Fig 2: High levels of sputum IFN? are associated with high levels of IL-18, IL-1a or IL-1ß. a The concentrations of IL-18, IL-1a and IL1ß in 35 sputum samples from patients either stable, having an exacerbation or convalescent were determined using the human MAPv1.6 multiplex assay. Results are colored by patient ID, and the data were fitted with a linear mixed effects model to account for patient variability and test for significant difference in exacerbation compared to stable or convalescence samples (see Additional file 1: Table S3). b Patients were grouped according to the median value at exacerbation of IL-18, IL-1a and IL-1ß and the corresponding sputum IFN? values were plotted. Lines indicate median. Statistical significance was assessed using a Student’s t-Test.

Fig 3: Telomere dysfunction activates YAP1.a Immunoblotting of intestinal enteroid lysate from G0 and G4 mice treated with or without tamoxifen for the indicated antibodies (n = 2). b Immunohistochemistry for YAP1 in the colonic crypts from the G0 and G4 mice treated with or without tamoxifen (n = 3). Scale bars, 50 µm. Insets are shown to illustrate the higher nuclear localization of YAP1 in the G4 mice than in others. c Immunohistochemistry with the colonic epithelium from healthy pediatric patients(control) and patients with telomeropathies for YAP1 and IL-18. (Control, n = 10; telomere dysfunctional patients, n = 3) d Histogram depicting the quantification of staining intensity for YAP1 and IL-18 in the colonic epithelium of healthy (control) and telomere dysfunctional patients. YAP1, p = 0.0035 IL-18, p = 0.0001. (Control, n = 10; telomere dysfunctional patients, n = 3). e qRT-PCR for the indicated genes from enteroids isolated from the YAP1(S127A) transgenic mouse treated with or without doxycycline to inhibit degradation and increase stabilization and nuclear localization (n = 3). P values were calculated using two sided t test between dox untreated and dox treated organoids. The following are the p-values for pro-Il18, p < .0001, Vgll3, p = 0.0067, Cyr61, p < 0.0001. f Microscopic images of the enteroids from a control vector transduced or YAP1 shRNAs transduced enteroids (n = 2). Scale bars, 30 µm. g qRT-PCR for the indicated genes from G4 enteroids transduced with either a control or YAP1 shRNAs (n = 2). Histogram shown with individual bar for each sample. h Western blot for the indicated proteins in CRL-1831 cells overexpressing TRF2?B?M plasmid or control vector 1-day post selection with puromycin. i Cell fractionation performed with CRL-1831 cells overexpressing TRF2?B?M plasmid or control vector 1-day post selection with puromycin. j Immunohistochemistry for YAP1 in irradiated or control G0 colon crypts showing higher nuclear positive staining for YAP1 after irradiation. Scale bars, 50 µm. k Western blot for the YAP1 and pYAPY357 phosphorylated proteins in irradiated or control G0 colon crypts showing higher levels of phosphorylation after irradiation (n = 3). l qRT-PCR of RNA from control or irradiated colonic G0 mouse crypts for pro-IL-18 (n = 3). P values were calculated using two sided t test, p = 0.0122. *statistically significant, p < 0.05 by unpaired Student’s t test, two-tailed and Fisher’s Exact Test. n represents number of mice used in the study. “Ab” denotes antibody and “I” denotes Input samples. Each experiment was conducted at least two times. Data are represented as mean ± SEM. Also refer to Supplementary Fig. 3.

Fig 4: Effect of rapamycin treatment in vivo on a patient with pVal341Leu NLRC4 mutation. (A) Reported NLRC4 mutations associated with macrophage activation syndrome are located in the Nucleotide Binding Domain. Mutation p.Val341Leu and p.His392del are framed. (B) Evaluation of circulating monocytes subsets in the patient peripheral blood before and after initiation of therapy by flow cytometry. (C) Ferritin, C-reactive protein (CRP) and IL-18 levels in sera of the patient. MAS (macrophage activation syndrome). Line infection was caused by staphylococcus epidermidis. Viral infection was rhinopharyngitis without microbial documentation. (D) Hematoxylin and eosin staining of rectal biopsy from the patient. (E) Stool microscopy. Arrow point to necrotic intestinal mucosa. (F) IL-18 staining of rectal biopsy from healthy control (left panel), patient with NLRC4 p.V341L mutation (middle panel) and colon biopsy of a patient with ulcerative colitis (left panel).

Fig 5: Induction of IFN? by endogenous IL-18 required the IL-18-producing cells to be in close proximity to NK cells. IFN? release was induced by stimulating either PBMC with IL-12 and LPS (a) or by stimulating NK cells with IL-12, LPS and monocytes in a co-culture system (b) or segregating NK and monocytes in a transwell system (c). IFN? production by NK cells was determined in the presence of Anakinra (IL-1 antagonist), IL-18BP (IL-18 antagonist), or a control IgG1. Due to donor to donor variations in the level of IFN? response, the mean values across experiments were determined after normalization to IFN? levels observed in the supernatant of NK cells stimulated in absence of antagonists. Data show the individual data points and median from n = 5 donors (a) and n = 4 donors (b-c). p’ values calculated with Friedman test with Dunn’s multiple comparison test (d) Bright field micrograph exemplifying the typical and common close proximity of IL-18+ cells (Vina green chromogen) and CD56+ cells (brown DAB) in a lung section. Scale bar denotes 18 µm