- Open Access
Soluble epoxide hydrolase modulates immune responses in activated astrocytes involving regulation of STAT3 activity
- Chia-Chi Hung†1, 3,
- Yi-Hsuan Lee†1,
- Yi-Min Kuo1,
- Pei-Chien Hsu1,
- Huey-Jen Tsay2,
- Ying-Ting Hsu2,
- Chih-Chin Lee3,
- Jia-Jun Liang3 and
- Feng-Shiun Shie3Email authorView ORCID ID profile
© The Author(s). 2019
- Received: 28 January 2019
- Accepted: 21 May 2019
- Published: 8 June 2019
Astrocyte activation is a common pathological feature in many brain diseases with neuroinflammation, and revealing the underlying mechanisms might shed light on the regulatory processes of the diseases. Recently, soluble epoxide hydrolase (sEH) has been proposed to affect neuroinflammation in brain injuries. However, the roles of astrocytic sEH in brains with neurodegeneration remain unclear.
The expression of astrocytic sEH in the brains of APPswe/PSEN1dE9 (APP/PS1) mice developing Alzheimer’s disease (AD)-like pathology was evaluated by confocal imaging. LPS-activated primary astrocytes with mRNA silencing or overexpression of sEH were used to investigate its regulatory roles in astrocyte activation and the induction of pro-inflammatory markers. Primary astrocytes isolated from a sEH knockout (sEH−/−) background were also applied.
The immunoreactivity of sEH was increased in activated astrocytes in parallel with the progression of AD in APP/PS1 mice. Our data from primary astrocyte cultures further demonstrate that the overexpression of sEH ameliorated, while the silencing of sEH mRNA enhanced, the lipopolysaccharides (LPS)-induced expression of pro-inflammatory markers, such as inducible nitric oxide, cyclooxygenase 2 (COX-2), and pro-inflammatory cytokines. These findings suggest that sEH negatively regulates astrocyte immune responses. Enhanced immune responses found in LPS-activated sEH−/− astrocytes also support the notion that the expression of sEH could suppress the immune responses during astrocyte activation. Similarly, sEH−/− mice that received intraperitoneal injection of LPS showed exacerbated astrocyte activation in the brain, as observed by the elevated expression of glial fibrillary acidic protein (GFAP) and pro-inflammatory markers. Moreover, our data show that the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was upregulated in activated astrocytes from sEH mouse brains, and the pharmacological blockade of STAT3 activity alleviated the pro-inflammatory effects of sEH deletion in LPS-activated primary astrocytes.
Our results provide evidence, for the first time, showing that sEH negatively regulates astrocytic immune responses and GFAP expression, while the underlying mechanism at least partly involves the downregulation of STAT3 phosphorylation. The discovery of a novel function for sEH in the negative control of astrocytic immune responses involving STAT3 activation confers further insights into the regulatory machinery of astrocyte activation during the development of neurodegeneration.
- Astrocyte activation
- Soluble epoxide hydrolase
- Immune responses
Astrocytes are major glial cells residing in the brain. Astrocytes become activated during the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease (AD). In AD, activated astrocytes are in proximity to β-amyloid (Aβ) plaques, one of the pathological hallmarks of AD, which have been implicated in the progression of AD [1, 2]. A plethora of effectors derived from activated astrocytes are involved in various physiological functions, including neuronal migration, synaptogenesis, and neuroplasticity, as well as in many pathological conditions [3–5]. Conjointly with the impacts derived from microglial activation, the uncontrolled release of these effectors from activated astrocytes causes neuroinflammation and instigates oxidative stress in the diseased brain . Oxidative stress increases the expression of pro-inflammatory genes, which leads to the increased production of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and prostaglandins, through cyclooxygenase 2 (COX2), and nitric oxide (NO) through the activity of inducible NO synthase (iNOS) [7–9]. The resulting consequences, in turn, release more unwanted effectors that repeatedly propagate oxidative stress, ultimately leading to neurotoxicity. Among the many signaling pathways involved in the induction of pro-inflammatory genes, the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was recently reported to be critical for the induction of glial fibrillary acidic protein (GFAP) and pro-inflammatory genes such as TNFα and IL-6 [10–12]. These results suggest that STAT3 may play an important role in the control of astrocyte activation and the associated immune responses.
Glia-mediated neuro-inflammation and subsequent neurodegeneration are common pathological features shared by many neurodegenerative diseases, and the inflammatory responses derived from activated astrocytes exacerbate the pathogenesis of these diseases . However, compelling evidence shows that astrocyte activation can also be beneficial for disease recovery and survival [14, 15]. Indeed, the ablation of activated astrocytes disrupts glial scar formation, leading to the persistent infiltration of inflammatory cells and the failure to recover the integrity of the blood brain barrier [16, 17]. In AD, it has been shown that activated astrocytes mediate Aβ degradation, suggesting that they may function against Aβ accumulation [18–20]. These findings suggest that astrocyte activation is a double-edged sword that can be both beneficial and detrimental to neuronal functions , thus, revealing the regulatory mechanism of astrocyte activation may lead to a better understanding of neuronal survival and death in the diseased brain. The fine-tuning of astrocyte activation that enhances neuronal survival and ablates the self-reinforcing cycles of neuro-inflammation is critical for developing treatments for diseased brain with robust neuro-inflammation .
Soluble epoxide hydrolase (sEH) has recently gained increasing attention because the inhibition of hydrolase activity by sEH can result in sustained levels of epoxyeicosatrienoic acids (EETs) and provide neuroprotection in cardiovascular disease and brain injuries [23–28]. Epoxygenases, a subgroup of enzymes in the cytochrome P450 family, metabolize arachidonic acid into hydroxyeicosatetraenoic acids (HETEs) and EETs , the latter of which can be further metabolized into less active forms of DiHETEs by sEH, which is encoded by the gene Ephx2. The C-terminus of sEH is the site of the epoxide hydrolysis activity responsible for producing DiHETEs, while the N-terminal domain of sEH possesses phosphatase activity, with lysophosphatidic acid as its endogenous substrate . sEH is primarily found in the liver, the kidney, the cardiovascular system, and the brain [31, 32]. The expression level of sEH was increased in mice with pilocarpine-induced epilepsy, while the genetic knockout of sEH in mice made them more susceptible to inducible seizures . However, the mechanism through which astrocytic sEH regulates glia-mediated neuroinflammation remains unclear. In this study, we demonstrate that the increased expression of sEH in activated astrocytes in the vicinity of Aβ plaques was associated with the progression of AD in a mouse model. The immune regulatory roles of astrocytic sEH in LPS-activated astrocytes were investigated using in vitro and in vivo models.
Lipopolysaccharide (LPS, Escherichia coli O55:B5) was purchased from Calbiochem. Antibodies for inducible nitric oxide synthase (iNOS) and β-actin were purchased from BD Transduction Lab and Novus Biologicals, respectively. Cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP) were purchased from Abcam. Total STAT3 and phospho-STAT3 (p-STAT3) were purchased from Cell Signaling Technology. ELISA kits for TNFα and IL-6 were purchased from Invitrogen. 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-acetyl-S-farnesyl-L-cysteine (AFC) were purchased from Sigma-Aldrich. Poly-ornithine was purchased from BD Biosciences. Papain and DNase I were purchased from Worthington Biochemical. Fetal bovine serum was purchased from HyClone and heat-inactivated. Culture media and penicillin/streptomycin were purchased from Gibco, and other common chemicals were from Sigma-Aldrich, unless stated otherwise.
Breeding pairs of wild-type (Wt) controls and APPswe/PSEN1dE9 (APP/PS1) transgenic mice, overexpressing both human APP695 Swedish mutations (HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9), were originally obtained from the Jackson laboratory. The transgenic genotypes of both APP and PS1 were detected using PCR, according to the manufacturer’s instruction. APP/PS1 transgenic mice that were heterozygous for the transgenes and Wt littermates were used as controls. Given that LPS has been widely used to induce inflammatory responses [9, 12, 22], a single dose of LPS was administered intraperitoneally to mice at 5 mg/kg body weight. Wt and sEH−/− mice that received a single intraperitoneal injection of saline were served as sham control. Forty-eight hours after LPS injection, brain tissues were harvested for later experiments. For primary astrocytic cultures, Wt mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan), and sEH−/− mice (C57BL/6/B6.129X-Ephx2tm1Gonz/J) were generously provided by Dr. Tzong-Shyuan Lee. Mice were maintained at the NHRI Laboratory Animal Center with free access to food and water. All experiments were performed as approved by the NHRI IACUC.
Primary astrocytic cultures
Primary astrocytic cultures were derived from the cortices of P1 to P3 neonates. Cells were dissociated using an enzyme solution, containing Dulbecco’s modified Eagle’s medium (DMEM), ethylenediamine tetraacetic acid (0.5 mmol/L), L-cysteine (0.2 mg/ml), papain (15 U/ml), and DNase I (200 μg/ml), followed by trituration. Culture medium (DMEM with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin) was changed after 24 hours of initial seeding. To obtain enriched primary astrocytes, the astrocytic monolayer was repeatedly trypsinized (at 90% confluence) and reseeded twice. On the 14th day in vitro (14 DIV), primary astrocytes were reseeded in poly-ornithine-coated 24-well culture plates for experiments, and a fraction of the cells was cultivated on chambered slides at 1 × 105 cells per well, followed by GFAP staining, to determine the purity of the astrocytes. The purity of primary astrocyte cultures was approximately 95%. Primary cultures were treated with 10 ng LPS/ml of culture medium for 24 h. To inhibit STAT3 phosphorylation in LPS-activated astrocytes, 1-h pretreatment with 10 μM of the STAT3 inhibitor, stattic (Abcam), was applied.
Genetic manipulations of sEH by using siRNA and over-expression plasmid
Small interfering RNA duplexes targeting sEH (si-sEH), consisting of a pool of 3 target-specific siRNAs designed to knock down sEH gene expression, and a scramble control were purchased from Santa Cruz Biotechnology, and an Ephx2 mouse cDNA clone for the overexpression of sEH and a vehicle control were purchased from OriGene. Primary cell cultures were transfected with siRNAs, plasmid, or controls at a final concentration of 30 nM in serum-free Opti-MEM by using the TransIT®-siQUEST™ transfection reagents (Mirus) for 48 h, followed by 24-h treatments of LPS. Quantification of sEH gene expression was evaluated by quantitative polymerase chain reaction (qPCR), as described below.
RNA extraction, reverse-transcription, and qPCR
Total RNA was extracted with an RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA (1 μg) was reverse transcribed into cDNA using a random primer and a SuperScript III Reverse Transcriptase Kit (Invitrogen). Quantitative PCR was conducted using Luminaris Color HiGreen qPCR Master Mix (Thermo) and an ABI PRISM 7500 Real-Time PCR System. Forward and reverse primer sets for each cDNA were used as follows: 5′-TGGTGGTGACAAGCACATTT-3′ and 5′-AAGGCCAAACACAGCATACC-3′ (for Nos2, NM_010927.3); 5′-GGCCATGGAGTGGACTTAAA-3′ and 5′-CACCTCTCCACCAATGACCT-3′ (for COX-2, NM_011198.3); 5′-GAGGGACAACTTTGCACAGG-3′ and 5′-TCCTGTCTATACGCAGCCAG-3′ (for GFAP, NM_001131020.1); 5′-ATCTCATACCAGGAGAAAGTCAACCT-3′ and 5′-TGGGCTCATACCAGGGTTTG-3′ (for TNF-α, NM_013693.3); 5′-GACCAAGACCATCCAATTCATCTT-3′ and 5′-GGAATGTCCACAAACTGATATGCT-3′ (for IL-6, NM_031168.1); 5′-TGGTGTGGAACATGGCTCTCT-3′ and 5′-ACTGGGATAGATCGGATAACTTTCA-3′ (for sEH, NM_007940.4); and 5′-TGTGTCCGTCGTGGATCTGA-3′ and 5′-GATGCCTGCTTCACCACCTT-3′ (for GAPDH, NM_008084.3). The average cycle threshold (Ct) value was normalized using the GAPDH signal. Relative transcript levels were calculated as x = 2−ΔCt, in which ΔCt = Cttarget gene − CtGADH. For cell cultures, each experimental condition was collected from at least 3 independent cultures.
Confocal microscopy was used for semi-quantification and qualitative analysis. Brain samples were subjected to paraformaldehyde (4% in PBS) fixation, overnight, followed by cryoprotection with sucrose (30% in PBS). Cryosections, at 30 μm thick, were subjected to immunohistochemical analysis using antibodies against Aβ (6E10, 1:200, Biolegend), GFAP (astrocyte marker, 1:200, Abcam), p-STAT3 (1:200, Cell Signaling Technology), or sEH (1:100) for overnight incubation at 4 °C. The corresponding secondary antibodies conjugated with Alexa Fluor (Invitrogen) as indicated in the results were applied for 2 h. Tissues were coverslipped with mounting medium (Vector Lab) containing 4,6-diamiino-2-phenylindole (DAPI) for nuclei counterstaining. Images were acquired using a Leica confocal microscopy imaging system. GFAP immunoreactivity has been widely used for the evaluation of morphological alterations in activated astrocytes in vivo and thus the quantification of GFAP immunoreactivity was used as a measure of astrocyte activation in this study. Semi-quantification of the immunoreactivity from two sections per mouse was performed using MetaMorph imaging software.
Measurements of sEH activity
Homogenates of the hippocampus or cortex were subjected to measurements of sEH activity, using an epoxide hydrolase activity assay kit (Cayman Chemistry) according to the manufacturer’s instructions. Briefly, the assay utilized Expox Fluor 7, a sensitive fluorescent substrate for sEH that can be used to monitor the activity of sEH. Hydrolysis of the substrate epoxide yielded a highly fluorescent product, 6-methoxy-2-naphthaldehyde, that was monitored at excitation and emission wavelengths of 330 and 465 nm, respectively, using a microplate reader. sEH activity was presented as pmol/min per mg of protein.
The expression levels of COX-2, iNOS, GFAP, total STAT3, p-STAT3, and β-actin were determined using cell lysates or brain homogenates. Samples were lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.5 % sodium dodecyl sulfate and protease inhibitor cocktail) and were subjected to electrophoresis, followed by protein transfer onto PVDF membranes and Western blot analyses using antibodies against COX-2 (1:750), iNOS (1:750), GFAP (1:1000), total STAT3 (1:1000), p-STAT3 (1:1000), and β-actin (1:5000). Targets were detected by the incubation of HRP-labeled secondary antibodies. The corresponding bands, revealed by electrochemiluminescence (ECL) reaction, were analyzed using ImageJ.
Measurements of cytokines
The culture media from primary astrocytic cultures was subjected to measurements of pro-inflammatory cytokines, including IL-6 and TNFα, using ELISA kits. The assay was performed according to the manufacturers’ instructions. Detection of the results was performed using an ELISA plate reader (SpectraMaxM2, Molecular Devices) at a wavelength of 450 nm.
A two-tailed independent Student’s t test was used to test significance. For ANOVA, significance for post hoc multiple comparisons between groups was determined with the Bonferroni test using GraphPad Prism software. Data are presented as the mean ± SEM. Statistical significance was set at p < 0.05.
Immunoreactivity of sEH in activated astrocytes increased in parallel with the progression of AD
Expression of sEH in primary astrocytes regulated the LPS-induced immune response
The LPS-induced astrocyte activation was exacerbated in the brains of sEH−/− mice
Regulation of astrocyte activation by sEH involves the suppression of STAT3 phosphorylation
It has been reported that sEH possesses phosphatase and hydrolase activities. We then investigated whether the regulation of STAT3 phosphorylation during astrocyte activation was differentially affected by these two functional domains of sEH. Pretreatments of AUDA and AFC were applied, to inhibit the sEH hydrolase activity and phosphatase activity, respectively, in LPS-activated astrocytes. In Fig. 7b, the data show that STAT3 phosphorylation was upregulated in LPS-activated astrocytes and was further enhanced by pretreatments of AFC (138 ± 6% and 141 ± 4% of the control with LPS for 1 μM and 10 μM of AFC, respectively) but not by pretreatments of AUDA. In contrast, LPS-induced STAT3 phosphorylation was reduced in astrocytes overexpressing sEH (59 ± 5% of the control with LPS, p < 0.05) compared to the control with LPS (100 ± 5%) (Fig. 7c). These data suggest that the phosphatase, but not the hydrolase, activity of sEH may contribute to the negative regulation of STAT3 phosphorylation.
Astrocyte activation is an important pathological feature in AD and many other neurodegenerative diseases [34, 35]. Understanding the underlying regulatory mechanism may confer a molecular basis for the development disease therapies. Our data show that the expression of sEH was positively associated with the progression of astrocyte activation in APP/PS1 transgenic mice, while sEH immunoreactivity was barely detectable in resting astrocytes with a quiescent phenotype. Importantly, we demonstrated, for the first time, that sEH plays a role in the negative regulation of LPS-induced astrocyte immunity, as evidenced by data from in vivo and in vitro models. Data suggest that the genetic deletion of sEH, either sEH−/− or si-sEH, enhanced the production of LPS-induced pro-inflammatory markers, whereas the overexpression of sEH ameliorated the immune response. However, the involvement of microglial activation in the enhanced pro-inflammatory markers found in sEH−/− mice cannot be excluded due to the constant interactions between microglia and astrocytes in the brain. Furthermore, our findings identify sEH as a novel inhibitory effector in the regulation of STAT3 activity and demonstrate the critical role of STAT3 in astrocyte activation. Based on these findings, we propose that the immune-suppressing function of sEH involving STAT3 inhibition may play an important role in the regulation of astrocyte activation.
The expression of sEH in astrocyte activation in AD pathogenesis
The number of GFAP-positive astrocytes near Aβ plaques increases as AD progresses. Similarly, our data show that astrocytic sEH immunoreactivity increased in parallel with the extent of astrogliosis and was limited to the activated astrocytes in proximity to Aβ plaques. However, astrocytes in the brain of AD mice appear to express different levels of sEH, and less activated astrocytes that were close to Aβ plaques expressed little or no sEH, as shown in the abovementioned results. One possible explanation for these findings is that the different levels of sEH expression in response to an inflammatory microenvironment may be due to the heterogeneity of astrocytes . Indeed, it has been reported that subtypes of activated astrocytes, with distinct functionality in the diseased brain, exist . Alternatively, the increase of sEH expression in astrocytes may be associated with the progression of chronic disease status, and Aβ exposure per se may not necessarily instigate the expression of sEH in activated astrocytes. Given that various soluble Aβ species, including Aβ oligomers, are widespread in the brain, the exposure of activated astrocytes to Aβ is inevitable. Moreover, as demonstrated in our data, acute stimulation by LPS reduced, rather than increased, sEH in mice and primary astrocytes. One explanation is that the unspecified LPS-activated signaling might be involved in the acute effect of LPS on reducing the expression of sEH, whereas chronic activation leading to severe astrogliosis triggers upregulation of astrocytic sEH to counteract the inflammatory state. Therefore, the more the astrocytes are in an advanced and chronic activation, the more sEH is expressed. This speculation echoes our observation showing that intensive sEH immunoreactivity was commonly found within the enlarged processes of highly activated astrocytes in aged APP/PS1 mice. In contrast, activated astrocytes in younger APP/PS1 mice showed less sEH immunoreactivity. Whether the extent of sEH expression in activated astrocytes could be indicative of disease progression and perhaps serves as a biomarker for AD remains to be explored.
Nevertheless, the fact that levels of sEH modulate immune response in activated astrocytes may have significant impacts on AD pathogenesis. We speculate that high sEH-expressing astrocytes could be a subtype of astrocytes that are attempting to cope with inflammation because the expression of sEH could be anti-inflammatory in activated astrocytes. Therefore, it is conceivable to propose that activated astrocytes with high expression levels of sEH could be beneficial for counteracting the over-activation of astrocyte. However, sEH-overexpressing astrocytes, on the other hand, may reduce their beneficial functions of immune response against the disease progression. In fact, the beneficial functions of activated astrocytes have been recently reported, showing that Aβ plaques with little or no activated astrocytes were associated with dementia status in AD patients [37, 38]. Therefore, the possible adverse effects of sEH overexpression on over-reducing astrocytic functions remained to be elucidated.
Regulation of astrocyte activation by sEH involving STAT3 inhibition
Our study support the notion that STAT3 activation can be pro-inflammatory and is involved in triggering the expression of pro-inflammatory markers in activated astrocytes . In addition, we further revealed a novel function of sEH in the regulation of STAT3 activation in activated astrocytes. Based on our findings, the genetic deletion or pharmacological blockade of sEH led to an increase in LPS-induced STAT3 phosphorylation in activated astrocytes, which can be significantly attenuated by the inhibition of STAT3 using stattic in LPS-activated astrocytes. These data suggest that the STAT3-mediated astrocyte activation plays an important role in the regulation of LPS-activated astrocyte activation, whereas the pro-inflammatory properties of STAT3 are likely to be at least partly negatively regulated by sEH. In addition to its pro-inflammatory properties, many studies have reported that STAT3 activity can be anti-inflammatory and might promote the expressions of many anti-inflammatory genes, resulting in the indirect inhibition of pro-inflammatory gene expression in dendritic cells or macrophages [39, 40]. Indeed, our data show that stattic alone triggered the expression of TNFα and IL-6 in astrocytes at resting condition. It is conceivable to speculate that the upregulation of the pro-inflammatory cytokines by stattic in astrocytes at basal levels might result from the blockade of the anti-inflammatory mode of STAT3. However, the possibility that effects of stattic on the expression of pro-inflammatory cytokines are independent of STAT3 activation cannot be excluded. Taken together, the sEH-mediated STAT3 inhibition may play multiple roles in the regulation of astrocyte activation. Further research is needed to clarify how sEH interacts with STAT3 and how the sEH-mediated STAT3 inhibition contributes to the pathogenesis of AD.
In conclusion, we demonstrated, for the first time, that the deletion of sEH in astrocytes was pro-inflammatory, whereas the overexpression of sEH suppressed the immune response. The underlying mechanism may be at least partly attributed to the regulation of STAT3 activation. The sEH-mediated STAT3 inhibition in a cellular context suggests that astrocytic sEH may play an important role in the regulatory machinery of pro-inflammatory response in activated astrocytes during the progression of the brain diseases. Revealing the underlying mechanisms through which sEH orchestrates STAT3 activity and developing means to manipulate astrocytic sEH expression could facilitate the future development of treatment for brain diseases.
This work was supported by the grants NP-PP03 from the National Health Research Institutes, Central Government S&T grant, Taiwan (107-1901-01-10-02), and by the grant MOST 105-2320-B400-001. This work was also financially supported by the Brain Research Center, National Yang-Ming University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan (107BRC-B405, 108BRC-B404). The authors thank Prof. Tzong-Shyuan Lee at National Yang-Ming University for providing sEH-/- mice and acknowledge the technical assistance by J.C. Lu and W.T. Zhang and the technical support provided by the Imaging Core Facility of Nanotechnology of the UST-YMU. All authors declare no conflicts of interest.
NP-PP03 from National Health Research Institutes, Central Government S&T grant, Taiwan (107-1901-01-10-02), and MOST 105-2320-B400-001.
C-CH, Y-HL, H-JT, and F-SS designed research. C-CH, P-CH, Y-TH, C-CL, JJL, and F-SS performed research. C-CH, Y-MK, P-CH, JJL, and F-SS analyzed data. Y-HL and F-SS wrote the paper. All authors read and approved the final manuscript.
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