Rianne C. Scholman

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, …

Increased Presence of FOXP3+ Regulatory T Cells in Inflamed Muscle of Patients with Active Juvenile Dermatomyositis Compared to Peripheral Blood

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.

A novel FcγRIIa Q27W gene variant is associated with common variable immune deficiency through defective FcγRIIa downstream signaling

We identified a novel Q27W FcγRIIa variant that was found more frequently in common variable immunodeficiency (CVID) or CVID-like children. We analyzed the possible functional consequence of the Q27W FcγRIIa mutation in human cells. We used peripheral blood mononuclear cells from Q27W FcγRIIa patients and healthy controls, and cultured cells that overexpress the Q27W and common FcγRIIa variants. The Q27W FcγRIIa mutation does not disrupt FcγRIIa surface expression in peripheral blood mononuclear cells. Mononuclear cells express multiple FcγR, precluding careful analysis of Q27W FcγRIIa functional deviation. For functional analysis of FcγRIIa function, we therefore overexpressed the Q27W FcγRIIa and common FcγRIIa variant in IIA1.6 cells that are normally deficient in FcγR. We show that FcγRIIa triggering-induced signaling is obstructed, as measured by both decrease in calcium flux and defective MAPK phosphorylation. In conclusion, we here describe a novel Q27W FcγRIIa variant that causes delayed downstream signaling. This variant may contribute to CVID.

Self‐Sustained Resistance to Suppression of CD8+ Teff Cells at the Site of Autoimmune Inflammation Can Be Reversed by Tumor Necrosis Factor and Interferon‐γ Blockade

Resistance of Teff cells to Treg cell–mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). However, unanswered questions are whether this resistant phenotype is self‐sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically.

T cell recognition of naturally presented epitopes of self-heat shock protein 70

Self-reactive T cells have shown to have a potential role as regulators of the immune system preventing or even suppressing autoimmunity. One of the most abundant proteins that can be eluted from human HLA molecules is heat shock protein 70 (HSP70). The aims of the current study are to identify HSP70 epitopes based on published HLA elution studies and to investigate whether T cells from healthy individuals may respond to such self-epitopes. A literature search and subsequent in silico binding prediction based on theoretical MHC binding motifs resulted in the identification of seven HSP70 epitopes. PBMCs of healthy controls proliferated after incubation with two of the seven peptides (H167 and H290). Furthermore H161, H290, and H443 induced CD69 expression or production of cytokines IFNγ or TNFα in healthy controls. The identification of these naturally presented epitopes and the response they elicit in the normal immune system make them potential candidates to study during inflammatory conditions as well as in autoimmune diseases.

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

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.

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children

Background Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children. Methods 13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2) and leptin receptor expression on peripheral blood mononuclear cell subsets was performed. Results Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI). The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also higher numbers of immature transition B-cells and intermediate CD14++CD16+ monocytes combined with lower total monocyte numbers, compared to controls. Furthermore, adiponectin receptor 1 expression on nonclassical CD14+CD16++ monocytes was consistently upregulated in obese children pre-intervention, compared to controls. However, none of the differences in leukocyte subset numbers and adipokine receptor expression profiles between obese children and controls remained significant after multiple testing correction. Conclusions First, the distinct adipokine receptor profiles of circulating leukocyte subsets may partly explain the differential impact of adipokines on leukocyte subsets. Second, the similarities in adipokine receptor expression profiles between obese children and normal weight controls suggest that adipokine signaling in childhood obesity is primarily modulated by circulating adipokine levels, instead of adipokine receptor expression.

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

HighlightsIn house validation shows inter‐assay variation <10%, and average inter‐assay variation of 12.2%Immune profiles remain stable after multiple freeze–thaw cycles.Only 19 out of 162 soluble proteins have similar expression in serum, EDTA plasma and sodium heparin plasma. &NA; 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.

Self-Sustained Resistance to Suppression of CD8+ Teff Cells at the Site of Autoimmune Inflammation Can Be Reversed by Tumor Necrosis Factor and Interferon-γ Blockade: REVERSAL OF SELF-SUSTAINED CD8+ Teff CELL RESISTANCE TO SUPPRESSION

Resistance of Teff cells to Treg cell–mediated suppression contributes to the breakdown of peripheral tolerance in the inflamed joints of patients with juvenile idiopathic arthritis (JIA). However, unanswered questions are whether this resistant phenotype is self‐sustained and whether CD8+ and CD4+ Teff cells share the same mechanism of resistance to suppression. We undertook this study to investigate intrinsic resistance of CD8+ Teff cells to suppression and to determine how this can be targeted therapeutically.

Monocytes and neutrophils in the inflammatory cascade of systemic onset Juvenile Idiopathic Arthritis

8th International Congress of Familial Mediterranean Fever and Systemic Autoinflammatory Diseases