Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase 1 (MKP-1) in innate immune responses

Abstract

Engagement of Toll-like receptors (TLRs) on macrophages leads to activation of the mitogen-activated protein kinases (MAPKs), which contribute to innate immune responses. MAPK activity is regulated negatively by MAPK phosphatases (MKPs). MKP-1, the founding member of this family of dual-specificity phosphatases, has been implicated in regulating lipopolysaccharide (LPS) responses, but its role in TLR-mediated immune responses in vivo has not been defined. Here, we show that mice deficient in MKP-1 were highly susceptible to endotoxic shock in vivo, associated with enhanced production of proinflammatory cytokines TNF-alpha and IL-6 and an anti-inflammatory cytokine, IL-10. We further examined the regulation and function of MKP-1 in macrophages, a major cell type involved in endotoxic shock. MKP-1 was transiently induced by TLR stimulation through pathways mediated by both myeloid differentiation factor 88 (MyD88) and TIR domain-containing adaptor inducing IFN-beta (TRIF). MKP-1 deficiency led to sustained activation of p38 MAPK and c-Jun N-terminal kinase (JNK) in LPS-treated macrophages. In response to TLR signals, MKP-1-deficient macrophages produced 5- to 10-fold higher IL-10, which could be blocked by a p38 MAPK inhibitor. Thus, p38 MAPK plays a critical role in mediating IL-10 synthesis in TLR signaling. TNF-alpha was found to be more abundant in MKP-1-deficient macrophages within 2 hours of TLR stimulation, but its production was rapidly down-regulated by IL-10. Our studies demonstrate that MKP-1 attenuates the activities of p38 MAPK and JNK to regulate both pro- and anti-inflammatory cytokines in TLR signaling. These results highlight the complex mechanisms by which the MAPKs regulate innate immunity.

Fig. 1.

MKP-1^−/− mice are hyperresponsive to endotoxic shock in vivo, associated with enhanced production of TNF-α, IL-6, and IL-10. (A) MKP-1^+/+ (solid line) and MKP-1^−/− (dotted line) mice were injected i.p. with LPS (10 mg/kg, Left, n ≥ 4; 2.5 mg/kg, Right, n ≥5), and lethality was monitored for up to 50 h. Both treatments resulted in significant differences between the two groups of mice (P < 0.005 and P < 0.001 for 10 and 2.5 mg/kg LPS challenges, respectively). (B) Serum from MKP-1^+/+ (open bars) and MKP-1^−/− (filled bars) mice was collected 3 h after 10 mg/kg LPS challenge, and cytokines were measured by Bio-plex assays or ELISA (IL-6). Data show mean ± SD for four or five mice from each group (∗, P < 0.05 determined using Student’s t test).

Fig. 2.

TLR stimulation induces MKP-1 expression in macrophages. (A) BMDM from WT mice were treated with 10 ng/ml LPS for the indicated time points. RNA was isolated and subjected to quantitative RT-PCR analysis of MKP-1 levels. (B) BMDM from WT, MyD88^−/−, and TRIF^Lps2 mice were treated with 10 ng/ml LPS, and whole-cell lysates were subjected to Western blot analysis using anti-MKP-1 and anti-Actin Abs. (C) BMDM from WT, MyD88^−/− (M), and TRIF^Lps2 (T) mice were stimulated with 1 μM CpG, 50 μg/ml poly(I-C), or 200 ng/ml Pam₃CSK₄ for 1 h, and MKP-1 protein expression was examined as above.

Fig. 3.

MKP-1^−/− macrophages exhibit higher p38 MAPK and JNK activation and increased AP-1 DNA-binding activity. (A) BMDM from MKP-1^+/+ and MKP-1^−/− mice were stimulated with 10 ng/ml LPS for the indicated time points, and activities of p38 MAPK, JNK, and ERK were examined by Western blot analysis using phosphospecific Abs. The total protein levels of p38 MAPK, JNK, and ERK also were measured. (B) BMDM from MKP-1^+/+ and MKP-1^−/− mice were stimulated with 10 ng/ml LPS, and AP-1 DNA-binding activity was measured by gel shift assay using AP-1 consensus oligonucleotide.

Fig. 4.

MKP-1^−/− macrophages show altered levels of IL-10 and TNF-α in response to LPS. (A) BMDM from MKP-1^+/+ (open bars) and MKP-1^−/− (filled bars) mice were stimulated with 10 ng/ml LPS for the indicated time points, and culture supernatants were harvested for ELISA analysis of IL-10 and TNF-α levels. (B) RNA was isolated and subjected to quantitative RT-PCR analysis of IL-10 and TNF-α levels. (C) BMDM from MKP-1^+/+ and MKP-1^−/− mice were incubated with (+) or without (−) an IL-10 neutralizing Ab (20 μg/ml) for 1 h, and activated with 10 ng/ml LPS for the indicated time points. Expression of TNF-α mRNA was measured by quantitative RT-PCR analysis. Shown is a representative result from three independent experiments. (D) BMDM from MKP-1^+/+ (open bars) and MKP-1^−/− (filled bars) mice were stimulated with 10 ng/ml LPS, and expression of Bcl-3 was examined by quantitative RT-PCR analysis.

Fig. 5.

Enhanced IL-10 production in MKP-1^−/− macrophages can be blocked by a p38 MAPK inhibitor. (A) BMDM from MKP-1^+/+ (open bars) and MKP-1^−/− (filled bars) mice were stimulated with 10 ng/ml LPS, 1 μM CpG, 50 μg/ml poly(I-C), 10 μg/ml peptidoglycan (PGN), 200 ng/ml Pam₃CSK₄, 100 ng/ml flagellin, or 200 μM loxoribine, or left untreated for 12 h. IL-10 production was measured by ELISA. (B Left) Serum from MKP-1^+/+ (white bars), MKP-1^+/− (gray bars), and MKP-1^−/− (black bars) mice (n ≥ 3 for each group) was collected 3 h after LPS challenge, and IL-10 levels were measured by ELISA. (Right) BMDM from MKP-1^+/+, MKP-1^+/−, and MKP-1^−/− mice were stimulated with 10 ng/ml LPS for 12 h, and IL-10 levels were measured by ELISA. (C) BMDM from MKP-1^+/+ (open bars) and MKP-1^−/− (filled bars) mice were treated with U0126 (ERK inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor), or vehicle alone (DMSO) at the indicated concentrations for 30 min and activated with 10 ng/ml LPS for 12 h. IL-10 levels were measured by ELISA.

Fig. 6.

Model of MKP-1-mediated temporal regulation of cytokine production in TLR signaling. Macrophage activation is divided into two phases: an initial phase characterized by rapid production of proinflammatory cytokines such as TNF-α and a second phase that involves the synthesis of IL-10. In the first phase, p38 MAPK and JNK are rapidly activated (≈20 min) and contribute to the expression of TNF-α. This early event is followed by the induction of MKP-1 (≈60 min) mediated by MyD88 and TRIF pathways, which serves as a negative feedback mechanism to down-regulate p38 MAPK and JNK activities and TNF-α production. In the second phase, activities of JNK and p38 MAPK are reduced because of MKP-1 but still higher than basal levels, whereas MKP-1 expression is suppressed to allow the remaining active p38 MAPK and JNK to promote IL-10 expression. IL-10, in turn, further limits TNF-α expression. For simplicity, only pathways relevant to MKP-1 are shown.