Fig 2: Inflammatory serum cytokine signature in CF.(A) ELISA assays were used to detect IL-18 (HC, n = 10, CF, n = 30, SAID, n = 10, NCFB, n = 4), (B) IL-1ß (HC, n = 10, CF, n = 30, SAID, n = 10, NCFB, n = 4), (C) IL-1Ra (HC, n = 10, CF, n = 30, SAID, n = 7, NCFB, n = 4) in patient sera. Outliers in SAID group for IL-1ß and IL-1Ra correspond to HIDS one and A20 deficiency (D) Flow cytometry was used to detect ASC specks (HC, n = 10, CF, n = 15, SAID, n = 10, NCFB, n = 4) in patient sera. (E) A colorimetric assay to detect caspase-1 activity in sera of patients with CF, SAID and NCFB as a percentage of HC (HC, n = 10, CF, n = 15, SAID, n = 4, NCFB, n = 4). Of note, an undetermined amount of detected IL-1Ra is attributed to circulating Anakinra (recombinant IL-1Ra) specifically in the SAID cohort. The Kruskal-Wallis non-parametric test, with Dunn’s multiple comparison test, was performed (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001; error bars ± S.E.M).10.7554/eLife.49248.008Figure 3—source data 1.Serum cytokine levels for all patient groups.

Fig 3: Pro-inflammatory response in A20-deficient patient. Cytokines were measured by ELISA using supernatant from patient and healthy donors. The median confidence interval presented from two separate patient samples. PBMCs were treated with or without MCC950 (NLRP3 inhibitor) and stimulated for 4 h with LPS (10 ng/mL) followed by 1 h ATP (1 mM). Pro-inflammatory responses to NLRP3-dependent activation of (A) IL-1ß and (B) IL-18, and in measurements of (C) IL-6, (D) TNFa, (E) IL-10, (F) IL-8 xtbf, (G) IL-17a, and (H) IL-1a. The median confidence interval or repeated patient measurements is presented. A two-way ANOVA statistical test was performed (*p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001). (I) ASC specks found in the HA20 patient at twice the normal range indicate NLRP3 inflammasome activation and pyroptosis. Treatment concentrations as described in panel (A). Measurements were taken for 7 CAPS patients which also represent a pro-inflammatory response although not as profound as the HA20 response. ASC specs were measured as previously described (11).

Fig 4: LPS-induced IL-1ß/IL-18 secretion in human monocytes is higher in CF and is NLRP3 inflammasome dependent.Primary monocytes from HC and CF (HC, n = 10; CF, n = 10) were unstimulated or stimulated with LPS which specifically targets TLR4 (10 ng/mL) for 4 hr before being stimulated for 4 hr with Flagellin (10 ng/mL with Lipofectamine 2000) for NLRC4 inflammasome, or TcdB (10 ng/mL) for Pyrin inflammasome or poly(dA:dT) dsDNA (1 µg/mL with Lipofectamine 2000) for AIM2 inflammasome. ELISA assays were used to detect (A) IL-18 and (B) IL-1ß cytokine secretion in supernatants. To monitor NLRP3 inflammasome activation, primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4) were pre-incubated with MCC950 (15 µM), OxPAPC (30 µg/mL) and YVAD (2 µg/mL) for 1 hr before a stimulation with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for the final 30 min. ELISA assays were used to detect (C) IL-18 and (D) IL-1ß cytokine secretion in supernatants and (E) a colourimetric assay was used to detect caspase-1 activity in protein lysates (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4). (F) Flow cytometry was used to detect ASC specks in supernatants of primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 6; NCFB, n = 4) for ±LPS/ATP and (HC, n = 5; CF, n = 5) for MCC950 with LPS/ATP. (G) Necrosis and pyroptosis are represented as superimposed bar charts. Total necrosis was measured using LDH release assay. For pyroptotic cell death, each sample/condition was repeated in parallel with a caspase-1 inhibitor (YVAD (2 mg/mL, 1 hr)) pre-treatment. The total necrosis level was taken away from the caspase-1 inhibited sample, or ‘caspase-1 independent’ necrosis, with the remaining LDH level termed ‘caspase-1 dependent necrosis’ or pyroptosis. Cells were then stimulated with LPS (10 ng/mL, 4 hr), and ATP (5 mM) for final 30 min. The assay was performed with primary monocytes from HC, CF, SAID and NCFB (HC, n = 10; CF, n = 10; SAID, n = 4; NCFB, n = 4). (?) Significance for Total Necrosis (?) Significance for pyroptosis. A 2-way ANOVA statistical test was performed, with Tukey post-hoc correction (p values * =< 0.05, ** =< 0.01, *** =< 0.001 and **** =< 0.0001; error bars ± SEM). Inhibitor treatments in panels a-c were found to significantly reduce cytokine secretion and caspase-1 activity to **p =< 0.01 or less, for CF and SAID groups respectively. Significance values not displayed on the graph.

Fig 5: Irg1 phenotype is associated with dysregulation of caspase-1 and GSDMD processing(A) BMDMs were stimulated as in Figure 1H. Nigericin (Nig) for 1 h was used to trigger inflammasome. Fixed and permeabilized cells were analyzed for ASC scattering properties by flow cytometry.(B) Protein levels analyzed by western blot in supernatants of BMDMs stimulated as in Figure 1H, ATP was added for the indicated time.(C) IL-1ß secretion in BMDMs treated as in Figure 1H. Caspase inhibitors (20, 50, and 100 µM) were added 30 min before ATP. Data are mean ± SEM, with n = 3 experiments.(D) GSDMD detection in cell lysates of BMDMs treated as in Figure 1H. VX-765 (caspase-1 inhibitor, 50 µM) was added 30 min before ATP. Western blots are representative of n = 3 experiments. GAPDH was used as loading control.See also Figure S2.