Mélanie Dieudé

Predictive value of antinuclear autoantibodies: the lessons of the systemic sclerosis autoantibodies.

Physicians who provide care to patients with systemic sclerosis (SSc, scleroderma) ask themselves several questions: Is SSc the correct diagnosis? Can the disease course be predicted? Is there a greater risk for involvement of certain organs? Can the vital prognosis be predicted? In this brief review, we answer these questions by excerpting data from the literature focused on the 4 major SSc specific autoantibodies (aAbs) to nuclear autoantigens (ANAs): anti-centromere (anti-CENP-B), anti-Th/To, anti-topoisomerase I (anti-topo I) and anti-RNA polymerase III (anti-RNAPIII). The data show that these aAbs are highly valuable as markers for the diagnosis of SSc, as biomarkers for phenotypic subsets and as prognostic markers. We also identify areas for future clinical research.

Antiperlecan antibodies are novel accelerators of immune-mediated vascular injury.

Acute vascular rejection (AVR) is characterized by immune‐mediated vascular injury and heightened endothelial cell (EC) apoptosis. We reported previously that apoptotic ECs release a bioactive C‐terminal fragment of perlecan referred to as LG3. Here, we tested the possibility that LG3 behaves as a neoantigen, fuelling the production of anti‐LG3 antibodies of potential importance in regulating allograft vascular injury. We performed a case–control study in which we compared anti‐LG3 IgG titers in kidney transplant recipients with AVR (n = 15) versus those with acute tubulo‐interstitial rejection (ATIR) (n = 15) or stable graft function (n = 30). Patients who experienced AVR had elevated anti‐LG3 titers pre and posttransplantation compared to subjects with ATIR or stable graft function (p < 0.05 for both mediators). Elevated pretransplant anti‐LG3 titers (OR: 4.62, 95% CI: 1.08–19.72) and pretransplant donor‐specific antibodies (DSA) (OR 4.79, 95% CI: 1.03–22.19) were both independently associated with AVR. To address the functional role of anti‐LG3 antibodies in AVR, we turned to passive transfer of anti‐LG3 antibodies in an animal model of vascular rejection based on orthotopic aortic transplantation between fully MHC‐mismatched mice. Neointima formation, C4d deposition and allograft inflammation were significantly increased in recipients of an ischemic aortic allograft passively transferred with anti‐LG3 antibodies. Collectively, these data identify anti‐LG3 antibodies as novel accelerators of immune‐mediated vascular injury and obliterative remodeling.

The Perlecan Fragment LG3 Is a Novel Regulator of Obliterative Remodeling Associated With Allograft Vascular Rejection

Rationale: Endothelial apoptosis is increased in association with acute and chronic vascular rejection (VR) of solid allografts. Apoptotic endothelial cells (EC) release LG3, a C-terminal fragment of perlecan of potential importance in vascular remodeling and neointima formation. Objective: Our 2 goals were to determine whether circulating levels of LG3 are increased in association with acute VR of renal allografts and to evaluate the impact of LG3 on vascular remodeling. Methods and Results: We conducted a case-control study to compare serum LG3 levels in human renal transplant patients with acute VR, tubulo-interstitial rejection (ATIR) and normal graft function. Aorta transplantation between fully MHC-mismatched mice in association with intravenous LG3 injection was used to characterize the impact of LG3 on vascular remodeling. Scratch assays evaluated the promigratory activity of LG3 on vascular smooth muscle cells (VSMC) in vitro. Serum LG3 levels were significantly elevated in human renal transplant patients with acute VR (n=16) compared to ATIR (n=16) and normal graft function (n=32, P=0.004). In patients with acute VR, graft loss was associated with elevated LG3 levels. Increasing LG3 serum levels in aortic allograft recipients significantly increased neointima formation. LG3 injection fostered accumulation of &agr;-smooth muscle actin–positive cells and decreased the number of CD31 positive EC. LG3 increased the migration of VSMC through extracellular signal-regulated kinases 1/2-dependent pathways. Conclusion: These results indicate that LG3 is a novel regulator of obliterative vascular remodeling during rejection.

A comprehensive characterization of membrane vesicles released by autophagic human endothelial cells.

The stress status of the apoptotic cell can promote phenotypic changes that have important consequences on the immunogenicity of the dying cell. Autophagy is one of the biological processes activated in response to a stressful condition. It is an important mediator of intercellular communications, both by regulating the unconventional secretion of molecules, including interleukin 1β, and by regulating the extracellular release of ATP from early stage apoptotic cells. Additionally, autophagic components can be released in a caspase‐dependent manner by serum‐starved human endothelial cells that have engaged apoptotic and autophagic processes. The nature and the components of the extracellular vesicles released by dying autophagic cells are not known. In this study, we have identified extracellular membrane vesicles that are released by human endothelial cells undergoing apoptosis and autophagy, and characterized their biochemical, ultrastructural, morphological properties as well as their proteome. These extracellular vesicles differ from classical apoptotic bodies because they do not contain nucleus components and are released independently of Rho‐associated, coiled‐coil containing protein kinase 1 activation. Instead, they are enriched with autophagosomes and mitochondria and convey various danger signals, including ATP, suggesting that they could be involved in the modulation of innate immunity.

Detection and Quantification of Microparticles from Different Cellular Lineages Using Flow Cytometry. Evaluation of the Impact of Secreted Phospholipase A2 on Microparticle Assessment

Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

The 20S proteasome core, active within apoptotic exosome-like vesicles, induces autoantibody production and accelerates rejection

Exosome-like vesicles containing an active 20S proteasome core contribute to autoimmunity and vascular allograft inflammation. Friendly fire from organ failure Despite advances in organ transplantation, rejection still poses a substantial risk. Autoantibodies contribute to rejection, but how these autoantibodies are generated remains unknown. Dieudé et al. found that injection of apoptotic exosome-like vesicles apoExo stimulated autoantibody production in mice, which led to increased graft rejection after transplant. The apoExo contained active 20S proteasome core complexes, and inhibition of proteasome activity decreased the immunogenicity of apoExo and graft rejection in transplanted mice. Circulating apoExo and increased anti-autoantibody titers were also observed in mouse models of ischemia-reperfusion injury, suggesting that the same organ failure that necessitates the transplant might increase the risk of rejection. Therefore, proteasome inhibitors could provide a new therapeutic avenue for graft rejection. Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)–incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

The dual role of innate immunity in the antiphospholipid syndrome

The antiphospholipid syndrome (APS), as both a primary syndrome and a syndrome in association with systemic lupus erythematosus (SLE), can be a devastating disease. It is unclear what factors (genetic and/or environmental) lead to the generation of antiphospholipid antibodies (aPL). It is equally unclear why only certain individuals with aPL develop clinical events. We hypothesize that innate immune activation plays a critical role at two distinct stages of APS, namely, the initiation phase, in which aPL first appear, and the effector phase, in which aPL precipitate a thrombotic event. According to the model we propose, aPL alone are insufficient to cause thrombosis and a concomitant trigger of innate immunity, e.g. a toll-like receptor (TLR) ligand, must be present for thrombosis to occur. Here, we discuss our findings that mice immunized with β2-glycoprotein I (β2GPI) and lipopolysaccharide (LPS), a TLR ligand, produce high levels of aPL and other SLE-associated autoantibodies, and develop lupus-like glomerulonephritis. We also discuss our data showing that autoantibodies to heat shock protein 60 (HSP60), an ‘endogenous TLR ligand’, promote thrombus generation in a murine model of arterial injury. Thus, both pathogen-derived TLR ligands (e.g. LPS) and endogenous TLR ligands (e.g. HSP60) may contribute to the pathogenesis of APS. This putative dual role of innate immunity provides new insight into the generation of aPL as well as the enigma of why some individuals with aPL develop APS, while others do not.

Interaction of β2-Glycoprotein I with Lipopolysaccharide Leads to Toll-like Receptor 4 (TLR4)-dependent Activation of Macrophages

Background: β2-Glycoprotein I (β2GPI) binds to negatively charged lipids, but its physiological role remains unknown. Results: β2GPI containing LPS, but not β2GPI depleted of LPS activity, stimulated macrophages in a TLR4-dependent manner. Conclusion: β2GPI interacts specifically with LPS. Significance: Apparent TLR4-mediated activation of macrophages by β2GPI is due to the presence of LPS. β2-Glycoprotein I (β2GPI) 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 β2GPI 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-β2GPI antibodies activate endothelial cells and monocytes by signaling through TLR4, it is unclear how anti-β2GPI antibodies and/or β2GPI 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, β2GPI interacts specifically with LPS and that this interaction is responsible for apparent TLR4 activation by β2GPI. Here, we show that both LPS and TLR4 are required for β2GPI to bind to and activate macrophages. Untreated β2GPI stimulated TNF-α production in TLR4-sufficient (but not TLR4-deficient) macrophages. In contrast, neither polymyxin B-treated nor delipidated β2GPI stimulated TNF-α production. Furthermore, β2GPI bound to LPS in a specific and dose-dependent manner. Finally, untreated β2GPI bound to the surface of TLR4-sufficient (but not TLR4-deficient) macrophages. Polymyxin B treatment of β2GPI abolished macrophage binding. Our findings suggest a potential new biological activity for β2GPI 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.

Autophagy fosters myofibroblast differentiation through MTORC2 activation and downstream upregulation of CTGF.

Recent evidence suggests that autophagy may favor fibrosis through enhanced differentiation of fibroblasts in myofibroblasts. Here, we sought to characterize the mediators and signaling pathways implicated in autophagy-induced myofibroblast differentiation. Fibroblasts, serum starved for up to 4 d, showed increased LC3-II/-I ratios and decreased SQSTM1/p62 levels. Autophagy was associated with acquisition of markers of myofibroblast differentiation including increased protein levels of ACTA2/αSMA (actin, α 2, smooth muscle, aorta), enhanced gene and protein levels of COL1A1 (collagen, type I, α 1) and COL3A1, and the formation of stress fibers. Inhibiting autophagy with 3 different class I phosphoinositide 3-kinase and class III phosphatidylinositol 3-kinase (PtdIns3K) inhibitors or through ATG7 silencing prevented myofibroblast differentiation. Autophagic fibroblasts showed increased expression and secretion of CTGF (connective tissue growth factor), and CTGF silencing prevented myofibroblast differentiation. Phosphorylation of the MTORC1 target RPS6KB1/p70S6K kinase was abolished in starved fibroblasts. Phosphorylation of AKT at Ser473, a MTORC2 target, was reduced after initiation of starvation but was followed by spontaneous rephosphorylation after 2 d of starvation, suggesting the reactivation of MTORC2 with sustained autophagy. Inhibiting MTORC2 activation with long-term exposure to rapamycin or by silencing RICTOR, a central component of the MTORC2 complex abolished AKT rephosphorylation. Both RICTOR silencing and rapamycin treatment prevented CTGF and ACTA2 upregulation, demonstrating the central role of MTORC2 activation in CTGF induction and myofibroblast differentiation. Finally, inhibition of autophagy with PtdIns3K inhibitors or ATG7 silencing blocked AKT rephosphorylation. Collectively, these results identify autophagy as a novel activator of MTORC2 signaling leading to CTGF induction and myofibroblast differentiation.

The Emerging Importance of Non-HLA Autoantibodies in Kidney Transplant Complications

Antibodies that are specific to organ donor HLA have been involved in the majority of cases of antibody-mediated rejection in solid organ transplant recipients. However, recent data show that production of non-HLA autoantibodies can occur before transplant in the form of natural autoantibodies. In contrast to HLAs, which are constitutively expressed on the cell surface of the allograft endothelium, autoantigens are usually cryptic. Tissue damage associated with ischemia-reperfusion, vascular injury, and/or rejection creates permissive conditions for the expression of cryptic autoantigens, allowing these autoantibodies to bind antigenic targets and further enhance vascular inflammation and renal dysfunction. Antiperlecan/LG3 antibodies and antiangiotensin II type 1 receptor antibodies have been found before transplant in patients with de novo transplants and portend negative long-term outcome in patients with renal transplants. Here, we review mounting evidence suggesting an important role for autoantibodies to cryptic antigens as novel accelerators of kidney dysfunction and acute or chronic allograft rejection.