Flagellin induces antibody responses through a TLR5- and inflammasome-independent pathway

Abstract

Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1Rs and IL-18Rs, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. In this study, we define roles of known flagellin receptors and MyD88 in Ab responses generated toward flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype-specific Ab responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote Ab responses toward flagellin and coadministered OVA in C57BL/6 mice. We demonstrate IgG2c responses toward flagellin were TLR5 and inflammasome dependent; IgG1 was the dominant isotype and partially TLR5 and inflammasome dependent. Our data indicate a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5 and MyD88 dependent and caspase-1 independent. Unlike C57BL/6 mice, flagellin-immunized A/J mice induced codominant IgG1 and IgG2a responses. Furthermore, MyD88-independent, flagellin-induced Ab responses were even more pronounced in A/J MyD88(-/-) mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward coadministered OVA, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to Ab production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.

Figure 1. Flagellin induced cytokines are differentially regulated by TLR5, Naip5, Casp1, and MyD88

WT (n=7–12), TLR5^−/− (n=4–8), Naip5^−/− (n=3), Casp1^−/− (n=4–11), DKO (n=3–4), MyD88^−/− (n=3–6), and mock (n=8–10) mice were injected i.p. of FliC (30 μg) or PBS (mock). Serum was collected 2 hours after injections and cytokine levels were determined by ELISA. IL-12/23p40 (A), IL-18 (B), IL-6 (C) and Cxcl1 (D). All groups have a minimum n=3. Statistical analysis was done using one-way ANOVA with Bonferroni multiple comparisons post-test: ** = P<0.01, *** = P<0.001.

Figure 2. IgG1 anti-FliC responses are partially and IgG2c anti-FliC are entirely MyD88-dependent

WT (n=9) and MyD88^−/−(n=7) mice were immunized with 30 μg FliC on day 1 and 21 and sera collected on days 14 and 35. Naïve, day 14, and day 35 sera were analyzed for IgG1 (A) and IgG2c (B) antibody responses against FliC by ELISA. Data is a combination of two-independent experiments with n = 3–5 per experiment. Statistical analyses were done using Mann-Whitney analysis of individual groups: ** = P < 0.01.

Figure 3. IgG2c anti-FliC responses are partially TLR5- and inflammasome-dependent

TLR5^−/− (n=19) (A, D), Naip5^−/− (n=9) (B, E), and Casp1^−/− (n=14) (C, F) mice were immunized twice on day 1 and day 21 and sera collected on days 14 and 35. Naïve, day 14, and day 35 sera were analyzed for IgG1 (A–C) and IgG2c (D–F) isotype specific antibody responses against FliC by ELISA. Data is a combination of 2–3-independent experiments with n = 3–6 per experiment. Statistical analyses were done using Mann-Whitney analysis of individual groups: *= P < 0.05, ** = P < 0.01.

Figure 4. IgG1 anti-FliC responses are partially TLR5- & caspase-1-independent

TLR5^−/−/Casp1^−/−, DKO (n=10) mice were immunized twice on day 1 and day 21 and sera collected on days 14 and 35. Naïve, day 14, and day 35 sera were analyzed for IgG1 (A) and IgG2c (B) isotype specific antibody responses against FliC by ELISA. Data is representative of two-independent experiments with n= 4–5 per group. Statistical analyses were done using Mann-Whitney analysis of individual groups: ** = P < 0.01.

Figure 5. IgA anti-FliC responses are TR5- & MyD88-dependent

C57BL/6 (n=17) (A), TLR5^−/− (n=14) (B), Naip5^−/− (n=9) (C), and Casp1^−/− (n=11) (D), TLR5^−/−/Casp1^−/−, DKO (n=9) (E), MyD88^−/− (n=11) (F) mice were immunized on day 1 and day 21 and sera was collected on days 0 (naïve), 14 and 35. Naïve and day 35 sera (boost) were analyzed for IgA specific antibody responses against FliC by ELISA. Data is a combination of 2–3-independent experiments with n = 3–6 per experiment. Statistical analyses were done using Mann-Whitney analysis of individual groups: *= P < 0.05, *** = P < 0.001.

Figure 6. Flagellin induced IgG1 anti-OVA responses are partially MyD88-dependent

WT, mice were immunized twice on day 1 and day 21 with OVA alone (n=5) or OVA plus FliC (n=5), and sera collected on days 14 and 35. A) Naïve, day 14, and day 35 sera were analyzed for IgG1 specific antibody responses against OVA by ELISA. B–F) WT, TLR5^−/− (n=19) (B), Naip5^−/− (n=9) (C), Casp1^−/− (n=14) (D), MyD88^−/− (n=7) (E) and TLR5^−/−/Casp1^−/−, DKO (n=10) (F), mice were immunized twice on day 1 and day 21 with OVA plus FliC, and sera collected on days 14 and 35. Naïve, day 14, and day 35 sera were analyzed for IgG1 specific antibody responses against OVA by ELISA. B–F) Data is a combination of 2–3-independent experiments with n=3–6 per experiment. Statistical analyses were done using Mann-Whitney analysis of individual groups: *= P < 0.05, *** = P < 0.001.

Figure 7. Defective flagellin-induced IL-18 in A/J mice

C57BL/6 (n = 5) and A/J (n=5) mice were injected i.p. with FliC (30μg). Serum was collected 2 hours after injections and cytokine levels were determined by ELISA: Cxcl1 (A), IL-6 (B), IL-12/23p40 (C), IL-18 (D), IL-1β (E), and TNF (F). Statistical analysis was done using one-way ANOVA with Bonferroni multiple comparisons post-test: * = P<0.05.

Figure 8. Augmented IgG1 and IgG2a anti-FliC responses in A/J mice are partially MyD88-dependent

C57BL/6 (n=9) and A/J mice (n=7), and their MyD88-defiecient counterparts (n=7–9) were immunized twice on day 1 and day 21 with FliC plus OVA and sera collected on days 14 and 35 were analyzed for IgG1 (A), and IgG2a or IgG2c (B) responses against FliC by ELISA. C) Naïve, day 14, and day 35 sera were assessed for IgG1 anti-OVA responses by ELISA. Data is a combination of two-independent experiments with n = 3–5 per experiment. Statistical analyses were done using Mann-Whitney analysis of individual groups: *= P<0.05, ** = P<0.01.

Figure 9. A/J MyD88^−/− mice generate robust IgA anti-FliC responses

A/J mice (n=8), and their MyD88-defiecient counterparts (n=7) were immunized twice on day 1 and day 21 with FliC plus OVA and sera collected on days 0 (naïve), 14 and 35. Naïve and day 35 sera were analyzed for IgA specific antibody responses against FliC by ELISA (A, B). Data is a combination of two-independent experiments with n = 3–4 per experiment. Statistical analyses were done using Mann-Whitney analysis of individual groups: ** = P<0.01.