Fig 1: FeTPPS attenuated caspase-11-dependent immune responses and lethality in sepsis.A–D WT or Casp11-/- mice were pretreated with FeTPPS 6 mg/kg or saline by intraperitoneal injection 1 h before intraperitoneal challenge with 25 mg/kg LPS. Serum levels of IL-1a, IL-1ß, TNFa, IL-6 (A) (unpaired t-test, n = 5, ****P < 0.0001), western blots for GSDMD-NT, caspase-11 in lung (B), hematoxylin and eosin (H&E) stained sections of lung. Scale bar: 50 µm (C) and kaplan–Meier survival curves (D). Significance assessed using Log-rank test: LPS (WT n = 14) vs. LPS + FeTPPS (WT n = 14), *P = 0.0422; WT LPS (n = 14) vs. Casp11-/- LPS (n = 10), ****P < 0.0001. E–H WT or Casp11-/- mice were pretreated with FeTPPS 6 mg/kg or saline by intraperitoneal injection 1 h before subjected to cecum ligation and puncture (CLP) or sham operation. Serum levels of IL-1a, IL-1ß (E), western blots for GSDMD-NT,Caspase-11 in lung (F), hematoxylin and eosin (H&E) stained sections of lung. Scale bar: 50 µm (G) and kaplan–Meier survival curves (H). Significance assessed using Log-rank test: CLP (WT n = 10) vs. CLP + FeTPPS (WT n = 10), **P = 0.0036; CLP WT (n = 10) vs. Casp11-/- (n = 10), ****P < 0.0001. I WT or Casp11-/- mice were injected with FeTPPS 6 mg/kg 1 h before intraperitoneal challenge with 25 mg/kg LPS. Plasma concentrations of PAI-1 and TAT were measured. An unpaired t-test was used in PAI-1 LPS(WT n = 5) vs. LPS+FeTPPS (WT n = 5), ****P < 0.0001; WT LPS (n = 5) vs. Casp11-/- LPS (n = 5) ***P = 0.0001 and TAT LPS (WT n = 5) vs. LPS + FeTPPS (WT n = 5), *P < 0.0299; WT LPS (n = 5) vs. Casp11-/- LPS (n = 5) ****P < 0.0001.
Fig 2: PERK inactivation and cytokines expression.(A) IL-10 and TNF secretion was measured by Legendplex in Flt3-L BMDCs activated with LPS and treated or not with ISRIB or GSK2656157 for 4 h. (B) IL-1ß mRNA expression measured by qRT-PCR (left) and secretion by ELISA (right) in WT and PERK?K Flt3-L BMDCs stimulated with LPS during 4 h and with ATP for the last 30 min of treatment. (C) IL-1ß mRNA expression measured by qRT-PCR (left) and secretion by ELISA (right) in Flt3-L BMDCs activated with LPS and treated with GSK2656157 and/or ISRIB for 4 h and with ATP for the last 30 min of treatment. (A, B, C) Statistical analysis was performed using Dunnett’s multiple comparison (A, B) and Wilcoxon test (C). Data are mean ± SD (n = 3). independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).
Fig 3: Anti-TNF ameliorates severe systemic phenotype induced in Card14LSL-E138A/LSL-E138ARosa26CreERT2/+ mice Card14LSL-E138A/LSL-E138ARosa26CreERT2/+ mice were intraperitoneally injected with tamoxifen on d0, 1 and 2 and intraperitoneally injected with either isotype control, anti-TNF, or anti-GR1 on days -1, 1 and 3.Mice were sacrificed on d5. (A) Mouse weight was monitored from d0 until the experiment end. Isotype (n = 24, from four independent experiments), anti-TNF (n = 11, from two independent experiments), anti-GR1 (n = 11, from two independent experiments). For each day, differences between groups were analysed by one-way ANOVA. (B) Mouse temperatures were taken by rectal thermometer on d5. Card14+/+ control mice are a mixture of Cre-, Rosa26CreERT2/+ and Krt14CreERT2 mice (from four independent experiments). Card14LSL-E138A/LSL-E138A Rosa26CreERT2/+ mice: isotype (from four independent experiments), anti-TNF (from two independent experiments), anti-GR1 (from two independent experiments). (C) Numbers of S100A9+ myeloid cells were calculated from stained sections of kidney, liver, and lung tissue taken from isotype, anti-TNF or anti-GR1 treated Card14LSL-E138A/LSL-E138A Rosa26CreERT2/+ mice and Card14+/+ Rosa26CreERT2/+ (not antibody treated) controls. Data pooled from three independent experiments. (D) qRT-PCR was performed on d5 ear and lung tissue from Card14+/+Rosa26CreERT2/+ control mice and Card14LSL-E138A/LSL-E138A Rosa26CreERT2/+ mice treated with either isotype control IgG or anti-TNF. Fold changes were calculated by comparison with the Card14LSL-E138A/LSL-E138A Rosa26CreERT2/+ isotype treated group One representative experiment of 2 is shown. (E) Serum quantification of biochemistry for indicators of liver (ALT: alanine aminotransferase and GLDH: glutamate dehydrogenase) and kidney (inorganic phosphate) damage. Sera were collected from nine independent experiments. Differences between groups analysed by one-way ANOVA (A-E). *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001.
Fig 4: Conditional expression of CARD14E138A in Card14LSL-E138A/+ Rosa26CreERT2/+ mice induces rapid changes in ear skin histology.(A) CARD14-FLAG protein expression analysed in total extracts from different organs from control (WT) and Card14E138A-LSL (KI) mice by immunoblotting. (B) Localisation of Card14 mRNA expression in the skin assessed by RNAscope. (C) Timeline of the Card14E138A induction by tamoxifen and sample collection. (D) Representative histology images of ears on d5 after tamoxifen injection: (first panel) H and E staining and acanthosis quantification over time; (second panel) Ki67 staining at d5 and quantitation; (third panel) S100A9 staining at d5 and quantitation; (fourth panel) involucrin staining at d5 and (bottom panel) endomucin staining at d5. (E) qRT-PCR analysis of the expression of IL17a, Tnfa, S100a9, Il17c and Il1f9 mRNAs. (F) Quantification and characterisation of the immune cell infiltrate of the ears at d5 after tamoxifen by FACS. Data pooled from 4 independent experiments; Card14+/+ Rosa26CreERT2/+n = 22, Card14LSL-E138A/+ Rosa26CreERT2/+ 5d n = 22. Data collected from a mixture of male and female mice. (B, D) Scale bar = 100 µm. Differences between groups analysed by one-way ANOVA (D and E) or Student’s t-test (F). *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. For clarity, only statistical analyses between the two genotypes at day 4 and day 5 were noted.
Fig 5: Evaluation of the effect of pirfenidone on early events in acute pancreatitis.Pancreatic acini were isolated from healthy C57BL/6J mice and treated with supramaximal carbachol, with or without pirfenidone pretreatment (0.5 mg/mL for 30 minutes before supramaximal stimulation) in vitro. (A) Treatment of pancreatic acini with supramaximal carbachol (1 mM) leads to trypsin activation. Pirfenidone pretreatment is unable to inhibit supramaximal carbachol–induced trypsin activation (n = 3). (B) Treatment with caerulein in vivo (50 µg/kg, i.p) results in I?Ba degradation at 1 hour, as shown by Western blotting in pancreatic tissue. Pretreatment with pirfenidone gavage (400 mg/kg, 30 minutes prior to caerulein injection) does not affect I?Ba degradation (n = 3). (C and D) Pretreatment with pirfenidone resulted in decreased secretion of TNF-a (C) and IL-6 (D) from acini treated with supramaximal caerulein (100 nM) for 4 hours (n = 3). (E and F) Pirfenidone disrupts acinar cell–macrophage crosstalk. Pretreatment with pirfenidone resulted in attenuation of the acinar cell mediated activation of macrophages as shown by decreased secretion of TNF-a (E) and IL-6 (F) (n = 3). (G–J) Splenocytes isolated from C57BL/6J mice with L-arginine AP (G and H) (n = 6) or caerulein 2-day model AP (I and J) (n = 3) at the peak of injury were cultured in duplicate, with or without pirfenidone (0.5 mg/mL) for 12 hours, and cells were analyzed using flow cytometry. Pirfenidone treatment led to a significant decrease in TNF-a+Ly6C+ cells (G and I) and MHCII+ Ly6C+ cells (H and J) in the L-arginine model, as well as caerulein 2-day model. Pirf., pirfenidone. Data represent mean ± SEM. *P <.05 by ordinary 1-way ANOVA for (C and D); 2-way ANOVA for (E and F), and Mann-Whitney U test for (G–J).
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