Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A) RT-qPCR showing up-regulation of metabolic genes in mDM (above) or BMDM (below) treated with LPS or MSUc for 5h (n≥4/condition). (B) WB of lysates of BMDM treated with LPS or MSUc in control PBS, LPS or MSUc at 8h or 24h showing up-regulation of SLC2A1 and PFKBP3 in BMDM treated with LPS or MSUc and down-regulation of LDHB in BMDM treated with MSUc.

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A) Bar plot showing up-regulation of genes involved in glucose metabolism in macrophages treated with MSUc or LPS. (B) Protein analysis by IF showing up-regulation of SLC2A1 in macrophages treated with MSUc or LPS at 5h. (C-D) Analysis of the concentration of glycolytic metabolites by 1D 1 H-NMR in the pellet (C) or supernatant (D) or BMDM treated with LPS or MSUc for 4h or 8h showing activation of glycolysis in macrophages treated with MSUc to a higher degree than LPS (n≥4/condition). (E) 1D 1 H-NMR showing increased levels of acetate, citrate and lactate and reduced levels of succinate in the supernatant of mDM treated with MSUc for 4h or 8h (n=3 donors/condition). (F) Bar graphs showing up-regulation of several amino acid transporters (top) or genes involved in glycine/serine/threonine metabolism (bottom) in macrophages treated with MSUc or LPS for 5h assessed by RNA-Seq. (G,H) 1D 1 H-NMR showing increased levels of glycine, threonine and tryptophan in the pellet (G) and alanine, glutamate, phenylalanine and tryptophan in supernatant (H) of BMDM treated with MSUc or LPS for 4h or 8h (n≥4/condition). (I) 1D 1 H-NMR showing increased levels of glutamate and glycine, and reduced levels of aspartate, glutamine, isoleucine and leucine in the supernatant of mDM treated with MSUc for 4h or 8h (n=3 donors/condition). (#=P<0.10;*=P<0.05; **=P<0.01).

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Concentration Assay, Activation Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A,B) Quantification of pJNK expression in BMDM treated with LPS or MSUc at various time points showing increased total pJNK expression in BMDM treated with LPS for 30 min (A) but prolonged pJNK expression in BMDM treated with MSUc (A,B)(n=2 replicates/condition). (C) Pie chart showing the degree of amelioration of gene expression in BMDM treated with LPS+JNKi vs. LPS or MSUc+JNKi vs. MSUc for 5h. (D) HOMER analysis of the promoter [-2000;+500 bp, TSS] of genes up-regulated by LPS or MSUc and further down-regulated by treatment with JNKi (>50% left, 33%-50% center, <33% right). Data shows enrichment in IRFs motifs in genes ameliorated by JNKi in LPS and motifs for AP-1, MYC, NRF2 and circadian clock proteins in genes ameliorated by JNKi in MSUc. (E) GSEA analysis using REACTOME of genes between > 50% reduccion of expression in MSUc+JNKi vs. MSUc or LPS+JNKi vs. LPS. Data shows enrichment in inflammatory gene sets in LPS and MSUc and metabolic gene sets in MSUc. (F,G) Protein analysis by IF of JUN, pJUN Ser63 , FOSL1, SLC2A1 and PFKBP3 in BMDM treated with MSUc or MSUc+JNKi for 5h. Quantification of G corresponds to experiment shown in F and shows downregulation of protein expression in MSUc+JNKi vs. MSUc (n=3/condition). (#=P<0.10;*=P<0.05; **=P<0.01). Broken lines in E represents the cutoff for significance –log 10 (0.05)=1.30.

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Expressing, Gene Expression

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A,B) Protein analysis by IF of JUN, pJUN Ser63 , FOSL1, SLC2A1, FOSB in mDM treated with MSUc or MSUc+JNKi for 5h. Quantification of B corresponds to experiment shown in A, and shows downregulation of protein expression in MSU+JNKi vs. MSUc (n=3/condition). (C) Protein analysis by ELISA of cytokines in the supernatant of BMDM treated with LPS or MSUc w/wo JNKi showing complete reduction in MSUc+JNKi vs. MSUc and partial reduction in LPS+JNKi vs. LPS (n=3/group). (D) Analysis of metabolites by 1D 1 H-NMR in the culture media of BMDM treated with MSUc or MSUc+JNKi showing varying degree of recovery (n=3/condition). (E,F) JUN ChIP in BMDM (E) or mDM (F) treated with LPS, MSUc, LPS+JNKi or MSUc+JNKi and qPCR over regulatory regions of genes up-regulated by LPS or MSUc. Data shows higher levels of JUN binding in macrophages treated with MSUc that is reduced upon treatment with JNKi (n=4/condition). (#=P<0.10;*=P<0.05; **=P<0.01).

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: bioRxiv

Article Title: Monosodium Urate Crystals regulate a unique JNK-dependent macrophage metabolic and inflammatory response

doi: 10.1101/2021.04.14.439881

Figure Lengend Snippet: (A,B) Protein analysis by IF showing expression of SLC2A1 (A) and pJNK (B) in macrophages in the subcutaneous cavity of the air pouch when injected with MSUc (n=2/group). (C) Histological analysis by HE showing the recruitment of inflammatory cells (arrows) induced by MSUc in the air pouch is reduced upon treatment with JNKi (n≥4/group). (D,E) Pathological assessment of inflammatory cell infiltrates of the air pouch (D) and neutrophil count of the air pouch lavage (E) showing reduction in MSUc+JNKi vs. MSUc (n≥4/group). (F) Histological analysis by HE showing the recruitment of inflammatory cells (arrows) induced by MSUc in the air pouch is reduced upon treatment with BAY-876 (n≥5/group). (G,H) Pathological assessment of inflammatory cell infiltrates of the air pouch (G) and neutrophil count of the air pouch lavage (H) showing reduction in MSUc+BAY-876 vs. MSUc (n≥10/group). (I,J) Histological analysis by H&E showing reduction in the recruitment of inflammatory cells (arrow) in the synovial cavity of mice injected with MSUc+JNKi vs. MSUc (n≥10/group). (K,L) Histological analysis by H&E showing reduction in the recruitment of inflammatory cells (arrow) in the synovium of mice injected with MSUc+BAY-876 vs. MSUc (n≥7/group). (M) Diagram showing that MSUc induces a unique transcriptional program with up-activation of inflammatory –but not interferon stimulated genes- and metabolic genes, which is regulated through up-regulation of AP-1 via JNK but not IRFs. (#=P<0.10;*=P<0.05; **=P<0.01).

Article Snippet: PBS, DMEM (w/5% glutamine, 10% FBS, 5% penicillin), trypsin 0.25%, LPS (100 ng/mL), MSUc were prepared as described ( ) suspended at 25 mg/mL in sterile, endotoxin-free phosphate buffered saline (PBS) and verified to be free of detectable lipopolysaccharide contamination by Limulus lysate assay (Lonza, Walkersville, Maryland, USA), MCSF (10 ng/mL, Peprotech), GLUT1 inhibitor BAY-876 (20nM, Selleckchem, #S8452), p38 MAPK inhibitor SB203580 (3 uM, Tocris, #1202), JNK inhibitor SP600125 (20uM, Tocris, #1496), beta-hydroxybutyric acid (BHA; 10uM, Sigma, #B1253), NLRP3 inflammasome inhibitor MCC950 (1uM, Selleckchem, #S7809).

Techniques: Expressing, Injection, Activation Assay