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Macrophage activation syndrome (MAS) is a life-threatening complication of inflammatory disorders like systemic juvenile idiopathic arthritis (sJIA). MAS is characterized by systemic inflammation which includes liver pathology and is thought to be caused by excessive activation of T cells and macrophages leading to a cytokine storm. The role of natural killer (NK) cells in MAS is unclear. In this thesis, we characterized conventional NK (cNK) cells and liver tissue-resident (trNK) NK cells in a MAS mouse model that rely on injection of CpG oligonucleotide, a toll like receptor 9 agonist. We confirmed development of MAS by CpG injection and found that the disease was associated with a decrease of cNK cells in liver and spleen. In contrast, trNK cells were expanded in liver and became significantly present in spleen and other organs. trNK cells produced high levels of IFN-, TNF  and IL-10 and exhibited potent cytotoxic capacity. In line with the potential pathogenic role of IFN  in MAS, trNK cells were not expanded in IFN  deficient mice and animals did not exhibit liver pathology. In contrast, dramatically expanded trNK cells and severe disease pathology were observed in transgenic mice generated to produce more active IL-18, a well described IFN  inducing factor in NK cells and a cytokine that is abundantly present in plasma of sJIA and MAS patients. In conclusion, characterization of NK cells in a MAS model identified trNK cells as an important source of the cytokine storm and as a subset of cytotoxic cells that may drive liver pathology.

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Systemic juvenile idiopathic arthritis (sJIA) is a rare but severe childhood immune disorder and is a subtype of JIA, a heterogeneous group of arthritic diseases. In contrast to other JIA subtypes, sJIA is characterised by systemic features, such as fever, rash and enlargement of lymph nodes, spleen or liver, which are often more prominent than joint inflammation. The aetiology of sJIA remains largely unknown. One hypothesis is that sJIA results from the inappropriate control of the immune response to an initially harmless trigger in predisposed children. In line with this, polymorphisms in the anti-inflammatory cytokine IL-10, resulting in a lower IL-10 production, are associated with sJIA disease development.In the first part of this thesis, we investigated whether a defective IL-10 production underlies the pathogenesis of sJIA. To this end, we took advantage of a recently developed mouse model for sJIA and used plasma and blood cells from sJIA patient. In the mouse model, injection of IFN-γ deficient (KO) mice with Complete Freund's Adjuvant (CFA) results in the development of sJIA-like symptoms, while CFA-injected wild type (WT) mice only develop a subtle inflammatory reaction and serve as a control. In the diseased CFA-injected IFN-γ KO mice, we observed a cell-specific defect in IL-10 production in B cells, regulatory T cells and NK cells, with B cells being the major source of IL-10. Furthermore, neutralisation of IL-10 signalling in CFA-injected WT mice resulted in an immune-inflammatory disorder that is clinically and haematologically reminiscent of sJIA, indicating that a defective IL-10 production contributes to the development of sJIA symptoms. In line with the observations in the mouse model, we observed low levels of IL-10 in the plasma of sJIA patients and demonstrated a decreased IL-10 production by B cells, both ex vivo and after in vitro stimulation. Together, our data show that a decreased IL-10 production may underlie sJIA development.The defective IL-10 production in sJIA mice which genetically lack IFN-γ came as a surprise considering the traditional paradigm of IL-10 and IFN‑γ as each other's antagonists. Since the sJIA mouse model is induced with CFA, an adjuvant that contains several Toll-like receptor (TLR) ligands, the influence of IFN-γ on TLR-induced IL-10 production was explored in the second part of this thesis. The study was performed by using different cell types obtained from naïve mice and from human healthy donors. In agreement with the traditional viewpoint of IL-10 and IFN-γ as antagonists, we confirmed that IFN-γ inhibits TLR-induced IL‑10 production by monocytes and macrophages. In contrast, we observed an opposite scenario when B cells were stimulated with CpG, a TLR9 ligand. Thus, IFN-γ stimulated TLR9-induced IL-10 production in B cells, and this observation is in line with the defective IL-10 production in the CFA-injected IFN-γ KO mice. Further research into the mechanisms involved revealed that the increase of TLR9-induced IL-10 by IFN-γ was restricted to B cells. We excluded involvement of B cell proliferation, increased TLR9 expression, type I IFN production, IL-6 production, IL-10 mRNA stabilisation and B cell activating factor but demonstrated that the mitogen-activated protein kinases (MAPK) p38 and JNK are essential players in the stimulatory effect of IFN-γ on CpG-induced IL-10. Furthermore, we showed that the MAPK phosphatase MKP1, which is an inhibitor of p38 and JNK, is downregulated by IFN-γ, potentially contributing to the increased IL-10 production. Together, these data may represent a novel anti-inflammatory property of the traditionally considered pro-inflammatory IFN-γ, by stimulating TLR-induced IL-10 production in B cells.

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Systemische juveniele idiopathische artritis (sJIA) is een unieke vorm van artritis bij kinderen en wordt geclassificeerd als een subtype van juveniele idiopathische artritis (JIA). Naast artritis wordt de ziekte gekenmerkt door koorts, huiduitslag en hepatosplenomegalie. Het aangeboren immuunsysteem speelt een grote rol in de pathogenese van sJIA. De afwezigheid van auto-antilichamen leidt samen met het systemische karakter tot classificatie onder de term ‘auto-inflammatoire aandoeningen’. Desondanks werd de exacte rol van neutrofielen in sJIA nog niet eerder onderzocht. In deze thesis werd bijgevolg gezocht naar de rol van neutrofielen in de pathogenese van sJIA. Hiervoor werd gebruik gemaakt van een sJIA-muismodel, eerder gekarakteriseerd in het labo van Immunobiologie (Rega-instituut, Leuven). Neutrofielen werden lang aanzien als eenvoudige, kortlevende, fagocyterende granulocyten, maar recente bevindingen bewijzen het tegendeel. Zo werden er hoge aantallen mature en immature neutrofielen teruggevonden in de milt van Complete Freund’s adjuvant (CFA)-geïmmuniseerde muizen. Deze immature neutrofielen worden in muizen visueel waargenomen als donutcellen. Hiernaast werden er in de milt en longen zogenaamde ‘neutrofiel-dendritische cel hybriden’ aangetroffen. Bovendien lijken neutrofielen een anti-inflammatoire rol te hebben in de initiële fase van de ziekte, maar exacte mechanismen blijven echter ongekend. Functionele karakterisatie toont aan dat neutrofielen onder inflammatoire condities in staat zijn om meer fagocytose uit te voeren. Dit in tegenstelling tot de gedaalde ROS-productie in het sJIA-muismodel. Deze resultaten zijn een eerste stap in het achterhalen van de biologische rol van neutrofielen in sJIA. Nieuwe inzichten kunnen leiden tot een bredere kennis over de ziekte en tot nieuwe aangrijpingspunten voor farmacologische therapieën.

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