Diversity of gut commensal flagellins results in distinct intestinal epithelial cell immune responses

Abstract Flagellins from bacteria in the Lachnospiraceae family are immunodominant antigens in inflammatory bowel disease. Flagellin is the basic unit of flagella which is used for bacterial motility and is recognized by toll-like receptor 5 (TLR5). Due to the diversity in flagellated organisms that reside in proximity to the intestinal epithelium, we asked whether this resulted in distinct interactions between commensal flagellins and the immune system. Analysis of over 200 flagellins, primarily from Lachnospiraceae organisms, revealed a divergence in peptide sequences in specific non-conserved regions of the D0 domain that broadly separate the flagellins examined into 2 clusters which we refer to as Group 1 and Group 2 flagellins. We stimulated HEK-Blue mTLR5 cells and murine intestinal epithelial cells (IECs) with selected Group 1 or Group 2 flagellins to assess TLR5 activity and IEC production of lipocalin-2 (Lcn-2) - a biomarker of inflammation - respectively. Group 2 flagellin strongly activated TLR5 signaling in the mTLR5 HEK cell line, even more so than Salmonella typhimurium (S. typhi) flagellin. Conversely, cells stimulated with the Group 1 flagellin exhibited significantly reduced TLR5 signaling relative to both Group 2 and S. typhi flagellins. While IEC stimulation with Group 1 flagellin resulted in undetectable Lcn-2, stimulation with Group 2 flagellin led to robust Lcn-2 production; more so than stimulation with S. typhi flagellin. These findings demonstrate that flagellins of commensal bacteria elicit differential IEC response via selective activation of TLR5. These functional differences are dependent on specific motifs in non-conserved regions of their D0 domains. Supported by R01 AI162736, R21 DK118386, T32 AI007051.