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.

Figure 1. GM-CSF upregulates CCL17 expression in human monocytes and in vitro–derived and ex vivo murine macrophages.

(A–C) Human monocytes were treated with either PBS or GM-CSF (10 ng/ml) for 16 hours. (A) Heat map of significantly (P < 0.05) regulated cytokines (n = 3). (B) mRNA expression (qPCR) and (C) secreted CCL17 protein (ELISA) (n = 5). (D and E) Murine bone marrow cells were cultured in M-CSF (CSF-1) (5,000 U/ml) for 7 days to derive BMM before treating with either PBS or GM-CSF (20 ng/ml) for 16 hours in the absence of M-CSF. (D) mRNA expression and (E) secreted CCL17 protein (n = 4). (F) mRNA expression in thioglycolate-elicited peritoneal macrophages following treatment with PBS or GM-CSF (20 ng/ml) for 24 hours (n = 4). Graphs are plotted as mean ± SEM. ND, not detected. P values were obtained using a t test.

Figure 2. CCL17 is required for GM-CSF–driven inflammatory pain and can induce such pain.

(A–C) i.pl. injection of GM-CSF (20 ng) or saline in (A) WT mice treated with/without indomethacin (12.5 μg/paw i.pl. at 2 hours) (n = 10 per group), (B) WT and Ccl17^E/E mice (n = 5 per group), and (C) WT mice treated with anti-CCL17 or isotype control (2 μg/paw i.pl. at t = 0) (n = 5 per group). Pain development (incapacitance meter — ratio of weight bearing on injected relative to noninjected hindlimb — a value of < 100 indicates pain) was measured. (D–F) i.pl. injection of CCL17 (50 ng) or saline in (D) WT mice treated with and without indomethacin (12.5 μg/paw i.pl. at t = 0) (n = 5 per group), (E) WT mice treated with/without the COX2 inhibitor, SC58125 (5 mg/kg i.p. at t = –30 minutes) (n = 10 per group), and (F) WT and GMCSF^–/– mice. Pain development was measured. (n = 6 per group). Results are shown as mean ± SEM. P values were obtained using a 2-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001, WT saline vs. WT GM-CSF or WT CCL17 (+ vehicle). ^#P < 0.05; ^##P < 0.01; ^###P < 0.001, WT GM-CSF vs. Ccl17^E/E GM-CSF; GM-CSF + isotype vs. GM-CSF + anti-CCL17 mAb; GM-CSF or CCL17 + vs. – indomethacin (Indo) or COX2 inhibitor. ^ζP < 0.05, GMCSF^–/– saline vs. GMCSF^–/– CCL17.

Figure 3. CCL17 is required for GM-CSF–driven arthritic pain and disease.

(A and B) mBSA/GM-CSF arthritis (mBSA i.a.) (day 0) and GM-CSF or saline (days 0 to 2) was induced in (A) WT and Ccl17^E/E mice (n = 10 per group) and (B) WT mice treated with anti-CCL17 or isotype control (150 μg i.p., days –2 and 0) (n = 10 per group). Pain (incapacitance meter) and arthritis (histology) were measured. Original magnification, ×125. Results are shown mean ± SEM. P values were obtained using a 2-way ANOVA test for pain (weight distribution) readings and a 2-way (A) or 1-way (B) ANOVA test for histology quantification. **P < 0.01; ***P < 0.001; ****P < 0.0001, WT saline vs. WT GM-CSF. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001; ^####P < 0.0001, WT GM-CSF vs. Ccl17^E/E GM-CSF; GM-CSF + isotype vs. GM-CSF + anti-CCL17 mAb.

Figure 4. CCL17 can drive arthritic pain and disease.

(A–C) mBSA/CCL17 arthritis (mBSA i.a. [day 0] and CCL17 or saline [days 0 to 2]) was induced in (A) WT mice treated with indomethacin (1 mg/kg i.p.) or PBS from day 5 (n = 5 per group), (B) WT and Rag1^–/– mice (n = 6 per group), and (C) WT and GMCSF^–/– mice (n = 6 per group). Pain (incapacitance meter) and arthritis (histology) were measured. Original magnification, ×125. Results are shown as mean ± SEM. P values were obtained using a 2-way ANOVA test for pain (weight distribution) readings and a 1-way (A) or 2-way (B and C) ANOVA test for histology quantification. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, WT saline vs. WT CCL17. ^#P < 0.05; ^##P < 0.01, CCL17 + vs. – indomethacin. ^ζP < 0.05; ^ζζP < 0.01; ^ζζζP < 0.001; ^ζζζζP < 0.0001, Rag1^–/– or GMCSF^–/– saline vs. Rag1^–/– or GMCSF^–/– CCL17.

Figure 5. The requirement for CCL17 in GM-CSF–dependent inflammatory pain.

(A and B) WT and GMCSF^–/– mice received an i.pl. injection of zymosan, and (A) pain (incapacitance meter) and (B) footpad mRNA expression (qPCR) (6 hours zymosan or saline) were measured (n = 10 per group). (C and D) Ccl17^E/+ and Ccl17^E/E mice received an i.pl. injection of zymosan, and (C) pain and (D) footpad mRNA expression (6 hours zymosan or saline) were measured (n = 7–8 per group). Results are shown as mean ± SEM. P values were obtained using 2-way ANOVA tests. **P < 0.01; ****P < 0.0001, WT or Ccl17^E/+ vs. GMCSF^–/– or Ccl17^E/E mice.

Figure 6. The requirement for CCL17 in GM-CSF–dependent arthritic pain and disease.

(A and B) WT and GMCSF^–/– mice received an i.a. injection of zymosan, and (A) pain and arthritis (histology, day 7) and (B) joint mRNA expression (day 7) were measured (n = 10 per group). (C) Ccl17^E/+ and Ccl17^E/E mice received an i.a. injection of zymosan, and pain and arthritis (histology, day 7) were measured (n = 5 per group). (D) Pain and arthritis (histology, day 14) development in WT and Ccl17^E/E mice with AIA (n = 15 per group). Results are shown as mean ± SEM. P values were obtained using a 2-way ANOVA test (A, C, and D) for pain readings, Mann-Whitney U test (A, C, and D) for histology, and a t test (B) for gene expression. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, WT or Ccl17^E/+ vs. GMCSF^–/– or Ccl17^E/E mice. Original magnification, ×60.

Figure 7. GM-CSF–induced CCL17 expression in monocytes/macrophages is IRF4 dependent.

(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 Ccl17^E/+ 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.

Figure 8. IRF4 is required for GM-CSF–driven inflammatory pain and GM-CSF–driven arthritic pain and disease.

(A and B) i.pl. injection of GM-CSF (20 ng) in WT and Irf4^–/– mice. (A) Pain (incapacitance meter) and (B) footpad mRNA expression (qPCR) (4 hours) (n = 6 per group). (C) Pain (incapacitance meter) following i.pl. CCL17 (50 ng) in WT and Irf4^–/– mice (n = 5 per group). (D) Irf4 mRNA expression (day 7) in the joints of WT mice with mBSA/GM-CSF arthritis (n = 6 per group). (E and F) mBSA/GM-CSF arthritis was induced in WT and Irf4^–/– mice and (E) pain and arthritis (histology, day 7) and (F) joint Ccl17 mRNA expression (day 7) were measured (n = 6 per group). (G) mBSA/CCL17 arthritis model was induced in WT and Irf4^–/– mice, and pain and arthritis (histology, day 7) were measured (n = 6 per group). Original magnification, ×125. Results are shown as mean ± SEM. P values were obtained using a 2-way ANOVA test (A, C, D, E, F, G) and a t test (B). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, WT saline vs. WT GM-CSF or WT CCL17; ^##P < 0.01 ^###P < 0.001; ^####P < 0.0001, WT GM-CSF vs. Irf4^–/– GM-CSF; ^ζζP < 0.01, Irf4^–/– saline vs. Irf4^–/– CCL17.

Figure 9. IRF4 is required for GM-CSF–dependent inflammatory and arthritic pain and disease.

(A–C) WT and Irf4^–/– mice received i.pl. zymosan, and (A) pain, (B) swelling, and (C) mRNA expression (6 hours zymosan or saline) in the footpad were measured (n = 8 per group). (D) Pain and arthritis (histology, day 7) development following zymosan-induced arthritis in WT and Irf4^–/– mice (n = 5 per group). Results are shown as mean ± SEM. P values were obtained using a 2-way ANOVA test (A–D) for pain and gene expression and Mann-Whitney U test (D) for histology. Original magnification, ×60. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, WT vs. Irf4^–/– mice.

Figure 10. GM-CSF regulates IRF4 expression via JMJD3 demethylase in human monocytes, and inhibition of JMJD3 demethylase ameliorates GM-CSF–driven inflammatory pain.

(A and B) Human monocytes were treated with GM-CSF (10 ng/ml) for indicated periods of time. (A) KDM6B, IRF4, and CCL17 mRNA expression (qPCR) and (B) JMJD3 activity were determined (n = 5). (C and D) Human monocytes were pretreated with JMJD3 inhibitor GSK-J4 (10 nM) or DMSO for 30 minutes before treatment with GM-CSF (10 ng/ml) for 16 hours. (C) Whole cell lysates were subjected to Western blotting with anti-JMJD3, anti-IRF4, anti–β-actin, anti-H3K27me3, and anti-H3 antibodies and (D) secreted CCL17 measured by ELISA (n = 4). (E) Human monocytes were pretreated with JMJD3 inhibitor GSK-J4 (10 nM) or DMSO for 30 minutes before treatment with GM-CSF (10 ng/ml) for 1 hour. ChIP analysis of the association of RNA Pol II, H3K27me3, and total H3 with the IRF4 TSS is expressed as percentage of input DNA (n = 4). (F) i.pl. injection of GM-CSF (20 ng) or saline in WT mice treated with/without GSK-J4 (25 mg/kg i.p. at t = –30 minutes). Pain development was measured (n = 11 per group). Graphs are plotted as mean ± SEM. P values were obtained using a 1-way ANOVA test (A, B, D, and E) and a 2-way ANOVA test (F). *P < 0.05; **P < 0.01, vs. t = 0; ***P < 0.001, saline vs. GM-CSF + vehicle. ^####P < 0.0001, vehicle vs. GSK-J4.