Chemokine CCL2-CCR2 Signaling Induces Neuronal Cell Death Via STAT3 Activation and IL-1Β Production After Status Epilepticus

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Research Articles: Neurobiology of Disease

Chemokine CCL2-CCR2 signaling induces neuronal cell death via STAT3 activation and IL-1β production after status epilepticus

Dai-Shi Tian1,2, Jiyun Peng2,3, Madhuvika Murugan2,3, Lijie Feng2,3, Jun-Li Liu2,4, Ukpong B Eyo2,3, Li-Jun Zhou2, Rochelle Mogilevsky2, Wei Wang1 and Long-Jun Wu2,3

1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China 2Department of Cell Biology and Neuroscience, School of Arts and Sciences, Rutgers University, Piscataway, NJ 08854, USA 3Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA 4Cancer center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China

DOI: 10.1523/JNEUROSCI.0315-17.2017

Received: 2 February 2017

Revised: 20 June 2017

Accepted: 12 July 2017

Published: 17 July 2017

Author contributions: D.-S.T., J.P., W.W., and L.-J.W. designed research; D.-S.T., J.P., M.M., L.-J.F., J.-L.L., U.B.E., L.-J.Z., and R.M. performed research; D.-S.T., J.P., M.M., L.-J.F., and J.-L.L. analyzed data; D.-S.T., J.P., M.M., U.B.E., W.W., and L.-J.W. wrote the paper.

Conflict of Interest: The authors declare no competing financial interests.

We thank Dr. Richard Ransohoff (Biogen) for critical reading of the manuscript. This work was supported by National Institutes of Health (R01NS088627; R21DE025689), New Jersey Commission on Spinal Cord Research (CSCR15ERG015), National Natural Science Foundation of China (81101766, 81171157, 61327902) and National Basic Research Development Program (973 Program) of China (2011CB504403).

Correspondence should be addressed to Correspondance: Dr. Wei Wang, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430030, China TEL: 86-27-83663657; FAX: 86-27-83662600 E-MAIL: [email protected]; Dr. Long-Jun Wu, Department of Cell Biology and Neuroscience, Rutgers University, Department of Neurology, Mayo Clinic, 604 Allison Road, Piscataway, NJ 08854, TEL: (848) 445-2182; FAX: (732) 445-5870, E-MAIL: [email protected]

Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0315-17.2017

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1 Chemokine CCL2-CCR2 signaling induces neuronal cell death via STAT3

2 activation and IL-1β production after status epilepticus

3 Dai-Shi Tian1,2, #, Jiyun Peng2,3, #, Madhuvika Murugan2,3, Lijie Feng2,3, Jun-Li

4 Liu2,4, Ukpong B Eyo2, 3, Li-Jun Zhou2, Rochelle Mogilevsky2, Wei Wang1*,

5 Long-Jun Wu2, 3*

7 1Department of Neurology, Tongji Hospital, Tongji Medical College,

8 Huazhong University of Science and Technology, Wuhan 430030, China

9 2Department of Cell Biology and Neuroscience, School of Arts and Sciences,

10 Rutgers University, Piscataway, NJ 08854, USA

11 3Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA

12 4Cancer center, Union Hospital, Tongji Medical College, Huazhong University

13 of Science and Technology, Wuhan 430022, China

15 Running Title: CCL2-CCR2 signaling in epilepsy

16 Text Pages: 48; Figures: 8

17 Abstract : 199 ; Introduction : 640 ; Discussion : 1499

18 Financial interests: none

20 ACKNOWLEDGEMENTS

21 We thank Dr. Richard Ransohoff (Biogen) for critical reading of the manuscript.

22 This work was supported by National Institutes of Health (R01NS088627;

23 R21DE025689), New Jersey Commission on Spinal Cord Research

24 (CSCR15ERG015), National Natural Science Foundation of China (81101766,

25 81171157, 61327902) and National Basic Research Development Program (973

26 Program) of China (2011CB504403).

29 *Correspondance:

30 Dr. Wei Wang

31 Tongji Medical College, Huazhong University of Science and Technology

32 Wuhan 430030, China

33 TEL: 86-27-83663657; FAX: 86-27-83662600

34 E-MAIL: [email protected]

35 36 Dr. Long-Jun Wu

37 Department of Cell Biology and Neuroscience

38 Rutgers University

39 Department of Neurology

40 Mayo Clinic

41 604 Allison Road, Piscataway, NJ 08854

42 TEL: (848) 445-2182; FAX: (732) 445-5870

43 E-MAIL: [email protected]

44 45 46 47 48 49 50 51 52 53 54 55 56 57 2

58 ABSTRACT 59 60 Elevated levels of chemokine C-C motif ligand 2 (CCL2) and its receptor

61 CCR2 have been reported in patients with temporal lobe epilepsy and in

62 experimental seizures. However, the functional significance and molecular

63 mechanism underlying CCL2-CCR2 signaling in epileptic brain remains

64 largely unknown. In this study, we found that the upregulated CCL2 was

65 mainly expressed in hippocampal neurons and activated microglia from mice

66 1d after kainic acid (KA)-induced seizures. Taking advantage of

67 CX3CR1GFP/+:CCR2RFP/+ double transgenic mice, we demonstrated that

68 CCL2-CCR2 signaling participated in resident microglial activation and

69 blood-derived monocyte infiltration. Moreover, seizure-induced degeneration

70 of neurons in the hippocampal CA3 region was attenuated in mice lacking

71 CCL2 or CCR2. We further showed that CCR2 activation induced STAT3

72 phosphorylation and IL-1β production, which are critical for promoting

73 neuronal cell death after status epilepticus. Consistently, pharmacological

74 inhibition of STAT3 by WP1066 reduced seizure-induced IL-1β production

75 and subsequent neuronal death. Two weeks after KA-induced seizures, CCR2

76 deficiency not only reduced neuronal loss, but also attenuated

77 seizure-induced behavioral impairments, including anxiety, memory decline,

78 and recurrent seizure severity. Taken together, we demonstrated that

79 CCL2-CCR2 signaling contributes to neurodegeneration via STAT3 activation

80 and IL-1β production after status epilepticus, providing potential therapeutic

81 targets for the treatment of epilepsy. 82 83 Keywords: Neuroinflammation, CCL2, MCP-1, CCR2, microglia, monocytes,

84 IL-1β, STAT3, epilepsy

86 SIGNIFICANCE STATEMENT

88 Epilepsy is a global concern and epileptic seizures occur in many neurological

89 conditions. Neuroinflammation associated with microglial activation and

90 monocyte infiltration are characteristic of epileptic brains. However,

91 molecular mechanisms underlying neuroinflammation in neuronal death

92 following epilepsy remains to be elucidated. Here we demonstrate that

93 CCL2-CCR2 signaling is required for monocyte infiltration, which in turn

94 contributes to KA-induced neuronal cell death. The downstream of CCR2

95 activation involves STAT3 phosphorylation and IL-1β production. Two weeks

96 after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss,

97 but also attenuated seizure-induced behavioral impairments, including

98 anxiety, memory decline, and recurrent seizure severity. The current study

99 provides a novel insight on the function and mechanisms of CCL2-CCR2

100 signaling in KA-induced neurodegeneration and behavioral deficits.

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113 INTRODUCTION

114 Epilepsy is a global concern with an estimated 50-65 million living with

115 the condition worldwide (Thurman et al., 2011; Vezzani et al., 2011), causing

116 an average mortality of 2-10 years earlier than the general population

117 (Gaitatzis et al., 2004). The disorder is characterized by increased occurrence

118 of seizures resulting from excessive or uncontrolled increase in neuronal

119 activity (Fisher et al., 2005). Epileptic seizures are known to occur in many

120 neurological conditions including as a consequence of stroke and traumatic

121 brain injury (Vezzani et al., 1999; Temkin, 2009). Current antiepileptic

122 strategies primarily target neuronal mechanisms. These medical therapies

123 have proven insufficient in at least a third of patients with recurrent seizures,

124 raising the need for alternative and/or complementary strategies (Devinsky et

125 al., 2013). Aside from neuronal hyperactivity, neuroinflammation associated

126 with activation of resident microglia and infiltration of peripheral monocytes

127 are characteristic of epileptic brains compared to non-epileptic ones (Shapiro

128 et al., 2008). Indeed, autopsy analysis of human patients with intractable

129 seizures revealed an 11-fold increase in microglia/macrophage reactivity in

130 the hippocampus from epileptic brains (Beach et al., 1995).

131 Although there is a strong correlation between the severity of epilepsy

132 and degree of microglial/macrophage accumulation (Boer et al., 2006), the

133 morphological and proliferative signs of their activation are generally delayed

134 (Avignone et al., 2008; Drexel et al., 2012; Eyo et al., 2017). Therefore, it is still

135 unclear whether microglia/macrophage activation is a secondary reaction to

136 neuronal injury following the primary seizure or is actively involved in

137 epileptogenesis. Interestingly, both beneficial and detrimental roles have been

138 attributed to microglia/macrophages in epilepsy. For example, recent studies

139 suggest a neuroprotective role of microglia in epilepsy by a microglial

140 ablation strategy as well as using P2Y12 knockout (P2Y12-/-) mice (Mirrione et

141 al., 2010; Eyo and Wu, 2013; Eyo et al., 2014; Eyo et al., 2016). On the other

142 hand, reactive microglia/macrophages are known to release a variety of

143 pro-inflammatory mediators that aggravate seizure activities and

144 excitotoxicity under epileptic conditions (Ding et al., 2014; Miskin and

145 Hasbani, 2014). Indeed, broad microglial inhibitors such as minocycline and

146 macrophage inhibitory factor (MIF) reduce neurodegeneration following

147 epilepsy (Rogove and Tsirka, 1998; Wang et al., 2012). However, the molecular

148 mechanism underlying microglia/macrophage-induced neuronal cell death

149 following epilepsy remains to be elucidated.

150 Chemokines are pro-inflammatory mediators that are critical in recruiting

151 leukocytes into the brain following seizures. Recent studies have shown the

152 upregulation of several chemokines including chemokine C-C motif ligand 2

153 (CCL2) in patients with temporal lobe epilepsy and in rodents after

154 experimental seizures (Manley et al., 2007; Wu et al., 2008). CCL2 is a potent

155 chemoattractant protein for monocytes, hence alternatively referred to as

156 monocyte chemoattractant protein-1 (MCP-1). Elevated levels of CCL2

157 expression in an inflamed brain were associated with activation and

158 recruitment of macrophages/microglia to the injury sites (Selenica et al., 2013).

159 CCL2 attracts and acts through chemokine C-C motif receptor 2 (CCR2)

160 expressing cells, thereby regulating migration and infiltration of monocytes,

161 T-lymphocytes and natural killer cells to regions of inflammation (Rollins,

162 1991; Semple et al., 2010). In addition to CCL2 upregulation, the expression of

163 CCR2 was also significantly increased in the hippocampus in a rat model of

164 pilocarpine-induced status epilepticus (Foresti et al., 2009). The results

165 obtained in experimental animal models and in human patients point to a key

166 role for CCL2-CCR2 signaling in epilepsy.

167 Despite the well-characterized regulation of CCL2 following epilepsy, the

168 molecular mechanisms underlying the CCL2-CCR2 signaling and its

169 functional significance in the pathogenesis of epilepsy still remains largely

170 unknown. Here we show direct evidence using the double transgenic

171 CX3CR1GFP/+:CCR2RFP/+ mice that CCL2-CCR2 signaling is required for

172 macrophage infiltration and neurodegeneration in the hippocampus in a

173 mouse model of kainic acid (KA)-induced status epilepticus. The underlying

174 neurotoxic mechanism of CCL2-CCR2 signaling in activated

175 microglia/macrophages involves the STAT3 pathway and IL-1β production.

176 The current study provides a novel therapeutic strategy targeting CCL2-CCR2

177 signaling against neuroinflammation and hippocampal neurodegeneration

178 under epileptic conditions.

179

180

181 MATERIALS AND METHODS

182 Animals and seizure models

183 Mice were used in accordance with institutional guidelines as approved by

184 the animal care and use committee at Rutgers University. Adult C57BL/6

185 wild-type (WT) mice, CCR2RFP/RFP (CCR2-/-) mice, CX3CR1GFP/GFP mice,

186 CX3CR1GFP/+ mice, CCL2-/- mice were all purchased from Jackson Laboratory.

187 Double transgenic mice CCR2RFP/+:CX3CR1GFP/+ mice were obtained by crossing

188 CCR2RFP/RFP mice with CX3CR1GFP/GFP mice. For all seizure experiments, 6-8

189 week old mice of both genders were used. All animal procedures performed

190 in this study were in accordance with institutional guidelines, as approved by

191 the animal care and use committee at Rutgers University. The KA-induced

192 seizure models in mice were described in our previous studies (Eyo et al.,

193 2014; Eyo et al., 2015). Briefly, animals were anesthetized with isoflurane,

194 stereotaxically implanted with a 26-gauge, stainless steel, guide cannula

195 aimed towards the lateral cerebral ventricle [from the bregma: −0.2 mm

196 anteroposterior (AP), +1.0 mm mediolateral (ML), −2.0 mm dorsoventral

197 (DV)], and then were intracerebroventricularly (i.c.v) injected with KA at 0.15

198 μg in 5 μl of aCSF. In order to study recurrent seizure activity, a second dose

199 of KA (0.15 μg/5 μl) was administered 14 d after the first dose and the seizure

200 scores were monitored. Sham controls were injected with 5 μl of aCSF.

201

202 Seizure Quantification

203 Seizure behavior was monitored under a modified Racine scale as follows

204 (Racine, 1972; Eyo et al., 2014): (1) freezing behavior; (2) rigid posture with

205 raised tail; (3) continuous head bobbing and forepaws shaking; (4) rearing,

206 falling, and jumping; (5) continuous level 4; and (6) loss of posture and

207 generalized convulsion activity. Mice progressed at least to stage 3 and were

208 killed at 1d or 3d after seizure. For EEG recordings, stainless steel bone screw

209 recording electrodes (Pinnacle Technologies) were implanted together with

210 the ICV guide cannula. Four screws were placed on the dura through burr

211 holes in the skull, electrode leads were attached to a microplug. The icv guide

212 tube was modified to be implanted at 30º from the vertical angle to avoid the

213 position conflict with the microplug. Dental cement was applied to fix both

214 electrodes and guide cannula in place. Animals were connected to the

215 recording system for baseline recording and habituation to the environment

216 after 1d recovery. After 2d, KA was injected through the guide cannula to

217 induce seizures. Seizure spell was defined as high frequency, high voltage

218 synchronized polyspike or paroxysmal sharp waves with amplitude > 2 fold

219 background and lasted more than 6s. The EEG recordings were made

220 continuously from -1d to 14d after i.c.v KA. Seizure spells were not detected

221 in any mice after 2 hr of KA injection.

222

223 Behavioral Assays

224 Open field test: The open field test was used to detect seizure-related

225 anxiety-like behavior. Mice received a 5 min session in an empty open field

226 box equipped with a video camera (50 x 50 cm). The overall distance travelled

227 by the mice, the distance from the center and the time spent in the center of the

228 field were used to evaluate the anxiety levels. The center region is designated

229 as 25 x 25 cm in the open field.

230

231 Novel object recognition: To determine whether seizure-induced neuronal

232 damage impairs hippocampal-dependent cognitive function, novel object

233 recognition test was used. This test measures recognition memory and is

234 performed as previously described with minor modifications (Matsuda et al.,

235 2015). Briefly, each mouse was subject to a 10-min acquisition trial, during

236 which they were placed in the open field in presence of two identical objects A

237 (acquisition task). On completion of 10 min of exploration, the mouse was

238 returned to its cage for 1 h. The mouse was then placed back in the box and

239 introduced to the familiar object A and a novel object B for another 5 min (test

240 task). The total time spent exploring each of the two objects was recorded. A

241 preference index for novel object used to measure recognition memory and is

242 defined as the percentage of time exploring the novel object (B) divided by the

243 total time spent exploring both objects [B/(A+B)] × 100.

244

245 Immunohistochemistry

246 At 1d and 3d after seizures, animals were deeply anesthetized with

247 isoflurane and perfused with PBS, followed by 4% paraformaldehyde with 0.5%

248 picric acid. After the perfusion, the brain were removed and postfixed in the

249 same fixative overnight. Brain sections (15 μm) were cut in a cryostat and

250 processed for immunofluorescence. All histological data were obtained from

251 sections taken from coronal planes -1.0 - 2.0 mm AP, and the center of the

252 field of view was focused on the hippocampal CA3 region (+ 2.3 - 2.8 mm ML;

253 + 2.5 - 3.0 mm DV). The sections were first blocked with 5% goat or donkey

254 serum for 1 h at room temperature, and then incubated overnight at 4°C with

255 the following primary antibodies: Iba-1 antibody (rabbit, 1:1000; Wako),

256 NeuN antibody (rabbit, 1:500; Abcam), GFAP (rabbit, 1:500; Chemicon),

257 MCP-1 antibody (mouse, 1:200; Novus Biologicals), RFP (mouse, 1;500;

258 Abcam), Iba-1 antibody (goat, 1:200; Abcam), IL-1β (rabbit, 1:500; Abcam),

259 pSTAT3 or total-STAT3 (1:200, rabbit; Cell Signaling Technology). The

260 sections were then incubated for 1 h at room temperature with Cy3- or

261 FITC-conjugated secondary antibodies (1:500; Jackson ImmunoResearch). For

262 double immunofluorescence, sections were incubated with a mixture of

263 polyclonal and monoclonal primary antibodies, followed by a mixture of

264 FITC- and Cy3-congugated secondary antibodies. The stained sections were

265 examined and captured with a fluorescence microscope (EVOS® FL Cell

266 Imaging System, Life Technologies). For co-localization analysis of CCL2,

267 confocal images of double immunostained hippocampus sections were

268 analyzed using a macro program designed by Daniel Shriwarski at the

269 University of Pittsburg in Image J software.

270

271 Fluoro-Jade B staining

272 The anionic fluorochrome Fluoro-Jade B (FJB) was used to stain

273 degenerating neurons in brain sections. Briefly, tissue sections were air-dried,

274 and then immersed in 0.006% potassium permanganate solution for 15

275 minutes. Slides were washed briefly in water and then placed in 0.001% FJB

276 solution (Chemicon) in 0.1% acetic acid for 30 minutes. The sections were

277 dehydrated and cleared in xylene, air dried, and mounted with a coverslip. To

278 quantify Fluoro-Jade B-positive cells, three sections per mouse were selected.

279 The numbers of FJB-positive cells from 9 locations per mouse (3 fields per

280 section × 3 sections per mouse) were averaged and results are reported as the

281 number of positive neurons per field of view and expressed as mean ± SEM.

282

283 Nissl staining

284 For Nissl staining, hippocampal tissue sections were mounted and

285 stained with 0.5% cresyl violet. The number of Nissl stained neurons in a

286 defined area CA1/ CA3 (1.0 mm × 0.25 mm) was counted in 3 fields of view per

287 region per section in at least three sections per mouse brain. All assessments of

288 histological sections were blindly performed.

289

290 Western blot

291 Hippocampus samples were extracted and homogenized in lysis buffer

292 containing protease and phosphatase inhibitors, and the homogenates were

293 centrifuged at 7000× g for 15 minutes at 4°C as described previously (Wu et al.,

294 2012). The supernatants were collected and the protein concentration was

295 determined by Bradford method, and 30 μg of proteins were loaded for each

296 lane and separated by SDS-PAGE. After the transfer, the blots were incubated

297 overnight at 4°C with one of the primary antibodies listed below: β-actin

298 (1:5000, rabbit; Santa Cruz Biotechnology), RFP (1;1000, rabbit; Sicgen), IL-1β

299 (1;1000, rabbit; Abcam), Iba-1 (1:1000, rabbit; Wako), pSTAT3 (1:1000, rabbit;

300 Cell Signaling Technology, detects tyrosine phosphorylation at Tyr705 of

301 STAT3) or STAT3 (1:1000, rabbit; Cell Signaling Technology). These blots

302 were then washed, incubated with HRP-conjugated secondary antibody and

303 developed in an enhanced chemiluminescent solution. Specific bands were

304 evaluated by apparent molecular size. Finally, images were captured using a

305 Fuji-Film LAS-300 (Fuji, Sheffield, UK) and the intensity of the selected bands

306 was analyzed using NIH Image J software. For experiments to test blockage of

307 CCL2 on monocyte infiltration, CCL2 neutralizing antibody or its isotype 308 (i.c.v., 100 ng in 5 μl aCSF, Millipore) was administered at 15min before and

309 2hr and 8hr after KA injection and the mice were sacrificed at 1d after KA.

310

311 Cytokine array

312 The mouse cytokine array kit pre-coated with 40 cytokine/chemokine

313 antibodies was purchased from R & D Systems. Protein samples were

314 prepared in the same way as for Western blot analysis. Each reaction was

315 performed according to the manufacturer’s instructions using 150 μg of

316 proteins collected from mouse hippocampus.

317

318 Statistical analysis

319 Data were expressed as mean ± SEM. All statistical tests were performed

320 using GraphPad Prism 5.0 (GraphPad Software). Quantitative data were

321 compared between different groups by two-sample (unpaired Student’s)

322 two-tailed t test assuming equal variance. Data for multiple comparisons were

323 analyzed by two-way Analysis of variance (ANOVA) followed by post

324 hoc Student–Newman–Keuls (SNK) tests. Instances for which a statistically

325 significant difference was detected were indicated by asterisks: *, p < 0.05; **,

326 p < 0.01; ***, p < 0.001.

327

328

329 RESULTS

330 Chemokine CCL2 was upregulated and mainly expressed in microglia and

331 neurons after status epilepticus

332 Inflammatory cytokines are known to be critical for the pathogenesis of

333 epilepsy (Dedeurwaerdere et al., 2012). To determine the change of cytokine

334 expression after status epilepticus, we induced seizure activities in mice by

335 intracerebroventrical (i.c.v.) injection of KA (0.15 μg in 5 Pl aCSF, artificial

336 cerebrospinal fluid) and then performed a nonbiased protein screening using

337 a cytokine/chemokine array that contains 40 different cytokine and

338 chemokine antibodies (Fig. 1A). Hippocampal tissue lysates from control or at

339 1d after KA-induced seizure group were examined using the

340 cytokine/chemokine array. We found a marked increase in the levels of several

341 chemokines including CCL2, CCL3, CCL5, CCL12 and CXCL10, as well as

342 cytokines including IL-1α and IL-1β. These results clearly indicate that

343 KA-induced seizures significantly upregulated cytokine/chemokine

344 expression 1d after treatment.

345 To further study the function of seizure-associated cytokines and

346 chemokines, we chose CCL2 for further characterization for the following

347 reasons: (1) CCL2 expression is enriched in microglia (Leung et al., 1997;

348 Zhang et al., 2014) (2) CCL2 is one of the most elevated inflammatory

349 mediators in patients with epilepsy, but its contribution to seizure generation

350 remains largely unknown (Cerri et al., 2016); (3) The etiology of seizures is

351 mostly idiopathic, however, infection is one of the few known risk factors of

352 acquired epilepsy which is accompanied by increased macrophage infiltration

353 in the brain (Marchi et al., 2014); (4) The blood brain barrier is compromised

354 following epilepsy (Marchi et al., 2012); (5) The availability of both CCL2-/-

355 mice and CCR2-/- mice enhances our ability to fully investigate CCL2 signaling

356 pathway. Using Western blot, we confirmed that CCL2 was significantly

357 upregulated at 1d after i.c.v. KA and maintained at high levels until 3d after

358 i.c.v. KA treatment (Fig. 1B, P<0.001).

359 Next, to define the cellular distribution of CCL2, we performed

360 immunofluorescence double staining of CCL2 with neuronal/glial cell

361 markers in hippocampal CA3 area where neuronal cell death is known to be

362 prominent after i.c.v. KA treatment (Lee et al., 2003). Microglial marker Iba1,

363 astrocyte marker GFAP, and neuronal marker NeuN were used. In the control

364 group, we found very weak expression of CCL2 (data not shown). However,

365 CCL2 was dramatically upregulated and primarily expressed in microglia

366 and neurons but not in astrocytes in hippocampal CA3 area 1d after i.c.v. KA

367 (Fig. 1C). The specificity of CCL2 immunostaining was confirmed by lack of

368 positive signals in KA-injected CCL2-/- mice (data not shown). Taken together,

369 these results indicate that CCL2 is one of the key components that are

370 upregulated after KA-induced seizures, and increased CCL2 is mainly

371 expressed by hippocampal microglia and neurons.

372

373 CCL2 chemokine induced macrophage infiltration in the hippocampus

374 after seizures

375 Next, we determined the role of CCL2 upregulation after KA-induced

376 status epilepticus. Given that CCL2 acts through its receptor CCR2 in

377 mediating macrophage infiltration (Cazareth et al., 2014), we hypothesized

378 that the upregulated CCL2 after seizures induced the infiltration of blood

379 derived CCR2-positive monocytes. To test this hypothesis, we took advantage

380 of double transgenic CCR2RFP/+:CX3CR1GFP/+mice, in which resident

381 CX3CR1-positive microglia are labeled with GFP while CCR2-positive

382 monocytes are labeled with RFP, to investigate the timeline of monocyte

383 infiltration and microglia activation after KA-induced seizures. In the aCSF

384 control group, we observed the occurrence of ramified GFP-positive resting

385 microglia but not RFP-positive macrophages in the hippocampus. However,

386 in mice after KA-induced seizures, we identified that the RFP-positive cells,

387 presumably infiltrated CCR2-positive macrophages were increased at 1-3d

388 and started declining by 7d, whereas microglial activation increased

389 progressively from 1-7d after i.c.v. KA (Fig. 2A-B). Using Western blot, we

390 confirmed the upregulation of both Iba1 expression and RFP levels at 1d and

391 3d after KA injection (Fig. 2D-E, P<0.05). These results directly demonstrated

392 the infiltration of CCR2-positive macrophages after KA-induced seizures.

393 We further tested the idea that CCL2 is the chemokine that induced the

394 infiltration of CCR2-positive macrophages. To this end, we administered

395 CCL2 neutralizing antibody (i.c.v., 100 ng in 5 μl aCSF, three successive doses

396 at 15min before and 2hr and 8hr after KA injection) in

397 CCR2RFP/+:CX3CR1GFP/+mice after status epilepticus. Indeed, we found that the

398 infiltration of CCR2RFP/+ cells after seizure was largely reduced (Fig. 2C).

399 Moreover, resident microglial activation after seizure was also diminished

400 after the application of the CCL2 neutralizing antibody as evidenced

401 morphologically (Fig. 2C). Consistently, we found that the treatment with

402 CCL2 neutralizing antibody but not isotype control antibody suppressed

403 KA-induced upregulation of both Iba1 and RFP levels in the hippocampus

404 (Fig. 2D-E, P<0.05). To further confirm that CCL2-CCR2 signaling was

405 required for CCR2-positive macrophage infiltration, CCR2-/- mice were used

406 (Fig. 2C). As expected, we found a reduced occurrence of Iba1-positive

407 microglia/macrophages in CCR2-/- mice at 1d after KA-induced seizures.

408 Taken together, our data indicated that upregulated levels of CCL2 after

409 seizures induced the recruitment of CCR2-positive macrophages and

410 subsequently caused, at least in part, microglial activation in the hippocampal

411 CA3 area.

412

413 CCR2 receptor deficiency did not alter the severity of acute seizures but

414 attenuated neuronal degeneration after status epilepticus

415 CCL2-CCR2 signaling has been shown to increase neuronal activity and

416 neuronal cell death in a variety of neurological diseases, such as acute

417 ischemic stroke and chronic Alzheimer disease (Chen et al., 2003; Severini et

418 al., 2014) . However, little is known about the functional significance of

419 CCL2-CCR2 signaling in status epilepticus. Here, we report the upregulation

420 of CCL2 and infiltration of CCR2 macrophage at 1d after KA-induced seizures.

421 Therefore, we surmised that CCL2-CCR2 signaling may participate in the

422 pathology of seizure-associated events, such as neuronal cell death. To

423 address this question, we examined neurodegeneration and seizure behavior

424 in CCR2-/- and CCL2-/- mice after i.c.v. injection of KA.

425 To begin, we used Fluoro-Jade B (FJB), a fluorescent green dye that labels

426 degenerating neurons, to detect neuronal degeneration in the hippocampus

427 after seizures. In wild-type (WT) mice, we noted characteristic FJB staining

428 localized mostly in the ipsilateral CA3 region of hippocampus at 1 d after i.c.v

429 KA (Fig. 3A). However, CCR2-/- and CCL2-/- mice both displayed reduced FJB

430 staining in the hippocampal CA3 area 1d after KA injection (Fig. 3B-C). We

431 counted FJB positive neurons and found dramatically fewer degenerating

432 neurons in the epileptic brains of CCR2-/- and CCL2-/- mice compared with that

433 of WT mice (Fig. 3D, P<0.01).

434 Next, we compared KA-induced acute seizure behaviors in age-matched

435 WT, CCR2-/- mice and CCL2-/- mice. Interestingly, during the initial 2hr of

436 seizure observations, we found that neither CCR2-/- nor CCL2-/- mice displayed

437 a significant difference in seizure Racine scores compared to WT mice (Fig. 3E,

438 P>0.05). In line with this, electroencephalograpic (EEG) measurements

439 showed no significant difference in EEG scores between the WT, CCR2-/- and

440 CCL2-/- mice during the acute phase (0-2 hours after i.c.v. KA) (Fig 3F-G).

441 Moreover, no recurrent seizures were observed during the chronic phase

442 (7-14d after i.c.v. KA) in either WT, CCR2-/-, or CCL2-/- mice in our model of

443 KA induced seizure (data not shown). Taken together, the results suggest that

444 CCL2-CCR2 signaling may not be directly relevant in acute seizure behaviors,

445 but the reduced KA-induced neuronal death in CCR2-/- and CCL2-/- mice

446 implicates a critical role of the signaling in neuronal degeneration after status

447 epilepticus.

448

449 CCR2 is required for production of IL-1β after seizures

450 To study the molecular mechanisms underlying CCL2-CCR2 signaling in

451 neuronal degeneration after epilepsy, we turned to cytokine IL-1β because (1)

452 it was upregulated after seizures in our cytokine/chemokine array (Fig. 1A),

453 and (2) IL-1β is known to increase neuronal activities and neuronal

454 degeneration in many disease conditions, such as epilepsy (Patel et al., 2006),

455 Parkinson's disease (Koprich et al., 2008), and Alzheimer’s disease (Kitazawa

456 et al., 2011). Using Western blot, we indeed observed an increase in IL-1β

457 levels at 1d after KA-induced status epilepticus (Fig. 4A), consistent with that

458 seen in the cytokine/chemokine array (Fig. 1A). To test the role of CCR2 in

459 IL-1β upregulation, we compared IL-1β levels in WT and CCR2-/- mice after

460 i.c.v. KA treatment. Interestingly, we found that KA-induced IL-1β

461 upregulation was largely abolished in CCR2-/- mice (Fig. 4A-B, P<0.05). These

462 results suggest that CCR2 is required for IL-1β upregulation after status

463 epilepticus.

464 Next, we wanted to determine the cellular distribution of IL-1β after

465 KA-induced seizures. To this end, we performed double immunostaining of

466 IL-1β with neuronal and glial markers. We found that in WT aCSF control

467 mice, IL-1β expression was low in the mouse hippocampus (Fig. 4C-D).

468 However, there was dramatic upregulation in IL-1β levels after KA-induced

469 seizures and the expression was strictly co-localized with Iba1 (Fig. 4C-D).

470 Consistently, IL-1β did not colocalize with GFAP or NeuN in the

471 hippocampus after seizure (data not shown). In addition, using

472 CCR2RFP/+:CX3CR1GFP/+mice, we found that IL-1β was expressed in both

473 CX3CR1-GFP microglia and CCR2-RFP macrophages (Fig. 4D). These results

474 suggest that both activated resident microglia and infiltrated macrophages are

475 the source for upregulated IL-1β after KA-induced seizures. This is in contrast

476 to another study that reported IL-1β release by reactive astrocytes in a rat

477 model of temporal lobe epilepsy (Akin et al., 2011). We believe this

478 discrepancy may be due to the difference in epilepsy model, animal species

479 and time course investigated, given that even minor strain differences can

480 affect epilepsy outcome (Schauwecker, 2012). To confirm CCR2 is required for

481 IL-1β production after status epilepticus, we examined the expression of IL-1β

482 in CCR2-/- mice. Indeed, we found compromised IL-1β expression in

483 association with reduced microglial activation and macrophage infiltration in

484 CCR2-/- mice (Fig. 4C-D). Our results collectively showed that KA seizures

485 induced production of IL-1β from microglia and macrophages is

486 CCR2-dependent.

487 Given the reduced neuronal degeneration and the decreased IL-1β

488 production in CCR2-/- mice, we hypothesized that IL-1β may promote

489 neuronal cell death after seizures. To test this hypothesis, we used IL-1

490 receptor antagonist (IL-1ra) to neutralize IL-1β and tested its effect on

491 KA-induced neuronal degeneration. IL-1ra was applied 15min before and 2hr

492 after the injection of KA (i.c.v., 0.5 μg in 5 μl aCSF, using the same

493 stereotaxically-placed cannula for KA) and then neuronal cell death was

494 examined at 1d after seizures using FJB staining. We found that IL-1ra

495 significantly decreased the number of degenerated neurons damaged by

496 seizures (Fig. 4E-F, P<0.05). Therefore, IL-1β upregulation and probably

497 release may promote neuronal cell death after status epilepticus and thus

498 inhibition of IL-1β was able to limit the hippocampal degeneration.

499

500 STAT3 signaling in CCR2-dependent IL-1β production after seizures

501 We have shown that CCR2 is required for IL-1β production from

502 activated microglia and infiltrated macrophages after KA-induced seizures.

503 To further examine the possible downstream signaling of CCR2 that leads to

504 IL-1β production, we tested signal transducer and activator of transcription 3

505 (STAT3) signaling. STAT3 is critical to promote the production of

506 inflammatory cytokines (Qin et al., 2012; Przanowski et al., 2014) and is

507 coupled to CCR2 activation in cancer cells (Izumi et al., 2013). First, we set out

508 to investigate whether STAT3 is activated after status epilepticus. Since STAT3

509 activation requires its phosphorylation (Wen et al., 1995), we first performed

510 immunostaining of phosphorylated STAT3 (pSTAT3) in the mouse

511 hippocampus from aCSF control and 1d after KA injection. Consistent with

512 the low basal STAT3 activity, we found pSTAT3 staining was low in aCSF

513 control mice (Fig. 5A). However, pSTAT3 was highly expressed in the

514 hippocampal CA3 after i.c.v. KA (Fig. 5A). In addition, pSTAT3 expression

515 mainly colocalized (90.85 ± 1.64 %) with the microglial/macrophage marker

516 Iba-1. To further examine pSTAT3 expression in resident microglia or

517 infiltrated macrophages, we performed pSTAT3 staining in

518 CCR2RFP/+:CX3CR1GFP/+mice. Our results showed that pSTAT3 was expressed in

519 both CX3CR1-GFP microglia and CCR2-RFP macrophages. These results

520 suggest STAT3 signaling is upregulated in both activated resident microglia

521 and infiltrated macrophages after KA-induced seizures.

522 Next, we determined whether CCR2 was involved in the activation of

523 STAT3 signaling pathway after seizures using CCR2-/- mice. Interestingly, we

524 found that CCR2-/- mice had decreased pSTAT3 levels compared with WT

525 mice after KA-induced seizures (Fig. 5A). Western blot analysis was further

526 used to assess the role of CCR2 in the regulating the expression of pSTAT3.

527 Consistently, we found that pSTAT3 level was up-regulated significantly and

528 reached more than 3 folds in KA injected mice as compared to control group.

529 However, the pSTAT3 upregulation after seizures was attenuated in CCR2-/-

530 mice (Fig. 5B-C, P<0.05). Taken together, these results indicate that activation

531 of microglia and macrophages after seizures was associated with the initiation

532 of STAT3 signaling pathway, which might be partially dependent on CCR2

533 activation.

534

535 Inhibition of STAT3 signaling decreased IL-1β production and neuronal

536 degeneration after seizures

537 To further determine the role of STAT3 signaling following seizures, a

538 potent STAT3 inhibitor WP1066 (100 mg/kg, i.p.) was used 30min before and

539 2h after i.c.v. KA treatment and the expression of pSTAT3 was tested at 1d

540 after i.c.v KA. Despite the short half-life of WP1066, a previous study showed

541 that transient levels of WP1066 in vivo in the brain and plasma was sufficient

542 to reduce chronic seizure severity in a pilocarpine model of epilepsy

543 (Grabenstatter et al., 2014). Consistent with their study, we confirmed that

544 WP1066 effectively inhibited the levels of pSTAT3 without reducing the

545 expression of total STAT3 (Fig. 6A-B) (Grabenstatter et al., 2014). Since we

546 have shown that both STAT3 activation and IL-1β production require CCR2

547 after status epilepticus, we tested whether STAT3 signaling acts upstream of

548 IL-1β. To test this idea, we examined the effect of STAT3 inhibition by WP1066

549 on the IL-1β production after seizures. Indeed, we found that in addition to

550 suppression of STAT3 phosphorylation, WP1066 significantly attenuated

551 IL-1β production and Iba1 levels (Fig. 6A-B, P<0.05). Therefore, these results

552 indicate that STAT3 signaling is the upstream for IL-1β production and

553 microglial activation after seizures.

554 Since STAT3 signaling is required for IL-1β production, we surmised that

555 the inhibition of STAT3 by WP1066 would rescue KA-induced neuronal

556 degeneration. To this end, we studied the effect of WP1066 on neuronal

557 degeneration after status epilepticus by FJB staining. WP1066 was applied

558 30min before and 2h after i.c.v. KA and neuronal cell death was assessed 1d

559 after KA treatment. We found that the number of FJB positive neurons in the

560 CA3 region was decreased significantly after WP1066 treatment compared to

561 those with vehicle treatment (Fig. 6C-D, P<0.05). However, the severity of the

562 acute seizure scores after WP1066 treatment was similar to that in the

563 vehicle-treated group (Fig. 6E, P>0.05). This is in contrast to a previous study

564 which reported that WP1066 did not affect cell death, although it affected

565 chronic seizure severity (Grabenstatter et al 2014). The discrepancy of WP1066

566 in neuronal cell death and seizure severity might be due to the different SE

567 models that were used. Indeed, i.c.v KA-induced SE causes localized neuronal

568 death in hippocampal CA3, while pilocarpine-induced SE is more widespread

569 in hippocampal CA1, CA3, and hilus. Also, as opposed to the pilocarpine

570 model, i.c.v KA model failed to induce chronic seizures, as recorded by EEGs

571 even in WT mice (data not shown). Therefore, the molecular mechanisms

572 underlying the cell death and seizure severity in the two models could be

573 different. Taken together, our results show that although WP1066 treatment

574 did not prevent the development or reduce the severity of acute seizure

575 behaviors, it was able to inhibit neuronal cell death after KA-induced status

576 epilepticus. Together, these results indicate that STAT3 signaling may act as a

577 mediator for CCR2-induced IL-1β production in activated microglia and

578 infiltrated macrophages, leading to neuronal degeneration after seizures.

579

580 CCR2 deficiency attenuates seizure-induced behavioral impairments

581 beyond the acute injury

582 To study the functional significance of CCL2-CCR2 dependent acute

583 neuronal death after KA-induced status epilepticus, we examined the

584 pathology and behaviors at 14 d post-KA treatment in WT and CCR2-/- mice.

585 Nissl staining was used to investigate the neuronal loss in the CA1 and CA3

586 region of the hippocampus at 14 d after KA, since FJB method more accurately

587 measures acute neuronal degeneration. We observed a characteristic loss of

588 pyramidal neurons in WT mice, which is consistent with increased FJB

589 staining 1d after KA. There was no significant difference in neuronal survival

590 between WT and CCR2-/- mice in the CA1 region (data not shown). However,

591 there were more surviving neurons in the CA3 of CCR2-/- mice compared with

592 WT mice at 14d post-KA treatment (Fig. 7A, p < 0.001). Therefore, the tendency

593 of neuronal loss shown by Nissl staining in the chronic seizure stage correlated

594 with that seen in the acute stage shown by FJB staining at 1d post seizure (Fig.

595 3B), suggesting that CCL2-CCR2 signaling contribution to neuronal death

596 persists in the chronic phase of epilepsy.

597 Epilepsy is often associated with behavior alterations, such as anxiety

598 and impaired cognitive performance (Caplan et al., 2008). Hence, we wanted

599 to test the implications of CCL2-CCR2 dependent hippocampal

600 neurodegeneration in anxiety and cognition in KA-induced seizure model.

601 Anxiety-related behavior was evaluated by analyzing the time/distance that

602 mice traveled in the center of the open field. Under control condition with

603 aCSF injection, there was no significant difference in the time spent in the

604 center between WT and CCR2-/- mice. However, at 7d or 14d after injection of

605 KA, the WT mice spent less time in the center than that in CCR2-/- mice,

606 suggesting that epilepsy-induced anxiety-like behavior is reduced in CCR2-/-

607 mice (Fig. 7B, p < 0.05). Similarly, the distance travelled in the central area was

608 shorter in WT mice than that in CCR2-/- mice (data not shown). As a control, we

609 found no significant difference in total distance between WT and CCR2-/- mice

610 (data not shown). Next, we used novel object recognition to examine

611 hippocampal-dependent cognitive function and investigate whether cognitive

612 decline is more evident in WT mice as compared to CCR2-/- mice.

613 Administration of KA to WT mice induced cognitive decline in agreement

614 with others previous reports (Matsuda et al., 2015). Indeed, CCR2 deficiency

615 attenuated this seizure-induced cognitive decline, although in the control

616 group WT and CCR2-/- mice showed no significant difference (Fig. 7C).

617 We showed no significant difference in acute seizure scores between

618 the WT and CCR2-/- following the KA injection, but CCR2 deficiency reduced

619 KA-induced neuronal cell death (Fig. 3). Here we tested the severity of

620 recurrence of seizures following KA re-injection at 14d after the first KA

621 injection (Fig. 7D). Interestingly, we found that the second dose of KA (14d

622 after the first KA injection) evoked more severe seizures in WT mice compared

623 with CCR2-/- mice. The WT group presented a drastically higher mortality rate

624 and seizure score within the first hour after the second KA injection (Fig. 7D).

625 Taken together, these results suggest that CCL2-CCR2 signaling exacerbates

626 neuronal loss, seizure-induced cognitive decline, and increased seizure

627 severity upon secondary seizure induction in the chronic stage of KA-induced

628 epilepsy.

629

630

631 DISCUSSION

632 In the current study, we delineate an intriguing molecular cascade

633 underlying CCL2-CCR2 signaling in neuronal cell death after status

634 epilepticus (Fig. 8). At 1d following KA-induced seizures, hyperactive

635 neurons and microglia release CCL2, triggering CCR2-positive monocyte

636 infiltration. Activation of CCR2 is coupled to the STAT signaling pathway and

637 subsequent production of IL-1β by both microglia and macrophages, which in

638 turn promotes neuronal cell death after seizures. Our results suggest a critical

639 role for CCL2-CCR2 signaling in neuronal cell death after status epilepticus,

640 identifying it as a potential therapeutic target for the management of epilepsy.

641

642 CCL2-CCR2 signaling in macrophage infiltration after seizures

643 Increasing evidence based on observations made from experimental

644 animals and human patients suggests that the neuroinflammatory response

645 may be a potential contributor to seizure-related pathologies (Vezzani et al.,

646 2011; Janigro et al., 2013). It is not clearly known whether the inflammatory

647 response underlies epileptogenesis or it exacerbates the damage after the

648 onset of seizures. Seizure-induced inflammatory responses, including

649 activation of glial cells and secretion of inflammatory mediators, were known

650 to increase neuronal hyperactivities, thereby promoting neuronal cell death

651 during epilepsy (Vezzani and Viviani, 2014). However, the molecular

652 mechanisms underlying the role of inflammation in epileptic neuronal death

653 are still largely unknown. In this study, we found an array of chemokines

654 including CCL2, CCL3, CCL5, CCL10, and CXCL10 to be upregulated at 1d

655 following KA-induced seizures. This is consistent with previous studies that

656 reported an increase in CCL2 levels in rodent brains after acute seizures

657 (Manley et al., 2007; Foresti et al., 2009) and in brain tissue of epilepsy patients

658 (Wu et al., 2008; Choi et al., 2009). Our study further determined the cell types

659 expressing CCL2 and showed that the elevated CCL2 expression was

660 predominantly localized in microglia and neurons. Future studies are needed

661 to address how CCL2 is upregulated and whether common mechanisms are

662 employed to increase the CCL2 expression in neurons and microglia.

663 It has been reported that CCL2 is able to induce monocyte recruitment

664 into the CNS after experimental autoimmune encephalomyelitis and ischemic

665 stroke (Che et al., 2001; Huang et al., 2001; Hughes et al., 2002; Semple et al.,

666 2010). Indeed, our study directly demonstrated the role of CCL2 in

667 macrophage infiltration through CCR2 activation after seizures using two

668 complimentary approaches: (1) We observed blood-born CCR2RFP/+ cells in the

669 hippocampus after seizures, which was inhibited by a CCL2 neutralizing

670 antibody; (2) There is absence of macrophage infiltration in CCR2-/- mice after

671 KA-induced status epilepticus. In addition to CCL2 upregulation, the

672 expression of its receptor CCR2 was also notably increased after seizures.

673 Considering the infiltration of CCR2-poistive macrophages in the

674 hippocampus, the upregulated CCR2 was likely due to the increased

675 macrophages after seizures. Interestingly, we also noted that blocking CCL2

676 not only reduced macrophage infiltration as evidenced by reduced Iba1

677 expression, but also reduced the activation of resident microglia as evidenced

678 morphologically. This suggests that macrophage infiltration may trigger

679 activation of endogenous microglia after seizure induction and is worthy of

680 further investigation. Microglia and CCR2-poistive macrophages are shown to

681 play differential roles in the inflamed brain with the monocyte-derived

682 macrophages being highly phagocytic and inflammatory, whereas

683 microglia demonstrate an unexpected signature of globally suppressed

684 cellular metabolism at disease onset (Yamasaki et al., 2014). The deficiency of

685 CCR2 reduced overall pSTAT3 and IL-1E which indicates that CCL2-CCR2

686 signaling is critical to trigger the cascade in infiltrated macrophages as well as

687 in resident microglia. Interestingly, a recent study identified CCR2 expression

688 in neuronal progenitors (Hung et al., 2013). However, we did not observe

689 CCR2RFP/+ cells in the dentate gyrus, the region of origin of neuronal

690 progenitors in our transgenic mice. The discrepancy might be due to the

691 differences in animal species used, methods for CCR2 detection or models for

692 the seizure induction.

693 Previous studies have demonstrated that CCL2 can activate macrophages

694 by binding to CCR2 expressed on the surface of these cells (Rollins, 1991).

695 CCR2 activation triggers monocyte adhesion to inflamed endothelium via

696 E-integrin pathways (Shi and Pamer, 2011). Therefore, monocyte CCR2

697 promotes its attachment to endothelial cells and subsequent infiltration into

698 the brain parenchyma. We have shown that seizures induced the upregulation

699 of CCL2, activated CCR2 and triggered monocyte infiltration. Indeed, the

700 number of CCR2 positive macrophage was suppressed by neutralizing CCL2,

701 as well as in CCR2-/- mice. However, it is still unknown whether neuronal

702 CCL2 and microglial CCL2 may play different roles in attracting

703 CCR2-positive monocytes into the injured CA3 area. Nevertheless, our

704 findings indicate a critical role for CCL2-CCR2 signaling in macrophage

705 infiltration in the mouse hippocampus after seizures.

706

707 CCL2-CCR2 signaling and neuronal cell death in epilepsy

708 Status epilepticus results in neurodegeneration in the hippocampus, a

709 feature that is prominent in clinical epilepsy (Thom, 2014). Previous studies

710 have associated CCL2 with excitotoxic neurodegeneration (Kalehua et al.,

711 2004; Sheehan et al., 2007). However, the underlying molecular mechanism

712 remained to be elucidated. Elevated levels of STAT3 signaling has been

713 documented in rats following pilocarpine-induced SE (Grabenstatter et al.,

714 2014). In the present study, we found that CCL2-CCR2 signaling activates the

715 STAT3 pathway and subsequent IL-1β production. Consistently, the

716 upregulation of pSTAT3 was attenuated in CCR2-/- mice. In addition, WP1066,

717 a potent STAT3 inhibitor suppressed the seizure-induced increase in

718 expression of the microglial maker Iba-1 and inflammatory cytokine IL-1β.

719 Although our study does not clearly differentiate the contribution of pSTAT3

720 on microglial activation versus monocyte infiltration, our results indicated a

721 critical role for pSTAT3 in CCL2/CCR2-mediated microglial activation and

722 IL-1β production. In line with our study, recent studies also demonstrated

723 that CCL2-CCR2 signaling plays an important role in STAT3 activation in

724 cancer metastasis (Izumi et al., 2013; Lin et al., 2013) as well as in

725 epithelial-mesenchymal transition (Chen et al., 2015).

726 We further showed that increased IL-1β production in

727 microglia/macrophages may promote neuronal cell death based on several

728 lines of evidence: (1) IL-1 receptor antagonist decreased the number of

729 degenerated neurons after seizures, which is consistent with previous reports

730 (Allan et al., 2005); (2) decrease in IL-1β production and cell death after

731 seizures in CCR2-/- mice and CCL2-/- mice; (3) inhibition of STAT3

732 accompanied by attenuation in IL-1β production and neuronal cell death.

733 Consistently, recent studies have shown that IL-1β enhanced seizure

734 susceptibility and neuronal cell death after prolonged febrile seizures

735 (Fukuda et al., 2014); whereas, pharmacological blockade of IL-1β/IL-1

736 receptor type 1 axis during epileptogenesis provided neuroprotection (Noe et

737 al., 2013). Our cytokine array shows an increase in a repertoire of cytokines 738 and chemokines following epilepsy. This is consistent with a recent report 739 that showed an upregulation of CCL2, CCL3, CCL5 and IL-1β in a pilocarpine 740 model of epilepsy in rats (Arisi et al., 2015). It is important to note in our data 741 that although there is an upregulation in endogenous IL-1ra following 742 epilepsy, it seems to be insufficient to counteract the large increase of both

743 IL1α and IL-1β after epilepsy. How is IL-1β able to promote neuronal cell

744 death after epilepsy? Several possibilities are proposed according to previous

745 studies (Vezzani and Baram, 2007; Choi and Friedman, 2014). Firstly, IL-1β is

746 known to directly excite neurons via NMDA receptor activation (Vezzani et al.,

747 1999). Not surprisingly, this increased neuronal excitability by IL-1β

748 aggravates the neuronal hyperactivities and excitotoxicity that lead to severe

749 hippocampal degeneration. Secondly, IL-1β is reported to regulate expression

750 of the p75 neurotrophin receptor (p75NTR) and induce neuronal cell death by

751 activating the intrinsic caspase cascade (Troy et al., 2002). Thirdly, IL-1β is able

752 to directly trigger apoptosis signaling by activating NF-kB/NO and

753 MAPK/caspase-3 signaling (Wang et al., 2005; Vetere et al., 2014). Collectively,

754 our results suggested that CCL2-CCR2 signaling triggers STAT3 activation in

755 microglia/macrophages, which in turn produces inflammatory cytokine IL-1β

756 resulting in neuronal cell death after seizures.

757 In addition to being involved in seizure-induced neurodegeneration, our

758 study reports a role for CCL2-CCR2 signaling in chronic seizure outcomes

759 such as anxiety, memory and tolerance to secondary seizure episode.

760 Evidence from previous studies revealed that CCR2-mediated trafficking of

761 monocytes to the brain and activation of resident microglia promoted the 28

762 establishment of anxiety-like behaviors (Wohleb et al., 2013; Sawada et al.,

763 2014). Consistently, our results revealed reduced epilepsy-induced

764 anxiety-like behavior in CCR2-/- mice, in which both monocyte infiltration and

765 microglial activation is suppressed. In line with our findings another report

766 showed that CCR2 deficiency prevented neuronal cognitive impairments

767 induced by cranial irradiation (Belarbi et al., 2013). In these studies, CCR2

768 mediated monocyte infiltration and their subsequent inflammatory repertoire

769 was implicated in causing anxiety-like behavior and cognitive decline (Belarbi

770 et al., 2013; Wohleb et al., 2013; Sawada et al., 2014). However, the exact

771 mechanism underlying CCR2-mediated chronic seizure outcomes needs to be

772 further investigated.

773 In conclusion, our current study revealed a novel CCL2-CCR2 signaling

774 cascade in hippocampal degeneration through STAT3 activation and IL-1β

775 production after status epilepticus. Such information could contribute greatly

776 to our understanding of this component of neuroinflammatory response,

777 including microglial activation, macrophage recruitment and production of

778 pro-inflammatory mediators in causing epilepsy after a precipitating injury.

779 Our study identifies CCL2-CCR2 signaling as a novel candidate for future

780 therapeutic target to ameliorate neuroinflammatory damage following status

781 epilepticus.

782 783 784 785

786

787

788

789

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1000 Nature reviews Neurology 7:31-40. 1001 Vezzani A, Conti M, De Luigi A, Ravizza T, Moneta D, Marchesi F, De Simoni MG 1002 (1999) Interleukin-1beta immunoreactivity and microglia are enhanced in the 1003 rat hippocampus by focal kainate application: functional evidence for 1004 enhancement of electrographic seizures. J Neurosci 19:5054-5065. 1005 Wang DD, Englot DJ, Garcia PA, Lawton MT, Young WL (2012) Minocycline- and 1006 tetracycline-class antibiotics are protective against partial seizures in vivo. 1007 Epilepsy & behavior : E&B 24:314-318. 1008 Wang XJ, Kong KM, Qi WL, Ye WL, Song PS (2005) Interleukin-1 beta induction of 1009 neuron apoptosis depends on p38 mitogen-activated protein kinase activity 1010 after spinal cord injury. Acta Pharmacol Sin 26:934-942. 1011 Wen Z, Zhong Z, Darnell JE, Jr. (1995) Maximal activation of transcription by Stat1 1012 and Stat3 requires both tyrosine and serine phosphorylation. Cell 82:241-250. 1013 Wohleb ES, Powell ND, Godbout JP, Sheridan JF (2013) Stress-induced recruitment of 1014 bone marrow-derived monocytes to the brain promotes anxiety-like behavior. 1015 J Neurosci 33:13820-13833. 1016 Wu LJ, Wu G, Akhavan Sharif MR, Baker A, Jia Y, Fahey FH, Luo HR, Feener EP, 1017 Clapham DE (2012) The voltage-gated proton channel Hv1 enhances brain 1018 damage from ischemic stroke. Nat Neurosci 15:565-573. 1019 Wu Y, Wang X, Mo X, Xi Z, Xiao F, Li J, Zhu X, Luan G, Wang Y, Li Y, Zhang J (2008) 1020 Expression of monocyte chemoattractant protein-1 in brain tissue of patients 1021 with intractable epilepsy. Clinical neuropathology 27:55-63. 1022 Yamasaki R et al. (2014) Differential roles of microglia and monocytes in the inflamed 1023 central nervous system. J Exp Med 211:1533-1549. 1024 Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, O'Keeffe S, Phatnani HP, 1025 Guarnieri P, Caneda C, Ruderisch N, Deng S, Liddelow SA, Zhang C, 1026 Daneman R, Maniatis T, Barres BA, Wu JQ (2014) An RNA-sequencing 1027 transcriptome and splicing database of glia, neurons, and vascular cells of the 1028 cerebral cortex. J Neurosci 34:11929-11947.

1029

1030

1031

1032

1033

1034

1035

1036

1037 FIGURE LEGENDS

1038 Figure 1. Elevated levels of CCL2 in the hippocampus after seizures. (A) The

1039 upper panel is a schematic representation of the cytokine array containing 40

1040 different antibodies with duplicates. The array contains three positive controls

1041 (PC) and one negative control (NC). The expression level of

1042 cytokines/chemokines in the aCSF control (middle panel) and KA group

1043 (lower panel) is shown. KA-induced seizures upregulated the expression of

1044 cytokines/chemokines including CCL2 (D9,10), IL-1β (B19,20), TIMP1 (E3,4),

1045 CCL10 (D11,12), CCL3 (D15,16), CCl5 (D21,22), CXCL10 (D1,2), IL-1a (B17,18)

1046 and IL-1ra (B21,22) compared to those in the control group (n=3 for each

1047 group). The red box highlights CCL2 (D9,10) expression in aCSF control and

1048 KA treatment group. (B) Western blotting analysis shows an increase in CCL2

1049 expression at 1d and 3d after KA-induced seizures (left). The summarized

1050 results showing relative CCL2/E-actin in KA groups to the aCSF control

1051 condition (right) n = 4 for each group, ***p < 0.001. (C) Representative

1052 immunostaining images shows that the expression of CCL2 colocalized with

1053 microglial marker, Iba-1, and neuronal marker, NeuN, but not with astrocytic

1054 marker, GFAP in hippocampal CA3 area after seizures. DAPI staining was

1055 used to identify the cell nuclei. Scale bars, 100 μm, and 50μm in magnified

1056 merged figures. Summarized histogram shows co-localization of CCL2 with

1057 neuronal and microglial markers. Arbitrary unit (A.U) is defined as

1058 percentage area of CCL2 stained region (Pm2) that co-localized with the

1059 neuron and glial markers (n = 3 for each group).

1060

1061 Figure 2. Microglial activation and macrophage infiltration in the

1062 hippocampus after status epilepticus. (A) Representative confocal images of

1063 hippocampus (CA3) in which CX3CR1 positive resident microglia are labeled

1064 with GFP and CCR2-positive macrophages are labeled with RFP in

1065 CX3CR1GFP/+:CCR2RFP/+ double transgenic mice at 1d, 3d and 7d after KA

1066 induced seizures and aCSF control group. Note that resident microglia

1067 become activated and CCR2-positive macrophages accumulate in

1068 hippocampal CA3 area at 1 d after seizures. Scale bar, 100 μm. (B)

1069 Quantitative analysis showing time course of CCR2-RFP macrophage

1070 infiltration and CX3CR1-GFP microglia activation at different timepoints

1071 following i.c.v KA. N.T., not detection. Bars represent mean ± SEM. (C)

1072 Administration of CCL2 neutralizing antibody effectively suppressed

1073 infiltration of CCR2 RFP-positive cells, accompanied by the reduction in

1074 microglia activation after 1d KA. Similarly, no infiltration of CCR2

1075 RFP-positive cells was found in CCR2-/- (CX3CR1GFP/+:CCR2RFP/RFP) mice 1d

1076 after KA. Scale bar, 50 μm. (D) Western blots of Iba-1, RFP and β-actin in the

1077 hippocampus of aCSF control, 1d post KA, 3d post KA, CCL2 mAb and CCL2

1078 isotype mice groups. The CCL2 neutralizing antibody or the isotype (i.c.v., 100

1079 ng in 5 μl aCSF) was administered at 15min before and 2hr and 8hr after KA

1080 injection and the mice were sacrificed at 1d after KA. β-actin was used as

1081 loading control. (E) The bar graphs represent densitometric analysis of the

1082 immunoreactive blots. All data were normalized to aCSF control. n = 3 for

1083 each group, *p < 0.05, **p<0.01 compared with aCSF group; # p < 0.05

1084 compared with KA 1d group.

1085

1086 Figure 3. Decrease in the number of degenerated neurons in CCR2-/- mice

1087 and CCL2-/- mice 1d after KA treatment. Fluoro-Jade B (FJB) staining was

1088 used to detect neuronal degeneration in the hippocampus at 1 d post KA

1089 treatment in WT (A), CCR2-/- (B) and CCL2-/- (C) mice. Scale bar, 100 μm. A’, B’

1090 and C’ are the magnified images for the rectangles shown in A, B and C,

1091 respectively. Scale bar, 50 μm. (D) Quantification of number of FJB-positive

1092 neurons in WT, CCR2-/- and CCL2-/- mice 1d after KA-induced seizures is

1093 shown. n=4 for each group, **p < 0.01. (E) Seizure scores 0-2 hours after

1094 intracerebroventricular (i.c.v.) delivery of KA in WT, CCR2-/- and CCL2-/- mice.

1095 n = 9 in WT, n = 6 in CCR2-/-, and n = 6 in CCL2-/- mice, p > 0.05. (F)

1096 Representative EEG traces from awake WT, CCR2-/- and CCL2-/- mice before

1097 i.c.v KA injection (baseline) and with stage 4 seizures after i.c.v KA injection

1098 (seizure). (G) The histogram depicts total seizure spells detected between 0-2h

1099 after KA administration in WT, CCR2-/- and CCL2-/- mice. Seizure spells were

1100 defined as high frequency, high voltage synchronized polyspike or

1101 paroxysmal sharp waves with amplitude > 2 fold background and lasting > 6

1102 sec. The peak-to-peak amplitude cutoff was set at 1000 PV. n = 3 for each

1103 genotype of mice.

1104 1105

1106 Figure 4. CCR2 deficiency reduces the production of proinflammatory

1107 cytokine IL-1β after status epilepticus. (A-B) Western blots of IL-1β level in

1108 the hippocampus of WT and CCR2-/- group. β-actin was used as internal

1109 control. The graph represents densitometric analysis of the blots at 1 d post

1110 KA treatment normalized to WT aCSF control. n = 4 for each group, *p < 0.05.

1111 (C) Immunofluorescence images of CA3 hippocampal area from WT and

1112 CCR2-/- mice at 1 d after seizures showing co-localized expression of Iba-1 and

1113 IL-1β. DAPI was used for nuclear staining. Scale bar: 50 μm. (D)

1114 Quantification of number of IL1E+ Iba1+ cells in WT mice as well as IL1E+ RFP+

1115 cells in CX3CR1GFP/+:CCR2RFP/+ double transgenic mice before and after i.c.v.

1116 KA. KA-induced IL1E upregulation was reduced in CCL2-/- mice. n = 4 for

1117 each group. (E-F) Administration of IL-1ra decreased the number of FJB

1118 positive neurons in the hippocampus at 1 d post seizure. Scale bar: 50 μm. n =

1119 4 for each group, *p < 0.05.

1120

1121 Figure 5. Microglial activation and STAT3 phosphorylation is attenuated in

1122 CCR2-/- mice 1d after KA-induced seizures. (A) Immunofluorescence images

1123 of CA3 hippocampal area showing co-localized expression of pSTAT3 with

1124 Iba-1 in WT and CCR2-/- mice 1 d after KA-induced seizures and in aCSF WT

1125 controls. DAPI was used for nuclear staining. Note attenuated pSTAT3

1126 immunofluorescence in the CCR2-/- mice. Scale bar: 100 μm. (B) Western blots

1127 of Iba-1 and pSTAT3 level in the hippocampus of WT and CCR2-/- group at 1 d

1128 after seizures. β-actin was used as loading control. (C) The summarized

1129 results of the densitometric analysis with each bar representing the ratio of

1130 optical density of pSTAT3 level to that of STAT3 (left) or Iba-1 to that of

1131 β-actin. All data were normalized to WT aCSF control. n = 4 for each group, *p

1132 < 0.05.

1133

1134 Figure 6. Inhibition of pSTAT3 reduces production of pro-inflammatory

1135 cytokine IL-1β and neuronal degeneration induced by seizures. (A) Western

1136 blots of pSTAT3, STAT3, Iba-1 and IL-1β in the hippocampus of aCSF,

1137 KA+vehicle and KA+WP1066 treatment group. β-actin was used as loading

1138 control. (B) The summarized results showing the bar graphs represent

1139 densitometric analysis with each bar representing the ratio of optical density

1140 of pSTAT3 level to that of STAT3 (left), IL-1β to that of β-actin (middle), or

1141 Iba-1 to that of β-actin. All data were normalized to aCSF control. n = 3 for

1142 each group, *p < 0.05, **p < 0.01. (C-D) FJB staining was used to detect

1143 neuronal degeneration in the hippocampus at 1 d post-KA treatment and the

1144 data is represented qualitatively (C) and quantitatively (D) showing that

1145 WP1066 reduces seizure-induced FJB staining. Scale bar, 50μm. (E) The graph

1146 represents seizure scores of mice treated with vehicle and WP1066 at 1d post

1147 KA injections. n = 6 mice each group, p > 0.05.

1148

1149 Figure 7. CCR2 deficiency attenuates seizure-induced behavioral

1150 impairments. (A) The number of surviving neurons in Nissl stained sections

1151 was counted in the CA3 area of ipsilateral side of the hippocampus in WT and

1152 CCR2-/- mice 14d after KA injection. n = 5 for each group, ***p < 0.001. Scale

1153 bars, 50 μm. (B) Open field test was used to examine anxiety behaviors of WT

1154 and CCR2-/- mice at 7d and 14d after KA treatment. Representative traces (left)

1155 and summarized data (right) showing that WT mice spent less time in the

1156 center of the open field than that of CCR2-/- mice at both 7d and 14d after KA

1157 injection. n = 8 and 5 for aCSF-injected and KA-injected group (WT mice and

1158 CCR2-/- mice) respectively, *p < 0.05, **p < 0.01. (C) Novel object recognition

1159 was used to detect hippocampal-dependent memory change following

1160 KA-induced seizures. The preference for novel object was significantly higher

1161 in CCR2-/- mice than in WT mice. n = 8 and 5 for aCSF-injected and KA-injected

1162 group (WT mice and CCR2-/- mice) respectively, *p < 0.05,**p< 0.01. (D) A

1163 second dose of KA (i.c.v., 0.15Pg/5Pl) 14 d after first KA injection (i.c.v.,

1164 0.15Pg/5Pl) induced recurrent seizure. The rate of live and died mice (left) and

1165 the seizure score of the recurrent seizure (right) was examined in WT and

1166 CCR2-/- mice. Since all the WT mice died within 60 min of KA injection, the

1167 mean seizure score was also counted within the first hour after second KA

1168 injection. n = 5 for each group, **p < 0.01.

1169

1170 Figure 8. Schematic representation of CCL2-CCR2 signaling in causing

1171 neuronal cell death via STAT3 activation and IL-1β production after status

1172 epilepticus. After KA-induced seizure, (1) CCL2 is upregulated and released

1173 from neurons/microglia; (2) increased CCL2 aids in CCR2-positive monocyte

1174 infiltration; (3) CCL2-CCR2 dependent expression of pSTAT3 in infiltrating

1175 macrophages and microglia; (4) upregulation and release of IL-1β from

1176 activated microglia/macrophages; (5) IL-1β produced by microglia /

1177 macrophages induces neuronal cell death after epilepsy. Consistently, both

1178 CCL2-/- and CCR2-/- have the phenotype of less neuronal death at 1d after

1179 KA-induced seizures.

1180