Granulocyte macrophage colony-stimulating factor induces CCL17 production via IRF4 to mediate inflammation
Adrian Achuthan, … , Stephen J. Turner, John A. Hamilton
Adrian Achuthan, … , Stephen J. Turner, John A. Hamilton
Published August 15, 2016
Citation Information: J Clin Invest. 2016;126(9):3453-3466. https://doi.org/10.1172/JCI87828.
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Granulocyte macrophage colony-stimulating factor induces CCL17 production via IRF4 to mediate inflammation

Abstract

Data from preclinical and clinical studies have demonstrated that granulocyte macrophage colony-stimulating factor (GM-CSF) can function as a key proinflammatory cytokine. However, therapies that directly target GM-CSF function could lead to undesirable side effects, creating a need to delineate downstream pathways and mediators. In this work, we provide evidence that GM-CSF drives CCL17 production by acting through an IFN regulatory factor 4–dependent (IRF4-dependent) pathway in human monocytes, murine macrophages, and mice in vivo. In murine models of arthritis and pain, IRF4 regulated the formation of CCL17, which mediated the proinflammatory and algesic actions of GM-CSF. Mechanistically, GM-CSF upregulated IRF4 expression by enhancing JMJD3 demethylase activity. We also determined that CCL17 has chemokine-independent functions in inflammatory arthritis and pain. These findings indicate that GM-CSF can mediate inflammation and pain by regulating IRF4-induced CCL17 production, providing insights into a pathway with potential therapeutic avenues for the treatment of inflammatory diseases and their associated pain.

Authors

Adrian Achuthan, Andrew D. Cook, Ming-Chin Lee, Reem Saleh, Hsu-Wei Khiew, Melody W.N. Chang, Cynthia Louis, Andrew J. Fleetwood, Derek C. Lacey, Anne D. Christensen, Ashlee T. Frye, Pui Yeng Lam, Hitoshi Kusano, Koji Nomura, Nancy Steiner, Irmgard Förster, Stephen L. Nutt, Moshe Olshansky, Stephen J. Turner, John A. Hamilton

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Figure 7

GM-CSF–induced CCL17 expression in monocytes/macrophages is IRF4 dependent.

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GM-CSF–induced CCL17 expression in monocytes/macrophages is IRF4 depende...
(A and B) Human monocytes were treated with either PBS or GM-CSF (10 ng/ml) for 16 hours. (A) IRF4 and IRF5 mRNA expression (qPCR) and (B) whole cell lysates were subjected to Western blotting with anti-IRF4, anti-IRF5, and anti–β-actin antibodies (n = 4). (C and D) Human monocytes were nucleofected with IRF4, IRF5, or a control nontargeting (CNT) siRNA before being stimulated with GM-CSF (10 ng/ml) for 16 hours. (C) IRF and cytokine/chemokine mRNA expression plotted relative to that in CNT siRNA, which was given an arbitrary value of 1.0, and (D) secreted CCL17 by ELISA (n = 5). (E and F) Murine bone marrow cells were cultured in M-CSF (5,000 U/ml) for 7 days to derive BMM followed by GM-CSF treatment in the absence of M-CSF. (E) Irf4 mRNA expression (24 hours) and (F) whole cell lysates from BMM treated with GM-CSF for the indicated periods of time were subjected to Western blotting with anti-IRF4 and anti–β-actin antibodies (n = 4). (G) Representative histograms of IRF4 in CCL17/EGFP+ and CCL17/EGFP– populations of BMM (from Ccl17E/+ mice) treated with fresh medium containing either M-CSF or GM-CSF for 72 hours (n = 3). (H and I) BMM from WT or Irf4–/– mice were stimulated with GM-CSF (10 ng/ml) for 24 hours in the absence of M-CSF. (H) Cytokine mRNA expression and (I) secreted CCL17 protein in the supernatant (ELISA) (n = 4). (J and K) Thioglycolate-elicited peritoneal macrophages were treated with PBS (vehicle) and GM-CSF (20 ng/ml) for 24 hours. (J) Irf4 mRNA expression in WT macrophages and (K) cytokine mRNA expression in WT and Irf4–/– macrophages (n = 4). Graphs are plotted as mean ± SEM. P values were obtained using a t test.