We aimed to study the role of MKP-2 in regulating the inflammatory response in sepsis by utilizing the MKP-2 null mouse. Initially, we anticipated that MKP-2 would play a similar and perhaps redundant role to MKP-1 in negatively regulating cytokine production in response to a canonical inflammatory trigger (e.g., LPS). Contrary to our initial hypothesis, we observed that mice lacking the DUSP MKP-2 exhibited a decreased inflammatory response following LPS stimulation. Furthermore, MKP-2−/−
mice were conferred a survival benefit not only in this “sterile” in vivo
model but also in the polymicrobial CLP model of sepsis (Fig. ). This survival benefit correlated with significantly reduced production of pro- and anti-inflammatory cytokines (Fig. ). These findings are intriguing, as it suggests that the functional role of MKP-2 in regulating the host's immune response can involve modulating the cytokine response, but preliminarily, without affecting the ability to contain and/or eradicate a viable pathogen. Ongoing studies are examining the effect of MKP-2 in regulating components of this immune response that are beyond the scope of these current studies, including antigen presentation, phagocytosis, and oxidative burst. Immune responsive cells in the form of isolated BMDMs also exhibited decreased production of archetypal pro- and anti-inflammatory cytokines TNF-α and IL-10 in in vitro
studies. Mechanistically, this regulation of cytokine production by MKP-2 was associated with altered phosphorylation of ERK, JNK, and p38 as well as an increased production of the negative regulatory DUSP, MKP-1. LPS stimulation of MKP-2−/−
BMDMs increased phosphorylation of ERK and decreased phosphorylation of both JNK and p38 compared to that of BMDMs derived from WT mice (Fig. ). Additionally, MKP-1 was increased in MKP-2−/−
BMDMs starting at 30 min following LPS stimulation (Fig. ). This increase in MKP-1, which is a negative regulator of both pro- and anti-inflammatory cytokine production via JNK and p38 regulation (10
) likely explains the decreased cytokine production in the MKP-2−/−
cells compared to WT cells. As further evidence of this mechanistic link between MKP-2 and MKP-1 expression, we also found that silencing MKP-1 in the MKP-2 null BMDMs reversed this phenotype, as we observed increased production of TNF-α from MKP-1-silenced MKP-2−/−
BMDMs (Fig. ). These data suggest that increased expression of MKP-1 driven by augmented ERK activation in the absence of MKP-2 was involved in the attenuated cytokine production.
Given these observations, we believe that our data demonstrate a critical mechanism of cross talk between MKP-2 and MKP-1 that is mediated through ERK activation. This is highly plausible given prior findings that ERK activation is important for maximal induction of MKP-1 (24
) and stabilization of the MKP-1 protein (6
). Furthermore, this link likely explains our findings that all three terminal MAPKs were altered in the absence of MKP-2. This was initially not expected, as several studies have shown that MKP-2 has much a higher specificity for ERK than for either JNK or p38 (8
). Thus, it is not surprising that in the absence of MKP-2, we observed increased activation of ERK; however, given that we also observed increased expression of MKP-1, a known regulator of JNK and p38, it is likely that MKP-1 is responsible for the significant reduction in phosphorylation of JNK and p38 observed in the LPS-stimulated MKP-2 null BMDMs. We therefore propose a mechanism of MKP-2 regulation of cytokine production in which, following LPS binding to TLR4, the three arms of the MAPK pathway are simultaneously activated (Fig. ). JNK and p38 are directly involved in the induction and production of proinflammatory cytokines, while ERK activation leads to induction and stabilization of MKP-1, which serves as a negative regulator of proinflammatory cytokine production by inhibiting the action of JNK and p38 (10
). Subsequently, MKP-2 is induced to deactivate ERK and thus destabilize MKP-1, resetting the cellular mechanism for cytokine production.
FIG. 7. Proposed regulatory mechanism of MKP-2 impacting MKP-1 levels via ERK deactivation. Following LPS binding to TLR4, the three MAPK pathways are activated. Activation of ERK results in the induction and stabilization of MKP-1. The activation of JNK and (more ...)
Since MKPs remove phosphates on activated MAPKs, this putative role of a phosphatase as a positive regulator of the inflammatory response is surprising; however, such a role has been demonstrated for a related MKP, PAC-1 (16
). Similar to our results, Jeffrey et al. showed decreased cytokine production by PAC-1−/−
macrophages in response to LPS. MKP-2 is present in a variety of tissues (19
) whereas PAC-1 is limited to immune cells (16
). It is plausible that the redundancy of the positive regulation on the inflammatory response for these two MKPs is necessary in immune cells, but the ubiquitous nature of MKP-2 may provide a more global positive regulation. The complex nature of regulation of the MAPK pathway by the DUSPs is not completely understood but may impact the regulation of scaffolding and cellular trafficking (7
) as well as the flexibility of the cellular response to amount and type of stimuli (3
). Although our data support a positive regulatory role for MKP-2 involving ERK-mediated cross talk with MKP-1, further studies are under way to define the precise interactions involved between MKP-2, ERK, and MKP-1. We are additionally pursuing the effects of other TLR agonists on MKP-2 induction as well as the effects of MKP-2 overexpression in order to gain more insight into the regulatory role of MKP-2. Finally, we acknowledge that the altered cytokine production may be the result of preconditioned or altered phenotypic macrophages. Further investigation is needed to understand the impact MKP-2 has on macrophage development as well as involvement of other regulators besides MKP-1 that are responsible for the altered cytokine response.
An additional limit to the current studies is the complex nature of cytokine production in vivo in response to LPS. Although we used BMDMs to investigate the regulatory role of MKP-2 in cytokine production, the exact role of macrophages in the improved survival noted in our MKP-2−/− mice is unknown. Further studies are under way investigating both potential alterations in the hematopoietic differentiation and impact on the function of various tissue macrophages (e.g., lung, spleen, and peritoneum), as well as lymphocytes in the MKP-2−/− mice and its impact on modifying the response to LPS. These investigations will strengthen our understanding of the regulatory role of MKP-2 in both hematopoietic differentiation and inflammatory/immune response.
In summary, our studies demonstrated a survival benefit associated with decreases in both pro- and anti-inflammatory cytokines in the absence of MKP-2 and an increase in the phosphorylation of ERK, while there is a decrease in phosphorylation of JNK and p38 concomitant with an increase in MKP-1 induction. These data suggest a cross talk mechanism (Fig. ) by which MKP-1 is involved in the global regulation of cytokine production by MKP-2.