were shipped to the NYU clinical lab for confirmation of anti-dsDNA. Clinical endpoints included demographics, ACR criteria, SLEDAI score, SLICC damage index, mortality, vital signs, and laboratory values as available.
Result(s): The frequencies of the G1, and G2 alleles were 0.24, and 0.12 respectively- lower than would be expected given the reported regional frequencies. Subjects were 100% female, with an average age of 32.1 years and disease duration of 2.9 years. There were no differences in demographics across the genotypes. The RV associated with higher BP: 108/71, 108/ 73, and 120/82 in the G0/G0, RV/G0, and RV/RV groups respectively (p=0.04 systolic; 0.009 diastolic). Among those with nephritis, RV/RV carriers presented with higher dipstick proteinuria than G0/G0 and RV/G0 carriers (2.6 vs 1.2 and 1.3 respectively; p=0.05; F=3.1). While proteinuria responded most robustly to therapy in RV/RVs, it remained higher at each time point compared to the other genotypes (p=0.002; F=5.4). SLEDAI scores were comparable across the genotypes, however RV/RV carriers had lower dsDNA titers (10.7 IU/mL) than G0/G0 (57.1 IU/mL) and RV/G0 (95.6 IU/mL) carriers (p: 0.03). RV homozygosity associated with elevated SLICC damage index: G0/G0 or RV/G0: 0.95 vs RV/RV: 1.7; driven by renal, CVD, and neurologic manifestations G0/G0: 0.46, RV/G0: 0.39, RV/RV: 1.25; (p=0.03). There were 5 deaths during the study period: 1 death in the G0/G0 group (ESRD), 1 in the RV/G0 group (post-partum sepsis plus renal), and 3 in the RV/RV group (myocarditis/heart failure; ESRD; unknown). RV carrier status associated with mortality with 25% of the RV/RV group succumbing to disease vs 2% in the G0/G0 and RV/G0 groups (p=0.003; F=6.3).
Conclusion(s): Taken together, APOL1 RV associates with renal progression, organ damage accrual, and mortality in this Ghanaian SLE cohort. Despite having poorer outcomes, RV homozygotes exhibited lower dsDNA titers and similar SLEDAI scores compared to G0/G0 or RV/G0 patients suggesting a genetic effect independent of SLE activity. APOL1 genotyping could have important prognostic implications in ancestrally African SLE patients

Background/Purpose: Towards understanding the molecular mechanisms that link maternal anti-Ro antibodies to the development of conduction system disease in a second trimester fetus, single cell (scRNA-seq) and bulk RNA-seq were applied to a fetal heart dying with complete congenital heart block (CHB) and a gestational age-matched healthy heart from an elective termination.
Method(s): The CHB heart was obtained from a 20-week fetus identified to have complete block at 19 weeks. The mother (35 y/o Asian with SS on no hydroxychloroquine) declined dexamethasone or IVIG and elected to terminate, thus no exposure to maternal medications confounded interpretation of findings. Both hearts were obtained under identical conditions. Freshly collected single-cell suspensions were generated using a Langendorff preparation with cannulation and perfusion of the aorta with collagenase and trypsin enzymes. Two approaches were taken to mine the transcriptome in the resulting cell suspensions: agnostic evaluation applying 10X Genomics platform-based scRNA-seq and low input RNA-seq of flow sorted cells upon leukocytes (DAPI negative, CD45+) and fibroblasts (DAPI negative, CD45-, podoplanin-positive).
Result(s): For scRNA-seq, we obtained 2,693 and 5,408 high-quality scRNA-seq profiles from the control and CHB hearts, respectively. We applied a graph-based clustering method and identified 13 and 14 major clusters of cells from the control and CHB hearts, respectively, as visualized by t-distributed stochastic neighbor embedding (t-SNE). Differential gene expression analysis guided by established lineage markers revealed four cardiomyocyte clusters (CM1-CM4), three fibroblast clusters (FB1-FB3), endothelial cells (EC), erythroblasts (EB), macrophages (MAC), dendritic cells (DC), Tcells (TC) and B cells (BC). Ranked by abundance, the control heart exhibited CM>FB>EC>MAC>DC>EB, BC, TC; the CHB heart exhibited CM>FB>EC, MAC>TC, BC, EB. The CHB heart also contained natural killer cells (NK) and mast cells (MC, lowest abundance). Given the high abundance of MACs among the immune cells (control:108;CHB:606) and the consistent identification of MACs on histologic analysis of CHB hearts, differential expression analysis demonstrated overexpression of interferon-induced genes (4-fold or greater, i.e. log2(CHB-control)>2) in CHB MACs. In CHB, most cell types expressed high levels of ISG1, IFITM1 and IFITM3, whereas in the control only IFITM3 showed widespread expression. For SIGLEC1, expression was restricted to MACs and was expressed by 18% of CHB MACs and only 6% of control MACs. While the transcriptome using low input RNA-seq of anti-CD45 flow-sorted CHB leukocytes did not allow granular analysis of leukocyte subpopulations, expression of SIGLEC1 and interferon-related genes were increased in CHB versus control. Applying 10X Genomics, proliferating fibroblasts expressed MKI67 and TOP2A in CHB but not control fibroblasts.
Conclusion(s): This unprecedented opportunity to obtain CHB tissue absent any exposure to maternal medications support scRNA-seq's utility to survey landscape and heterogeneity not possible with low input RNA-seq of flow-sorted cells. IFNand SIGLEC1-positive macrophages may contribute to fibrosis

Background/Purpose: Apolipoprotein L1 (APOL1) risk variants (RV), G1 and G2, associate with CKD in African Americans (AA) and are evolutionarily preserved due to improved infectious resistance. Interferons (IFN) in SLE, have been shown to increase APOL1 expression and RV toxicity in endothelial cells and podocytes. Though RV homozygotes with SLE nephritis demonstrate advanced renal progression, associations with renal histopathologies have not been validated in SLE.
Method(s): Herein, this cohort study tested the hypothesis that RV homozygosity (RV/RV) associates with specific clinical and biopsy features compared to reference allele (G0) homozygosity (G0/G0) or RV heterozygosity (RV/G0). Whole blood DNA for genotyping, kidney biopsy slides, and clinical reports from 77 AA SLE patients with biopsy-proven nephritis reviewed for: biopsy class, activity index (AI), chronicity index (CI) and clinical features across APOL1 genotype. RVattributed mitochondrial morphology, was assessed on electron microscopy (EM) images. Analysis was confirmed by two blinded pathologists. As proof of concept, primary endothelial cells across genotype were given IFN to over-express APOL1, and features on EM were compared.
Result(s): The G0/G0, RV/G0, and RV/RV groups comprised 35%, 52%, and 12% of the cohort. There were no genotype differences in SLE history or demographics. Compared to G0/G0, and RV/G0 groups, the RV/RV had higher urine protein to creatinine ratios (uPCR) and creatinine (Cr) at biopsy (mean uPCR: 2.5; 2.7; 4.3 mg/L p=0.06 and Cr: 1.3; 1.03; 2.3 mg/dL p=0.01 respectively). Adjusting for dsDNA, AI, CI, and percent sclerotic glomeruli, the RVs independently associated with proteinuria at biopsy (OR=2.1, p=0.05). Paradoxically, the G0/G0 and RV/G0 vs the RV/RV group displayed higher AI and CI with a trend toward higher dsDNAs at biopsy (G0/G0 or RV/G0: AI: 5.3/24; CI: 2.6/12 dsDNA: 447 vs RV/RV: AI: 1.2/24; CI: 1.3/12; dsDNA: 69, p=AI: 0.004; CI: 0.01; dsDNA: 0.1). In 30% of the RV/RV cases, the reading pathologist commented that clinical severity was out of proportion to the biopsy lesion. The RVassociated with ESRD in 7.9%, 3.9%, and 20% of the G0/ G0, RV/G0, and RV/RV cases (OR: 5.7; p=0.03). On EM, RVs associated with mitochondrial condensation (mitochondrial area: G0/G0: 0.17; RV/G0: 0.09; RV/RV: 0.06 mum²; p<0.01); this result was recapitulated in our cell culture model. IFNtreated endothelial cells increased APOL1 expression 18 fold across genotypes (p<0.01). Compared to G0/G0 and RV/G0 cells, RV/RV cells had mitochondrial areas: G0/G0: 0.08; RV/G0: 0.07; RV/RV: 0.04 mum²; (p<0.01).
Conclusion(s): In this SLE cohort, APOL1 RVs associated with poorer prognosticators, initial proteinuria and creatinine, and ultimately progressive nephritis. These features were paradoxically out of proportion to the SLE lesion on biopsy. The literature supports RV-conferred podocyte and endothelial cell mitochondrial defects owing to a mitophagy deficiency. As evidenced by EM images from both SLE patient biopsies and primary cell cultures, these genes may conferrer intrinsic renal pathology. Consequently, traditional scoring of histopathologic severity may underestimate injury that associates with RValleles

Photosensitivity, or skin sensitivity to ultraviolet radiation (UVR), is a feature of lupus erythematosus and other autoimmune and dermatologic conditions, but the mechanistic underpinnings are poorly understood. We identify a Langerhans cell (LC)-keratinocyte axis that limits UVR-induced keratinocyte apoptosis and skin injury via keratinocyte epidermal growth factor receptor (EGFR) stimulation. We show that the absence of LCs in Langerin-diphtheria toxin subunit A (DTA) mice leads to photosensitivity and that, in vitro, mouse and human LCs can directly protect keratinocytes from UVR-induced apoptosis. LCs express EGFR ligands and a disintegrin and metalloprotease 17 (ADAM17), the metalloprotease that activates EGFR ligands. Deletion of ADAM17 from LCs leads to photosensitivity, and UVR induces LC ADAM17 activation and generation of soluble active EGFR ligands, suggesting that LCs protect by providing activated EGFR ligands to keratinocytes. Photosensitive systemic lupus erythematosus (SLE) models and human SLE skin show reduced epidermal EGFR phosphorylation and LC defects, and a topical EGFR ligand reduces photosensitivity. Together, our data establish a direct tissue-protective function for LCs, reveal a mechanistic basis for photosensitivity, and suggest EGFR stimulation as a treatment for photosensitivity in lupus erythematosus and potentially other autoimmune and dermatologic conditions.

Background Given that diseases associated with anti-Ro suchas SLE and Sjogren's syndrome associate with an upregulationof type I interferons, recent attention has focused on a potential role for IFN in the pathogenesis of congenital heart block(CHB). Based on the consistent demonstration of macrophagesand multinucleated giant cells in areas of injury, it is relevantthat Sialic Acid Binding Ig Like Lectin 1 (SIGLEC1), a receptor on monocytes/macrophages is upregulated by IFN. Functionally, Siglec-1 expressing macrophages might play animportant role as effector cells in fibrosis. Accordingly, thisstudy leveraged both autopsy tissue and freshly isolated macrophages from a fetal heart dying with CHB to address whetherIFN-a contributes to the pathogenesis of CHB by regulatingactivated macrophages in affected cardiac tissue.Methods Three approaches were taken to evaluate Siglec-1expression. Transcriptomic analysis was performed on macrophages freshly isolated from a fetal heart dying with CHB at 19weeks and a heart from an otherwise healthy electively terminatedfetus using (DAPI negative cells with isolation by flow using antibodies to CD45). Immunohistochemistry was performed onanother fetal heart dying with CHB. In vitro experiments utilizedcultured healthy human macrophages transfected with anti-SSA/Ro-associated ssRNA as a proxy for the in vivo conditions.Results Transcriptomes of the two hearts for each isolated leukocyte fraction were compared. By following 213 IFN inducible genes, there was enrichment of targeted transcripts inCHB vs control (p=0.0001) and SIGLEC1, which was 200-fold more abundant in CHB vs control and ranked among thetop three differentially expressed candidates. In another fetalheart dying with CHB, Siglec1 staining as detected by antibody HPA053457 was prominent in areas of injury. By morphology, the two cell types expressing Siglec 1 weremacrophages and dendritic cells. In vitro experiments wereperformed in accordance with previous laboratory work, inwhich a model of anti-SSA/Ro-associated injury exploits macrophages stimulated with the ssRNA component (hY3) of theSSA/Ro immune complex. IFN inducible genes (15 transcripts)were among the 30 most highly upregulated genes in hY3stimulated conditions and SIGLEC1 was two-fold more abundant in CHB vs control. Given the enrichment of type I IFNresponsive genes in the macrophage transcriptome, a WISHcell line was selected to evaluate supernatants from macrophages transfected with hY3. IFIT1 MX1, and EIF2AK2transcripts were significantly increased in the WISH cellstreated with hY3 macrophage supernatants, but not macrophage supernatants alone (n=7, p=0.02).Conclusion These data now provide a link between IFN and theinflammatory and possibly fibrosing component of CHB and position Siglec-1 positive macrophages as integral to the process

BACKGROUND:Immunoglobulin M (IgM) autoreactivity to malondialdehyde (MDA) protein modifications is part of the natural antibody repertoire in health and may have beneficial functions. In contrast, IgG anti-MDA are increased in chronic inflammation and autoimmunity and may instead have pathogenic properties. METHODS:Herein, we investigated serum IgG anti-MDA levels by enzyme-linked immunosorbent assay (ELISA) in 398 systemic lupus erythematosus (SLE) patients in the Swedish Karolinska SLE cohort and compared these to findings in 225 US SLE patients from New York University and Johns Hopkins University. RESULTS:In two independent cohorts, IgG anti-MDA levels correlated positively with disease activity by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI; p < 0.0001, Spearman R = 0.3). Meta-analysis found an odds ratio of 2.7 (confidence interval (CI) 1.9-3.9; p < 0.0001) for high anti-MDA IgG levels with active disease (SLEDAI ≥ 6). Furthermore, IgG anti-MDA correlated directly with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), soluble tumor necrosis factor receptors (sTNFR-1, sTNFR-2), and vascular cell adhesion molecule 1 (VCAM-1) measurements, and inversely with complement factors (C1q, C2, C3, C4). Importantly, IgG anti-MDA levels were significantly elevated in SLE patients with active nephritis (p = 0.0005) and correlated with cystatin C estimated glomerular filtration rate and albuminuria. CONCLUSIONS:Elevated IgG anti-MDA in SLE patients was associated with high disease activity, with active lupus nephritis, and with biomarkers of systemic inflammation. This natural antibody reactivity may have potential prognostic utility, and may also actively contribute to pathogenesis.

Systemic lupus erythematosus (SLE) is a significant risk factor for cardiovascular disease. The relationship between SLE and perioperative cardiovascular risks following non-cardiac surgery is uncertain. We investigated associations between a diagnosis of SLE and outcomes following major non-cardiac surgery in a large national database from the United States. Patients age ≥ 18 years requiring major non-cardiac surgery were identified from Healthcare Cost and Utilization Project's National Inpatient Sample data from 2004 to 2014. Systemic lupus erythematosus and perioperative major adverse cardiovascular events (MACE; myocardial infarction, ischemic stroke or death) were defined by ICD-9 diagnosis codes. Perioperative MACE were reported for SLE patients stratified by age and sex. From 2004 to 2014, a total of 17,853,194 hospitalizations for major non-cardiac surgery met study inclusion criteria. SLE was identified in 70,578 (0.4%) hospitalizations. Overall, the frequency of perioperative MACE was higher in patients with vs. without SLE [2.4 vs. 2.0%, p < 0.001; adjusted OR (aOR) 1.25; 95% CI 1.18-1.31]. Perioperative MACE associated with SLE was largely driven by increased death (aOR 1.58 95% CI 1.40-1.77) and myocardial infarction (aOR 1.32; 95% CI 1.05-1.66) in younger patients with SLE. The increased risk of perioperative MACE associated with SLE in younger patients was attenuated with increasing age. A diagnosis of SLE is associated with increased risk of perioperative MACE, particularly among younger patients. Efforts to improve the perioperative management and outcomes of patients with SLE are needed.

OBJECTIVE: Fetal exposure to maternal anti-SSA/Ro antibodies is necessary but not sufficient for the development of autoimmune congenital heart block (CHB), suggesting that other factors, such as fetal genetic predisposition, are important. Given the previously described association between major histocompatibility complex alleles and CHB risk, we undertook the present study to test the hypothesis that a variant form of HLA-C Asn80Lys, which binds with high affinity to an inhibitory killer cell immunoglobulin-like receptor (KIR) and thus renders natural killer (NK) cells incapable of restricting inflammation, contributes to the development of CHB. METHODS: Members of 192 pedigrees in the US and Europe (194 cases of CHB, 91 unaffected siblings, 152 fathers, 167 mothers) and 1,073 out-of-study controls were genotyped on the Immunochip single-nucleotide polymorphism microarray. Imputation was used to identify associations at HLA-C Asn80Lys (Asn, C1; Lys, C2) and KIR. Tests for association were performed using logistic regression. McNemar's test and the pedigree disequilibrium test (PDT) were used for matched analyses between affected and unaffected children. RESULTS: Compared with out-of-study controls of the same sex, the C2 allele was less frequent in the mothers (odds ratio [OR] 0.63, P = 0.0014) and more frequent in the fathers (OR 1.40, P = 0.0123), yielding a significant sex-by-C2 interaction (P = 0.0002). The C2 allele was more frequent in affected siblings than in unaffected siblings (OR 3.67, P = 0.0025), which was consistent with the PDT results (P = 0.016); these results were observed in both sexes and across the US and European cohorts. There was no difference in the frequency of the inhibitory KIR genotype (KIR AA) between affected and unaffected children (P = 0.55). CONCLUSION: These data establish C2 as a novel genetic risk factor associated with CHB. This observation supports a model in which fetuses with C2 ligand expression and maternal anti-SSA/Ro positivity may have impaired NK cell surveillance, resulting in unchecked cardiac inflammation and scarring.