Fig 2: Deficiency of DROSHA suppresses AIM2 inflammasome activation in alveolar macrophages. (A) Quantification of interleukin (IL)-1β (left), IL-18 (middle), and TNF-α (right) secretion from wild-type (WT) alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with lipopolysaccharide (LPS) and poly(dA:dT). (n = 9 mice per group). (B) Representative immunoblot analysis for caspase-1 and IL-1β (left) and densitometry quantification of caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin) (right) from WT alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). For immunoblots, β-actin was used as loading control. (n = 3 mice per group). (C) Quantification of IL-1β and IL-18 secretion from WT alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with LPS and either ATP, flagellin, or MDP (n = 9 mice per group). (D) Representative immunofluorescence images (total 100 cells in 10 individual images per group) (left) and quantification (right) of ASC speck formation (white arrows) (the number of ASC speck positive cells in 10 individual images per group) in WT alveolar macrophages transduced with DROSHA-targeting gRNA (Drosha gRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). (n = 6 mice per group). Scale bars, 20 μm. Data are mean ± SEM. ** p <0.01; by Student’s two-tailed t-test or ANOVA.

Fig 3: MicroRNA (miRNA) promotes AIM2 inflammasome activation by ASC speck formation in macrophages. (A) Quantification of IL-1β (left), IL-18 (middle), and TNF-α (right) secretion from WT BMDMs transduced with five independent miRNA (miRNA#1, miRNA#2, miRNA#3, miRNA#4 and miRNA#5), or with a control RNA (Control), and stimulated with LPS and poly(dA:dT) (n = 9 mice per group). (B) Representative immunoblot analysis for caspase-1 and IL-1β (left) and densitometry quantification of caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin) (right) from WT BMDMs transduced with two independent miRNA (miRNA#1 and miRNA#5), or with a control RNA (Control), and stimulated with LPS and poly(dA:dT). For immunoblots, β-actin was used as loading control. (n = 3 mice per group). (C) Representative immunofluorescence images (total 100 cells in 10 individual images per group) (left) and quantification (right) of ASC speck formation (white arrows; the number of ASC speck-positive cells in 10 individual images per group) in WT BMDMs transduced with miRNA (miRNA#5), or with a control RNA (Control), and stimulated with LPS and poly(dA:dT) (n = 6 mice per group). Scale bars, 20 μm. Data are mean ± SEM. ** p <0.01, * p <0.05; by Student’s two-tailed t-test or ANOVA.

Fig 4: GH-R-Deficient Mice Are Protected from Nlrp3 Inflammasome Activation in the Visceral Adipose and from T Cell Diversity Restriction in Aging(A–D) VAT from young (3 month) and old (30 month) WTand Ghr−/− mice was analyzed for Nlrp3, Casp-1, Asc, and Il1β gene expression by real-time PCR (data are expressed as mean ± SEM; n = 3/age group/genotype).(E and F) The pro IL-1β and the adaptor protein ASC (E) and the active p20 Casp-1 with its proform (F) were analyzed by immunoblot analysis in the VAT of young (3 month) and old (30 month) WT and Ghr−/− mice (each band is representative of a single mouse).(G and H) FACS analysis depicting spleen naive (CD4+/CD8+ CD62L+ CD44−) and effector memory (E/M) (CD4+/CD8+ CD62L− CD44+)-enriched T cells from 3 months and 36 months WT and Ghr−/− mice.(I and J) Percentage of gated CD4+ and CD8+ naive T cells and effector memory T cells in the spleens of 3-month and 36-month WT and Ghr−/− mice.(K) Luminex analysis of IFNγ secretion from CD4+ CD44+ effector-memory-sorted and activated T cells from splenocytes in 12-month WT and Ghr−/− mice siblings.Data are expressed as mean ± SEM; n = 3–9/age group/genotype. Statistical differences were calculated by two-way ANOVA with Tukey’s test (*p < 0.05; A–D) and by two-tailed paired Student’s t test (*p < 0.05; I–K).

Fig 5: Tumor cell-derived ASC induces IL-1β secretion by macrophages and indirectly TSLP secretion by CAFs. a IL-1β secretion by THP-1 cells in the presence of the indicated stimuli, i.e. none (NT), PMA alone, PMA + LPS, PMA+ tumor cell sup (untreated sup), ASC-depleted or iso-rab-depleted tumor cell sups. The experiments were performed with BxPC3 (left) and A8184 (right) cell lines. Error bars represent standard deviations of triplicate values. Significance was determined using Students’ t test. b Cumulative results of independent experiments (each symbol refers to a single experiment). Percentage of inhibition was calculated on the values of THP1 in the presence of untreated sup. Significance was determined using paired Students’ t test. c IL-1β secretion by THP-1 cells in the presence of the indicated stimuli, i.e. none (NT), PMA alone, PMA + LPS, the supernatant (sup) of tumor cells (BxPC3 and A8184) either mock transfected (mock sup) or transfected with negative control siRNA (neg ctrl siRNA) or ASC siRNA. Error bars represent standard deviations of triplicate values. Significance was determined using Students’ t test. d Cumulative results of independent experiments (each symbol refers to a single experiment). Percentage of inhibition was calculated on the values of THP1 in the presence of mock-sup. Significance was determined using paired Students’ t test. e TSLP secretion by CAFs stimulated with the supernatant of THP1 cells treated with the supernatant of the indicated tumor cell lines (BxPC-3 and A8184) either mock transfected (mock sup) or transfected with control siRNA (neg ctrl sup) or ASC siRNA sup (n = 3). Error bars represent standard deviations of triplicate values. Significance was determined using unpaired Students’ t test. Values significantly different were indicated as: *p < 0.05, **p < 0.01 and ***p < 0.001