Objective
Many Native American (NA) patients with systemic lupus erythematosus (SLE) do not exhibit the classical SLE autoantibody profiles of European American (EA) and African American (AA) ...patients with SLE. The poorer SLE disease outcomes noted in NA patients highlights a need for more equitable diagnostic and prognostic tools for NA patients with SLE. The objective was to identify informative autoantibody profiles for NA, AA, and EA patients with SLE using an expanded set of autoantigens.
Methods
Sera from 49 NA, 49 AA, and 49 EA age‐, sex‐, and antinuclear autoantibody titer–matched patients with SLE who met the American College of Rheumatology classification criteria and 10 ethnicity‐, sex‐, and age‐matched controls were tested for autoantibody reactivity by autoantigen microarrays. Autoantibodies that were significantly elevated in patients with SLE compared with ethnicity‐specific controls were selected for hierarchical clustering. Differences in clinical criteria between patient clusters were determined by Fisher's exact test and corrected for multiple comparisons.
Results
NA, AA, and EA patients with SLE each had a cluster distinguished by higher levels of anti‐Ro52 and another cluster distinguished by nucleic acid–specific autoantibodies. Additional clusters were distinguished in NA patients by elevated extracellular matrix autoantibodies and were distinguished in AA patients by elevated Sm/RNP autoantibody and elevated nucleolin/histone autoantibody. Two EA patient clusters with similar nucleic acid– and Ro52‐specific autoantibodies were distinguished by either high or low histone 2A reactivity. Renal manifestations trended higher in the NA Ro52 cluster and were significantly enriched in the AA nucleolin/histone cluster. The AA nucleolin/histone cluster and EA H2A cluster had higher disease activity.
Conclusion
Expanded autoantibody profiles can identify informative subsets of patients with SLE.
The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, vascular thrombosis, and thrombocytopenia occurring in the presence of antiphospholipid (aPL) antibodies. The ...pathogenesis of fetal loss and tissue injury in APS is incompletely understood, but is thought to involve platelet and endothelial cell activation as well as procoagulant effects of aPL antibodies acting directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation in the placenta leads to fetal death in utero. We hypothesized that aPL antibodies activate complement in the placenta, generating split products that mediate placental injury and lead to fetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. We found that inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1-related gene/protein y (Crry)-Ig, blocks fetal loss and growth retardation. Furthermore, mice deficient in complement C3 were resistant to fetal injury induced by aPL antibodies. While antigenic epitopes recognized by aPL antibodies are important in the pathogenesis of APS, our data show that in vivo complement activation is required for aPL antibody-induced fetal loss and growth retardation.
Pleiotropism is a hallmark of cytokines and growth factors; yet, the underlying mechanisms are not clearly understood. We have identified a motif in the granulocyte macrophage‐colony‐stimulating ...factor receptor composed of a tyrosine and a serine residue that functions as a binary switch for the independent regulation of multiple biological activities. Signalling occurs either through Ser585 at lower cytokine concentrations, leading to cell survival only, or through Tyr577 at higher cytokine concentrations, leading to cell survival as well as proliferation, differentiation or functional activation. The phosphorylation of Ser585 and Tyr577 is mutually exclusive and occurs via a unidirectional mechanism that involves protein kinase A and tyrosine kinases, respectively, and is deregulated in at least some leukemias. We have identified similar Tyr/Ser motifs in other cell surface receptors, suggesting that such signalling switches may play important roles in generating specificity and pleiotropy in other biological systems.
The common beta chain (beta(c)) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ...ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of beta(c). However, the contribution of serine phosphorylation in beta(c) to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of beta(c) that interacts with the adaptor protein 14-3-3zeta. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3zeta fusion protein showed that 14-3-3 directly associates with beta(c) but not the GM-CSF receptor alpha chain. C-terminal truncation mutants of beta(c) further showed that a region between amino acids 544 and 626 in beta(c) was required for its association with 14-3-3zeta. This region contains the sequence (582)HSRSLP(587), which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of (582)HSRSLP(587) for EFAAAA completely abolished interaction of beta(c) with GST-14-3-3zeta. Furthermore, the interaction of beta(c) with GST-14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when (585)Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated (585)Ser bound 14-3-3zeta with an affinity of 150 nmol/L. To study the regulation of (585)S phosphorylation in vivo, we raised antibodies that specifically recognized (585)Ser-phosphorylated beta(c). Using these antibodies, we showed that GM-CSF stimulation strongly upregulated (585)Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site ((577)Tyr) to the 14-3-3-binding site ((582)HSRSLP(587)) and their conservation between mouse, rat, and human beta(c) but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.
Systemic lupus erythematosus (SLE) affects 1 in 537 Black women, which is >2-fold more than White women. Black patients develop the disease at a younger age, have more severe symptoms, and have a ...greater chance of early mortality. We used a multiomics approach to uncover ancestry-associated immune alterations in patients with SLE and healthy controls that may contribute biologically to disease disparities. Cell composition, signaling, epigenetics, and proteomics were evaluated by mass cytometry; droplet-based single-cell transcriptomics and proteomics; and bead-based multiplex soluble mediator levels in plasma. We observed altered whole blood frequencies and enhanced activity in CD8+ T cells, B cells, monocytes, and DCs in Black patients with more active disease. Epigenetic modifications in CD8+ T cells (H3K27ac) could distinguish disease activity level in Black patients and differentiate Black from White patient samples. TLR3/4/7/8/9-related gene expression was elevated in immune cells from Black patients with SLE, and TLR7/8/9 and IFN-α phospho-signaling and cytokine responses were heightened even in immune cells from healthy Black control patients compared with White individuals. TLR stimulation of healthy immune cells recapitulated the ancestry-associated SLE immunophenotypes. This multiomic resource defines ancestry-associated immune phenotypes that differ between Black and White patients with SLE, which may influence the course and severity of SLE and other diseases.
Systemic lupus erythematosus (SLE) is a multisystem, autoimmune disease that predominantly affects women. Previous findings that duplicated Toll-like receptor 7 (Tlr7) promotes lupus-like disease in ...male BXSB mice prompted us to evaluate TLR7 in human SLE. By using a candidate gene approach, we identified and replicated association of a TLR7 3'UTR SNP, rs3853839 (G/C), with SLE in 9,274 Eastern Asians (P combined = 6.5 × 10 −10 ), with a stronger effect in male than female subjects odds ratio, male vs. female = 2.33 (95% CI = 1.64–3.30) vs. 1.24 (95% CI = 1.14–1.34); P = 4.1 × 10 −4 . G-allele carriers had increased TLR7 transcripts and more pronounced IFN signature than C-allele carriers; heterozygotes had 2.7-fold higher transcripts of G-allele than C-allele. These data established a functional polymorphism in type I IFN pathway gene TLR7 predisposing to SLE, especially in Chinese and Japanese male subjects.
In the hemopoietic compartment, IL-3, GM-CSF, and IL-5 receptors are major transducers of survival signals; however, the receptor-proximal events that determine this vital function have not been ...defined. We have found that IL-3 stimulation induces phosphorylation of Ser-585 of β
c. This promotes the association of phospho-Ser-585 of β
c with 14-3-3 and the p85 subunit of PI 3-K. Mutation of Ser-585 specifically impairs the PI 3-K signaling pathway and reduces cell survival in response to IL-3. These results define a distinct IL-3 receptor–mediated survival pathway regulated by site-specific receptor serine phosphorylation and 14-3-3 binding and suggest that this novel mode of signaling may be utilized by disparate transmembrane receptors that have as a common theme the transduction of survival signals.
Vitamin D deficiency is widespread and has been associated with many chronic diseases, including autoimmune disorders. A study was undertaken to explore the impact of low vitamin D levels on ...autoantibody production in healthy individuals, as well as B cell hyperactivity and interferon α (IFNα) activity in patients with systemic lupus erythematosus (SLE).
Serum samples from 32 European American female patients with SLE and 32 matched controls were tested for 25-hydroxyvitamin D (25(OH)D) levels, lupus-associated autoantibodies and serum IFNα activity. Isolated peripheral blood mononuclear cells were tested for intracellular phospho-ERK 1/2 as a measure of B cell activation status.
Vitamin D deficiency (25(OH)D <20 ng/ml) was significantly more frequent among patients with SLE (n=32, 69%) and antinuclear antibody (ANA)-positive controls (n=14, 71%) compared with ANA-negative controls (n=18, 22%) (OR 7.7, 95% CI 2.0 to 29.4, p=0.003 and OR 8.8, 95% CI 1.8 to 43.6, p=0.011, respectively). Patients with high B cell activation had lower mean (SD) 25(OH)D levels than patients with low B cell activation (17.2 (5.1) vs 24.2 (3.9) ng/ml; p=0.009). Patients with vitamin D deficiency also had higher mean (SD) serum IFNα activity than patients without vitamin D deficiency (3.5 (6.6) vs 0.3 (0.3); p=0.02).
The observation that ANA-positive healthy controls are significantly more likely to be deficient in vitamin D than ANA-negative healthy controls, together with the finding that vitamin D deficiency is associated with certain immune abnormalities in SLE, suggests that vitamin D plays an important role in autoantibody production and SLE pathogenesis.
Objective
Systemic lupus erythematosus (SLE) is a multifaceted disease characterized by immune dysregulation and unpredictable disease activity. This study sought to evaluate the changes in plasma ...concentrations of soluble mediators that precede clinically defined disease flares.
Methods
Fifty‐two different soluble mediators, including cytokines, chemokines, and soluble receptors, were examined using validated multiplex bead‐based or enzyme‐linked immunosorbent assays in plasma from 28 European American patients with SLE who developed disease flare 6 or 12 weeks after a baseline assessment (preflare), 28 matched SLE patients without impending flare (nonflare), and 28 matched healthy controls. In a subset of 13 SLE patients, mediators within samples obtained preceding disease flare were compared with those within samples from the same individual obtained during a clinically stable period without flare.
Results
Compared to SLE patients with clinically stable disease, SLE patients with impending flare had significant alterations (P ≤ 0.01) in the levels of 27 soluble mediators at baseline; specifically, the levels of proinflammatory mediators, including Th1‐, Th2‐, and Th17‐type cytokines, were significantly higher several weeks before clinical flare. Baseline levels of regulatory cytokines, including interleukin‐10 and transforming growth factor β, were higher in nonflare SLE patients, whereas baseline levels of soluble tumor necrosis factor receptor type I (TNFRI), TNFRII, Fas, FasL, and CD40L were significantly higher (P ≤ 0.002) in preflare SLE patients. The normalized and weighted combined soluble mediator score was significantly higher (P ≤ 0.0002) in preflare samples from SLE patients compared to samples from the same patients obtained during periods of stable disease.
Conclusion
The levels of proinflammatory adaptive cytokines and shed TNF receptors are elevated prior to disease flare, while the levels of regulatory mediators are elevated during periods of stable disease. Alterations in the balance between inflammatory and regulatory mediators may help identify patients at risk of disease flare and help decipher the pathogenic mechanisms of SLE.
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