CDDO-Me Inhibits Microglial Activation and Monocyte Infiltration by Abrogating NFκB- and p38 MAPK-Mediated Signaling Pathways Following Status Epilepticus

Following status epilepticus (SE, a prolonged seizure activity), microglial activation, and monocyte infiltration result in the inflammatory responses in the brain that is involved in the epileptogenesis. Therefore, the regulation of microglia/monocyte-mediated neuroinflammation is one of the therapeutic strategies for avoidance of secondary brain injury induced by SE. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me; RTA 402) is an activator of nuclear factor-erythroid 2-related factor 2 (Nrf2), which regulates intracellular redox homeostasis. In addition, CDDO-Me has anti-inflammatory properties that suppress microglial proliferation and its activation, although the underlying mechanisms have not been clarified. In the present study, CDDO-Me ameliorated monocyte infiltration without vasogenic edema formation in the frontoparietal cortex (FPC) following SE, accompanied by abrogating monocyte chemotactic protein-1 (MCP-1)/tumor necrosis factor-α (TNF-α) expressions and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Furthermore, CDDO-Me inhibited nuclear factor-κB (NFκB)-S276 phosphorylation and microglial transformation, independent of Nrf2 expression. Similar to CDDO-Me, SN50 (an NFκB inhibitor) mitigated monocyte infiltration by reducing MCP-1 and p38 MAPK phosphorylation in the FPC following SE. Therefore, these findings suggest, for the first time, that CDDO-Me may attenuate microglia/monocyte-mediated neuroinflammation via modulating NFκB- and p38 MAPK-MCP-1 signaling pathways following SE.


Introduction
Microglia are unique non-neuroepithelial and myeloid cells found in the brain parenchyma as the brain is separated from the systemic immune system by a brain-blood barrier (BBB) [1]. Under physiological conditions, microglia have small cell bodies with slender ramified processes (resting states). In response to various stresses, microglia show hypertrophic and/or elongated morphologies with hyper-ramified processes (intermediate states) and finally amoeboid shape indicating phagocytic and cytotoxic activity (activated states) [2]. Microglial activation is mediated by various receptors and ion channels (such as purinergic receptors, C-X3-C motif chemokine receptor, transient receptor potential channel (TRPC) and K + channels), which activate diverse signaling molecules including the nuclear factor of activated T-cells, nuclear factor-κB (NFκB), mitogen-activated protein kinases (MAPKs) and phosphatidylinositol-3-kinase (PI3K)/AKT [3]. This microglial activation seems to play an important role in epileptogenic processes. Activated microglia secrete various cytokines such as at a concentration of 0.4 nM in vitro and~0.5 nmol/kg/day (i.c.v.) over 7 days in vivo [31,40]. Thus, the anti-cancer concentrations of CDDO-Me in vitro and in vivo are~250-25,000 and 30,000 times higher than anti-inflammatory doses, respectively. Thus, it is noteworthy to explore the effects of CDDO-Me on microglial activation and monocyte infiltration following SE, although once-daily administration of 20 mg of CDDO-Me increases the risk of the cardiovascular dysfunctions in patients with prior history of heart failure without evidence of direct cardiotoxicity [42][43][44].
Here, we demonstrate that CDDO-Me effectively attenuated SE-induced microglial activation and monocyte infiltration in the FPC by inhibiting NFκB-and p38 mitogen-activated protein kinase (p38 MAPK)-mediated inductions of MCP-1 and TNF-α, independent of Nrf2 activity. Therefore, our findings propose an underlying anti-inflammatory mechanism of CDDO-Me by regulating microglial functions, and its availability for neuroinflammation.

Experimental Animals and Chemicals
Adult male Sprague-Dawley (SD) rats (7 weeks old) were used in the present study. Rats were in-housed under controlled conditions (22 ± 2 • C, humidity 55 ± 5%, a light-dark cycle on a 12-h on-off cycle) and freely accessed to water and food throughout the experiments (See Supplementary Materials). All experimental protocols were approved by the Institutional Animal Care and Use Committee of Hallym University (Hallym 2018-2, April 2018). All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), except as noted.

SE Induction
Three days after surgery, rats were treated with atropine methylbromide (5 mg/kg, i.p.). Twenty min after atropine treatment, rats were given pilocarpine (380 mg/kg, i.p.). Two h after onset of SE, diazepam (Valium; Hoffman la Roche, Neuilly sur-Seine, France; 10 mg/kg, i.p.) was administered. Age-matched controls received the same volume of saline instead of pilocarpine.

Tissue Processing and Immunohistochemistry
Three days after SE, we transcardially perfused animals with 0.9% saline followed by 4% paraformaldehyde in 0.1M phosphate buffer (PB, pH 7.4) under urethane anesthesia (1.5 g/kg, i.p.), since this time point is the best to observe microglial activation and monocyte infiltration [8,25]. The brains were removed and post-fixed in the same fixative overnight. Next, the brains were stored in 30% sucrose/0.1M PBS for cryoprotection. Coronal sections were made at a 30-µm thickness with a cryo-microtome. After 3 times-wishing with PBS (0.1M, pH 7.3), tissues were incubated in 3% H 2 O 2 and 10% methanol in PBS (0.1 M) for 20 min at room temperature. Later, sections were incubated Cells 2020, 9, 1123 4 of 19 in 0.1% bovine serum albumin and subsequently primary antibody (Table 1). Tissue sections were  developed in 3,3 -diaminobenzidine in 0.1M Tris buffer and mounted on gelatin-coated slides. Some  sections were reacted with a cocktail solution containing the primary antibodies or isolectin B4 (IB4,  Table 1) in PBS containing 0.3% Triton X-100 and visualized with appropriate Cy2-and Cy3-conjugated secondary antibodies. A negative control test was carried out with preimmune serum instead of the primary antibody, which showed no immunoreactivity in any structures.

Cell Count and Measurements of Iba-1 Positive Area and Fluorescent Intensity
For cell counts, sections (10 sections per each animal) were captured and areas of interest (1 × 10 4 µm 2 ) were selected from the FPC using an AxioImage M2 microscope [8,25]. Thereafter, cell counts and ionizing calcium-binding adaptor molecule 1 (Iba-1) positive area were performed using AxioVision Rel. 4.8 Software. For measurement of fluorescent intensity, 30 areas/rat (300 µm 2 /area) were randomly selected within the FPC (15 sections from each animal, n = 7 in each group), and mean fluorescence intensities (a 256 grayscale) were measured using AxioVision Rel. 4.8 software (Carl Zeiss Korea, Seoul, South Korea). Fluorescent intensity was normalized by setting the mean background obtained from five image inputs.

Western Blot
For Western blot, animals were decapitated under urethane anesthesia (1.5 g/kg, i.p.). The FPC was rapidly dissected out and homogenized in lysis buffer. After the measurement of the protein concentration using a Micro BCA Protein Assay Kit (Pierce Chemical, Dallas, TX, USA), standard Western blot was performed (n = 7 in each group) using each primary antibody ( Table 1). The band was detected and quantified using ImageQuant LAS4000 system (GE Healthcare Korea, Seoul, South Korea). The values of each sample were normalized with the amount of β-actin. The ratio of phospho-protein to total protein was described as the protein phosphorylation level.

Data Analysis
Comparisons between groups were performed using Student t-test and one-way ANOVA followed by Bonferroni's post hoc comparisons. A p-value of less than 0.05 was considered to be significant. SPSS 18.0 software was used for all analyses.

CDDO-Me Influences Monocyte Infiltration and Microglial Morphogenesis Induced by SE
In control animals, Iba-1 microglia had small cell bodies with thin ramified processes in the FPC. Following SE, Iba-1 microglia were transformed to hypertrophic and/or elongated cell bodies ( Figure 1A). Thus, the Iba-1 positive area was increased to 3.1 ± 0.2-fold of control level (p < 0.05 vs. control, one-way ANOVA, n = 7, respectively; Figure 1B). CDDO-Me reduced the Iba-1 positive area to 1.9 ± 0.3-fold of control level in the FPC following SE (p < 0.05 vs. vehicle, one-way ANOVA, n = 7, respectively; Figure 1B).

CDDO-Me Abolishes Microglial MCP-1 Production Independent of Nrf2 Activation Following SE
As CDDO-Me reduces MCP-1 production in blood immune cells and monocytes via Nrf2 activation [41], we validated whether CDDO-Me inhibits microglial MCP-1 production through Nrf2-mediated pathway. Western blot data revealed that SE increased Nrf2 protein level to 1.7 ± 0.2-fold of control level in the FPC (p < 0.05 vs. control, one-way ANOVA, n = 7, respectively; Figure 3A,B). CDDO-Me more enhanced the SE-induced Nrf2 up-regulation to 2.1 ± 0.2-fold of control level (p < 0.05 vs. vehicle, one-way ANOVA, n = 7, respectively; Figure 3A,B). In control animals, Nrf2 expression was mainly observed in neurons and astrocytes, but not microglia, in the FPC ( Figure 3C,D). SE increased Nrf2 fluorescent intensity to 2.6 ± 0.2-fold of the control level without altering the fractions of Nrf2 positive cells in total microglia and astrocytes (p < 0.05 vs. control, Student t-test, n = 7, respectively; Figure 3C-F). CDDO-Me enhanced Nrf2 fluorescent intensity in the FPC more than vehicle following SE (p < 0.05 vs. vehicle, one-way ANOVA, n = 7, respectively; Figure 3C,D,F). However, CDDO-Me did not influence microglial Nrf2 expression ( Figure 3C). These findings indicate that CDDO-ME may attenuate SE-induced microglial activation and monocyte infiltration in Nrf2-independent manners. Cells 2020, 9, x 9 of 22

Discussion
In the present study, we found that CDDO-Me ameliorated SE-induced microglial activation and monocyte infiltration in the FPC, accompanied by inhibiting MCP-1 expression and phosphorylation of NFκB-S276 and p38 MAPK, independent Nrf2 activity. Thus, these findings suggest that CDDO-Me may attenuate SE-induced monocyte infiltration and microglial activation by inhibiting NFκB-and p38 MAPK-MCP-1 signaling pathways (Figure 9). MAPK positive microglia (E) and p38 MAPK phosphorylation (F) following SE. (G) Representative images for CD68 positive cells. (H) Quantification of the effect of SN50 on the number of CD68 amoeboid and ramified cells following SE. Open circles indicate each value. Horizontal bars indicate the mean value. Error bars indicate SEM (* ,# p < 0.05 vs. control and vehicle, respectively; n = 7, respectively).

Discussion
In the present study, we found that CDDO-Me ameliorated SE-induced microglial activation and monocyte infiltration in the FPC, accompanied by inhibiting MCP-1 expression and phosphorylation of NFκB-S276 and p38 MAPK, independent Nrf2 activity. Thus, these findings suggest that CDDO-Me may attenuate SE-induced monocyte infiltration and microglial activation by inhibiting NFκB-and p38 MAPK-MCP-1 signaling pathways (Figure 9). Figure 9. Scheme of the effect of CDDO-Me on monocyte infiltration in FPC following SE. Following SE, the increased NFκB S276 phosphorylation in microglia initiate microglial transformation and up-regulation of TNF-α and MCP-1 expression, which are abolished by CDDO-Me and SN50. In activated microglia, p38 MAPK activation (phosphorylation) also triggers microglial MCP-1 production, which leads to monocyte infiltration. Both CDDO-Me and SN50 inhibits p38 MAPKmediated MCP-1 expression, which abrogates monocyte infiltration.
Microglia are the innate immune effector cells in the central nervous system. Following harmful stresses, microglia change their shape to amoeboid and acquire phagocytic capacity [2]. In addition, activated microglia secrete pro-inflammatory mediators such as TNF-α and MCP-1, which result in monocyte infiltration into the damaged tissue with/without the altered BBB integrity [49]. Together with resident microglia, CD68-positive infiltrating monocytes have the phagocytic ability and aggravate brain lesions by generating ROS, proteolytic enzymes, and pro-inflammatory cytokines [8,11,13,25,50]. Thus, the modulations of microglial activation and monocyte infiltration may ameliorate secondary damage induced by neuroinflammatory responses. In the present study, CDDO-Me inhibited microglia-mediated inflammatory responses to SE insults. Indeed, CDDO-Me Figure 9. Scheme of the effect of CDDO-Me on monocyte infiltration in FPC following SE. Following SE, the increased NFκB S276 phosphorylation in microglia initiate microglial transformation and up-regulation of TNF-α and MCP-1 expression, which are abolished by CDDO-Me and SN50. In activated microglia, p38 MAPK activation (phosphorylation) also triggers microglial MCP-1 production, which leads to monocyte infiltration. Both CDDO-Me and SN50 inhibits p38 MAPK-mediated MCP-1 expression, which abrogates monocyte infiltration.
Microglia are the innate immune effector cells in the central nervous system. Following harmful stresses, microglia change their shape to amoeboid and acquire phagocytic capacity [2]. In addition, activated microglia secrete pro-inflammatory mediators such as TNF-α and MCP-1, which result in monocyte infiltration into the damaged tissue with/without the altered BBB integrity [49]. Together with resident microglia, CD68-positive infiltrating monocytes have the phagocytic ability and aggravate brain lesions by generating ROS, proteolytic enzymes, and pro-inflammatory cytokines [8,11,13,25,50]. Thus, the modulations of microglial activation and monocyte infiltration may ameliorate secondary damage induced by neuroinflammatory responses. In the present study, CDDO-Me inhibited microglia-mediated inflammatory responses to SE insults. Indeed, CDDO-Me prevents high fat diet-induced impairments in recognition memory by reducing inflammation in the PFC [51] and improves neurological functions following focal cerebral ischemia [28,32]. Thus, our findings provide the preclinical evidence concerning the usefulness of CDDO-Me in the treatment/prevention of inflammatory reactions in various neurological diseases.
The present data reveal that CDDO-ME effectively alleviated SE-induced monocyte infiltration by inhibiting MCP-1 induction. MCP-1 is the first discovered chemokine and functions by recruiting monocytes and T cells to sites of inflammation. CCR2 (the main receptor of MCP-1) is primarily expressed on CD68-positive monocytes [8,25,52,53]. Following SE, resident microglia are the principal cellular sources of MCP-1 [8,10,25]. Recently, we have reported that roscovitine (a cyclin-dependent kinase 5 inhibitor) abrogates SE-induced monocyte infiltration by inhibiting microglial MCP-1 induction without affecting microglial transformation [25]. However, roscovitine did not affect p65 NFκB-S276 phosphorylation and microglial transformation following SE [25]. In the present study, CDDO-Me mitigated monocyte infiltration accompanied by microglial MCP-1/TNF-α inductions. Unlike roscovitine, CDDO-Me inhibited microglial transformation into the elongated/enlarged soma with less ramified processes covering thorny spine following SE. Furthermore, CDDO-Me attenuated p65-Ser276 NFκB phosphorylation induced by SE. As the NFκB inhibition by SN50 abolished microglial transformation in the present study, p65-Ser276 NFκB phosphorylation may likely play an important role in microglial transformation during their activations.
As the modulation of the Nrf2 pathway attenuates microglial activation in vitro [62,63], we explored whether CDDO-Me inhibits SE-induced microglial activation by increasing Nrf2 activity. In the present study, however, CDDO-Me did not affect Nrf2 expression in microglia following SE but increased it in neurons and astrocytes. These findings are consistent with previous studies demonstrating the up-regulation of astroglial Nrf2 expression induced by CDDO-Me [28,31,32]. However, the cell-type-specific expression of Nrf2 is still controversial in vivo: Nrf2 expresses in neurons but not glial cells [64]; in neurons, astrocytes, and microglia [65]; in neurons and astrocytes [28,32]; or in astrocytes [66]. These discrepancies would be the consequence of lower Nrf2 expression in various cells as Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and facilitates Nrf2 degradation via the ubiquitin-proteasome system under physiological condition [67]. CDDO-Me dissociates Keap1 from Nrf2 by interacting with the reactive cysteine 151 residue of Keap1 through a Michael addition [68], which abrogates Keap1-mediated Nrf2 ubiquitination and results in Nrf2 activation [69]. In addition, CDDO-Me itself exerts Nrf2 transcription [70,71]. Regardless of cell-type-specific expression of Nrf2, therefore, our findings suggest that CDDO-Me may attenuate microglia-mediated neuroinflammatory responses independent of Nrf2 activity following SE. On the other hand, CDDO-Me reduced p38 MAPK phosphorylation in both microglia and neurons following SE, although SN50 selectively abolished it in microglia, but not neurons. With respect to the Nrf2-mediated p38 MAPK regulation [72], our findings indicate that CDDO-Me may inhibit p38 MAPK phosphorylation in neurons via Nrf2 activation, independent of NFκB activity. To elucidate the underlying mechanisms of these phenomena, further studies are needed.

Conclusions
In the present study, we validated, for the first time, the anti-inflammatory effects of CDDO-Me against SE-induced microglial activation and monocyte infiltration (Figure 9). CDDO-Me ameliorated NFκB S276 and p38 MAPK phosphorylation in microglia, which inhibited microglial transformation and TNF-α production. Furthermore, CDDO-Me abolished microglial MCP-1 expression, which mitigated monocyte infiltration. Therefore, these findings propose an underlying pharmacological mechanism of CDDO-Me and its availability for neuroinflammation.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4409/9/5/1123/s1, Figure S1: The whole gel images of Western blot in Figures 2A, 3A and 4A. Figure S2, The whole gel images of Western blot in Figures 5A and 6A. Figure S3, The whole gel images of Western blot in Figure 7A. Table S1: Average of weight and consumptions of food and water in each group. Funding: This study was supported by a grant of Hallym University (No. HRF-202001-007). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.