MAP kinase phosphatase-1, a gatekeeper of the acute innate immune response

Abstract

Mitogen-activated protein kinase (MAPK)^§ cascades are crucial signaling pathways in the regulation of the host immune response to infection. MAPK phosphatase (MKP)-1, an archetypal member of the MKP family, plays a pivotal role in the down-regulation of p38 and JNK. Studies using cultured macrophages have demonstrated a pivotal role of MKP-1 in the restraint of the biosynthesis of both pro-inflammatory and anti-inflammatory cytokines as well as chemokines. Using MKP-1 knockout mice, several groups have not only confirmed the critical importance of MKP-1 in the regulation of the cytokine synthesis in vivo during the acute host response to bacterial infections, but also revealed novel functions of MKP-1 in maintaining bactericidal functions and host metabolic activities. RNA-seq analyses on livers of septic mice infected with E coli have revealed that MKP-1 deficiency caused substantial perturbation in the expression of over 5000 genes, an impressive > 20% of the entire murine genome. Among the genes whose expression are dramatically affected by MKP-1 deficiency are those encoding metabolic regulators and acute phase response proteins. These studies demonstrate that MKP-1 is an essential gate-keeper of the acute innate immune response, facilitating pathogen killing and regulating the metabolic response during pathogenic infection. In this review article, we will summarize the studies on the function of MKP-1 during acute innate immune response in the regulation of inflammation, metabolism, and acute phase response. We will also discuss the role of MKP-1 in the actions of numerous immunomodulatory agents.

The innate immune system serves as the frontline defense against invading pathogenic organisms or damages as a result of injury. The host recognizes pathogen-associated molecular patterns evolutionarily conserved in pathogens via a variety of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), RIG-I-like receptors, cyclic GMP-AMP synthase, and nucleotide-binding oligomerization domainlike receptors [1–5]. Interaction of PRRs of the innate immune cells with microbial components leads to the activation of multiple signal transduction pathways, resulting in the activation of transcription factors such as nuclear factor (NF)-κB and interferon regulatory factors (IRFs), and the transcriptional induction of numerous immune-related genes [6]. NF-κB binds to the promoters of many cytokine and chemokine genes and activates their transcription [7,8], whereas IRFs can, sometimes in corporation with NF-κB, interact with the promoters of various interferon and interferon-regulated genes to mediate the interferon responses. Those cytokines and chemokines are crucial for the orchestration of the adaptive immune response, recruitment of microbicidal leukocyte effectors cells, and fortification of pathogen-resistant mechanism for the containment and ultimate clearance of the invading microbial pathogens [9,10]. For example, these cytokines can further stimulate a broad inflammatory response and induce the expression of many downstream genes to facilitate the antimicrobial host defense [6]. Interleukin (IL)-1 and IL-6 induce the expression of a group of acute phase response proteins in the liver, including proteins mediating pathogen opsonization and complement activation as well as blood coagulation [4,5,11,12]. Additionally, pathogen infections also cause a broad shift in the metabolism of the host, likely contributing to the complex host responses during pathogenic infections. Although host response to tissue damage is not fully understood, numerous studies have provided evidence that the PPRs used for pathogen recognition are also involved in the detection of damage-associated molecules characteristic of tissue injury [1]. For example, high mobility group box 1 protein, a non-histone DNA-binding nuclear protein released as the result of injury triggers inflammatory response through TLR2, TLR4, and the multi-ligand receptor for advanced glycation end products [13]. Although the immune responses to pathogens and/or injuries are critical in the host defense and tissue repair [8,14,15], exaggerated host responses can have devastating consequences to the host, leading to excessive organ damage and mortality. Because of the critical importance of the inflammatory response, through millions of years of evolution the mammalian organisms have developed intricate regulatory mechanisms to control the inflammatory response with multiple checkpoints in almost every step of the biological processes, including signal transduction.

MAPKs, including extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 subfamilies, play crucial roles in the host defense process [16]. First, MAPKs can regulate the transcription of many pro-inflammatory cytokine genes by phosphorylating certain transcription factors and promoting chromatin remodeling. Second, MAPKs may also control the transport, stability, and translation of mRNA transcripts containing AU-rich elements (ARE) in their 3’ untranslated regions, which are present in many cytokine mRNAs. This review article focuses on the role of a negative regulator of the MAPK pathway, MKP-1, in the innate immune response. We will summarize major studies on the function and regulation of MKP-1 in the innate immune response to microbial infection. We will concentrate on studies using MKP-1 knockout mice in bacterial infection models, particularly on aspects related to bacterial containment, host metabolism, acute phase response, and physiology. We will discuss the role of MKP-1 as an effector molecule in the actions of some pro- and anti-inflammatory agents. We apologize to colleagues whose important studies are not covered by this review because of the space limitation.

1. The function and regulation of MAPKs during immune responses

MAPKs are a group of serine/threonine protein kinases evolutionarily conserved across eukaryotic species. In mammalian cells, there are four well-defined MAPK subfamilies: ERK, JNK, p38, and BMK1 [17–19]. The MAPK pathway is activated via a cascade of sequential phosphorylation events, beginning with the activation of MAPK kinase kinase (MAP3K). MAP3K activates MAPK kinase (MAP2K) by phosphorylating two serine residues. MAP2K, a tyrosine/threonine dual specificity kinase, in turn activates MAPK by phosphorylating the adjacent threonine and tyrosine residues in a conserved TXY motif, where X represents a specific amino acid defining each of the four MAPK subfamilies. While both the ERK and BMK1 subfamilies have a TEY motif, the JNK and p38 MAPK subfamilies have a TPY and a TGY motif, respectively. The TXY motif resides in a regulatory loop between the kinase subdomains VII and VIII of the MAPK [20], and phosphorylation of the TXY motif results in a conformational change and > 1000-infold increase in catalytic activity [21]. Once activated, MAP kinases can phosphorylate a wide array of downstream targets, including protein kinases and transcription factors which facilitate the transcription of MAPK-regulated genes [22–24]. MAPK also regulate gene expression by facilitating chromatin remodeling and modulating the half-lives and translation of certain mRNA transcripts [25–31]. Additionally, MAPKs can directly modulate the enzymatic activities of a variety of enzymes, including downstream protein kinases, cytosolic phospholipase A2, and NADPH oxidase. Therefore, MAPKs play a pivotal role in the orchestration of a variety of cellular processes including cell proliferation, differentiation, stress response, apoptosis, and host immune defense.

In innate immune cells, MAPKs, p38 subfamily in particular, are crucial for the initiation of the inflammatory responses [16,32]. The p38 MAPK subfamily has at least 4 isoforms, p38α, p38β, p38γ, and ρ38δ. p38α and p38β are essential for the biosynthesis of numerous cytokines, chemokines, and other inflammatory mediators which are necessary for the immune system to combat pathogenic infections [33,34]. In addition to controlling the transcription of a variety of pro-inflammatory mediators, MAPK also regulate protein expression by altering the stability, transport, and translation of mRNA transcripts that contain ARE in their 3’ untranslated regions [16]. It has been demonstrated that tristetraprolin (TTP) binds to the ARE of many cytokine transcripts and promotes deadenylation and destabilization of these cytokine mRNAs [35]. p38 MAPK, particularly p38a and β, can activate a downstream protein kinase, MAPK-activated protein kinase (MK) 2 that in turn phosphorylates TTP at Ser-52 and Ser-178. Phosphorylation of TTP by MK2 decreases its affinity to ARE, thus favoring the interaction of the ARE-containing mRNAs with another RNA-binding protein HuR and facilitating the translation of these mRNAs [28–31]. Many pro-inflammatory cytokine and chemokine transcripts, including TNF-α, IL-1β, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-2, CXCL1, CXCL2, as well as proinflammatory protein COX2, contain ARE in their mRNAs [36], and are targets of TTP-mediated mRNA decay and translational depression [37,38]. In addition to the regulation of the expression of inflammatory mediators, MAPKs are also implicated in the regulation of reactive oxygen and nitrogen species [39,40], which are critical for the killing of microbes engulfed by phagocytes. In adaptive immune cells, MAPKs serve as critical regulators in the maturation of T-lymphocytes and clonal expansion of effector T- and B-lymphocytes through modulation of cytokine production, cell proliferation, and survival [16,41].

MAPK pathways are activated through phosphorylation. Thus, it is unsurprising that dephosphorylation of MAPKs by phosphatases is the primary mode of negative regulation. A number of protein phosphatases are known to deactivate MAPK, including tyrosine phosphatases, serine/threonine phosphatases, and dual specificity phosphatases (DUSPs) that are capable of removing the phosphate groups from phosphoserine, phosphothreonine, and phosphotyrosine residues [42]. In fact, strong evidence has accumulated to support the role of DUSPs with a N-terminal MAPK-binding domain as the most important phosphatases responsible for dephosphorylation of MAPKs [42]. This specific group of DUSPs are often referred to as MAPK phosphatases (MKPs). To date, at least 10 MKPs have been identified in mammalian cells [42], with MKP-1 being the archetypal member.

2. The role of MKP-1 in the regulation of host inflammatory response to pathogens

2.1. The biochemistry

MKP-1, also referred to as DUSP1, is the first member of the MKP family identified and the best characterized with regard to its functions during immune response. Initially cloned as an immediate-early gene highly induced by mitogen and oxidative stress [43–45], MKP-1 contains a (I/V)HCXAGXXR(S/T)A signature motif, characteristic of the catalytic domain of tyrosine phosphatases, in the C-terminal region (Fig. 1A). The C-terminal region of MKP-1 also shares a considerable homology with VH1, a vaccinia virus DUSP. Compared to VH1, MKP-1 is substantially larger due to a novel N-terminal domain, which is absent in VH1. Since this protein exhibited relatively high enzymatic activity towards ERKs both in vitro and in cultured cells, capable of dephosphorylating phosphotyrosine and phosphothreonine residues, it was designated as MKP-1 [46–49]. Because MKP-1 is robustly induced by mitogenic stimulation and oncogenic Ras which also activates ERKs, it was proposed that MKP-1 acts as a feedback control mechanism to switch off the ERK pathway. Subsequent studies have demonstrated that in addition to ERK, MKP-1 also inactivates the stress-activated JNK and p38 MAPKs. Liu et al. found that MKP-1 was robustly induced in HeLa cells by genotoxic stress which potently activated JNK but had little effect on ERK, suggesting that MKP-1 plays an important role in the feedback control of the stress-induced genetic responses [50]. Raingeaud et al. demonstrated that MKP-1 was highly effective in the inhibition of p38 activity [51]. To address the substrate preference of MKP-1, Franklin and Kraft established a U937 cell line which could express MKP-1 at different levels. By titrating the expression level of MKP-1, they demonstrated that p38 and JNK were much more sensitive than ERK to dephosphorylation by MKP-1 [52]. Several subsequent studies conducted using MKP-1 knockout cells further support the conclusion that p38 and JNK, but not ERK, are the preferred substrates of MKP-1 [53–55]. However, these studies do not exclude the possibility that MKP-1 may also participate in the inactivation of ERK, particularly when MKP-1 is expressed in high levels. In mice, the lungs are one of the organs with the highest levels of MKP-1 expression [44]. Following systemic E. coli infection, substantial increases in ERK activities were seen in the lungs of MKP-1 knockout mice relative to wildtype mice, indicating an important role of MKP-1 in ERK regulation in some organs [40].

Fig. 1.

Diagram of the structure and function of MKP-1. (A) The primary structure of MKP-1. MKP-1 has an amino terminal region which contains a basic amino acid cluster surrounded by a CDC25-homology (CH) 2 domain. The basic cluster (marked in blue as kinase-interaction motif) is required for interaction with substrate MAPKs. The catalytic domain characteristic of all tyrosine phosphatases is located at the carboxyl region, with a cysteine at the center of the catalytic site. The carboxyl terminus of MKP-1 contains a degradation signal. Phosphorylation of the two serine residues at the carboxyl terminus by ERK masks the degradation signal, leading to stabilization. (B) The predicted MKP-1 structure. The 3D structure of MKP-1 was predicted the template-based protein structure modeling using the RaptorX web server [131,132]. The N-terminal kinase-interaction motif was marked in blue, while the CH2 domain was marked in yellow. The catalytic site was marked in red. Note Cys-258, Arg-264, and Asp227 locate closely in the catalytic site. Binding of substrate with MKP-1 via the kinase-interaction motif may cause a conformational change, bringing Asp-227 closer to Cys-258. Asp-227 acts as a general acid in the dephosphorylation reaction. Dephosphorylation of the substrate is initiated by a nucleophilic attack of the active site cysteine (Cys-258) on the phosphorus atom of the bound substrate. As the ester bond is cleaved, Asp-227 donates its proton to the leaving group oxygen of the substrate side chain. Closer positioning of Asp-227 to Cys-258 enhances the catalytic activity of the phosphatase. The degradation signal at the carboxyl terminus is marked in magenta. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.2. Control the inflammatory response to microbial components

2.2.1. MKP-1 function at the cellular level

The earliest experimental evidence implicating MKP-1 in innate immune response was obtained from an unbiased study to identify differentially expressed genes in macrophages after phagocytosis of Gram-positive Listeria monocytogenes by differential display [56]. Schwan et al. identified MKP-1 as a highly induced gene in immortalized murine J774 macrophage-like cells exposed to L. monocytogenes. They further observed that MKP-1 was also substantially induced by Shigella flexneri and Salmonella typhimurium, and slightly stimulated by a non-pathogenic strain of E. coli (HB101, a strain widely used in laboratories for molecular cloning) or latex beads [56]. Overexpression of MKP-1 significantly attenuated the phagocytosis of L. monocytogenes, suggesting that MKP-1 may inhibit this innate immune function [57]. Using macrophages derived from mouse bone marrow, Valledor et al. demonstrated that MKP-1 was potently induced by bacterial lipopolysaccharide (LPS) through a transcriptional mechanism positively regulated by protein kinase Ce and a tyrosine kinase(s) [58]. They also found that MKP-1 induction coincided with ERK inactivation, and suggested that MKP-1 may be responsible for modulating ERK in this system.

To understand whether MKP-1 controls cytokine expression during bacterial infection, Chen et al. examined the kinetics of MAPK activation and MKP-1 expression in RAW264.7 macrophages following LPS stimulation. They found that stimulation of RAW264.7 macrophages with LPS resulted in a spike in the activity of both JNK and p38 [59,61]. The activities of these MAPKs reached peak levels within 15 min, and returned to nearly basal levels by 60 min, while MKP-1 protein levels were increased dramatically from undetectable basal levels. The kinetics of p38 and JNK deactivation correlated closely with the accumulation of MKP-1 protein such that the increasing level of MKP-1 was temporally associated with the diminution of p38 and JNK activities. ERK activity was also enhanced in response to LPS stimulation. However, unlike JNK and p38, ERK activity did not change significantly with the accumulation of MKP-1 protein. As with LPS, stimulation of RAW264.7 macrophages with peptidoglycan also elicited transient activation of JNK and p38 [60]. The deactivation of these kinases also occurred concomitantly with MKP-1 induction [60]. The importance of MKP-1 in the deactivation of p38 and JNK was demonstrated by blocking of MKP-1 expression pharmacologically with triptolide, a diterpenoid triepoxide [59,60]. Triptolide is a potent non-specific transcriptional blocker [61], stopping gene transcription by reacting with a cysteine residue of XPB, a subunit of the general transcription factor TFIIH [62,63]. Blockade of MKP-1 induction by triptolide in LPS-stimulated macrophages prolonged p38 and JNK activation, but had little effect on ERK activity [59,60]. These results illustrated the importance of MKP-1 in the deactivation of p38 and JNK in these cells. A modest increase in MKP-1 expression in RAW264.7 cells shortened the window of p38 and JNK activation in LPS- and peptidoglycan-stimulated cells, and substantially inhibited the production of both TNF-α and IL-6 [59,60].

To delineate the physiological function of MKP-1 in microbial infection, several laboratories, including our own, have studied the effects of MKP-1 deficiency on the inflammatory response of primary innate immune effector cells isolated from wildtype and MKP-1 knockout mice [53–55,64]. Note, MKP-1 knockout mice were referred to as Duspl knockout mice in several studies that adopted the Human Genome Organization nomenclature [54,65–68]. MKP-1 knockout mice do not exhibit any immunological phenotype under normal housing conditions [69], although subsequent studies showed that these mice have lower body weight and are leaner than wildtype mice [70]. Compared to primary macrophages isolated from wild type mice, peritoneal and alveolar macrophages originating from MKP-1 knockout mice exhibited prolonged p38 and JNK activation after stimulation with LPS [53–55,64,71 ]. The kinetics of ERK activation were not altered by MKP-1 deficiency. Similar findings were found with peptidoglycan and lipoteichoic acid, two important cell wall components of Gram-positive bacteria [72]. These results corroborate the observations made with immortalized macrophages [59,60], and have firmly established MKP-1’s role as the primary phosphatase for p38 and JNK in innate immune cells. Macrophages, splenocytes, and bone marrow-derived dendritic cells originated from MKP-1-deficient mice produced substantially larger quantities of pro-inflammatory cytokines and chemokines, including TNF-α, IL-6, macrophage inflammatory protein (MIP)-1α, MIP-1β, and MIP-2, than their wildtype counterparts [53–55,64]. It is important to note that in addition to augmented production of pro-inflammatory cytokines and chemokines, deletion of the MKP-1 gene also profoundly enhanced the synthesis of a potent anti-inflammatory cytokine, IL-10. However, the expression of two classic T_H-1 cytokines, IL-12 and interferon (IFN)-γ, was decreased in MKP-1-deficient splenocytes and dendritic cells [53], suggesting a shift in cytokine production profiles. Decreased IL-12 and IFN-γ production is likely caused by over-production of IL-10, as IL-10 is a potent anti-inflammatory cytokine known to inhibit IL-12 and IFN-γ through an autocrine system [73–75].

To understand the function that MKP-1 plays in the regulation of the inflammatory response systemically, Hammer et al. challenged wild type and MKP-1 knockout mice (referred to as Dusp1^−/− mice in the article) with endotoxin and analyzed splenic gene expression profiles using microarray [54]. They found that in the spleens of wild type mice, LPS challenge caused > 2-fold increases in the expression of approximately 160 genes, whereas approximately 3 times the number of genes in the spleens of the MKP-1 knockout mice exhibited > 2-fold increase in expression levels. The augmented inflammatory responses in MKP-1-deficient innate immune cells are also seen in cells exposed to other microbial components, including ligands for TLR2, TLR3, TLR5, TLR7 and TLR9 [55,60,76].

Together, the studies carried out using both immortalized and primary innate immune cells have firmly established the concept that MKP-1 is a pivotal negative regulator of the acute innate immune response. During the acute innate immune response, microbial infections are detected by pathogen-sensing receptors such as TLRs, and such information is conveyed through a set of adaptor proteins and protein kinases, resulting in activation of NF-κB and MAPK pathways. NF-κB plays a pivotal role in the transcriptional induction of cytokine genes. MAPKs, particularly p38, enhance the stability and translation of cytokine mRNAs by activating MK-2 and stimulating TTP phosphorylation. Simultaneously, signals initiated at the pathogen-sensing receptors also stimulate the transcription of MKP-1 gene, at least partially through the ERK pathway. ERK can phosphorylate MKP-1 protein, leading to a drastic increase in MKP-1 protein stability and an accelerated MKP-1 protein accumulation. MKP-1 will inactivate p38 and JNK, turning off the production of cytokines. In this sense, MKP-1 serves as a servo controller of the acute inflammatory response. The very low basal level of MKP-1 in quiescent innate immune cells permits a narrow window of robust inflammatory response necessary for cytokine production. Yet the rapid induction of MKP-1 following stimulation allows the system to tune down the inflammatory response, preventing the harmful consequences of overzealous inflammation. Thus, by modulating the activities of both p38 and JNK, MKP-1 limits the magnitude and duration of the p38/JNK signals that drive the production of inflammatory cytokines. In other words, MKP-1 serves as an intrinsic feedback restraining mechanism of the MAPKs to prevent overreaction of the innate immune system (Fig. 2). Interestingly, using a highly sensitive Forster resonance energy transfer (FRET)-based p38 activity reporter, Tomida et al. examined the dynamics of p38 activation in individual HeLa cells stimulated with IL-1β [77]. They found that after an initial burst activity, p38 activity undergoes an asynchronous oscillation following IL-1β stimulation for > 8 h [77]. By using mathematical modeling coupled with FRET-based p38 reporter experiments, they demonstrated that the asynchronous p38 activity oscillation under a constant inflammatory stimulus is the result of a negative feedback loop mediated by MKP-1 [77]. While such regulation is demonstrated with IL-1β-stimulated HeLa cells, p38 oscillation also appears to occur in LPS-stimulated macrophages [78], suggesting that MKP-1-mediated p38 oscillation may be involved in the time-dependent cytokine production in leukocytes following pathogenic infections.

Fig. 2.

Restraint of pro-inflammatory cytokine biosynthesis by MKP-1. (A). Diagram illustrating the role of MKP-1 in the regulation of the innate immune response. In response to microbial infection, TLRs engage various adaptor proteins, including MyD88, TRAM, TRIF, via the Toll/IL-1 receptor (TIR) homology domains. While MyD88 can activate TRAF6 through IL-1 receptor-associated kinases (IRAKs), TRIF can also activate TRAF6 through a mechanism independent of IRAKs. TRAF6 can turn on both the IKK/NF-κB and MAPK pathways. IKKs will phosphorylate IκB leading to its degradation and resulting translocation of NF-κB transcription factor to the nucleus. MAPKs can also phosphorylate certain transcription factors to modulate their activities. In the nucleus, NF-κB regulate the transcription of cytokine genes, sometimes in cooperation with transcription factors controlled by MAPKs. p38 phosphorylates/activates MK-2, which in turn phosphorylates TTP, leading to both enhanced cytokine mRNA stability and accelerated cytokine mRNA translation, ultimately resulting in enhanced cytokine production. Simultaneously, activation of TLRs also induce MKP-1 gene transcription through a signaling process, at least partially mediated by ERK, leading to the production of MKP-1 protein. ERK also increases MKP-1 protein stability via phosphorylating MKP-1, leading to a rapid accumulation of MKP-1 protein. The MKP-1 protein in turn dephosphorylates MAPKs, particularly JNK and p38, thus stopping the perpetuation of the inflammatory cascades and terminating cytokine production. DD, death domain. (B) Dynamic shifting of the inflammatory signaling events as well as cytokine synthesis and levels at different phases after microbial infection.

2.2.2. The phenotypes of MKP-1 knockout mice following exposure to TLR ligands

Consistent with the notion that the absence of MKP-1 knockout induces hyper-inflammation in innate immune effector cells during the bacterial ligand response, LPS challenged MKP-1-deficient mice produced substantially greater amounts of TNF-α, IL-1β, monocyte chemoattractant protein (MCP)-1/CCL2, GM-CSF, IL-6, and IL-10, than wild type mice [53–55,64]. The excessive production of the pro-inflammatory mediators was associated with a markedly increased LPS sensitivity. Compared to wild type mice, MKP-1 deficient mice more readily succumbed to LPS challenge, as indicated by injury and dysfunction in multiple organs as well as an increased rate of mortality [53–55,64]. Additionally, in response to either cell wall components isolated from Staphylococcus aureus or heat-killed S. aureus, MKP-1 knockout mice exhibited more robust cytokine production, more severe injury and greater mortality than did similarly treated wildtype mice [76].

Severe hypotension is a clinical characteristic of sepsis and plays a direct role in the development of shock and multi-organ dysfunction syndrome [53]. MKP-1 knockout mice exhibited impaired cardiovascular responses after LPS challenge, indicated by a substantial and long-lasting decrease in systemic blood pressure associated with cardiac dysfunction [79]. Underlying the severe decrease in blood pressure in MKP-1 knockout mice, a marked increase in circulating NO was detected [53]. Analysis of the lungs and livers of these MKP-1 knockout mice have indicated that iNOS expression levels were substantially greater than in similarly treated wild type mice [40]. To address the molecular mechanism underlying the enhanced iNOS induction, Wang et al. isolated peritoneal macrophages from wildtype and MKP-1 knockout mice, and examined iNOS induction by LPS with or without IFN-γ [40]. They found that knockout of MKP-1 clearly enhanced the induction of iNOS. Both heightened iNOS gene transcription and increased mRNA stability contributed to the elevated iNOS expression in macrophages following LPS stimulation. Interestingly, MKP-1 deficiency substantially enhanced the tyrosine phosphorylation of STAT1, which mediates iNOS induction and is activated by IFN-γ, but had no effect on NF-κB. MicroRNA array analysis indicated that microRNA (miR)-155 expression was increased in MKP-1-deficient macrophages relative to wildtype macrophages. Transfection of miR-155 attenuated the expression of Suppressor of Cytokine Signal (SOCS)-1 and enhanced the expression of iNOS. These results suggest that MKP-1 suppresses miR-155 expression, leading to enhanced SOCS-1 expression, ultimately resulting in a restraint of the JAK/STAT1 pathway and iNOS expression. As MiR-155 is a known facilitator of the inflammatory response [80], inhibition of miR-155 by MKP-1 represents an additional mechanism by which MKP-1 negatively regulates the inflammatory response and protects the host against excessive harmful inflammation.

2.2.3. The role of MKP-1 in mice during bacterial infection

2.2.3.1. On inflammation.

Several studies have been carried out to define the physiological function of MKP-1 during bacterial infection, using live bacterial pathogens. We infected wildtype and MKP-1 knockout with both E. coli and Staphylococcus aureus in a systemic bacterial sepsis model, and examined bacterial burden, pathology, cytokine production, and mortality [76,81]. In response to both E. coli and S. aureus infection, MKP-1 knockout mice produced substantially more pro-inflammatory cytokines (TNF-α and IL-6) and chemokines (MIP-1α/CCL3), as well as the anti-inflammatory cytokine IL-10. Enhanced iNOS and COX-2 expression were seen in several organs [76,81]. Hammer et al. investigated the consequences of MKP-1 deficiency during systemic bacterial infection using colon ascendens stent peritonitis (CASP) and caecal ligation and puncture (CLP) models [66]. Following CASP, MKP-1 knockout mice (referred to as Dusp1^−/− mice in the articles) had increased serum levels of CCL4, IL-10 and IL-6 relative to wildtype mice [66]. These cytokines, along with iNOS mRNA, were also expressed at higher levels in spleen and liver tissues. Similar over-production of these cytokines was detected in the CLP model, with even larger differences from wildtype mice [66]. In a pulmonary infection model, Rodriguez et al. infected wildtype and MKP-1 knockout with an intracellular pathogen Chlamydophila pneumoniae [82]. They found that following nasal infection, MKP-1-deficient mice mounted an augmented pulmonary cytokine (IL-1β, IL-6) and chemokine response (CCL3, CCL4, CXCL1, CXCL2), which was associated with an increased granulocyte but not monocyte/ macrophage infiltration to the lungs. Although the expression of Thl cytokines, IL12p40 and IFN-γ, in the lungs was similar between C. pneumonia-infected wildtype and MKP-1 mice, iNOS expression in the lungs of MKP-1 knockout mice was elevated relative to wildtype mice. Thus, amplified inflammatory response appears to be a general phenomenon of MKP-1 knockout following bacterial infections.

2.2.3.2. On bacterial burden and host mortality.

While MKP-1 knockout mice displayed enhanced inflammatory response to bacterial infections, the effect of MKP-1 on the outcome of the infection appears to be dependent on the type of bacterial pathogens, at least in the case of systemic infections. Our group found that the mortality of wildtype and MKP-1 knockout mice after systemic S. aureus infection were similar, despite the enhanced inflammatory response in the MKP-1 knockout mice [76]. The bacterial burden was also comparable between the S. aureus-infected wildtype and MKP-1 knockout mice. In contrast, following systemic E. coli infection MKP-1 knockout mice exhibited increased mortality and substantially greater bacterial burden relative to wildtype mice [81]. Heightened inflammatory response in the MKP-1 deficient mice in response to both E. coli and S. aureus was associated with increased neutrophil infiltration to the lung, lung edema, and organ damage. Interestingly, following E. coli infection MKP-1 deficient mice had reduced blood volume, profound hemoconcentration, and substantially decreased blood pressure relative to wildtype mice, suggesting severe capillary leakage [81]. Additionally, these MKP-1 knockout mice also displayed profound abnormality in metabolism. Wildtype mice developed hyperlipidemia and had liver glycogen depletion following E. coli infection, while MKP-1 knockout mice failed to mobilize triglycerides to the blood and utilize liver glycogens (See details in the later sections). Since IL-10 was also dramatically increased in the blood of E. coil-infected MKP-1 knockout mice, we examined IL-10 deficiency by using a neutralizing antibody and in a IL-10 knockout mice model [81]. IL-10 depletion substantially decreased the bacterial burden in the E. coil-infected MKP-1 knockout mice.

Similar to our observation, Hammer et al. found that despite the increased inflammatory response, MKP-1 knockout mice (referred to as Dusp1^−/− mice in their articles) suffered increased lethality and elevated bacterial burden in both the CASP and CLP peritonitis models [66]. In a lung infection model, Rodriguez et al. found that MKP-1 knockout mice had more severe weight loss than the wildtype mice after C. pneumonia infection [67]. Rodriguez et al. found that in the C. pneumonia infection model, MKP-1 knockout mice displayed elevated IL-6 as well as soluble IL-6 receptor a (sIL-6Ra) in lung homogenates [67]. IL-6 can form a complex with sIL-6Ra, and together they bind to the signaling receptor chain gp130, which is expressed on many cell types. This process is termed IL-6 trans-signaling and renders IL-6Ranegative cells, such as endothelial cells, responsive to IL6 [83]. Rodriguez et al. took the advantage of a fusion protein of soluble gpl30 with a Fc fragment (sgp130-Fc) to block IL-6 trans-signaling in vivo [67]. They found that a single injection of sgp130-Fc one day post C. pneumonia infection attenuated bacterial burden and decreased cytokine levels to those in C. pneumonia-infected wildtype mice. These results clearly demonstrate the critical role of IL-6 signaling in the immunological defect of MKP-1 knockout mice in this model. As MKP-1 knockout substantially enhanced the production of multiple anti-inflammatory cytokines, including IL-10, IL-1Ra (Wang and Liu, unpublished findings), and dual-functional cytokine IL-6, these cytokines could all contribute to the defects of the host defense during bacterial infections.

2.2.3.3. On liver metabolism and acute phase response.

The liver plays a central role in the metabolic processes in the body. It also produces acute phase response proteins (APP) following pathogenic infections. MKP-1 appears to regulate both metabolic processes and the acute phase response. Frazier et al. found that following systemic E. coli infection wildtype mice mobilized large amounts of triglycerides to the blood and depleted glycogen in the liver upon systemic E. coli infection, while MKP-1 knockout mice failed to mobilize fat to blood and utilize liver glycogen [81]. Liver lipid contents in wildtype and MKP-1 knockout mice either before or after E. coli infection were measured [84]. Consistent with the report that MKP-1 knockout mice in normal housing condition are leaner than wildtype mice due to increased energy expenditure [70], we found that in the absence of E. coli infection, MKP-1 knockout mice had lower liver triglyceride content than wildtype mice. E. coli infection resulted in a significant increase in liver triglyceride levels in wildtype mice, but not in MKP-1 knockout mice [84]. To understand the molecular mechanisms involved, we examined the global gene expression profile in the livers of wildtype and MKP-1 knockout mice either before or after E. coli infection by RNA-seq [84]. In wildtype mice E. coli infection resulted in a profound change in gene expression landscape: the expression of 2519 genes was enhanced and the expression of 2850 genes was attenuated. Impressively, over 5400 genes exhibited > 2-fold change in expression levels between E. coli-infected wildtype and MKP-1 knockout mice. As expected, E. coli infection-elicited TLR and inflammatory cytokine/chemokine pathways were dramatically enhanced in the absence of MKP-1. We also analyzed the RNA-seq data using DESeq2 algorithm to identify the pathways differentially affected by E. coli infection and MKP-1 knockout. We found that MKP-1 caused profound changes in the expression of large number of genes encoding various metabolic processes, including retinol metabolism, steroid synthesis, and carbon metabolism, suggesting an important function of MKP-1 in broad metabolic functions. The most surprising finding of the RNA-seq analysis is profoundly attenuated gene expression of a large number of lipogenic proteins in wildtype mice after E. coli infection. These results suggest that the hyperlipidemia in E. coli-infected septic wildtype mice is probably a result of lipid mobilization from fat tissues. These findings also support the notion that MKP-1 plays a significant role in the regulation of the host lipid metabolism and mobilization in response to bacterial infection.

Acute phase response is a systemic response against infection or injury [85,86]. Inflammatory cytokines, particularly IL-1 and IL-6, play a central role in the initiation of the acute phase response [87]. Acute phase response is an important host defense mechanism involving fever, leukocytosis, increased vascular permeability, and rapid changes in the serum levels of acute phase proteins, including c-reactive protein (CRP), serum amyloid P component (APCS), and serum amyloid A (SAA) proteins. Many of the acute phase proteins, such as CRP and APCS directly mediate the capture and destruction of microbial pathogens through activating the complement and coagulation pathways, while others such as SAA proteins facilitate the recruitment of leukocytes to the inflammatory sites and stimulate phagocytosis. RNA-seq analysis of wildtype and MKP-1 knockout mice livers, after systemic E. coli infection, offered us a unique opportunity to examine the role of MKP-1 in the regulation of the acute phase response (Fig. 3). The genes encoding the acute phase proteins are listed in the left column, and the expression levels were expressed using a heat map, with red indicating high expression and green depicting low expression. Each column represents an individual mouse. Several differences were seen in the mRNA expression of acute phase proteins between the wildtype and MKP-1 knockout mice: 1) Major acute phase proteins: As expected, in the livers of the wildtype mice the mRNA levels of the major acute phase proteins, SAA1-3, APCS, and CRP were lower in un-infected controls and substantially elevated following systemic E. coli infection. Although SAA1 and SAA2 mRNA levels in the liver of E. coii-infected MKP-1 knockout mice were increased to levels comparable to those in the E. coli-infected wildtype mice, SAA3 mRNA levels in the E. coli-infected MKP-1 knockout mice were significantly lower than those in similarly infected wildtype mice. Unlike the modest up-regulation seen in the E. coli-infected wildtype mice, CRP mRNA expression did not change in the MKP-1 knockout mice following E. coli infection. Unlike APCS expression in wildtype mice that was amplified by > 20-fold following E. coli infection, APCS mRNA expression was only enhanced by 2-3-fold in E. coli-infected MKP-1 knockout mice. 2) Moderate/minor acute phase proteins: Overall there appeared to be an enhancement in the expression of this group of genes in the E. coli-infected MKP-1 knockout mice relative to similarly infected wildtype mice, except for complement component 3 (C3), complement factor b (Cfb), ceruloplasmin (Cp), and several members of the phospholipase A2 group (Plag6, 7, 12b, 15, and 16). Interestingly, several genes in this group were down-regulated after E. coli infection in wildtype mice, including fibronectin 1 (Fn1), Von Willebrand factor homolog (Vwf), complement components 4B (C4b) and 9 (C9), although these genes are reported to be up-regulated in humans during acute phase response [88,89]. 3) Negative acute phase proteins: In the wildtype mice, liver mRNA levels of the negative acute phase proteins, except transferrin (Trf), were markedly down-regulated after E. coli infection in wildtype mice. The basal expression levels of these genes appeared to be elevated in control MKP-1 knockout mice relative to wildtype mice. These genes also failed to tone down their expression in MKP-1 knockout mice following infection. The increased IL-1 and IL-6 levels in the blood of E. coli-infected MKP-1 knockout mice [81] could be responsible for the up-regulation of the acute phase proteins. Liver dysfunction and simultaneous elevation of some pro-inflammatory cytokines in the E. coli-infected MKP-1 knockout mice may also contribute to the abnormality in the acute phase protein expression.

Fig. 3.

Liver mRNA expression levels of acute phase proteins in control and E. coli-infected wildtype and MKP-1 knockout mice. Wildtype and MKP-1 knockout mice were infected with either E coli (2.5 × 10⁷ CFU/g body weight) or given PBS via the tail veins. Total RNA was isolated from the livers 24 h post infection, and analyzed by RNA-seq. mRNA transcript numbers were log-transformed and used to generate the heat map. High, intermediate, and low expression are indicated by red, white, and green respectively for each gene. Each column displays a separate animal. APP, acute phase protein; WT, wildtype; KO, MKP-1 knockout. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3. The regulation of MKP-1 during innate immune responses

The activity of MKP-1 can be regulated at multiple levels, including gene transcription, protein stability, and catalytic activation as a result of substrate binding-induced conformational changes.

3.1. Transcriptional regulation

MKP-1 transcription can be robustly induced by a variety of growth factors, microbial components, and stressful insults [42]. As one of the immediate-early genes, MKP-1 induction occurs rapidly after extracellular stimuli occurs in a fashion independent of de novo protein synthesis [43]. MKP-1 mRNA levels can be increased by 10–100 fold within 15–60 min in response to extracellular stimulus. Because the stability of MKP-1 mRNA does not appear to change significantly [90], the induction of MKP-1 expression is very likely mediated by a transcriptional mechanism. In leukocytes, transcriptional induction of MKP-1 gene is also a major contributing factor to the increases in MKP-1 protein during the innate immune response. MKP-1 mRNA can be detected within 15 min after exposure of macrophages to bacterial components, with maximal mRNA levels reaching > 100-fold above basal levels within 1 h. Valledor et al. demonstrated that MKP-1 was potently induced in bone marrow-derived macrophages by LPS through a transcriptional mechanism mediated by protein kinase Cε and a tyrosine kinase(s) [58]. In RAW264.7 macrophages, the transcriptional induction of MKP-1 by LPS was substantially inhibited by the MEK1/2 inhibitor U0126, suggesting that ERK plays an important role in the induction of MKP-1 transcription [59]. Since the ERK pathway is regulated by the protein kinase Cε and a tyrosine kinase(s) [91], it is tempting to speculate that at least some of the effects of these upstream regulators on MKP-1 are mediated by ERK. However, Neither ERK activation alone induced MKP-1 expression [92] nor did U0126 completely block MKP-1 induction, suggesting that other pathways also contribute to MKP-1 induction [59]. Consistent with this postulation, Sanchez-Tillo et al. demonstrated that JNK1 is required for the induction of MKP-1 in macrophages in response to LPS [93]. We also found that p38 contributes to the induction of MKP-1 in RAW264.7 macrophages [59]. Chi et al. examined the upstream regulators controlling MKP-1 induction by TLR ligands in primary macrophages lacking either MyD88 or TRIF [55]. The ligands of TLR9 and TLR2 only signal through MyD88-mediated pathways. In contrast, the TLR3 ligands, such as poly (I-C), trigger a signaling pathway through TRIF. The TLR4 ligand, LPS, initiates innate immune responses through both a signaling cascade mediated by MyD88 and a signaling pathway mediated by TRIF. Chi et al. found that the induction of MKP-1 by LPS was reduced in both the MyD88- and TRIF-deficient cells, as compared with wildtype cells, indicating that both MyD88 and TRIF contribute to optimal MKP-1 induction. In response to ligands of TLR9 and TLR2, MKP-1 induction was completely ablated in MyD88-deficient macrophages, but was normal in TRIF-deficient cells. Conversely, loss of TRIF, but not of MyD88 function, eliminated MKP-1 expression induced by poly(I-C). Thus, during the response to pathogens multiple signaling pathways can contribute to MKP-1 induction.

While transcriptional induction plays a pivotal role in the induction of MKP-1 during the inflammatory response, the transcriptional factors and signaling pathways involved remain elusive. Several putative transcription factor-binding elements were identified in the promoter region of MKP-1 gene, including cAMP-responsive elements (CRE), SP-1, AP-1, AP-2, and NF-1-like sites, within a ~400-bp region of the MKP-1 promoter [94]. Ananieva et al. demonstrated that CREB and/or ATF1 are implicated in the induction of MKP-1 (referred to as DUSP1 in the article) in macrophages after stimulation with TLR ligands [95]. They showed that ligands for TLR2, 4, and 9 activate both ERK and p38 MAPKs, leading to activation of downstream mitogen- and stress-activated protein kinase (MSK) 1 and MSK2. MSK1/2 potently phosphorylates transcription factors occupying the CRE sequences in the MKP-1 promoter, including CREB and ATF1. Since abolishing the MSK1/2-targeted phosphorylation site of CREB had no effect on MKP-1 induction, Ananieva et al. speculated that MKP-1 induction in response to TLR ligands is likely mediated by ATF1. While this study proposed a frame work in the transcriptional regulation of the MKP-1 gene, more studies are still needed to advance our understanding of the transcriptional regulation of MKP-1 gene during the innate immune response.

3.2. Regulation of MKP-1 stability

The stability of MKP-1 can be altered dramatically via phosphorylation. Bronello et al. showed that MKP-1 protein can be phosphorylated at two serine residues (Ser-359 and Ser-364) near the C-terminus by ERK [96]. They also demonstrated that phosphorylation enhances MKP-1 protein stability. Their studies suggest that MKP-1 protein is degraded by the ubiquitin-directed proteasome complex. We found that MKP-1, at least in vitro, can also be phosphorylated by both JNK and p38 (Chen and Liu, unpublished findings), although the phosphorylation site(s) has not been fully characterized. Whether such phosphorylation is biologically significant, and if so, how MKP-1 phosphorylation by JNK and p38 affects MKP-1 stability await further examination. It is worth noting that findings on MKP-1 stability made with the use of pharmacological inhibitors of MAPK pathways should be viewed with caution, because there appear to be extensive cross-talks between distinct MAPK pathways in macrophages. For example, inhibition of the p38 pathway actually enhanced the ERK pathway [97]. Conversely, inhibiting the ERK pathway also led to an enhancement of the p38 pathway [59].

Although it is clear that MKP-1 phosphorylation by ERK enhances MKP-1 protein stability, the mechanism involved is still unclear. We found that mutation of the two C-terminal serine residues in MKP-1 to alanine decreased their half-lives, while mutating these residues to aspartates increased half-life of MKP-1 protein by 18-fold [97]. Deletion of the C-terminus from MKP-1 also increased the half-life of MKP-1 from ~30–40 min to ~150 min. These results suggest that the MKP-1 carboxyl terminus contains a degradation signal and phosphorylation of the serine residues masked this degradation signal (Fig. 1), resulting in an increased stability. Brondello et al. proposed that phosphorylation of MKP-1 at the C-terminus by ERK inhibits ubiquitin-mediated degradation, thus enhancing MKP-1 stability [96]. However, our own studies did not support this theory. Enhanced stabilities of either the full length phosphor-mimic (Ser-359 → Asp/Ser-364 → Asp) or the C-terminustruncated MKP-1 mutants were not associated with decreased polyubiquitination. In fact, more ubiquitination was seen with the more stable MKP-1 mutants. For both the wildtype and mutant MKP-1 proteins, ubiquitinated MKP-1 primarily contained a single ubiquitin molecule. Most importantly, the more stable phosphor-mimic or C-terminus-truncated MKP-1 mutants actually had more, rather than less ubiquitination.

3.3. Catalytic activation

Finally, in addition to transcriptional induction and increased protein stability, the catalytic activity of MKP-1 protein can be enhanced by interaction with its substrate MAPKs [98–100]. We have shown that inclusion of any of the three MAPKs, ERK, JNK, or p38, increases the catalytic activity of MKP-1 protein by 6–8 fold in in vitro biochemical assays [98]. Analysis of the crystal structure of a related phosphatase, MKP-3, has suggested that interaction between MKP-3 and its substrate ERK MAPK enables the phosphatase to adopt a more efficient conformation at the catalytic site [101]. Subsequent studies have demonstrated that the interaction between MAPKs and the MKP family is dependent on the kinase-interaction motif (KIM) at the amino terminus of the phosphatase and the acidic domain located at the carboxyl terminus of the kinase [102]. The kinase-interaction motif of all MKPs has the consensus sequence of ΨΨXRRΨXXG (where Ψ represents a hydrophobic residue and X is any amino acid), which is located insides of a Cdc25-homology 2 domain (Fig. 1A). Such as what was proposed for MKP-3 [101], interaction of a substrate MAPK with MKP-1 probably results in a conformational change, bringing the “general acid” Asp-227 closer to Cys-258 at the catalytic site, thus facilitating substrate dephosphorylation (Fig. 1B). Since physical interaction between MKP-1 and MAPKs were detected in LPS-stimulated macrophages (Chen and Liu, unpublished observations), catalytic activation of MKP-1 upon binding to its MAPK substrates is likely an important mechanism regulating MKP-1 activity during the innate immune response.

4. MKP-1 and immunomodulatory agents

4.1. Anti-inflammatory agents

4.1.1. Glucocorticoids

MKP-1 serves as an important negative regulator of the inflammatory response, raising an intriguing question of whether MKP-1 is implicated in the actions of some immunomodulatory agents. A number of studies have provided compelling evidence to support a significant role of MKP-1 in actions of some immunomodulatory agents. Perhaps the best studied example is the function of MKP-1 in the anti-inflammatory action of glucocorticoids. Chen et al. examined the effects of a panel of commonly used anti-inflammatory drugs on the expression of MKP-1. They found that MKP-1 is significantly induced by dexamethasone, an anti-inflammatory glucocorticoid, in RAW264.7 macrophages [59]. Such induction provided a mechanistic explanation to an earlier observation made by Swantek et al. that dexamethasone inhibited LPS-induced JNK activation [103]. While Liu’s group were the first to link induction of MKP-1 to the anti-inflammatory action of glucocorticoid in macrophages, other studies have addressed the mechanism by which glucocorticoid inhibit COX2 expression in human cervical cancer HeLa cells. Lasa et al. showed that dexamethasone enhanced MKP-1 expression in HeLa cells, which was linked to the inhibition of p38 and attenuated COX2 expression [104]. Kassel addressed the anti-allergic mechanism of glucocorticoid [105]. They demonstrated that dexamethasone-induced MKP-1 was responsible for the drug’s inhibitory effect on ERK activity in mast cells. Zhao et al. compared eight commonly used synthetic corticosteroids with different anti-inflammatory potencies, with regard to their capacity to induce MKP-1 expression. Some of the corticosteroids are routinely used systemically, such as dexamethasone and betamethasone, while others, e.g. clobetasol propionate, flunisolide, and beclomethasone, are primarily used in topical, inhaler, or nasal spray forms. Zhao et al. found that in MH-S alveolar macrophages the relative anti-inflammatory potencies of synthetic glucocorticoids were closely associated with their potencies to induce MKP-1 expression [71]. Using macrophages isolated from wildtype and MKP-1 knockout mice, Abraham et al. demonstrated that dexamethasone-mediated inhibition of p38 and JNK was abrogated in MKP-1-deficient macrophages. Additionally, dexamethasone-mediated suppression of several pro-inflammatory genes, including TNF-α, IL-1α, IL-1β, and COX2, was impaired in MKP-1-deficient macrophages, whereas other pro-inflammatory genes were inhibited by dexamethasone in a manner dependent on MKP-1 (referred to as DUSP1 in the article) [68]. Furthermore, in vivo anti-inflammatory effects of dexamethasone on zymosan-induced inflammation were impaired in MKP-1 knockout mice. Our studies partially corroborated their findings. We found that the inhibitory effects of dexamethasone on p38 and JNK were compromised, but not totally abolished in MKP-1-deficient macrophages [106]. Acceleration of p38 and JNK deactivation by dexamethasone was dependent on MKP-1, while the inhibitory effect of dexamethasone on p38 and JNK activation immediately (< 15 min) after LPS stimulation was independent of MKP-1. Dexamethasone treatment completely protected wildtype mice from the mortality caused by a relatively high dose of LPS, while dexamethasone only offered a partial protection to MKP-1 knockout mice. Dexamethasone attenuated TNF-α production in both wildtype and MKP-1 knockout mice challenged with LPS in vivo, although TNF-α production in MKP-1 knockout mice was significantly more robust than that in wildtype mice. Our results are consistent with the observation of Meier et al. who demonstrated TNF-α production in MKP-1/DUSPl-deficient mast cells is still sensitive to glucocorticoid inhibition [107]. It is important to note that MKP-1 is not only involved in the anti-inflammatory action of exogenously administered synthetic glucocorticoids, but also mediates the immunosuppressive effects of the endogenous stress hormone cortisol [108]. Since glucocorticoids are immunosuppressive substances released endogenously upon exposure to stress, MKP-1 induction by corticosteroids may represent a potential mechanism underlying the immunosuppressant property of stress. It should be pointed out that, while MKP-1 is required for the optimal anti-inflammatory activity of glucocorticoids, corticosteroids inhibit inflammatory responses through multiple mechanisms. MKP-1 induction constitutes only one of the antiinflammatory mechanisms of glucocorticoids. For example, glucocorticoids potently inhibit the transcription factor NF-κB [109]. We also showed that dexamethasone can inhibit p38 and JNK activation immediately after LPS stimulation in the absence of MKP-1 gene [106].

Mapracorat is a novel small molecule selective glucocorticoid receptor agonist (SEGRA) developed by Bausch and Lomb, though it is structurally distinct from corticosteroids [110–112]. Mapracorat was tested in clinical trials for the treatment of inflammatory skin and eye diseases. In preclinical studies, mapracorat potently inhibits the production of a variety of inflammatory mediators including cytokines and prostaglandin E2, with limited side effects compared to traditional corticosteroids. Vollmer et al. studied the mechanisms underlying the anti-inflammatory properties of mapracorat [78]. They found that mapracorat potently inhibited the production of GM-CSF and TNF-α in LPS-stimulated RAW264.7 macrophages. Mapracorat also substantially attenuated the expression of COX-2 and the production of prostaglandin E2. Examination of the activation kinetics of p38 and its downstream target MK-2 revealed a shortened activation course after LPS stimulation in cells pretreated with mapracorat. Mapracorat enhanced the expression of MKP-1 following LPS stimulation, explaining the accelerated p38 deactivation and the inhibition on the production of inflammatory mediators. Blocking MKP-1 expression by triptolide also abolished the accelerating effects of mapracorat on p38 and MK-2 deactivation, further supporting a role of MKP-1 in the anti-inflammatory mechanism of mapracorat.

4.1.2. IL-10

Hammer et al. performed a systematic analysis of genes whose expression was altered in response to LPS exposure, and found that several MKP/DUSP genes were induced in macrophages by LPS [65]. Interestingly, they found that MKP-1/DUSP1 expression was transiently up-regulated after stimulation with LPS alone, and its expression was augmented and sustained when cells were stimulated with both IL-10 and LPS. They further showed that IL-10 synergized with dexamethasone to enhance MKP-1/DUSP1 expression and to inhibit IL-6 and IL-12 production. Augmentation of MKP-1/DUSP1 expression by IL-10 in LPS-stimulated macrophages was associated with accelerated p38 deactivation, indicating that enhancement of MKP-1/DUSP1 may constitute an important part of the anti-inflammatory mechanism of IL-10 [65].

4.1.3. Vitamin D

Vitamin D deficiency has been implicated in various inflammatory diseases [113], although the mechanism by which vitamin D reduces inflammation remains poorly understood. Zhang et al. investigated the inhibitory effects of physiologic levels of vitamin D on LPS-stimulated inflammatory response in human blood monocytes, and explored potential mechanisms of vitamin D action [114]. They found that vitamin D, at physiologic concentrations, dose-dependently inhibited LPS-induced p38 phosphorylation as well as IL-6 and TNF-α production by human monocytes. Vitamin D also significantly enhanced the MKP-1 expression in human monocytes and murine bone marrow-derived macrophages. Both the murine and the human MKP-1 promoters have a vitamin D response element. Vitamin D treatment increased vitamin D receptor binding to the vitamin D response element and stimulated histone H4 acetylation at the MKP-1 promoter [114]. In MKP-1-deficient murine macrophages, the inhibition of LPS-induced p38 phosphorylation by vitamin D was completely abolished. Accordingly, vitamin D inhibition of LPS-induced IL-6 and TNF-α production by MKP-1-deficient macrophages was significantly attenuated. This study identified the up-regulation of MKP-1 by vitamin D as a novel potential mechanism by which vitamin D inhibits inflammation and exhibits therapeutic benefits to patients with certain inflammatory diseases.

4.1.4. Endocannabinoids

Endocannabinoids are endogenous lipid-based retrograde neurotransmitters that bind to cannabinoid receptors expressed throughout the vertebrate central nervous system (CNS) in the brain and peripheral nervous system. Endocannabinoids are released after brain injury and are believed to attenuate neuronal damage by binding to CB₁ cannabinoid receptors and protecting against excitotoxicity. After brain injury, excitotoxic brain lesions initially lead to primary destruction of brain parenchyma, which in turn attracts macrophages and microglia. Upon arrival, these macrophages and microglia produce toxic cytokines and free radicals, resulting in secondary neuronal damage. Eljaschewitsch et al. showed that the endocannabinoid system was highly activated during CNS inflammation [115]. They further demonstrated that the endocannabinoid anandamide protects neurons from inflammatory damage by CB_1/2 receptor-mediated rapid induction of MKP-1 in microglial cells. Induction of MKP-1 in response to anandamide was associated with histone H3 phosphorylation at the MKP-1 gene locus. As a result, anandamide-induced MKP-1 switches off ERK MAPK signal transduction and NO production in microglial cells that are activated by stimulation of pattern recognition receptors. They proposed that induction of MKP-1 by the released endocannabinoid anandamide in injured CNS tissue represents a new mechanism of neuro-immune communication during CNS injury, which controls and limits immune response after primary CNS damage.

4.1.5. Rapamycin

Rapamycin, a natural product of the bacterium Streptomyces hygroscopicus and a macrolide antibiotic, is a potent anti-proliferative medication with immunosuppressive properties [116]. In clinics, rapamycin is used to coat coronary stents, prevent organ transplant rejection, and to treat a rare lung disease called lymphangioleiomyomatosis [117–119]. Rastogi et al. investigated the effect of rapamycin on MKP-1 expression and found that low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression [120]. Rapamycin treatment led to phosphorylation of CREB, ATF1, and ATF2, three transcription factors that bind to CRE on the MKP-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. Rapamycin treatment induced MKP-1 in wildtype macrophages but not in macrophages lacking AKT1 or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation, and decreased nitric oxide and IL-6 production. These studies provide a novel conceptual framework for the immune modulatory effect of rapamycin.

5. Pro-inflammatory agents

5.1. IFN-γ

As MKP-1 acts to restrain inflammatory responses, it is unsurprising that some cytokines known to boost inflammation can inhibit MKP-1 expression. As a potent T_H-1 cytokine, IFN-γ can enhance the antimicrobial activity of macrophages. It has been demonstrated that priming resident peritoneal macrophages with IFN-γ dramatically increases the production of NO and TNF-α upon stimulation with LPS [121,122]. Interestingly, we found that priming of peritoneal macrophages with IFN-γ significantly attenuates the MKP-1 expression induced by LPS, which is associated with prolonged activation of p38 and JNK [53]. Although LPS by itself does not significantly induce iNOS expression in wildtype resident macrophages, it induces a substantial iNOS expression in MKP-1-deficient resident macrophages [40]. This finding suggests that the inflammation-enhancing effect of IFN-γ is achieved, at least in part, by its inhibition on MKP-1 expression, and knockout of MKP-1 partially alleviated the requirement of IFN-γ for the induction of iNOS [40]. In fact, in the absence of MKP-1, LPS was able to stimulate a robust STAT1 activation, further highlighting the function of MKP-1 in the crosstalk between TLR4-mediated inflammatory pathway and IFN-γ-stimulated signaling pathway. In addition to the role of MKP-1 in the pro-inflammatory function of IFN-γ, MKP-1 also appears to mediate the growth inhibitory and pro-maturation function of IFN-γ on macrophages. Valledor et al. showed that inhibition of MKP-1 by IFN-γ is responsible for the prolonged MAPK activation and underlies the growth inhibitory effects of IFN-γ on M-CSF-stimulated macrophages [99,100,123,124]. These studies suggest that inhibition of MKP-1 by IFN-γ may be an important part of the immunomodulatory mechanism of IFN-γ.

5.2. Macrophage migration inhibitory factor (MIF)

MIF is a potent pro-inflammatory cytokine which enhances the expression of other pro-inflammatory cytokines in macrophages. MIF is tightly associated with mortality in experimental bacterial sepsis models [125,126], as either knockout of the MIF gene or depletion of MIF protein protects animals from septic shock. MIF is considered to be a counter-regulator of the immunosuppressive effects of glucocorticoids [127]. Rogers et al. found that MIF inhibited the induction of MKP-1 by LPS and dexamethasone, and alleviated the inhibition on TNF-α and IL-8 production caused by dexamethasone in macrophages [127]. Conversely, blockade of MIF expression augmented MKP-1 induction by dexamethasone, leading to decreased TNF-α production. Aeberli et al. also independently demonstrated that endogenous MIF modulates glucocorticoid sensitivity in macrophages via inhabiting MKP-1 [128]. These studies demonstrate that MIF overrides inhibition of cytokine production by glucocorticoids in innate immune effector cells through attenuating MKP-1 expression. Thus, it appears that a number of immunomodulatory agents influence the MAPK-mediated inflammatory responses in part through regulating MKP-1 expression (Fig. 4).

Fig. 4.

Regulation of MKP-1 expression by immunomodulatory agents. Microbial infection or tissue injury (referred to as inflammatory insult) activates the inflammatory response through NF-κB and MARK pathways. MARK pathways also elicit a feedback control mechanism to restrain MAPK-mediated cytokine production by inducing MKP-1 expression. Anti-inflammatory/immunosuppressive agents, including glucocorticoids, IL-10, vitamin D, endocannabinoids, and rapamycin, induce/augment MKP-1 expression, leading to inhibition of p38, JNK, and possibly also ERK cascades and attenuation of the inflammatory response. In contrast, pro-inflammatory cytokines, such as IFN-γ and MIF, inhibit MKP-1 expression, thereby prolonging the p38 and JNK activation and enhancing the inflammatory response.

6. Closing remarks

The balance between prompt initiation and subsequent attenuation of the inflammatory response is critical during the host immune defense against microbial infection and tissue injury. While prompt initiation of the inflammatory response is pivotal for mounting an aggressive immune defense to invading pathogens and tissue injury, deactivation of the signaling pathways limits the potentially harmful effects of excessive inflammation on the host and preventing collateral damage. Moreover, deactivation of the inflammatory cascades also “resets” the regulatory circuits, allowing the immune system to react effectively to subsequent pathogenic challenges. A number of negative regulators have been discovered in the past few years that operate at nearly every step of the signal transduction pathways to moderate and turn off the immunological responses. These negative regulators act together to restrain the magnitudes and durations of the positive inflammatory signals, thereby modulating the production of inflammatory cytokines and other inflammatory mediators and ultimately shaping the course of the adaptive immune responses [129]. The discovery of MKP-1 as a crucial gatekeeper of the acute innate immune response, both in vivo and in vitro, places it in the center of the complex negative regulatory mechanism. Because of the critical role of the innate immune response in the development of the adaptive immunity, it is not surprising that the knockout of MKP-1 has a profound impact on the shape of the adoptive immune response [130]. The fact that many known immunomodulatory agents exert their immuno-regulatory actions at least partially through adjusting MKP-1 activity highlights the potential of MKP-1 as a therapeutic target in the treatment of immunological disorders. Thus, small molecule chemicals capable of enhancing or inhibiting MKP-1 activity could be novel drug candidates for the treatment of certain human diseases including arthritis, inflammatory bowel diseases, infection, and cancer as well as injury.

Abbreviations

MAPK

mitogen-activated protein kinase

AP-1

activating protein-1

AP-2

activating protein-2

ATF

activating transcription factor

APCS

serum amyloid P component;ARE AU-rich element

ARE

AU-rich element

BMK1

big MAPK 1

C3

complement 3

C9

complement 9

C4b

complement component 4B

CASP

colon ascendens stent peritonitis

CCL

chemokine C-C motif ligand

CD36

cluster of differentiation 36

CLP

cecal ligation and puncture

COX

cyclooxygenase

CRE

camp-response element

CREB

cAMP-response element-binding protein

CRP

c-reactive protein

CXCL

chemokine C-X-C motif ligand

DGAT2

diglyceride acyltransferase 2

DUSP

dual specificity phosphatase

ERK

extracellular signal-regulated kinase

Fn1

fibronectin 1

FRET

Förster resonance energy transfer

GM-CSF

granulocyte macrophage colony stimulating factor

IκB

inhibitor κB

IFN

interferon

IKK

IκB kinase

IL

interleukin

IL-1Ra

IL-1 receptor a

iNOS

inducible nitric oxide synthase

IRAK

IL-1 receptor-associated kinase

IRF

interferon regulatory factors

JAK

Janus kinase

JNK

c-Jun N-terminal kinase

LPS

lipopolysaccharides

MAP2K

MAPK kinase

MAP3K

MAPK kinase kinase

MCP

monocyte chemoattractant protein

MIF

macrophage migration inhibitory factor

MIP

macrophage inflammatory protein

miR

microRNA

MK-2

MAPK-activated protein kinase-2

MKP-1

MAPK phosphatase-1

MSK

mitogen-and stress-activated protein kinase

MyD88

myeloid differentiation factor 88

NF-1

nuclear factor-1

NF-κB

nuclear factor-κB

NO

nitric oxide

Plag

phospholipase A2 group

PRR

pattern recognition receptor

SAA

serum amyloid A

SOCS-1

suppressor of cytokine signal-1

sIL-6Ra

soluble IL-6 receptor a

SP-1

stimulating protein-1

STAT

signal transducer and activator of transcription

TFIIH

transcription factor II H

TLR

toll-like receptor

TIR

TLR/IL-1 receptor domain

TNF

tumor necrosis factor

TRAF

TNF receptor-associated factor

TRAM

TRIF-related adaptor molecule

TRIF

Toll-IL-1 receptor domain containing adaptor inducing IFN-β

Trf

transferrin

TTP

tristetraprolin

Vwf

von Willebrand factor

XPB

xeroderma pigmentosum type B-a subunit of TFIIH