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. 2020 Jan 10;11:179. doi: 10.1038/s41467-019-13984-8

Fig. 5. FXII W268R induces and increases IL-1β secretion and expression.

Fig. 5

a–c Peripheral blood mononuclear cells were isolated from healthy control subjects by density-gradient centrifugation. Interleukin-1β (IL-1β) secretion requires a two-step process: (i) transcriptional upregulation of pro-IL-1β via toll-like receptors and (ii) conversion of pro-IL-1β into its active form by inflammasome activation. Cells were pre-stimulated with toll-like receptor agonist lipopolysaccharide (LPS), and nucleotide binding like receptor protein 3 (NLRP3) inflammasome activation was induced by the danger molecule ATP (LPS + ATP = blue symbols). Red symbols denote LPS-stimulated cells only, and green symbols represent ATP-activated cells without priming by LPS. Experiments were performed in the presence of FXII-depleted plasma (control) and addition of recombinant FXII wild-type (rFXII wt) and W268R (rFXII W268R) protein (a) or rFXII W268R-S544A (b). Experiments in (c) were conducted with purified FXII, FXIIa, and different doses of the small peptide-based inhibitor PPACK. Black arrows indicate priming effect by the mutant rFXII W268R (a) and the active site incapacitated rFXII W268R-S544A (b), which was absent in rFXII wt and control. Also, the presence of FXIIa (with or without PPACK) did not induce changes in IL-1β release (c). IL-1β secretion was assessed by ELISA. Pooled data of n = 3 different (a) or n = 2 (b, c) experiments. Bar graphs show individual data points and the mean ± s.e.m. Statistical significance in is indicated by one-way ANOVA (a, c) with Tukey’s multiple comparisons test for ATP between the groups control vs. rFXII W268R and rFXII wt vs. rFXII W268R (a) or unpaired Student’s t test (b). d, e Representative image from serial sections of the lesional skin from FACAS patient following cold exposure exhibits IL-1β immunoreactivity. IL-1β expression is pronounced around blood vessels (yellow arrows) and co-localizes with neutrophils (d) and macrophages (e) (white arrows). Original magnification ×400, n = 2 technical replicates. Source data are provided as a source data file.