Aspirin-triggered lipoxin A₄attenuates LPS-induced pro-inflammatory responses by inhibiting activation of NF-κB and MAPKs in BV-2 microglial cells

There is increasing awareness that inflammation may play a role in various neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, HIV-associated dementia, trauma, multiple sclerosis and stroke [1, 2]. Microglial cells are generally considered to be the immune cells of the central nervous system (CNS). They respond to neuronal injury or immunologic challenges with a reaction termed microglial activation. Activated microglial cells can serve diverse beneficial functions essential to neuron survival, which include cellular maintenance and innate immunity [3, 4]. However, overactivated microglia can induce significant and highly detrimental neurotoxic effects through excess production of a large array of cytotoxic factors such as superoxide, nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) [1]. Overactivation of microglia followed by overproduction of neurotoxic factors results in deleterious and progressive neurotoxic consequences [5, 6]. In several studies it has been shown that reduction of pro-inflammatory mediators produced by microglia may attenuate the severity of neuronal damage [7]. Therefore, inhibiting inflammatory cytokine production by activated microglia may be useful for preventing neurodegeneration [8‐10].

Lipoxins (LXs) are endogenous lipid mediators with potent anti-infiammatory and pro-resolving actions [11]. Of special interest, aspirin can also trigger transcellular biosynthesis of 15-epimers of LX, termed aspirin-triggered LX (ATL) [12], that share the potent anti-infiammatory actions of LX but are more resistant to metabolic inactivation [13]. LXs and ATL elicit multicellular responses via a specific G protein-coupled receptor termed the LXA₄ receptor (ALX) that has been identified in human [14], mouse [15] and rat [16] tissues. In our previous papers, we evaluated the anti-inflammatory activity of an LXA₄ analogue, 5(S), 6(R)-LXA₄ methyl ester, in a rat model of permanent focal cerebral ischemia and focal cerebral ischemia reperfusion [17, 18]. Our results showed that this LXA₄ analogue could attenuate focal ischemia-induced inflammatory responses and inhibit activation of microglia in vivo. Expression of functional ALXs was identified in neural stem cells, neurons, astrocytes and microglia [19‐23]. Microglial cells are key sensors and versatile effectors in normal and pathologic brain [24]. These findings suggest that microglia may be a target for LXs in brain. However, the effects of LXs on expression of inflammation-related genes and molecular mechanisms in microglia have not been demonstrated.

Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, initiates a number of major cellular responses that play critical roles in the pathogenesis of inflammatory responses and has been commonly used to model proinflammatory and neurotoxic activation of microglia [25, 26]. We used LPS as a stimulant of the microglial reactivity in the current study.

In the present study, we investigated the impact of ATL on the infiammatory response induced by LPS in murine microglial BV-2 cells, as well as the signaling pathways involved in these processes. Our data suggest that ATL inhibits NO and pro-inflammatory cytokine production in LPS-activated microglia at least in part via NF-κB, ERK, p38 MAPK and AP-1 signaling pathways.

Our present data provide the first evidence that ATL inhibits the infiammatory activation of microglia. To date, two separate LXA₄ receptors (ALX1/FPR-rs1 and ALX2/FPR2) have been identified in mice [15, 29]. Mouse ALX2/FPR2 is expressed by neutrophils, monocytes, macrophages, dendritic cells, and microglial cells, and its transcripts are detected at high levels in spleen and lung [30]. ALX1/FPR-rs1 and ALX2/FPR2 are both expressed in the mouse pituitary gland, hypothalamic tissue and vomeronasal organ [31, 32]. As demonstrated by RT-PCR analysis, ALX1/FPR-rs1 and ALX2/FPR2 are both expressed in BV-2 microglial cells. ATL reduced LPS-induced production of NO, IL-1β and TNF-α in BV-2 microglial cells. This is a receptor-mediated effect as it disappeared when microglial cells were pretreated with Boc-2 before ATL treatment. Quantitative PCR analysis showed that ATL markedly suppresses iNOS, IL-1β and TNF-α gene expression in BV-2 microglia cells. Similarly, this effect was abrogated by the use of Boc-2. NF-κB, ERK and p38 MAPK pathways are at least partly involved in the anti-infiammatory mechanisms of ATL in BV-2 cells. Thus, ATL is a promising agent for preventing and treating neuroinflammation and may be useful for mitigating a dysregulated linkage between the immune system and brain.

Although microglial activation has important repairative functions in the CNS, microglial cell activation in infection, infiammation, or injury may go beyond control and eventually produce detrimental effects that override the beneficial effects. Activation of microglia leads to release of various toxic molecules such as superoxide, NO, IL-1β and TNF-α, contributing to neuronal damage in various neurodegenerative disorders [1].

LX possesses dual anti-inflammatory and pro-resolution activities that have been demonstrated in a multitude of acute and chronic inflammatory conditions [11]. Previously, LXA₄, ATL and their stable analogues have been shown to play a major role in important functional properties of the central nervous system, such as neural stem cell proliferation and differentiation, pain, and cerebral ischemia [17‐19, 33]. In primary murine microglia or N9 microglial cells, expression of ALX2/FPR2 has been identified and is up-regulated by inflammatory stimuli [20, 21]. In the present study, the expression of ALX2/FPR2 and another murine high-affinity ALX1/FPR-rs1 were confirmed in BV-2 microglial cells. These findings suggest that ATL could work as a modulator of the inflammatory reaction of the brain immune system, eventually acting as a microglial activation repressor.

NO and pro-infiammatory cytokines such as IL-1β and TNF-α are known to be important mediators in the process of infiammation. These proinfiammatory mediators are thought to be responsible for some of the harmful effects of brain injuries and diseases, including ischemia, Alzheimer's disease, Parkinson's disease and multiple sclerosis [34]. Under various pathological conditions associated with infiammation, large amounts of NO are produced in the brain as a result of the induced expression of iNOS in glial cells [35]. High levels of NO exert their toxic effects through multiple mechanisms, including lipid peroxidation, mitochondrial damage, protein nitration and oxidation, depletion of antioxidant reserves, activation or inhibition of various signaling pathways, and DNA damage [35]. Therefore, the effect of ATL on NO production and iNOS expression in LPS-stimulated microglia cells was examined. As shown in previous research [36, 37], NO is produced at low levels in unstimulated microglia. Stimulation of BV-2 microglial cells with LPS induced strong NO production and iNOS expression. The magnitude of the NO/iNOS response to LPS in BV-2 microglial cells is different in different studies with different concentrations as well as durations of LPS treatment. In the present study, ATL markedly reduced NO production and mRNA and protein expression of iNOS in dose-dependent manners without significant cytotoxicity. This indicates that inhibition of NO production by ATL is a result of inhibition of iNOS gene expression. Previous studies also have shown that LXA₄ and ATL analogues inhibit LPS-induced NO production and peroxynitrite formation in human leukocytes [38] and in mouse lung [39].

Pro-infiammatory cytokines produced by activated microglia, including IL-1β and TNF-α, play an important role in the process of neuroinfiammatory diseases [34]. IL-1β is a potent pro-infiammatory cytokine that acts through IL-1 receptors found on numerous cell types, including neurons and microglia. TNF-α can cause cell death directly by binding to neuronal TNF receptors linked to death domains that activate caspase-dependent apoptosis [40] or by potentiating glutamate release, thereby enhancing excitotoxicity [41]. IL-1β and TNF-α also drive self-propagating cycles of microglial activation and neuroinflammation by inducing activation of NF-κB, cytokine generation and further activation of NF-κB. Thus, inhibition of cytokine production or function serves as a key mechanism in the control of neurodegeneration. Our results showed that ATL markedly attenuates the production of IL-1β and TNF-α, and their mRNA expressions; induced by LPS in BV-2 cells. Consistent with our findings, similar results have shown that LXA₄ and ATL inhibit LPS-induced production of IL-1β and TNF-α in uvea and in macrophages and endothelial cells [42‐44].

In subsequent studies, we found that ATL has a strong inhibitory effect on infiammatory signaling pathways that include NF-κB and MAPK/AP-1. NF-κB activity increases in acute neurodegenerative disorders such as stroke, severe epileptic seizures, and traumatic brain injury; and in chronic neurodegenerative conditions, including Alzheimer's disease, Parkinson's disease, Huntington disease, and amyotrophic lateral sclerosis [45]. In general, activation of NF-κB in microglia contributes to neuronal injury and promotes the development of neurodegenerative disorders [45]. NF-κB is known as a pleiotropic regulator of various genes involved in the production of many proinfiammatory cytokines and enzymes. NF-κB is also a central regulator of microglial responses to activating stimuli, including LPS and cytokines [46]. In this study, ATL was able to inhibit the LPS-evoked degradation of IκB-α, nuclear translocation of NF-κB p65 and the DNA-binding activities of NF-κB in BV-2 cells. Previous studies have shown that LXs reduce nuclear translocation of NF-κB in human neutrophils, mononuclear leukocytes [38] and macrophages [43]. It has also been reported that ATLs reduce NF-κB-mediated transcriptional activation in an ALX-dependent manner, and inhibit the degradation of IκB [47]. Therefore, induction of anti-inflammatory responses by LXs may be dependent on the NF-κB signaling pathway.

In addition, LPS also activates MAPK pathways which lead to the induction of another transcription factor, AP-1. MAPKs are a group of signaling molecules that appear to play key roles in infiammatory processes [48]. We found that phosphorylation of ERK and p38 MAPK in response to LPS is decreased by ATL treatment. Our results also show that ATL treatment of BV-2 microglia results in decreased DNA-binding activities of AP-1 following LPS stimulation. This observation is in line with studies in mesangial cells, endothelial cells, neutrophils, fibroblasts and T cells, which have shown that ERK and/or p38 MAPK activation is attenuated in the presence of LXs [42, 49‐51]. In the present study, ATL failed to inhibit LPS-induced phosphorylation of JNK. A previous study in primary astrocytes found that an ATL analogue prevents ATP-evoked JNK phosphorylation, but has no effect on TNF-α-induced JNK phosphorylation [33]. Strikingly, our results show that ATL induces JNK phosphorylation, but has no effect on ERK and p38 MAPK activity. In another study, LXA₄ attenuated microvascular fluid leaks caused by LPS partly mediated by the JNK signaling pathway [52]. LXA₄ and ATL analogues could promote ERK phosphorylation in macrophages and monocytes [53, 54]. The reasons for these discrepancies are mainly due to differences in experimental models, cell types and stimulators.

In summary, our results show that ATL inhibits release of NO and pro-inflammatory cytokines in a concentration-dependent manner. Moreover, ATL acts at the level of transcription in LPS-stimulated microglia. A possible mechanism for this effect involves ATL's ability to activate a signaling cascade that results in repression of NF-κB, ERK and p38 MAPK activation in activated microglia. Given the fact that microglial activation contributes to the pathogenesis of neurodegenerative diseases, ATL may be considered as a potential therapeutic agent for neurodegenerative diseases involving neuroinflammation.