Background
Microglia, the resident macrophages of the central nervous system (CNS), monitor their environment through a constant movement of their processes, and respond to local stressors and immune disturbances [
1,
2]. Microglia express a complement of pattern recognition receptors (PRR) that can respond to pattern associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs), such as Lipopolysaccharides (LPS) and β-amyloid (Aβ). A major class of PRRs includes the Toll-like receptors (TLRs) that play a pivotal role in host defense by regulating innate immunity and linking with adaptive immune responses (for reviews, see: [
3,
4]). Activation of TLRs on microglia leads to the production of inflammatory mediators, such as IL-1β, IL-6, TNFα, and nitric oxide. TLR engagement and signaling in the CNS provide an important defense mechanism by which microglia respond to external pathogens or host-derived ligands. Microglia can also be activated by inflammatory mediators (e.g. cytokines and chemokines) from autocrine, paracrine, and endocrine sources (for detailed reviews on microglia, see: [
5,
6]). The local environment, and possibly intrinsic changes to the microglia determine how the cells will respond to the activating signals [
7,
8]. Like peripheral immune cells, microglia can adopt a number of activated phenotypes, and the functional outcome depends on a complex balance between beneficial protective responses and detrimental harmful responses [
9]. Tight regulation of microglial activation pathways is essential for appropriate responses to stressor stimuli and maintenance of CNS homeostasis, because uncontrolled or dysregulated inflammatory responses can lead to propagation of detrimental and neurotoxic responses.
A relevant example is the control of microglia proinflammatory cytokine production in response to various ligands. Proinflammatory cytokines have many important physiological functions in the CNS, from protection against pathogens to acting as neuromodulators affecting cognition [
10]. However, clinical studies and preclinical animal models have implicated dysregulation and overproduction of proinflammatory cytokines from activated microglia in the CNS as a contributor to pathophysiology progression in both chronic neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease, and multiple sclerosis, as well as acute neurodegenerative conditions such as traumatic brain injury and stroke [
11‐
13]. Taken in its entirety, the evidence is consistent with the hypothesis that proinflammatory cytokine overproduction is a comparatively early event in the progression of pathophysiology that is causally linked to synaptic dysfunction, behavior deficits and, in the more extreme case, neuronal death. This raises the possibility that up-regulation of proinflammatory cytokine production could be targeted in new therapeutic development strategies with potential for disease modification in multiple diseases and clinical presentations.
One approach to targeting CNS cytokine dysregulation is to modulate the intracellular signal transduction cascades that regulate the production of proinflammatory cytokines. This requires that we explore which specific signal transduction pathways are involved in cytokine overproduction in microglia exposed to different stressors, and which of these pathways are amenable to intervention. A major signaling pathway that contributes quantitatively to up-regulated cytokine production in peripheral inflammation is the p38 mitogen activated protein kinase (MAPK) pathway, especially the key regulatory enzyme p38α MAPK [
14,
15]. The p38α MAPK is amenable to therapeutic intervention in peripheral inflammatory diseases, and treatment with p38α MAPK-targeted inhibitors can suppress cytokine levels back towards basal. There is increasing evidence that the p38 MAPK signaling cascade also contributes to CNS cytokine overproduction and neurodegenerative sequelae, and that it may be a good therapeutic target for CNS disorders characterized by increased proinflammatory cytokine production as a contributor to neurologic dysfunction or susceptibility to injury [
11,
16]. However, studies are needed to determine a direct linkage between the p38α MAPK-mediated pathway in microglia and proinflammatory cytokine production in response to different stressors. In addition, whether the pathway in microglia is druggable; i.e., responsive to intervention with CNS-penetrant kinase inhibitors, needs to be determined.
The field has been limited in its ability to pursue these questions because of the lack of selective p38α MAPK inhibitors with sufficient brain penetrance and metabolic stability for use in CNS disorders [
17]. We recently developed a selective, orally bioavailable, CNS-penetrant, small molecule p38α inhibitor, MW01-2-069A-SRM (069A) [
18]. Compound 069A attenuates hippocampal proinflammatory cytokine overproduction and leads to improved neurologic outcomes in an AD-relevant mouse model when administered orally at a low dose and in a clinically relevant time window of disease progression; specifically, 069A suppressed the Aβ
1-42-induced hippocampal IL-1β and TNFα up-regulation back towards basal and attenuated the resultant synaptic dysfunction and behavioral deficits [
18]. These data provided an initial causative link between p38α MAPK and CNS disease-related endpoints. Moreover, pharmacological downregulation of IL-1β production may have important protective consequences, as IL-1β can induce the expression and activation of p38 MAPK in neurons, promoting tau phosphorylation and loss of the synaptic protein synaptophysin [
19,
20]. The availability of this p38α MAPK inhibitor, as well as a novel mouse model where p38α MAPK is genetically deleted in microglia, provide the opportunity to test whether p38α MAPK is a major regulator of microglial proinflammatory cytokine overproduction in response to specific stressors, and whether this signaling pathway is amenable to intervention. We report here that p38α MAPK is a key contributor to microglia proinflammatory cytokine production in response to a variety of stressor stimuli, including Aβ
1-42 and different TLR ligands. In addition, inhibition of p38α MAPK by either pharmacological or genetic approaches leads to a reduction in microglia cytokine production. The results indicate the feasibility of targeting p38α MAPK in attempts to modulate proinflammatory cytokine overproduction, a potential contributor to CNS disease progression and susceptibility.
Discussion
We report here several findings with important implications for the treatment of CNS inflammation. First, we show using both pharmacological and genetic approaches that the p38α MAPK isoform is sufficient for blocking a substantial portion of the IL-1β and TNFα produced by microglia following LPS stimulation. Second, using primary microglia and a microglia cell line we show that the brain-penetrant p38α inhibitor, 069A, is able to block the cytokine response to diverse, disease-relevant stimuli. Third, we demonstrate in vivo that a single oral dose of 069A can block the IL-1β response in the brain to a peripheral LPS insult. Fourth, we show that 069A inhibits the activation of known downstream targets of p38α, namely MK2 and MSK1, and demonstrates selectivity in the signal transduction pathways that it affects. Altogether, our data support the idea that the p38α MAPK pathway is quantitatively important for microglia proinflammatory cytokine up-regulation in response to a variety of stressors, and that the kinase may be a viable drug discovery target for CNS disorders where overproduction of proinflammatory cytokines has been implicated in disease progression.
Cytokines are inflammatory mediators that act throughout the body, including the CNS, via specific receptors and signal transduction pathways. Clinical studies and preclinical animal models have provided extensive evidence to support the hypothesis that overproduction of proinflammatory cytokines contributes to the progression of chronic neurodegenerative disorders (for review, see [
11]), as well as to increased susceptibility to later-in-life disease or secondary injuries [
35]. Microglia are the primary cell type in the CNS responsible for the production of cytokines. We were able to inhibit the production of two key proinflammatory cytokines, IL-1β and TNFα, with the p38α MAPK inhibitor 069A at concentrations consistent with its ability to block p38α kinase activity. Interestingly, we found that 069A was equally effective at blocking the production of cytokines when the compound was given up to two hrs after the LPS treatment; however, the mechanism underlying this observation was not pursued as part of this study. The observation that 069A also suppresses the phosphorylation of two direct p38 substrates, MK2 and MSK1, provides additional evidence of target engagement in the microglial cells. The inhibition of the active phosphorylated form of MK2 upon 069A treatment is consistent with the observation [
36] that MK2 kinase activity is dependent on p38α MAPK for its phosphorylation and activation. MK2 has also been reported to directly regulate TNFα at the 3'-untranslated region (UTR) of mRNA in the AU-rich elements [
37], suggesting a potential mechanistic explanation for the action of 069A in suppression of cytokine production. MSK1 can be phosphorylated and activated by both p38α MAPK and ERK1/2 [
29]. However, our observation that inhibition of p38α MAPK by 069A or by genetic deletion leads to suppression of phosphorylated MSK1 in the microglia cultures suggests that the p38α MAPK pathway is playing a key role in transducing the stressor stimulus to cytokine production in microglia.
In AD-relevant models, TLR2 and TLR4 have been shown to be required for microglia cytokine response to fibrillar Aβ [
38]. Microglia associated with senile plaques exhibit elevated levels of TLR2/4/5/7/9 [
33]. While the role of TLRs in AD may be the best defined for any CNS disorder to date, the potentially neurotoxic role of TLRs is not limited to AD (for a recent review see: [
39,
40]). The inflammatory cytokine response to TLR ligands is typically through NF-κB and MAPK signal transduction pathways [
41]. However, the response to the ligands can vary depending on the cell type activated. In BV-2 cells we found that 069A was highly effective at blocking the inflammatory cytokine response to TLR2/3/4/7/8/9 ligands. This suggests that p38α plays an important role in the signal transduction pathways that lead to inflammatory cytokines in microglia in response to a variety of ligands, and that this kinase may prove to be an effective convergence point that can be targeted by small molecule inhibitors, such as 069A, to block cytokine overproduction. Blocking p38α MAPK in microglia, a principle source of inflammatory cytokines such as IL-1β, may ameliorate the detrimental sequelae of increases in neuronal tau phosphorylation, synaptic loss, or other cytokine-induced neuronal damage responses [
42‐
45]. In addition, inhibition of p38 MAPK in the neuron may also have beneficial consequences. Particularly relevant to AD, for example, are reports showing that activation of neuronal p38 MAPK contributes to Aβ-induced impairment of cortical LTP [
46], and that p38 MAPK can phosphorylate tau
in vitro at sites seen in AD brain [
47,
48].
It should be emphasized that microglia responses to stimuli can be neuroprotective and assist with phagocytosis or protein aggregate clearance, or can be detrimental and contribute to a progression of pathology [
13,
49‐
51]. Therefore, attempts to develop disease-modifying therapeutics that target microglial activation responses must be selective in their action (e.g., NOT be pan-suppressors of glial activation such as steroids), must consider the stage of disease progression and the relative contribution of a given endpoint or signaling pathway to the particular disease stage, and the appropriate dosing.
Dosing is the pharmacological foundation to selective therapeutic intervention. Dosing includes the amount of drug given normalized to body weight or volume and includes the therapeutic time window for administration. The desired effect of a drug, therefore, requires a combination of timing of administration based on mechanism of action and the amount administered. It is a given that all drugs will have an adverse effect at some dose, so safety with efficacy depends on finding the appropriate concentration range over which the desired effects are observed in the absence of undesired effects. However, one can also obtain pharmacological selectivity with dosing, or one pharmacological effect at one dose range and additional desired effects across a higher dose range. Examples from prior art relative to modulating inflammation are instructive in this regard. Steroids are used as anti-inflammatories, but are pleiotropic in their actions. The pleiotropic effect contributes to a diversity of pharmacological actions across efficacious doses and to a comparatively narrow therapeutic range in the absence of adverse events. In contrast, non-steroidal anti-inflammatories (NSAIDs) are more selective in their action due to their targeting of the cyclooxygenases and altering biologically active eicosanoids such as prostaglandins and thromboxanes. However, the NSAIDs require appropriate dosing for safe and effective use due to the fundamental importance of the widely distributed cyclooxgenase targets in physiological processes (e.g., [
52,
53]). In the case of the NSAID drug aspirin, it can be administered daily at a very low-dose for cardiovascular disease modification or 'as needed' dosing at higher concentrations for CNS symptomatic effects (for reviews, see [
54,
55]). It is anticipated that the pharmacodynamic (what the drug does to the body) effects of p38 MAPK inhibitors will be determined by the dosing regimen which must be empirically determined. The concentration-dependent target engagement in microglia treated with a p38 MAPK inhibitor as shown in this report is consistent with the achievement of a desired pharmacodynamic effect through appropriate dosing. However, the potential for dosing microglial product endpoints studied here must be placed in the context of other potential roles, such as effects on phagocytosis/clearance or neuronal functions, which remain to be dissected.
It is important to note that compound 069A is not a NSAID as it does not target cyclooxgenases. In terms of anti-inflammatory actions, 069A's pharmacological effects more closely resemble those of macromolecular Biological Response Modifier drugs, such as the TNFα blockers Etanercept or Infliximab, that alter inflammation-related pathology and exhibit extended pharmacodynamic effects after each administration. A goal of p38α MAPK targeted drug development for CNS indications is to generate small molecule drugs that partially mimic the biological actions of macromolecular biological response modifiers, yet are bioavailable, CNS-penetrant compounds that modulate disease-relevant endpoints. The data presented here demonstrate that pharmacological inhibition of p38α MAPK effectively suppresses the microglial cytokine upregulation response to a number of different activating ligands, raising the possibility for an extended pharmacodynamic effect due to alteration of cytokine production.
In order to explore the potential for an
in vivo effect of 069A on cytokine levels, we screened for an effect on the proinflammatory cytokine surge induced by LPS administered
ip. The results reveal a clear pharmacodynamic effect on brain cytokine level. Further exploration of the mechanisms for this
in vivo effect was not done as part of this study, but deserves a cautionary comment. LPS administered
ip will elicit a strong peripheral inflammatory response, which is then transduced to the brain via many pathways (for a recent review see: [
56]). Therefore, in the LPS model, one cannot distinguish between a direct effect of a compound on microglia cytokine induction or a more indirect effect that involves reduction of the inflammatory response in the periphery which then leads to a reduction in the CNS response (or a combination of the two mechanisms). Regardless, we previously reported [
18] that oral administration of 069A attenuated excessive proinflammatory cytokine production in the brain of an AD-relevant mouse model stimulated centrally with a disease-relevant stressor (oligomeric Aβ
1-42). Therefore, viewing both our current and previous
in vivo results in the context of the glial biology results indicates a probable direct effect on microglia as one component of the therapeutic outcome. In addition, our results provide evidence that oral administration of 069A can reduce the levels of IL-1β in the brain brought about by more than one class of
in vivo stressor.
Competing interests
DMW and LVE are principal investigators on project funding from the NIH and non-profit disease foundations with development of CNS new molecular entities as the long-term goal. Patents and patent applications covering novel compounds, including the one described here, have been filed by Northwestern University's technology transfer office and licensed to industry.
Authors' contributions
ADB, DMW, LVE designed the research studies. ADB, BX, LA, ERD performed the experiments. ADB, DMW and LVE drafted the manuscript with the assistance of the other authors. All authors read and approved the final manuscript.