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Objective. Plasmacytoid dendritic cells (PDCs) are a critical source of type I interferons (IFNs) that can contribute to the onset and maintenance of autoimmunity. Molecular mechanisms leading to PDC dysregulation and a persistent type I IFN signature are largely unexplored, especially in patients with systemic sclerosis (SSc), a disease in which PDCs infiltrate fibrotic skin lesions and produce higher levels of IFN alpha than those in healthy controls. This study was undertaken to investigate potential microRNA (miRNA)-mediated epigenetic mechanisms underlying PDC dysregulation and type I IFN production in SSc. Methods. We performed miRNA expression profiling and validation in highly purified PDCs obtained from the peripheral blood of 3 independent cohorts of healthy controls and SSc patients. Possible functions of miRNA-618 (miR-618) on PDC biology were identified by overexpression in healthy PDCs. Results. Expression of miR-618 was up-regulated in PDCs from SSc patients, including those with early disease who did not present with skin fibrosis. IFN regulatory factor 8, a crucial transcription factor for PDC development and activation, was identified as a target of miR-618. Overexpression of miR-618 reduced the development of PDCs from CD34+ cells in vitro and enhanced their ability to secrete IFN alpha, mimicking the PDC phenotype observed in SSc patients. Conclusion. Up-regulation of miR-618 suppresses the development of PDCs and increases their ability to secrete IFN alpha, potentially contributing to the type I IFN signature observed in SSc patients. Considering the importance of PDCs in the pathogenesis of SSc and other diseases characterized by a type I IFN signature, miR-618 potentially represents an important epigenetic target to regulate immune system homeostasis in these conditions.

Radboud Universiteit Nijmegen, 03 juni 2015, Promotores : Radstake, T.R.D.J., Berg, W.B. van den Co-promotores : Wagener, F.A.D.T.G., Vonk, M.C., Contains fulltext : 141096.pdf (publisher's version ) (Open Access)

Radboud Universiteit Nijmegen, 03 juni 2015, Promotores : Radstake, T.R.D.J., Berg, W.B. van den Co-promotores : Wagener, F.A.D.T.G., Vonk, M.C., Contains fulltext : 141096.pdf (publisher's version ) (Open Access)

Radboud Universiteit Nijmegen, 03 juni 2015, Promotores : Radstake, T.R.D.J., Berg, W.B. van den Co-promotores : Wagener, F.A.D.T.G., Vonk, M.C., Contains fulltext : 141096.pdf (publisher's version ) (Open Access)

Contains fulltext : 96523.pdf (publisher's version ) (Open Access), Population-based studies have investigated the relation between beta-amyloid levels in cerebrospinal fluid or plasma and white matter lesions (WMLs). However, these circulating levels of beta-amyloid in cerebrospinal fluid or plasma may not reliably reflect the actual degree of amyloid present in the brain. Therefore, we investigated the relation between WMLs and beta-amyloid plaques and amyloid angiopathy in brain tissue. WML on MRI or CT were rated in 28 nondemented patients whose neuroimaging was available prior to death. beta-amyloid in plaques and arterioles were immunohistochemically stained and quantified in postmortem brain necropsies. WMLs were present in 43% of the total population. Both cortex and periventricular region showed no differences for beta-amyloid deposition in either plaques or blood vessel walls in patients with WMLs compared to those without WMLs. Thus, our results indicate that there is no relation between the degree of WMLs and beta-amyloid deposition in the brain.

Contains fulltext : 96523.pdf (publisher's version ) (Open Access), Population-based studies have investigated the relation between beta-amyloid levels in cerebrospinal fluid or plasma and white matter lesions (WMLs). However, these circulating levels of beta-amyloid in cerebrospinal fluid or plasma may not reliably reflect the actual degree of amyloid present in the brain. Therefore, we investigated the relation between WMLs and beta-amyloid plaques and amyloid angiopathy in brain tissue. WML on MRI or CT were rated in 28 nondemented patients whose neuroimaging was available prior to death. beta-amyloid in plaques and arterioles were immunohistochemically stained and quantified in postmortem brain necropsies. WMLs were present in 43% of the total population. Both cortex and periventricular region showed no differences for beta-amyloid deposition in either plaques or blood vessel walls in patients with WMLs compared to those without WMLs. Thus, our results indicate that there is no relation between the degree of WMLs and beta-amyloid deposition in the brain.

Contains fulltext : 96523.pdf (publisher's version ) (Open Access), Population-based studies have investigated the relation between beta-amyloid levels in cerebrospinal fluid or plasma and white matter lesions (WMLs). However, these circulating levels of beta-amyloid in cerebrospinal fluid or plasma may not reliably reflect the actual degree of amyloid present in the brain. Therefore, we investigated the relation between WMLs and beta-amyloid plaques and amyloid angiopathy in brain tissue. WML on MRI or CT were rated in 28 nondemented patients whose neuroimaging was available prior to death. beta-amyloid in plaques and arterioles were immunohistochemically stained and quantified in postmortem brain necropsies. WMLs were present in 43% of the total population. Both cortex and periventricular region showed no differences for beta-amyloid deposition in either plaques or blood vessel walls in patients with WMLs compared to those without WMLs. Thus, our results indicate that there is no relation between the degree of WMLs and beta-amyloid deposition in the brain.

1 december 2010, Contains fulltext : 89215.pdf (publisher's version ) (Closed access), OBJECTIVES: > To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs). METHODS: 217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+ CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry. RESULTS: Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r = 0.45, p < 0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p = 0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p < 0.001) associated with less efficient suppressive activity (p=0.012). CONCLUSIONS: Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.

01 december 2010, Contains fulltext : 89215.pdf (publisher's version ) (Closed access), OBJECTIVES: > To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs). METHODS: 217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+ CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry. RESULTS: Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r = 0.45, p < 0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p = 0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p < 0.001) associated with less efficient suppressive activity (p=0.012). CONCLUSIONS: Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.

01 december 2010, Contains fulltext : 89215.pdf (publisher's version ) (Closed access), OBJECTIVES: > To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs). METHODS: 217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+ CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry. RESULTS: Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r = 0.45, p < 0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p = 0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p < 0.001) associated with less efficient suppressive activity (p=0.012). CONCLUSIONS: Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.

Background: Systemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc. Methodology and Principal Findings: Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGF beta and IFN gamma using flow cytometry. Levels of IL-17, IL-6, IL-1a and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFN gamma and TGFb were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1a were increased in all or subsets of SSc patients. Conclusion and Significance: The combination of IL-17, IFN gamma and TGF beta levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.

TLR2 plays an important role in the removal of Gram-positive bacteria; contrastingly, it also appears to have important protective effects against unrestrained inflammation and subsequent organ injury during infection and autoimmunity. We hypothesized that TLR2 tunes the phenotype of dendritic cells (DCs) activated through other TLRs, thereby fulfilling a crucial role in the modulation of the immune response. TLR2 potently inhibited TLR4- and TLR7/8-induced cytokine production by human DCs. The inhibitory effect of TLR2 on the release of TNF-alpha but not of IL-12p70 was mediated by PI3K. TLR2 inhibits the production of IL-12p70 by dampening the type 1 IFN amplification loop. When DCs were triggered with the potent synergistic combination of LPS (TLR4) and R848 (TLR7/8) in conjunction with a TLR2 ligand, a clear shift to more Th2- and Th17-prone responses in the naive and memory T cell subpopulations was observed. This shift in T cell responses was inherent to the inability of TLR2-stimulated DCs to produce IL-12p70 and was dependent on the production of IL-1 and IL-6. The Journal of Immunology, 2009,183: 6960-6970.