Jenny Meerding

Natural killer T cells in adipose tissue prevent insulin resistance

Lipid overload and adipocyte dysfunction are key to the development of insulin resistance and can be induced by a high-fat diet. CD1d-restricted invariant natural killer T (iNKT) cells have been proposed as mediators between lipid overload and insulin resistance, but recent studies found decreased iNKT cell numbers and marginal effects of iNKT cell depletion on insulin resistance under high-fat diet conditions. Here, we focused on the role of iNKT cells under normal conditions. We showed that iNKT cell-deficient mice on a low-fat diet, considered a normal diet for mice, displayed a distinctive insulin resistance phenotype without overt adipose tissue inflammation. Insulin resistance was characterized by adipocyte dysfunction, including adipocyte hypertrophy, increased leptin, and decreased adiponectin levels. The lack of liver abnormalities in CD1d-null mice together with the enrichment of CD1d-restricted iNKT cells in both mouse and human adipose tissue indicated a specific role for adipose tissue-resident iNKT cells in the development of insulin resistance. Strikingly, iNKT cell function was directly modulated by adipocytes, which acted as lipid antigen-presenting cells in a CD1d-mediated fashion. Based on these findings, we propose that, especially under low-fat diet conditions, adipose tissue-resident iNKT cells maintain healthy adipose tissue through direct interplay with adipocytes and prevent insulin resistance.

Functional human regulatory T cells fail to control autoimmune inflammation due to PKB/c-akt hyperactivation in effector cells

During the last decade research has focused on the application of FOXP3(+) regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.

Cytokine assays: an assessment of the preparation and treatment of blood and tissue samples.

Cytokines are key components of the innate and adaptive immune system. As pivotal players in the progression or regression of a pathological process, these molecules provide a window through which diseases can be monitored and can thus act as biomarkers. In order to measure cytokine levels, a plethora of protocols can be applied. These methods include bioassays, protein microarrays, high-performance liquid chromatography (HPLC), sandwich enzyme-linked immunosorbent assay (ELISA), Meso Scale Discovery (MSD) electrochemiluminescence and bead based multiplex immunoassays (MIA). Due to the interaction and activity of cytokines, multiplex immunoassays are at the forefront of cytokine analysis by allowing multiple cytokines to be measured in parallel. However, even with optimized protocols, sample standardization needs to occur before these proteins can optimally act as biomarkers. This review describes various factors influencing the levels of cytokines measured in plasma, serum, dried blood spots and tissue biopsies, focusing on sample collection and handling, long term storage and the repetitive use of samples. By analyzing how each of these factors influences protein levels, it is concluded that samples should be stored at low temperatures in order to maintain cytokine stability. In addition, within a study, sample manipulations should be kept the same, with measurement protocols being chosen for their compatibility with the research in question. By having a clear understanding of what factors influence cytokine levels and how to overcome these technical issues, minimally confounded data can be obtained and cytokines can achieve optimal biomarker activity.

Local IL-17A Potentiates Early Neutrophil Recruitment to the Respiratory Tract during Severe RSV Infection

Respiratory syncytial virus (RSV) bronchiolitis triggers a strong innate immune response characterized by excessive neutrophil infiltration which contributes to RSV induced pathology. The cytokine IL-17A enhances neutrophil infiltration into virus infected lungs. IL-17A is however best known as an effector of adaptive immune responses. The role of IL-17A in early immune modulation in RSV infection is unknown. We aimed to elucidate whether local IL-17A facilitates the innate neutrophil infiltration into RSV infected lungs prior to adaptive immunity. To this end, we studied IL-17A production in newborns that were hospitalized for severe RSV bronchiolitis. In tracheal aspirates we measured IL-17A concentration and neutrophil counts. We utilized cultured human epithelial cells to test if IL-17A regulates RSV infection-induced IL-8 release as mediator of neutrophil recruitment. In mice we investigated the cell types that are responsible for early innate IL-17A production during RSV infection. Using IL-17A neutralizing antibodies we tested if IL-17A is responsible for innate neutrophil infiltration in mice. Our data show that increased IL-17A production in newborn RSV patient lungs correlates with subsequent neutrophil counts recruited to the lungs. IL-17A potentiates RSV-induced production of the neutrophil-attracting chemokine IL-8 by airway epithelial cells in vitro. Various lung-resident lymphocytes produced IL-17A during early RSV infection in Balb/c mice, of which a local population of CD4 T cells stood out as the predominant RSV-induced cell type. By removing IL-17A during early RSV infection in mice we showed that IL-17A is responsible for enhanced innate neutrophil infiltration in vivo. Using patient material, in vitro studies, and an animal model of RSV infection, we thus show that early local IL-17A production in the airways during RSV bronchiolitis facilitates neutrophil recruitment with pathologic consequences to infant lungs.

A Multiplex Immunoassay for Human Adipokine Profiling

BACKGROUND Adipose tissue secretory proteins, called adipokines, play pivotal roles in the pathophysiology of obesity and its associated disorders such as metabolic syndrome, type 2 diabetes, and cardiovascular disease. Because methods for comprehensive adipokine profiling in patient plasma and other biological samples are currently limited, we developed a multiplex immunoassay for rapid and high-throughput measurement of 25 adipokines in only 50 microL of sample. METHODS (Pre)adipocyte and ex vivo cultured adipose tissue supernatants were generated and together with plasma from 5 morbidly obese patients and 5 healthy and normal weight controls used to develop the adipokine multiplex immunoassay and test its usefulness in biological samples. We assessed adipokine dynamic ranges, lower limits of detection and quantification, cross-reactivity, intra- and interassay variation, and correlation with adipokine ELISAs. RESULTS The limits of quantification and broad dynamic ranges enabled measurement of all 25 adipokines in supernatants and patient plasmas, with the exception of TNF-alpha in plasma samples. Intraassay variation was <10% for all adipokines; interassay variation was < 15%. The multiplex immunoassay results correlated significantly with ELISA measurements. Plasma adipokine profiling showed significantly higher concentrations of the novel adipokines cathepsin S (5.1 x 10(4) vs 4.3 x 10(4) ng/L, P = 0.003) and chemerin (4.1 x 10(5) vs 2.7 x 10(5) ng/L, P = 0.0008) in morbidly obese patients than normal weight controls, besides the established differences in adiponectin and leptin concentrations. CONCLUSIONS Our findings underscore the relevance of the novel adipokines cathepsin S and chemerin, but foremost the potential of this novel method for both comprehensive adipokine profiling in large patient cohorts and for biological discovery.

Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells

Autologous hematopoietic stem cell transplantation (HSCT) is increasingly considered for patients with severe autoimmune diseases whose prognosis is poor with standard treatments. Regulatory T cells (Tregs) are thought to be important for disease remission after HSCT. However, eliciting the role of donor and host Tregs in autologous HSCT is not possible in humans due to the autologous nature of the intervention. Therefore, we investigated their role during immune reconstitution and re-establishment of immune tolerance and their therapeutic potential following congenic bone marrow transplantation (BMT) in a proteoglycan-induced arthritis (PGIA) mouse model. In addition, we determined Treg T-cell receptor (TCR) CDR3 diversity before and after HSCT in patients with juvenile idiopathic arthritis and juvenile dermatomyositis. In the PGIA BMT model, after an initial predominance of host Tregs, graft-derived Tregs started dominating and displayed a more stable phenotype with better suppressive capacity. Patient samples revealed a striking lack of diversity of the Treg repertoire before HSCT. This ameliorated after HSCT, confirming reset of the Treg compartment following HSCT. In the mouse model, a therapeutic approach was initiated by infusing extra Foxp3(GFP+) Tregs during BMT. Infusion of Foxp3(GFP+) Tregs did not elicit additional clinical improvement but conversely delayed reconstitution of the graft-derived T-cell compartment. These data indicate that HSCT-mediated amelioration of autoimmune disease involves renewal of the Treg pool. In addition, infusion of extra Tregs during BMT results in a delayed reconstitution of T-cell compartments. Therefore, Treg therapy may hamper development of long-term tolerance and should be approached with caution in the clinical autologous setting.

CD30 Discriminates Heat Shock Protein 60-Induced FOXP3+CD4+ T Cells with a Regulatory Phenotype

In many animal models, the manifestations of inflammatory diseases can be prevented by the adoptive transfer of CD4+FOXP3+ regulatory T cells (Tregs). CD4+FOXP3+ Tregs can be obtained by isolation and expansion of polyclonal naturally occurring Tregs or by Ag-specific activation of CD4+CD25−FOXP3− T cells. Two major obstacles are hampering the translation of this latter protocol into therapeutic application. First, there is a lack of knowledge on relevant autoantigens. Second, the resulting population is contaminated with activated CD4+ T cells that transiently express Forkhead box P3 but gain no regulatory function. Therefore, these cells may not be safe for clinical application. In this study, we demonstrate that highly suppressive FOXP3+ Tregs can be induced in vitro by the activation of CD4+CD25− T cells with the self-Ag human 60-kDa heat shock protein (HSP60). The activation induced suppressive FOXP3+ Tregs can be distinguished by surface expression of CD30 from nonsuppressive FOXP3+ effector cells. We confirm that the induced CD30+FOXP3+ Tregs recognize HSP60 epitopes and that the induction of Tregs by HSP60 is enhanced by signaling via TLR4 on APCs. These findings have implications for the generation and isolation of pure populations of Ag-specific Tregs, with the potential to prevent and treat human inflammatory diseases.

Brief Report: Anti–Tumor Necrosis Factor α Targets Protein Kinase B/c‐Akt–Induced Resistance of Effector Cells to Suppression in Juvenile Idiopathic Arthritis

OBJECTIVE To determine whether therapeutic strategies that block interleukin-6 (IL-6) or tumor necrosis factor α (TNFα) can improve the responsiveness of Teff cells to suppression in patients with juvenile idiopathic arthritis (JIA). METHODS Synovial fluid mononuclear cells (SFMCs) from the inflamed joints of patients with JIA were cultured in the presence of etanercept or anti-IL-6 in vitro, and protein kinase B (PKB)/c-Akt activation and responsiveness to suppression were measured. In addition, the in vivo effects of TNFα blockade were investigated using peripheral blood mononuclear cells obtained from patients before and after the start of etanercept therapy. RESULTS In vitro treatment of SFMCs with anti-IL-6 led to improved Treg cell-mediated suppression of cell proliferation in some but not all patients. Blocking TNFα with etanercept, however, clearly enhanced suppression, especially that of CD8+ T cells. In the presence of etanercept, PKB/c-Akt activation of Teff cells was reduced, and cells became more susceptible to transforming growth factor β-mediated suppression, indicating that anti-TNFα directly targets resistant Teff cells. CONCLUSION This study is the first to show that anti-TNFα targets the resistance of Teff cells to suppression, resulting in improved regulation of inflammatory effector cells.

TLR9 agonist CpG enhances protective nasal HSP60 peptide vaccine efficacy in experimental autoimmune arthritis

Objectives Peptide-based immune tolerance induction is considered an attractive treatment option for autoimmune diseases. The authors have developed a novel method that can enhance the induction of protective peptide-specific T-cell responses, using a rat arthritis model. The authors focused on the Toll-like receptor 9 ligand CpG, which was shown to stimulate regulatory T-cell proliferation when added to plasmacytoid dendritic cells (pDC) using in-vitro cultures. Methods The peptide used is a heat shock protein 60 epitope (p1) that elicits tolerogenic peptide-specific immune responses in human arthritis patients and was recently shown to have protective capacity as a bystander antigen in the rat adjuvant arthritis model. Rats were treated with three nasal doses of p1, CpG or a combination of p1 and CpG. Antigen-presenting cells were studied in nose-draining lymph nodes (mandibular lymph nodes; MLN) after nasal treatment, and T-cell responses were analysed in joint-draining lymph nodes after arthritis induction. Results Nasal co-administration of p1/CpG significantly augmented the arthritis-protective effect of p1, while CpG treatment alone did not. Co-treatment of p1/CpG increased both the number and activation status of pDC in draining MLN, which was accompanied by amplified p1-specific T-cell proliferation and interleukin (IL)-10 production. During early arthritis, p1-specific IL-10 production was identified at the site of inflammation. P1 and p1/CpG-treated rats showed a greater amount of CD4+FoxP3+ regulatory T cells in the joint-draining lymph nodes, which correlated with lower arthritis scores. Conclusions These clinical and immunological data suggest the use of CpG as a potent adjuvant for mucosal peptide-specific immune therapy in arthritis.

Cytokine profiling at disease onset: support for classification of young antinuclear antibody-positive patients as a separate category of juvenile idiopathic arthritis

The current classification for juvenile idiopathic arthritis (JIA) separates the disease entity JIA into seven disease categories based on clinical and laboratory features1 It is postulated that the combination of early onset of disease and antinuclear antibody (ANA) positivity better classifies a homogeneous subset of patients than the current International League of Associations for Rheumatology (ILAR) categories, displaying asymmetric arthritis, female predominance and an increased risk of developing iridocyclitis.2–5 The classification by the number of joints involved might not be an adequate criterion for identifying homogeneous disease entities. We sought out to determine if this homogenous group of young ANA-positive patients could be distinguished from other JIA patients based on their soluble inflammatory profile. These profiles could aid in the differentiation of patient groups by identifying similar underlying inflammatory processes. This study prospectively followed 40 patients with JIA at onset of disease at their first visit to the paediatric rheumatology clinic for oligoarthritis (n=24) and rheumatoid factor-negative polyarthritis (n=16). Ethics approval was given by the regional review boards. Informed consent was obtained from all patients. The occurrence of iridocylitis was determined by reviewing total clinical history; the time of follow-up did not differ between the groups analysed (table 1). Apart from nonsteroidal anti-inflammatory drugs (NSAID) therapy, …