Celiac Disease Monocytes Induce a Barrier Defect in Intestinal Epithelial Cells

Intestinal epithelial barrier function in celiac disease (CeD) patients is altered. However, the mechanism underlying this effect is not fully understood. The aim of the current study was to evaluate the role of monocytes in eliciting the epithelial barrier defect in CeD. For this purpose, human monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from active and inactive CeD patients and healthy controls. PBMCs were sorted for expression of CD14 and co-cultured with intestinal epithelial cells (IECs, Caco2BBe). Barrier function, as well as tight junctional alterations, were determined. Monocytes were characterized by profiling of cytokines and surface marker expression. Transepithelial resistance was found to be decreased only in IECs that had been exposed to celiac monocytes. In line with this, tight junctional alterations were found by confocal laser scanning microscopy and Western blotting of ZO-1, occludin, and claudin-5. Analysis of cytokine concentrations in monocyte supernatants revealed higher expression of interleukin-6 and MCP-1 in celiac monocytes. However, surface marker expression, as analyzed by FACS analysis after immunostaining, did not reveal significant alterations in celiac monocytes. In conclusion, CeD peripheral monocytes reveal an intrinsically elevated pro-inflammatory cytokine pattern that is associated with the potential of peripheral monocytes to affect barrier function by altering TJ composition

Intestinal Barrier Function in Gluten-Related Disorders

Gluten-related disorders include distinct disease entities, namely celiac disease, wheat-associated allergy and non-celiac gluten/wheat sensitivity. Despite having in common the contact of the gastrointestinal mucosa with components of wheat and other cereals as a causative factor, these clinical entities have distinct pathophysiological pathways. In celiac disease, a T-cell mediate immune reaction triggered by gluten ingestion is central in the pathogenesis of the enteropathy, while wheat allergy develops as a rapid immunoglobulin E- or non-immunoglobulin E-mediated immune response. In non-celiac wheat sensitivity, classical adaptive immune responses are not involved. Instead, recent research has revealed that an innate immune response to a yet-to-be-defined antigen, as well as the gut microbiota, are pivotal in the development in this disorder. Although impairment of the epithelial barrier has been described in all three clinical conditions, its role as a potential pathogenetic co-factor, specifically in celiac disease and non-celiac wheat sensitivity, is still a matter of investigation. This article gives a short overview of the mucosal barrier of the small intestine, summarizes the aspects of barrier dysfunction observed in all three gluten-related disorders and reviews literature data in favor of a primary involvement of the epithelial barrier in the development of celiac disease and non-celiac wheat sensitivity

Differential subcellular expression of MICA alleles in a celiac disease cell culture model

Einführung: Die Expression von MICA, einem atypischen MHC-Klasse-I-Molekül, wird in der Zöliakie durch Gliadinpeptide und sekundär durch Interleukin-15 erhöht. Als Ligand des NKG2D-Rezeptors auf intraepithelialen Lymphozyten (IELs) führt die MICA/NKG2D- Interaktion zur Lyse des intestinalen Epithels. MICA-Allele zeigen einen hohen Polymorphismus. In der Literatur ist beschrieben, dass viele Zöliakiepatienten das trunkierte MICA-Allel A5.1 aufweisen und die Klinik oft einen abgemilderten, symptomatischen Verlauf annimmt. Zur Untersuchung der differenziellen zellulären Kompartimentierung des trunkierten MICA-Allels MICA*008 im Vergleich zu dem nicht trunkierten Allel MICA*019 und der pathophysiologischen Folgen auf die Interaktion mit IELs, exprimierten wir die verschiedenen MICA-Allele in einem Zellmodell aus intestinalen Epithelzellen (IEC). Ziele: In dieser Arbeit soll untersucht werden, ob die differenzielle Kompartimentierung der MICA-Isoformen MICA*008 und MICA*019 in einem humanen IEC-Zellsystem nachvollziehbar ist. Stabil MICA*008-, MICA*019- und MICB-exprimierende Zellklone sollten generiert und hierbei die mögliche Interaktion von MICA-exprimierenden Epithelzellen und IELs diskutiert werden. Methoden: Mittels Subklonierung wurden MICA/-B-Konstrukte (MICA*008 als trunkiertes Allel, MICA*019 als nicht-trunkiertes Allel sowie MICB als Kontrolle) generiert und in Caco-2- bbe-Zellen transient sowie stabil exprimiert. Neben Wildtypkonstrukten wurden Konstrukte mit einem HA-Tag hergestellt. In der konfokalen Mikroskopie nach Immunfärbung sowie durchflusszytometrisch wurde die differenzielle Kompartimentierung charakterisiert, mittels Western Blotting wurde die Expression des trunkierten MICA*008 untersucht. Stabil MICA*008- und MICA*019-exprimierende Caco-2-bbe-Zellen wurden hinsichtlich ihrer epithelialen Barrierefunktion (transepithelialer Widerstand, TER) untersucht. Resultate: Die nicht-trunkierten Allele MICA*019 und MICB werden membranös exprimiert, während das trunkierte MICA*008 hauptsächlich intrazellulär lokalisiert. MICA*008 zeigte sich im Zytosol sowie submembranös in vesikelartigen Formationen, eine Kolokalisation mit Zellmembranproteinen fand sich nicht. Eine apikale Sortierung von MICA*008 konnte nicht eindeutig beobachtet werden. MICA*019 fand sich in der basolateralen Zellmembran und zeigte deutliche Kolokalisationen mit basolateralen Membranproteinen. Im Western Blot zeigten Banden das Vorliegen kleinerer MICA*008-Moleküle im Vergleich zu den nicht-trunkierten MICA/-B-Proteinen. In der Durchflusszytometrie sahen wir ein vom Genotyp abhängiges Verhältnis von intrazellulärer zu oberflächlicher MICA-Expression. In TER-Messungen zeigten MICA*008-exprimierende Zellen geringere Werte. Schlussfolgerungen: Es konnte gezeigt werden, dass die subzelluläre Kompartimentierung von MICA Genotyp-abhängig ist. Das bei Zöliakiepatienten häufig vorkommende Allel MICA*008 wird – anders als das Wildtyp-Allel des MICA – nicht transmembranös exprimiert, sondern lokalisiert im Zytosol und submembranös. Der gehäuft vorkommende symptomatische Verlauf der Zöliakie bei Trägern des MICA-A5.1-Allels könnte somit als Konsequenz der verminderten Aktivierung des NKG2D-Rezeptors verstanden werden. Perspektivisch sollten die stabil MICA*008- und MICA*019-exprimierenden IEC-Konstrukte hinsichtlich ihrer Empfindlichkeit gegenüber NK-Rezeptor-verursachter Lyse untersucht werden.Introduction: In celiac disease, MICA is upregulated in the presence of gliadin peptides and secondarily by IL-15. MICA acts as ligand for the NKG2D receptor expressed on intraepithelial lymphocytes (IELs). The interaction of the ligand with its receptor leads to epithelial lysis and is a crucial factor in the development of an epithelial barrier defect in celiac disease. As shown in a preceding study, many celiacs are carriers of the truncated allele MICA-A5.1 (MICA*008). Since the clinical course of these carriers is often oligosymptomatic, we expressed this allele in intestinal epithelial cells (IEC) and compared the subcellular localization of MICA*008 to the non-truncated MICA*019 and MICB alleles. Aims of the study: This study examines the differential subcellular localization of MICA proteins by visualizing MICA in transfected Caco-2-bbe cells. We aimed to reconstruct the interaction between IELs and MICA-expressing intestinal epithelial cells and thus to shed a light on how the MICA genotype affects the subcellular MICA localization. Methods: MICA*008 (MICA-A5.1, truncated allele), MICA*019 (non-truncated allele), and MICB were subcloned into the vector pCI-puro, and transiently as well as stably expressed in Caco-2-bbe cells. The subcellular localization of MICA/-B was examined by confocal microscopy after immunostaining, western blotting and flow cytometry. Stably MICA*008- and MICA*019-expressing Caco-2-bbe cells were examined for barrier function (transepithelial resistance, TER). Results: Confocal microscopy confirmed the differential compartmentalization of the MICA alleles: MICA*008 localized intracellularly, whereas MICA*019 and MICB were predominantly expressed on the cell membrane. Furthermore we observed a submembranous vesicular arrangement of MICA*008. Colocalization with apical or basolateral membrane proteins has not been observed in MICA*008-expressing cells. MICA*019 localized in the basolateral cell membrane and displayed colocalizations with membrane proteins. Western blotting revealed a specific band of the truncated molecule MICA*008 in comparison to the larger MICA*019 protein. Flow cytometry confirmed that the ratio of intracellular to surface MICA expression is genotype-dependent. In TER measurements, MICA*008-expressing cells showed significantly lower values. Conclusion: The localization of MICA is dependent on the MICA genotype. MICA*008 localized in the cytosol whereas MICA*019 was found in the cell membrane. This supports the hypothesis that an altered clinical course with absent or reduced gastrointestinal symptoms in MICA-A5.1 carriers is secondary to a lack of activation of the NKG2D receptor. In perspective, stable constructs of MICA*008 and MICA*019 should be analyzed regarding their viability in the presence of NK receptor-bearing cells to examine the interaction

Gluten-Free Diet in Celiac Disease—Forever and for All?

The gluten-free diet is the only effective treatment available for celiac disease. However, it is difficult to adhere to and a closer look on the diet’s implementation and indications reveals several ambiguities: Not only is there controversy on the threshold of gluten that can be tolerated in the frame of a strict gluten-free diet, but it is also unclear whether the gluten-free diet is an appropriate treatment in patient subgroups with asymptomatic or potential celiac disease. Reports from a number of research groups suggest that a certain proportion of patients may effectively develop tolerance to gluten and thus become suitable for gluten reintroduction over time. In this review, we set out to create an overview about the current state of research as regards the definition of a strict gluten-free diet in terms of the gluten thresholds considered tolerable and the indication for a gluten-free diet in the absence of histological abnormalities or symptoms. Furthermore, we discuss the concept that a gluten-free diet must be followed for life by all patients