Blockade of αEβ7 integrin suppresses accumulation of CD8+ and Th9 lymphocytes from patients with IBD in the inflamed gut in vivo

Objective: Therapeutically targeting lymphocyte adhesion is of increasing relevance in IBD. Yet, central aspects of the action of anti-adhesion compounds are incompletely understood. We investigated the role of αEβ7 and α4β7 integrins and their blockade by vedolizumab and etrolizumab for trafficking of IBD T lymphocytes in an in vivo model of homing to and retention in the inflamed gut. Design: We explored integrin expression in IBD patients by flow cytometry and immunohistochemistry, while regulation of integrins was studied in T cell cultures. The functional relevance of integrins was assessed by adhesion assays and a recently established humanized mouse model in DSS-treated immunodeficient mice. Results: High expression of αEβ7 was noted on CD8+ and CD4+ Th9 cells, while α4β7 was expressed on CD8+, Th2 and Th17 cells. TCR stimulation and TGF-β were key inducers of αEβ7 on human T cells, while butyric acid suppressed αEβ7. In comparison to α4β7 blockade via vedolizumab, blockade of β7 via etrolizumab surrogate antibody superiorly reduced colonic numbers of CD8+ and Th9 cells in vivo after 3 hours, while no difference was noted after 0.5 hours. AEβ7 expression was higher on CD8+ T cells from IBD patients under vedolizumab therapy. Conclusion: AEβ7 is of key relevance for gut trafficking of IBD CD8+ T cells and CD4+ Th9 cells in vivo and mainly retention might account for this effect. These findings indicate that blockade of αEβ7 in addition to α4β7 may be particularly effective in intestinal disorders with expansion of CD8+ and Th9 cells such as IBD

Internal Hernia Through Foramen of Winslow a Rare Cause of Small Bowel Obstruction: A Case Report

Internal hernia through foramen of Winslow (FoW) is rare condition as there are only 200 cases reported so far in the literature. Our patient a 78 years man presented with a clinical picture suggestive of small bowel obstruction for 5 days. Patient underwent emergency laparotomy following suspicion of internal hernia on imaging. On exploratory laparotomy there was grossly dilated bowel loops and a small segment of terminal ileum and omentum was found herniating through FoW in to the lesser sac. The bowel segment was reduced with gentle traction and herniated segment of omentum was excised due to questionable viability. Opening of FoW was unusually large and to prevent hernia recurrence it was closed partially. Postoperative period was uneventful. This unusual case presented to us diagnostic confusion and management challenge considering the previous history, multiple comorbidities and geriatric profile

The α4β1 homing pathway is essential for ileal homing of Crohn's disease effector T cells in vivo

The precise mechanisms controlling homing of T effector (Teff) cells to the inflamed gut in Crohn’s disease (CD) are still unclear and clinical outcome data from inflammatory bowel disease (IBD) patients treated with the anti-α4β7 integrin antibody vedolizumab suggest differences between ulcerative colitis (UC) and CD. Methods: Expression of homing molecules was studied with flow cytometry and immunohistochemistry. Their functional role was investigated in in vitro adhesion assays and in a humanized mouse model of T cell homing to the inflamed gut in vivo. Results: Despite in vitro blockade of CD Teff adhesion to MAdCAM-1 and in contrast to previous oberservations in UC, anti-α4β7 treatment did not result in reduced Teff cell homing to the gut in vivo. However, the integrin α4β1 was expressed in higher levels on Teffs from CD patients compared with controls, while its expression in the peripheral blood declined and its expression in the intestine increased during the course of clinical vedolizumab treatment. Consistently, adhesion of CD Teffs to VCAM-1 was blocked by inhibition of α4 and α4β1 in vitro. Moreover, in vivo homing of CD Teffs to the inflamed ileum was reduced by inhibition of α4 and α4β1 integrins, but not α4β7 integrins. Conclusions: Our findings suggest that Teff cell homing to the ileum via the axis α4β1 – VCAM-1 is an essential and non-redundant pathway in CD in vivo possibly affecting efficacy of clinical treatment with anti-adhesion compounds

Physiological Intermolecular Modification Spectroscopy for the Prediction of Response to Anti-Tumor Necrosis Factor Therapy in Patients with Inflammatory Bowel Diseases

Background/Aims: Anti-tumor necrosis factor (TNF) antibodies have clinical efficiency only in a subgroup of patients with inflammatory bowel diseases (IBD). Prediction of clinical response is a critical clinical problem. Physiological intermolecular modification spectroscopy (PIMS) is a label-free technology performed in physiological conditions. PIMS enables real-time monitoring of dynamic molecular resonance of entire proteins and macromolecules of an individual. The aim of this study was to explore the capacity of PIMS to discriminate IBD patients regarding response to anti-TNF treatment. Methods: Protein extracts of peripheral blood mononuclear cells (PBMC) from 30 outpatients diagnosed with ulcerative colitis (UC) or Crohn's disease (CD) and treated with infliximab were subjected to PIMS analysis in a blinded transversal study. Total protein from each patient's PBMCs was challenged with infliximab. Dynamic changes in macromolecular interaction were registered while the temperature rose from -37 to 37°C. Individual macromolecular volume and molecular elasticity were determined for each patient. Results: Clinical data revealed that 67% of UC and 79% of CD patients responded to infliximab therapy during the 3-month study period based on their respective clinical activity score. These results confirm that PIMS data predicted response to anti-TNF therapy with an accuracy of 96%. Conclusion: PIMS stratified IBD patients into two groups, responders and nonresponders, which correlated with the clinical efficacy of anti-TNF therapy. PIMS seems to be a powerful technology to adapt IBD treatment to the individual patient. Further studies with PIMS might enable to predict clinical response to biological treatment in IBD patients before the therapy is initiated

Differential effects of α4β7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo

Objective: Gut homing of lymphocytes via adhesion molecules has recently emerged as new target for therapy in inflammatory bowel diseases. We aimed to analyze the in vivo homing of effector (Teff) and regulatory (Treg) T cells to the inflamed gut via α4β7 and GPR15. Design: We assessed the expression of homing receptors on T cells in peripheral blood and inflamed mucosa. We studied the migration pattern and homing of Teff and Treg cells to the inflamed gut using intravital confocal microscopy and FACS in a humanized mouse model in DSS-treated NSG (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) mice. Results: Expression of GPR15 and α4β7 was significantly increased on Treg rather than Teff cells in peripheral blood of patients with ulcerative colitis (UC) as compared to Crohn´s disease and controls. In vivo analysis in a humanized mouse model showed augmented gut homing of UC Treg cells as compared to controls. Moreover, suppression of UC (but not control) Teff and Treg cell homing was noted upon treatment with the α4β7 antibody vedolizumab. In contrast, siRNA blockade of GPR15 had only effects on homing of Teff cells but did not affect Treg homing in UC. Clinical vedolizumab treatment was associated with marked expansion of UC Treg cells in peripheral blood. Conclusion: α4β7 rather than GPR15 is crucial for increased colonic homing of UC Treg cells in vivo, while both receptors control UC Teff homing. Vedolizumab treatment impairs homing of UC Treg cells leading to their accumulation in peripheral blood with subsequent suppression of systemic effector T cell expansion

Oligonucleotides—A Novel Promising Therapeutic Option for IBD

Inflammatory Bowel Diseases (IBD), whose denomination comprehends Crohn's Disease (CD) and Ulcerative Colitis (UC), are intestinal chronic diseases that often require lifelong medical therapy. In the last two decades monoclonal antibodies against the cytokine TNF have become integral parts in the treatment of IBD patients, however there are unwanted side-effects and one third of patients show primary non-response while another subgroup loses response over time. Finding novel drugs which could act as therapies against precise pro-inflammatory molecular targets to avoid unwanted systemic side effects and additionally the process of immunization, represents an important aim for subsequent therapeutic approaches. Oligonucleotide based therapies represent a promising novel concept for the treatment of IBD. The molecular action of oligonucleotides ranges from inhibition of the translational process of mRNA transcripts of pro-inflammatory molecules, to mimicking bacterial DNA which can activate cellular targets for immunomodulation. Alicaforsen, selectively targets ICAM-1 mRNA. ICAM-1 is an adhesion molecule which is upregulated on endothelial cells during IBD, thereby mediating the adhesion and migration of leucocytes from blood to sites of active inflammation. In CD parenteral application of alicaforsen did not show therapeutic efficacy in phase II trials, but it demonstrated an improved efficacy as a topical enema in distal UC. Topical application of alicaforsen might represent a therapeutic perspective for refractory pouchitis as well. SMAD7 is a protein that inhibits the signaling of TGFβ, which is the mainstay of a regulatory counterpart in cellular immune responses. An antisense oligonucleotide against SMAD7 mRNA (mongersen) demonstrated pre-clinical and phase II efficacy in CD, but a phase III clinical trial was stopped due to lack of efficacy. Cobitolimod is a single strand oligonucleotide, which mimics bacterial DNA as its CpG dinucleotide sequences can be recognized by the Toll-like receptor 9 on different immune cells thereby causing induction of different cytokines, for example IL10 and IFNα. Topical application of cobitolimod was studied in UC patients. We will also discuss two other novel oligonucleotides which act on the GATA3 transcription factor (SB012) and on carbohydrate sulfotransferase 15 (STNM01), which could both represent novel promising therapeutic options for the treatment of UC

Treatment of Allergic Airway Inflammation and Hyperresponsiveness by Antisense-Induced Local Blockade of Gata-3 Expression

Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 (Th2)-mediated allergic airway inflammation in mice. However, it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness. Here, we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma. We could suppress GATA-3 expression in interleukin (IL)-4–producing T cells in vitro and in vivo by an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3, whereas nonsense control oligonucleotides were virtually inactive. In a murine model of asthma associated with allergic pulmonary inflammation and hyperresponsiveness in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate–labeled GATA-3 antisense oligonucleotides led to DNA uptake in lung cells associated with a reduction of intracellular GATA-3 expression. Such intrapulmonary blockade of GATA-3 expression caused an abrogation of signs of lung inflammation including infiltration of eosinophils and Th2 cytokine production. Furthermore, treatment with antisense but not nonsense oligonucleotides induced a significant reduction of airway hyperresponsiveness in OVA-sensitized mice to levels comparable to saline-treated control mice, as assessed by both enhanced pause (PenH) responses and pulmonary resistance determined by body plethysmography. These data indicate a critical role for GATA-3 in the effector phase of a murine asthma model and suggest that local delivery of GATA-3 antisense oligonucleotides may be a novel approach for the treatment of airway hyperresponsiveness such as in asthma. This approach has the potential advantage of suppressing the expression of various proinflammatory Th2 cytokines simultaneously rather than suppressing the activity of a single cytokine

Cellular Mechanisms of Etrolizumab Treatment in Inflammatory Bowel Disease

Background: Anti-integrin therapy is a new frontline strategy in the treatment of inflammatory bowel diseases (IBD). The anti-β7 integrin antibody etrolizumab is currently being investigated for safety and efficacy in Crohn’s disease (CD) and ulcerative colitis (UC) in several phase III trials. Mechanistically, etrolizumab is known to block β7 integrin ligand binding and reduces intestinal trafficking of β7-expressing cells. Etrolizumab blocks β7 integrin ligand binding and reduces β7-positive lymphocyte migration and retention in the inflamed gut mucosa, but the exact mechanisms by which this inhibition occurs are not fully understood.Methods: Cellular effects of etrolizumab or etrolizumab surrogate antibody (etrolizumab-s) were investigated in cell culture models and analyzed by flow cytometry, fluorescence microscopy, ImageStream®, stimulated emission depletion (STED) microscopy and functional dynamic in vitro adhesion assays. Moreover, effects on α4β7 integrin were compared with the pharmacodynamically similar antibody vedolizumab.Results: As demonstrated by several different approaches, etrolizumab and etrolizumab-s treatment led to internalization of β7 integrin. This resulted in impaired dynamic adhesion to MAdCAM-1. Internalized β7 integrin localized in endosomes and re-expression of β7 was dependent on de novo protein synthesis. In vitro etrolizumab treatment did not lead to cellular activation or cytokine secretion and did not induce cytotoxicity. Internalization of α4β7 integrin was increased with etrolizumab compared with vedolizumab.Discussion: Our data suggest that etrolizumab does not elicit secondary effector functions on the single cell level. Integrin internalization may be an important mechanism of action of etrolizumab, which might explain some but not all immunological effects observed with etrolizumab