Engineered Lactococcus lactis Secreting IL-23 Receptor-Targeted REX Protein Blockers for Modulation of IL-23/Th17-Mediated Inflammation.

Tina Vida Plavec,Petr Malý,Veronika Lišková,Jiří Černý,Anja Benko,Milan Kuchař,Aleš Berlec

doi:10.3390/microorganisms7050152

Abstract

Lactococcus lactis, a probiotic bacterium of food origin, has recently been demonstrated as a suitable strain for the production and in vivo delivery of therapeutically important proteins into the gut. We aimed to engineer recombinant L. lactis cells producing/secreting REX binding proteins that have been described as IL-23 receptor (IL-23R) blockers and IL-23R antagonists suppressing the secretion of cytokine IL-17A, a pivotal step in the T-helper Th17-mediated pro-inflammatory cascade, as well as in the development of autoimmune diseases, including inflammatory bowel disease (IBD). To reach this goal, we introduced cDNA sequences coding for REX009, REX115, and REX125 proteins into plasmid vectors carrying a Usp45 secretion signal, a FLAG tag sequence consensus, and a LysM-containing cA surface anchor (AcmA), thus allowing cell–surface peptidoglycan anchoring. These plasmids, or their non-FLAG/non-AcmA versions, were introduced into L. lactis host cells, thus generating unique recombinant L. lactis–REX strains. We demonstrate that all three REX proteins are expressed in L. lactis cells and are efficiently displayed on the bacterial surface, as tested by flow cytometry using an anti-FLAG antibody conjugate. Upon 10-fold concentration of the conditioned media, a REX125 secretory variant can be detected by Western blotting. To confirm that the FLAG/non-FLAG REX proteins displayed by L. lactis retain their binding specificity, cell-surface interactions of REX proteins with an IL-23R-IgG chimera were demonstrated by flow cytometry. In addition, statistically significant binding of secreted REX009 and REX115 proteins to bacterially produced, soluble human IL-23R was confirmed by ELISA. We conclude that REX-secreting L. lactis strains were engineered that might serve as IL-23/IL-23R blockers in an experimentally induced mouse model of colitis.

Highlights

Inflammatory bowel disease (IBD) is a debilitating chronic inflammation of the gastrointestinal (GI) tract
Genes for REX binders were fused with a Usp45 secretion signal, and optionally with a cAcmA surface anchor [19,33] or a FLAG-tag for detection
The characteristic microbial fingerprint of IBD microbiota consists of an increase in phylum Bacteroidetes and family Enterobacteriaceae, as well as a decrease in the phylum Firmicutes [36]

Summary

Introduction

Inflammatory bowel disease (IBD) is a debilitating chronic inflammation of the gastrointestinal (GI) tract. It can manifest itself either as Crohn’s disease (CD) or ulcerative colitis (UC). T helper Th17 cells are characteristically increased in IBD and contribute to inflammation by producing IL-17, a key pro-inflammatory cytokine of IBD [2]. The expansion and maintenance of Th17 cells is promoted by IL-23, and the whole cascade is referred to as the IL-23/IL-17 axis. The importance of the latter was substantiated by animal trials, in which the inactivation of certain components of the axis protected against IBD [1]. In patients with IBD, levels of IL-23 and Th17 cell cytokines were increased in the intestinal mucosa, plasma, and serum [3]

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