The A4-Fla2 flagellin, a dominant antigen in Crohnʼs Disease, is a poor TLR5 agonist

Approximately half of patients with Crohn's disease (CD) have measurable serum antibodies to CBir1, a flagellin protein from a nonculturable member of the Lachnospiraceae group of Clostridiales order bacteria. A taxonomically related organism, designated A4, is found in high numbers in the normal mouse colon and expresses a flagellin, Fla2, which also strongly reacts with CD sera. Inflammatory responses to flagellin in the intestinal epithelium are mediated by Toll-like receptor 5 (TLR5) but the role of TLR5 in the generation of pathologic anti-flagellin antibodies in CD remains unknown and has been called into doubt by recent findings that TLR5 is not required for the colitigenic effects of flagellin in the DSS colitis model. It has previously been reported that CBir1 causes cytokine release from hemopoietic cells, but its direct TLR5 agonist activity has never been quantified. To determine if Fla2 and other flagellins that react with CD sera have different TLR5-stimulatory activity than flagellins from Enterobacteriaceae. 1) Recombinant Fla2 and E. coli FliC were used to stimulate HEK 293T cells transiently expressing murine or human TLR5, and IL-8 and CCL20 production were measured. 2) C57Bl/6 mice were injected intraperitoneally with Fla2 or FliC, and serum IL-8 measured after 3h. 3) The fla2 and fliC genes were cloned into a low-copy expression system under control of the native E. coli fliC promoter in E. coli with a chromosomal fliC deletion, and motility was measured. As previously reported, FliC was a potent agonist for both human and murine TLR5 in HEK 293T cells, with an EC50 of 1-5 ng/ml. In contrast, Fla2 was a much weaker agonist, with an EC50 of 100-500 ng/ml for murine TLR5, and greater than 1 μg/ml for human TLR5. This potency difference was also evident in the in vivo model, where IL-6 production was markedly higher in FliC treated mice (4332±1563 pg/ml) than in Fla2-treated mice (111±66 pg/ml). Finally, unlike FliC, Fla2 was unable to confer motility to E. coli:fliC- in the native expression system. Motility of the A4 organism itself was not measured due to the requirement for strict anaerobic conditions. The A4-Fla2 flagellin is a poor agonist for TLR5, with higher activity for murine than human TLR5. This manifests in vivo as a weak inflammatory response to IP injection compared to E. coli FliC. Fla2 also appears to lack the ability to confer motility to E. coli, although the motility of the native A4 strain remains unknown. These results support the hypothesis that TLR5 stimulation and the development of pathological adaptive immune responses in IBD may be inversely correlated.

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