Background
Stroke is the second leading cause of death and the most frequent cause of permanent disability worldwide [
1]. Inflammatory mechanisms that are activated within hours after brain ischemia represent a key target of current translational ischemic stroke research [
2]. It has been reported that the levels of proinflammatory cytokines and chemokines are increased after focal ischemia. Chemokines are cytokines that have the ability to induce chemotaxis on neighboring cells, particularly those involved in inflammatory actions [
3,
4]. While some cytokines may offer protection, many cytokines and most chemokines have been shown to participate in the neuronal damage processes [
4,
5]. Upregulation of cerebral proinflammatory cytokines, activation of local microglia, astrocytes and systemic lymphocytes and invasion of leukocyte in the brain contribute substantially to ischemic brain damage [
6].
Published data have shown that lipopolysaccharide (LPS) preconditioning is a powerful neuroprotective phenomenon by which a sublethal injurious stimulus renders the brain resistant to a subsequent damaging ischemic insult [
7‐
11]. LPS preconditioning-induced neuroprotection is related to the suppression of the inflammatory response in the ischemic area of the brain, but the mechanisms involved in LPS preconditioning are poorly understood [
12,
13].
MCPIP1 (also known as ZC3H12A) is a recently identified protein in human peripheral blood monocytes treated with monocyte chemotactic protein 1 (MCP-1) [
14]. In our previous studies, MCPIP1 was shown to be a negative regulator of macrophage activation [
15]. Further investigations by our group and others indicated that MCPIP1 can play a significant anti-inflammatory role by inhibiting the generation of a set of major proinflammatory cytokines [
16,
17]. MCPIP1 was also found to be inducibly expressed in monocytes, macrophages, and endothelial cells with LPS stimulation [
13,
17‐
19]. However, the role of MCPIP1 in ischemic stroke has not been examined. In this study we examined MCPIP1 gene expression in human and mouse microglia, and in mouse brain under LPS treatment or preconditioning. We also examined MCPIP1 gene expression in mouse brain undergoing MCAO. We studied whether there is loss of LPS preconditioning-induced ischemic stroke tolerance in MCPIP1 knockout mice and whether such effects involve regulation of expression of proinflammatory cytokines. Furthermore we investigated the effects of MCPIP1 on JNK signal pathway under brain ischemia conditions and the effects of JNK inhibitor on the production of proinflammatory cytokines in MCPIP1 knockout mice after brain ischemia. Our data indicate that MCPIP1 is upregulated under LPS preconditioning or after brain ischemia stress and MCPIP1 participates in LPS preconditioning-induced ischemic stroke tolerance by modulating gene expression of proinflammatory cytokines.
Discussion
It is becoming increasingly clear that inflammation and innate immune response play an important role in the brain injury after ischemic stroke [
26,
27]. Inflammatory mechanisms that are activated within hours after brain ischemia represent a key target for stroke intervention. Although it has been known that LPS preconditioning can induce significant tolerance to ischemic brain injury and inhibit inflammatory responses such as activation of microglia, brain neutrophil infiltration and proinflammatory cytokine generation, the molecular mechanisms that contribute to brain ischemia tolerance by LPS preconditioning are not well-understood. The present study is the first to examine the role of MCPIP1 in acute cerebral ischemia. We found that MCPIP1 can be significantly induced in human and mouse microglia and mouse brain with LPS stimulus or preconditioning and that MCPIP1 gene expression significantly increased in mouse brain after MCAO. These findings are consistent with the significance of inflammatory processes contributing to the brain damage in ischemic stroke [
26] and the anti-inflammatory properties of MCPIP1 [
16,
17]. Similarly we also found the upregulation of MCPIP1 in cultured mice astrocytes following LPS stimulation (data not shown) and activated astrocytes also play an important role in neuroinflammation [
28]. Thus, we conclude that MCPIP1 is involved in LPS preconditioning-induced ischemic stroke tolerance by its anti-inflammatory activities and MCPIP1 may play an important role against ischemic brain injury. Consistent with other reports [
7,
12] we found that a small dose of LPS given systemically confers protection against ischemia in mouse brain with respect to infarct volume. More interestingly, we found that there was loss of LPS-induced ischemic stroke tolerance by MCPIP1 deficiency. There was much larger ischemic brain infarct area in MCPIP1 deficient mice compared to the wild type. Higher mortality in MCPIP1 knockout mice subjected to focal brain ischemia/reperfusion injury compared with wild type indicated that MCPIP1 deficient mice is more sensitive to ischemic brain injury than wild type. This result was not due to the possible general physiological weakness of MCPIP1 knockout mice to resist stress of surgery as mortality of sham group of MCPIP1 knockout mice was much less than the animals subjected to ischemic injury.
Clinical data showed that the principal cause of stroke death in patients with malignant middle cerebral artery infarction was focal brain edema [
29]. Experimental models of cerebral infarction show an increase in water content beginning within hours and peaking at 48 hours [
30]. Progressive postischemic edema would result in increased intracranial pressure with compression of the brain stem, occlusive hydrocephalus and secondary ischemic damage finally followed by clinical deterioration, coma, and death within 2 to 5 days after stroke [
31,
32]. In this study, our data showed that absence of MCPIP1 significantly increased the infarct volume and brain edema compared to wild type after ischemia/reperfusion injury. Thus, severe brain edema may be the major cause of higher mortality in MCPIP1 knockout mice subjected to ischemic stroke.
Inflammatory response after stroke significantly contributes to ischemic brain damage, which can be sub-divided into the cellular response and the cytokine response [
6]. Neutrophils accumulate in the brain as early as 30 min after permanent middle cerebral artery occlusion (MCAO). Transmigration is mediated by cell adhesion molecules such as vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, and selectin, which contribute to the recruitment of inflammatory cells to endothelial blood vessel wall [
33,
34]. When adhered to cerebral blood vessel walls, neutrophils transmigrate into the cerebral parenchyma, which can cause tissue damage by releasing reactive oxygen species and proteolytic enzymes. MCPIP1 expression is induced in human blood endothelial cells and overexpression of MCPIP1 suppresses cytokine-induced expression of VCAM-1, as well as monocyte adhesion to human endothelial cells [
19]. Such properties of MCPIP1 helps to explain the present results that absence of MCPIP1 exacerbates ischemia induced brain injury.
Proinflammatory cytokines and chemokines contribute to stroke-related brain injury [
6,
35]. During ischemia, cytokines, such as TNF-α, IL-1β, IL-6, and chemokines such as CINC and MCP-1 are produced by a variety of activated cell types, including endothelial cells, microglia, astrocytes and neurons [
36]. The deleterious effects of these cytokines include fever, arachidonic acid release, enhancement of NMDA mediated excitotoxicity, and stimulation of nitric oxide synthesis. TNFα and IL-1 have been shown to cause up-regulation of E-selectin, ICAM-1, ICAM-2, and VCAM-1 on cerebral endothelial cells and the induction of such adhesion molecules may explain the elevation of TNFα and IL-1 levels after ischemia increases neutrophil infiltration [
37]. Additionally TNFα can stimulate acute-phase protein production, including plasminogen, disrupt the blood-brain barrier and stimulate the induction of other inflammatory mediators. LPS preconditioning can induce significant tolerance to ischemic brain injury and inhibit inflammatory responses such as activation of microglia, brain neutrophil infiltration and proinflammatory cytokine generation. Some studies attributed LPS preconditioning to reprograming of cellular response to ischemia via genomic changes that render the brain refractory to ischemic injury [
11,
38]. However, the molecular mechanisms that mediate the beneficial effects in LPS-induced ischemic tolerance remains poorly understood. In this study we observed that knocking out MCPIP1 increased the brain damage after ischemic stroke and significantly enhanced the expression of proinflammatory cytokines in the brain, which may account for the severe brain damage resulting from ischemia in MCPIP1 deficient mice. It has been established that endotoxin-preconditioning by LPS, TNFα and IL-1β can induce ischemic tolerance. Less well studied is what mediates such beneficial tolerance in preconditioning process i.e. the necessary intermediate links for completion of preconditioning tolerance. If this link is lost, the chain from cytokines to tolerance would be broken; in other words, even if cytokines still exist the tolerance would be not induced or would be inhibited. We hypothesized that MCPIP1 is such an inducible factor or link which mediates, at least in part, the translation from cytokine stimulation to tolerance. When MCPIP1 is deficient, tolerance is reduced even in the presence of more cytokines, as the key mediator is missing. Our results suggest that MCPIP1 is such a mediating factor and MCPIP1-deficiency results in the loss of LPS-induced ischemia tolerance in the brain and higher level of proinflammatory cytokines in ischemic brain. How does MCPIP1 regulate these inflammatory cytokine responses remains to be fully elucidated. It has been reported that MCPIP1 might be functioning as an RNase to promote the degradation of some inflammatory mRNA such as IL-6 and IL-1β [
17]. We have found that MCPIP1 can also act as a deubiquitinase to negatively regulate JNK and NF-κB signaling by targeting TNF receptor-associated factors (TRAFs) [
16,
39], which suggests that MCPIP1 may control inflammatory response by multiple mechanisms. Activation of JNK signaling pathways leads to c-Jun mediated inflammatory cytokine production [
24,
40,
41]. In this study we found that knocking out MCPIP1 significantly enhanced the activation of JNK kinase and phosphorylation of c-jun in mouse brain after ischemic/reperfusion injury and that administration of the JNK inhibitor SP600125 reduced hyperphosphorylation of JNK and c-Jun signaling after cerebral ischemia and significantly decreased the production of proinflammatory cytokines such as TNFα and IL-1β. Our study suggests that increased activation of JNK signaling pathway in MCPIP1 knockout mice leads to increased proinflammatory cytokine production.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JL designed the experiments, performed all experiments, analyzed the data, generated the figures, and wrote the manuscript. JW did parts of the animal surgery. JW, YS, LW, EB performed the experiments. PEK provided advice in the design of the study and in interpreting of data and critically read and corrected the manuscript. All authors have read and approved the manuscript.