Publications by authors named "Jenny Meerding"

22 Publications

  • Page 1 of 1

Reversal of Sepsis-Like Features of Neutrophils by Interleukin-1 Blockade in Patients With Systemic-Onset Juvenile Idiopathic Arthritis.

Arthritis Rheumatol 2018 06 7;70(6):943-956. Epub 2018 May 7.

University Medical Center Utrecht, Wilhelmina Children's Hospital and Utrecht University, Utrecht, The Netherlands.

Objective: Neutrophils are the most abundant innate immune cells in the blood, but little is known about their role in (acquired) chronic autoinflammatory diseases. This study was undertaken to investigate the role of neutrophils in systemic-onset juvenile idiopathic arthritis (JIA), a prototypical multifactorial autoinflammatory disease that is characterized by arthritis and severe systemic inflammation.

Methods: Fifty patients with systemic-onset JIA who were receiving treatment with recombinant interleukin-1 receptor antagonist (rIL-1Ra; anakinra) were analyzed at disease onset and during remission. RNA sequencing was performed on fluorescence-activated cell-sorted neutrophils from 3 patients with active systemic-onset JIA and 3 healthy controls. Expression of activation markers, apoptosis, production of reactive oxygen species (ROS), and degranulation of secretory vesicles from neutrophils were assessed by flow cytometry in serum samples from 17 patients with systemic-onset JIA and 15 healthy controls.

Results: Neutrophil counts were markedly increased at disease onset, and this correlated with the levels of inflammatory mediators. The neutrophil counts normalized within days after the initiation of rIL-1Ra therapy. RNA-sequencing analysis revealed a substantial up-regulation of inflammatory processes in neutrophils from patients with active systemic-onset JIA, significantly overlapping with the transcriptome of sepsis. Correspondingly, neutrophils from patients with active systemic-onset JIA displayed a primed phenotype that was characterized by increased ROS production, CD62L shedding, and secretory vesicle degranulation, which was reversed by rIL-1Ra treatment in patients who had achieved clinical remission. Patients with a short disease duration had high neutrophil counts, more immature neutrophils, and a complete response to rIL-1Ra, whereas patients with symptoms for >1 month had normal neutrophil counts and an unsatisfactory response to rIL-1Ra. In vitro, rIL-1Ra antagonized the priming effect of IL-1β on neutrophils from healthy subjects.

Conclusion: These results strongly support the notion that neutrophils play an important role in systemic-onset JIA, especially in the early inflammatory phase of the disease. The findings also demonstrate that neutrophil numbers and the inflammatory activity of systemic-onset JIA are both susceptible to IL-1 blockade.
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http://dx.doi.org/10.1002/art.40442DOI Listing
June 2018

Effect of anticoagulants on 162 circulating immune related proteins in healthy subjects.

Cytokine 2018 06 28;106:114-124. Epub 2017 Oct 28.

Department of Pediatric Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; Multiplex Core Facility, Laboratory of Translational Immunology, University Medical center Utrecht, Utrecht, The Netherlands. Electronic address:

Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89-107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.
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http://dx.doi.org/10.1016/j.cyto.2017.10.021DOI Listing
June 2018

Serum Cytokines as Biomarkers in Islet Cell Transplantation for Type 1 Diabetes.

PLoS One 2016 11;11(1):e0146649. Epub 2016 Jan 11.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands.

Background: Islet cell transplantation holds a potential cure for type 1 diabetes, but many islet recipients do not reach long-lasting insulin independence. In this exploratory study, we investigated whether serum cytokines, chemokines and adipokines are associated with the clinical outcome of islet transplantation.

Methods: Thirteen islet transplant patients were selected on basis of good graft function (reaching insulin independence) or insufficient engraftment (insulin requiring) from our cohort receiving standardized grafts and immune suppressive therapy. Patients reaching insulin independence were divided in those with continued (>12 months) versus transient (<6 months) insulin independence. A panel of 94 proteins including cytokines and adipokines was measured in sera taken before and at one year after transplantation using a validated multiplex immunoassay platform.

Results: Ninety serum proteins were detectable in concentrations varying markedly among patients at either time point. Thirteen markers changed after transplantation, while another seven markers changed in a clinical subpopulation. All other markers remained unaffected after transplantation under generalized immunosuppression. Patterns of cytokines could distinguish good graft function from insufficient function including IFN-α, LIF, SCF and IL-1RII before and after transplantation, by IL-16, CCL3, BDNF and M-CSF only before and by IL-22, IL-33, KIM-1, S100A12 and sCD14 after transplantation. Three other proteins (Leptin, Cathepsin L and S100A12) associated with loss of temporary graft function before or after transplantation.

Conclusions: Distinct cytokine signatures could be identified in serum that predict or associate with clinical outcome. These serum markers may help guiding patient selection and choice of immunotherapy, or act as novel drug targets in islet transplantation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146649PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713434PMC
July 2016

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

Blood 2016 Jan 19;127(1):91-101. Epub 2015 Oct 19.

Laboratory of Translational Immunology, Department of Paediatric Immunology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands;

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.
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http://dx.doi.org/10.1182/blood-2015-06-649145DOI Listing
January 2016

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

Ann Rheum Dis 2015 Feb 10;74(2):470-2. Epub 2014 Nov 10.

Laboratory of Translational Immunology, Department of Pediatric Immunology, University Medical Center Utrecht, Utrecht, The Netherlands.

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http://dx.doi.org/10.1136/annrheumdis-2014-206424DOI Listing
February 2015

Increased presence of FOXP3+ regulatory T cells in inflamed muscle of patients with active juvenile dermatomyositis compared to peripheral blood.

PLoS One 2014 26;9(8):e105353. Epub 2014 Aug 26.

Laboratory of Translational Immunology, Pediatric Immunology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.

Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4+ CD25+ FOXP3+ regulatory T cells (Tregs) are key regulators of immune homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne's Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne's muscular dystrophy. Both in JDM and Duchenne's muscular dystrophy the proportion of FOXP3+ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105353PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144849PMC
May 2015

Local IL-17A potentiates early neutrophil recruitment to the respiratory tract during severe RSV infection.

PLoS One 2013 23;8(10):e78461. Epub 2013 Oct 23.

Department of Pediatric Immunology and Infectious Diseases, University Medical Center Utrecht, Wilhelmina Children's Hospital, Utrecht, the Netherlands.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078461PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3806820PMC
February 2015

Alterations in regulatory T cells induced by specific oligosaccharides improve vaccine responsiveness in mice.

PLoS One 2013 20;8(9):e75148. Epub 2013 Sep 20.

Department of Pediatric Immunology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands ; Department of Immunology, Nutricia Research, Utrecht, The Netherlands.

Unlabelled: Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4(+)CD25(+)Foxp3(+) regulatory T-cells (Tregs) in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet(+) (Th1) activated CD69(+)CD4(+) T cells (p<0.001) and reduced percentage of Gata-3(+) (Th2) activated CD69(+)CD4(+)T cells (p<0.001) was detected in the mesenteric lymph nodes (MLN) of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4(+)Foxp3(+)) could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 (+) /T-bet(+) (Th1-Tregs) was significantly reduced (p<0.05) in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.

In Conclusion: These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0075148PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779252PMC
May 2014

Anti-tumor necrosis factor α targets protein kinase B/c-Akt-induced resistance of effector cells to suppression in juvenile idiopathic arthritis.

Arthritis Rheum 2013 Dec;65(12):3279-84

Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1002/art.38132DOI Listing
December 2013

Stabilization of the transcription factor Foxp3 by the deubiquitinase USP7 increases Treg-cell-suppressive capacity.

Immunity 2013 Aug;39(2):259-71

Department of Immunology, University Medical Center Utrecht, Utrecht 3584EA, The Netherlands.

Stable Foxp3 expression is required for the development of functional regulatory T (Treg) cells. Here, we demonstrate that the expression of the transcription factor Foxp3 can be regulated through the polyubiquitination of multiple lysine residues, resulting in proteasome-mediated degradation. Expression of the deubiquitinase (DUB) USP7 was found to be upregulated and active in Treg cells, being associated with Foxp3 in the nucleus. Ectopic expression of USP7 decreased Foxp3 polyubiquitination and increased Foxp3 expression. Conversely, either treatment with DUB inhibitor or USP7 knockdown decreased endogenous Foxp3 protein expression and decreased Treg-cell-mediated suppression in vitro. Furthermore, in a murine adoptive-transfer-induced colitis model, either inhibition of DUB activity or USP7 knockdown in Treg cells abrogated their ability to resolve inflammation in vivo. Our data reveal a molecular mechanism in which rapid temporal control of Foxp3 expression in Treg cells can be regulated by USP7, thereby modulating Treg cell numbers and function.
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http://dx.doi.org/10.1016/j.immuni.2013.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133784PMC
August 2013

Canonical Wnt signaling negatively modulates regulatory T cell function.

Immunity 2013 Aug 15;39(2):298-310. Epub 2013 Aug 15.

Department of Immunology, University Medical Center Utrecht, Utrecht 3584EA, The Netherlands.

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.
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http://dx.doi.org/10.1016/j.immuni.2013.07.019DOI Listing
August 2013

Der p 1-induced CD4⁺FOXP3⁺GATA3⁺ T cells have suppressive properties and contribute to the polarization of the TH2-associated response.

J Allergy Clin Immunol 2013 Dec 27;132(6):1440-44. Epub 2013 Jul 27.

Center for Molecular and Cellular Intervention, Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children's Hospital, Utrecht, The Netherlands. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2013.06.028DOI Listing
December 2013

Defective TH17 development in human neonatal T cells involves reduced RORC2 mRNA content.

J Allergy Clin Immunol 2013 Sep 29;132(3):754-756.e3. Epub 2013 May 29.

Department of Pediatric Immunology, Center for Molecular and Cellular Intervention CMCI, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.

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http://dx.doi.org/10.1016/j.jaci.2013.04.014DOI Listing
September 2013

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

Methods 2013 May 19;61(1):10-7. Epub 2013 Apr 19.

Department of Pediatric Immunology (KC01.069.0), Centre for Molecular and Cellular Intervention, University Medical Centre Utrecht, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1016/j.ymeth.2013.04.005DOI Listing
May 2013

Cerebral ischemia initiates an immediate innate immune response in neonates during cardiac surgery.

J Neuroinflammation 2013 Feb 7;10:24. Epub 2013 Feb 7.

Department of Pediatric Cardiothoracic Surgery, Wilhelmina Children's Hospital, University Medical Center Utrecht, Lundlaan 6, Utrecht, EA 3584, The Netherlands.

Background: A robust inflammatory response occurs in the hours and days following cerebral ischemia. However, little is known about the immediate innate immune response in the first minutes after an ischemic insult in humans. We utilized the use of circulatory arrest during cardiac surgery to assess this.

Methods: Twelve neonates diagnosed with an aortic arch obstruction underwent cardiac surgery with cardiopulmonary bypass and approximately 30 minutes of deep hypothermic circulatory arrest (DHCA, representing cerebral ischemia). Blood samples were drawn from the vena cava superior immediately after DHCA and at various other time points from preoperatively to 24 hours after surgery. The innate immune response was assessed by neutrophil and monocyte count and phenotype using FACS, and concentrations of cytokines IL-1β, IL-6, IL-8, IL-10, TNFα, sVCAM-1 and MCP-1 were assessed using multiplex immunoassay. Results were compared to a simultaneously drawn sample from the arterial cannula. Twelve other neonates were randomly allocated to undergo the same procedure but with continuous antegrade cerebral perfusion (ACP).

Results: Immediately after cerebral ischemia (DHCA), neutrophil and monocyte counts were higher in venous blood than arterial (P = 0.03 and P = 0.02 respectively). The phenotypes of these cells showed an activated state (both P <0.01). Most striking was the increase in the 'non-classical' monocyte subpopulations (CD16(intermediate); arterial 6.6% vs. venous 14%; CD16+ 13% vs. 22%, both P <0.01). Also, higher IL-6 and lower sVCAM-1 concentrations were found in venous blood (both P = 0.03). In contrast, in the ACP group, all inflammatory parameters remained stable.

Conclusions: In neonates, approximately 30 minutes of cerebral ischemia during deep hypothermia elicits an immediate innate immune response, especially of the monocyte compartment. This phenomenon may hold important clues for the understanding of the inflammatory response to stroke and its potentially detrimental consequences.

Trial Registration: ClinicalTrial.gov: NCT01032876.
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http://dx.doi.org/10.1186/1742-2094-10-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599234PMC
February 2013

Natural killer T cells in adipose tissue prevent insulin resistance.

J Clin Invest 2012 Sep 6;122(9):3343-54. Epub 2012 Aug 6.

Department of Metabolic Diseases, University Medical Center Utrecht, Utrecht, the Netherlands.

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.
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http://dx.doi.org/10.1172/JCI62739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428087PMC
September 2012

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

Ann Rheum Dis 2012 Oct 5;71(10):1706-15. Epub 2012 May 5.

Department of Pediatric Immunology, University Medical Centre Utrecht, Centre for Molecular and Cellular Intervention, Lundlaan 6, 3584 EA Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1136/annrheumdis-2011-201131DOI Listing
October 2012

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

Blood 2011 Sep 9;118(13):3538-48. Epub 2011 Aug 9.

Center for Molecular and Cellular Intervention, Department of Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.1182/blood-2010-12-328187DOI Listing
September 2011

CD30 discriminates heat shock protein 60-induced FOXP3+ CD4+ T cells with a regulatory phenotype.

J Immunol 2010 Aug 14;185(4):2071-9. Epub 2010 Jul 14.

Center for Molecular and Cellular Immunology, University Medical Center Utrecht, The Netherlands.

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.
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http://dx.doi.org/10.4049/jimmunol.0901901DOI Listing
August 2010

A multiplex immunoassay for human adipokine profiling.

Clin Chem 2010 Aug 8;56(8):1320-8. Epub 2010 Jun 8.

Department of Metabolic and Endocrine Diseases, UMC Utrecht, the Netherlands.

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.
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http://dx.doi.org/10.1373/clinchem.2010.146118DOI Listing
August 2010