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: 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.

Fig 4: PKD kinase inhibition down-regulates NLRP3 activation.(A) THP-1 cells were treated with CRT0066101 (10 µM) or 0.1% DMSO (vehicle control) for one hour before challenge with nigericin (15 µM) for 20 min. Immunoblot of whole cell lysates probed for auto-phosphorylated PKD. Tubulin is used as loading control. One of three experiments with similar results is shown (B) Left, FACS analysis of ASC speck formation in THP-1 cells treated with 15 uM nigericin for up to 3h in the presence of 20 µM CGP084892 (used to prevent pyroptosis and the resulting speck leakage), in the presence or absence of CRT0066101 (10 µM), MCC950 (1 µM) or vehicle control. Average values of two determinations ±SD; FACS plot data are provided as supplementary material (S1 Fig). Right, IL-1ß secretion measured using PMA-primed THP-1 cells, treated with CRT0066101 (10 µM), MCC950 (1 µM) or vehicle control for one hour, before a time course of stimulation with nigericin (15 µM). This is one of three experiments with similar results. Statistical significance between inhibitor-treated samples and DMSO-treated samples was calculated with a Student T-test (C) Left, IL-1ß secretion measured in THP-1 cells after 3h of incubation with a concentration range of nigericin; corresponding immunoblots displaying auto-phosphorylated PKD and cleaved gasdermin D levels obtained upon a kinetics of stimulation with 5 µM and 15 µM nigericin. One of two similar experiments is shown. Right, Effect of concentration ranges of MCC950 and CRT0066101 on IL-1ß secretion, tested upon stimulation with nigericin (5 µM for 5h, or 15 µM for 3h). Means of triplicate determinations ±SEM. IC50 (µM) for both compounds under both experimental conditions are indicated in the table. Statistical significance was calculated with a Student T-test.

Fig 5: Inhibition of amiloride-sensitive sodium channels modulates inflammation in cells with CF-associated mutations.ELISA assays were used to detect IL-18 (A) and (B) IL-1ß in monocytes from HC (n = 9 amiloride, n = 10 S18), patients with CF (n = 10), SAID (n = 4) and NCFB (n = 4) and IL-18 (E) HBEC (n = 3, amiloride independent experiments) (F) HBEC (n = 3, S18 independent experiments). (C) Colourimetric assay was used to detect caspase-1 activity in protein lysates (HC n = 11, CF n = 11) and (D) flow cytometry was used to detect ASC specks in supernatant of primary monocytes (HC n = 5, CF n = 5). Cell stimulation was as follows: Amiloride (100 µM or 10 µM, 1 hr) or S18 derived peptide (25 µM, 4 hr) were used as a pre-treatment before a stimulation with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min. (E, F) SCNN1B over-expression in BEAS-2B cells increases pro-inflammatory cytokine secretion. (E) BEAS-2B cells were transiently transfected with 10µg SCNN1B cDNA (+) or a pcDNA3.1 vector only control (-) for 48 hr then stimulated with LPS (10 ng/mL, 4 hr) and ATP (5 mM) for the final 30 min (n = 3 independent experiments). Cells were lysed and immunoblotted for ß-ENaC and ß-actin. (F) ELISA assays were used to detect IL-18 in the supernatant fraction. A 2-way ANOVA with Tukey’s multiple comparison test was performed (p values * == 0.05, ** == 0.01, *** == 0.001 and **** == 0.0001) (*) indicate significance, when comparing HC with CF. (•) indicate significance between treatments within the same cell line.