Roman Volchenkov

The complexity of Sjögren's syndrome: Novel aspects on pathogenesis

In Sjögrens syndrome, like in most other autoimmune diseases, the enigma leading to a pathogenic attack against self has not yet been solved. By definition, the disease must be mediated by specific immune reactions against endogenous tissues to qualify as an autoimmune disease. In Sjögrens syndrome the autoimmune response is directed against the exocrine glands, which, as histopathological hallmark of the disease, display persistent and progressive focal mononuclear cell infiltrates. Clinically, the disease in most patients is manifested by two severe symptoms: dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). A number of systemic features have also been described and the presence of autoantibodies against the ubiquitously expressed ribonucleoprotein particles Ro (Sjögrens-syndrome-related antigen A - SSA) and La (SSB) further underline the systemic nature of Sjögrens syndrome. The original explanatory concept for the pathogenesis of Sjögrens syndrome proposed a specific, self-perpetuating, immune mediated loss of acinar and ductal cells as the principal cause of salivary gland hypofunction. Although straightforward and plausible, the hypothesis, however, falls short of accommodating several Sjögrens syndrome-related phenomena and experimental findings. Consequently, researchers considered immune-mediated salivary gland dysfunction prior to glandular destruction and atrophy as potential molecular mechanisms underlying the symptoms of dryness in Sjögrens syndrome. Accordingly, apoptosis, fibrosis and atrophy of the salivary glands would represent consequences of salivary gland hypofunction. The emergence of advanced bio-analytical platforms further enabled the identification of potential biomarkers with the intent to improve Sjögrens syndrome diagnosis, promote the development of prognostic tools for Sjögrens syndrome and the long-term goal to identify possible processes for therapeutic treatment interventions. In addition, such approaches allowed us to glimpse at the apparent complexity of Sjögrens syndrome.

Type 1 Regulatory T Cells and Regulatory B Cells Induced by Tolerogenic Dendritic Cells

Dendritic cells (DC) are professional antigen‐presenting cells that are capable of both activating immune responses and inducing tolerance. Several studies have revealed efficiency of therapeutic vaccination with tolerogenic DC (tolDC) in inhibition of experimental autoimmunity. The purpose of this study was to compare four different protocols for generation of tolDC – the antidiabetic drug troglitazone (TGZ DC), NF‐κB inhibitor BAY 11‐7082 (BAY DC), prostaglandin D2 metabolite 15d‐PGJ2 (PGJ DC) and a combination of dexamethasone and 1α,25‐dihydroxyvitamin D3 (DexVD3 DC) regarding phenotype, cytokine production and T cell stimulatory capacity. TGZ DC and BAY DC had a phenotype comparable to immature DC, while DexVD3 DC were more macrophage like. Analysis of cytokine production using cell culture supernatants from all DC populations revealed that DexVD3 DC were efficient producers of IL‐10 and produced less pro‐inflammatory cytokines. T cells primed with DexVD3 DC showed reduced proliferation, and further analyses of these T cells revealed that functionally effective type 1 regulatory T cells (Tr1) but not FoxP3+ Treg were induced. Furthermore, DexVD3 DC promoted the induction of regulatory B cells (Breg). Together, these results indicate that DexVD3 DC have the best potential to be used in a tolerogenic antigen‐presenting cell‐based immunotherapy setting.

In vitro suppression of immune responses using monocyte-derived tolerogenic dendritic cells from patients with primary Sjögren's syndrome

IntroductionTherapeutic vaccination with antigen-specific tolerogenic dendritic cells (tolDC) might become a future option of individualized therapy for patients with autoimmune diseases. In this study, we tested the possibility of generating monocyte-derived tolDC from patients with primary Sjögrens syndrome (pSS). We analyzed phenotype, cytokine production and ability to suppress Ro/La-specific immune responses.MethodsMonocyte-derived tolDC from patients with pSS were generated in the presence of dexamethasone, vitamin D3 and lipopolysaccharide (DexVD3 DC). The phenotype was analyzed by flow cytometry and the cytokine profile was investigated using a 25-plex Luminex assay and ELISA. The capacity to both stimulate Ro/La-specific T cells and suppress this response was evaluated by autologous mixed lymphocyte reaction (MLR).ResultsDC generated from patients with pSS had a similar phenotype and cytokine profile to those from healthy controls. DexVD3 DC from pSS patients induced little antigen-specific T cell proliferation, but DexVD3 DC-primed lymphocytes successfully suppressed Ro/La-specific T cell responses.ConclusionsDexVD3 DC presenting Ro/La antigens might be a promising new therapeutic option for patients with pSS.

Anti-Ro and anti-La autoantibody profiling in Norwegian patients with primary Sjögren’s syndrome using luciferase immunoprecipitation systems (LIPS)

Primary Sjögren’s syndrome (pSS) is a chronic systemic autoimmune disease characterized by mononuclear cell infiltrations preferentially in salivary and lacrimal glands leading to xerostomia and keratoconjunctivitis sicca, respectively. The aetiology and pathogenesis of SS are still not clear and diagnosis is complicated (1). The presence of antibodies to Ro and La antigens is strongly indicative of SS (2), although they are also found in other autoimmune diseases. In clinically used laboratory tests and most of the published studies, solid-phase enzyme-linked immunosorbent assays (ELISAs) with native bovine or recombinant human Ro and La proteins are used. However, these assays can give false-positive results and have low sensitivity (3). Tests with full-length Ro52 can give false-positive results because its C-terminal part contains a B30.2 domain (4). As an alternative, a novel method was developed based on solution-phase luciferase immunoprecipitation systems (LIPS) technology (5). LIPS harnesses recombinant light-emitting Renilla luciferase (Ruc) fusion proteins to efficiently detect antibody responses to linear and conformational epitopes and can detect anti-Ro and anti-La autoantibodies in serum and saliva (6, 7). In our study we assessed the performance and diagnostic accuracy of the test in a Norwegian cohort of pSS patients. Serum samples from 140 patients (133 females and seven males) fulfilling the American–European classification criteria for pSS (2) and 50 ageand gender-matched healthy controls from Bergen, Norway, were investigated. Mean age was 56 (range 23–81) years for patients and 54 (range 27–73) years for controls. The study was approved by the Ethical Committee at the University of Bergen (Approval number 242.06). LIPS was performed as described previously (8). Mammalian Ruc expression vectors for Ro60, La, Ro52, and C-terminal truncated Ro52 (Ro52d1, aa2-273) were kindly provided by Dr Peter D Burbelo, National Institute of Dental and Craniofacial Research, NIH, USA. LIPS detected anti-Ro60 autoantibodies in 81/140 pSS patients (58%), and 56/140 (40%) had anti-La autoantibodies. A total of 75 out of 140 pSS patients (54%) had anti-Ro52 autoantibodies, and no difference was observed between reactivity to full-length Ro52 and Ro52d1. The diagnostic specificity of LIPS with respect to healthy controls was 100% for Ro60, La, and fulllength Ro52, and 98% for Ro52d1. The individual autoantibody spectrum was as follows: 39 patients had all three autoantibodies, 32 patients had anti-Ro52 and anti-Ro60 autoantibodies, two patients had anti-Ro60 and anti-La autoantibodies, 10 patients had only anti-Ro60 autoantibodies, four patients had only anti-Ro52 autoantibodies, and one patient had only anti-La autoantibodies. According to the clinically used ELISA (Varelisa ReCombi ANA Profile, Phadia, Freiburg, Germany), 74/ 140 (54%) pSS patients were anti-Ro positive (without differentiation between Ro52 and Ro60) and only 41/140 (29%) anti-La positive (Table 1). Thus, LIPS showed a better detection rate (11% increase in detected anti-La autoantibodies, 4% increase in anti-Ro60 autoantibodies) together with a clear distinction between positive and negative samples (Figure 1). Our data confirm the results published previously by Burbelo et al (6) and validate the method in a large cohort of pSS patients. In contrast to our study, Burbelo et al observed a higher increase in the detection rate of anti-La autoantibodies whereas anti-Ro60 and anti-Ro52 autoantibodies were detected at approximately the same levels as using ELISA. Furthermore, LIPS is superior to an ELISA using recombinant proteins produced in Escherichia coli previously performed in our laboratory: only 33% of

The 2011 Nobel Prize in Physiology or Medicine

The 2011 Noble Prize in Physiology or Medicine was awarded to Bruce A. Beutler, Jules A. Hoffmann and Ralph M. Steinman for their groundbreaking research within immunology. Bruce A. Beutler and Jules A. Hoffmann were recognized for their discoveries on Toll and Toll‐like receptor activation of innate immunity in fruit fly and mammals, respectively. Ralph M. Steinman received the award for the discovery of dendritic cells, a cell type bridging innate and adaptive immunity, and how these cells orchestrate immune responses.

Metabolic profiling of synovial tissue shows altered glucose and choline metabolism in rheumatoid arthritis samples

metabolism in rheumatoid arthritis samples R Volchenkov, M Dung Cao, KB Elgstøen, GL Goll, K Eikvar, O Bjørneboe, TF Bathen, HL Holen, TK Kvien, BS Skålhegg Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Norway, Department of Circulation and Medical Imaging, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway, Department of Medical Biochemistry, Oslo University Hospital, Rikshospitalet, Oslo, Norway, Department of Rheumatology, Diakonhjemmet Hospital, Oslo, Norway, Department of Surgery, Diakonhjemmet Hospital, Oslo, Norway, Martina Hansen’s Hospital, Gjettum, and Rheumatech AS, Oslo, Norway

T Helper Cell Activation and Expansion Is Sensitive to Glutaminase Inhibition under Both Hypoxic and Normoxic Conditions

Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability.

Th17 Polarization under Hypoxia Results in Increased IL-10 Production in a Pathogen-Independent Manner

The IL-17-producing CD4+ T helper cell (Th17) differentiation is affected by stimulation of the aryl hydrocarbon receptor (AhR) pathway and by hypoxia-inducible factor 1 alpha (HIF-1α). In some cases, Th17 become non-pathogenic and produce IL-10. However, the initiating events triggering this phenotype are yet to be fully understood. Here, we show that such cells may be differentiated at low oxygen and regardless of AhR ligand treatment such as cigarette smoke extract. Hypoxia led to marked alterations of the transcriptome of IL-10-producing Th17 cells affecting genes involved in metabolic, anti-apoptotic, cell cycle, and T cell functional pathways. Moreover, we show that oxygen regulates the expression of CD52, which is a cell surface protein that has been shown to suppress the activation of other T cells upon release. Taken together, these findings suggest a novel ability for Th17 cells to regulate immune responses in vivo in an oxygen-dependent fashion.

Peritumoral dermis of squamous cell carcinomas in renal transplant recipients contains less CD11c+ myeloid dendritic cells and FoxP3+ T cells compared to immunocompetent controls.

Renal transplant recipients (RTR) have an increased risk of developing cutaneous squamous cell carcinomas (SCC). These SCC are often more aggressive than SCC in immunocompetent individuals.

Ablation of the Cβ2 subunit of PKA in immune cells leads to increased susceptibility to systemic inflammation in mice

Protein kinase A (PKA) is a holoenzyme composed of a regulatory subunit dimer and two catalytic subunits and regulates numerous cellular functions including immune cell activity. There are two major catalytic subunit genes, PRKACA and PRKACB encoding the catalytic subunits Cα and Cβ. The PRKACB gene encodes several splice variants including Cβ2, which is enriched in T‐, B‐ and natural killer cells. Cβ2 is significantly larger (46 kDa) than any other C splice variant. In this study we characterized mice ablated for the Cβ2 protein demonstrating a significantly reduced cAMP‐induced catalytic activity of PKA in the spleenocytes, lymphocytes and thymocytes. We also observed a significantly increased number of CD62L‐expressing CD4+ and CD8+ T cells in LNs, accompanied by increased susceptibility to systemic inflammation by the Cβ2 ablated mice. The latter was reflected in an elevated sensitivity to collagen‐induced arthritis (CIA), as well as higher concentration of TNF‐α and lower concentration of IL‐10 in response to LPS challenges. We suggest a role of Cβ2 in regulating innate as well as adaptive immune sensitivity in vivo.