Amino acids 89-96 of Salmonella typhimurium flagellin represent the major domain responsible for TLR5-independent adjuvanticity in the humoral immune response

Abstract

Toll-like receptor 5 (TLR5) signaling in response to flagellin is dispensable for inducing humoral immunity, but alterations of aa 89-96, the TLR5 binding site, significantly reduced the adjuvanticity of flagellin. These observations indicate that the underlying mechanism remains incompletely understood. Here, we found that the native form of Salmonella typhimurium aa 89-96-mutant flagellin extracted from flagella retains some TLR5 recognition activity, indicating that aa 89-96 is the primary, but not the only site that imparts TLR5 activity. Additionally, this mutation impaired the production of IL-1β and IL-18. Using TLR5KO mice, we found that aa 89-96 is critical for the humoral adjuvant effect, but this effect was independent of TLR5 activation triggered by this region of flagellin. In summary, our findings suggest that aa 89-96 of flagellin is not only the crucial site responsible for TLR5 recognition, but is also important for humoral immune adjuvanticity through a TLR5-independent pathway.

Figure 1

Characterization of mutant flagellin proteins. (a) SDS–PAGE analysis of the recombinant mutant flagellin proteins expressed from pTrc99a. (b) Western blot analysis of the recombinant mutant flagellin proteins expressed from pTrc99a. (c) Western blot analysis of the aa 89–96 fliC→flaA mutant flagellin protein expressed from pET30a. M indicates the molecular size markers in kDa.

Figure 2

TLR5 dose–response curves to the purified recombinant mutant flagellin proteins. (a) Dose–response curve of IL-8 production by HEK293-mTLR5 cells stimulated with purified recombinant mutant flagellin proteins at concentrations of 0.1–1000 ng/ml. (b) Dose–response curves of IL-8 production by wild-type HEK293 cells stimulated with purified recombinant mutant flagellin proteins at concentrations of 0.1–1000 ng/ml. (c) NF-κB pathway activation in response to stimulation with the recombinant mutant flagellin proteins. Cells were stimulated 0.5 ng/ml flagellin. Data are presented as the means±s.d. of three independent experiments that we performed in triplicate. Error bars represent one s.d. Statistical analyses were done using Mann–Whitney analysis relative to the unstimulated well; *P<0.05. TLR, Toll-like receptor.

Figure 3

IL-6 and TNF-α pro-inflammatory cytokine levels in mouse serum induced by flagellin. One hour after i.p. injection with 1-µg dose mutant flagellin protein, sera were collected for ELISA. Error bars represent one s.d. Data are presented as the means±s.d. of three independent experiments. N=5 mice per group. (a) IL-6. (b) TNF-α.

Figure 4

Deletion of the hypervariable region in flagellin significantly enhances the secretion of the inflammatory cytokines IL-1β and IL-18 by ex vivo stimulated PECs. Each mutant flagellin was prepared 10 µg/ml concentration, except for the mutant flagellin pTrc99a-fliC-SJW61 (7.73 µg/ml), and cells were stimulated for 24 h. Data are presented as the means±s.d. of three independent experiments that were performed in triplicate. Error bars represent one s.d. Statistical analyses were done using Mann–Whitney analysis; *P<0.05. (a) IL-1β inflammatory cytokine production. (b) IL-18 inflammatory cytokine production. PEC, peritoneal exudate cell.

Figure 5

The adjuvant activity of mutant flagellin proteins expressed from pTrc99a. (a) Comparison of the adjuvant activities of the groups immunized with 0.5 µg pTrc99a-expressed flagellin mixed with 50 µg OVA. Mice were immunized with OVA alone or with 50 µg of OVA and a mutant flagellin protein on days 0 and 14. OVA-specific IgG titers were assayed on day 28 by ELISA. (b) A comparison of the adjuvant activities of the groups immunized with pTrc99a-expressed mutant flagellins mixed with inactivated H5N1 vaccine strain virions. Mice were immunized with H5N1 Re-6 alone or were co-injected with 0.2 µg H5N1 Re-6 and 0.5 µg mutant flagellin on days 0 and 14. The HA titer was assayed on day 28. Lines indicate the arithmetic means. Each point represents the serum HI titer for an individual mouse. Error bars represent one s.d. Data are presented as the means±s.d. of three independent experiments. N=5 mice per group. Statistical analyses were performed using Mann–Whitney analysis; *P<0.05.

Figure 6

The amino acids 89–96 of flagellin represents the key site for the adjuvant effect in humoral immunity, but is independent of typical TLR5 binding characteristics. A comparison of the adjuvant activities in mice immunized with 20 µg pTrc99a-fliC-89–96 flagellin or pTrc99a-fliC-WT flagellin mixed with 50 µg OVA in TLR5KO and WT mice. Mice were injected on days 0 and 14. OVA-specific IgG titers were measured on day 28 by ELISA. Data are presented as the means±s.e.m. of two independent experiments. Error bars represent one s.d. Statistical analyses were carried out using Mann–Whitney analysis; *P<0.05. For PBS-treated TLR5KO mice, n=3 per group; for other cohorts, n=5 per group.