Interaction of the antiphospholipid syndrome autoantigen beta-2 glycoprotein I with DNA and neutrophil extracellular traps.

Beta-2 glycoprotein I (ß2GPI) is a phospholipid-binding plasma protein and prominent autoantigen in antiphospholipid syndrome (APS). Here, we tested the hypothesis that ß2GPI might bind to not only phospholipids, but also cell-free DNA and neutrophil extracellular traps (NETs). We report that ß2GPI interacts with cell-free DNA from different species, as well as NETs, in a dose-dependent manner, retarding their migration in an agarose-gel electrophoretic mobility shift assay. We confirm the direct binding interaction of ß2GPI with DNA and NETs by ELISA. We also demonstrate that ß2GPI colocalizes with NET strands by immunofluorescence microscopy. Finally, we provide evidence that ß2GPI-DNA complexes can be detected in the plasma of APS patients, where they positively correlate with an established biomarker of NET remnants. Taken together, our findings indicate that ß2GPI interacts with DNA and NETs, and suggest that this interaction may play a role as a perpetuator and/or instigator of autoimmunity in APS.

Predictors and clinical complications associated with antiphospholipid antibodies in sickle cell disease.

Although a higher prevalence of antiphospholipid autoantibodies (aPL) has been observed in some cohorts of sickle cell disease (SCD) patients, the clinical risk factors for the development of aPL and its associated complications remain unclear. In a retrospective study of 63 SCD patients, a lower hemoglobin concentration and higher white blood cell count were independently associated with an elevated aPL. SCD patients with elevated aPL had increased pregnancy complications (≥3 miscarriages, preterm delivery, pre-eclampsia) and venous thrombotic events. Our findings suggest that SCD may predispose to the generation of aPL and that aPL itself may contribute to the vasculopathy of SCD. Prospective testing for aPL is warranted in patients with SCD.

Outcomes associated with antiphospholipid antibodies in COVID-19: A prospective cohort study.

Background: The significance of antiphospholipid antibodies (aPL) in COVID-19 remains uncertain. Objectives: We determined whether aPL are associated with COVID-19 and/or thrombosis or adverse outcomes during hospitalization for COVID-19. Methods: Symptomatic adults tested for SARS-CoV-2 for clinical reasons (March-July 2020) with either ≥1 positive polymerase chain reaction (COVID-19+) or all negative (non-COVID-19) results were recruited to a biobank collecting plasma, clinical data, and outcomes. We tested baseline plasma samples (days 0-7) of all subjects (and day-30 samples in the COVID-19+ subjects, when available) for aPL (anticardiolipin immunoglobulin [Ig]M/IgG, anti-ß2-glycoprotein I IgM/IgG, antiphosphatidylserine/prothrombin IgM/IgG, and lupus anticoagulant). We compared the baseline prevalence of aPL between the COVID-19+ and non-COVID-19 subjects. Among hospitalized COVID-19+ subjects, multivariable logistic regression was used to evaluate the association of aPL (and their subtypes) with arterial or venous thromboembolic events, acute kidney injury, intensive care unit admission, mechanical ventilation, and death after adjusting for potential confounders. Results: At baseline, 123 of 289 (43%) COVID+ subjects had ≥1 aPL versus 116 of 261 (32%) non-COVID-19 subjects (difference, 10%; 95% CI, 3%-18%). Among 89 COVID+ subjects with repeated samples, aPL persisted on day 30 in 15 of 34 (44%) subjects with baseline aPL positivity, and half of those without aPL at baseline developed one or more new aPL. In hospitalized COVID-19 subjects (n = 241), baseline aPL positivity was associated with acute kidney injury (odds ratio [OR], 1.8; 95% CI, 1.1-3.2) and mechanical ventilation (OR, 3.2; 95% CI, 1.5-6.8) but not death (OR, 1.2; 95% CI, 0.6-2.5). In secondary analyses, medium-to-high titers of anticardiolipin IgG (>40) were associated with thromboembolic events (OR, 7.3; 95% CI, 1.8-30.1). Conclusion: In patients with COVID-19, aPL may help identify an increased risk of thrombosis and other adverse outcomes.

Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis.

BACKGROUND: Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). In vivo, ACPAs target peptidyl-citrulline epitopes (cit-) in a variety of proteins (cit-prot-ACPAs) and derived peptides (cit-pept-ACPAs) generated via the peptidylarginine deiminase (PAD) isoenzymes. We aimed to identify a cell line with self-citrullination capacity, to describe its autoantigenic citrullinome, and to test it as a source of autocitrullinated proteins and peptides. METHODS: Human cell lines were screened for cit-proteins by Western blot. PAD isoenzymes were identified by RT-PCR. Autocitrullination of ECV304 was optimized, and the ECV304 autocitrullinomes immunoprecipitated by sera from three RA patients were characterized by mass spectrometry. Cit-pept-ACPAs were detected using anti-CCP2 ELISA and cit-prot-ACPAs, by an auto-cit-prot-ECV304 ELISA. Sera from 177 RA patients, 59 non-RA rheumatic disease patients and 25 non-disease controls were tested. RESULTS: Of the seven cell lines studied, only ECV304 simultaneously overexpressed PAD2 and PAD3 and its extracts reproducibly autocitrullinated self and non-self-proteins. Proteomic analysis of the cit-ECV304 products immunoprecipitated by RA sera, identified novel cit-targets: calreticulin, profilin 1, vinculin, new 14-3-3 protein family members, chaperones, and mitochondrial enzymes. The auto-cit-prot-ECV304 ELISA had a sensitivity of 50% and a specificity of 95% for RA diagnosis. CONCLUSIONS: ECV304 cells overexpress two of the PAD isoenzymes capable of citrullinating self-proteins. These autocitrullinated cells constitute a basic and clinical research tool that enable the detection of cit-prot-ACPAs with high diagnostic specificity and allow the identification of the specific cit-proteins targeted by individual RA sera.

FcγRIIA expression accelerates nephritis and increases platelet activation in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by deposits of immune complexes (ICs) in organs and tissues. The expression of FcγRIIA by human platelets, which is their unique receptor for immunoglobulin G antibodies, positions them to ideally respond to circulating ICs. Whereas chronic platelet activation and thrombosis are well-recognized features of human SLE, the exact mechanisms underlying platelet activation in SLE remain unknown. Here, we evaluated the involvement of FcγRIIA in the course of SLE and platelet activation. In patients with SLE, levels of ICs are associated with platelet activation. Because FcγRIIA is absent in mice, and murine platelets do not respond to ICs in any existing mouse model of SLE, we introduced the FcγRIIA (FCGR2A) transgene into the NZB/NZWF1 mouse model of SLE. In mice, FcγRIIA expression by bone marrow cells severely aggravated lupus nephritis and accelerated death. Lupus onset initiated major changes to the platelet transcriptome, both in FcγRIIA-expressing and nonexpressing mice, but enrichment for type I interferon response gene changes was specifically observed in the FcγRIIA mice. Moreover, circulating platelets were degranulated and were found to interact with neutrophils in FcγRIIA-expressing lupus mice. FcγRIIA expression in lupus mice also led to thrombosis in lungs and kidneys. The model recapitulates hallmarks of human SLE and can be used to identify contributions of different cellular lineages in the manifestations of SLE. The study further reveals a role for FcγRIIA in nephritis and in platelet activation in SLE.

Necroptotic cell binding of ß2 -glycoprotein I provides a potential autoantigenic stimulus in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies against diverse self-antigens with damage to multiple organs. Immunization with the SLE autoantigen ß2 -glycoprotein I (ß2 GPI) and lipopolysaccharide (LPS), a known trigger of necroptosis, induces a murine model of SLE. We hypothesized that necroptotic cells, like apoptotic cells, provide a "scaffold" of cellular self-antigens, but, unlike apoptotic cells, necroptotic cells do so in a proinflammatory and immunogenic context. We demonstrate that ß2 GPI indeed binds to necroptotic cells and serves as a target for anti-ß2 GPI autoantibodies. We further demonstrate that necroptotic, but not apoptotic, cells promote antigenic presentation of ß2 GPI to CD4 T cells by dendritic cells. Finally, we show that ß2 GPI/LPS-immunized mice deficient in RIPK3 (receptor-interacting serine/threonine-protein kinase 3) or MLKL (mixed lineage kinase domain like), and consequently unable to undergo necroptosis, have reduced SLE autoantibody production and pathology. RIPK3-/- mice had low levels of SLE autoantibodies and no renal pathology, while MLKL-/- mice produced low levels of SLE autoantibodies initially, but later developed levels comparable with wild type (WT) mice and pathology intermediate to that of WT and RIPK3-/- mice. Serum cytokine levels induced by LPS tended to be lower in RIPK3-/- and MLKL-/- mice than in WT mice, suggesting that impaired proinflammatory cytokine production may impact the initiation of autoantibody production in both strains. Our data suggest that self-antigen (i.e. ß2 GPI) presented in the context of necroptosis and proinflammatory signals may be sufficient to overcome immune tolerance and induce SLE.

Autoantibodies in Systemic Lupus Erythematosus Target Mitochondrial RNA.

The mitochondrion supplies energy to the cell and regulates apoptosis. Unlike other mammalian organelles, mitochondria are formed by binary fission and cannot be directly produced by the cell. They contain numerous copies of a compact circular genome that encodes RNA molecules and proteins involved in mitochondrial oxidative phosphorylation. Whereas, mitochondrial DNA (mtDNA) activates the innate immune system if present in the cytosol or the extracellular milieu, it is also the target of circulating autoantibodies in systemic lupus erythematosus (SLE). However, it is not known whether mitochondrial RNA is also recognized by autoantibodies in SLE. In the present study, we evaluated the presence of autoantibodies targeting mitochondrial RNA (AmtRNA) in SLE. We quantified AmtRNA in an inducible model of murine SLE. The AmtRNA were also determined in SLE patients and healthy volunteers. AmtRNA titers were measured in both our induced model of murine SLE and in human SLE, and biostatistical analyses were performed to determine whether the presence and/or levels of AmtRNA were associated with clinical features expressed by SLE patients. Both IgG and IgM classes of AmtRNA were increased in SLE patients (n = 86) compared to healthy controls (n = 30) (p < 0.0001 and p = 0.0493, respectively). AmtRNA IgG levels correlated with anti-mtDNA-IgG titers (rs = 0.54, p < 0.0001) as well as with both IgG and IgM against ß-2-glycoprotein I (anti-ß2GPI; rs = 0.22, p = 0.05), and AmtRNA-IgG antibodies were present at higher levels when patients were positive for autoantibodies to double-stranded-genomic DNA (p < 0.0001). AmtRNA-IgG were able to specifically discriminate SLE patients from healthy controls, and were negatively associated with plaque formation (p = 0.04) and lupus nephritis (p = 0.03). Conversely, AmtRNA-IgM titers correlated with those of anti-ß2GPI-IgM (rs = 0.48, p < 0.0001). AmtRNA-IgM were higher when patients were positive for anticardiolipin antibodies (aCL-IgG: p = 0.01; aCL-IgM: p = 0.002), but AmtRNA-IgM were not associated with any of the clinical manifestations assessed. These findings identify mtRNA as a novel mitochondrial antigen target in SLE, and support the concept that mitochondria may provide an important source of circulating autoantigens in SLE.

Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations.

Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.

T cells from induced and spontaneous models of SLE recognize a common T cell epitope on ß2-glycoprotein I.

Systemic lupus erythematosus is a prototypic model for B-cell epitope spread in autoimmunity. Autoantibodies to numerous molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting phospholipid-binding proteins, particularly ß2-glycoprotein I. Notably, mice immunized with ß2-glycoprotein I and lipopolysaccharide develop a strong T cell response to ß2-glycoprotein I that is associated with autoantibody production and renal disease, similar to that seen in human SLE. Here we hypothesized that mice with murine systemic lupus erythematosus, whether induced or spontaneous, should have T cells that recognize ß2-glycoprotein I. We evaluated the response of splenic T cells from mice with induced (C57BL/6 and C3H/HeN) and spontaneous (MRL/lpr) systemic lupus erythematosus to peptides spanning the entire sequence of human ß2GPI. We found that mice with induced and spontaneous systemic lupus erythematosus recognize a common T cell epitope (peptide 31; LYRDTAVFECLPQHAMFG) in domain III of ß2-glycoprotein I. ß2GPI-reactive CD4+ T cells from the two models differed primarily in cytokine production: T cells from mice with induced SLE expressed IFN-γ, while T cells from MRL/lpr mice expressed both IL-17 and IFN-γ, indicating that IL-17-expressing T cells are not necessary for generating a ß2GPI-reactive T cell response. These data suggest that the generation of a ß2-glycoprotein I-reactive T cell response is shared by both induced and spontaneous models of systemic lupus erythematosus and that this T cell response may mediate epitope spread to autoantibodies in both models.

Repeated exposure of epithelial cells to apoptotic cells induces the specific selection of an adaptive phenotype: Implications for tumorigenesis.

The consequences of apoptosis extend beyond the mere death of the cell. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits PTEC proliferation, growth, and survival. Here, we tested the hypothesis that continual exposure to apoptotic targets can induce a phenotypic change in responding PTECs, as in other instances of natural selection. In particular, we demonstrate that repeated exposure to apoptotic targets leads to emergence of a PTEC line (denoted BU.MPTSEL) resistant to apoptotic target-induced death. Resistance is exquisitely specific. Not only are BU.MPTSEL responders fully resistant to apoptotic target-induced death (∼85% survival versus <10% survival of nonselected cells) but do so while retaining sensitivity to all other target-induced responses, including inhibition of proliferation and growth. Moreover, the resistance of BU.MPTSEL responders is specific to target-induced apoptosis, as apoptosis in response to other suicidal stimuli occurs normally. Comparison of the signaling events induced by apoptotic target exposure in selected versus nonselected responders indicated that the acquired resistance of BU.MPTSEL cells lies in a regulatory step affecting the generation of the pro-apoptotic protein, truncated BH3 interacting-domain death agonist (tBID), most likely at the level of BID cleavage by caspase-8. This specific adaptation has especial relevance for cancer, in which the prominence and persistence of cell death entail magnification of the post-mortem effects of apoptotic cells. Just as cancer cells acquire specific resistance to chemotherapeutic agents, we propose that cancer cells may also adapt to their ongoing exposure to apoptotic targets.

ß2-Glycoprotein I-Reactive T Cells in Autoimmune Disease.

Anti-phospholipid syndrome (APS) and systemic lupus erythematosus (SLE) are autoimmune diseases characterized by autoantibody production and autoantibody-related pathology. Anti-phospholipid antibodies (aPL) are found in all patients with APS and in 20-30% of individuals with SLE. aPL recognize a number of autoantigens, but the primary target in both APS and SLE is ß2-glycoprotein I (ß2GPI). The production of IgG aPL in APS and SLE, as well as the association of aPL with certain MHC class II molecules, has led to investigation of the role of ß2GPI-reactive T helper (Th). ß2GPI-reactive CD4 Th cells have been associated with the presence of aPL and/or APS in both primary APS and secondary APS associated with SLE, as well as in SLE patients and healthy controls lacking aPL. CD4 T cells reactive with ß2GPI have also been associated with atherosclerosis and found within atherosclerotic plaques. In most cases, the epitopes targeted by autoreactive ß2GPI-reactive CD4 T cells in APS and SLE appear to arise as a consequence of antigenic processing of ß2GPI that is structurally different from the soluble native form. This may arise from molecular interactions (e.g., with phospholipids), post-translational modification (e.g., oxidation or glycation), genetic alteration (e.g., ß2GPI variants), or molecular mimicry (e.g., microbiota). A number of T cell epitopes have been characterized, particularly in Domain V, the lipid-binding domain of ß2GPI. Possible sources of negatively charged lipid that bind ß2GPI include oxidized LDL, activated platelets, microbiota (e.g., gut commensals), and dying (e.g., apoptotic) cells. Apoptotic cells not only bind ß2GPI, but also express multiple other cellular autoantigens targeted in both APS and SLE. Dying cells that have bound ß2GPI thus provide a rich source of autoantigens that can be recognized by B cells across a wide range of autoantigen specificities. ß2GPI-reactive T cells could potentially provide T cell help to autoantigen-specific B cells that have taken up and processed apoptotic (or other dying) cells, and subsequently present ß2GPI on their surface in the context of major histocompatibility complex (MHC) class II molecules. Here, we review the literature on ß2GPI-reactive T cells, and highlight findings supporting the hypothesis that these T cells drive autoantibody production in both APS and SLE.

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Apoptotic cells activate AMP-activated protein kinase (AMPK) and inhibit epithelial cell growth without change in intracellular energy stores.

Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses.

ß2-Glycoprotein I-specific T cells are associated with epitope spread to lupus-related autoantibodies.

Systemic lupus erythematosus (SLE) is a prototypic model for B cell epitope spread in autoimmunity. Autoantibodies to numerous and molecularly distinct self-antigens emerge in a sequential manner over several years, leading to disease manifestations. Among the earliest autoantibodies to appear are those targeting the apoptotic cell-binding protein ß2-glycoprotein I (ß2GPI). Notably, mice immunized with ß2GPI and LPS display a remarkably similar pattern of autoantibody emergence to that seen in human SLE. Here, we used this model to investigate whether epitope spread to SLE-related autoantibodies is associated with a unique or limited ß2GPI-specific T cell response. We ask whether MHC class II haplotype and its associated T cell epitope restriction impact epitope spread to SLE-related autoantibodies. We found that ß2GPI/LPS-immunized mice produced similar SLE-related autoantibody profiles regardless of their ß2GPI T cell epitope specificity or MHC class II haplotype. Although ß2GPI T cell epitope specificity was clearly determined by MHC class II haplotype, a number of different ß2GPI T cell epitopes were associated with epitope spread to SLE-related autoantibodies. Notably, one ß2GPI T cell epitope (peptide 23, NTGFYLNGADSAKCT) was also recognized by T cells from an HLA-DRB1*0403(+) autoimmune patient. These data suggest that the generation of a ß2GPI-reactive T cell response is associated with epitope spread to SLE-related autoantibodies, independent of epitope specificity or MHC class II restriction. On the basis of these findings, we propose that factors enabling a ß2GPI-reactive T cell response may predispose individuals to the development of SLE-related autoantibodies independent of their MHC class II haplotype.

Autoimmunity and antibody affinity maturation are modulated by genetic variants on mouse chromosome 12.

Autoimmune diseases result from a break in immune tolerance leading to an attack on self-antigens. Autoantibody levels serve as a predictive tool for the early diagnosis of many autoimmune diseases, including type 1 diabetes. We find that a genetic locus on mouse chromosome 12 influences the affinity maturation of antibodies as well as autoantibody production. Thus, we generated a NOD.H2(k) congenic strain bearing B10 alleles at the locus comprised within the D12Mit184 and D12Mit12 markers, which we named NOD.H2(k)-Chr12. We determined the biological relevance of the Chr12 locus on the autoimmune process using an antigen-specific TCR transgenic autoimmune mouse model. Specifically, the 3A9 TCR transgene, which recognizes a peptide from hen egg lysozyme (HEL) in the context of I-A(k), and the HEL transgene, which is expressed under the rat-insulin promoter (iHEL), were bred into the NOD.H2(k)-Chr12 congenic strain. In the resulting 3A9 TCR:iHEL NOD.H2(k)-Chr12 mice, we observed a significant decrease in diabetes incidence as well as a decrease in both the quantity and affinity of HEL-specific IgG autoantibodies relative to 3A9 TCR:iHEL NOD.H2(k) mice. Notably, the decrease in autoantibodies due to the Chr12 locus was not restricted to the TCR transgenic model, as it was also observed in the non-transgenic NOD.H2(k) setting. Of importance, antibody affinity maturation upon immunization and re-challenge was also impeded in NOD.H2(k)-Chr12 congenic mice relative to NOD.H2(k) mice. Together, these results demonstrate that a genetic variant(s) present within the Chr12 locus plays a global role in modulating antibody affinity maturation.

A polymorphism in TLR2 is associated with arterial thrombosis in a multiethnic population of patients with systemic lupus erythematosus.

OBJECTIVE: Thrombosis is a serious complication of systemic lupus erythematosus (SLE). Studies that have investigated the genetics of thrombosis in SLE are limited. We undertook this study to assess the association of previously implicated candidate genes, particularly Toll-like receptor (TLR) genes, with pathogenesis of thrombosis. METHODS: We genotyped 3,587 SLE patients from 3 multiethnic populations for 77 single-nucleotide polymorphisms (SNPs) in 10 genes, primarily in TLRs 2, 4, 7, and 9, and we also genotyped 64 ancestry-informative markers (AIMs). We first analyzed association with arterial and venous thrombosis in the combined population via logistic regression, adjusting for top principal components of the AIMs and other covariates. We also subjected an associated SNP, rs893629, to meta-analysis (after stratification by ethnicity and study population) to confirm the association and to test for study population or ethnicity effects. RESULTS: In the combined analysis, the SNP rs893629 in the KIAA0922/TLR2 region was significantly associated with arterial thrombosis (logistic P = 6.4 × 10(-5) , false discovery rate P = 0.0044). Two additional SNPs in TLR2 were also suggestive: rs1816702 (logistic P = 0.002) and rs4235232 (logistic P = 0.009). In the meta-analysis by study population, the odds ratio (OR) for arterial thrombosis with rs893629 was 2.44 (95% confidence interval 1.58-3.76), without evidence for heterogeneity (P = 0.78). By ethnicity, the effect was most significant among African Americans (OR 2.42, P = 3.5 × 10(-4) ) and European Americans (OR 3.47, P = 0.024). CONCLUSION: TLR2 gene variation is associated with thrombosis in SLE, particularly among African Americans and European Americans. There was no evidence of association among Hispanics, and results in Asian Americans were limited due to insufficient sample size. These results may help elucidate the pathogenesis of this important clinical manifestation.

Recognition-dependent signaling events in response to apoptotic targets inhibit epithelial cell viability by multiple mechanisms: implications for non-immune tissue homeostasis.

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular ß-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses.

Interaction of ß2-glycoprotein I with lipopolysaccharide leads to Toll-like receptor 4 (TLR4)-dependent activation of macrophages.

ß(2)-Glycoprotein I (ß(2)GPI) is an abundant plasma protein that binds to the surface of cells and particles expressing negatively charged lipids, but its physiological role remains unknown. Antibodies to ß(2)GPI are found in patients with anti-phospholipid syndrome, a systemic autoimmune disease associated with vascular thrombosis and pregnancy morbidity. Although it has been suggested that anti-ß(2)GPI antibodies activate endothelial cells and monocytes by signaling through TLR4, it is unclear how anti-ß(2)GPI antibodies and/or ß(2)GPI interact with TLR4. A number of mammalian proteins (termed "endogenous Toll-like receptor (TLR) ligands") have been reported to bind to TLR4, but, in most cases, subsequent studies have shown that LPS interaction with these proteins is responsible for TLR activation. We hypothesized that, like other endogenous TLR ligands, ß(2)GPI interacts specifically with LPS and that this interaction is responsible for apparent TLR4 activation by ß(2)GPI. Here, we show that both LPS and TLR4 are required for ß(2)GPI to bind to and activate macrophages. Untreated ß(2)GPI stimulated TNF-α production in TLR4-sufficient (but not TLR4-deficient) macrophages. In contrast, neither polymyxin B-treated nor delipidated ß(2)GPI stimulated TNF-α production. Furthermore, ß(2)GPI bound to LPS in a specific and dose-dependent manner. Finally, untreated ß(2)GPI bound to the surface of TLR4-sufficient (but not TLR4-deficient) macrophages. Polymyxin B treatment of ß(2)GPI abolished macrophage binding. Our findings suggest a potential new biological activity for ß(2)GPI as a protein that interacts specifically with LPS and point to the need to evaluate newly discovered endogenous TLR ligands for potential interactions with LPS.

Association of autoantibodies to heat-shock protein 60 with arterial vascular events in patients with antiphospholipid antibodies.

OBJECTIVE: Anti-heat shock protein 60 autoantibodies (anti-Hsp60) are associated with cardiovascular disease and are known to affect endothelial cells in vitro, and we have recently shown that anti-Hsp60 promote thrombosis in a murine model of arterial injury. Based on those findings, we undertook the present study to investigate the hypothesis that the presence of anti-Hsp60, alone or in combination with other thrombogenic risk factors, is associated with an elevated risk of vascular events. METHODS: The study population was derived from 3 ongoing cohort studies: 2 independent systemic lupus erythematosus (SLE) registries and 1 cohort comprising SLE patients and non-SLE patients. Data from a total of 402 participants were captured; 199 of these participants had had confirmed vascular events (arterial vascular events in 102, venous vascular events in 76, and both arterial and venous vascular events in 21). Anti-Hsp60 were detected by enzyme-linked immunoassay, and association with vascular events was assessed by regression analysis. RESULTS: Multiple regression analysis revealed that arterial vascular events were associated with male sex, age, and hypertension. Analyses of the vascular events according to their origin showed an association of anti-Hsp60 with arterial vascular events (odds ratio 2.26 [95% confidence interval 1.13-4.52]), but not with venous vascular events. Anti-Hsp60 increased the risk of arterial vascular events (odds ratio 5.54 [95% confidence interval 1.89-16.25]) in antiphospholipid antibody (aPL)-positive, but not aPL-negative, individuals. CONCLUSION: We demonstrate that anti-Hsp60 are associated with an increased risk of arterial vascular events, but not venous vascular events, in aPL-positive individuals. These data suggest that anti-Hsp60 may serve as a useful biomarker to distinguish risk of arterial and venous vascular events in patients with aPL.

Cardiolipin binds to CD1d and stimulates CD1d-restricted γδ T cells in the normal murine repertoire.

Cardiolipin (CL), a major phospholipid in bacterial cell walls, is sequestered from the immune system in mammalian mitochondria and is, therefore, a potential danger signal. Based on growing evidence that phospholipids constitute natural ligands for CD1 and that CD1d-restricted T cells recognize phospholipids, we hypothesized that CD1d binds and presents CL and that T cells in the normal immune repertoire respond to CL in a CD1d-restricted manner. We determined the murine CD1d-CL crystal structure at 2.3 Šresolution and established through additional lipid loading experiments that CL, a tetra-acylated phospholipid, binds to murine CD1d with two alkyl chains buried inside the CD1d binding groove and the remaining two exposed into the solvent. We furthermore demonstrate the functional stimulatory activity of CL, showing that splenic and hepatic γδ T cells from healthy mice proliferate in vitro in response to mammalian or bacterial CL in a dose-dependent and CD1d-restricted manner, rapidly secreting the cytokines IFN-γ and RANTES. Finally, we show that hepatic γδ T cells are activated in vivo by CD1d-bearing dendritic cells that have been pulsed with CL, but not phosphatidylcholine. Together, these findings demonstrate that CD1d is able to bind and present CL to a subset of CL-responsive γδ T cells that exist in the spleen and liver of healthy mice and suggest that these cells could play a role in host responses to bacterial lipids and, potentially, self-CL. We propose that CL-responsive γδ T cells play a role in immune surveillance during infection and tissue injury.