Fig 2: Deletion of IL-1α in pancreatic tissue impairs whole body glucose tolerance as male mice age. (A) Il1a and (B) cre expression in islets isolated from IL-1αfl/fl and IL-1αPdx1−/− mice (n = 8–14 per group). (C) IL-1α abundance was determined in islets isolated from IL-1αfl/fl and IL-1αPdx1−/− mice that were exposed to 10 ng/mL recombinant IL-1β for 4 h (n = 3 per group pooled and run in duplicate). (D) Body mass, (E) fat mass, (F) lean mass, and (G) fluid mass in male IL-1αfl/fl and IL-1αPdx1−/− mice from 2 to 9 months of age. (H) GTT performed in 4-month-old male mice. (I) Area under the curve (AUC) for GTT shown in panel H. (J) ITT conducted in 2 month old male mice. (K) GTT performed in 8 month old male mice. (L) Area under the curve (AUC) for GTT shown in panel K. (A–B) n = 8–14 per group; (D–L) n = 9–16 per group. ∗∗∗∗, p < 0.0001; ∗∗, p < 0.01; ∗, p < 0.05; #, p < 0.10.

Fig 3: Expression of the Il1a gene is enhanced in response to IL-1β in lean mice and increased in the islets of obese rodents. (A) Islets isolated from 8-week-old male fa/+ and fa/fa rats, and (B) male (8 week old) db/+ and db/db mice. (C) Islets isolated from female IL-1Rfl/fl and IL-1RPdx1−/− mice were untreated (NT) or treated with 10 ng/mL IL-1β for 3 h. (D) 832/13 rat insulinoma cells and (E) INS-1E insulinoma cells were either untreated (NT) or treated with 1 ng/mL of IL-1β for the indicated times. (F) Isolated rat and (G) human islets were either untreated or stimulated with 10 ng/mL IL-1β for 3 h. (A–G) Il1a transcript data are shown as means ± SEM from 3 to 6 individual experiments. ∗∗, p < 0.01 vs. NT; ∗, p < 0.05 vs. NT.

Fig 4: FeTPPS selectively inhibits recombinant HMGB1-mediated caspase-11 activity in vitro.A, B Production of IL-1α, IL-1β, TNFα, and IL-6 (as measured by ELISA) of WT or Casp11−/− peritoneal macrophages stimulated with LPS alone (1 μg/mL) or LPS (1 μg/mL) + HMGB1 (400 ng/mL) in the absence or presence of indicated doses of FeTPPS for 16 h. Data presented as mean ± SD of technical replicates. ****P < 0.0001 (unpaired t-test). C Western blots for IL-1α, caspase-11, and caspase-1 in the supernatants or GSDMD-NT, caspase-11, IL-1α, IL-1β, caspase-1 in the cell lysates from WT or Casp11−/− peritoneal macrophages stimulated with LPS alone (1 μg/mL) or LPS (1 μg/mL) + HMGB1 (400 ng/mL) in the absence or presence of indicated doses of FeTPPS for 16 h. D Necrotic cell lysate preparation is shown in the left panel. Western blots for HMGB1, IL-1α, caspase-11, and caspase-1 in the supernatants or GSDMD-NT, Caspase-11, IL-1α, IL-1β, and caspase-1 in the cell lysates from WT or Casp11−/− peritoneal macrophages stimulated with LPS alone (1 μg/mL), LPS (1 μg/mL) + HMGB1+/+, or HMGB1−/− MEF cells in the absence or presence of FeTPPS for 16 h. E Primary hepatocytes isolated from mice of indicated genotypes co-cultured with WT mouse peritoneal macrophages are shown in the left panel. IL-1a and IL-1β released from macrophages after the stimulation of LPS (1 μg/mL) in the absence or presence of FeTPPS are shown in the right panel. Data presented as mean ± SD of technical replicates. Two-way ANOVA was used (IL-1α hmgb1f/f Albcre− LPS vs. LPS + FeTPPS **P = 0.004; hmgb1f/f Albcre− + LPS vs. hmgb1f/f Albcre+ + LPS ****P < 0.0001; IL-1β hmgb1f/f Albcre− LPS vs. LPS + FeTPPS **P = 0.0025; hmgb1f/f Albcre− + LPS vs. hmgb1f/f Albcre+ + LPS ****P < 0.0001). F Production of IL-1β (as measured by ELISA), Western blots of supernatants and cell lysates to detected IL-1β, Caspase-1 cleavage from WT or NLRP3−/− peritoneal macrophages primed with LPS 100 ng/mL and indicated stimulation. G Western blots for IL-1α, caspase-4 (Casp4), GSDMD-NT in the supernatants or cell lysates. LDH assay and ELISA for IL-1α in the supernatants of PMA-primed human monocytic THP-1 cells transfected with scrambled siRNA or CASP4-specific siRNA upon HMGB1 (400 ng/mL) and LPS (1 μg/mL) stimulation in the absence or presence of FeTPPS. Data presented as mean ± SD of technical replicates. An unpaired t-test (two-sided) was used (LDH LH vs. LH + FeTPPS 2.5 μM **P = 0.0085; LH vs. LH + FeTPPS 5 μM **P = 0.0071; LH scramble vs. LH casp4 siRNA **P = 0.0043; IL-1α LH vs. LH + FeTPPS 2.5 μM *P = 0.0328; LH vs. LH + FeTPPS 5 μM *P = 0.0198; LH scramble vs. LH casp4 siRNA ***P = 0.004). Graphs are representative of at least three independent experiments.