Acute graft versus host disease (GvHD) is a severe complication after hematopoietic stem cell transplantation. The impaired integrity of the gastrointestinal tract during GvHD (GI-GvHD) seems to play an important role in the amplification of the systemic disease.
In this study, we show for the first time that GI-GvHD leads to tight junction impairment with subsequently increased paracellular permeability. In the murine T-cell mediated acute GI-GvHD model used in this study, the transmembrane tight junction protein occludin is downregulated and its mesh wire-like appearance within the apical tight-junction complex is lost due to cytoplasmic re-localization. Furthermore, we observed an increased expression of TNF-α which might in turn lead to the observed changes in occludin expression and/or localization.
Our findings are in line with other studies on T-cell mediated small bowel diseases which found increased paracellular permeability [
14‐
19]. Tight-junction proteins are essential for the integrity of the intestinal barrier by building the most apical structure and regulating paracellular permeability and polarity of the cell. The tight-junction composition is a highly dynamic process and different tight-junction components can be divided into the transmembrane proteins (occludin, the claudin protein family, junctional adhesion molecules, coxsackie adenovirus receptor and tight-junction associated marvel proteins) and cytoplasmatic proteins (ZO-1 and cingulin). Furthermore, F-actin and myosin, as part of the cytoskeleton [
20,
21], are involved in the composition of the tight junctions. Interactions between tight-junction proteins, kinase activation and cytokine release influence the regulation of two transepithelial molecular pathways: a high-capacity, charge selective pore pathway for small uncharged molecules influenced primarily by claudin expression and a low-capacity leak pathway for larger ions regardless of charge regulated by the cytoskeleton involving ZO-1 and occludin. We observed a downregulation of occludin and a shift from the membrane to the cytoplasm without any significant changes in ZO-1 expression. Hypothetically, this could be an important cause of the observed increased permeability. Occludin is known to interact with other tight-junction proteins like ZO-1 and F-actin, mediating signal transduction, tissue growth and differentiation as well as cytoskeleton mediated leak pathway regulation [
22]. Furthermore, the function and the localisation seem to differ depending on posttranslational phosphorylation by enzymes [
20]. Fluorescence recovery after photobleaching analyses showed that occludin is very mobile at the tight-junction undergoing constant remodelling [
23]. In line with our observations, recent data in colitis associated impaired barrier function indicated that alterations in the expression level and localization of occludin are pivotal for increased paracellular permeability. Interestingly, the promoter of the occludin gene harbours a TNF-α response element, negatively regulating the transcription of occludin [
24]. Referring to the pivotal role of TNF-α in the regulation of gastrointestinal barrier function and permeability [
10,
13,
16,
25‐
28] and the fact that we observed a strong upregulation of TNF-α expression, it is tempting to speculate, that occludin is a central downstream target of TNF-α in GI-GvHD. It is well known, that IFN-γ production by the donor T cells, endotoxins and LPS as a component of the normal gut flora who penetrate the impaired intestinal barrier can lead to an excessive TNF-α production by monocytes and macrophages [
4,
29‐
31]. This increased TNF-α expression is associated with the severity of intestinal GvHD [
32] and an elegant recent work showed the pivotal role of TNF tight junction remodeling and barrier maintenance [
13]. Thus, increased TNF-α may impair the barrier function through disturbance of the tight junction which in turn results in increased paracellular permeability, enforced translocation of antigens from the gut into the circulation and an uncontrolled systemic inflammatory response.
By immune fluorescence microscopy, we were able to show that f-actin was released from the tight organisation of the terminal web beneath the apical plasma membrane and showed a mostly diffuse, spreaded appearance with some more densely packed areas in irregular patterns in GvDH. This could be due to a contraction of the cytoskeleton with tension on the tight-junction followed by an augmented paracellular channel as described by other studies [
17] or part of the response of intestinal epithelia to TNF-α induced shedding [
13] as reported recently.