Role of Macrophages in Status Epilepticus Predisposing to Alzheimer’s Disease

Abstract

Continuous epileptic seizures hallmark status epilepticus, leading to preferential neuronal cell loss in the hippocampus that can progress into Alzheimer’s disease. Previous studies have shown that status epilepticus prompts an overproduction of nitric oxide (NO) by upregulation of NO synthase II (NOS II) to induce apoptosis of neuronal cells in the hippocampus, in a nuclear factor-kappaB (NF-κB) signaling dependent manner. Here, in an experimental rat model for status epilepticus, elicitation of sustained seizure activity was achieved by microinjection of kainic acid (KA) into the hippocampal CA3 subfield. We found that KA induced features of status epilepticus, which could be attenuated by blocking NF-κB signaling through a specific inhibitor. Interestingly, infiltration of macrophages of primarily pro-inflammatory subtype was detected in the hippocampal CA3 region immediately after KA injection. Experimental elimination of macrophages by an anti-CD115 antibody significantly attenuated the features of status epilepticus, likely through suppressing activation of NF-κB signaling. Together, these data suggest that macrophages play a critical role in NF-κB signaling-mediated status epilepticus that predisposes to Alzheimer’s disease.

Keywords

Alzheimer’s disease kainic acid macrophages status epilepticus

INTRODUCTION

Status epilepticus is a condition of continuous epileptic seizures, which could lead to a clinic emergency associated with essential glutamate-mediated brain damage and preferential neuronal cell death in the hippocampus and extrahippocampal temporal lobe to cause high morbidity and mortality in the patients and could potentially cause Alzheimer’s disease [1].

Nuclear factor-kappa B (NF-κB) is composed of p65, p50, RelB, c-Rel, and p52 that diversely combine to form different transcriptional dimers for signal transduction [2]. NF-κB is normally sequestered in the cytoplasm due to its binding to its inhibitor, iκB. Phosphorylation of iκB leads to its ubiquitination and subsequent degradation, resulting in release of NF-κB from the complex, phosphorylation and nuclear translation to regulate expression of critical genes involved in inflammation, immune response, cell adhesion, cell growth, cell migration, and cell death [3 –5].

NF-κB signaling was recently found to play a critical role in the neuronal survival at a physiological level likely through its protective effect against oxidative and ischemic stress [6]. However, activation of NF-κB signaling was also found to contribute to inflammatory reaction and apoptotic cell death after brain injury, stroke, and status epilepticus [7, 8]. Of note, previous studies have shown that status epilepticus prompts an overproduction of nitric oxide (NO) by upregulation of NO synthase II (NOS II) to induce apoptosis of neuronal cells in the hippocampus, in a NF-κB signaling dependent manner [9].

Macrophages are important inflammatory cells involved in immunity. Alongside the macrophages that display the classical pro-inflammatory phenotype, designated “M1” macrophages, another macrophage sub-type, designated “M2”, is responsible for wound healing and tissue-remodeling functions. The degree to which a given macrophage bears M1 or M2 characteristics is termed “polarization” [10]. M1 macrophages are characterized by high levels of reactive oxygen species (ROS), NO, iNOS, CD11c, TNFα, IL-6, IL-1β, and MHC-II, while M2 macrophages are characterized by high levels of arginase 1 (ARG1), CD206 (Mac1), CD163, CD301, IL-10, Fizz1 (Retnla: resistin-lke-α), and Ym1 (Chi3l3: chitinase 3-like 3) [11]. It is known now that macrophages can “polarize” into a wide spectrum of phenotypes that do not fit rigidly into the definition of “M1” or “M2” [12]. Microenvironmental signals together with epigenetic changes influence macrophage activation and function [13]. So far, a possible role of macrophages in the NF-κB signaling-mediated development of status epilepticus has not been acknowledged and thus addressed in the current study.

Here, status epilepticus was induced in an experimental rat model by microinjection of kainic acid (KA) into the hippocampal CA3 subfield. We found that KA induced features of status epilepticus, which could be attenuated by blocking NF-κB signaling through a specific inhibitor. Interestingly, infiltration of macrophages of primarily pro-inflammatory subtype was detected in the hippocampal CA3 region immediately after KA injection. Experimental elimination of macrophages by an anti-CD115 antibody significantly attenuated the features of status epilepticus, likely through suppressing activation of NF-κB signaling.

MATERIALS AND METHODS

Protocol approval

Experimental protocol and approaches including ethic issues on animal work have been approved by the research committee at the Shanghai Jiao Tong University.

Rat treatment

Experiments were carried out in pathogen-free female Sprague-Dawley rats (at 20 weeks of ago, weighing 245–300 g) obtained from SLAC Laboratory Animal Co. Ltd (Shanghai, China). Standard housing, laboratory rat chow, and tap water were applied. An experimental Temporal Lobe Status Epilepticus protocol was used to induce status epilepticus in rats. Briefly, microinjection of KA (Sigma-Aldrich, St Louis, MO, USA) was performed unilaterally into the hippocampal CA3 subfield that results in a progressive buildup of bilateral seizure-like hippocampal electroencephalographic (hEEG) activity, which was recorded for 210 min from the CA3 subfield on the right side. The wound was then closed in layers, and rats were returned to the animal room for recovery in individual cages after waking-up. Rats that received unilateral microinjection of same volume of saline were served as controls (saline). Diethyldithiocarbamate (DDTC) is a major metabolite of disulfiram, and is a specific NF-κB inhibitor, functioning through inhibiting NF-κB nuclear translocation, inhibition of IκB phosphorylation, and proteasome degradation [14, 15]. DDTC was given intraperitoneally at a dose of 150 mg/kg 30 min prior to KA injection.

Assessment of seizure activities

Seizure activity was assessed using behavioral observation and seizure scoring according to the Racine scale (Racine 1972). Class 5 limbic motor seizures composed of rearing and falling were considered with status epilepticus or prolonged continuous seizure activity. Moreover, time-course alternations in root mean square (RMS) and mean power frequency (MPF) values of hEEG signals were also recorded from the right hippocampal CA3 subfield.

Immunohistochemistry

Perfusion with 4% paraformaldehyde (PFA) was done at the end of the experiments before collection of brain samples. Hippocampus CA3 region was removed from the sampled rats and fixed in 30% sucrose and then in 10% formaldehyde-saline solution for 48 h at 4°C. The sectioning was done at 6μm sections. The slides were stained with ABC method (Sigma-Aldrich) with anti-rat CD68 antibody (Millipore, Billerica, MA, USA), followed by hematoxylin counterstain. Quantification was done with NIH ImageJ software.

Flow cytometry

Flow cytometry on dissociated brain cells was done using FITC-conjugated anti-CD68 and PE-cy7-conjugated anti-CD86 antibodies (Becton-Dickinson Biosciences, San Jose, CA, USA) on a FACSAria cell sorter (Becton-Dickinson Biosciences). Data were quantified and presented using Flowjo software v11 (Flowjo LLC, Ashland, OR, USA).

Western blot and ELISA

The total protein was extracted with a RIPA buffer (Sigma-Aldrich) and the protein concentration was determined with a BCA protein assay (Sigma-Aldrich). The rabbit anti-p50, rabbit anti-p65, and rabbit anti-GAPDH (Cell Signaling, Billerica, MA, USA) were used as primary antibodies and HRP-labeled anti-rabbit antibody was used as second antibody (Dako, Carpinteria, CA, USA). Each condition has 5 repeats and the mean value was quantified with NIH ImageJ. ELISA for iNOS and arginase was performed using specific kits (R&D System, Los Angeles, CA, USA).

Statistical analysis

GraphPad prism version 8.0 (GraphPad Software, Inc. La Jolla, CA, USA) was used to analyze the data with a one-way analysis of variance (ANOVA) test followed by the Fisher’s Exact Test to compare two groups. All values represent the mean±standard deviation (SD). A value of p < 0.05 was considered statistically significant after Bonferroni correction.

RESULTS

KA induces status epilepticus in rats

Here, we used an experimental rat model for inducing status epilepticus, in which elicitation of sustained seizure activity was achieved by microinjection of KA into the hippocampal CA3 subfield of the rats. We found that KA induced features of status epilepticus, including increases in RMS (Fig. 1A), increases in MPF (Fig. 1B), increases in Latency to forelimb clonus (Fig. 1C) and increases in Latency to status epilepticus (Fig. 1D). However, all these features were significantly attenuated by blocking NF-κB signaling through a prior administration of a specific NF-κB inhibitor, DDTC (Fig. 1A-D). These data suggest that KA induces status epilepticus in rats in a NF-κB-signaling-dependent manner, consistent with previous reports.

Fig.1

KA induces status epilepticus in rats. Sustained seizure activity was achieved by microinjection of kainic acid (KA) into the hippocampal CA3 subfield of the rats. Injection of saline was used a control. DDTC was injected 30 min before KA for determining role of NF-κB signaling. A) RMS. B) MPF. C) Latency to forelimb clonus. D) Latency to status epilepticus. ^*/&p < 0.05. In A and B, *KA versus saline; ^&KA+DDTC versus KA. N = 5.

Increases in pro-inflammatory macrophages are detected in the KA-region

We studied the role of macrophages in KA-induced status epilepticus. First, we did immunostaining for a pan-macrophage marker CD68 in the hippocampal CA3 region. We detected increased infiltration of macrophages in the hippocampal CA3 region immediately after KA injection, shown by representative images (Fig. 2A), and by quantification (Fig. 2B). Moreover, DDTC did not affect the increased infiltration of macrophages (Fig. 2A, B), suggesting that NF-κB signaling is not a trigger for KA-induced macrophage infiltration. To confirm it and further detect macrophage subtypes, we did flow cytometric analysis on dissociated cells from the hippocampal CA3 region. First, we confirmed the increases in total CD68+ macrophages by KA, shown by representative flow charts (Fig. 2C), and by quantification for CD68+ cells (Fig. 2D). Moreover, more increases in macrophages appeared to be attributable to increases in CD86+ macrophages, a pro-inflammatory subtype generally called M1, shown by representative flow charts (Fig. 2C), and by quantification for CD86+ cells (Fig. 2E). Again, DDTC did not affect neither the level of increases in CD68+ macrophages nor the macrophage subtype distribution (Fig. 2C-E), which was confirmed by analysis of iNOS and arginase levels in total CD68+ macrophages (Fig. 2F-G), suggesting that NF-κB signaling does not regulate KA-induced macrophage infiltration and polarization.

Fig.2

Increases in pro-inflammatory macrophages are detected in the KA-region. Immunostaining for a pan-macrophage marker CD68 in the hippocampal CA3 region, shown by representative images (A) and by quantification (B). Flow cytometric analysis for CD68 and CD86 on dissociated cells from the hippocampal CA3 region, shown by representative flow charts (C), by quantification for CD68+ cells (D), and by quantification for CD86+ cells (E). ELISA for iNOS (F) and arginase (G) in total CD68+ macrophages. ^*p < 0.05. NS, non-significant. N = 5. Scale bars are 50μm.

AFS98 eliminates macrophages in rat brain

In order to evaluate the importance of macrophage infiltration in KA-induced status epilepticus, we used a specific anti-CD115 antibody (AFS98) to eliminate macrophages and examined the effects on KA-induced status epilepticus. CD115 is the cell-surface receptor for colony-stimulating factor-1 (CSF-1) that regulates myeloid lineage cells, and is encoded by the c-fms proto-oncogene, a member of class III receptor tyrosine kinase family [16]. First, we checked the effects of AFS98 on macrophage infiltration. We did immunostaining for a pan-macrophage marker CD68 in the hippocampal CA3 region. We detected significantly reduced infiltration of macrophages in the hippocampal CA3 region immediately after KA injection, shown by representative images (Fig. 3A), and by quantification (Fig. 3B). To confirm it and further detect macrophage subtypes, we did flow cytometric analysis on dissociated cells from the hippocampal CA3 region. First, we confirmed the reduced increases in total CD68+ macrophages by AFS98, shown by representative flow charts (Fig. 3C), and by quantification (Fig. 3D). Moreover, the CD86+ pro-inflammatory macrophages were primarily affected (Fig. 3C, D). Thus, AFS98 effectively eliminates macrophages in rat brain.

Fig.3

AFS98 eliminates macrophages in rat brain. In order to evaluate the importance of macrophage infiltration in KA-induced status epilepticus, we used a specific anti-CD115 antibody (AFS98) to eliminate macrophages and examined the effects on KA-induced status epilepticus. Immunostaining for a pan-macrophage marker CD68 in the hippocampal CA3 region, shown by representative images (A) and by quantification (B). Flow cytometric analysis for CD68 and CD86 on dissociated cells from the hippocampal CA3 region, shown by representative flow charts (C) and by quantification for CD86+ cells (D). ^*p < 0.05. N = 5. Scale bars are 50μm.

Macrophages play a critical role in KA-induced status epilepticus

Next, we checked the effects of macrophage depletion on KA-induced status epilepticus. We found that KA-induced features of status epilepticus, including increases in RMS (Fig. 4A), increases in MPF (Fig. 4B), increases in Latency to forelimb clonus (Fig. 4C) and increases in Latency to status epilepticus (Fig. 4D), were also significantly attenuated by macrophage depletion by AFS98 (Fig. 4A-D). Thus, these data suggest that KA-induced status epilepticus in rats requires macrophages.

Fig.4

Macrophages play a critical role in KA-induced status epilepticus. We checked the effects of macrophage depletion on KA-induced status epilepticus. A) RMS. B) MPF. C) Latency to forelimb clonus. D) Latency to status epilepticus. ^*p < 0.05. N = 5.

Macrophages play a critical role in NF-κB signaling-mediated status epilepticus

Finally, we assessed if the effects of macrophages on KA-induced status epilepticus may be mediated through NF-κB signaling. Two key NF-κB factors, p50 and p65, and an NF-κB inhibitor, iκB, were examined in tissue from hippocampal CA3 region, showing that both of p50 and p65 increased by KA, but attenuated by macrophage depletion (Fig. 5A, B), but the levels of iκB were not affected (Fig. 5C). Hence, macrophages are required for the KA-induced status epilepticus in a NF-κB-signaling-dependent manner, consistent with previous reports.

Fig.5

Macrophages play a critical role in NF-κB signaling-mediated status epilepticus. Key NF-κB factors, p65 (A), p50 (B), and iκB (C) were examined in tissue from hippocampal CA3 region by western blot. ^*p < 0.05. N = 5.

DISCUSSION

Previous studies have demonstrated that prolonged seizures may cause an overproduction of NO by upregulation of NOS II in the hippocampal CA3 subfield, which is regulated by NF-κB signaling through direct transcriptional activation of NOS II [9]. Activation of NF-κB signaling occurs early after KA-induced status epilepticus, and has been shown to promote apoptotic neuronal cell death in the hippocampus and extrahippocampal temporal lobe [17]. Since p50 and p65 have been shown as key factors involved in KA-induced status epilepticus in these studies, here we also examined their levels after macrophage depletion. Our results suggest that p50 and p65 upregulation were both inhibited by elimination of macrophages. On the other hand, blocking NF-κB signaling did not affect macrophage infiltration and polarization, suggesting that macrophage infiltration should act upstream of NF-κB signaling activation.

We have eliminated macrophages using a monoclonal antibody to CD115, or the colony stimulating factor-1 (CSF-1 receptor), named AFS98 [18]. This antibody has been shown to induce loss of loss of macrophages in many tissues and major lost macrophages are resident macrophages [19]. Thus, in our study, the macrophage population that was removed should also be mainly resident macrophages, or microglia, rather than macrophages recruited from circulation. It is thus explained that the macrophage-associated factors are upstream of NF-κB signaling, which occurs early after KA. It is expected that the resident macrophages first respond to KA and immediately proliferate and differentiate, release cytokines to induce the activation of NF-κB signaling in neuronal cells, leading to the apoptotic cell death and loss of the latter to cause status epilepticus.

The DDTC was systemically administrated and thus may affect the tissue other than brain. Non-specific effects may be induced outside the target brain and could somehow influence the interpretation of data. This is a limitation of use of DDTC as a loss of function approach.

To the best of our knowledge, it is the first study to show the involvement of macrophages in the pathogenesis of status epilepticus. Our study should provide new insights into the mechanisms underlying development and progression of status epilepticus.

DISCLOSURE STATEMENT

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/19-0994r1).

References

Gronheit

, Popkirov

, Wehner

, Schlegel

, Wellmer

(2018) Practical management of epileptic seizures and status epilepticus in adult palliative care patients. Front Neurol 9, 595.

Tobon-Velasco

, Cuevas

, Torres-Ramos

(2014) Receptor for AGEs (RAGE) as mediator of NF-kB pathway activation in neuroinflammation and oxidative stress. CNS Neurol Disord Drug Targets 13, 1615–1626.

Tsai

, Li

, Wu

, Chang

, Chan

(2016) Sumoylation of IkB attenuates NF-kB-induced nitrosative stress at rostral ventrolateral medulla and cardiovascular depression in experimental brain death. J Biomed Sci 23, 65.

Carloni

, Nasuti

, Fedeli

, Montani

, Vadhana

, Amici

, Gabbianelli

(2013) Early life permethrin exposure induces long-term brain changes in Nurr1, NF-kB and Nrf-2. Brain Res 1515, 19–28.

Perez-Otano

, McMillian

, Chen

, Bing

, Hong

, Pennypacker

(1996) Induction of NF-kB-like transcription factors in brain areas susceptible to kainate toxicity. Glia 16, 306–315.

Wooten

(1999) Function for NF-kB in neuronal survival: Regulation by atypical protein kinase C. J Neurosci Res 58, 607–611.

Sarnico

, Lanzillotta

, Benarese

, Alghisi

, Baiguera

, Battistin

, Spano

, Pizzi

(2009) NF-kappaB dimers in the regulation of neuronal survival. Int Rev Neurobiol 85, 351–362.

Sarnico

, Lanzillotta

, Boroni

, Benarese

, Alghisi

, Schwaninger

, Inta

, Battistin

, Spano

, Pizzi

(2009) NF-kappaB p50/RelA and c-Rel-containing dimers: Opposite regulators of neuron vulnerability to ischaemia. J Neurochem 108, 475–485.

Chuang

, Chen

, Lin

, Chang

, Lu

, Liou

, Chan

, Chang

(2010) Transcriptional upregulation of nitric oxide synthase II by nuclear factor-kappaB promotes apoptotic neuronal cell death in the hippocampus following experimental status epilepticus. J Neurosci Res 88, 1898–1907.

10.

Taylor

, Martinez-Pomares

, Stacey

, Lin

, Brown

, Gordon

(2005) Macrophage receptors and immune recognition. Annu Rev Immunol 23, 901–944.

11.

Lawrence

, Natoli

(2011) Transcriptional regulation of macrophage polarization: Enabling diversity with identity. Nat Rev Immunol 11, 750–761.

12.

Nahrendorf

, Swirski

(2016) Abandoning M1/M2 for a network model of macrophage function. Circ Res 119, 414–417.

13.

Ginhoux

, Schultze

, Murray

, Ochando

, Biswas

(2016) New insights into the multidimensional concept of macrophage ontogeny, activation and function. Nat Immunol 17, 34–40.

14.

Matsuno

, Kariya

, Yano

, Morino-Koga

, Taura

, Suico

, Shimauchi

, Matsuyama

, Okamoto

, Shuto

, Kai

, Okada

(2012) Diethyldithiocarbamate induces apoptosis in HHV-8-infected primary effusion lymphoma cells via inhibition of the NF-kappaB pathway. Int J Oncol 40, 1071–1078.

15.

Cvek

, Dvorak

(2007) Targeting of nuclear factor-kappaB and proteasome by dithiocarbamate complexes with metals. Curr Pharm Des 13, 3155–3167.

16.

Sherr

(1990) The colony-stimulating factor 1 receptor: Pleiotropy of signal-response coupling. Lymphokine Res 9, 543–548.

17.

Lubin

, Ren

, Xu

, Anderson

(2007) Nuclear factor-kappa B regulates seizure threshold and gene transcription following convulsant stimulation. J Neurochem 103, 1381–1395.

18.

Murayama

, Yokode

, Kataoka

, Imabayashi

, Yoshida

, Sano

, Nishikawa

, Kita

(1999) Intraperitoneal administration of anti-c-fms monoclonal antibody prevents initial events of atherogenesis but does not reduce the size of advanced lesions in apolipoprotein E-deficient mice. Circulation 99, 1740–1746.

19.

Sudo

, Nishikawa

, Ogawa

, Kataoka

, Ohno

, Izawa

, Hayashi

, Nishikawa

(1995) Functional hierarchy of c-kit and c-fms in intramarrow production of CFU-M. Oncogene 11, 2469–2476.