Manon E. Wildenberg

Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn's disease: results of a phase I study

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 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 Crohns disease. Patients and intervention 10 adult patients with refractory Crohns 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 1–2×106u2005cells/kg body weight, intravenously, 7u2005days apart. During follow-up, possible side effects and changes in patients Crohns disease activity index (CDAI) scores were monitored. Colonoscopies were performed at weeks 0 and 6, and mucosal inflammation was assessed by using the Crohns disease endoscopic index of severity. Results MSCs isolated from patients with Crohns disease showed similar morphology, phenotype and growth potential compared to MSCs from healthy donors. Importantly, immunomodulatory capacity was intact, as Crohns 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) 6u2005weeks 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 Crohns disease. No serious adverse events were detected during bone marrow harvesting and administration.

Pretreatment with interferon-γ enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohns 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‐γ (IFN‐γ). In this article, we assessed the effects of IFN‐γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN‐γ 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‐γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN‐γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;29:1549–1558

Loss of Infliximab Into Feces Is Associated With Lack of Response to Therapy in Patients With Severe Ulcerative Colitis

BACKGROUND & AIMSnIt is not clear why some patients with ulcerative colitis (UC) do not respond to treatment with anti-tumor necrosis factor (TNF) agents, such as infliximab. It could be that some patients have high level of inflammation, with large quantities of TNF to be neutralized by the drug. We investigated whether loss of anti-TNF agents through ulcerated intestinal mucosa reduces the efficacy of these drugs in patients with severe UC.nnnMETHODSnWe collected fecal samples from 30 consecutive patients with moderate to severely active UC during the first 2 weeks of infliximab therapy at the University of Amsterdam hospital. Infliximab concentrations were measured in serum and supernatants of fecal samples using an enzyme-linked immunosorbent assay (Sanquin Biologicals Laboratory, Amsterdam, The Netherlands). Clinical and endoscopic responses were assessed 2 and 8 weeks and 3 months after treatment began.nnnRESULTSnInfliximab was detected in 129 of 195 fecal samples (66%); the highest concentrations were measured in the first days after the first infusion. Patients that were clinical nonresponders at week 2 had significantly higher fecal concentrations of infliximab after the first day of treatment than patients with clinical responses (median concentration, 5.01 μg/mL in nonresponders vs 0.54 μg/mL in responders; Pxa0= .0047). We did not observe a correlation between fecal and serum concentrations of infliximab.nnnCONCLUSIONSnInfliximab is lost into stools of patients with UC. High fecal concentrations of infliximab in the first days after therapy begins are associated with primary nonresponse. Additional studies are needed to determine how therapeutic antibodies are lost through the intestinal mucosa and how this process affects treatment response. Clinical trial ID: NL41310.018.12.

Anti–Tumor Necrosis Factor-α Antibodies Induce Regulatory Macrophages in an Fc Region-Dependent Manner

BACKGROUND & AIMSnAnti-tumor necrosis factor (TNF)α antibodies are effective in treating patients with Crohns disease whereas soluble TNFα receptors have not shown clinical efficacy; the mechanism that underlies these different effects is not clear. We examined the immunosuppressive effects of different anti-TNFα reagents on activated T cells.nnnMETHODSnWe studied the effects of anti-TNFα antibodies infliximab and adalimumab, the soluble TNFα 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 (3)H 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.nnnRESULTSnInfliximab 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.nnnCONCLUSIONSnRegulatory 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.

Regulatory macrophages induced by infliximab are involved in healing in vivo and in vitro.

Background: Regulatory macrophages play an important role in wound healing and gut homeostasis and have antiinflammatory properties. Induction of this cell type (M&psgr;ind) by the anti‐tumor necrosis factor (TNF) antibodies, infliximab and adalimumab, has recently been shown in vitro. Also, the superiority of infliximab/azathioprine combination therapy over infliximab or azathioprine monotherapy has recently been established, but the mechanism behind this remains unclear. The aim of this study was to examine the induction of regulatory macrophages in patients with and without mucosal healing in response to infliximab. In addition, we studied the effect of infliximab/azathioprine combination treatment on the differentiation and function of regulatory macrophages. Methods: Inflammatory bowel disease (IBD) patients (n = 10) underwent endoscopy before and after first infliximab treatment. Immunohistochemical staining of CD68 and CD206 was performed in all patients. Mixed lymphocyte reactions (MLRs) were treated with infliximab, azathioprine, or both. Macrophage phenotype was evaluated by flow cytometry and inhibition of T‐cell proliferation was measured in a secondary MLR containing macrophages and third‐party lymphocytes. Results: A significant induction of regulatory macrophages was observed in patients with mucosal healing after treatment with infliximab; this induction was absent in patients without mucosal healing. In addition, M&psgr;ind have the ability to induce wound healing in an in vitro model, further suggesting a key role for infliximab‐induced macrophages in mucosal healing. Upon infliximab/azathioprine combination treatment, an increased number of regulatory macrophages was observed. These macrophages also displayed stronger immunosuppressive properties than macrophages induced by infliximab monotherapy. Conclusions: These data show that regulatory macrophages may be involved in mucosal healing and provide a rationale for the superiority of infliximab/azathioprine combination treatment observed in the clinic. (Inflamm Bowel Dis 2012;)

Antibody‐mediated activation of the classical pathway of complement may compensate for mannose‐binding lectin deficiency

Deficiency of mannose‐binding lectin (MBL), a recognition molecule of the lectin pathway of complement, is associated with increased susceptibility to infections. The high frequency of MBL deficiency suggests that defective MBL‐mediated innate immunity can be compensated by alternative defense strategies. To examine this hypothesis, complement activation by MBL‐binding ligands was studied. The results show that the prototypic MBL ligand mannan can induce complement activation via both the lectin pathway and the classical pathway. Furthermore, antibody binding to mannan restored complement activation in MBL‐deficient serum in a C1q‐dependent manner. Cooperation between the classical pathway and the lectin pathway was also observed for complement activation by proteinu200460 from Listeria monocytogenes. MBL pathway analysis at the levels of C4 and C5b–9 in the presence of classical pathway inhibition revealed a large variation of MBL pathway activity, depending on mbl2 gene polymorphisms. MBL pathway dysfunction in variant allele carriers is associated with reduced MBL ligand binding and a relative increase of low‐molecular‐mass MBL. These findings indicate that antibody‐mediated classical pathway activation can compensate for impaired target opsonization via the MBL pathway in MBL‐deficient individuals, and imply that MBL deficiency may become clinically relevant in absence of a concomitant adaptive immune response.

Blimp1 regulates the transition of neonatal to adult intestinal epithelium.

In many mammalian species, the intestinal epithelium undergoes major changes that allow a dietary transition from mothers 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.

Statins augment the chemosensitivity of colorectal cancer cells inducing epigenetic reprogramming and reducing colorectal cancer cell ‘stemness’ via the bone morphogenetic protein pathway

Background Promoter hypermethylation is an important and potentially reversible mechanism of tumour suppressor gene silencing in cancer. Compounds that demethylate tumour suppressor genes and induce differentiation of cancer cells, but do not have toxic side effects, would represent an exciting option in cancer therapy. Statins are cholesterol-lowering drugs with an excellent safety profile and associated with a reduced incidence of various cancers including colorectal cancer (CRC). The authors have previously shown that statins act by activating tumour suppressive bone morphogenetic protein (BMP) signalling in CRC, increasing expression of BMP2. BMP2 is silenced by hypermethylation in gastric cancer. Aim To investigate whether BMP2 is methylated in CRC, whether statins can reverse this, and what implications this has for the use of statins in CRC. Methods Methylation-specific PCR, bisulphite sequencing, immunoblotting, reverse transcription PCR, quantitative PCR, fluorescence-activated cell sorting analysis, an in vitro DNA methyltransferase (DNMT) assay, and cell viability studies were performed on CRC cells. The effect of statins was confirmed in a xenograft mouse model. Results BMP2 is silenced by promoter hypermethylation in cell lines with the hypermethylator phenotype and in primary tumours. Treatment with lovastatin downregulates DNMT activity, leading to BMP2 promoter demethylation and to upregulation of expression of BMP2 as well as other genes methylated in CRC. Statins alter gene expression, indicating a shift from a stem-like state to a more differentiated state, thereby sensitising cells to the effects of 5-fluorouracil. In a xenograft mouse model, simvastatin treatment induces BMP2 expression, leading to differentiation and reduced proliferation of CRC cells. Conclusions Statins act as DNMT inhibitors, demethylating the BMP2 promoter, activating BMP signalling, inducing differentiation of CRC cells, and reducing ‘stemness’. This study indicates that statins may be able to be used as differentiating agents in combined or adjuvant therapy in CRC with the CpG island methylator phenotype.

Autophagy Attenuates the Adaptive Immune Response by Destabilizing the Immunologic Synapse

BACKGROUND & AIMSnVariants in the genes ATG16L1 and IRGM affect autophagy and are associated with the development of Crohns disease. It is not clear how autophagy is linked to loss of immune tolerance in the intestine. We investigated the involvement of the immunologic synapse-the site of contact between dendritic cells (DCs) and T cells, which contains molecules involved in antigen recognition and regulates immune response.nnnMETHODSnDC autophagy was reduced using small interfering RNAs or pharmacologic inhibitors. DC phenotype and function were analyzed by confocal microscopy, time-lapse microscopy, and flow cytometry. We also examined DCs isolated from patients with Crohns disease who carried the ATG16L1 risk allele.nnnRESULTSnImmunologic synapse formation induced formation of autophagosomes in DCs; the autophagosomes were oriented toward the immunologic synapse and contained synaptic components. Knockdown of ATG16L1 and IRGM with small interfering RNAs in DCs resulted in hyperstable interactions between DCs and T cells, increased activation of T cells, and activation of a T-helper 17 cell response. LKB1 was recruited to the immunologic synapse, and induction of autophagy in DC required inhibition of mammalian target of rapamycine signaling by the LKB1-AMP activated protein kinase (AMPK) pathway. DCs from patients with Crohns disease who had an ATG16L1 risk allele had a similar hyperstability of the immunologic synapse.nnnCONCLUSIONSnAutophagy is induced upon formation of the immunologic synapse and negatively regulates T-cell activation. This mechanism might increase adaptive immunity in patients with Crohns disease who carry ATG16L1 risk alleles.

Mechanism of Action of Anti-TNF Therapy in Inflammatory Bowel Disease.

Several anti-tumour necrosis factor [TNF] blocking strategies have been evaluated in patients with Crohns disease. Compounds that have been tested included the full monoclonal IgG1 antibodies infliximab and adalimumab, the pegylated anti-TNF F[ab]2 fragment certolizumab, an IgG4 anti-TNF CDP571 with reduced affinity for the Fc receptor, the soluble TNF receptor I onercept, and the TNF receptor II-Fc fusion protein etanercept. The endpoints of these studies suggest that not all methods of blocking TNF are equal. Here we will review the differences in the clinical, biochemical, and endoscopic endpoints of the major clinical studies. Collectively the data suggest that only IgG1 monoclonal antibodies have the ability to induce complete clinical, biochemical, and endoscopic remission. We discuss the potential multiple modes of action that may contribute to the response to full IgG1 anti-TNFs, focusing on the rapid induction of lamina propria T cell apoptosis and Fc receptor-dependent induction of M2-type wound-healing macrophages. We discuss how novel insights into the mechanism of action of anti-TNFs in Crohns disease may contribute to the development of novel anti-TNFs with improved efficacy.