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. 2015 Apr 27;13(4):514–523. doi: 10.1038/cmi.2015.33

Figure 4.

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.