Siglec-7 Mediates Immunomodulation by Colorectal Cancer-Associated Fusobacterium nucleatum ssp. animalis

Fusobacterium nucleatum is involved in the development of colorectal cancer (CRC) through innate immune cell modulation. However, the receptors of the interaction between F. nucleatum ssp. and immune cells remain largely undetermined. Here, we showed that F. nucleatum ssp. animalis interacts with Siglecs (sialic acid–binding immunoglobulin-like lectins) expressed on innate immune cells with highest binding to Siglec-7. Binding to Siglec-7 was also observed using F. nucleatum-derived outer membrane vesicles (OMVs) and lipopolysaccharide (LPS). F. nucleatum and its derived OMVs or LPS induced a pro-inflammatory profile in human monocyte-derived dendritic cells (moDCs) and a tumour associated profile in human monocyte-derived macrophages (moMϕs). Siglec-7 silencing in moDCs or CRISPR-cas9 Siglec-7-depletion of U-937 macrophage cells altered F. nucleatum induced cytokine but not marker expression. The molecular interaction between Siglec-7 and the LPS O-antigen purified from F. nucleatum ssp. animalis was further characterised by saturation transfer difference (STD) NMR spectroscopy, revealing novel ligands for Siglec-7. Together, these data support a new role for Siglec-7 in mediating immune modulation by F. nucleatum strains and their OMVs through recognition of LPS on the bacterial cell surface. This opens a new dimension in our understanding of how F. nucleatum promotes CRC progression through the generation of a pro-inflammatory environment and provides a molecular lead for the development of novel cancer therapeutic approaches targeting F. nucleatum-Siglec-7 interaction

Dectin-2 recognises mannosylated O-antigens of human opportunistic pathogens and augments lipopolysaccharide activation of myeloid cells

Lipopolysaccharide (LPS) consists of a relatively conserved region of lipid A and core-oligosaccharide, and a highly variable region of O-antigen polysaccharide. While lipid A is known to bind to the toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and the mannosylated O-antigen found in a human opportunistic pathogen Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared to Salmonella enterica O66 LPS which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-β1,3]GalNAc-α1,3-GalNAc-β1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognise H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2 dependent, as Dectin-2 knockout BM-DCs failed to do so. This receptor crosstalk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a, also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several gram-negative bacteria augment TLR4 responses through interaction with Dectin-2

Mechanistic insights into the interaction between gut bacteria polysaccharides and lectins of the host innate immune system

Colorectal cancer (CRC) is one of the most common cancer types worldwide, with cases showing the dominance of Bacteroides fragilis and Fusobacterium nucleatum species. While B. fragilis is involved in tumorigenesis through the toxin that it secretes, F. nucleatum is implicated in CRC progression through the recruitment of tumour infiltrating immune cells. However, the mechanisms underpinning the interaction between F. nucleatum ssp. and immune cells remain to be determined. Mammalian lectins expressed by immune or epithelium cells are glycan-binding proteins playing an important role in host cell physiology. The Siglec superfamily and Galectin-3 have been shown to play a role in tumour progression, and Dectin-2 has been shown to activate immune response. Here, we tested the hypothesis that the interaction of cell surface glycoconjugates present on B. fragilis and F. nucleatum ssp. with host lectins may mediate immune response and tumour progression. Our results showed that, among B. fragilis and the three F. nucleatum subspecies (ATCC 10953, ATCC 25586, ATCC 51191) tested, F. nucleatum ssp. bound to Siglecs and specifically to Siglec-7. Further, we showed that the lipopolysaccharide (LPS) derived from F. nucleatum ssp. bound to Siglec-7 through novel sugar epitopes contained in the LPS structures and we also uncovered a novel LPS structure for F. nucleatum ATCC 51191 strain. Moreover, we purified and characterised the outer membrane vesicles (OMVs) from F. nucleatum ssp. and showed that they interact with Siglec-7, as we demonstrated with the whole bacteria or LPS. We then showed that F. nucleatum ssp. and their derived LPS and OMVs induced a pro-inflammatory phenotype in dendritic cells and a tumour-promoting phenotype in macrophages. Depletion of Siglec-7 in human myeloid cells led to a change in bacteria internalisation and immune response induced by F. nucleatum ssp. Finally, we showed that F. nucleatum ssp. abundance was elevated in the on-tumour as compared to the off-tumour site across CRC clinical samples tested. In addition, our preliminary data reported the presence of anti-F. nucleatum ssp. IgG antibodies and high levels of the Siglec-7 protein in the serum of CRC patients, which further support F. nucleatum-Siglec-7 interaction as a novel underlying mechanism implicated in CRC