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A gardian of gut epithelial barrier from inflammation: the elastase inhibitor ELAFIN
Author(s) -
Deraison Celine,
Jaeger Marie Charlotte,
Guiraud Laura,
Rolland Claire,
Bellot Audrey,
Rolland Corinne,
Sebbag Mireille,
Bermudez Luis,
Langella Philippe,
Vergnolle Nathalie
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05293
Subject(s) - elafin , elastase , neutrophil elastase , inflammation , chemistry , colitis , slpi , immunology , biology , biochemistry , enzyme
In the gastrointestinal tract (GIT), proteolytic activity is tightly regulated to prevent inappropriate and potentially harmful proteolysis. In IBD this regulation is broken and our recent work has demonstrated that elastolytic activity is significantly increased in colonic epithelium from IBD patients. We have shown that the expression of the only elastase released by epithelial cells, ELA2 is up‐regulated; and the selective elastase inhibitor ELAFIN is severely down regulated in colonic epithelium from IBD patients. We showed that oral treatments with ELAFIN‐expressing L. Lactis bacteria restored proteolytic balance in the inflamed gut and was protective against acute and chronic colitis. ELAFIN is a low‐molecular weight molecule (10‐kDa), which specifically inhibits elastases. At N‐terminal terminus, a cationic helix showed anti‐microbial activities and could interact with phospholipids. Moreover ELAFIN is also able to inhibit NFkB and AP‐1 activation but this domain is not localized on protein. Therefore, several mechanisms of action are thus possible to explain ELAFIN’s protective property. Objectives If ELAFIN‐recombinant L. lactis has to be considered as a possible treatment for IBD in human, there is an absolute need to define the mechanisms by which ELAFIN delivery protects against intestinal inflammation. Methods We generated and expressed in LAB ELAFIN’s mutants allowing investigation the biological properties of ELAFIN individually. These different forms of ELAFIN‐recombinant LAB were co‐culture with thapsigargin (10mg/ml) and ELAFIN (10 7 CFU/well). After 6H, cells were additionally exposed to gentamicin (100 mg/ml) to limit bacterial development. The barrier function was measured using FITC‐dextran passage through the epithelial layer. The elastolytic activity and CXCL8 release were quantified from the culture media. Expression of inflammatory mediators was quantified by qPCR. Results Addition of thapsigargin for 24H on Caco2 cells induced ER‐stress and disruption of elastolytic balance. ELAFIN expression was decreased with up‐regulation of ELA2, leading to increase of elastolytic activity in medium, disclosed in IBD patients. Concomitantly, barrier function was reduced, and expression of CXCL8, CXCL10 and hBD2 was increased. Co‐culture with ELAFIN expressing‐LAB normalized all these parameters confirming anti‐inflammatory properties of ELAFIN. Neutralization of a‐helix (mutant K44D/K50G) did not modify ELAFIN’s properties. However, disruption of protease inhibitory function (mutant A62G/M63G) led to unability of ELAFIN mutant to modify permeability and inflammatory phenotype induced by exposure of intestinal epithelial cells to thapsigagin. Conclusion This suggests that anti‐inflammatory activity of ELAFIN is based on the control of elastase activity released by epithelial cells.