Evasin-displaying lactic acid bacteria bind different chemokines and could be used for therapy of inflammatory bowel disease
published: May 23, 2017, recorded: April 2017, views: 1
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Chemokines, small chemoattractant cytokines, are key signals in the intestinal immune system. They play an important role in the recruitment and activation of immune cells in mucosa, which is a fundamental event in the pathogenesis of both forms of inflammatory bowel disease (IBD), Crohn’s disease and ulcerative colitis. The development of chemokine binding proteins as potential therapeutic agents in IBD is therefore of great interest.
Recombinant lactic acid bacteria (LAB) were engineered to bind different families (CC and CXC) of chemokines by displaying chemokine binding proteins (CKBPs) evasins-1, -3 and -4 on their surface. We used CKBPs produced by the salivary gland of the brown tick Rhipicephalus sanguineus, named evasins. Evasins are small proteins, which have the ability to bind and neutralize chemokines of different families (CC and CXC) and inhibit the chemokine-mediated recruitment of leukocytes. Evasin genes were cloned into a lactococcal surface display vector and over-expressed in L. lactis NZ9000 and NZ9000ΔhtrA in fusion with secretion signal and surface anchor. Expression of evasin fusion proteins was confirmed by Western blot and the surface localization was indirectly confirmed with flow cytometry and whole cell ELISA. Functionality of evasins on the bacterial surface was confirmed via the ability to remove chemokines from the solution with ELISA and Luminex multiplexing system assays. Using different evasins-displaying bacteria we have demonstrated the binding from 15% to 90% of 11 different chemokines. The binding was largely dependent on bacterial concentration. The evasin fusion protein-containing growth medium of L. lactis was used for heterologous coating of non-recombinant bacterial cells of Lactobacillus salivarius ATCC 11741 and its ability to bind chemokines was also confirmed. Evasin- displaying L. lactis NZ9000ΔhtrA cells had superior chemokine binding ability. Evasin-3-displaying L. lactis removed 88.0% of IL-1β-induced CXCL8 from the supernatant of Caco-2 epithelial cells. It also prevented secretion of CXCL8 from Caco- 2 cells in a time dependent manner when added before induction with IL-1β. Evasin-displaying LAB have the ability to bind multiple chemokines simultaneously and exert synergistic activity. This innovative treatment approach therefore has the potential for mucosal therapy of IBD and will be tested in an animal model.
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