Journal: The FASEB Journal

Article Title: Timing of Glucocorticoid Treatment Dictates Glucocorticoid Receptor Actions Modulating the NLRP3‐Inflammasome Activation in Macrophages

doi: 10.1096/fj.202504083R

Figure Lengend Snippet: Glucocorticoids modulate inflammation and pyroptosis by regulating late NLRP3‐inflammasome activation through ACOD1 and iNOS. (A) Schematic representation of the experimental setup for conventional NLRP3‐inflammasome activation (upper), and late NLRP3‐inflammasome activation regulated by Dex and MCC950 (lower). (B) and (C) Quantification of IL‐1β concentration determined by ELISA in the supernatant of WT and GRKO BDMDs stimulated with LPS for 3 h (conventional) and 24 h (late), with Dex and vehicle added during the final 2 and 8 h of treatment, followed by ATP (5 mM) or Nigericin (10 μM) ( n = 3). (D) Immunoblot analysis depicting Dex effects on the protein levels of ACOD1, iNOS, NLRP3, pro‐IL‐1β, Caspase‐1 and GSDM‐D during late LPS‐induced NLRP3‐inflammasome activation and using ATP as signal 2 in WT and GRKO BMDMs lysates, detected with specific antibodies using western blotting. (E) Densitometry values of the immunoreactive bands quantified for protein content, normalized to β‐actin as depicted in (D) ( n = 4). (F) Nitrite levels in the supernatants of WT and GRKO BMDMs as depicted in (D) determined by colorimetric assay ( n = 4). (G) Assessment of lactate dehydrogenase (LDH) activity in the supernatants of WT and GRKO BMDMs as depicted in (D) ( n = 4). (H) Immunoblot analysis depicting the protein levels of NRF2 as depicted in (D) in presence of the proteasome inhibitor MG‐132 (2 μM) 6 h before harvesting cells ( n = 3). (I) Immunoblot analysis depicting the protein levels of HIF‐1α as depicted in (D) in presence of the prolyl‐hydroxylase inhibitor Roxadustat (RXD; 10 μM) 2 h before adding Dex ( n = 3). (J) Quantification of IL‐1β concentration determined by ELISA in the supernatant of WT BDMDs as depicted in (H) and (I) ( n = 3). (K) Assessment of lactate dehydrogenase (LDH) activity in supernatants of WT BMDMs subjected to the conditions described in (H) and (I) ( n = 3). Data represented as mean ± SEM. Blots shown are representative of a minimum of 3 independent experiments. Statistical analysis was performed using 2‐way ANOVA with Sidak's multiple‐comparison test (B–I); one‐way ANOVA with Tukey's multiple‐comparison test (J) and (K). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Lipopolysaccharide from E. coli 0111: B4 strain (LPS‐EB), NLRP3 inhibitor MCC950, Caspase‐1 inhibitor Ac‐Y‐VAD‐cmk, 4‐octyl‐itaconate (4‐OI), adenosine 5′‐triphosphate disodium salt (ATP), nigericin, and monosodium urate (MSU) crystals were purchased from InvivoGen (San Diego, CA, USA).

Techniques: Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Colorimetric Assay, Activity Assay, Comparison