Jonathan Cremer

Neutralization of membrane TNF, but not soluble TNF, is crucial for the treatment of experimental colitis.

Background:Agents neutralizing membrane tumor necrosis factor (mTNF) and soluble TNF (sTNF) are widely used for the treatment of inflammatory bowel disease (IBD). Neutralization of mTNF, however, is associated with increased susceptibility to infectious diseases. The aim of this study was to determine whether neutralization of sTNF exclusively, by the use of a dominant negative mutant of TNF (XENP1595), could reduce the severity of colitis in mice. Methods:Colitis was induced in immunodeficient mice by transfer of CD45RBhi CD25− T-cells. Once the disease had developed, mice were treated twice a week with XENP1595, phosphate-buffered saline (PBS), anti-TNF monoclonal antibody (mAb), or isotype control. The anti-TNF mAb blocks both mTNF and sTNF. Weights, disease activity index, macroscopic inflammation of the colon, and histological sections were evaluated. T-cell populations from the colon were analyzed by flow cytometry. Results:Treatment of mice with XENP1595 did not change the course of the disease, whereas mice treated with anti-TNF mAb recovered weight soon after the first treatment dose. Inflammation in the colon was reduced in mice treated with anti-TNF mAb compared to isotype control-treated animals. Mice treated with XENP1595 had a similar degree of inflammation in the colon as PBS-treated animals. The number of effector and regulatory T-cells in the colon remained unaffected by all treatments. Conclusions:Neutralization of sTNF exclusively was unable to induce remission in T-cell-mediated colitis, suggesting that neutralization of mTNF is crucial for the treatment of IBD.

Unique gene expression and MR T2 relaxometry patterns define chronic murine dextran sodium sulphate colitis as a model for connective tissue changes in human Crohn's disease.

Introduction Chronically relapsing inflammation, tissue remodeling and fibrosis are hallmarks of inflammatory bowel diseases. The aim of this study was to investigate changes in connective tissue in a chronic murine model resulting from repeated cycles of dextran sodium sulphate (DSS) ingestion, to mimic the relapsing nature of the human disease. Materials and Methods C57BL/6 mice were exposed to DSS in drinking water for 1 week, followed by a recovery phase of 2 weeks. This cycle of exposure was repeated for up to 3 times (9 weeks in total). Colonic inflammation, fibrosis, extracellular matrix proteins and colonic gene expression were studied. In vivo MRI T 2 relaxometry was studied as a potential non-invasive imaging tool to evaluate bowel wall inflammation and fibrosis. Results Repeated cycles of DSS resulted in a relapsing and remitting disease course, which induced a chronic segmental, transmural colitis after 2 and 3 cycles of DSS with clear induction of fibrosis and remodeling of the muscular layer. Tenascin expression mirrored its expression in Crohn’s colitis. Microarray data identified a gene expression profile different in chronic colitis from that in acute colitis. Additional recovery was associated with upregulation of unique genes, in particular keratins, pointing to activation of molecular pathways for healing and repair. In vivo MRI T2 relaxometry of the colon showed a clear shift towards higher T2 values in the acute stage and a gradual regression of T2 values with increasing cycles of DSS. Conclusions Repeated cycles of DSS exposure induce fibrosis and connective tissue changes with typical features, as occurring in Crohn’s disease. Colonic gene expression analysis revealed unique expression profiles in chronic colitis compared to acute colitis and after additional recovery, pointing to potential new targets to intervene with the induction of fibrosis. In vivo T2 relaxometry is a promising non-invasive assessment of inflammation and fibrosis.

CD28/CTLA-4/B7 costimulatory pathway blockade affects regulatory T-cell function in autoimmunity.

Naïve T cells require B7/CD28 costimulation in order to be fully activated. Attempts to block this pathway have been effective in preventing unwanted immune reactions. As B7 blockade might also affect Treg cells and interfere with negative signaling through membrane CTLA‐4 on effector T (Teff) cells, its immune‐modulatory effects are potentially more complex. Here, we used the mouse model of multiple sclerosis (MS), EAE, to study the effect of B7 blockade. An effective therapy for MS patients has to interfere with ongoing inflammation, and therefore we injected CTLA‐4Ig at day 7 and 9 after immunization, when myelin‐reactive T cells have been primed and start migrating toward the CNS. Surprisingly, B7 blockade exacerbated disease signs and resulted in more severe CNS inflammation and demyelination, and was associated with an enhanced production of the inflammatory cytokines IL‐17 and IFN‐γ. Importantly, CTLA‐4Ig treatment resulted in a transient reduction of Ki67 and CTLA‐4 expression and function of peripheral Treg cells. Taken together, B7 blockade at a particular stage of the autoimmune response can result in the suppression of Treg cells, leading to a more severe disease.

Impaired neurogenesis, learning and memory and low seizure threshold associated with loss of neural precursor cell survivin

BackgroundSurvivin is a unique member of the inhibitor of apoptosis protein (IAP) family in that it exhibits antiapoptotic properties and also promotes the cell cycle and mediates mitosis as a chromosome passenger protein. Survivin is highly expressed in neural precursor cells in the brain, yet its function there has not been elucidated.ResultsTo examine the role of neural precursor cell survivin, we first showed that survivin is normally expressed in periventricular neurogenic regions in the embryo, becoming restricted postnatally to proliferating and migrating NPCs in the key neurogenic sites, the subventricular zone (SVZ) and the subgranular zone (SGZ). We then used a conditional gene inactivation strategy to delete the survivin gene prenatally in those neurogenic regions. Lack of embryonic NPC survivin results in viable, fertile mice (SurvivinCamcre) with reduced numbers of SVZ NPCs, absent rostral migratory stream, and olfactory bulb hypoplasia. The phenotype can be partially rescued, as intracerebroventricular gene delivery of survivin during embryonic development increases olfactory bulb neurogenesis, detected postnatally. SurvivinCamcrebrains have fewer cortical inhibitory interneurons, contributing to enhanced sensitivity to seizures, and profound deficits in memory and learning.ConclusionsThe findings highlight the critical role that survivin plays during neural development, deficiencies of which dramatically impact on postnatal neural function.

Characterization of PD-1 upregulation on tumor-infiltrating lymphocytes in human and murine gliomas and preclinical therapeutic blockade

Blockade of the immune checkpoint molecule programmed‐cell‐death‐protein‐1 (PD‐1) yielded promising results in several cancers. To understand the therapeutic potential in human gliomas, quantitative data describing the expression of PD‐1 are essential. Moreover, due the immune‐specialized region of the brain in which gliomas arise, differences between tumor‐infiltrating and circulating lymphocytes should be acknowledged. In this study we have used flow cytometry to quantify PD‐1 expression on tumor‐infiltrating T cells of 25 freshly resected glioma cell suspensions (10 newly and 5 relapsed glioblastoma, 10 lower grade gliomas) and simultaneously isolated circulating T cells. A strong upregulation of PD‐1 expression in the tumor microenvironment compared to the blood circulation was seen in all glioma patients. Additionally, circulating T cells were isolated from 15 age‐matched healthy volunteers, but no differences in PD‐1 expression were found compared to glioma patients. In the murine GL261 malignant glioma model, there was a similar upregulation of PD‐1 on brain‐infiltrating lymphocytes. Using a monoclonal PD‐1 blocking antibody, we found a marked prolonged survival with 55% of mice reaching long‐term survival. Analysis of brain‐infiltrating cells 21 days after GL261 tumor implantation showed a shift in infiltrating lymphocyte subgroups with increased CD8+ T cells and decreased regulatory T cells. Together, our results suggest an important role of PD‐1 in glioma‐induced immune escape, and provide translational evidence for the use of PD‐1 blocking antibodies in human malignant gliomas.

Centrally Determined Standardization of Flow Cytometry Methods Reduces Interlaboratory Variation in a Prospective Multicenter Study

Objectives:Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies.Methods:Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor.Results:Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4–102.1%; LGCS, 10.9–65.6%; CG, 1.8–20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG.Conclusions:Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays.

IL-13 is a central mediator of chemical-induced airway hyperreactivity in mice

Background While the importance of the Th2 cytokine IL-13 as a central mediator of airway hyperreactivity (AHR) has been described in allergic protein-induced asthma, this has never been investigated in chemical-induced asthma. Objective We examined the importance of IL-13 in a mouse model of chemical-induced AHR, using toluene-2,4-diisocyanate (TDI). Methods In a first set-up, wild type (WT) and IL-13 knockout (KO) C57Bl/6 mice were dermally treated on days 1 and 8 with 1% TDI or vehicle (acetone/olive oil) on both ears. On day 15, mice received an intranasal instillation with 0.1% TDI or vehicle. In a second set-up, WT mice sensitized with 1% TDI or vehicle, received i.v. either anti-IL-13 or control antibody prior to the intranasal challenge. Results TDI-sensitized and TDI-challenged WT mice showed AHR to methacholine, in contrast to TDI-sensitized and TDI-challenged IL-13 KO mice, which also showed lower levels of total serum IgE. TDI-sensitized and TDI-challenged IL-13 KO mice had lower numbers of T-cells in the auricular lymph nodes. TDI-treated WT mice, receiving anti-IL-13, showed no AHR, in contrast to those receiving control antibody, despite increased levels of IgE. Anti-IL-13 treatment in TDI-treated WT mice resulted in lower levels of serum IL-13, but did not induce changes in T- and B-cell numbers, and in the cytokine production profile. Conclusion and clinical relevance We conclude that IL-13 plays a critical role in the effector phase of chemical-induced, immune-mediated AHR. This implicates that anti-IL-13 treatment could have a beneficial effect in patients with this asthma phenotype.

P052 Inhibition of Th17 cells but not of Tregs affects extracellular matrix protein deposition in chronic DSS colitis

Background: Chronic intestinal inflammation that occurs in Inflammatory Bowel Disease (IBD) predisposes to colitisassociated colorectal cancer (CAC). Tumor-induced lymphangiogenesis is one of the mechanisms that promotes metastasis to regional lymph nodes, but no mechanistic data is available on the molecules involved in modulating lymphangiogenesis and tumor development in CAC. VEGF-C is a key molecule in lymphangiogenesis that signals through its cognat receptor VEGFR-3 and its blockade has been successful in several models of cancer and metastasis. We hypothesized that lymphatic vessel-directed therapy may be therapeutically effective in CAC. Methods: We addressed these issues in vivo by systemic inhibition of the VEGFR-3 or delivery of the lymphangiogenic factor VEGF-C in the AOM/DSS model of CAC by a blocking antibody or adenovirus transfer, respectively. Tumor density and size were measured at the end of the protocol by visual, endoscopical and histological inspection. Whole mounts of colons were stained with antibodies against LYVE-1 and CD31 to analyze area density and dimension of the lymphatic vessels within tumoral and peritumoral regions. Moreover, in vitro tubulogenesis assays were used to assess human tumor-isolated intestinal lymphatic endothelial cell (HILEC) organization into capillary tubules using a Matrigel system. Results: Systemic administration of an anti-VEGFR-3 antibody inhibited lymphangiogenesis in tumoral and peritumoral regions, reducing both area density (from 48±1.1 to 10±0.6 vessels/mm2) and lymphatic vessel dimension (from 78±1.8mm to 24±0.6mm, p < 0.01), while increasing inflammatory edema formation. In addition, it reduced tumor density (from 15±0.8 to 3±0.3 tumors/mouse, p < 0.01) and size (from 6.4±0.4mm to 2.3±0.1mm, p < 0.01), in comparison with untreated mice. In contrast, tumor density and growth was enhanced by systemic delivery of the lymphangiogenic factor VEGF-C, which in turn increased lymphangiogenesis within the same regions. HILEC isolated from colorectal cancer biopsies showed increased growth rate and capacity to undergo tubulogenesis in vitro (38±1.5 number of tumoral tubes vs 14±0.5 number of NL tubes, p < 0.01) compared to healthy control tissues. Conclusions: Our findings demonstrate that VEGFC/VEGFR3-dependent lymphangiogenesis plays a role not only in metastasis dissemination, but also in colitis-associated tumor growth. Therefore, the lymphatic vasculature may be a promising target for the prevention and the treatment of CAC.

P024 Repeated cycles of DSS inducing a chronically relapsing inflammation: A novel model to study fibrosis using in vivo MRI T2 relaxometry

P023 Targeting metallothionein in Dextran Sulfate Sodium-induced colitis points to new therapeutic strategies for patients suffering from inflammatory bowel diseases L. Devisscher1 *, P. Hindryckx1, K. Olivier1, H. Peeters2, M. Lynes3, C. Cuvelier4, M. De Vos1, D. Laukens5. 1Ghent University, Department of Gastroenterology, Ghent, Belgium, 2Ghent University Hospital, Department of Gastroenterology, Ghent, Belgium, 3University of Connecticut, Department of Molecular and Cell Biology, Connecticut, United States, 4Ghent University, Department of Pathology, Ghent, Belgium, 5Ghent University Hospital, Department of gastroenterology, Ghent, Belgium

Sequential T2 Relaxometry as a Non-Invasive Assessment of Transmural Inflammation in a Murine Model of Chronically Relapsing Colitis

Background Anti-TNF therapy induces mucosal healing in patients with Crohns disease, but the effects on transmural inflammation, stenosis and complications are largely unknown. MRI offers excellent imaging of transmural and peri-visceral lesions in Crohns disease. Objective To assess the evolution of small bowel disease activity and complications in Crohns disease patients treated with anti-TNF agents using serial MRI scans. Methods We performed a retrospective study of patients with Crohns disease who had undergone serial MRI scans (T1-weighted preand post-contrast and T2-weighted sequences) while receiving anti-TNF treatment (infliximab or adalimumab). Patients were grouped as clinical responders or nonresponders based on a review of medical records. MRI scoring was performed blinded to clinical, laboratory and endoscopic data. Stricturing lesions were evaluated using simplified scoring systems of inflammation (Steward et al.) and stenosis (Fornasa et al.). Inflammation was scored using mural thickness, contrast enhancement, pattern of enhancement, mural and perimural T2 signal. At the level of stenosis, T2 signal intensity, post-contrast T1 enhancement and prestenotic dilatation was quantified. Complications were scored evaluating penetrating lesions, abscesses and/or fistulas. Results Twenty-one consecutive patients were studied (71% female) with a median age of 33 years (range 19-58 years), the median disease duration was 12 years (range 3-35 years) and the median time between initial and first follow-up MRI was 110 weeks (range 14-299 weeks). Forty-two small bowel MRI scans were evaluated in 13 clinical non-responders and 8 responders. In all patients, median inflammation scores decrease from 10 (range 6-15) on initial MRI, to 8 (range 1-13) on follow up MRI (p=0.168). In the responder group, 7/8 (86%) of the patients had reduced inflammation on follow-up MRI during anti-TNF treatment (p=0.0571). In this group the first MRI detected complicated disease in 2/8 (25%) of the patients which resolved at followup MRI. Severity of stenosis was significantly reduced between serial MRI scans (p=0.0148). In the non-responder group only 23% showed decreased inflammatory scores between serial MRI scans (non-significant). 3/13 patients had stricturing and penetrating lesions which was unchanged during the disease course as indicated by serial MRI scans. No change was seen in severity of stenosis. Conclusion A reduction in both small bowel inflammation and inflammatory stenosis on serial MRI scans correlates with clinical responses to anti-TNF treatment in Crohns disease. Hence, anti-TNF antibodies not only produce mucosal, but also transmural healing, as well as reduction in stenosis. MRI can be used as a complementary approach to measure transmural healing in order to improve the clinical management in Crohns disease patients.