Barbara G. Fürnrohr

Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis

Systemic lupus erythematosus (SLE) is an autoimmune disease in which patients develop autoantibodies to DNA, histones, and often to neutrophil proteins. These form immune complexes that are pathogenic and may cause lupus nephritis. In SLE patients, infections can initiate flares and are a major cause of mortality. Neutrophils respond to infections and release extracellular traps (NETs), which are antimicrobial and are made of DNA, histones, and neutrophil proteins. The timely removal of NETs may be crucial for tissue homeostasis to avoid presentation of self-antigens. We tested the hypothesis that SLE patients cannot clear NETs, contributing to the pathogenesis of lupus nephritis. Here we show that serum endonuclease DNase1 is essential for disassembly of NETs. Interestingly, a subset of SLE patients’ sera degraded NETs poorly. Two mechanisms caused this impaired NET degradation: (i) the presence of DNase1 inhibitors or (ii) anti-NET antibodies prevented DNase1 access to NETs. Impairment of DNase1 function and failure to dismantle NETs correlated with kidney involvement. Hence, identification of SLE patients who cannot dismantle NETs might be a useful indicator of renal involvement. Moreover, NETs might represent a therapeutic target in SLE.

Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE

Autoantibodies against double-stranded DNA (dsDNA) and nucleosomes represent a hallmark of systemic lupus erythematosus (SLE). However, the mechanisms involved in breaking the immunological tolerance against these poorly immunogenic nuclear components are not fully understood. Impaired phagocytosis of apoptotic cells with consecutive release of nuclear antigens may contribute to the immune pathogenesis. The architectural chromosomal protein and proinflammatory mediator high mobility group box protein 1 (HMGB1) is tightly attached to the chromatin of apoptotic cells. We demonstrate that HMGB1 remains bound to nucleosomes released from late apoptotic cells in vitro. HMGB1–nucleosome complexes were also detected in plasma from SLE patients. HMGB1-containing nucleosomes from apoptotic cells induced secretion of interleukin (IL) 1β, IL-6, IL-10, and tumor necrosis factor (TNF) α and expression of costimulatory molecules in macrophages and dendritic cells (DC), respectively. Neither HMGB1-free nucleosomes from viable cells nor nucleosomes from apoptotic cells lacking HMGB1 induced cytokine production or DC activation. HMGB1-containing nucleosomes from apoptotic cells induced anti-dsDNA and antihistone IgG responses in a Toll-like receptor (TLR) 2–dependent manner, whereas nucleosomes from living cells did not. In conclusion, HMGB1–nucleosome complexes activate antigen presenting cells and, thereby, may crucially contribute to the pathogenesis of SLE via breaking the immunological tolerance against nucleosomes/dsDNA.

Factors masking HMGB1 in human serum and plasma

High mobility group box 1 protein (HMGB1) is a ubiquitously expressed architectural chromosomal protein. Recently, it has become obvious that HMGB1 can also act as a proinflammatory mediator when actively secreted during cell activation or passively released from necrotic cells. HMGB1 appears to play an important role in the pathogenesis of diseases, including sepsis and rheumatoid arthritis. However, easy, sensitive, and reliable detection systems are required to investigate the clinical significance of HMGB1 in clinical samples for diagnosis and prognosis of diseases. Here, we describe sensitive ELISAs for the detection of HMGB1 in cell culture medium and cell lysates. However, these assays failed to reliably quantitate HMGB1 in serum and plasma when compared with immunoblot analysis. We found that serum/plasma components bind to HMGB1 and interfere with its detection by ELISA systems. In most serum/plasma samples investigated, including those from healthy individuals, we detected IgG antibodies binding to HMGB1. The titers of these antibodies correlated with the capacity of sera to interfere with the detection of recombinant HMGB1 by ELISA. Furthermore, HMGB1 coimmunoprecipitated with several proteins including IgG1, as identified by mass spectrometry. These HMGB1 interacting proteins are currently characterized and may contribute to complex formation, masking, and possibly, modulation of cytokine activity of HMGB1.

The genetics of systemic lupus erythematosus and implications for targeted therapy

Observations of familial aggregation (λs=8–29) and a 40% identical twin concordance rate prompted recent work towards a comprehensive genetic analysis of systemic lupus erythematosus (SLE). Since 2007, the number of genetic effects known to be associated with human lupus has increased by fivefold, underscoring the complexity of inheritance that probably contributes to this disease. Approximately 35 genes associated with lupus have either been replicated in multiple samples or are near the threshold for genome-wide significance (p>5×10−8). Some are rare variants that convincingly contribute to lupus only in specific subgroups. Strong associations have been found with a large haplotype block in the human leucocyte antigen region, with Fcγ receptors, and with genes coding for complement components, in which a single gene deletion may cause SLE in rare familial cases and copy number variation is more common in the larger population of SLE patients. Examples of newly discovered genes include ITGAM, STAT4 and MECP2/IRAK1. Ongoing studies to build models in which combinations of associated genes might contribute to specific disease manifestations should contribute to improved understanding of disease pathology. In addition, pharmacogenomic components of ongoing clinical trials are likely to provide insights into fundamental disease pathology as well as contributing to informed patient selection for targeted treatments and biomarkers to guide dosing and gauge responsiveness. Besides these potentially valuable new insights into the pathophysiology of an enigmatic, potentially deadly, and, as yet, unsolved disease, genetic studies are likely to suggest novel molecular targets for strategic development of safer and more effective therapeutics.

Orphan nuclear receptor NR4A1 regulates transforming growth factor-β signaling and fibrosis

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

C-reactive protein in rheumatology: biology and genetics

Measurement of serum C-reactive protein (CRP) level is in widespread clinical use as a sensitive marker of inflammation. CRP has a role in the clearance of bacteria and of dying and altered cells, and might also have more complex immunomodulatory functions. Impaired clearance of apoptotic cells is important in the pathogenesis of systemic lupus erythematosus (SLE), raising the possibility that CRP dysregulation plays a part in this process. We review the available functional and genetic evidence supporting a role for CRP in the pathogenesis of SLE, but recognize that inconsistencies in the existing data mean that conclusions have to be interpreted with caution. More consistent is the evidence that the genetic variants influencing basal CRP level also influence the magnitude of the acute-phase rise in CRP level in active inflammation. Initial reports suggest that these genetic effects might be large enough to directly influence clinical decision-making processes that are based on an interpretation of CRP thresholds. This concept is explored further in this article, particularly in relation to the use of the CRP-based disease activity score in the evaluation of rheumatoid arthritis, where a systematic under-scoring or over-scoring of disease activity could result from a failure to consider the genetic influences on CRP level.

After shrinkage apoptotic cells expose internal membrane-derived epitopes on their plasma membranes

Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker™ Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.

Genome-wide association meta-analysis in Chinese and European individuals identifies ten new loci associated with systemic lupus erythematosus

Systemic lupus erythematosus (SLE; OMIM 152700) is a genetically complex autoimmune disease. Genome-wide association studies (GWASs) have identified more than 50 loci as robustly associated with the disease in single ancestries, but genome-wide transancestral studies have not been conducted. We combined three GWAS data sets from Chinese (1,659 cases and 3,398 controls) and European (4,036 cases and 6,959 controls) populations. A meta-analysis of these studies showed that over half of the published SLE genetic associations are present in both populations. A replication study in Chinese (3,043 cases and 5,074 controls) and European (2,643 cases and 9,032 controls) subjects found ten previously unreported SLE loci. Our study provides further evidence that the majority of genetic risk polymorphisms for SLE are contained within the same regions across both populations. Furthermore, a comparison of risk allele frequencies and genetic risk scores suggested that the increased prevalence of SLE in non-Europeans (including Asians) has a genetic basis.

Macrophages Discriminate Glycosylation Patterns of Apoptotic Cell-derived Microparticles

Background: Apoptotic cells release vesicles, which expose “eat-me” signals. Results: Vesicles originated from endoplasmic reticulum expose immature glycoepitopes and are preferentially phagocytosed by macrophages. Conclusion: Immature surface glycoepitopes serve as “eat-me” signals for the clearance of apoptotic vesicles originated from endoplasmic reticulum. Significance: Understanding the distinction by macrophages of apoptotic blebs may provide new insights into clearance-related diseases. Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Inactivation of the transcription factor STAT-4 prevents inflammation-driven fibrosis in animal models of systemic sclerosis

OBJECTIVEnThe transcription factor STAT-4 has recently been identified as a genetic susceptibility factor in systemic sclerosis (SSc) and other autoimmune diseases. The aim of this study was to investigate the contribution of STAT-4 in the development of a fibrotic phenotype in 2 different mouse models of experimental dermal fibrosis.nnnMETHODSnSTAT-4-deficient (stat4(-/-) ) mice and their wild-type littermates (stat4(+/+) ) were injected with bleomycin or NaCl. Infiltrating leukocytes, T cells, B cells, and monocytes were quantified in the lesional skin of stat4(-/-) and stat4(+/+) mice. Inflammatory and profibrotic cytokines were measured in sera and lesional skin samples from stat4(-/-) and stat4(+/+) mice. The outcome of mice lacking STAT-4 was also investigated in the tight skin 1 (TSK-1) mouse model.nnnRESULTSnStat4(-/-) mice were protected against bleomycin-induced dermal fibrosis, with a reduction in dermal thickening (mean ± SEM 65 ± 3% decrease; P = 0.03), hydroxyproline content (68 ± 5% decrease; P = 0.02), and myofibroblast counts (71 ± 6% decrease; P = 0.005). Moreover, the number of infiltrating leukocytes, especially T cells, was significantly decreased in the lesional skin of stat4(-/-) mice (mean ± SEM 63 ± 5% reduction in T cell count; P = 0.02). Stat4(-/-) mice also displayed decreased levels of inflammatory cytokines such as tumor necrosis factor α, interleukin-6 (IL-6), IL-2, and interferon-γ in lesional skin. Consistent with a primary role of STAT-4 in inflammation, STAT-4 deficiency did not ameliorate fibrosis in TSK-1 mice.nnnCONCLUSIONnThe results of this study demonstrate that the transcription factor STAT-4 exerts potent profibrotic effects by controlling T cell activation and proliferation and cytokine release. These findings confirm the results of genetics studies on the role of STAT-4 in the development of SSc.