A NOVEL LECTIN BASED LIVE BIOTHERAPEUTIC FOR THE TREATMENT OF IBD

Abstract INTRODUCTION Lectins are carbohydrate-binding proteins used by every form of life to secure nutrients, establish niches and shape host-microbe interactions. Bacterial lectins have traditionally been studied for their role in pathogenicity. Recently, we demonstrated that lectins from human associated microbiota (e.g. human microbial lectins) are common, diverse and may contribute to homeostatic rather than pathogenic interactions. Here we follow up our initial studies of the highly prevalent human microbial lectin Cbeg5 to understand its relationship to inflammatory bowel disease, myeloid differentiation and potential for therapeutic development.1 METHODS CBEG5 was aligned to a microbial gene dataset from the MetaHIT Consortium.2 Genes at 70% nucleotide identity to CBEG5 were aligned to individual sequencing reads from healthy patients and patients with IBD. Human PBMC were taken from healthy donors and exposed to Cbeg5 or a truncated Cbeg5 protein (Fn3) lacking the carbohydrate binding domain at 500 nM concentration for 24 and 48h then assayed by flow cytometry or mass cytometry. E. coli was engineered to express and secrete Cbeg5 under an inducible IPTG promoter (EC:Cbeg5) or an empty vector (EC:Con). Mouse experiments utilized 8 week C57BL/6 mice and were colonized with EC:Con or EC:Cbeg5 by oral gavage every 2 days after pre-treatment with ampicillin. Mice were administered IPTG and kanamycin in the drinking water to facilitate colonization and drive Cbeg5 expression. After 1 week of colonization mice were treated with 3.5% DSS for 5 days and monitored until recovery of weight. RESULTS CBEG5 was significantly enriched in stool samples from healthy patients compared to patients with IBD (Figure 1a). Analysis of PBMC exposed to CBEG5 demonstrated a loss of CD14+ and CD16+ monocytes and an increase in a population of CD11c+ positive myeloid cells (Figure 1b). Further analysis of PBMC using a myeloid specific flow cytometry panel demonstrated a specific loss of CD14hi monocytes and an increase in protective CD14loCD11chiCD206+ macrophages (Figure 1c). In the DSS colitis model mice treated with EC:Cbeg5 had significantly less weight loss (Figure 1d). CONCLUSION These studies suggest that Cbeg5 may play a role in the differentiation of mucosal monocytes into protective macrophages consistent with our previous mouse models. The depletion of CBEG5 expressing bacteria in patients with IBD may affect the balance of inflammatory and protective macrophage populations reinforcing mucosal inflammation. Colonization of mice with bacteria engineered to produce Cbeg5 protects against DSS colitis suggesting a novel live biotherapeutic strategy to treat IBD through restoration of lectins deficient in the IBD microbiome thereby restoring protective intestinal macrophages.