Journal: Frontiers in Immunology

Article Title: Histamine and Th2 cytokines independently and synergistically upregulate MMP12 expression in human M2 macrophages

doi: 10.3389/fimmu.2024.1429009

Figure Lengend Snippet: Attenuation of IL-4- or IL-13-induced upregulation of MMP12 mRNA expression in M2 macrophages by targeting IL-4R type I and IL-4/IL-13R type II with anti IL-4Rα or anti IL-13Rα1 antibodies. (A) Type I (IL-4) receptor, consisting of the IL-4 receptor alpha (IL-4Rα) and the common γ-chain subunits (IL-2Rγ), type II (IL-4/IL-13) receptor, consisting of the IL-4Rα and IL-13Rα1 subunits, trigger activation of signaling adaptor molecules that induce recruitment and phosphorylation of Signal Transducer and Activator of Transcription 6 (STAT6) to activate specific gene transcription programs. Schematic overview created with BioRender.com . (B, C) After differentiation in the presence of M-CSF M2 macrophages were pre-incubated for 20 min with antibodies [dupilumab (10 µM), a monoclonal antibody which blocks the IL-4Rα subunit a polyclonal goat IgG antibody against the common γ chain/IL-2Rγ (20 µM) a monoclonal antibody (10 μM) against the IL-13Rα1 subunit] targeting the extracellular domains of IL-4R type I or IL-4/IL-13R type II receptor complex subunits at day 8. After 20 minutes M2 macrophages were stimulated with IL-4 (20 ng/ml) or IL-13 (15 ng/ml) for 48 h. (B) MMP12 mRNA expression of IL-4-activated M2 macrophages, (C) MMP12 mRNA expression of IL-13-activated M2 macrophages.Target/reference ratios of MMP12 mRNA/rps 20 mRNA expression were analyzed by qPCR and normalized to the IL-4 or IL-13 stimulated samples calculated by the [delta] [delta] Ct method and expressed as normalized ratio. Data are shown as individual values with medians. Significant differences, as determined by the Friedman Dunn’s multiple comparison test (bars and stars in orange/pink) or by the Wilcoxon matched-pairs signed-rank test (bars and stars in black) are indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001. (A, B) (n = 11 independent donors and experiments), NS, non-stimulated.

Article Snippet: To block the IL-4R type I and IL-4/IL-13R type II receptor complex subunits, the Janus/Tyrosine kinases (JAK/TYK) and the transcription factor activator protein-1 (AP-1), the following reagents were used in the culture systems: i) Antibodies against the IL-4R type I and IL-4/IL-13R type II receptor complex subunits: Monoclonal mouse IgG4 antibody dupilumab (Dupixent, Sanofi-Aventis GmbH, Frankfurt am Main, Germany) inhibiting the IL-4Rα chain; polyclonal goat IgG antibody (common γ chain/IL-2Rγ R&D Systems) inhibiting the IL-4R common γ chain/IL-2Rγ; monoclonal mouse antibody (Aldevron, Freiburg, Germany) inhibiting the IL-13R subunit α1. ii) Inhibitors of the IL-4R type I and IL-4/IL-13R type II receptor down-stream signaling adaptor molecules: T-5224 (10 μM) (Chayman, Chemical, USA) for activator protein-1 (AP-1); PF 06651600 malonate (10 μM) (Tocris Bioscience, Bristol, UK) as a potent and selective Janus kinase 3 (JAK3) inhibitor; TC JL 37 (10 μM) (Tocris Bioscience, Bristol, UK) as a potent inhibitor of Tyrosine kinase 2 (TYK2).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Incubation, Comparison