Gastroenterology

Gastroenterology

Volume 127, Issue 2, August 2004, Pages 502-513
Gastroenterology

Basic-alimentary tract
Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice

https://doi.org/10.1053/j.gastro.2004.05.020Get rights and content

Abstract

Background & Aims: Effective therapeutics for treating acute colitis, caused by disruption of the intestinal epithelial barrier, are scarce. Trefoil factors (TFF) are cytoprotective and promote epithelial wound healing and reconstitution of the gastrointestinal tract, which makes them good candidate therapeutics for acute colitis. However, orally administered TFF stick to the mucus of the small intestine and are absorbed at the cecum. Methods: We have engineered the food-grade bacterium Lactococcus lactis to secrete bioactive murine TFF. The protective and therapeutic potentials of these TFF-secreting L. lactis were evaluated in parallel with purified TFF in the dextran sodium sulfate (DSS)-induced murine model for acute colitis and in established chronic colitis in interleukin (IL)-10−/− mice. Disease was evaluated by blinded macroscopic and microscopic inflammatory scores and by myeloperoxidase activity. Results: Intragastric administration of TFF-secreting L. lactis led to active delivery of TFF at the mucosa of the colon and, in contrast to administration of purified TFF, proved to be very effective in prevention and healing of acute DSS-induced colitis. The in situ secreted murine TFF significantly decreased morbidity and mortality and stimulated prostaglandin-endoperoxide synthase 2 expression, which represents a major therapeutic pathway. In addition, this approach was successful in improving established chronic colitis in IL-10−/− mice. Conclusions: We have positively evaluated a new therapeutic approach for acute and chronic colitis that involves in situ secretion of murine TFF by orally administered L. lactis. This novel approach may lead to effective management of acute and chronic colitis and epithelial damage in humans.

Section snippets

Bacteria

The L. lactis strain MG1363 was used throughout this study. Bacteria were cultured in GM17 medium (i.e., M17; Difco Laboratories, Detroit, MI; supplemented with 0.5% glucose). Stock suspensions of all strains were stored at −20°C in 50% glycerol in GM17 medium. For intragastric inoculations, stock suspensions were diluted 200-fold in fresh GM17 medium and incubated at 30°C. They reached a saturation density of 2 × 109 colony-forming units (CFU) per mL within 16 hours. Bacteria were harvested by

In vitro synthesis of TFF by L. lactis

We constructed 3 recombinant L. lactis strains, LL-mTFF1, LL-mTFF2, and LL-mTFF3, that secrete mTFF1, mTFF2, and mTFF3 containing amino-terminal Myc epitope tags, respectively. Synthesis of mTFF was evaluated by Western blot (Figure 1A) and enzyme-linked immunosorbent assay (Figure 1B). After 24 hours of growth, mTFF was found in the culture supernatant at concentrations ranging from 312 ± 68 ng/mL (LL-mTFF3) to 531 ± 92 ng/mL (LL-mTFF2) (Figure 1B). Constitutive mTFF secretion did not alter

Discussion

TFF are known for their cytoprotective properties, involvement in epithelial wound healing, and reconstitution of the gastrointestinal tract. This makes them good candidate therapeutics for the treatment of acute colitis. Due to their role as structural components, however, orally administered TFF stick to the mucus of the small intestine and are removed from the lumen at the cecum26 and can therefore never reach the colon. The approach presented here, active in situ delivery of TFF by

Acknowledgements

The authors thank I. Bruggeman, H. Devlies, A. Raeymaekers, and K. Van Laer for technical assistance; J. Wells for the vector pTREX1; M. C. Rio for the mtff1 complementary DNA; J. Vandekerckhove for Edman degradation; and A. Bredan, C. Cuvelier, and F. Shanahan for critically reviewing the manuscript.

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    Supported by Ghent University GOA (file no. 12050700). K.V. holds a predoctoral grant from the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen, and J.V.H. holds a predoctoral BOF grant from Ghent University (BOF011D1700). L.S. and S.N. are supported in part by Science Foundation Ireland (SFI/01/F.1/B036).

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