Mcp 1

Neutrophils were isolated from naive mouse bone marrow or human blood by using the magnetic-activated cell sorting kit for mouse or human neutrophil isolation (Miltenyi Biotec). The purity of the isolated neutrophils exceeded 90%. The freshly prepared neutrophils (1 × 10⁵ cells/well) were incubated overnight in a 96-well plate with RPMI 1640 medium (biowest) containing 10% FBS and 10 ng/mL of the recombinant proteins IL-23 (BioLegend), MCP-1 (BioLegend), TGF-β1 (BioLegend), and/or GM-CSF (BioLegend). After washing with PBS, the cells were analyzed with flow cytometry for Siglec-F expression or ELISA (Cusabio Technology) and immunofluorescence imaging for COL1A1 expression. For the coculture study, 3 × 10⁵ NIH 3T3 fibroblasts/well (ATCC) were plated in a 6-well plate and cultured in serum-starved RPMI 1640 medium a day before coculture. Neutrophils that had been primed in vitro with TGF-β1 or GM-CSF were then added (3 × 10⁶/well). After coculture for 24 hours, the neutrophils were washed out with 3 PBS washes, and the fibroblast lysates were subjected to Western blot to assess the amounts of COL1A1 and α-SMA.

Intracellular formation of ROS was monitored using the DCFDA Cellular ROS Detection Assay Kit (Abcam) according to the manufacturer’s instructions for non-adherent cells. Healthy donor neutrophils isolated as described above were resuspended in 1 × assay buffer containing 20 μM DCFDA. A total of 10⁵ cells/well/100 μl were seeded into sterile, black, flat-bottom 96-well plates and incubated for 30 min. Pre-warmed HBSS supplemented with 1.26 mM CaCl₂ was added to a final assay volume of 270 μl. The plate was transferred into a fluorescence plate reader with a dual injector function (Varioskan Flash, Thermo Scientific) and monitored at 37°C for 85 min reading fluorescence at an excitation wavelength of 490 nm and an emission wavelength of 525 nm. After baseline recording, MCP-1 was injected at medium speed (BioLegend, 0.5 ng/ml final concentration) and fluorescence was measured per well immediately afterward. The plate was scanned every minute for 20 min. Then, ionomycin was injected at medium speed (Sigma, 1.3 μM final concentration) and fluorescence was again measured per well immediately afterward. ROS formation was monitored for 15 min every minute and then for 50 min every 5 min. Results are presented as fold change to baseline.

ELISA kit was used to detect the expression of several inflammatory cytokines such as MCP-1 (Cat#432704, Biolegend), IL-17A (Cat#433007, Biolegend), TGF-β (Cat#432507, Biolegend), IL-6 (Cat#1210602, DAKEWE), IL-12p70 (Cat#1211202, DAKEWE) and TNFα (Cat#1217202, DAKEWE) in the sera of mice models. All analyses and calibrations were performed in duplicate. Optical densities were determined using an absorbance microplate reader (Tecan, Infinite M200, Switzerland) at 450 nm. Graph prism 8 Data Analysis software was used to analyze all data. Differences between two groups were determined as unpaired two-tailed Student’ s t-test.

Antibodies for Akt, phospho-Akt (Ser⁴⁷³), AMP-activated protein kinase (AMPKα), phospho-AMPKα (Thr¹⁷²), the Akt substrate regulating GLUT4 translocation (AS160), phospho-AS160 (Thr⁶⁴²), ERK1/2, phospho-ERK1/2 (Thr²⁰²/Tyr²⁰⁴), p38 mitogen-activated protein kinase (p38), phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²), IRS-1, IL-6, MCP-1, Na⁺/K⁺-ATPase, TNF-α, and α-Tubulin were purchased from Cell Signaling (Danvers, MA, USA). Ang II, IL-17A, COX-2, c-Jun-N-terminal kinase (JNK), phospho-JNK (Thr¹⁸³/Tyr¹⁸⁵), mineralocorticoid receptor (MCR), PPARɣ, C/EBPα, adipocyte protein (aP2), adiponectin, β-actin, α-adducin-1(ADD1), cytochrome P450 family 11-subfamily β-2 (CYP11β-2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz (Dallas, Texas, USA). Antibodies for leptin, mTOR, and SREBP-1c were obtained from Abcam (Cambridge, MA, USA). For rt-PCR, the primers of target cDNAs such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were purchased. (GenoTech Corp, Dajeon, Korea) ELISA kits for detecting the cytokines such as MCP-1, IL-6, and TNF-α were purchased from BioLegend Inc (San Diego, CA, USA). U0126 or PD98059, f MEK/ERK inhibitors were purchased from Sigma Aldrich (St. Louis, MO, USA)