Activation of NLRC4 downregulates TLR5-mediated antibody immune responses against flagellin

Abstract

Bacterial flagellin is a unique pathogen-associated molecular pattern (PAMP), which can be recognized by surface localized Toll-like receptor 5 (TLR5) and the cytosolic NOD-like receptor (NLR) protein 4 (NLRC4) receptors. Activation of the TLR5 and/or NLRC4 signaling pathways by flagellin and the resulting immune responses play important roles in anti-bacterial immunity. However, it remains unclear how the dual activities of flagellin that activate the TLR5 and/or NLRC4 signaling pathways orchestrate the immune responses. In this study, we assessed the effects of flagellin and its mutants lacking the ability to activate TLR5 and NLRC4 alone or in combination on the adaptive immune responses against flagellin. Flagellin that was unable to activate NLRC4 induced a significantly higher antibody response than did wild-type flagellin. The increased antibody response could be eliminated when macrophages were depleted in vivo. The activation of NLRC4 by flagellin downregulated the flagellin-induced and TLR5-mediated immune responses against flagellin.

Figure 1

Generation and characterization of recombinant flagellin proteins. (a) Schematic representation of the recombinant WT and variant proteins used in this study. (b) IL-8 and MCP-1 were detected to confirm the bioactivity of the recombinant proteins on TLR5 activation. (c) Peritoneal macrophages were stimulated and analyzed using an IL-1β assay to confirm the effects of the recombinant proteins on NLRC4 activation. Data are presented as means ± SEM from triplicates of one experiment that was repeated at least three times.

Figure 2

Flagellin-specific antibody response activated via the NLRC4 or TLR5 pathway (a–d). C57BL/6 WT mice were immunized with 10 μg FliCΔ90-97:L3A, FliCΔ90-97, FliC-L3A, or FliC (six mice per group) on days 0 and 28. Sera were taken from each mouse on day 21, and FliCspecific IgG (a), IgG1 (b), IgG2a (c), and IgG2b (d) production was analyzed using ELISA. The results are presented as means ±6 SEM, and the data are representative of three independent experiments.

Figure 3

The effect of FliC and FliC-L3A on cytokine production and macrophage death (a–d). The ability of recombinant flagellin proteins to stimulate cytokine production was tested in vivo. Mouse sera were collected to analyze the cytokine response 3 h after the i.p. injection of 10 μg of FliC-L3A or FliC or PBS, and the concentrations of MCP-1 (a), IL-6 (b), IL-1β (c), and IL-18 (d) in serum were assayed using ELISA. The results are presented as means ± SEM, and the data are representative of three independent experiments. (e) C57BL/6 WT mice were injected i.p. with 10 μg FliC-L3A or FliC (four mice per group), and the percentage and absolute numbers of live macrophages in the peritoneal lavage fluids were detected 12 h later. The results are presented as means ± SEM, and the data are representative of three independent experiments.

Figure 4

Depleting macrophages using Clo-LP abolishes the NLRC4- but not TLR5-mediated innate immune response. (a, b) Clo-LP depleted macrophages effectively. Splenocytes or peritoneal cells was isolated from C57BL/6 mice 1 day after treatment with Clo-LP, respectively, stained with F4/80-APC and 7AAD and analyzed using Accuri C6 (BD Biosciences). Naive and PBS-LP-treated mice were used as controls. (c–f) The depletion of macrophages did not abolish the TLR5-mediated innate immune response. Macrophage-depleted WT C57BL/6 mice were immunized i.p. with 10 μg FliCΔ90-97:L3A or FliC-L3A, and serum IL-6 (c) and MCP-1 (d) levels were assayed after 4 h using ELISA. Twenty-four hours later, splenocytes were isolated and CD80 (e) and CD86 (f) expression on CD11⁺ DCs were detected using Accuri C6 (BD Biosciences; n = 4). The results are presented as means ± SEM, and the data are representative of three independent experiments. (g, h) The effect of macrophage depletion on cytokine levels in the peritoneal lavage fluid via activation of the NLRC4 pathway. PBS-LP- or Clo-LP-pretreated C57BL/6 mice were injected i.p. with 10 μg FliC or FliC-L3A. Four hours later 600 μl PBS was injected into the abdominal cavity, and peritoneal lavage fluid was collected from each mouse and assayed for IL-18 (g) and IL-1β (h) levels (n = 4). The results are presented as means ± SEM, and the data are representative of three independent experiments.

Figure 5

The different flagellin-specific antibody responses induced by FliC-L3A and FliC were abolished after macrophage depletion. (a–d) Macrophage-depleted C57BL/6 mice and PBS-LP pretreated mice were immunized with 10 μg FliC-L3A or FliC (five mice per group). Sera were collected from each mouse on day 21 after immunization, and flagellin-specific IgG (a), IgG1 (b), IgG2a (c), and IgG2b (d) levels in serum were analyzed using ELISA. The results are presented as means ± SEM, and the data are representative of three independent experiments (n = 5).