Fig 2: Pretreatment with low-dose Poly(I:C) reduces GILZ expression paralleled by sensitization toward LPS and Pam3CSK4. (A,B) AMs were left untreated (Co) or treated with LPS (100 ng/mL) or Poly(I:C) (PIC, 1 µg/mL) for 24 h. GILZ levels were determined by Western blot using tubulin as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values (n = 3, duplicates; **p < 0.01 compared with Co). (C,D) AMs were either left untreated (NT) or pretreated with PIC (1 µg/mL), followed by stimulation with LPS (1 µg/mL, C) or Pam3CSK4 (Pam, 1 µg/mL, D) for another 2 h. TNF, IL6, and CXCL8 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene (n = 4, duplicates; **p < 0.01, ***p < 0.001).

Fig 3: Mechanisms of GILZ downregulation upon TLR activation. (A,B) AMs pretreated with BAY-11-7082 (BAY82, 5 µM), BAY-11-7085 (BAY85, 5 µM), or the solvent control DMSO (0.1%) for 1 h, followed by treatment with LPS (100 ng/mL), Pam3CSK4 (Pam, 1 µg/mL), Poly(I:C) (PIC, 10 µg/mL), or medium (Co) for 4 h. GILZ expression was determined by Western blot. Tubulin served as a loading control. (A) Representative blot. (B) GILZ signal intensities were quantified and normalized to tubulin values (n = 7). Values for unstimulated DMSO controls were set as 100%. (C,D) After preincubation with BAY-11-7082 or BAY-11-7085 (5 µM, 1 h), solvent (0.1% DMSO) or medium only (Co), AMs were treated with LPS (100 ng/mL) for 2 h. GILZ and ZFP36 mRNA expression was determined by qRT-PCR using ACTB as a housekeeping gene (n = 3, duplicates). (E) AMs were either left untreated (Co) or treated with LPS (100 ng/mL), Pam3CSK4 (Pam, 1 µg/mL), or Poly(I:C) (PIC, 10 µg/mL) for 4 h. TTP levels were determined by Western blot using tubulin as a loading control. GILZ signal intensities were normalized to tubulin and are shown as a percentage of untreated cells (n = 2, triplicates). (F) The number of miRNAs predicted to target GILZ, CXCL8, IL6, and TNF was assessed via the microRNA Data Integration Portal (mirDIP, accession date 02/02/2018). (G) AMs were either left untreated (Co) or treated with Poly(I:C) (PIC, 10 µg/mL), aurintricarboxylic acid (ATA, 25 µM), or a combination of both for 8 h. GILZ expression was quantified by Western blot. Signal intensities were normalized to tubulin and expressed as a percentage of untreated cells (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Loss of GILZ increases the responsiveness of macrophages toward Pam3CSK4 and Poly(I:C). (A,B) Wildtype- (WT) and GILZ Knockout-(KO) BMMs were treated with Pam3CSK4 (4 h, A) or Poly(I:C) (6 h, B) at the indicated concentrations. TNF secretion was quantified by bioassay (n = 4, triplicates). (C–F) WT and GILZ KO BMMs were treated with Pam3CSK4 (100 ng/mL, C and E) or Poly(I:C) (10 µg/mL, D,F) for the indicated periods of time. ERK activation was determined by Western blot. (C,D) Representative blots. (E,F) pERK signal intensities were quantified and normalized to total ERK signals. Values for untreated WT cells (0') were set as 1 (n = 3). (G, H): WT and GILZ KO BMMs were pretreated with medium only (Med), the solvent control DMSO (0.1%), or U0126 (10 µM) for 2 h, followed by activation with Pam3CSK4 (100 ng/mL, 4 h; G) or Poly(I:C) (10 µg/mL, 6 h). TNF production was assessed by bioassay (Co: untreated cells; n = 4, triplicates). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Anti-bacterial host defense is enhanced in GILZ knockout macrophages. (A,B) Wildtype- (WT) and GILZ Knockout- (KO) MPI macrophages were treated with LPS (10 or 100 ng/mL) Pam3CSK4 (Pam, 1 µg/mL, B), or lipoteichoic acid (LTA, 5 µg/mL, B) with or without co-stimulation by IFN-? (20 ng/mL) for 20 h. NO-production was measured by Griess assay. Data were normalized to total protein concentration and expressed as x-fold of untreated cells (n = 4, triplicates). (C–E) Phagocytic activity of WT and GILZ KO MPI cells. MPI cells were incubated with fluorescent latex particles (diameter 1.75 µm, 50 particles per cell) for 15 or 30 min and particle-associated fluorescence was quantified by flow cytometry (15 min: n = 2, 30 min: n = 6, triplicates). (C) Representative histogram for 15 min. 0 P, cells without particles; 1 P, 1 particle; 2 P, 2 particles; > 2 P, more than 2 particles. (G) Phagocytic activity as a total percentage of cells with particles. (E) The percentage of cells that engulfed 0, 1, 2 ore more than 2 particles was quantified as shown in (C). (F,G) WT and GILZ KO BMMs were left untreated (Co) or treated with LPS (1 µg/mL) and INF-? (IFN, 20 ng/mL), IL-4 (20 ng/mL) or dexamethasone (Dex, 1 µM) for 20 h, followed by incubation with fluorescent latex particles (diameter 1.75 µm, 50 particles per cell) for 1 h. Particle uptake was quantified by flow cytometry. (F) Total percentage of cells with particle-associated fluorescence. (D) Percentage of cells with 0, 1, 2 or more than 2 particles. (H) Relative number of viable bacteria in Salmonella typhimurium-infected WT and GILZ KO BMMs. Extracellular bacteria were removed after 30 min, and the remaining bacteria were killed using gentamicin. Eight hours following infection, monolayers were lysed, and the number of intracellular bacteria was determined as Colony Forming Units (CFU) (n = 8, triplicates). *p < 0.05, **p < 0.01, ***p < 0.001 as indicated or compared with equally treated WT cells (E,G).