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Epithelial barrier dysfunction as permissive pathomechanism in human intestinal graft-versus-host disease

Bone Marrow Transplantation volume 53, pages 1083–1086 (2018)Cite this article

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Acute graft-versus-host disease (A-GVHD) is characterized by a strong inflammatory reaction of the graft against the recipient, mainly in the skin, the intestine and the liver. The gastrointestinal manifestation of the disease is characterized by nausea, vomiting, abdominal cramps and diarrhea with extensive fluid loss. Histologically, diagnosis of A-GVHD in colonic biopsies is characterized by crypt abscesses and epithelial apoptosis [1]. Pathophysiologically, A-GVHD is described as a three-stage model with activation of antigen presenting cells in the host, donor T-cell activation and target tissue destruction [2]. Within this model barrier dysfunction with translocation of microbial products and luminal antigens is thought to be a major perpetuating factor and a major cause of the “cytokine storm” through immune activation [3]. However, epithelial barrier function in A-GVHD may not only be relevant for the pathophysiology of immune activation but also for the pathomechanism of diarrhea in this disease. Therefore, we investigated the epithelial barrier function in human biopsies ex vivo in Ussing-type chambers with subsequent molecular characterization of potential pathomechanisms.

In this study, 22 patients with diarrhea and clinical suspicion of intestinal GVHD after bone marrow transplantation underwent sigmoidoscopy at the Charité-Universitätsmedizin Berlin. Infectious causes for symptoms were previously ruled out including stool analysis for Clostridium difficile toxin and whole blood CMV-PCR and immunohistochemistry of biopsies. Diagnosis of A-GVHD was made from histological biopsies in 16 patients (A-GVHD; grade I: n = 6, grade II: n = 8, grade III: n = 1, grade IV: n = 1). The remaining six patients without histopathological diagnosis of GVHD served as a further control group (no GVHD). A group of 15 patients which underwent endoscopy within this period of time for tumor exclusion without signs of pathology served also as controls (control). All patients gave their written consent for taking biopsies for scientific purpose and the study was approved by the local ethics committee of the Charité Berlin (229-32). A miniaturized Ussing-type chamber was used for impedance spectroscopy. This technique can differentiate the epithelial and subepithelial portion of the transmural electrical resistance [4]. Paracellular permeability was determined by 3H-Mannitol flux. For macromolecular flux the transcellular passage of horse radish peroxidase (HRP; 44 kDa; Sigma-Aldrich) was analyzed in six consecutive patients with A-GVHD grade I/II. Tight junction protein expression was analyzed by Western blotting. For cryosections, tissues were fixed with 2% paraformaldehyde. Antibodies were rabbit anti-claudin-2, rabbit anti-occludin, mouse anti-claudin-5 (Life Technologies GmbH, Darmstadt, Germany) and mouse anti-ZO-1 (BD Biosciences, San Jose, CA). Secondary antibodies were Alexa Fluor 594 goat anti-mouse or rabbit IgG and 488 goat anti-rabbit or mouse IgG (Molecular Probes, Eugene, OR). Nuclei were counterstained with DAPI. Images were obtained with a confocal laser scanning microscope (Zeiss LSM 510 Meta, Jena, Germany). Results are given as means ± SEM. Differences between groups were tested by one way analysis of variance with Bonferroni-Holm post hoc test.

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Acknowledgements

We thank the team of the endoscopy unit (Campus Benjamin Franklin, Charité .- Universitätsmedizin Berlin) for the excellent cooperation and in memoriam D. Sorgenfrei for his remarkable technical assistance in the lab. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG): Schu 559/11-2

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Authors and Affiliations

  1. Department of Gastroenterology, Infectiology and Rheumatology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Berlin, Germany

    Hanno Troeger, Christian Bojarski & Britta Siegmund

  2. Department of General, Visceral and Vascular Surgery, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Berlin, Germany

    Nina A. Hering

  3. Institute of Clinical Physiology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany

    Anja Fromm, Christian Barmeyer, Michael Fromm & Jörg-Dieter Schulzke

  4. Department of Hematology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, Berlin, Germany

    Lutz Uharek & Kathrin Rieger

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  1. Hanno Troeger
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Correspondence to Jörg-Dieter Schulzke.

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These authors contributed equally: Kathrin Rieger, Jörg-Dieter Schulzke

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Troeger, H., Hering, N.A., Bojarski, C. et al. Epithelial barrier dysfunction as permissive pathomechanism in human intestinal graft-versus-host disease. Bone Marrow Transplant 53, 1083–1086 (2018). https://doi.org/10.1038/s41409-018-0144-2

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