Autoantibodies against the chemokine receptor 3 predict cardiovascular risk.

BACKGROUND AND AIMS: Chronic inflammation and autoimmunity contribute to cardiovascular (CV) disease. Recently, autoantibodies (aAbs) against the CXC-motif-chemokine receptor 3 (CXCR3), a G protein-coupled receptor with a key role in atherosclerosis, have been identified. The role of anti-CXCR3 aAbs for CV risk and disease is unclear. METHODS: Anti-CXCR3 aAbs were quantified by a commercially available enzyme-linked immunosorbent assay in 5000 participants (availability: 97.1%) of the population-based Gutenberg Health Study with extensive clinical phenotyping. Regression analyses were carried out to identify determinants of anti-CXCR3 aAbs and relevance for clinical outcome (i.e. all-cause mortality, cardiac death, heart failure, and major adverse cardiac events comprising incident coronary artery disease, myocardial infarction, and cardiac death). Last, immunization with CXCR3 and passive transfer of aAbs were performed in ApoE(-/-) mice for preclinical validation. RESULTS: The analysis sample included 4195 individuals (48% female, mean age 55.5 ± 11 years) after exclusion of individuals with autoimmune disease, immunomodulatory medication, acute infection, and history of cancer. Independent of age, sex, renal function, and traditional CV risk factors, increasing concentrations of anti-CXCR3 aAbs translated into higher intima-media thickness, left ventricular mass, and N-terminal pro-B-type natriuretic peptide. Adjusted for age and sex, anti-CXCR3 aAbs above the 75th percentile predicted all-cause death [hazard ratio (HR) (95% confidence interval) 1.25 (1.02, 1.52), P = .029], driven by excess cardiac mortality [HR 2.51 (1.21, 5.22), P = .014]. A trend towards a higher risk for major adverse cardiac events [HR 1.42 (1.0, 2.0), P = .05] along with increased risk of incident heart failure [HR per standard deviation increase of anti-CXCR3 aAbs: 1.26 (1.02, 1.56), P = .03] may contribute to this observation. Targeted proteomics revealed a molecular signature of anti-CXCR3 aAbs reflecting immune cell activation and cytokine-cytokine receptor interactions associated with an ongoing T helper cell 1 response. Finally, ApoE(-/-) mice immunized against CXCR3 displayed increased anti-CXCR3 aAbs and exhibited a higher burden of atherosclerosis compared to non-immunized controls, correlating with concentrations of anti-CXCR3 aAbs in the passive transfer model. CONCLUSIONS: In individuals free of autoimmune disease, anti-CXCR3 aAbs were abundant, related to CV end-organ damage, and predicted all-cause death as well as cardiac morbidity and mortality in conjunction with the acceleration of experimental atherosclerosis.

Autoimmune pre-disease.

Approximately 5% of the world-wide population is affected by autoimmune diseases. Overall, autoimmune diseases are still difficult to treat, impose a high burden on patients, and have a significant economic impact. Like other complex diseases, e.g., cancer, autoimmune diseases develop over several years. Decisive steps in the development of autoimmune diseases are (i) the development of autoantigen-specific lymphocytes and (often) autoantibodies and (ii) potentially clinical disease manifestation at a later stage. However, not all healthy individuals with autoantibodies develop disease manifestations. Identifying autoantibody-positive healthy individuals and monitoring and inhibiting their switch to inflammatory autoimmune disease conditions are currently in their infancy. The switch from harmless to inflammatory autoantigen-specific T and B-cell and autoantibody responses seems to be the hallmark for the decisive factor in inflammatory autoimmune disease conditions. Accordingly, biomarkers allowing us to predict this progression would have a significant impact. Several factors, such as genetics and the environment, especially diet, smoking, exposure to pollutants, infections, stress, and shift work, might influence the progression from harmless to inflammatory autoimmune conditions. To inspire research directed at defining and ultimately targeting autoimmune predisease, here, we review published evidence underlying the progression from health to autoimmune predisease and ultimately to clinically manifest inflammatory autoimmune disease, addressing the following 3 questions: (i) what is the current status, (ii) what is missing, (iii) and what are the future perspectives for defining and modulating autoimmune predisease.

Low-dose interleukin-2 therapy for the treatment of systemic lupus erythematosus.

PURPOSE OF REVIEW: To provide an overview behind the concept and recent advances of low-dose interleukin-2 (IL-2) therapy in systemic lupus erythematosus (SLE). RECENT FINDINGS: A disruption of regulatory T cell homeostasis caused by an acquired deficiency of IL-2 is a crucial event in the pathogenesis of SLE. Here, we highlight the key rationales for the clinical translation of low-dose IL-2 therapy in SLE and summarize the main findings from two independent, early phase uncontrolled clinical studies that investigated the immunological and clinical responses to low-dose IL-2 therapy in patients with active SLE. Important commonalities and differences between these studies with regard to study design and results are discussed. SUMMARY: Low-dose IL-2 therapy is capable to promote the selective expansion of a functionally competent regulatory T cell population in a well-tolerated way and may have the potential to influence the clinical course in patients with active SLE. Although a clearer proof for the clinical efficacy of low-dose IL-2 therapy in SLE is still outstanding, these early studies provide important rationales and the scientific basis for more comprehensive and placebo-controlled trials in the future.

GPCR-specific autoantibody signatures are associated with physiological and pathological immune homeostasis.

Autoantibodies have been associated with autoimmune diseases. However, studies have identified autoantibodies in healthy donors (HD) who do not develop autoimmune disorders. Here we provide evidence of a network of immunoglobulin G (IgG) autoantibodies targeting G protein-coupled receptors (GPCR) in HD compared to patients with systemic sclerosis, Alzheimer's disease, and ovarian cancer. Sex, age and pathological conditions affect autoantibody correlation and hierarchical clustering signatures, yet many of the correlations are shared across all groups, indicating alterations to homeostasis. Furthermore, we identify relationships between autoantibodies targeting structurally and functionally related molecules, such as vascular, neuronal or chemokine receptors. Finally, autoantibodies targeting the endothelin receptor type A (EDNRA) exhibit chemotactic activity, as demonstrated by neutrophil migration toward HD-IgG in an EDNRA-dependent manner and in the direction of IgG from EDNRA-immunized mice. Our data characterizing the in vivo signatures of anti-GPCR autoantibodies thus suggest that they are a physiological part of the immune system.

Pathogenetic and Clinical Aspects of Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides.

Anti-neutrophil cytoplasmic autoantibodies (ANCA) targeting proteinase 3 (PR3) and myeloperoxidase expressed by innate immune cells (neutrophils and monocytes) are salient diagnostic and pathogenic features of small vessel vasculitis, comprising granulomatosis with polyangiitis (GPA), microscopic polyangiitis, and eosinophilic GPA. Genetic studies suggest that ANCA-associated vasculitides (AAV) constitute separate diseases, which share common immunological and pathological features, but are otherwise heterogeneous. The successful therapeutic use of anti-CD20 antibodies emphasizes the prominent role of ANCA and possibly other autoantibodies in the pathogenesis of AAV. However, to elucidate causal effects in AAV, a better understanding of the complex interplay leading to the emergence of B lymphocytes that produce pathogenic ANCA remains a challenge. Different scenarios seem possible; e.g., the break of tolerance induced by a shift from non-pathogenic toward pathogenic autoantigen epitopes in inflamed tissue. This review gives a brief overview on current knowledge about genetic and epigenetic factors, barrier dysfunction and chronic non-resolving inflammation, necro-inflammatory auto-amplification of cellular death and inflammation, altered autoantigen presentation, alternative complement pathway activation, alterations within peripheral and inflamed tissue-residing T- and B-cell populations, ectopic lymphoid tissue neoformation, the characterization of PR3-specific T-cells, properties of ANCA, links between autoimmune disease and infection-triggered pathology, and animal models in AAV.

Mapping urinary chemokines in human lupus nephritis: Potentially redundant pathways recruit CD4+ and CD8+ T cells and macrophages.

Renal infiltration of inflammatory cells contributes to the pathogenesis of lupus nephritis (LN). Current knowledge on the recruitment mechanisms relies mainly on findings in rodent models. Here, we assess various chemokine pathways in human LN by comparing urinary chemokine concentrations (in 25 patients with acute LN and in 78 lupus patients without active LN) with the expression of corresponding chemokine receptors on urinary leukocytes (in ten acute LN patients). Nine urinary chemokines were significantly elevated in LN patients and correlated with renal disease activity and urinary cell counts; however, their concentrations displayed considerable interindividual heterogeneity. Analysis of the corresponding receptors revealed abundance of urinary CD8+ T cells for CCR5 and CXCR3, while CD4+ T cells were additionally enriched for CCR1, CCR6 and CXCR6. Urinary Treg showed similar CCR expression, and urinary CD14+ macrophages were enriched for CCR5 expressing cells. In conclusion, cell specific recruitment patterns seem to involve CCR5 and CXCR3 in all cells studied, while CD4+ T-cell subset recruitment is probably much more varied. However, urinary chemokine abundance in active LN is individually variable in our cohort and does not offer a singular chemokine usable as universal biomarker or potential future treatment target.

The cellular signature of urinary immune cells in Lupus nephritis: new insights into potential biomarkers.

INTRODUCTION: Urinary T cells represent a reliable noninvasive biomarker for proliferative Lupus nephritis (LN). Little is known about the presence of T cell subsets, B cells and macrophages in the urine although they may further improve the validity of urinary cellular biomarkers for LN. METHODS: We analyzed contemporaneous blood and urine samples of patients with active LN (n = 19), other Systemic Lupus Erythematosus (SLE) patients (n = 79) and urine samples of patients with diabetic nephropathy (DN; n = 14) and anti-neutrophil cytoplasmatic antibody (ANCA) associated vasculitis (AAV; n = 11) by flow cytometry. RESULTS: Numbers of urinary T cells, B cells and macrophages correlated with disease activity and were significantly higher in the active LN group. Urinary T cells showed excellent distinction of patients with active LN, CD8+ T cells (AUC of ROC = 1.000) and CD4+ T cells (AUC = 0.9969) alike. CD19+ B cells (AUC = 0.7823) and CD14+ macrophages (AUC = 0.9066), as well as the clinical standard proteinuria (AUC = 0.9201), failed to reach these high standards. Patients with DN or AAV also showed increased urinary cell counts, although the CD4/CD8-ratio was significantly lower in SLE compared to in DN (p = 0.0006). Urinary CD4+ T cells of active LN patients proved to be mainly of effector memory phenotype and expressed significantly more CD40L and ki67 than corresponding blood cells. Urinary Treg counts correlated with disease activity. CONCLUSIONS: Despite of detectable urinary cell counts for B cells and macrophages, T cells remain the best urinary cellular biomarker for LN. A low CD4/CD8-ratio seems to be characteristic for LN.

Urinary CD4 T cells identify SLE patients with proliferative lupus nephritis and can be used to monitor treatment response.

OBJECTIVES: Proliferative lupus nephritis (LN) is one of the major concerns in the treatment of systemic lupus erythematosus (SLE). Here we evaluate urinary CD4 T cells as a biomarker of active LN and indicator of treatment response. METHODS: Urinary CD3CD4 T cells were quantified using flow cytometry in 186 urine samples from 147 patients with SLE. Fourteen patients were monitored as follow-up. Thirty-one patients with other nephropathies and 20 healthy volunteers were included as controls. RESULTS: In SLE, urinary CD4 T cell counts ≥800/100 ml were observed exclusively in patients with active LN. Receiver operator characteristic analysis documented clear separation of SLE patients with active and non-active LN (area under the curve 0.9969). All patients with up-to-date kidney biopsy results showing proliferative LN had high urinary CD4 T cell numbers. In patients monitored under therapy, normalisation of urinary CD4 T cell counts indicated lower disease activity and better renal function. In contrast, patients with persistence of, or increase in, urinary T cells displayed worse outcomes. CONCLUSIONS: Urinary CD4 T cells are a highly sensitive and specific marker for detecting proliferative LN in patients with SLE. Furthermore, monitoring urinary CD4 T cells may help to identify treatment responders and treatment failure and enable patient-tailored therapy in the future.

CD4+Foxp3+ regulatory T cells prolong drug-induced disease remission in (NZBxNZW) F1 lupus mice.

INTRODUCTION: The ability to ameliorate murine lupus renders regulatory T cells (Treg) a promising tool for the treatment of systemic lupus erythematosus (SLE). In consideration to the clinical translation of a Treg-based immunotherapy of SLE, we explored the potential of CD4+Foxp3+ Treg to maintain disease remission after induction of remission with an established cyclophosphamide (CTX) regimen in lupus-prone (NZBxNZW) F1 mice. As a prerequisite for this combined therapy, we also investigated the impact of CTX on the biology of endogenous Treg and conventional CD4+ T cells (Tcon). METHODS: Remission of disease was induced in diseased (NZBxNZW) F1 mice with an established CTX regimen consisting of a single dose of glucocorticosteroids followed by five day course with daily injections of CTX. Five days after the last CTX injection, differing amounts of purified CD4+Foxp3+CD25+ Treg were adoptively transferred and clinical parameters, autoantibody titers, the survival and changes in peripheral blood lymphocyte subsets were determined at different time points during the study. The influence of CTX on the numbers, frequencies and proliferation of endogenous Treg and Tcon was analyzed in lymphoid organs by flow cytometry. RESULTS: Apart from abrogating the proliferation of Tcon, we found that treatment with CTX induced also a significant inhibition of Treg proliferation and a decline in Treg numbers in lymphoid organs. Additional adoptive transfer of 1.5×106 purified Treg after the CTX regimen significantly increased the survival and prolonged the interval of remission by approximately five weeks compared to mice that received only the CTX regimen. The additional clinical amelioration was associated with an increase in the Treg frequency in the peripheral blood indicating a compensation of CTX-induced Treg deficiency by the Treg transfer. CONCLUSIONS: Treg were capable to prolong the interval of remission induced by conventional cytostatic drugs. This study provides valuable information and a first proof-of-concept for the feasibility of a Treg-based immunotherapy in the maintenance of disease remission in SLE.

Unmasking of autoreactive CD4 T cells by depletion of CD25 regulatory T cells in systemic lupus erythematosus.

OBJECTIVE: Autoreactive CD4 T cells specific for nuclear peptide antigens play an important role in tolerance breakdown during the course of systemic lupus erythematosus (SLE). However, reliable detection of these cells is limited due to their low frequency in peripheral blood. The authors assess autoreactive CD4 T cells in a representative SLE collective (n=38) by flow cytometry and study the influence of regulatory T cells (Treg) on their antigenic challenge. METHODS: CD4 T-cell responses were determined according to intracellular CD154 expression induced after 6-h short-term in-vitro stimulation with the SLE-associated autoantigen SmD1(83-119). To clarify the influence of Treg on the activation of autoreactive CD4 T cells, CD25 Treg were depleted by magnetic activated cell sorting before antigen-specific stimulation in selected experiments. RESULTS: In the presence of Treg, autoreactive CD4 T-cell responses to SmD1(83-119) were hardly observable. However, Treg removal significantly increased the frequency of detectable SmD1(83-119)-specific CD4 T cells in SLE patients but not in healthy individuals. Consequently, by depleting Treg the percentage of SmD1(83-119)-reactive SLE patients increased from 18.2% to 63.6%. This unmasked autoreactivity of CD4 T cells correlated with the disease activity as determined by the SLE disease activity index (p=0.005*, r=0.779). CONCLUSIONS: These data highlight the pivotal role of the balance between autoreactive CD4 T cells and CD25 Treg in the dynamic course of human SLE. Analysing CD154 expression in combination with a depletion of CD25 Treg, as shown here, may be of further use in approaching autoantigen-specific CD4 T cells in SLE and other autoimmune diseases.

Class switching and consecutive loss of dsDNA-reactive B1a B cells from the peritoneal cavity during murine lupus development.

B1a B cells have been implicated in the pathogenesis of systemic lupus erythematosus; however, their precise contribution to the disease remains unclear. Here we analysed isotype expression, organ accumulation and autoreactivity of B1a cells in the NZB/W F1 murine model for systemic lupus erythematosus using flow cytometry, ELISPOT and adoptive cell transfer. In the course of lupus, the B1a compartment is expanded and B1a cells class switch to IgG. Class-switched B1a B cells were excluded from the peritoneal cavity but accumulated in the spleen and target organs such as kidneys and thymus. Autoreactive B1a B cells preferentially class switch, which leads to a subsequent loss of autoreactive B1a cells from the peritoneal cavity. We propose a model whereby autoreactive B1a B cells become activated early in the course of lupus and class switch to IgG. These autoreactive B1a B cells accumulate in the spleen and affected organs where they presumably secrete autoantibodies and contribute to the local and systemic pathogenesis.

Autoregulation of Th1-mediated inflammation by twist1.

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor kappaB (NF-kappaB)-dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-kappaB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-gamma, IL-2, and tumor necrosis factor-alpha, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.