Ubiquitin E3 Ligase A20 Contributes to Maintaining Epithelial Barrier Function
Cellular Physiology and Biochemistry
and Aims: Epithelial barrier dysfunction is involved in the pathogenesis of allergic diseases; the mechanism is to be further understood. Ubiquitin E3 ligase A20 (A20) plays a role in maintaining the homeostasis in the body. This study aims to investigate the role of A20 in maintaining the epithelial barrier function. Methods: Human intestinal epithelial cell line, Caco-2 cells, was cultured to monolayers to test the endocytosis and degradation of a model allergen, ovalbumin (OVA). The role of
... (OVA). The role of A20 in the endosome/lysosome fusion in epithelial cells was tested with A20-sufficient and A20-deficient Caco-2 cells and visualized by immunocytochemistry. Results: Caco-2 cells could endocytose exogenous allergens (OVA) in culture. The endocytic OVA was degraded in A20-sufficient Caco-2 cells via the mechanism of endosome/lysosome fusion, while the A20-deficient Caco-2 monolayers converted the OVA to the basal compartment of transwells, which conserved the antigenicity reflected by that it induced T cell proliferation in an allergen-specific manner. A20 was required in the fusion of endosomes and lysosomes. Conclusion: A20 contributes to maintaining the epithelial barrier function. Statistics Differences between two groups were analysed using unpaired two-tailed Student's t-test, or by oneway analysis of variance (ANOVA) for experimental groups were more than 2 groups. Data were expressed as the mean ± SD. Caco-2 monolayers within 24h. On the other hand, we knocked down the A20 gene from Caco-2 cells; these cells were cultured to monolayers with the TER similar to A20-suf�icient monolayers (data not shown). The lysosomes contain the acid hydrolase enzymes that can hydrolyze the endocytic allergens, which is dependent on a premise that the allergen-carrying endosomes fuse with lysosomes. Our data are in line with the information that about 90% OVA-carrying endosomes fused with the lysosomes in Caco-2 cells. Further evidence indicates that in A20-de�icient Caco-2 cells, although the OVA-carrying endosomes were abundant in the cytoplasm, less than 5% of the endosomes had fused with lysosomes. The evidence implies that A20 is required in the endosome/lysosome fusion. The data are in line with published information that A20 contributes to the tethering of endosome and lysosome  . Published data indicate that A20 plays a critical role in the prevention of in�lammation in the body. One of the features of A20 is that can suppress the activation of NF-κB and Toll like receptor activities, so as to prevent the gene transcription of a number of proin�lammatory cytokines [19, 20] . Our data have added new information to this point that A20 is also required in the degradation of endocytic allergens in epithelial cells to prevent the allergens to be converted to the basal chambers of transwells, an in vitro experimental model mimicking the subepithelial region in the intestine. Fig. 4 . Antigenicity test. OVA-speci�ic CD3 + CD4 + CD25 -T cells and DC were cultured for 3 days in the presence of commercial OVA (A), or supernatant protein (10µg/ml) from A20-de�icient group (B) or A20-suf�icient group (C), or medium alone (D), or BSA (E; 10µg/ml). Panel F shows the negative staining control. The histograms show the proliferated T cells (the gated cells); the numbers on each panel indicate the mean ± SD. The data represent 10 separate experiments. *, p<0.01, compared with the medium alone group (D). In summary, the present study reveals that intestinal epithelial cell line, Caco-2 cells, endocytose exogenous allergens, which can be degraded in the epithelial cells. The endosome/lysosome fusion is required in the degradation of endocytic allergens, in which A20 plays a critical role.