Reciprocal modulation of C/EBP-α and C/EBP-β by IL-13 in activated microglia prevents neuronal death

Authors

  • Hung Chuan Pan,

    1. Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan
    2. Department of Neurosurgery, Taichung Veterans General Hospital, Taichung, Taiwan
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  • Cheng Ning Yang,

    1. Institute of Neuroscience, School of Life Science, National Yang-Ming University, Taipei, Taiwan
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  • Yi Wen Hung,

    1. Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan
    2. Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan
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  • Wen Jane Lee,

    1. Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan
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  • Hsing Ru Tien,

    1. Institute of Biomedical Sciences, College of Life Science, National Chung Hsing University, Taichung, Taiwan
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  • Chin Chang Shen,

    1. Institute of Nuclear Energy Research, Atomic Energy Council, Longtan, Taoyuan, Taiwan
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  • Jason Sheehan,

    1. Department of Neurological Surgery, University of Virginia Health System, Charlottesville, VA, USA
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  • Chiang Ting Chou,

    1. School of Nursing, Chang Gung University of Science and Technology, Chiayi Campus, Taiwan
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  • Meei Ling Sheu

    Corresponding author
    1. Department of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan
    2. Institute of Biomedical Sciences, College of Life Science, National Chung Hsing University, Taichung, Taiwan
    • Full Correspondence Dr. Meei Ling Sheu, Institute of Biomedical Sciences, College of Life Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan

      Fax: +886-4-22853469

      e-mail: mlsheu@nchu.edu.tw

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Abstract

In response to aggravation by activated microglia, IL-13 can significantly enhance ER stress induction, apoptosis, and death via reciprocal signaling through CCAAT/enhancer-binding protein alpha (C/EBP-α) and C/EBP-beta (C/EBP-β). This reciprocal signaling promotes neuronal survival. Since the induction of cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor gamma/heme oxygenase 1 (PPAR-γ/HO-1) by IL-13 plays a crucial role in the promotion of and protection from activated microglia, respectively; here, we investigated the role of IL-13 in regulating C/EBPs in activated microglia and determined its correlation with neuronal function. The results revealed that IL-13 significantly enhanced C/EBP-α/COX-2 expression and PGE2 production in LPS-treated microglial cells. Paradoxically, IL-13 abolished C/EBP-β/PPAR-γ/HO-1 expression. IL-13 also enhanced ER stress-evoked calpain activation by promoting the association of C/EBP-β and PPAR-γ. SiRNA-C/EBP-α effectively reversed the combined LPS-activated caspase-12 activation and IL-13-induced apoptosis. In contrast, siRNA-C/EBP-β partially increased microglial cell apoptosis. By NeuN immunochemistry and CD11b staining, there was improvement in the loss of CA3 neuronal cells after intrahippocampal injection of IL-13. This suggests that IL-13-enhanced PLA2 activity regulates COX-2/PGE2 expression through C/EBP-α activation. In parallel, ER stress-related calpain downregulates the PPAR-γ/HO-1 pathway via C/EBP-β and leads to aggravated death of activated microglia via IL-13, thereby preventing cerebral inflammation and neuronal injury.

Introduction

Microglial cell activation is exquisitely sensitive to brain injury and diseases that contribute to neuronal cell death (e.g. repeated infection, traumatic brain damage, and stroke). Such activation likely plays a crucial role in inflammatory neuronal injury and chronic neurodegenerative diseases [1]. Anti-inflammatory medications may be protective against brain damage. Emerging evidence indicates that endoplasmic reticulum (ER) stress plays a pivotal role in the pathogenesis of neurodegeneration [2]. The ER activates the unfolded protein response, a signaling pathway for adaptive response, which initially exerts a protective effect by upregulating specific ER stress-regulated genes and inhibiting general protein translation [3, 4]. However, severe or prolonged ER stress results in cell death via apoptotic signaling, ultimately leading to neurodegeneration. A previous study has shown that IL-13 downregulates peroxisome proliferator-activated receptor gamma/heme oxygenase 1 (PPAR-γ/HO-1) via ER stress-stimulated calpain activation. Thus, IL-13 may reduce chronic brain inflammation [5]. This finding is consistent with the findings of Yang et al. [6] showing that IL-13 enhances cyclooxygenase-2 (COX-2) expression in activated rat brain microglia, thereby reducing brain inflammation. Recently, Kawahara et al. [7] suggested that intracerebral microinjection of IL-4/IL-13 reduces β-amyloid accumulation on the ipsilateral side and improves cognitive deficits in young amyloid precursor protein 23 mice. However, the mechanisms underlying how IL-13 regulates activated microglia and its relationship with the dampening of neuronal death have not been well elucidated.

Studies on the relationship between glial activation and neurotoxicity have identified several molecular targets for transcription factor research. The special transcription factor CCAAT/enhancer-binding protein (C/EBP) family regulates the transcription of genes that play important roles in glial activation [8]. Ejarque-Ortiz et al. [9] have also shown that the restoration of C/EBP-α levels may be a strategy for attenuating neurotoxic effects. Moreover, LPS can induce C/EBP-β expression by astrocytes and microglia in primary mouse glial cultures. It has been demonstrated by Straccia et al. [8] that C/EBP-β-null glial culture in activated microglia abrogates neurotoxicity, implying that C/EBP-β is a possible therapeutic target for ameliorating neuronal damage due to neuroinflammation. However, the relationships between the response of microglial cells to environmental damage or inflammatory processes and the profound changes of gene expression associated with ER stress-related signaling have not been clearly established [10, 11].

This study hypothesizes that enhancement of calpain-II-regulated C/EBP-β downregulation by IL-13 through the induction of ER stress-related signaling in activated microglia may exacerbate microglial cell death and lead to the inhibition of proinflammatory cytokines release from deteriorated microglia. Neuronal cells will no longer be exposed to toxic damage. Thus, this change may reduce neuronal damage due to neuroinflammation. The present study also shows that IL-13-enhanced ER stress-related calpain activation plays an important role in the downregulation of C/EBP-β-regulated PPAR-γ/HO-1 expression in activated microglia. In activated microglia, IL-13 may potentially confer functional and therapeutic benefits in neurologic disorders by abrogating neurodegeneration.

Results

IL-13-enhanced activated microglia PGE2 production is regulated by C/EBP-α, not C/EBP-β

Previously, PGE2 production was reportedly involved in activated microglial death [6]. Here, the role of C/EBP-α and C/EBP-β was analyzed using specific small interfering RNA (siRNA) to elucidate whether IL-13-enhanced activated microglia PGE2 expression using ELISA. IL-13 increased PGE2 expressions in LPS-induced primary and BV-2 microglial cells (Fig. 1A). C/EBP is thought to play a crucial role in the activation of microglia following brain injury. Moreover, transfection of siRNA targeting C/EBP-α significantly decreased PGE2 production, whereas silencing C/EBP-β alone resulted in minor effects. To more directly assess IL-13 enhancement on NO induction in activated microglia, NO production was examined by Griess reagents. NO production was detected in LPS-treated cells (Fig. 1B). The combination of IL-13 in LPS showed no effects. These suggested that C/EBP-α could be a factor mediating IL-13-induced PGE2 production and death of activated microglia.

Figure 1.

IL-13-enhanced PGE2 production by activated microglia is regulated by C/EBP-α but not C/EBP-β. (A) The effect of transfection of siRNA C/EBP-α or C/EBP-β in primary microglial cells and BV-2 microglial cell line later treated with LPS and IL-13 on PGE2 production at 24 h is shown. Scrambled RNA was used as negative control. (B) NO production was measured by Griess reagent for 24 h. Data are shown as mean + SEM of n = 4 and are from one experiment representative of at least three performed. *p < 0.05 compared to control, one-way ANOVA with the Fisher's LSD test.

IL-13 reciprocally controls and regulates the protein expression of C/EBP-α and C/EBP-β

IL-13-enhanced apoptotic cell death in activated microglia has been shown to be involved in neurodegenerative disorders [5-7, 12, 13]. Related genes in activated microglia were analyzed to determine whether they were regulated by C/EBP-α and C/EBP-β. LPS significantly increased C/EBP-α and C/EBP-β in primary microglia cells and BV-2 microglia (Fig. 2). IL-13 significantly enhanced LPS-induced C/EBP-α, but effectively reversed LPS-induced C/EBP-β protein expression. Promoter regulation in the COX-2 promoter-flanking region (−95∼−90) containing the cis-acting elements C/EBP DNA binding activity in silico was predicted in the laboratory. Notably, the C/EBP-α-regulated protein COX-2 showed a similar result to that observed in IL-13-treated conditions.

Figure 2.

IL-13 reciprocally regulates C/EBP-α and C/EBP-β protein expression. (A) Primary microglial cells and (B) BV-2 microglial cell line were treated with LPS (10 ng/mL or 0.1 μg/mL) in the presence or absence of IL-13 for 24 h. The protein expression of C/EBP-α, COX-1, COX-2 (left) or C/EBP-β, PPAR-γ, and HO-1 (right) was examined. The fold of image density is presented as a numerical value. GAPDH was used as a loading control. The results shown are from one experiment representative of at least five independent experiments performed.

The COX-1 protein was considered a constitutive isoform, equally expressed in almost all tissues, which did not have any effects. In contrast, a previous report demonstrated that IL-13 downregulates PPAR-γ/HO-1 via ER stress-stimulated calpain activation. Further examining the regulatory role of C/EBP-β in the expression of protective PPAR-γ and HO-1 signaling, we found that IL-13 regulated LPS-induced protein expression in a dose-dependent manner (Supporting Information Fig. 1). The data showed that IL-13 markedly decreased the induction of C/EBP-β and PPAR-γ/HO-1 expression by activated microglia cells, indicating that IL-13 reciprocally regulated C/EBP-α and C/EBP-β in activated microglia.

IL-13 enhances calpain expression and promotes an association with C/EBP-β and PPAR-γ

Calpain has been demonstrated to be involved in ER stress-induced activated microglia cell death [5]. Further investigating the possible mechanisms of IL-13 regulation of calpain in association with C/EBP-β, PPAR-γ, and HO-1, the results showed that IL-13 markedly enhanced calpain-II protein expression (Fig. 3A) and activity (Fig. 3B(i)) in primary activated microglia, but markedly reduced the functional activity of calpain inhibitors ALLN, ALLM, and Z-Leu-Leu-CHO (Fig. 3B(ii)). In terms of the role of calpain-II in IL-13-induced C/EBP-β, PPAR-γ, and HO-1 downregulation, calpain-II was shown to interact with C/EBP-β and PPAR-γ but not HO-1 with co-immunoprecipitation and Western blot in activated microglia. Calpain-II was specifically associated with C/EBP-β and PPAR-γ in activated BV-2 microglia cells with the presence of IL-13-treated cells compared with the IgG control (Fig. 3C). There was no direct interaction of HO-1 with calpain-II.

Figure 3.

IL-13 enhances a calpain expression and promotes a calpain association with C/EBP-β and PPAR-γ. (A) Primary microglia cells were treated with LPS (10 ng/mL) in the presence or absence IL-13 for 24 h. Calpain-1 and calpain-2 protein levels were detected by Western blot analysis. (B) Calpain activity was measured with the fluorescent calpain substrate Suc-LLVY-AMC in primary microglial cells and shown as 1.75 ± 0.15 of six samples from one experiment representative of six performed/pooled from six experiments performed. (i) Time course response to calpain activity. (ii) Calpain inhibitors ALLN, ALLM, and Z-Leu-Leu-CHO to calpain activity. (C) Interaction of calpain and C/EBP-β and PPAR-γ, but not HO-1, was detected in BV-2 microglial cell line. Immunoprecipitated proteins were collected at 16 h and subjected to SDS-PAGE and immunoblotting with anti-calpain-2 or anti-C/EBP-β, PPAR-γ, and HO-1 antibodies. Data shown are from one experiment representative of at least three independent experiments performed. (D) Proteolysis of C/EBP-β and PPAR-γ in the presence of calpain. Purified C/EBP-β and PPAR-γ were digested by the indicated concentrations of recombinant m-calpain-2 at 30°C for 4 h in the presence of 0.5 mM CaCl2. Samples were analyzed by 8% SDS-PAGE and stained with Coomassie blue. Data shown are from one experiment representative of at least three performed. (E) Microglial cells were pretreated with calpain inhibitor following LPS and IL-13, and protein expressions were determined by Western blotting. GAPDH was used as a loading control. The results shown are from one experiment representative of at least three independent experiments. *p < 0.05 compared to control, one-way ANOVA with the Fisher's LSD test in the sentence.

To clarify if calpain cleaved C/EBP-β and PPAR-γ, C/EBP-β or PPAR-γ were digested with recombinant calpain-II under various conditions in vitro cleavage assay. The incubation of C/EBP-β or PPAR-γ with recombinant m-calpain led to the complete digestion of C/EBP-β or PPAR-γ, as determined by Western blotting analysis (Fig. 3D). Moreover, the calpain inhibitor, Z-Leu-Leu-CHO, effectively reversed the IL-13-enhanced LPS-induced C/EBP-β downregulation, but not C/EBP-α and COX-2, in BV-2 microglia (Fig. 3E). These results indicated that calpain-II induction plays an important role in IL-13-triggered reduction of C/EBP-β and PPAR-γ in inflammation-activated microglia.

Silencing C/EBP-α abolishes IL-13-enhanced apoptosis in activated microglia

Death of activated microglia could act as an endogenous mechanism for the resolution of brain inflammation [6]. Thus, the effect of knockdown of C/EBP-α expression was investigated to determine if C/EBP-α abolishes IL-13-enhanced apoptosis in activated microglia. LPS significantly increased activated caspase-12 (cleavage of pro-caspase-12) in BV-2 microglia (Fig. 4A). Caspase-12 mediated ER-specific apoptosis and cytotoxicity in various stimulated cells. Knockdown of C/EBP-α expression efficiently inhibited activated caspase-12.

Figure 4.

Silencing of C/EBP-α abolishes IL-13-enhanced apoptosis in activated microglia. (A) Cells were transfected with siRNA C/EBP-α or C/EBP-β. BV-2 microglia cells were treated in presence or absence of LPS (0.1 μg/mL) with or without IL-13 (20 ng/mL). After 24 h, cell lysates were analyzed for C/EBP-α, COX-2, C/EBP-β, PPAR-γ, HO-1, and activated caspase 12 expression by Western blotting. GAPDH was used as a loading control. Data shown are from one experiment representative of at least three independent experiments. (B) Apoptosis was analyzed by annexin V/PI staining. Apoptotic cell populations were determined and quantified by densitometric analysis (Supporting Information Fig. 2). Data shown are from one experiment representative of at least three independent experiments.

Silencing of C/EBP-β by siRNA did not modify the expression of caspase 12, C/EBP-α, or COX-2 compared with IL-13 combined with LPS-treated apoptosis. Quantitative analysis of protein expression was determined by densitometry (Image-Pro Plus software, Supporting Information Fig. 2A). Silencing of C/EBP-α by siRNA reduced IL-13 combined with LPS-treated cell apoptosis, as determined by annexin-V and propidium iodide (PI) dual staining following ER stress induction in activated microglia (Fig. 4B and Supporting Information Fig. 2B). However, knockdown of C/EBP-β by siRNA presented with consistent results in LPS and IL-13-treated apoptotic response.

IL-13 induces PLA2 expression, which regulates C/EBP-α expression

PLA2 had been shown to be involved in inflammation of both acute and chronic neurodegeneration [14, 15]. Three groups of PLA2 were involved in AA generation, including secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), and calcium-independent PLA2 (iPLA2) [16]. The induction of iPLA2, cPLA2 activity, and protein expression in activated microglia was investigated. LPS increased the enzyme activity of iPLA2 and cPLA2 in primary and BV-2 microglia (Fig. 5A). IL-13 (20 ng/mL) also mildly enhanced iPLA2 and cPLA2 activity. LPS increased enzyme activity in microglia and this was significantly enhanced by IL-13. Protein expression was similarly affected (data not shown).

Figure 5.

IL-13-induced PLA2 expression further regulates C/EBP-α expression. (A) Primary microglial cells were treated with LPS and IL-13, then calcium-dependent cytoslic PLA2 (cPLA2) and calcium-independent cytoslic PLA2 (iPLA2) was determined by ELISA. Data are shown as mean ± SEM of n = 5 and are from one experiment representative of five performed. *p < 0.05 compared to control, #p < 0.01, one-way ANOVA with the Fisher's LSD test. (B) BV-2 was exposed to the cPLA2 inhibitor methyl arachidonyl fluorophosphates (MAFP, 10 μM) or iPLA2 inhibitor bromoenol lactone (BEL, 1 μM) (data not shown) and then treated with LPS and IL-13 for 24 h. The nuclear protein separation of C/EBP-α or C/EBP-β was determined by Western blotting. C23 was identified as a nucleolus control. (C) Primary microglial cells were incubated with LPS and IL-13 for 1 h and then fixed and incubated with mAb against for immunofluorescent staining. Original magnification: ×400. (D) BV-2 microglial cells were treated in the presence or absence of LPS (0.1 μg/mL) with or without IL-13 (20 ng/mL) for 60 min, and nuclear C/EBP DNA binding activity was analyzed by electrophoretic mobility shift assay. Nuclear proteins extracted from LPS or LPS+IL-13 were incubated with the labelled probe anti-C/EBP-α and anti-C/EBP-β antibody (long exposure image). Arrow indicates shifted DNA binding complex. The C/EBP DNA binding labeled probe as indicated. All experiments were repeated at least five times.

Further examining the regulatory role of PLA2 in the expression of C/EBP-α or C/EBP-β, treatment of microglia with LPS resulted in increased expression of C/EBP-α and C/EBP-β nuclear protein, by Western blot analysis (Fig. 5B). IL-13 effectively increased C/EBP-α expression but reversed C/EBP-β, while the PLA2 inhibitor, methyl arachidonyl fluorophosphates, markedly reduced C/EBP-α expression (Fig. 5B). LPS-activated microglia also showed marked C/EBP-α nuclear translocation, by immunofluorescent staining and confocal microscopy to capture the image and by Western blotting. However, IL-13 effectively reversed the LPS-induced C/EBP-β nuclear translocation. In contrast, C/EBP-α enhanced the nuclear proportion in activated microglia (Fig. 5C and Supporting Information Fig. 3A and B).

Moreover, IL-13 markedly increased C/EBP-α DNA binding activity in microglial cells, but this was effectively reversed by methyl arachidonyl fluorophosphates (10 μM) (Fig. 5D). IL-13 appeared to effectively promote LPS-induced C/EBP-α DNA binding activity in microglia. These findings imply that PLA2-upregulated, C/EBP-α-regulated cascade signaling pathway is involved in IL-13-enhanced LPS-triggered microglial activation.

LPS aggravates microglial and neuronal death, which is alleviated by IL-13 administration

To further establish the relationship between the deterioration of spatial memory cells and activation of microglia, as well as the subsequent hippocampus CA3 neuron dysfunction, PBS, LPS, IL-13, LPS+IL-13, and LPS+IL-13+NA (IL-13 neutralizing antibody (NA)) were stereotactically injected into CA3 of the hippocampus to assess microglial activation and the associated neuronal death. The injected dye was mostly located in the hippocampus CA1–3 region when injection time was longer than 30 min (Supporting Information Fig. 4).

In the water maze assessment, LPS injection resulted in neurologic deterioration at 3 days, with little improvement for up to 21 days. This deterioration of neurological function was restored by IL-13 injection (Fig. 6B and Supporting Information Fig. 5). Furthermore, injection of IL-13-neutralized antibody caused a similar neurologic outcome as that of the LPS group. Injection of IL-13 did not cause significant neurologic dysfunction compared with the PBS group.

Figure 6.

Aggregation of microglia/macrophage and neuronal loss by LPS and alleviation by IL-13 administration. Imaging of microglia/macrophage aggregation related to the number of neuronal cells in CA3 of the hippocampus. Deposition of NeuN (green), CD11b (green), C/EBP-α (red), and C/EBP-β (red) over CA3 in different treatment groups. Scale bar, 50 μm; magnification, ×400; PBS, IL-13, LPS, LPS+IL-13, LSP+IL-13+NA (IL-13 neutralizing antibodies). All experiments were repeated at least five times.

On the day of the worst neurologic dysfunction (3 days after stereotactic injection), the brain was harvested to assess the distribution of microglial/monocyte and neuronal survival (Fig. 6). LPS injection increased the deposition of CD11b with a reciprocal decrease in NeuN-positive cells. Co-injection of LPS with IL-13 decreased the number of CD11b positive cells and further restored the number of NeuN positive cells. Ablation of IL-13 with IL-13 NA exerted the same effect as LPS injection. LPS injection increased the expression of C/EBP-α and C/EBP-β in CD11b positive cells, while the combination of LPS and IL-13 only caused the expression of C/EBP-α in CD11b positive cells. The combined effect of LPS and IL-13 in C/EBP-α and C/EBP-β was abolished by IL-13 NA. Hence, microglia/macrophage (CD11b positive cells) was activated by LPS injection and IL-13 further aggravated the microglia/macrophage cell loss. Attenuation of microglia/macrophage cells increased the number of neuronal cells and provided a more favorable neuro-behavioral response in animals.

Discussion

A previous study reported that IL-13-enhanced ER stress-related calpain activation plays an important role in the downregulation of PPAR-γ-regulated HO-1 expression in activated microglia. The present study shows that IL-13 enhances COX-2/PGE2 expression through PLA2 and C/EBP-α regulation. More importantly, IL-13 simultaneously augments ER stress and calpain activity, and cleavage of C/EBP-β and PPAR-γ expression results in aggravation of activated microglia death. Finally, this study is the first to demonstrate that administration of IL-13 in activated microglia in an animal model enhances C/EBP-α expression, but abolished C/EBP-β expression, which diminishes neuronal cell loss and damage in regions associated with memory and the hippocampal CA3 region.

The ER is a major component of the protein quality control system. Emerging evidence indicates a potent association between accumulation of protein aggregates and ER stress induction in various important neurodegenerative conditions. Previous reports have shown that calpain inhibitors have impressive neuroprotective effects in in vivo models of cerebral ischemia. Calpains belong to a family of calcium-dependent thiol-proteases that proteolyze a wide variety of cytoskeletal, membrane-associated, and regulatory proteins [17]. Calpains do not generally function as destructive proteases, but act as calcium-dependent modulators that remove limited portions of protein substrates. Their proteolysis is usually a late-stage common pathway toward cell death induced by excitotoxic compounds.

Calpains can also cleave other potentially important apoptosis-related proteins, including caspase-12, Bax, Bcl-XL, GRP94, c-Fos, and p53 [18-21]. They are also thought to play a critical role in a form of neuronal cell death involved in the pathogenesis of several diseases [22-24]. However, calpain activity and expression are increased in activated glial and inflammatory cells [25-28]. The application of calpain inhibitor effectively reduces the frequency of spontaneous release of neurotransmitter in Alzheimer's disease.

C/EBP-β is one of the members of the C/EBP subfamily of bZIP transcription factors, which is thought to regulate proinflammatory gene expression primarily expressed by microglia with lower upregulation in astrocytes [8, 29, 30]. Raised C/EBP-β levels have also been demonstrated in vivo in situations wherein neuroinflammation occurs, such as systemic LPS injection, cerebral ischemia, excitotoxic insult, or aging. Straccia et al. [8] have reported that the lack of C/EBP-β results in greater attenuation of proinflammatory gene expression activated by LPS+IFN-γ compared with that with LPS alone in the activating stimulus. The neurotoxicity elicited by LPS+IFN-γ treated microglia is abrogated by the lack of C/EBP-β. Valente et al. [31] have also shown that C/EBP-β may control the expression of potentially neurotoxic genes in microglial cells in amyotrophic lateral sclerosis. Dasgupta et al. [32] showed that overexpression of ΔC/EBP-β, a truncated alternate C/EBP-β translation product, LIP, which acts as a dominant-negative inhibitor of C/EBP-β activity, inhibited the myelin basic protein primed T-cell-induced expressions of IL-1β, IL-1α, TNF-α, and IL-6 in microglial cells. Thus, C/EBP-β plays a role in the regulation of neurotoxic effects in activated glial cells.

Abrogation of C/EBP-β expression or its downregulation by gene regulation may serve as a therapeutic target to attenuate deleterious effects in neural tissue and ultimately prevent the development of neurodegenerative disorders. In the present study, IL-13 directly enhanced calpain and C/EBP-β interaction, resulting in decreased C/EBP-β. These findings imply that the anti-inflammatory cytokine IL-13 protects neurons by mechanisms probably involving the regulation of ER stress by calpain activation. These results provide evidence that IL-13-induced calpain activation in activated microglia under ER stress condition can aggravate microglia cell death and, consequently, promote neuronal cell survival.

Numerous reports have shown that PPAR-γ and HO-1 are master regulators that induce a battery of cytoprotective genes, including anti-oxidative enzymes, anti-inflammatory mediators, and a variety environmental activities involved in ER stress [33-35]. PPAR-γ has been proposed as a transcription factor that activates the HO-1 gene in silico [5]. Studies have shown that PPAR-γ and HO-1 exert beneficial effects in neurodegenerative disorders. Interestingly, functional binding sites for the transcription factor C/EBPβ can be found in the promoter regions of PPAR-γ and HO-1 [36, 37]. In the present study, IL-13 markedly abolishes LPS-induced C/EBP-β, PPAR-γ, and HO-1 expressions. Consistent with previous studies using brain injury models, the results here demonstrate that C/EBP-β regulation provides a potential new avenue for the development of therapeutic strategies to prevent hyperactivated microglia-induced neuronal damage.

Pathologic conditions and proinflammatory stimuli in the brain induce COX-2, a key enzyme in arachidonic acid metabolism. COX-2 mediates the production of prostanoids, such as PGE2, which induce fever and pain, increase vascular permeability, and recruit inflammatory cells to sites of inflammation. In previous studies, Yang et al. [6] found that COX-2 and PGE2 appear to be involved in IL-13-induced death of activated microglia. Their findings suggest that the death of activated microglia may act as an endogenous mechanism for the resolution and termination of brain inflammation [6].

The current study demonstrates that IL-13 enhances apoptosis in activated microglia and this plays a crucial role in reducing brain inflammation. C/EBP-α, a basic leucine zipper transcription factor, has been identified in many studies as playing a critical role in COX-2 expression in the transcriptional activation of the COX-2 promoter [38]. C/EBP-α binds to C/EBP enhancer elements and is essential for inducing COX-2 expression by LPS, TNF-α, and IL-1β. Hsieh et al. [39] showed that attenuated C/EBP-α expression in COX-2 activation in fat inflammation is important in the development of insulin resistance and fatty liver in high fat-induced obese rats. In addition, signaling cascade coupling of COX enzymes with PLA2s may be a key mechanism in the propagation of inflammatory reaction. Recently, PLA2s have been found to play a prominent role in the regulation of C/EBP-α gene expression. The activation of C/EBP-α is under the control of different signaling pathways and can be activated via PLA2 pathway more than the influence of COX-2 expression [40].

The hippocampus is part of a group of structures forming the limbic system and is also a part of the hippocampal formation, which also includes the dentate gyrus, subiculum, and entorhinal cortex. Different components of the limbic system play critical roles in various aspects of emotions, fear, learning, and memory [41-44]. Within the hippocampus, recent studies have suggest important differences in the functioning of areas CA1 and CA3 (the principal pyramidal cell fields in the hippocampus) in spatial and contextual memory [45, 46]. Neurotoxic lesions or pharmacological inactivation of hippocampal areas CA3 or CA1 have been reported to produce different effects on the encoding and retrieval of contextual memories [47, 48]. The CA3 region, with its extensive recurrent collateral system, is thought to be a crucial site for hippocampal function. This region supports processes involved in spatial pattern association, spatial pattern completion, novelty detection, and short-term memory. The CA1 region supports processes associated with temporal pattern completion and intermediate-term memory. Furthermore, CA3, in conjunction with CA1, supports temporal pattern separation [49]. In a water maze test, recent and remote memory are similarly impaired after hippocampal damage [50].

In the present study, the distribution of methylene blue dyes in stereotactic injection is time dependent. Stereotactic injection of dye with infusion time of 30 min resulted in distribution of dye to the whole hippocampus and some diffusion to the lateral ventricle, suggesting that LPS injection was not only detrimental to CA3 but also to the CA1 region. LPS injection causes microglia activation with subsequent neuronal death in CA3, which mirrors the impaired performance in the water maze test. In contrast, LPS combined with IL-13 injection triggered microglia death, reduced proinflammatory cytokine secretion [6], and decreased neuron death. This cascade of events improved performance on the water maze test. Due to the diffuse involvement of the whole hippocampus by stereotactic injection, the functional outcome is not solely attributable to CA3 but also to CA1 function.

In conclusion, this study reveals that IL-13 induces ER stress, resulting in reduced damage of neuronal cells through calpain activation cleavage of C/EBP-β and PPAR-γ, which parallel the PLA2-triggered C/EBP-α and COX-2 activation pathway. A proposed mechanism for IL-13-enhanced aggravated microglia death is shown in Figure 7. The current findings demonstrate the mechanisms involved in the regulation of IL-13 in activated microglia and point to new directions for therapeutic research on neuroinflammatory disorders.

Figure 7.

A proposed mechanism of IL-13-enhanced aggravated microglia death. IL-13 induces ER stress, resulting in reduced damage of neuronal cells through calpain activation cleavage of C/EBP-β and PPAR-γ, which parallels the PLA2-triggered C/EBP-α and COX-2 activation pathway.

Materials and methods

Many of the methods listed here have been published previously but are repeated here for clarity [5].

Chemicals and materials

LPS from Escherichia coli serotype 0111:B4 prepared by phenolic extraction and gel filtration chromatography obtained from Sigma-Aldrich. IL-13 was purchased from PeproTech. Calpain inhibitors were purchased from BIOMOL. Recombinant calpain was obtained from Merck Biosciences. Antibodies used in the present study were listed in Table 1. Lipofectin transfection reagent was purchased from Invitrogen. Specific siRNA and scrambled siRNA control were synthesized by Santa Cruz Biotechnology, Inc. or Dharmacon (Boulder, CO, USA). Other chemicals were of the best grade available from commercial sources.

Table 1. Additional antibodies used in the study
NameSp. (Clone number or code number)In theVendor
  work usage 
  1. WB: Western blot; IF: immunofluorescence.

Primary antibody    
 C/EBP-αSC-61 (14AA)Rabbit polyclonal IgGWB, IFSanta Cruz Biotechnology
 C/EBP-βSC-7962 (H-7)Mouse monoclonal IgG2aWB, IFSanta Cruz Biotechnology
 COX-2SC-1747 (M-19)Goat polyclonal IgGWBSanta Cruz Biotechnology
 COX-1SC-19998 (11)Mouse monoclonal IgG2bWBSanta Cruz Biotechnology
 calpain ISC-7531 (N-19)Goat polyclonal IgGWBSanta Cruz Biotechnology
 calpain IISC-7533 (N-19)Goat polyclonal IgGWBSanta Cruz Biotechnology
 C23SC-8031 (MS-3)Mouse monoclonal IgG1WBSanta Cruz Biotechnology
 Caspase-12No. 2202SPolyclonal antibodyWBCell Signaling Technology
 CD11b/cNo. 554859Monoclonal antibodyIFBD Pharmingen™
 GAPDHSC-32233Mouse monoclonal IgG1WBSanta Cruz Biotechnology
 HO-1SC-1797 (M-19)Goat polyclonal IgGWBSanta Cruz Biotechnology
 PPAR-γSC-7273 (E-8)Mouse monoclonal IgG1WBSanta Cruz Biotechnology
 NeuNA60 | MAB377Monoclonal antibodyIFMillipore Corporation (formerly Chemicon)
Secondary antibody    
 Peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L)115–035–003 WBJackson ImmunoResearch Laboratories, Inc.
 Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L)111–035–003 WBJackson ImmunoResearch Laboratories, Inc.
 Donkey anti-goat IgG-HRPsc-2020 WBSanta Cruz Biotechnology
 Alexa Fluor® 488 goat anti-mouse IgG (H+L)A-11001 IFMolecular Probes, Invitrogen
 Alexa Fluor® 594 goat anti-rabbit IgG (H+L)A-21207 IFMolecular Probes, Invitrogen
 Rhodamine-labeled affinity purified antibody to mouse IgG (H+L)03–18–06 IFKPL, Kirkegaard & Perry Laboratories, Inc.
 Fluorescein-labeled antibody to rabbit IgG (H+L)02–15–16 IFKPL, Kirkegaard & Perry Laboratories, Inc.

Cell culture and isolation of microglia

Microglial cells of BV-2 and primary rat microglia lines were cultured as previously described [5]. The purity of the cultures was 98–100% as determined by immunostaining with CD11b antibody.

Production of PGE2 and PLA2

The PGE2 levels in cell culture supernatants were determined by PGE2 enzyme immunoassay (Cayman Chemical). PLA2 production was measured by iPLA2 phospholipase A2 ELISA, Calcium Independent (iPLA2, Uscn Life Science Inc.) and cPLA2 activity was measured by commercially available assay (Cayman Chemicals).

Nitrite assay

Nitrite production was determined using the Griess reagent as reported before [5]. Absorbance was determined at 550 nm using a Thermo micro-plate reader (Molecular Devices).

Immunoblotting

Immunoblotting was performed as described previously [5]. Whole cell lysate proteins (60 μg) were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. After blocking, the blots were incubated with antibodies overnight. Membranes were then incubated for 1 h with secondary antibody. Detection was performed by ECL (Amersham) and by chemiluminescence using Kodak X-Omat film.

Calpain activity assays

Calpain activity assay was performed as described previously [5]. Assay was done using fluorogenic peptide substrate (Suc-Leu-Tyr-AMC) analyzed on a fluorescence plate reading system (HTS-7000 Plus Series BioAssay, Perkin Elmer) with filter settings of 380 ± 20 nm for excitation and 460 ± 20 nm for emission.

In vitro proteolysis of C/EBP-β or PPAR-γ by calpain

Cleavage of C/EBP-β or PPAR-γ by calpain-2 was analyzed by a modified procedure as described previously. Purified 100 μg C/EBP-β or PPAR-γ was incubated with 5 U/mL recombinant m-calpain-2 (Calbiochem) in a reaction buffer containing 40 mM Tris-HCl (pH 7.5) and 2 mM CaCl2 at 30°C for 4 h. The reactions were stopped by the addition of SDS-PAGE sample buffer. The reaction mixtures were then loaded on a 12% SDS-PAGE gel. The cleavage of C/EBP-β or PPAR-γ by calpain was analyzed by Coomassie blue staining of the gel and immunoblotting.

RNA interference and transfection assays

The delivery of siRNA pools into primary microglial cells or BV2 cells was performed using lipofectin (Invitrogen). siRNA duplexes specific for the inhibition of C/EBP-α and C/EBP-β expression in human cells were obtained from Santa Cruz Biotechnology, Inc. The pooled siRNA duplexes were dissolved in buffer (20 mM KCl, 6 mM HEPES, pH7.5, and 0.2 mM MgCl2). Cell transfection was conducted for 24 h at a final siRNA concentration of 1 μM, followed by normal growth medium. Scrambled siRNA, a nontargeting 20–25 nt siRNA, was used as negative control.

Annexin-V FITC and PI double staining

The annexin V/PI assay (Clontech, Mountain View, CA, USA) was used to quantify numbers of apoptotic cells as described previously [5]. Analysis was done on a FACSCalibur flow cytometer (Becton Dickinson, Rockville, MD, USA) and analyzed by CellQuest software (Becton Dickinson).

Immunocytochemical staining

Staining was conducted as previously described [5]. The cells were treated with as indicated for 60 min and then fixed with 1 mL 4% paraformaldehyde in PBS and further blocked and reacted with anti-mouse mAb antibody (1:1000 dilution in PBS; Santa Cruz Biotechnology) overnight at 4°C. After washing, the slides were incubated for 1 h at room temperature with anti-mouse-immunoglobulins-RPE or anti-rabbit-immunoglobulins-FITC, and then viewed on a fluorescence microscope.

Electrophoretic mobility shift assay

The electrophoretic mobility shift assay was performed as described previously [5]. The consensus sequence-specific oligo-nucleotide probes were end-labeled with γ-32P-ATP according to the manufacturer's recommendations. The oligonucleotide with the C/EBP consensus binding sequence used were 5′-GGTTCTTGCGCAACTCACTGAA-3′ and 3′-TTCAGTGAGTTGCGCAAGAACC-5′

For the binding reaction, 2 ng labeled oligonucleotide (approximately 20 000 cpm) and 2 μg poly dIdC (Amersham Pharmacia Biotech) carrier were incubated with 2 μg nuclear protein in a binding buffer (10 mM HEPES, 60 mM KCl, 1 mM DTT, 1 mM EDTA, 7% glycerol, and pH 7.6) for 30 min at room temperature. DNA–protein complexes were resolved on 6% nondenaturing polyacrylamide gels and visualized by exposure to autoradiographic films.

Stereotaxic surgery and drug injection

Sprague-Dawley rats (230–250 g) were anesthetized by i.p. injection of chloral hydrate (400 mg/kg), positioned in a stereotaxic apparatus, and either LPS (from Salmonella enteritidis; Sigma, St. Louis, MO), IL-13, IL-13 antibody, or a combination of 2–3 were stereotactically injected into the right cerebral cortex (AP+4.8 mm ML, −5.5 mm, DV −6.0 mm from the bregma) according to Paxinos’ atlas. The animals were categorized into to five groups: group I, PBS injection (30 μL); group II, LPS injection (10 μg); group III, IL-13 (100 μg) injection; group IV, LPS (10 μg) + IL-13 (100 μg) injection; and group V, LPS (10 μg) + IL-13 (100 μg) + IL-13 neutralized antibody (NA, 10 ng) in a final volume of 30 μL PBS injected at a rate of 0.15 μL/min using a 26-gauge Hamilton syringe attached to an automated pump and left in situ for an additional 5 min to avoid reflux along the injection tract.

Water maze test

A 1.5 m diameter, 45 cm deep Morris water maze was filled with water to a depth of 26.5 cm. The water temperature was kept at 26 ± 2˚C. A circular platform, 25 cm high, and 12 cm in diameter was placed into the tank at a fixed location in the centre of one of four imaginary quadrants. Approximately 1.5 L of milk was used to make the water opaque. The rat was then guided to swim to the platform. Activity in the water maze was recorded using a CCD camera on the ceiling above the centre of pool, which was attached to an automated tracking system (Noldus, Netherlands). A single experiment was performed with three rats. Behavioral measures included latency to targets, swing speed (cm/s), number of platform crosses, and percent time within the targeted area. Percent time in appositive object in reversal trial and in targeted object in extinction test was also conducted. Data were analyzed by Etho Vision 3.1.

Immunofluorescent staining

The animals were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/mL), followed by 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB). The brain was removed from the cranium and postfixed for 1 h, washed in 0.1 M PB, and then immersed in 30% sucrose solution until it sank. Tissues were sectioned on a sliding microtome at 40-μm thickness. Every sixth serial section was selected and processed for immunostaining. The primary antibodies used were against mouse CD11b (1:400), NeuN (1:500), C/EBP-α (1:300), and C/EBP-β (1:300). The following day, brain sections were rinsed with PBS 0.5% BSA and incubated with appropriate secondary antibodies. The immunoreactive signals were observed using Alexa Fluor® 488 goat anti-mouse and Alexa Fluor® 594 goat anti-rabbit (1:200) and viewed by confocal microscopy capture imaging.

Statistical analyses

The results are presented as mean ± standard error of the mean (SEM). All analyses of variance were followed by Fisher's least significant difference posthoc analyses. Statistical significance was set at p < 0.05.

Acknowledgements

The authors thank the Department of Education and Research, Taichung Veterans General Hospital for the excellent editing and technical assistance. This work was supported by grants from Taichung Veterans General Hospital, Taiwan (TCVGH-977304B) and the National Science Council of Taiwan (NSC96-2320-B-040-003-MY3 and NSC-101–2314-B-075A-003-MY2).

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Abbreviations
C/EBP-α

CCAAT/enhancer binding protein alpha

C/EBP-β

C/EBP–beta

COX-2

cyclooxygenase-2

cPLA2

cytosolic PLA2

HO-1

heme oxygenase 1

NA

neutralizing antibody

PPAR-γ

peroxisome proliferator-activated receptor-gamma

siRNA

small interfering RNA

Ancillary