Glucocorticoids target suppressor of cytokine signaling 1 (SOCS1) and type 1 interferons to regulate Toll-like receptor-induced STAT1 activation

Abstract

Endogenous and pharmacologic glucocorticoids (GCs) limit inflammatory cascades initiated by Toll-like receptor (TLR) activation. A long-standing clinical observation has been the delay between GC administration and the manifestation of GC's anti-inflammatory actions. We hypothesized that the GCs would have inhibitory effects that target late temporal pathways that propagate proinflammatory signals. Here we interrogated signal transducer and activator of transcription 1 (STAT1) regulation by GC and its consequences for cytokine production during activation of macrophages with TLR-specific ligands. We found that robust STAT1 activation does not occur until 2-3 h after TLR engagement, and that GC suppression of STAT1 phosphorylation first manifests at this time. GC attenuates TLR4-mediated STAT1 activation only through induction of suppressor of cytokine signaling 1 (SOCS1), which increases throughout the 6-h period after treatment. Inhibition of TLR3-mediated STAT1 activation occurs via two mechanisms, impairment of type I IFN secretion and induction of SOCS1. Our data show that SOCS1 and type I interferons are critical GC targets for regulating STAT1 activity and may account for overall GC effectiveness in inflammation suppression in the clinically relevant time frame.

Fig. 1.

TLR-induced cytokine secretion in control and STAT1-null macrophages. Effect of LPS (100 ng/mL) (A), Poly (I:C) (50 μg/mL) (B), and CpG (2 μM) (C) on proinflammatory cytokine secretion in C57BL/6 (□), 129S6 (■), and STAT1^−/− () macrophages. Cells were treated with TLR ligands for the indicated periods. Concentrations of TNF-α and IL-12p40 in the culture media were analyzed by ELISA. Data are mean ± SEM; n = 3. *P < 0.05; **P < 0.01 for STAT1^−/− macrophages compared with control 129S6 macrophages.

Fig. 2.

Effect of GCs on TLR-induced STAT1 phosphorylation. Control or MGRKO macrophages were treated with or without Dex (100 nM) for 3 h, followed by LPS (A), Poly (I:C) (B), and CpG (C) treatment for the indicated periods. Cell lysates were analyzed by Western blot analysis using anti-phospho STAT1 Ser⁷²⁷ (P-STAT1 Ser), phospho STAT1 Tyr⁷⁰¹ (P-STAT1 Tyr), and total STAT1 (Total-STAT1) antibodies. Representative of between three and five independent experiments.

Fig. 3.

(A) IFNAR1 activation and TLR-induced STAT1 phosphorylation. Macrophages were pretreated with anti-IFNAR1 antibody (MARI) for 1 h, followed by treatment with LPS, Poly (I:C), CpG, or control oligonucleotide (ODN) for the indicated periods. Cell lysates were analyzed by Western blot analysis as described in Fig. 2. Representative of three independent experiments. (B–F) GC modulation of TLR-induced type I IFN secretion and nuclear IRF3. Macrophages were treated with Dex, followed by treatment with Poly (I:C) (B and D) or LPS (C) for 3, 6, or 9 h for IFN-β and for 12 or 18 h for IFN-α. Concentrations of IFN-α and IFN-β in the culture media were analyzed by ELISA. Data are mean ± SEM; n = 3. *P < 0.05; **P < 0.01 for Dex-treated cells compared with the group treated with Poly (I:C) only. (E and F) Cells were treated as described previously, and nuclear extracts were analyzed by Western blot analysis using anti-IRF3 or anti–β-actin antibodies. (G and H) Effect of GCs on IFN-β–induced STAT1 phosphorylation. Cells were pretreated with Dex, followed by treatment with varying doses of IFN-β (31.25–500 U/mL) for 1 h (G) or 125 U/mL of IFN-β for the indicated periods (H). Cell lysates were analyzed as described in Fig. 2. Representative of three independent experiments.

Fig. 4.

(A–C) Effect of GCs on TLR-induced SOCS1 expression. Cells were treated with LPS (A), Poly (I:C) (B), and CpG (C) as described in Fig. 2. Cell lysates were analyzed by Western blot analysis using anti-SOCS1 or anti–β-actin antibodies. (D–G) SOCS1 mediates GC inhibition of STAT1. SOCS1^+/+ or SOCS1^−/− macrophages were treated with or without Dex for 3 h, followed by treatment with LPS (D) or Poly (I:C) (E) for the indicated periods. Cell lysates were analyzed by Western blot analysis using anti-phospho STAT1 Ser⁷²⁷ (P-STAT1 Ser), phospho STAT1 Tyr⁷⁰¹ (P-STAT1 Tyr), and total STAT1 (Total-STAT1) antibodies. Representative of two or three independent experiments. Similar experiments were performed with LPS for 18 h (F) and Poly (I:C) for 48 h (G). Concentrations of TNF-α and IL-12p40 in the culture media were analyzed by ELISA. Data are mean ± SEM; n = 3. *P < 0.05; **P < 0.01 for Dex-treated cells compared with the group treated with TLR ligand only. (H and I) SOCS1 inhibits phospho-JAK2 abundance to inhibit STAT1 activation. SOCS1^+/+ (H) or SOCS1^−/− (I) macrophages were treated as described previously. Cell lysates were analyzed by Western blot analysis using anti-phospho JAK2 (P-JAK2), and total JAK2 (Total-JAK2) antibodies. Representative of two independent experiments.