Background Epidemiological studies and meta-analyses show an association between statin use and a reduced incidence of colorectal cancer (CRC). We have shown that statins act on CRC through bone... Show moreBackground Epidemiological studies and meta-analyses show an association between statin use and a reduced incidence of colorectal cancer (CRC). We have shown that statins act on CRC through bone morphogenetic protein (BMP) signalling, but the exact cellular targets and underlying mechanism of statin action remain elusive. In this study, we set out to assess the influence of statins on global cancer cell signalling by performing an array-based kinase assay using immobilised kinase substrates spanning the entire human kinome. Methods CRC cells with or without Lovastatin treatment were used for kinome analysis. Findings on kinome arrays were further confirmed by immunoblotting with activity-specific antibodies. Experiments in different CRC cell lines using immunoblotting, siRNA-mediated knockdown and treatment with specific BMP inhibitor Noggin were performed. The relevance of in vitro findings was confirmed in xenografts and in CRC patients treated with Simvastatin. Results Kinome analysis can distinguish between non-specific, toxic effects caused by 10 mu M of Lovastatin and specific effects on cell signalling caused by 2 mu M Lovastatin. Statins induce upregulation of PTEN activity leading to downregulation of the PI3K/Akt/mTOR signalling. Treatment of cells with the specific BMP inhibitor Noggin as well as PTEN knockdown and transfection of cells with a constitutively active form of AKT abolishes the effect of Lovastatin on mTOR phosphorylation. Experiments in xenografts and in patients treated with Simvastatin confirm statin-mediated BMP pathway activation, activation of PTEN and downregulation of mTOR signalling. Conclusions Statins induce BMP-specific activation of PTEN and inhibition of PI3K/Akt/mTOR signalling in CRC. Show less
Koelink, P.J.; Bloemendaal, F.M.; Li, B.F.; Westera, L.; Vogels, E.W.M.; Roest, M. van; ... ; Brink, G.R. van den 2020
Objective Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti... Show moreObjective Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fc gamma-receptor (Fc gamma R) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined.Design We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rb(high) T-cell transfer model in combination with several genetic mouse models.Results Anti-TNF therapy increased macrophage IL-10 production in an Fc gamma R-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rb(high) T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF.Conclusion The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages. Show less
Hansen, I.S.; Krabbendam, L.; Bernink, J.H.; Loayza-Puch, F.; Hoepel, W.; Burgsteden, J.A. van; ... ; Dunnen, J. den 2018
Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohn's disease. MSCs are pluripotent cells with immunosuppressive... Show moreMesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohn's disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon-gamma (IFN-gamma). In this article, we assessed the effects of IFN-gamma prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN-gamma and assessed their therapeutic effects in dextran sodium sulfate (DSS)- and trinitrobenzene sulfonate (TNBS)-induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS-induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS-induced colitis. IMSC-treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN-gamma enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN-gamma activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;29:1549-1558 Show less
BACKGROUND AND AIMS Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in... Show moreBACKGROUND AND AIMS Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I-III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays. METHODS The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-α on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied. RESULTS MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-α antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment. CONCLUSIONS This study demonstrates that, in vitro, MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-α therapy. Show less
Background and aims. Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained... Show moreBackground and aims. Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I-III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays. Methods. The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-alpha on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied. Results. MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-alpha antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment. Conclusions. This study demonstrates that, in vitro, MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-alpha therapy. Show less
Muncan, V.; Heijmans, J.; Krasinski, S.D.; Buller, N.V.; Wildenberg, M.E.; Meisner, S.; ... ; Brink, G.R. van den 2011
In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mother's milk to the adult diet at the end of the suckling period. These complex... Show moreIn many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mother's milk to the adult diet at the end of the suckling period. These complex developmental changes are the result of a genetic programme intrinsic to the gut tube, but its regulators have not been identified. Here we show that transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1) is highly expressed in the developing and postnatal intestinal epithelium until the suckling to weaning transition. Intestine-specific deletion of Blimp1 results in growth retardation and excessive neonatal mortality. Mutant mice lack all of the typical epithelial features of the suckling period and are born with features of an adult-like intestine. We conclude that the suckling to weaning transition is regulated by a single transcriptional repressor that delays epithelial maturation. Show less
Vos, A.C.W.; Wildenberg, M.E.; Duijvestein, M.; Verhaar, A.P.; Brink, G.R. van den; Hommes, D.W. 2011
BACKGROUND & AIMS: Anti-tumor necrosis factor (TNF)alpha antibodies are effective in treating patients with Crohn's disease whereas soluble TNF alpha receptors have not shown clinical efficacy;... Show moreBACKGROUND & AIMS: Anti-tumor necrosis factor (TNF)alpha antibodies are effective in treating patients with Crohn's disease whereas soluble TNF alpha receptors have not shown clinical efficacy; the mechanism that underlies these different effects is not clear. We examined the immunosuppressive effects of different anti-TNF alpha reagents on activated T cells. METHODS: We studied the effects of anti-TNF alpha antibodies infliximab and adalimumab, the soluble TNF alpha receptor etanercept, the pegylated F(ab') fragment certolizumab, and certolizumab-immunoglobulin (Ig)G on primary activated T cells. T cells were grown in isolation or in a mixed lymphocyte reaction (MLR). Proliferation was measured by H-3 thymidine incorporation and apoptosis was examined using Annexin V labeling and a colorimetric assay for activated caspase-3. Macrophage phenotypes were assayed by flow cytometry and cytokine secretion. RESULTS: Infliximab and adalimumab reduced T-cell proliferation in an MLR whereas etanercept and certolizumab did not; this effect was lost after Fc receptors were blocked. The infliximab F(ab') 2 fragment did not inhibit proliferation whereas certolizumab-IgG did inhibit proliferation. In the MLR, the antibodies against TNF induced formation of a new population of macrophages in an Fc region-dependent manner; these macrophages had an immunosuppressive phenotype because they inhibit proliferation of activated T cells, produce anti-inflammatory cytokines, and express the regulatory macrophage marker CD206. CONCLUSIONS: Regulatory macrophages have immunosuppressive properties and an important role in wound healing. Antibodies against TNF induce regulatory macrophages in an Fc region-dependent manner. These functions of anti-TNFs might contribute to the resolution of inflammation. Show less
Background and aim Mesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive effects both in vitro and in experimental colitis. Promising results of MSC therapy have been... Show moreBackground and aim Mesenchymal stromal cells (MSCs) are pluripotent cells that have immunosuppressive effects both in vitro and in experimental colitis. Promising results of MSC therapy have been obtained in patients with severe graft versus host disease of the gut. Our objective was to determine the safety and feasibility of autologous bone marrow derived MSC therapy in patients with refractory Crohn's disease. Patients and intervention 10 adult patients with refractory Crohn's disease (eight females and two males) underwent bone marrow aspiration under local anaesthesia. Bone marrow MSCs were isolated and expanded ex vivo. MSCs were tested for phenotype and functionality in vitro. 9 patients received two doses of 1-2x10(6) cells/kg body weight, intravenously, 7 days apart. During follow-up, possible side effects and changes in patients' Crohn's disease activity index (CDAI) scores were monitored. Colonoscopies were performed at weeks 0 and 6, and mucosal inflammation was assessed by using the Crohn's disease endoscopic index of severity. Results MSCs isolated from patients with Crohn's disease showed similar morphology, phenotype and growth potential compared to MSCs from healthy donors. Importantly, immunomodulatory capacity was intact, as Crohn's disease MSCs significantly reduced peripheral blood mononuclear cell proliferation in vitro. MSC infusion was without side effects, besides a mild allergic reaction probably due to the cryopreservant DMSO in one patient. Baseline median CDAI was 326 (224-378). Three patients showed clinical response (CDAI decrease >= 70 from baseline) 6 weeks post-treatment; conversely three patients required surgery due to disease worsening. Conclusions Administration of autologous bone marrow derived MSCs appears safe and feasible in the treatment of refractory Crohn's disease. No serious adverse events were detected during bone marrow harvesting and administration. Show less
