Figure 6.

Circulating IL-18 levels are significantly reduced in interleukin (IL)-18BPKO mice, yet they produce comparable amounts of pro-form IL-18 as wild type (WT), and release similar amounts of IL-18 following stimulation. (A) IL-18 ELISA results from submandibular plasma from WT (N = 21) and IL-18BPKO (N = 23) male and female mice. IL-18 levels were below limit of detection among all tested IL-18BPKO animals. Bars represent median. Significance calculated with Mann–Whitney U test (****p < 0.0001). (B) Representative IL-18 immunohistochemistry results from splenic sections from WT (N = 10), IL-18BPKO (N = 10), and IL-18KO (control) animals. Splenic IL-18-producing cells are similarly distributed among WT and IL-18BPKO mice and are most abundant within the red pulp, likely among red pulp macrophage. However, IL-18-expressing cells can also be observed in the marginal zone as well as within the white pulp. (C) Splenocyte lysates from WT and IL-18BPKO mice contain similar amounts of pro-form IL-18. Western blot showing pro-form IL-18 (~23 kD) is present in both WT and IL-18BPKO splenic lysates. We observed no mature or cleaved forms of IL-18 in lysates from WT and IL-18BPKO mice. 1 ng of recombinant murine IL-18 (rmIL-18) was ran as a control. Representative blot from total of eight WT and seven IL-18BPKO splenic lysates. (D) IL-18BPKO thioglycollate-elicited peritoneal macrophage (TEM) release comparable amounts of IL-18 as WT TEM following stimulation. TEM were cultured in the presence or absence of LPS (1 µg/mL) overnight. Cultures were then washed and treated with increasing concentrations of nigericin for 90 min to activate the NLRP3 inflammasome and promote release of mature IL-18. We observed no difference in IL-18 release between WT and IL-18BPKO TEM at any concentration of nigericin. Data are from combined results of three separate experiments per genotype, using pooled TEM from three mice per experiment. Bars represent mean and error bars are SD.