Effekt von Makrophagen-Subpopulationen auf die intestinale Barriere

Einleitung. Makrophagen sind von entscheidender Bedeutung in der Pathogenese chronisch entzündlicher Darmerkrankungen (CED). Sie werden in ein Spektrum eingeteilt, das von pro- bis hin zu anti-inflammatorischen Makrophagen reicht. Neben dem inflammatorischen Zellinfiltrat der Lamina propria stellt die Störung der epithelialen Barriere einen weiteren zentralen Pathomechanismus der CED-Entstehung dar. Ziel dieser Arbeit war es, den spezifischen Effekt von Makrophagen-Subpopulationen auf die epitheliale Barriere zu untersuchen. Methodik. Durch Immunhistochemie an intestinalen Resektionspräparaten wurde das Vorkommen von Makrophagen-Subpopulationen in der Mukosa des entzündeten Darms bei Patienten mit Morbus Crohn und Colitis ulcerosa quantifiziert und mit gesunden Kontrollen verglichen. Humane CD14+ Zellen des peripheren Bluts wurden in vitro in pro- bzw. anti-inflammatorische Makrophagen polarisiert und der Einfluss dieser Subtypen sowie von Monozyten als Kontrollpopulation auf verschiedene Epithelzelllinien (Caco-2, T84, HT-29/B6) mittels Widerstandsmessungen analysiert. Die Epithelzellen wurden anschließend durch Immunfluoreszenzfärbungen weiter charakterisiert. Zytokinkonzentrationen im Überstand wurden mittels „cytometric bead array“ und enzyme-linked immunosorbent assay (ELISA) quantifiziert. Eine Inhibition von Tumor Nekrose Faktor (TNF), Interleukin (IL)-1β und IL 18 wurde durch Verwendung spezifischer Antikörper bzw. Rezeptor-Antagonisten erreicht. Ergebnisse. Sowohl bei Patienten mit Morbus Crohn als auch mit Colitis ulcerosa fand sich eine massive Invasion der pro-inflammatorischen CD68+/iNOS+/TNF+-Makrophagen in die Lamina propria des entzündeten Darms. So verschob sich die Balance der Makrophagen in Richtung des pro-inflammatorischen Subtyps, wobei dieser Effekt beim Morbus Crohn am stärksten ausgeprägt war. In-vitro polarisierte pro-inflammatorische Makrophagen führten zu einem Widerstandsverlust und somit zu einer Störung der epithelialen Integrität von Caco-2, T84 und HT29/B6 Zellen, was durch Stimulation von Toll-like-Rezeptoren mit Lipopolysaccharid nochmals verstärkt wurde. Mechanistisch ist dies durch parazelluläre Leckage mittels deregulierter tight junction-Proteine zu erklären: Vor allem das porenbildende Claudin-2 wurde nach Co-Kultur mit den pro-inflammatorischen Makrophagen vermehrt an der Epithelzellmembran exprimiert. Ferner kam es zu einer Umverteilung von tight junction-Proteinen innerhalb der Zelle. Die pro-inflammatorischen Makrophagen waren charakterisiert durch eine hohe Expression von TNF. Daher konnte der vermittelte Epithelschaden effektiv durch die Inhibition von TNF durch Infliximab reduziert werden. Der Monozyten-induzierte Epithelschaden hingegen ließ sich durch IL-1-Rezeptor- Inhibition bzw. einen spezifischen Antikörper gegen IL-18 effektiv antagonisieren. Schlussfolgerung. Pro-inflammatorische Makrophagen, die die Lamina propria von CED-Patienten infiltrieren, beeinträchtigen die epitheliale Barriere durch Deregulation von Tight Junction-Proteinen. Durch Zytokin-Blockade kann dies effektiv antagonisiert werden, wodurch einer Chronifizierung der Entzündung entgegengewirkt werden kann.Background. Macrophages are key players in inflammatory bowel disease (IBD). They can be classified into pro- and anti-inflammatory macrophages. Besides inflammatory cell infiltrates in the lamina propria of the gut, intestinal barrier dysfunction is a main feature of the pathogenesis of IBD. Hence, the aim of this study was to determine the specific effect of macrophage subtypes on epithelial barrier. Methods. The presence of macrophage subtypes in intestinal resection specimens of IBD patients was visualized by immunohistochemistry and compared to healthy controls. Human peripheral blood CD14+ cells were polarized into either pro- or anti-inflammatory macrophages in vitro. The influence of these subtypes and monocytes as control population on epithelial cell layers (Caco-2, T84, HT-29/B6) was analyzed using resistance measurements. Epithelial cells were then further examined by immunofluorescence staining. Cytokine concentrations were quantified applying cytometric bead array and enzyme-linked immunosorbent assay (ELISA). Inhibition of TNF, IL-1β and IL-18 was achieved by specific antibodies or receptor antagonists. Results. A massive invasion of pro-inflammatory CD68+/iNOS+/TNF+ macrophages into the lamina propria of the inflamed intestine occurred in IBD patients, predominantly in Crohn´s disease, thereby shifting the balance of macrophages to a pro-inflammatory state. In-vitro polarized pro-inflammatory macrophages caused a significant loss of epithelial resistance and thus integrity of Caco-2, T84 and HT29/B6 cell layers. This was further amplified by toll-like receptor stimulation via addition of LPS. The mechanism was paracellular leakage through deregulation and/or redistribution of tight junction proteins, most notably claudin-2, caused by monocytes and pro-inflammatory macrophages. Pro-inflammatory macrophages were characterized by a dominant production of TNF. Consequently, the epithelial damage mediated by this cell population was effectively reduced via inhibition of TNF through infliximab. Monocyte-mediated damage was antagonized by inhibition of the inflammasome effector cytokines IL-1β and IL-18. Conclusion. Pro-inflammatory macrophages, as found in the lamina propria of IBD patients, disrupt the epithelial barrier through deregulation of tight junction proteins. This can effectively be antagonized by cytokine inhibition

The Multifaceted Role of the Inflammasome in Inflammatory Bowel Diseases

Inflammasomes are intracellular multiprotein complexes that coordinate the maturation of interleukin (IL)-1β and IL-18 in response to pathogens and metabolic danger. Both cytokines have been linked to intestinal inflammation. However, recently evolving concepts ascribe a major role to the inflammasome in maintaining intestinal homeostasis. This review recapitulates its position in the development of inflammatory bowel disease, thereby outlining a model in which hypo- as well as hyperfunctionality can lead to an imbalance of the system, depending on the specific cell population affected. In the epithelium, the inflammasome is essential for regulation of permeability and epithelial regeneration through sensing of commensal microbes, while excessive inflammasome activation within the lamina propria contributes to severe intestinal inflammation

The multifaceted role of the inflammasome in inflammatory bowel diseases

Inflammasomes are intracellular multiprotein complexes that coordinate the maturation of interleukin (IL)-1β and IL-18 in response to pathogens and metabolic danger. Both cytokines have been linked to intestinal inflammation. However, recently evolving concepts ascribe a major role to the inflammasome in maintaining intestinal homeostasis. This review recapitulates its position in the development of inflammatory bowel disease, thereby outlining a model in which hypo-as well as hyperfunctionality can lead to an imbalance of the system, depending on the specific cell population affected. In the epithelium, the inflammasome is essential for regulation of permeability and epithelial regeneration through sensing of commensal microbes, while excessive inflammasome activation within the lamina propria contributes to severe intestinal inflammation. KEYWORDS: cytokines, inflammatory bowel diseases, inflammasome Interleukin (IL)-1β and IL-18, both belonging to the IL-1 family, represent two crucial cytokines involved in the pathogenesis of both acute and chronic inflammatory disorders The first description of what is now referred to as IL-1 dates back a long time This review recapitulates recently evolving concepts of inflammasome activation and its relevance in vivo, concentrating on its complex and controversially discussed role within intestinal inflammation and mucosal injury. Based on the literature, a model will be outlined in which the inflammasome, as an Lissner and Siegmund: Inflammasomes in Inflammatory Bowel Diseases TheScientificWorldJOURNAL (2011) 11, 1536-1547 1537 important member of the innate immune system, plays a decisive role in maintaining intestinal homeostasis, where hypo-as well as hyperfunctionality can result in an imbalance of the system, depending on the specific cell population affected. THE INFLAMMASOME Inflammasomes are high-molecular-weight platforms in the cytosol of diverse cell types. Because they represent a component of the innate immune system, most studies identifying inflammasome-expressing cells have focused on derivatives of the myeloid lineage, such as macrophages and dendritic cells As explained above, two signals are required for the secretion of active IL-1β and IL-18 (see For NLRP3 activation, several mechanisms have been proposed (see Since one fundamental function of the inflammasome is the coordinated response to pathogen infections, its dominant activators are whole microorganisms and PAMPs. Potential identifiable pathogens include fungi (Candida albicans, Saccharomyces cerevisiae) INFLAMMASOME-ASSOCIATED HUMAN DISEASES There is evidence for a number of inflammatory diseases to be associated with inflammasome dysfunction. For some of them, hyperfunctional genetic mutations within the NLRP3 gene are proven and lead to their name, cryopyrin-associated periodic syndromes (CAPS) In addition to these rare autoinflammatory diseases, there is increasing evidence for the involvement of the inflammasome pathway on other, more common, medical conditions for which hereditary as well as acquired components seem likely. One such disease is rheumatoid arthritis (RA), for which the combination of NLRP3 and CARD8 polymorphisms represents a risk factor GENETIC MUTATIONS WITHIN THE INFLAMMASOME PATHWAY ASSOCIATED WITH INFLAMMATORY BOWEL DISEASES In genome-wide association studies, single nucleotide polymorphisms (SNPs) in the NLRP3 gene have recently been linked to the susceptibility to develop Crohn's disease (CD) INFLAMMASOME AND INFLAMMATORY BOWEL DISEASES IBD represent a chronic inflammatory condition of the gut, for which the innate as well as the adaptive immune system play a crucial role. Although the exact mechanisms are not fully understood, the concept of an inadequate immune response against commensal microbiota in the gut lumen is widely accepted In vivo Studies: Neutralization Strategies The first evidence for effective IL-1β inhibition was reported in vivo by applying a soluble IL-1 receptor antagonist (IL-1Ra) to suppress immune-complex colitis in rabbit

Analysis of Circulating Food Antigen-Specific T-Cells in Celiac Disease and Inflammatory Bowel Disease

To demonstrate and analyze the specific T-cell response following barrier disruption and antigen translocation, circulating food antigen-specific effector T-cells isolated from peripheral blood were analyzed in patients suffering from celiac disease (CeD) as well as inflammatory bowel disease (IBD). We applied the antigen-reactive T-cell enrichment (ARTE) technique allowing for phenotypical and functional flow cytometric analyses of rare nutritional antigen-specific T-cells, including the celiac disease-causing gliadin (gluten). For CeD, patient groups, including treatment-refractory cases, differ significantly from healthy controls. Even symptom-free patients on a gluten-free diet were distinguishable from healthy controls, without being previously challenged with gluten. Moreover, frequency and phenotype of nutritional antigen-specific T-cells of IBD patients directly correlated to the presence of small intestinal inflammation. Specifically, the frequency of antigen specific T-cells as well as pro-inflammatory cytokines was increased in patients with active CeD or Crohn's disease, respectively. These results suggest active small intestinal inflammation as key for the development of a peripheral food antigen-specific T-cell response in Crohn's disease and celiac disease

PuraStat in gastrointestinal bleeding: results of a prospective multicentre observational pilot study

Background: A recently developed haemostatic peptide gel for endoscopic application has been introduced to improve the management of gastrointestinal bleeding. The aim of this pilot study was to evaluate the feasibility, safety, efficacy and indication profiles of PuraStat in a clinical setting. Methods: In this prospective observational multicentre pilot study, patients with acute non-variceal gastrointestinal bleeding (upper and lower) were included. Primary and secondary application of PuraStat was evaluated. Haemoglobin, prothrombin time, platelets and transfusion behaviour were documented before and after haemostasis. The efficacy of PuraStat was assessed during the procedure, at 3 days and 1 week after application. Results: 111 patients with acute gastrointestinal bleeding were recruited into the study. 70 percent (78/111) of the patients had upper gastrointestinal bleeding and 30% (33/111) had lower gastrointestinal bleeding. After primary application of PuraStat, initial haemostatic success was achieved in 94% of patients (74/79, 95% CI 88-99%), and in 75% of the patients when used as a secondary haemostatic product, following failure of established techniques (24/32, 95% CI 59-91%). The therapeutic success rates (absence of rebleeding) after 3 and 7 days were 91% and 87% after primary use, and 87% and 81% in all study patients. Overall rebleeding rate at 30 day follow-up was 16% (18/111). In the 5 patients who finally required surgery (4.5%), PuraStat allowed temporary haemostasis and stabilisation. Conclusions: PuraStat expanded the therapeutic toolbox available for an effective treatment of gastrointestinal bleeding sources. It could be safely applied and administered without complications as a primary or secondary therapy. PuraStat may additionally serve as a bridge to surgery in order to achieve temporary haemostasis in case of refractory severe bleeding, possibly playing a role in preventing immediate emergency surgery

Analysis of Circulating Food Antigen-Specific T-Cells in Celiac Disease and Inflammatory Bowel Disease

To demonstrate and analyze the specific T-cell response following barrier disruption and antigen translocation, circulating food antigen-specific effector T-cells isolated from peripheral blood were analyzed in patients suffering from celiac disease (CeD) as well as inflammatory bowel disease (IBD). We applied the antigen-reactive T-cell enrichment (ARTE) technique allowing for phenotypical and functional flow cytometric analyses of rare nutritional antigen-specific T-cells, including the celiac disease-causing gliadin (gluten). For CeD, patient groups, including treatment-refractory cases, differ significantly from healthy controls. Even symptom-free patients on a gluten-free diet were distinguishable from healthy controls, without being previously challenged with gluten. Moreover, frequency and phenotype of nutritional antigen-specific T-cells of IBD patients directly correlated to the presence of small intestinal inflammation. Specifically, the frequency of antigen specific T-cells as well as pro-inflammatory cytokines was increased in patients with active CeD or Crohn’s disease, respectively. These results suggest active small intestinal inflammation as key for the development of a peripheral food antigen-specific T-cell response in Crohn’s disease and celiac disease

Combination of Vedolizumab With Tacrolimus Is More Efficient Than Vedolizumab Alone in the Treatment of Experimental Colitis

BACKGROUND Vedolizumab is a widely used and safe therapy in inflammatory bowel disease, particularly in ulcerative colitis (UC), making it a promising candidate for enhanced efficacy by combining it with additional immunomodulatory medications. In this study, we studied the impact of vedolizumab monotreatment vs vedolizumab coadministration with other immunomodulatory drugs on intestinal inflammation and intestinal immune cells in vivo. METHODS Colon tissue from human patients with UC with active disease or in remission with or without vedolizumab treatment was stained by immunohistochemistry. We reconstituted NOD-SCID-SGM3 mice with human CD34+ cells and treated them with dextran sodium sulfate to induce acute colitis. Mice were treated with vedolizumab alone, or in combination with tacrolimus, ozanimid, or tofacitinib. RESULTS Vedolizumab reduced the number of CD3+ T cells and CD68+ monocytes/macrophages in the colon of patients with UC with active disease. Vedolizumab moderately decreased immune cell numbers in acute dextran sodium sulfate-induced colitis. The combination of vedolizumab with tacrolimus further reduced the number of infiltrating CD3+ T cells and CD68+ monocytes/macrophages and was superior in ameliorating intestinal inflammation when compared to vedolizumab monotreatment. In contrast, cotreatment using vedolizumab with ozanimod or tofacitinib had no additive effect. CONCLUSIONS Our data show that vedolizumab reduces the number of innate and adaptive immune cells in the mucosa of patients with UC. Further, the combination of vedolizumab with tacrolimus was more efficient to reduce immune cell numbers and to increase therapeutic efficacy than vedolizumab monotreatment. This finding indicates that combination treatment using these two drugs may be beneficial for patients who do not respond to vedolizumab monotherapy