Macrophage glucocorticoid receptors regulate Toll-like receptor 4-mediated inflammatory responses by selective inhibition of p38 MAP kinase

Abstract

To explore the role of glucocorticoids in regulation of kinase pathways during innate immune responses, we generated mice with conditional deletion of glucocorticoid receptor (GR) in macrophages (MGRKO). Activation of toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) caused greater mortality and cytokine production in MGRKO mice than in controls. Ex vivo, treatment with dexamethasone (Dex) markedly inhibited LPS-mediated induction of inflammatory genes in control but not GR-deficient macrophages. We show that Dex inhibits p38 MAPK, but not PI3K/Akt, ERK, or JNK, in control macrophages. Associated with p38 inhibition, Dex induced MAP kinase phosphatase-1 (MKP-1) in control, but not MGRKO, macrophages. Consistent with the ex vivo studies, treatment with a p38 MAPK-specific inhibitor resulted in rescue of MGRKO mice from LPS-induced lethality. Taken together, we identify p38 MAPK and its downstream targets as essential for GR-mediated immunosuppression in macrophages.

Figure 1

Deletion of GR in macrophages augments LPS-induced lethality and proinflammatory responses. (A) Kaplan-Meier plot of control (n = 8) and MGRKO (n = 8) mice treated with LPS (10 mg/kg). P < .01 for MGRKO versus control. (B) Plasma concentrations of TNF-α and IL-6 in control (n = 7-10) and MGRKO (n = 6-10) mice treated with LPS for the indicated periods of time. Error bars indicate SEM.

Figure 2

Dex suppresses LPS-mediated inflammatory gene expression. (A) Effect of Dex on LPS-mediated induction of TNF-α and IL-6 in control (□) and MGRKO (■) macrophages. Data are shown as mean ± SEM; n = 5; *P < .01 compared with treatment group without Dex in control macrophages. (B) Effect of Dex on LPS-mediated induction of COX-2. Representative of 3 independent experiments.

Figure 3

Dex selectively suppresses LPS-mediated activation of p38 MAPK. (A) Time-dependent effect of LPS (100 ng/mL) on p38 MAPK phosphorylation. Representative of 2 independent experiments. Effect of Dex (100 nM) on LPS-mediated phosphorylation of (B) p38 MAPK in control and MGRKO macrophages and (C) JNK (p46/p54), (D) ERK1/2, or (E) Akt in the control macrophages. Representatives of 3 to 4 independent experiments.

Figure 4

p38 MAPK inhibitors impair proinflammatory gene expression and effect of Dex on MKP-1. (A) Effect of SB (20 μM) and PD (20 μM) on LPS-mediated induction of TNF-α and IL-6 in control macrophages; n = 3; **P < .01 and *P < .02 compared with treatment group with LPS. Error bars indicate SEM. Effect of SB (20 μM) on LPS-mediated (B) phosphorylation of HSP27 and (C) induction of COX-2 for the indicated period of time in control macrophages. Representatives of 2 to 3 independent experiments.

Figure 5

Dex induction of MKP-1 expression is the major GR target for suppression of p38 MAPK phosphorylation. (A) Effect of Dex on MKP-1 induction in control and MGRKO macrophages treated with LPS for the indicated periods of time. (B) Effect of triptolide on p38 MAPK phosphorylation in the presence and absence of Dex. Representative of 2 to 3 independent experiments.

Figure 6

SB suppresses LPS-induced systemic secretion of proinflammatory cytokines and lethality in MGRKO mice. (A) Effect of SB (●) or vehicle (■) on LPS-mediated mortality in MGRKO mice. For the SB-treated group, mice were injected intraperitoneally with SB (2 mg/kg) 1 hour before LPS (10 mg/kg) treatment. P < .01 for SB-treated mice (n = 9) compared with vehicle-treated mice (n = 8). (B) Effect of SB (□) or vehicle (■) on LPS-mediated induction of TNF-α and IL-6 in MGRKO mice. Mice were injected intraperitoneally with SB or vehicle followed by treatment with LPS for 6 hours. *P < .05 for SB-treated group (n = 5) compared with vehicle-treated group (n = 11). Error bars indicate SEM.

Figure 7

Figure 7. GR attenuates proinflammatory responses in macrophages by impairing p38 MAPK. While TLR4 engagement activates PI3 kinase (PI3K) and multiple MAP kinase kinases (MKKs) and their downstream kinases, p38 MAPK is selectively glucocorticoid (GC) sensitive by transcriptional activation of MKP-1 through GR.