To explain the underlying mechanisms of Myelophil, we subsequently examined the stress-responsive system, particularly focusing on the HPA axis hyper-activation. The HPA axis abnormality has been implicated in the pathophysiology of major depressive disorder. Hypercortisolemia is commonly observed in 40% to 60% of depressed patients (Murphy, 1991). The glucocorticoid receptor-mediated negative regulation of cortisol release is impaired in conditions of psychiatric pathology (Pariante and Lightman, 2008). In a previous animal study, chronically dexamethasone-injected mice have shown depressive-like behaviors and a decrease of hippocampal gene expression for glucocorticoid receptor (Skupio et al., 2015). Consistently with previous reports, we verified the increase of serum corticosterone by UCMS. However, this over-release of corticosterone was significantly attenuated by Myelophil administration, which proposed the antidepressant-like activity of Myelophil *via* balancing the endogenous glucocorticoid system (**Figure 3A**).

High glucocorticoids lead to morphological and functional changes of microglial cells in the brain, for example, from resting state into reactive phenotype as a neuroinflammatory response (Nair and Bonneau, 2006). Microglia, resident immune cells in the central nervous system, plays a pivotal role in the pathogenesis of depression (Yirmiya et al., 2015). Antidepressants, such as imipramine, suppressed the M1 phenotype microglia in the hippocampus of mice exposed to chronic mild stress (Zhao et al., 2016). As expected, the administration of Myelophil predominantly attenuated the over-activation of microglia in the CA1, dentate gyrus, and CA3 of the hippocampus (**Figure 2A** and **B**). Microglial activation in the hippocampal granule cell layer, hilus, CA1, and CA3 regions was known to trigger the onset of depression (Iwata et al., 2016). We further confirmed the inhibitory effects of Myelophil against the LPS-induced production of nitric oxide in the BV2 murine microglia cell line (**Figure 3B**). Researchers in pharmacology are attempting to identify the antidepressant agents that involve the regulation of microglia-derived neuroinflammation (Chen et al., 2017). In our study, the hippocampal pro-inflammatory cytokines, including TNF-α and IL-1β, were also completely normalized by Myelophil treatment (**Figure 3C** and **D**).

Emerging evidence reinforces the importance of NLRP3 inflammasome in neuropsychiatric disorders, particularly in depression and anxiety (Velasquez and Rappaport, 2016; Song et al., 2017). One preclinical study showed that 4 weeks of UCMS in mice resulted in depressive-like behaviors, and its contributing factor was NLRP3 inflammasome in the hippocampal region (Zhang et al., 2015). As expected, we found that Myelophil (100 mg/kg) significantly inhibited the hippocampal protein expressions of NLRP3, ASC, and caspase-1 activity (**Figure 4A**–**C**). These results implied the possibility of an NLRP3-dependent antidepressant-like action of Myelophil. In a case–control study, 20 patients with major depressive disorder exhibited high anxiety score and increased levels of caspase-1 and NLRP3 expression in peripheral blood mononuclear cells than did the healthy group (Alcocer-Gomez et al., 2016). The glucocorticoidinduced NLRP3 inflammasome formation was observed in a primary microglia cell isolated from mouse hippocampus (Frank et al., 2014). Furthermore, one clinical study identified a high correlation between the glial activation level and depressive score in patients with CFS using positron emission

tomography (PET) (Nakatomi et al., 2014). These facts support that antidepressant-like effects of Myelophil are linked to anti-fatigue activity.

Excessive microglial activation causes an impairment of the hippocampal neurogenesis under conditions of stress and inflammation (Sierra et al., 2014). In addition, the reduced hippocampal neurogenesis is closely involved in the pathophysiology of major depression (Snyder et al., 2011). Our data also showed a notable suppression of the hippocampal neurogenesis, which was significantly ameliorated by Myelophil treatment (**Figure 5**). It is known that chronic antidepressant treatment (serotonergic modulator) increases adult hippocampal neurogenesis in both non-human primates and humans (Perera et al., 2011; Boldrini et al., 2012). Serotonin, 5-HT, as a monoamine neurotransmitter, is the most important target molecule in depressive disorder. Therefore, serotonin-target medications, such as SSRIs and SNRIs, are prescribed most frequently in approximately 90% of cases (Artigas, 2013). It is particularly interesting that dysregulation of the HPA axis attenuates 5-HT neurotransmission (Mahar et al., 2014). In our results, the

altered 5-HT signals in the dorsal raphe nuclei were restored by Myelophil treatment (**Figure 6A** and **B**).

Myelophil was developed for the treatment of fatiguerelated disorders on the basis of the theory of traditional Chinese/Korean medicine. *Astragali Radix* and *Salviae miltiorrhizae Radix* are the main materials in Myelophil, and these two herbal plants are commonly used to maintain qi and bloodstream homeostasis in the human body (Cho et al., 2009). From our previous studies, Myelophil showed pharmacological actions against fatigue-related pathology such as brain oxidative stress and memory deficit (Lee et al., 2012; Lee et al., 2015). Our present data supported the applicability of Myelophil to depressive symptoms, which clearly accompany chronic fatigue disorders. However, the present study has a limitation of unknown information regarding the active compounds that correspond to the antidepressive property. A study found that salvianolic acid B, a major compound in *Salviae Radix*, exhibits anti-inflammatory effects by modulating NLRP3 inflammasome (Jiang et al., 2017). Salvianolic acid B promotes microglial M2-polarization against UCMS-induced M1 phenotype (Zhang et al., 2017). Astragaloside IV, a main *Astragali Radix* compound, inhibits depressive-like behaviors by regulating the nuclear factor-κB/ NLRP3 axis (Song et al., 2018). Further studies are required to identify the major active compound in the future. We used NAC as a positive control because the antidepressant-like effects of NAC have been explored in several studies (Ferreira et al., 2008; Costa-Campos et al., 2013). One study group is planning to evaluate the inflammation-inhibiting NAC effects on depressive disorder in a randomized placebo-controlled trial (Yang et al., 2018).

Taken together, our findings comprise the first evidence for the antidepressant-like effects of Myelophil, and its underlying mechanism may involve the regulation of NLRP3-dependent

neuroinflammation and the serotonergic signal. This study would support the pharmacological applicability of Myelophil in chronic fatigue, as well as neuropsychiatric disorders.

### ETHICS STATEMENT

Animal care and experiments were conducted in accordance with the guidelines issued by the Institutional Animal Care and Use Committee of Daejeon University (Daejeon, Republic of Korea; Approval No. DJUARB 2017-017) and the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.

### AUTHOR CONTRIBUTIONS

JL wrote the main manuscript text and conducted the experiments. WK supported the behavioral test for depression and anxiety. YJ prepared **Figure 2** and other immunofluorescence staining results. SL performed a statistical analysis. DL advised on the mechanism of NLRP3 inflammasome activation. CS supervised the preparation of manuscript and directed the final version of all contents. All authors reviewed and approved this manuscript.

## FUNDING

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Oriental Medicine R&D Project, Ministry of Health & Welfare, and Republic of Korea (HI15C0112), as well as the Ministry of Education, Science and Technology (NRF-2018R1A6A1A03025221).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2019.00683/ full#supplementary-material

### REFERENCES

signaling, and the kynurenine pathway. *Biol Psychiatry* 80, 4–5. doi: 10.1016/j. biopsych.2016.04.019

**Conflict of Interest Statement:** 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.

*Copyright © 2019 Lee, Kim, Jeon, Lee, Lee and Son. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Annexin A1 Bioactive Peptide Promotes Resolution of Neuroinflammation in a Rat Model of Exsanguinating Cardiac Arrest Treated by Emergency Preservation and Resuscitation

#### Edited by:

Pietro Giusti, University of Padua, Italy

#### Reviewed by:

Harshini Sarojini, University of Louisville, United States Tomas Drabek, University of Pittsburgh, United States

#### \*Correspondence:

Qing Ma<EMAIL_ADDRESS>Zhiquan Zhang<EMAIL_ADDRESS>
†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience

Received: 01 March 2019 Accepted: 28 May 2019 Published: 14 June 2019

#### Citation:

Ma Q, Zhang Z, Shim J-K, Venkatraman TN, Lascola CD, Quinones QJ, Mathew JP, Terrando N and Podgoreanu MV (2019) Annexin A1 Bioactive Peptide Promotes Resolution of Neuroinflammation in a Rat Model of Exsanguinating Cardiac Arrest Treated by Emergency Preservation and Resuscitation. Front. Neurosci. 13:608. doi: 10.3389/fnins.2019.00608 Qing Ma1 \* † , Zhiquan Zhang2,3 \* † , Jae-Kwang Shim4 , Talaignair N. Venkatraman5 , Christopher D. Lascola5,6, Quintin J. Quinones1 , Joseph P. Mathew7 , Niccolò Terrando2,3 and Mihai V. Podgoreanu1

1 Systems Modeling of Perioperative Organ Injury Laboratory, Department of Anesthesiology, Duke University, Durham, NC, United States, 2 Neuroinflammation and Cognitive Outcomes Laboratory, Department of Anesthesiology, Duke University, Durham, NC, United States, 3 Center for Translational Pain Medicine, Duke University, Durham, NC, United States, 4 Department of Anesthesiology and Pain Medicine, Yonsei University College of Medicine, Seoul, South Korea, 5 Departments of Radiology and Neurobiology, Duke University, Durham, NC, United States, 6 Duke-UNC Brain Imaging and Analysis Center, Duke University, Durham, NC, United States, 7 Department of Anesthesiology, Duke University, Durham, NC, United States

Neuroinflammation initiated by damage-associated molecular patterns, including high mobility group box 1 protein (HMGB1), has been implicated in adverse neurological outcomes following lethal hemorrhagic shock and polytrauma. Emergency preservation and resuscitation (EPR) is a novel method of resuscitation for victims of exsanguinating cardiac arrest, shown in preclinical studies to improve survival with acceptable neurological recovery. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has emerged as a key regulator of metabolic and energy stress response pathways in the brain and a pharmacological target to induce a neuronal pro-survival phenotype. This study aims to examine whether systemic administration of an Annexin-A1 bioactive peptide (ANXA1sp) could resolve neuroinflammation and induce sirtuin-3 regulated cytoprotective pathways in a novel rat model of exsanguinating cardiac arrest and EPR. Adult male rats underwent hemorrhagic shock and ventricular fibrillation, induction of profound hypothermia, followed by resuscitation and rewarming using cardiopulmonary bypass (EPR). Animals randomly received ANXA1sp (3 mg/kg, in divided doses) or vehicle. Neuroinflammation (HMGB1, TNFα, IL-6, and IL-10 levels), cerebral cell death (TUNEL, caspase-3, pro and antiapoptotic protein levels), and neurologic scores were assessed to evaluate the inflammation resolving effects of ANXA1sp following EPR. Furthermore, western blot analysis and immunohistochemistry were used to interrogate the mechanisms involved. Compared to vehicle controls, ANXA1sp effectively reduced expression of cerebral HMGB1, IL-6, and TNFα and increased IL-10 expression, which were associated with improved neurological scores.

ANXA1sp reversed EPR-induced increases in expression of proapoptotic protein Bax and reduction in antiapoptotic protein Bcl-2, with a corresponding decrease in cerebral levels of cleaved caspase-3. Furthermore, ANXA1sp induced autophagic flux (increased LC3II and reduced p62 expression) in the brain. Mechanistically, these findings were accompanied by upregulation of the mitochondrial protein deacetylase Sirtuin-3, and its downstream targets FOXO3a and MnSOD in ANXA1sp-treated animals. Our data provide new evidence that engaging pro-resolving pharmacological strategies such as Annexin-A1 biomimetic peptides can effectively attenuate neuroinflammation and enhance the neuroprotective effects of EPR after exsanguinating cardiac arrest.

Keywords: sirtuins, autophagy, apoptosis, neuroprotection, HMGB1

### INTRODUCTION

Exsanguinating hemorrhage, leading to cardiac arrest and multiple organ failure, remains the most common cause of death among trauma patients without traumatic brain injury, and neurologic outcomes in survivors are poor. An new therapeutic paradigm – termed EPR – involves rapidly cooling victims of exsanguinating cardiac arrest to deep or profound hypothermia levels (≤20◦C), in an effort to extend ischemic time, maintain organ viability during severe shock, allow operative repair of injuries and resuscitation, and ultimately improve survival and preserve neurological function (Tisherman et al., 2017). Following initial characterization of EPR, a growing body of proof-of-concept large animal studies have investigated the optimal depth and rate of cooling, duration of EPR, and physiological conditions prior to induction of EPR (Rhee et al., 2000; Alam et al., 2002, 2004, 2005, 2006a,b; Wu et al., 2006). Rapid cooling and subsequent controlled resuscitation and rewarming is accomplished through the use of CPB (Sailhamer et al., 2007; Alam et al., 2008). The development of rodent models of HS and EPR enabled characterization of key cellular and molecular changes involved in exsanguination cardiac arrest (Drabek et al., 2007a,b) – chief among those are neuroinflammation and apoptosis – which constitute targets for adjunctive therapies (Han et al., 2008; Alam et al., 2010).

Neuroinflammation is a complex immune response commonly observed following acute brain insults such as HS and associated warm ischemia, ischemia-reperfusion injury (I/RI), therapeutic hypothermia, exposure to CPB, and post-cardiac arrest syndrome. Multiple rodent models of surgical trauma, cardiac arrest and resuscitation have identified upregulation of pro-inflammatory cytokines and inflammatory mediators in both peripheral tissues and the central nervous system (CNS) (Terrando et al., 2010a, 2011). Acute neuroinflammation is characterized by increased cytokines, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), HMGB1 and their cognate receptors ultimately leading to neuron-glia dysfunction and blood–brain barrier impairments (Cao et al., 2010; Terrando et al., 2010b, 2011; He et al., 2012; Liu and McCullough, 2013; Wohleb et al., 2014; Xiang et al., 2016; Skvarc et al., 2018). Oxidative stress and mitochondrial dysfunction converge with the neuroinflammatory pathway, creating a positive feedback loop (Netto et al., 2018). Collectively, these mechanisms actively contribute to neuronal death and cognitive impairment. There is evidence that the neuroinflammatory milieu persists following surgical trauma, HS, I/RI, and resuscitation from cardiac arrest because is not efficiently controlled by endogenous anti-inflammatory mechanisms, thereby contributing to secondary injury, CNS dysfunction, and neuronal network hyperexcitability (Tahsili-Fahadan et al., 2018). Despite improvements in resuscitation science for victims of HS cardiac arrest, including EPR, total body cooling is technically and logistically challenging and the development of effective therapies to decrease morbidity and improve long-term neurologic outcomes following exsanguinating cardiac arrest remains a critical need.

Dysfunction of inflammation-resolving pathways have been described in experimental rodent trauma models, leading to exaggerated post-injury cognitive decline (Su et al., 2013). In recent years, a number of lipid mediators such as resolvins, lipoxins, and maresins have begun to receive attention as possible resolvers of neuroinflammation (Terrando et al., 2013; Vacas et al., 2013). Resolvins act to block neutrophil and monocyte migration and reduce the oxidative burst of neutrophils (Serhan et al., 2007; Perretti et al., 2015). Similarly, the glucocorticoid-regulated protein Annexin A1 and its peptide mimetics display pro-resolution effects, and have been shown to mitigate cerebral I/RI by attenuating neuroinflammation in many experimental models (Ansari et al., 2018) including cardiac surgery with deep hypothermic circulatory arrest (Zhang et al., 2017). The aim of this study was to determine the neuroprotective efficacy of a bioactive Annexin A1 short peptide (ANXA1sp) in a small animal model of exsanguination cardiac arrest and EPR. We hypothesized that ANXA1sp treatment would attenuate neuroinflammation and neuronal cell death, in part through upregulation of the mitochondrial protein deacetylase SIRT3 and downstream cytoprotective pathways.

**Abbreviations:** ANXA1sp, Annexin A1 short peptide; CPB, cardiopulmonary bypass; EPR, emergency preservation and resuscitation; HMGB1, high mobility group box 1; HS, hemorrhagic shock; LC3-II, microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate; MnSOD, manganese superoxide dismutase; p62/SQSTM1, sequestosome-1; ROS, reactive oxygen species; SIRT3, sirtuin-3.

### MATERIALS AND METHODS

fnins-13-00608 June 13, 2019 Time: 17:39 # 3

### Animals

The experimental protocol was approved by the Duke University Animal Care and Use Committee. All procedures met the guidelines of the National Institutes of Health for animal care (Guide for the Care and Use of Laboratory Animals, Health and Human Services, National Institute of Health Publication No. 86-23, revised 1996). Adult male Sprague–Dawley rats (age 13–15 weeks; weight 400–450 g; Charles River Laboratories, Wilmington, MA, United States) were housed (two animals per cage) in a 12-h light-dark cycle environment with free access to food and water. Rats were acclimated for at least 1 week before starting the experiment.

### Surgical Preparation for Experimental EPR

Experimental procedures are summarized in **Figure 1**. Fasted rats were anesthetized with isoflurane, intubated, and mechanically ventilated to maintain the arterial PaCO2 between 35 and 45 mmHg. Anesthesia was provided with isoflurane (1.5–2%) with an oxygen and air mixture at a FiO2 of 0.5. Bupivacaine 0.25% was injected at the operative sites to achieve local anesthesia. Routine physiologic parameters, and rectal and pericranial [hypodermic needle probe (Omega R , Stamford, CT, United States) placed beneath the temporalis muscle] temperatures were monitored. The right caudal epigastric artery was cannulated with a polyethylene catheter (PE10, Clay Adams, Sparks, MD, United States) and used for systemic mean arterial pressure (MAP) monitoring. The tail artery was cannulated with a 20-gauge catheter, which served as blood sampling and inflow CPB cannula. The right internal jugular vein was cannulated with a customized 4.5-F multi-orifice catheter advanced into the right atrium and used for withdrawing blood and venous out-flow CPB cannula. The external carotid artery was cannulated with a polyethylene catheter (PE50; Clay Adams, Sparks, MD, United States) advanced into the aortic arch and used for induction of profound hypothermia by flushing ice-cold normal saline. During surgical preparation, normothermia was maintained using a heat lamp.

### Induction of Hemorrhagic Shock and Ventricular Fibrillation

After a 30-min equilibration period, rapid exsanguination (50% blood volume, 0.034 ml/g body weight, 12–15 ml of blood) over 10 min was performed via the jugular venous cannula and MAP was allowed to drop below 20 mmHg. For the next 35 min (simulating pre-hospital transport time), the MAP remained <20 mm Hg without any resuscitative efforts HS. The shed blood was salvaged in a 20 ml syringe prefilled with 2 ml citrate phosphate dextrose solution (Fenwal Inc., Lake Zurich, IL, United States) and stored at 4◦C for later transfusion/resuscitation. At the end of HS, ventricular fibrillation (VF) was induced by transesophageal burst pacing at 50 V (S48 stimulator, RI, United States) for 90 s, and confirmed electrocardiographically and echocardiographically (Phillips Sonos7500 system, Andover, Mass 01810). During HS, anesthesia was maintained using 0.4% isoflurane. No neuromuscular blocker was administered until just prior to VF. A dose of vecuronium bromide (0.1 mg/kg) was given intravenously to prevent muscle contraction during VF and cooling.

### Induction of Profound Hypothermia

After the induction of VF, the animals were rapidly cooled to a core temperature of 10–15◦C for 45 min by flushing ice-cold solution [total volume 45 ml = 40 ml normal saline + 2 ml 5% human plasma protein fraction (Plasmanate R , Grifols Therapeutics, Inc., NC, United States) + 3 ml 8.5% Sodium Bicarbonate] into the ascending aorta/aortic arch and returning from right jugular vein using a peristaltic pump (Masterflex, model 770201-60, Cole-Parmer, Vernon Hills, IL, United States) at rate of 20 ml/kg/min (T208 transonic volume flow meter, Transonic Systems Inc. Ithaca, NY, United States). A water bath with ice water as well as topical cooling with ice bags was also applied.

### Resuscitation and Rewarming With Cardiopulmonary Bypass

Following 45 min of profound hypothermia, rats were rewarmed and resuscitated using CPB. The CPB circuit consisted of a peristaltic pump (MasterflexC, Cole-Parmer, Vernon Hills, IL, United States), a custom-designed oxygenator, and a venous reservoir, as previously described (Bartels et al., 2014; Shim et al., 2014; Zhang et al., 2017). The reservoir was primed with 3 ml of 6% hydroxyethylstarch 130/0.4 and 2 ml of previously shed and salvaged blood. Heparin (200 IU) and vecuronium bromide (0.1 mg/kg) were added to the venous reservoir. CPB was initiated at a flow rate of 20–30 ml/min, which was gradually increased to 50–60 ml/min upon reaching 33–34◦C. The gradient between the water bath and core body temperatures was not allowed to exceed 10◦C. Once core temperature increased from 10 to 34◦C, all salvaged blood was gradually re-transfused to keep up with the increased oxygen demand. The animals were rewarmed for 80 min until core temperatures of 34◦C were achieved; subsequently, CPB was terminated. Rewarming rate was maintained at approximately 0.4◦C/min. Acid-base abnormalities were corrected as needed. During rewarming, MAP was kept above 50 mmHg once core temperature reached >30◦C using intermittent administration of epinephrine.

After decannulation, animals were kept ventilated for 2 h under anesthesia with 0.5–1% isoflurane (core temperature 36–37◦C). To increase the hematocrit value to greater than 30%, the remaining autologous blood in the CPB circuit was collected, concentrated by centrifugation (3000 rpm for 5 min), and re-transfused. Heparin-induced anticoagulation was not reversed and allowed to dissipate spontaneously. Routine blood gas analysis was conducted serially (GEM Premier 3000 analyzer, Instrumentation Laboratory, Bedford, MA, United States). After spontaneous ventilation had resumed, animals were extubated and allowed to recover in an oxygen-enriched and humidified environment for 24 h, with free access to water and food.

To harvest the brain, 24 h after EPR rats were re-anesthetized, intubated, and mechanically ventilated. One sample of brain tissue was immediately fixed in 10% buffered formalin and paraffin-embedded for immunostaining. The remaining brain tissue was frozen in liquid nitrogen and stored at −80◦C until further use. Blood samples were also collected before and after induction of HS and at 0–120 min and 24 h after CPB and stored at −80◦C until analysis. Naïve rats were sacrificed under 5% isoflurane.

### Drug Treatments

fnins-13-00608 June 13, 2019 Time: 17:39 # 4

Annexin A1 biomimetic tripeptide (ANXA1sp or Ac-QAW, Ac = acetyl, MW = 445.47 Da) was synthesized and purified (>98% purity) by GenScript (Piscataway, NJ, United States). The peptide was suspended in 100% DMSO. For experiments, this stock solution was diluted in saline to a final dose of 1 mg/kg ANXA1sp and a concentration of 1% DMSO in saline as vehicle (control). ANXA1sp treatment solutions were freshly prepared immediately before use.

Rats were randomly assigned to three groups (EPR + ANXA1sp, n = 14; EPR+ vehicle, n = 15; naïve controls, n = 3) and terminated for histologic and biochemical analyses at 24 h after EPR. Rats received ANXA1sp (1 mg/kg iv) or vehicle (1% DMSO iv) in 1 ml saline immediately after induction of VF, then again at 1 h after CPB. Additionally, blood salvaged during exsanguination HS was also treated ex vivo with ANXA1sp 1 mg/kg or 1% DMSO and re-transfused during CPB rewarming and resuscitation (**Figure 1**). All treatments were administered in a blinded manner.

### Cell Death Assessment

Apoptosis was determined by terminal deoxynucleotidyl nick-end labeling (TUNEL) per assay manufacturer's protocol

(Roche Diagnostics, Indianapolis, IN, United States). Briefly, sections of paraffin-embedded brain tissue samples (5 µm thick) were deparaffinized using xylene and descending grades of ethanol, and pretreated with microwave radiation (350 W, in 200 mL of 0.1 M Citrate buffer, pH 6.0) for 5 min. Tissue sections were then incubated with terminal deoxynucleotidyl transferase (TdT) for 1.5 h at 37◦C and then rinsed with PBS. Slides of five representative areas of the retrosplenial and posterior parietal cortex and CA1 area of the hippocampus were mounted using UltraCruzTMMounting Medium with DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Negative controls were incubated in label solution without TdT. A separate set of brain tissue sections was stained with acid fuchsin-celestine blue to identify possible necrotic cells. Cell counting was performed in a blinded manner across five representative areas of the cerebral cortex and CA1 areas using fluorescence microscopy (Leica DM IRB, Germany) with a 20×/0.4 PH objective at 1.5-fold magnification. Data obtained in every field were added together to make a final data count for each slide and expressed as percentage of total cell number within the relevant fields.

### Western Blots

Frozen brain samples were homogenized and protein quantified by BCA assay (Thermo Fisher Scientific). Western blotting was performed using SDS–PAGE 4–20 and 8–16% gels gradient gels (Bio-Rad) with the following antibodies: rabbit anti-HMGB1 (Bioss Antibodies Inc., Woburn, MA, United States), rabbit polyclonal antibodies against SIRT3 (28-kDa isoform, Cell Signaling and Abcam), FOXO3a (Cell Signaling), Mn-SOD (Santa Cruz), cleaved caspase-3 (Cell Signaling), Bax and Bcl-2 (Santa Cruz), rabbit anti-LC3B (cell signaling), mouse anti-p62/SQSTM1 (R&D Systems, Minneapolis, MN, United States), and rabbit monoclonal antibody against GAPDH (Cell Signaling Technology, Boston, MA, United States). The bands were detected by Super-signal West Dura Extended Duration Substrate (Thermo Scientific Fisher, Rockford, IL, United States). Band intensities of HMGB1, SIRT3, FOXO3a, Mn-SOD, cleaved caspase-3, Bax, Bcl-2, LC3II, p62/SQSTM1 were all normalized with a GAPDH loading control.

### Cytokine Measurements

Concentrations of IL-10, IL-6, and TNFα in brain homogenates were measured using rat-specific ELISA kits per manufacturer's protocol (Thermo Fisher Scientific, Grand Island, NY, United States). Brain homogenates were separated by centrifugation at 14,000 g for 10 min at 4◦C to remove cellular debris. In addition, left ventricular myocardial concentrations of IL-6 and TNFα were analyzed by ELISA. Change in absorbance in every well was detected at 450 nm on a microplate reader. All measurements were performed in triplicate.

### Confocal and Fluorescence Microscopy

After deparaffinization, brain tissue sections were treated with 10 mM citrate buffer (pH 6.0) for antigen retrieval. After blocking with 10% normal goat serum at RT for 1 h, the sections were incubated with rabbit anti-SIRT3 antibody (1:300) and mouse anti-COXIV (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, United States), or rabbit anti-LC3B (1:400) and mouse anti-p62/SQSTM1 (1:400), respectively, at 4◦C overnight. The sections were then incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; Invitrogen, Carlsbad, CA, United States) and Alexa Fluor 550-conjugated goat anti-mouse IgG (1:500; Invitrogen, Carlsbad, CA, United States) at RT for 1 h. After washing with PBS, slides were prepared and mounted using UltraCruzTM Mounting Medium with DAPI (Santa Cruz Biotechnology, Santa Cruz, CA, United States) to detect nuclei. Images were captured on a Leica SP5 confocal microscope (Leica Microsystems, Germany) using a 40×/1.25–0.75 Plan APO oil objective, and the images were analyzed by NIH ImageJ software (version 1.51).

To assess biodistribution of systemically administered ANXA1sp, rats were anesthetized, intubated and mechanically ventilated, and the tail vein cannulated as described above. Fluorescein isothiocyanate (FITC)-conjugated ANXA1sp (FITC-QAW, MW = 905.48, >98% purity) was synthesized and purified by GenScript (Piscataway, NJ, United States) and injected via the tail vein (0.5 mg/kg). Brain tissue was harvested at 1 h after injection, fixed with 10% neutrally buffered formalin and embedded in paraffin. The 5 µm sections were deparaffinized and evaluated by fluorescence microscopy as above.

### Neurologic Evaluation

On postoperative day 1, rats underwent standardized functional neurologic testing by an observer blinded to group assignment, using an established neurologic scoring system that evaluates motor deficit (Homi et al., 2010). Briefly, rats were first placed on a 35 × 31 cm screen (grid size 0.6 × 0.6 cm) that could be rotated from horizontal (0◦ ) to vertical (90◦ ). The length of time that the rat could hold onto the screen after being rotated from 0 to 90◦ was recorded to a maximum of 15 s (0–3). Rats were then tested for balance on a horizontal wooden rod, and the time lapse before falling off the rod was recorded to a maximum of 30 s (0–3). Finally, rats underwent a prehensile traction test, and the length of time that the rat could cling to a horizontal rope was recorded to a maximum of 5 s (0–3). Animals received a score for each of the three tests. The final score was the sum of the individual test scores, with 0 the best score, and 9 the worst score.

### Statistical Analysis

Statistical analysis was performed using Prism 8 (GraphPad Software, San Diego, CA, United States). Results are expressed as mean ± standard deviation (SD). Parametric values, including physiologic values, data from western blots, ELISA were compared between groups using one-way analysis of variance (ANOVA) with post hoc Tukey's multiple comparison test, or a Student's t-test (equal variance not assumed), according to the characteristics of each experiment. Statistical significance was defined as p < 0.05.

## RESULTS

The 24 h survival rates were 75.0% (9/12) and 61.5% (8/13) for the ANXA1sp and vehicle treated groups, respectively. One rat

in vehicle group and three in ANXAsp1 group died from acute cardiac failure during the early postoperative phase. Additionally, three rats died of severe spinal cord injury and one of severe brain injury, all in the vehicle treated group. Two rats in each group died of technical failures and were excluded from analyses.

Baseline and intraoperative physiological parameters were similar between groups and are shown in **Figures 2A,B** and **Table 1**. Profound systemic hypotension (MAP < 20 mmHg) occurred during rapid exsanguination and cardiac arrest (**Figure 2A**) and was associated with severe metabolic acidosis (**Table 1**). All animals underwent the same cooling and rewarming protocol (**Figure 2B**). Rats exhibited spontaneous hypothermia (from 37◦C to 32◦C) before the onset of systemic cooling (shock-induced hypothermia). Following induction of hypothermia, pericranial temperature rapidly dropped to 23◦C at 5 min (rate of cooling 1.8◦C/min) and to 15◦C at 20 min (rate of cooling 0.85◦C/min). After 45 min of profound hypothermia (10–15◦C), animals were resuscitated and slowly rewarmed

using CPB to 34–35◦C over 80 min (rate of rewarming 0.4◦C/min).

### Systemically Administered ANXA1sp Traverses the Blood-Brain Barrier and Attenuates Neuroinflammation After EPR

Relative abundance of HMBG1, a key initiator of neuroinflammation, was assessed by Western analysis in brain homogenates. We found a 10% (p < 0.05) attenuation in cerebral expression of HMGB1 in ANXA1sp compared to HS-EPR vehicle treated animals (**Figures 3A,B**). Moreover, brain levels of the pro-inflammatory cytokines IL-6 and TNFα were reduced by 55% (p < 0.01) and 27% (p = 0.059), respectively, with ANXA1sp treatment (**Figures 3C,D**). Conversely, brain levels of the anti-inflammatory cytokine IL-10 were increased by 25% (p < 0.05) in ANXA1sp-treated compared to vehicle controls at 24 h after EPR (**Figure 3E**).

Fluorescence-conjugated peptide (FITC-QAW) was detectable in all areas of brain parenchyma at 1 h following intravenous injection (**Supplementary Figure 1**).

### Regulation of Cell Death by ANXA1sp After EPR

Acid fuchsin-celestine blue staining revealed acidophilic neurons and possible necrosis in the cortex at 24 h after EPR in vehicle-treated animals, which were reduced by 72% (p < 0.05) following ANXA1sp treatment (**Figures 4A,B**). ANXA1sp treatment was also associated with a 62% (p < 0.05) reduction in TUNEL-positive cells in the cerebral cortex, but not in the hippocampus, at 24 h after EPR (**Figures 4C,D**). This was corroborated by a 56% (p < 0.05) reduction in cerebral expression of cleaved caspase-3 in ANXA1sp-treated animals (**Figures 4E,F**).

We used Western analysis of brain homogenates to assess changes in ratios of death and survival factors. At 24 h, a modest but significant (10%, p < 0.05) increase in the relative abundance of the anti-cell death protein Bcl-2 was observed in ANXA1sp compared to vehicle-treated animals after HS-EPR (**Figures 5A,B**). Conversely, a marked 5-fold (p < 0.01) increase in expression of the pro-cell death protein Bax was seen after HS-EPR in vehicle-treated animals, with a significant 33% (p < 0.05) reduction following ANXA1sp treatment (**Figures 5A,C**). Consequently, the near 5-fold surge in Bax:Bcl-2 ratio detected after HS-EPR in vehicle-treated compared to naïve animals (p < 0.001) was attenuated by 38% (p < 0.01) with ANXA1sp treatment (**Figure 5D**).

### Modulation of SIRT3 Expression and SIRT3 Pathway by ANXA1sp

Western blot analysis in brain homogenates revealed a 33% (p < 0.01) reduction in relative abundance of the primary mitochondrial deacetylase SIRT3 (28 kDa isoform) in vehicle-treated HS-EPR compared to naïve animals, which was partially restored with ANXA1sp treatment (increased by 25%, p < 0.05) (**Figures 6A,B**). Increased SIRT3 expression with ANXA1sp treatment in both cortical and hippocampal

Results are shown as Mean ± SD; ∗p < 0.05 between groups. ANXA1sp, Annexin A1 short peptide; Hct, hematocrit; BE, base excess; End of HS, at the end of 45 min of hemorrhagic shock; End of cooling, at the end of 45 min cooling; End of CPB, at the end of cardiopulmonary bypass.

cells was further confirmed by confocal microscopy, with SIRT3 immunoreactivity colocalizing with the mitochondrial protein COXIV (**Figure 6E**). We further measured expression of FOXO3a and MnSOD, two downstream targets of SIRT3. EPR resulted in an 87% (p < 0.01) reduction in relative abundance of the transcription factor FOXO3a and a 42% (p < 0.001) reduction its regulated antioxidant enzyme MnSOD compared to naïve animals, and were both partially restored with ANXA1sp treatment (a 4-fold increase in FOXO3a expression, p < 0.05; and a 18% increase in MnSOD expression, p < 0.05) (**Figures 6A,C,D**).

### ANXA1sp Promotes Autophagy Following EPR

Western blot was performed to detect the expression of autophagy associated proteins in brain homogenates. Compared to vehicle, ANXA1sp treatment was associated with a 35% (p < 0.01) increased expression of LC3II and a 25% (p < 0.05) reduced expression of p62/SQSTM1 (**Figures 7A,B,C**). In addition, we detected expression of LC3B by immunohistochemistry, with LC3B and p62 colocalizing in cortical cells (**Figure 7D**).

### Neurologic Outcome After EPR and ANXA1sp Treatment

Finally, we evaluated neurologic changes at 24 h after EPR and ANXA1sp treatment. Compared to vehicle controls, ANXA1sp-treated animals had significantly lower neurologic severity scores, showing improved sensory-motor functions (including processing involving retrosplenial and posterior parietal cortex, **Figure 4G**).

### Systemically Administered ANXA1sp Also Attenuates Myocardial Inflammation

To evaluate the effects of ANXA1sp in organs that are not shielded by the blood-brain barrier, we assessed pro-inflammatory cytokines in left ventricular myocardial homogenates between groups. At 24 h after EPR, heart levels of IL-6 and TNFα were reduced in ANXA1sp-treated animals by 10% (p < 0.05) and 17% (p = 0.054), respectively (**Supplementary Figure 2**).

### DISCUSSION

In this study, we used a novel experimental model of acute neuroinflammation triggered by exsanguinating cardiac arrest and treated by emergent preservation and resuscitation (EPR) to test the neuroprotective effects and mechanisms of ANXA1sp administration. Together with our previous findings that ANXA1sp exerts pro-resolving effects in a rat model of CPB with global ischemia-reperfusion via deep hypothermic circulatory arrest (Zhang et al., 2017), we continue to illustrate how this small peptide impacts neuroinflammation. Here, we show that ANXA1sp treatment is superior to induction of profound hypothermia (EPR) alone by increasing cortical cell viability, promoting a favorable expression of pro-survival (Bcl-2) versus pro-death (Bax) factors in the brain, reducing caspase-3 activation, and overall improving neurological performance at 24 h after EPR.

High mobility group box 1 is a key endogenous danger associated molecular pattern which acts as a mediator of neuroinflammation resulting from a variety of conditions such as cerebral ischemia-reperfusion (Kim et al., 2006; Yang et al., 2011), septic shock, and traumatic brain injury (Parker et al., 2017). HMGB1 is actively released by neurons and glial cells upon inflammasome activation, and in turn activates two pattern recognition receptors on target cells (TLR4 and RAGE) (Gao et al., 2012), leading to NF-κB mediated production of pro-inflammatory cytokines (Paudel et al., 2018). HMGB1 also plays a pivotal role in BBB disruption (He et al., 2012; Festoff et al., 2016; Yang et al., 2018), either directly via cytokine mediated activation of metalloproteinase or via disruption of tight junctions (Gloor et al., 2001; Utech et al., 2010), although the precise mechanism remains elusive. Nonetheless, altered BBB permeability is known to then amplify neuroinflammation and neuronal excitation (Frank et al., 2015, 2016). We provide primary evidence that HS and exsanguinating cardiac arrest treated with induction of profound hypothermia and EPR

elicit neuroinflammation. Further, we show ANXA1sp treatment to favorably modulate the neuroinflammatory response, by attenuating the HS-EPR induced increase in cerebral levels of the proinflammatory alarmin HMGB1 and altering the balance of pro-inflammatory (IL-6 and TNFα) and anti-inflammatory cytokines (IL-10) (**Figures 3A–E**). This is consistent with the pleiotropic immunomodulatory functions of IL-10, to polarize the inflammatory system toward an anti-inflammatory phenotype (Ip et al., 2017), aiding in the resolution of neuroinflammation (Garcia et al., 2017). The pro-resolving effects were accompanied by improved cell survival (**Figures 4A–E**) and neurological function (**Figure 4G**). This study builds on our previous findings demonstrating beneficial effects of ANXA1sp on neuroinflammation, microglial activation, NF-κB activation

arrest/EPR and ANXA1sp or vehicle treatment. (A,B) Necrosis, detected by acid fuchsin-celestine blue, was reduced in the cerebral cortex of ANXA1sp treated rats. Arrowheads in panel A identify acidophilic neurons in the cortex and hippocampus (Hpc). (C,D) Terminal deoxynucleotidyl nick-end labeling (TUNEL) staining and quantification of apoptotic cells. Apoptosis was significantly reduced in the cerebral cortex of ANXA1sp-treated rats on postoperative day 1. Arrowheads in panel (C) identify pyknotic positive cells. Scale bar: 20 µm. (E,F) Western blot analysis of cleaved caspase-3 revealed significantly reduced levels in brain homogenates from ANXA1sp-treated animals compared to vehicle (controls). (G) Neurologic score (sensory-motor function) was significantly improved at postoperative day 1 in ANXA1sp rats. Data presented as mean ± SD (n = 5–7/group), ∗P < 0.05 and ∗∗P < 0.01 compared to vehicle (controls) analyzed by unpaired t-test (B,D,F) or Mann–Whitney U-test (G).

and postoperative neurocognitive performance following global I/RI associated with deep hypothermic circulatory arrest (Zhang et al., 2017). While our experimental model is complex, sequentially involving HS, warm cardiac arrest, induction of profound hypothermia followed by rewarming and resuscitation, the protective effects of ANXA1sp recapitulate those seen in other experimental models of cerebral I/RI (Relton et al., 1991; Gavins et al., 2007; Smith et al., 2015; Vital et al., 2016).

Systemically administered ANXA1sp was also associated with anti-inflammatory effects in the heart (reduced levels of IL-6 and TNFα) following HS-EPR. Consistent with previous reports in experimental models of regional myocardial I/RI (La et al., 2001; Qin et al., 2015), these results support the broader therapeutic roles attributed to ANXA1 and its peptide mimetics in reducing systemic inflammation and conferring organ protection from a variety of insults involving IR/I.

To better understand the added neuroprotective effects and mechanisms of action of ANXA1sp in the setting of HS-EPR with

profound hypothermia, we focused on SIRT3, a mitochondrial protein deacetylase known to be widely and abundantly expressed in most cell types in the CNS (Kim et al., 2011) and differentially regulated in brain regions and across stages of development in the rat (Sidorova-Darmos et al., 2014). SIRT3 is part of the silent information regulator of transcription (SIRT) family of NAD+-dependent protein deacetylases, which have emerged as key regulators of metabolic and energy stress-response pathways including neuroinflammation (Someya et al., 2010; Lee et al., 2013; Hattori et al., 2015; Liu L. et al., 2015; Yuan et al., 2016), but also systemic inflammation (Preyat and Leo, 2013; Vachharajani et al., 2014; Liu T. F. et al., 2015; Traba et al., 2015). Notably, SIRT3 exerts neuroprotective effects. For instance, SIRT3 overexpression rescues mutant SOD1-induced neuronal cell death (Song et al., 2013), increases neuronal lifespan under mitochondrial oxidative stress (Weir et al., 2012), protects against excitotoxic injury (Kim et al., 2011), mediates adaptive neuronal responses (resistance to oxidative stress, apoptotic cell death) to in vitro bioenergetic, oxidative and excitatory stress (Cheng et al., 2016), and protects mice against noise-induced hearing loss in vivo (Someya et al., 2010). Specifically, SIRT3 has been shown to have a major involvement in CNS ROS metabolism by regulating FOXO3a transcription factor and contributing to transactivation of key antioxidant enzymes including MnSOD and catalase (Rangarajan et al., 2015). Together, even though results from rodent studies are not completely consistent (Novgorodov et al., 2016), most data

regulated proteins after exsanguination cardiac arrest/EPR and ANXA1sp or vehicle treatment. (A,B) A significant reduction in SIRT3 (28 kDa short, processed, active form) was seen after EPR + vehicle treatment compared to naïve animals, and was rescued in ANXA1sp-treated animals. This was accompanied by similar expression changes for the forkhead box O3a (FOXO3a) transcription factor (C), known to transactivate the antioxidant enzyme manganese superoxide dismutase (MnSOD) (D). GAPDH was used as loading control (E). At 24 h postoperatively, colocalization of sirtuin-3 (SIRT3) with the mitochondrial protein cytochrome c oxidase complex IV (COXIV) in cortical and hippocampal slides from ANXA1sp-treated animals was visualized via double immunofluorescence staining and confocal microscopy. Data presented as mean ± SD (n = 3–7/group), ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared to either naïve or vehicle controls analyzed by one-way ANOVA with Tukey's multiple comparisons test.

suggest that SIRT3 expression is vital for neuronal survival and suppression of neuroinflammation following physical stressors. In this study, we noted that relative abundance of SIRT3 – the 28 kDa processed active isoform, known to possess deacetylase activity (Schwer et al., 2002; Sundaresan et al., 2008) – as well as FOXO3a and MnSOD, were all reduced in brain homogenates following HS-EPR, consistent with previous reports in experimental models of cerebral I/R (Zhao et al., 2018), but were rescued with systemic administration of ANXA1sp (**Figure 6**). HS, ischemia-reperfusion, and EPR incite neuroinflammation, and SIRT3-mediated upregulation of FOXO3a-dependent antioxidant gene expression could be

implicated in the attenuation of ROS neurotoxicity associated with acute neuroinflammation and activation of microglia. This may translate into attenuation of secondary brain injury and decreased cerebral edema. Along with downregulation of pro-apoptotic proteins, these findings suggest that ANXA1sp treatment offers an element of neuroprotection.

Modulation of protein acetylation is emerging as a therapeutic strategy to create a pro-survival and anti-inflammatory phenotype in various shock states, including lethal HS (Li and Alam, 2011). Intriguingly, promoting histone acetylation via administration of the histone deacetylase (HDAC) inhibitor valproic acid has been shown to decrease neuronal apoptosis, neuroinflammation, and brain lesion size while improving neural plasticity and resulting in faster neurocognitive and neurologic recovery in pre-clinical models of traumatic brain injury, HS, and polytrauma (Nikolian et al., 2017, 2018; Chang et al., 2019). An imbalance in histone acetyltransferase/deacetylase activity has been reported in both HS (Lin et al., 2006) and neurodegeneration (Rouaux et al., 2003). Valproic acid administration results in epigenetic and posttranslational modifications that induce differential gene expression (Dekker et al., 2014; Bambakidis et al., 2016), metabolic changes (Hwabejire et al., 2013) and alterations of the proteome. It appears that promoting acetylation of histones and other target transcription factors through the use of HDAC inhibitors (like valproic acid), as well as promoting mitochondrial protein deacetylation via induction of SIRT3 (in this study using ANXA1sp) can both exert neuroprotective effects in the setting of lethal HS. The precise mechanisms by which modulation of protein acetylation results in attenuation or resolution of neuroinflammation remain to be elucidated.

We found that ANXA1sp treatment promoted cerebral autophagy following EPR. This was accompanied by increased SIRT3 protein expression, reduced cell apoptosis and improved neuro severity scores. Importantly, upregulation of SIRT3 in response to ANXA1sp treatment was associated with increased expression of LC3II and a reduced level of p62/SQSTM1, indicating increased autophagic flux (**Figure 7**). These results are consistent with previous reports implicating SIRT3 as an activator of autophagy in neuronal cells (Dai et al., 2017; Yan et al., 2018) and macrophages (Liu et al., 2018) under various stress conditions in vitro, and in vivo studies showing sirtuin activation to be protective in spinal cord injury, brain trauma, and cerebral ischemia (He et al., 2017; Zhao et al., 2017). A schematic representation of the relationships between ANXA1sp, the SIRT3 pathway and neuroinflammation in the context of cerebral I/RI is shown in **Figure 8**.

Although the therapeutic effects of hypothermia in HS in general, and those of EPR for exsanguinating cardiac arrest in particular, have been previously reported in large animal experiments (Alam et al., 2002, 2004, 2005, 2006a,b; Chen et al., 2005; Wu et al., 2006), benefits of the clinically realistic rat model described in this study include lower costs to test efficacy of selective pharmacological neuroprotective interventions (such as the pro-resolving ANXA1sp) in enhancing clinical outcomes after EPR, as well as availability of molecular biology tools to understand the mechanisms at play. To increase clinical relevance, we modified previously described rodent models of EPR (Drabek et al., 2007a,b) in several ways: first, we introduced a prolonged period of HS followed by induction of fibrillatory cardiac arrest; second, we rapidly induced profound hypothermia via aortic flushing of a relatively low volume (1.5–2 fold the estimated blood volume) of ice-cold saline-based solution using a light weight cycling system at 20 ml/min; finally, we re-transfused salvaged autologous blood during resuscitation, thereby avoiding additional confounding effects from exposure to allogeneic blood transfusions. In this severe model, administration of a pro-resolving ANXA1 bioactive peptide during EPR was superior to EPR alone in attenuating neuroinflammation, increasing pro-survival protein expression and autophagic flux.

Several limitations of this study follow. HS was achieved by closely controlled exsanguination of blood, which was salvaged and re-transfused. Although we designed the model to closely mimic the clinical scenario, HS inevitably requires surgical bleeding control and transfusion of allogeneic blood products, which would significantly amplify the inflammatory response. Rats serve as imperfect surrogates for human subjects, and only male young adult animals were used. An injured but un-resuscitated control group was not used, as the survival without resuscitation is extremely poor. Instead, we used a naïve uninjured control group, to allow assessing whether ANXA1sp effects are restorative or de novo. We have not measured directly markers of resolution of inflammation (neutrophil apoptosis/efferocytosis, functional macrophage switches), or microglial activation. However, microglial activation was attenuated by ANXA1sp treatment in our previously reported rat deep hypothermic circulatory arrest study (Zhang et al., 2017). This study focuses on ANXA1sp-induced upregulation of the SIRT3 pathway, and thus we have not explored the traditional mechanisms implicated in the pro-resolving effects of Annexin A1 and its bioactive peptides (via the FPR2/ALX receptor pathway). The neuroinflammatory response to hypothermic cardiac arrest is not uniform, displaying important spatio-temporal differences between brain regions (Drabek et al., 2015). Our investigation focused on the cortex and hippocampus as two vulnerable brain regions, but other areas displaying robust early neuroinflammatory responses (e.g., the striatum) were not studied. Temporally, our observation period was limited to 24 h, and longer-term evaluations are required to fully assess ANXA1sp's protective effects, including neurocognitive tests. Although previous reports from experimental focal cerebral I/RI in the rat suggest neutrophil accumulation in the infarct area (which correlates closely with cortical lesion size) to be maximal at 24 h post-reperfusion (Zhang et al., 1994), it should be noted that our results may represent a delay rather than elimination of neuronal death in the selectively vulnerable brain regions studied.

## CONCLUSION

Using a clinically relevant rodent model of lethal HS and EPR-induced acute neuroinflammation, we show that administration of a pro-resolving ANXA1 peptide mimetic results in a pro-survival phenotype with attenuated cortical cell death, reduction of several common functional biomarkers of neuroinflammation (HMGB1, IL-6, and TNFα), and improved early neurological outcomes, compared to induction of profound hypothermia (EPR) alone. The protective effects were associated with increased expression of SIRT3 and corresponding upregulation of the FOXO3a-MnSOD antioxidant pathway. In addition, we note increased cerebral autophagy in ANXA1sp treated animals. Further studies need to refine the mechanisms involved and evaluate the use of SIRT3-activating drugs to resolve acute neuroinflammation and attenuate the uncontrolled production of ROS by activated microglia, as well as the long-term benefits of the proposed therapeutics.

### DATA AVAILABILITY

fnins-13-00608 June 13, 2019 Time: 17:39 # 12

All datasets generated for this study are included in the manuscript and/or the **Supplementary Files**.

### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the National Institutes of Health for animal care (Guide for the Care and Use of Laboratory Animals, Health and Human Services, National Institute of Health Publication No. 86-23, revised 1996). The protocol was approved by the Duke University Animal Care and Use Committee.

### AUTHOR CONTRIBUTIONS

ZZ, QM, J-KS, and MP designed and performed the research. TV, CL, JM, and NT contributed new reagents and analytic tools. ZZ,

### REFERENCES

QM, QQ, and MP analyzed the data. QM, ZZ, and MP wrote the manuscript. QQ, JM, and NT provided the critical edits to the manuscript. All authors read and approved the final draft of the manuscript.

### FUNDING

This work was supported by grants from the American Heart Association (AHA)-11BGIA (ZZ), 2009 SCA-IARS Mid-Career Grant (QM), NIH R01 HL092071 (MP), and the Department of Anesthesiology, Duke University Medical Center.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnins. 2019.00608/full#supplementary-material

preservation and resuscitation for cardiac arrest from trauma clinical trial. J. Trauma Acute Care Surg. 83, 803–809. doi: 10.1097/TA.000000000000 1585

fnins-13-00608 June 13, 2019 Time: 17:39 # 14

pathway. Neuroscience 348, 241–251. doi: 10.1016/j.neuroscience.2017. 02.027

Zhao, H., Luo, Y., Chen, L., Zhang, Z., Shen, C., Li, Y., et al. (2018). Sirt3 inhibits cerebral ischemia-reperfusion injury through normalizing Wnt/β-catenin pathway and blocking mitochondrial fission. Cell Stress Chaperones 23, 1079–1092. doi: 10.1007/s12192-018- 0917-y

**Conflict of Interest Statement:** ZZ, QM, NT, and MP are co-inventors on patents for the use of Annexin A1 peptides for activation of sirtuins and to attenuate neuroinflammation. NT is Associate Editor for Frontiers in Immunology.

The remaining 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.

Copyright © 2019 Ma, Zhang, Shim, Venkatraman, Lascola, Quinones, Mathew, Terrando and Podgoreanu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Scopoletin Suppresses Activation of Dendritic Cells and Pathogenesis of Experimental Autoimmune Encephalomyelitis by Inhibiting NF-**κ**B Signaling

*Fei Zhang†, Yuan Zhang†, Ting Yang†, Ze-Qing Ye, Jing Tian, Hai-Rong Fang, Juan-Juan Han, Zhe-Zhi Wang\* and Xing Li\**

### *Edited by:*

*Pietro Giusti, University of Padova, Italy*

#### *Reviewed by:*

*Jose Martinez-Navio, University of Miami Health System, United States Narendra Prasad Singh, University of South Carolina, United States*

#### *\*Correspondence:*

*Zhe-Zhi Wang<EMAIL_ADDRESS>Xing Li<EMAIL_ADDRESS>
*†These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology*

*Received: 27 February 2019 Accepted: 08 July 2019 Published: 02 August 2019*

#### *Citation:*

*Zhang F, Zhang Y, Yang T, Ye Z-Q, Tian J, Fang H-R, Han J-J, Wang Z-Z and Li X (2019) Scopoletin Suppresses Activation of Dendritic Cells and Pathogenesis of Experimental Autoimmune Encephalomyelitis by Inhibiting NF-κB Signaling. Front. Pharmacol. 10:863. doi: 10.3389/fphar.2019.00863*

*National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, The Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, The Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi'an, China*

Scopoletin, a phenolic coumarin derived from many medical or edible plants, is involved in various pharmacological functions. In the present study, we showed that Scopoletin effectively ameliorated experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), through novel regulatory mechanisms involving inhibition of NF-κB activity in dendritic cells (DCs). Scopoletin treatment significantly improved the severity of the disease and prominently decreased inflammation and demyelination of central nervous system (CNS) in EAE mice. Disease alleviation correlated with the downregulation of major histocompatibility complex (MHC) class II, CD80 and CD86, expressed on DCs of CNS or spleens, and the infiltration and polarization of encephalitogenic Th1/Th17 cells. Consistent with the *in vivo* data, Scopoletin-treated, bone marrow-derived dendritic cells (BM-DCs) exhibited reduced expression of MHC class II and costimulatory molecules (e.g., CD80 and CD86) and reduced NF-κB phosphorylation. These findings, for the first time, demonstrated the ability of Scopoletin to impair DC activation, downregulating pathogenic Th1/Th17 inflammatory cell responses and, eventually, reducing EAE severity. Our study demonstrates new evidence that natural products derived from medical or edible plants, such as Scopoletin, will be valuable in developing a novel therapeutic agent for MS in the future.

Keywords: scopoletin, experimental autoimmune encephalomyelitis, multiple sclerosis, dendritic cells, NF-**κ**B signaling

### INTRODUCTION

Multiple sclerosis (MS) is a T-cell-mediated chronic autoimmune disease featured by neuroinflammation and demyelination in the central nervous system (CNS), affecting 2.5 million people worldwide, and the incidence continues to increase (Hemmer et al., 2015). Experimental autoimmune encephalomyelitis (EAE) is a well-known animal model of MS that is used to study the underlying mechanism and that provides the theoretical basis for developing new therapies of MS (Procaccini et al., 2015; Lassmann and Bradl, 2016).

Dendritic cells (DCs) are antigen-presenting cells that connect the innate and adaptive immune response. Depending on the variety activation status, DCs can either activate or modulate naive T lymphocytes. When DCs are activated, costimulatory molecules are upregulated and produce cytokines that drive T-cell priming and effector differentiation (Amodio and Gregori, 2012). In the absence of activation, antigen presentation by steady-state DCs can result to T-cell unresponsiveness and tolerance (Besusso et al., 2015). Therefore, modulation of DCs has been shown to be a promising area for the treatment of autoimmune disease. For instance, it was proved that DCs treated with natural compounds or synthetic drugs blocked the inflammatory signal and halted disease development (Ginwala et al., 2016; Kornberg et al., 2018). DC-induced suppression is mainly dependent on inhibiting Th17 cell differentiation or promoting Treg cell generation (Thome et al., 2014a; Thome et al., 2018). It is known that naive CD4+ T cells can develop into various effector subsets containing Th1, Th2, Th17, and Treg cells, etc.; among these, Th17 cells are crucially related to the pathogenesis of MS/EAE (He et al., 2017; Malik et al., 2017). Suppression of the cellular response, Th17 cells in particular, can therefore act as a therapeutic target for controlling autoimmune and inflammatory diseases.

NF-κB is a broad transcription factor which can regulate various biological functions containing inflammation, immunity, cell growth, and survival, and its signaling pathway plays a role in DC activation and maturation (Ardeshna et al., 2000). Phosphorylation of NF-κB in innate immune cells induces inflammation reaction, cytokine secretion, and tissue damage, thus promoting to the progress of autoimmune diseases including EAE (Lu et al., 2013). In addition, immature DCs respond to danger signals or antigen and, when mature, produce abundance of proinflammatory cytokines, such as type I interferons (IFN). Subsequently, mature DCs present antigens to T lymphocytes to stimulate a sustained immune response, given the interferon regulatory factor (IRF) family's important role in DC development and maturation. For example, IRF-7 is necessary for producing IFN-α and IFN-β, and it serves as the prime regulator of type-I interferon-dependent immune responses (Honda et al., 2005; Gabriele and Ozato, 2007; Yasuda et al., 2007).

Scopoletin is a phenolic coumarin extracted from many medical plants, including *Erycibe obtusifolia*, *Aster tataricus*, *Foeniculum vulgare*, and *Artemisia iwayomogi*, as well as some edible plants, such as *Lycium barbarum* and *Morinda citrifolia* (Dou et al., 2013; Lee et al., 2014; Forino et al., 2016; Shalan et al., 2016)*.*  Scopoletin has been shown to possess anti-inflammatory, antioxidant, antidepressant, hypouricemic, and neuroprotective effects (Ding et al., 2005; Basu et al., 2016). Moreover, a recent report suggests that Scopoletin is effective in an adjuvant-induced arthritis rat model, increasing the possibility of its application as a therapeutic agent for autoimmune diseases (Pan et al., 2010). However, little is known about the regulatory properties and underlying mechanism of Scopoletin relative to innate immune cells such as DCs and its effect on the pathogenic progression of MS and EAE. In light of the pharmacological profile of Scopoletin and its reported immunoregulatory properties, we hypothesized that Scopoletin might have a therapeutic effect on the animal model of MS and EAE. The purpose of this study was, therefore, to test the therapeutic activity of Scopoletin on EAE and to elucidate the underlying therapeutic mechanisms of its action, as part of our ongoing search for development of immunomodulation medicine extracted from medical plants or edible plants (Zhao et al., 2018).

### MATERIALS AND METHODS

### Reagents and Animals

Scopoletin was purchased from Sigma (Sigma-Aldrich, St. Louis, MO). C57BL/6 mice, female, 8 weeks old, were obtained from the Fourth Military University (Xi'an, China). Studies were carried out by a protocol approved by the Animal Ethics Experimental Committee at of Shaanxi Normal University, and all experiments were operated strictly according to approved institutional guidelines and regulations.

### EAE Induction and Treatment

EAE was induced by subcutaneously injecting an emulsion containing 200 μg Myelin Oligodendrocyte Glycoprotein35–55 (MOG35–55) (Genescript, Piscataway, NJ), complete Freund's adjuvant (CFA) (Sigma-Aldrich), and 5 mg/ml *Mycobacterium tuberculosis* H37Ra (Difco Laboratories, Lawrence, KS), and intraperitoneally (i.p.) injected 200 ng pertussis toxin (Sigma-Aldrich) in PBS on day 0 and 2 postimmunization (p.i.) (Zhang et al., 2015). Disease progression of EAE mouse was evaluated daily in a blind way by two researchers, scoring standard for EAE disease progression as follows: 0, no symptoms; 0.5, stiff tail; 1, limp tail; 1.5, limp tail and waddle; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of one other limb; 3, complete paralysis of two hind limbs; 4, moribund; and 5, death. (Li et al., 2016). Scopoletin was prepared in dimethylsulfoxide (DMSO) at 100 mg/ml for stock, which was further diluted 20 times with PBS as a working solution. DMSO (5%) was dissolved in PBS as vehicle. Vehicle or Scopoletin (50 mg/kg) was injected i.p. daily from the day 0 p.i.

### Histopathology

Mice were killed at day 20 postimmunization (p.i.). To assess CNS histopathological, mice were perfused through the heart with cold PBS; spinal cord was then fixed with 4% paraformaldehyde, cut into 7-μm sections, and stained with hematoxylin and eosin (H&E) or Luxol fast blue (LFB) for evaluation of inflammation or demyelination, respectively. Slides were evaluated in a blinded fashion for inflammation using a 0–3 scale as description, and two investigators selected 10 areas in the white matter of the spinal cord and scored for slides, evaluation methods, and criteria as description followed our previous studies (Li et al., 2016). The white matter area was manually outlined, and the Image-Pro Plus software was used to calculated areas (%) of demyelination for evaluation of demyelination (Li et al., 2017). Demyelination area percentage was quantified as the area without LFB staining (white) in white matter divided by the area white matter of spinal cord slices. Fixed spinal cords were embedded in optimum cutting temperature (OCT) solution (Tissue-Tek, Sakura Finetek, Japan) and then sectioned coronally cut into 12 μm. Finally, sections stained with primary and secondary antibodies. Results were acquired by Nikon Eclipse E600 fluorescent microscopy (Melville, NY).

### Mononuclear Cells (MNCs) and BMDCs Preparation

Spleens were mechanically grinded and filtrated with a 100 µm cell strainer (Falcon, Tewksbury, MA) to harvest splenic cells, subsequently incubated with red blood cell lysis buffer (Biolegend, San Diego, CA) for 60 s. After washing cells with cold PBS, cells were stimulated with MOG35–55 *in vitro*. To acquire CNS infiltrating cells, the protocol was followed as previously described (Li et al., 2017). To acquire the bonemarrow-derived dendritic cells (BM-DCs), femurs and tibias were separated from the naive adult C57BL/6 mice, and the cells were blown out of the bone marrow with a syringe full of the precooled PBS. After filtering with 100-µm cell strainer, the harvested cells were subsequently cultured with PRMI1640 containing granulocyte colony-stimulating factor (GM-CSF) cytokine (10 ng/ml) until the ninth day to obtain mature BM-DCs. Dendritic cells are cultured in medium without GM-CSF cytokine after ninth day.

### Drug Preparation for Cell Experiments

Stock solution of Scopoletin was dissolved into the concentration of 30 mM for cell experiments with DMSO.

### T-cell Proliferation Assay

Splenic CD4+ T cells were isolated from the naive adult C57BL/6 mice using a CD4+ T Cell Isolation Kit (Miltenyi Biotec) and labeled with CFSE. Both BM-DCs and T cells were cocultured at 1:10 ratio in the presence of 10 μg/ml of MOG35–55 (Genescript, Piscataway, NJ) and 100 ng/ml of lipopolysaccharide (LPS) (Sigma-Aldrich). T-cell proliferation was measured by analyzing CFSE intensity by flow cytometry after 72 h.

### ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

DCs culture medium or supernatants from splenocytes were harvested at 18 or 72 h, and sandwich ELISA was performed to determine the concentrations of cytokine production by ELISA kits (R&D Systems, Minneapolis, MN). according to the manufacturer's instructions, including IFN-γ, IL-17, GM-CSF, IL-1β, IL-6, and IL-23. Briefly, the capture antibody was used to precoat overnight. Then, the plate was washed with wash buffer (R&D Systems, Minneapolis, MN). for three times. Blocking with 1% BSA in PBS for 1 h, gradient standard, and samples were added to the plate for 2 h incubation. Next, the samples and standard were removed clearly, and the detection antibody was incubated for 2 h. The wells were again washed, a TMB substrate solution was added to the wells, and color developed in proportion to the amounts of cytokines. The Stop Solution changed color from blue to yellow, and the intensity of the color was measured at 450 nm. A standard curve was run for each microwell plate. According to the standard curve, the concentration of cytokine was determined.

### Flow Cytometry Analysis

For surface-marker staining, cells were washed with PBS and incubated 30 min at 4°C with anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-CD80, and anti-CD86 (BD Biosciences, San Jose, CA) or isotype control Abs according to the manufacturer's instructions to dilute. With 25 or 10 μg/ml MOG35–55 to stimulate splenocytes or infiltrating MNCs in CNS for 72 h or overnight to analyze Th1 and Th17 response to MOG35–55. For intracellular staining, cells were stimulated with 50 ng/ml PMA, 500 ng/ml ionomycin, and GolgiPlug for 5 h. For blocking the nonspecific staining, 1 μg Fc Block reagent for the FcγII/III receptor (BD Biosciences, San Jose, CA) was addded to 1 × 106 cells in 100 μl staining buffer (BD Biosciences) and incubated for 15 min at 4°C. The cells are then washed. For surface staining, cells were incubated with anti-CD4, anti-CD8, anti-CD11b, anti-CD11c, anti-MHC class II, anti-CD80, and anti-CD86. Then, cells were fixed and permeabilized by Fix & Perm Medium (Invitrogen, Waltham, MA), using Abs anti-IL-17, anti-IFN-γ, or anti-GM-CSF (BD Biosciences, San Jose, CA) and were incubated overnight at 4°C to stain intracellular cytokines. FACS Aria was used for flow cytometric analysis (BD Biosciences, San Jose, CA). The results were evaluated by FlowJo (Treestar, Ashland, OR).

### Western Blot

Mature BM-DCs were placed in six-well plates with a density of 1.5 × 106 cells/ml and stimulated with 100 ng/ml LPS to induce DC activation. Meanwhile, 100 μM Scopoletin was added to DCs. After 18 h, proteins were extracted from BM-DCs as described. The total protein contents were measured by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, MA). DC proteins were equally loaded onto SDS-PAGE, electrophoresed, transferred onto PVDF membranes, and after blocking with 5% nonfat milk in TBS, the PVDF membranes were incubated with primary antibody over 12 h at 4°C (Li et al., 2018), including JNK, p-JNK, NF-κB, p-NF-κB, p38, p-p38 (cell signal technology), IRF-7, ERK1/2, p-ERK1/2 (Abcam, Cambridge, UK), and p-IRF-7 (Signalway Antibody, College Park, MD). After washing, the membranes were incubated with 0.5% horseradish peroxidase-labeled IgG. Temembrane-bound antibodies were detected with an Immolilon™ Western chemiluminescent HRP Substrate (Millipore, Billerica, MA) and analyzed with an ImageJ (ChemiDoc XRS System; Bio-Rad).

### Quantitative RT-PCR

Total RNA was extracted by RNeasy Mini Kit (Qiagen, Valencia, CA). cDNA was subsequently synthesized by QuantiTect® Reverse Transcription Kit (QIAGEN). Quantitative RT-PCR was performed with the QuantiFast™ SYBR® Green PCR Kit (QIAGEN) under standard thermocycler conditions (Applied Biosystems, Foster City, CA). Quantification was performed by normalization with housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and relative expression level as fold changes *via* the 2−ΔΔCt method. Primers are summarized in **Supplementary Table 1.**

### Statistical Analysis

Results are described as mean ± SD. Statistical differences between two or multiple groups were implemented using Student's *t*-test or the ANOVA. *P* < 0.05 was considered statistically significant. All statistical analyses were performed by GraphPad Prism 6 (GraphPad, La Jolla, CA).

### RESULTS

### Scopoletin Suppressed the Development of EAE

Previous studies have shown that Scopoletin is safe at the dosage of 10–100 mg/kg daily with no other side effects in *in vivo*  experiment (Jamuna et al., 2015; Basu et al., 2016; Zeng et al., 2017). To assess the effect of Scopoletin on the disease course of EAE, EAE mice were i.p. injected Scopoletin (50 mg/kg) from day 0 p.i. Vehicle-treated EAE mice demonstrated a progressive increase in disease severity after day 10 p.i., while the Scopoletintreated group showed a marked decrease in mean disease course from day 14 to 20 p.i. (**Figure 1A**). Consistent with the disease development, mice treated with vehicle demonstrated abundant inflammatory cell infiltrated in the white matter compared with the Scopoletin-treated EAE mice (**Figures 1B, C**). At the same time, there was a dramatic decrease in demyelination areas in the spinal cord of Scopoletin-treated EAE mice compared to control (**Figures 1D, E**). In the therapeutic regimen, Scopoletin administration starting from disease onset (day 11 p.i.) effectively ameliorated EAE progression (**Supplementary Figure 1**). Together, these results indicate that Scopoletin effectively suppressed EAE pathogenesis.

### Scopoletin Treatment Inhibited DC Activation *In Vivo*

To assess the impact of Scopoletin on peripheral immune responses in EAE, splenocytes were harvested from EAE mice treated with Scopoletin or vehicle at day 20 p.i. and cultured in the presence of MOG35–55 (25 μg/ml) for 72 h. Expression of costimulatory molecules on CD11c+ DCs was examined by flow cytometry, which showed significantly decreased levels of MHC class II, CD80 and CD86 in Scopoletin-treated EAE mice compared to the vehicle group (**Figures 2A–D**). These results indicate that Scopoletin inhibited DC activation during the development of EAE.

Consistent with the observed effect of Scopoletin in EAE and DC activation, there was a remarkable diminishment in the percentages of CD4+ and CD8+ T cells in the periphery (**Figures 2E, F**). The percentages of Th1 and Th17 cells were also determined by intracellular staining and showed an obvious inhibition under Scopoletin treatment (**Figures 2G–I**). When stimulated with MOG35–55 *ex vivo*, splenocytes of Scopoletin-treated EAE mice secreted significantly lower levels of MOG-induced IFN-γ, IL-17, and GM-CSF (**Figure 2J**). When EAE symptoms is at its peak, spleen cells were harvested at 15 days p.i. and stimulated with MOG35–55 for 3 days. The concentration of IFN-γ, IL-17, and GM-CSF in cells culture supernatants were significantly decreased in Scopoletin-treated group (**Supplementary Figure 3A**). Furthermore, the inflammatory cytokine expression levels from sera were also detected by ELISA and RT-PCR. Although we did not detected protein expression by ELISA due to limited samples from sera, RT-PCR results showed that IL-17A, GM-CSF, and IL-1β expression were significantly decreased after Scopoletin treatment (**Supplementary Figure 3B**). These results indicated that Scopoletin alleviated the disease development of EAE by altering expression of costimulatory molecules and reduced activation of DCs, inhibiting Th1 and Th17 cells development, as well as suppressing proinflammatory cytokine secretion.

### Scopoletin Treatment Alleviated CNS Inflammation

In the CNS, effector T cells produce a number of molecules that recruit inflammatory leukocytes, ultimately leading to demyelination (Thome et al., 2014b). To evaluate the effect of Scopoletin on EAE-related CNS pathology, thoracic spinal cords were harvested from Scopoletin- or vehicle-treated EAE mice. Immunofluorescence evaluation of CNS tissues showed that Scopoletin markedly decreased infiltration of CD45+ cells compared with control mice (**Figures 3A, B**). The number of MNCs in the CNS of Scopoletin-treated mice was dramatically decreased compared to the vehicle group (**Figure 3C**). Furthermore, flow cytometry was used to analyze CD45+ (leukocyte), CD11c+ (DC), and CD11b+ (macrophage/ microglia) cell populations. Results showed that the percentages and absolute numbers of these cells were significantly reduced in the CNS of Scopoletin-treated mice compared with vehicletreated EAE mice. In addition, Scopoletin-treated mice have lower percentages and absolute numbers of CD4+ and CD8+ T cells in the CNS (**Figures 3D–F**).

Emerging data suggest that both interferon-γ-producing (Th1) and interlukin-17-producing Th17 (Th17) cells contribute to CNS autoimmunity and mediate disease pathogenesis in EAE (El-Behi et al., 2010). In addition, although various cell types produce GM-CSF, myelin-specific CD4+ T cells are essential to EAE development (Rasouli et al., 2015). To explore potential mechanisms underlying the therapeutic effect of Scopoletin in EAE, we measured the numbers of Th1, Th17, and pathogenic CD4+GM-CSF+ (Rasouli et al., 2015) cells in the CNS of EAE mice. As shown in **Figures 4A–C**, the percentages and absolute numbers of Th17 cells were reduced in the Scopoletin-treated group in comparison to the vehicle-treated mice. While the percentages of CD4+IFN-γ+ and CD4+IL-17+ T cells were not significantly changed, the absolute numbers of these cells were decreased in the Scopoletin-treated group (**Figures 4A–C**) due to the reduced total numbers of CD4+ T cells in the CNS after Scopoletin treatment

FIGURE 1 | Scopoletin suppressed the development of experimental autoimmune encephalomyelitis (EAE). (A) Mice were injected i.p. with vehicle or Scopoletin (50 mg/kg) daily from day 0 p.i. to day 20 p.i. experimental autoimmune encephalomyelitis (EAE) development were evaluated and recorded daily following a 0–5 scale. (B) Hematoxylin and eosin (H&E), below is high magnification of H&E analysis of the spinal cord sections, and (D) Luxol fast blue staining, below is high magnification of Luxol fast blue (LFB) analysis of the spinal cord sections. (C) Mean score of inflammation in H&E staining. (E) Quantification of demyelination area was analyzed by Image-Pro Plus software. Scale bar = 100 µm. *n* = 8 mice each group. Symbols represent mean ± SD, \*\* *P* < 0.01 and \*\*\* *P* < 0.001, determined by two-way ANOVA (A), or Student's *t*-test (B, D, F). Data are combined from three independent experiments.

(**Figures 3D–F**). CD4+GM-CSF+ T cells infiltration in the CNS was found in these groups. Our data therefore suggest that the effect of Scopoletin in alleviating EAE development may be due to an alteration in the differentiation of Th1, Th17, and CD4+GM-CSF+ T cells (**Figures 4A–C**).

### Scopoletin Inhibited the Activation of Murine BM-DCs *In Vitro*

We have shown the inhibitory effect of Scopoletin on DC activation *in vivo* in EAE mice (**Figures 2A–D**); here, we evaluated its direct effects on DCs *in vitro*. To that end, BM-DCs were generated. Upon

DCs, CD4+, and CD8+ T cells were measured by flow cytometry. (F) Absolute numbers of different subtypes of CNS infiltrates were calculated by multiplying the percentages of these cells with total numbers of MNCs in each spinal cord and brain tissue. Figures (A, D) are one representative of three independent experiments. Data were determined by Student's *t*-test, and symbols represent mean ± SD, \**P* < 0.05, \*\**P* < 0.01, and \*\*\**P* < 0.001.

LPS stimulation, DCs expressed relatively high levels of MHC class II, CD80, and CD86, the markers of DCs activation, and their expression was significantly inhibited by Scopoletin treatment (**Figures 5A–F**). Scopoletin, thus, showed a potential for restricting DCs activation and inhibiting expression of costimulatory molecules in BM-DCs cultures.

by flow cytometry. (C) Absolute numbers of these cells were calculated by multiplying their percentages of cytokine-producing cells and total CD4+ cell numbers in each CNS. Data are representative of three independent experiments. Symbols represent mean ± SD (n = 5 each group), \*\**P* < 0.01 and \*\*\**P* < 0.001, determined by Student's t-test.

To further examine the inhibitory effect of Scopoletin on BM-DCs activation, we then determined the mRNA levels of multiple inflammatory-associated genes expressed by BM-DCs, such as IL-6, IL-12p35, IL-12/IL23p40, IL-23p19, IL-1β, and TNF-α. Previous studies have shown that IL-12 is probably related to Th1 cells differentiation, and IL-23 and IL-6 are crucial to the Th17 cells differentiation (Luckheeram et al., 2012). Importantly, Scopoletin treatment caused a remarkably inhibition in the expression of these genes except for IL-1β (**Figure 6A**). ELISA analysis indicated the suppressive effects of Scopoletin on the secretion of IL-6, IL-23, and TNF-α by BM-DCs (**Figure 6B**). These findings suggest that the effect of Scopoletin treatment may be through inhibiting production of proinflammatory cytokines and expression of costimulatory molecules in DCs.

To test the effects of Scopoletin in DC-induced T-cell proliferation assays, we performed the DC and T-cell coculture and quantified the T-cell proliferation with CFSE; the results showed that Scopoletin reduced the T-cell proliferation *via* DCs induction (**Supplementary Figure 3C**).

### Scopoletin Inhibited NF-**κ**B Signaling During LPS-Stimulated DC Activation *In Vitro*

It is well known that NF-κB and MAPK signaling pathways play a central role in the coordinated regulation of gene expression during DC activation (Lu et al., 2014; Baratin et al., 2015). Thus, expression of the total and phosphorylation forms of IRF-7, p38, JNK, and NF-κB in BM-DCs was determined by Western blot. The level of phosphorylation of NF-κB was significantly lower in Scopoletin-treated BM-DCs when compared to DCs stimulated with LPS alone. In contrast, expression of IRF-7, p38, and JNK were not markedly affected by Scopoletin treatment (**Figures 7A**, **B**). In addition, BM-DCs were pretreated with SN50 (20 μM) for 1 h, a specific NF-κB peptide inhibitor (Lin et al., 1995), before LPS (100 ng/ml) stimulation and Scopoletin (100 μM) treatment, after 4 h cells were collected, and then, expression level of cytokines were determined by quantitative real time PCR (qRT-PCR). The expression of NF-κB downstream genes such as IL-1β and TNF-α was significantly decreased by Scopoletin and SN50 treatment. When combined with SN-50 and Scopoletin, TNF-α expression

Statistical data are expressed as mean ± SD of three independent experiments. \**P* < 0.05 by Student's *t*-test.

level was significantly enhanced compared with Scopoletin only (**Supplementary Figure 3D**). These data suggest that Scopoletin may exert its action and ameliorate the progression of EAE specifically through inhibiting NF-κB signaling.

### DISCUSSION

In this study, we provide evidence, for the first time, of the antiinflammatory activities and protective effects of Scopoletin in the mouse model of MS, EAE. Our data present a promising bioactive drug, derived from a natural source, for the treatment of MS and possibly other autoimmune diseases.

In addition, Scopoletin is also the main coumarin constituent occurring in the stems of *Erycibe obtusifolia*, a classical medicinal plant that has demonstrated various biological activities mainly used for rheumatoid arthritis with a long history (Pan et al., 2010). Scopoletin has been reported to have anti-inflammatory properties, e.g., inhibition of lymphocyte proliferation and reduction of cytokine production (Arcos et al., 2006). *In vitro* studies showed that Scopoletin inhibited proinflammatory cytokine secretion from RAW 246.7 and HMC-1 cell lines (Moon et al., 2007; Connell et al., 2017). Scopoletin also exhibited antiinflammatory, antioxidant, and antiacetylcholinesterase potential against Alzheimer's disease (Garcia-Morales et al., 2015). However, its role is unknown in the regulation of APC functions in an autoimmune disease such as MS.

In autoimmune diseases, DCs as professional APCs have a potent ability to trigger naive T-cell reaction and activate an

autoreactive response (Thompson et al., 2018), and decreasing CD80/86 prevented T-cell activation and Th-cell differentiation by inhibiting the costimulatory second signal (Grimbert et al., 2011; Yamamoto et al., 2012). Our results showed that the therapeutic effects of Scopoletin were largely dependent on the suppression of DC activation. Scopoletin reduced the expression of costimulatory molecules CD80 and CD86 as well as MHC class II *in vivo* and *in vitro.* Furthermore, in MS patients and EAE mice, there is a marked increase in the levels of IFN-γ and IL-17, signature cytokines for Th1 and Th17 cells, respectively (Arellano et al., 2017). As drivers of autoimmune response, pathogenic Th1 and Th17 cells are therefore considered to be the main culprits in MS and EAE (Grigoriadis et al., 2015). Our data demonstrated that Scopoletin treatment remarkably decreased the percentages of Th1 and Th17 cells. On the other hand, GM-CSF, which can be produced by both Th1 and Th17 cells, is required for encephalitogenicity of these pathogenic T-cell subsets (Codarri et al., 2011; El-Behi et al., 2011). Reduced numbers of CD4+GM-CSF+ T cells in the spleen and CNS following Scopoletin treatment would, therefore, be an important mechanism underlying the effect of Scopoletin on EAE. When the direct effect of Scopoletin on Th1 and Th17 cell differentiation was further investigated *in vitro*, our results showed that differentiation of Th1 and Th17 was not significantly altered by Scopoletin treatment (**Supplementary Figure 2**). Furthermore, the inhibitory effect of Scopoletin treatment on Th1/Th17 cells *in vivo* in EAE mice must therefore be through an indirect mechanism, i.e., by inhibition of DC activation during EAE progression.

It has been well documented that IL-6 plays a key role in determination of Th17 polarization, and knockout of these cytokines completely inhibits EAE development (Giralt et al., 2013). In addition, IL-23 is important for expanding and maintaining the developing Th17 population (Smith and Colbert, 2014) and is required for differentiation of Th17 cells and the pathogenic role of these T cells (El-Behi et al., 2011). Given that DCs are major producers of these cytokines, differentiation of naive T cells into Th17 phenotype is dependent on activated DCs (Agalioti et al., 2018). IL-6 signaling plays a key role in the differentiation of CD4 + T cells, in combination with TGF-β, IL-1β, IL-23, and IL-6, promotes the differentiation of Th17 cells by activating transcription factors containing signal transducers and activators of transcription 3 (STAT3), retinoic acid-related orphan receptors gamma t (RORγt) (Yao et al., 2014). Here, we found that Scopoletin treatment significantly decreased expression of activation and costimulation markers of DCs and their IL-6 and IL-23 production, further

100 μM. After 18 h, cells were harvested and homogenized for Western blot. (A) Protein expression levels of nuclear transcription factors and protein kinase were measured by Western blot. (B) Densitometry was determined by ImageJ, and data were analyzed by GraphPad Prism 6. Data are expressed as mean ± SD of three biological replicates. \*\**P* < 0.01 by Student's *t*-test. Figure A is one representative of three independent experiments.

indicating that Scopoletin blocked DC secretion of Th17-inducing cytokines, thus resulting in inhibited Th17 phenotype formation.

When antigen presentation occurs, epitope-major histocompatibility complexes identified by T-cell receptors and costimulatory signals are sent by interaction of CD80, CD86 of DCs and CD28 expressing on T cells, as well as the cytokine milieu formed by DCs and other inflammatory cells in the microenvironment (Thome et al., 2014b). These signals working together results in T cell polarization into Th1, Th17, and other effector CD4+ T cells. Studies have shown that the activation of DCs is possibly regulated by three intracellular signaling pathways, including NF-κB, MAPKs, and PI3K (Chesi et al., 2016; Rescigno et al., 1998). To further explore the mechanism of Scopoletin-induced amelioration of EAE, we investigated the effects of Scopoletin on BM-DCs *in vitro,* which indicated decreased expression of a series of inflammation-related genes. While the activation of MAPKs containing ERK, p38, and JNK was not prominently influenced in LPS-stimulated DCs, Scopoletin treatment exerted a profound inhibitory effect on NF-κB activity during DC activation. It was shown that Scopoletin markedly suppressed the phosphorylation of NF-κB, which plays a significant role in the activation and maturation of DCs. Previous studies showed that IL-6 gene expression was reduced through downregulating of NF-κB signaling (Chiang et al., 2014), and the promoter activity of IL-23 was modulated by the NF-κB pathway. Our results indicated that the therapeutic effect of Scopoletin may be mediated by inhibiting the NF-κB signaling pathway, which is vitally involved in producing proinflammatory cytokines.

In summary, we reported, for the first time, that natural compound Scopoletin ameliorated the severity of EAE by regulating DC activation and reducing CNS inflammation *via* suppression of NF-κB signaling. Scopoletin treatment could therefore be a potential candidate for the modulation of inflammatory conditions of autoimmune diseases.

### CONCLUSION

Our study demonstrates that natural products Scopoletin derived from medical or edible plants will be valuable in developing a novel therapeutic agent for MS in the future.

### DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

### REFERENCES

### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of 'institutional guidelines and regulations approved by the Animal Ethics Experimental Committee at of Shaanxi Normal University', 'the Animal Ethics Experimental Committee at of Shaanxi Normal University'. The protocol was approved by the 'Animal Ethics Experimental Committee at of Shaanxi Normal University'.

### AUTHOR CONTRIBUTIONS

FZ, YZ, TY, and XL conceived and designed the experiments. FZ, Z-QY, TY, H-RF, and J-JH carried out the experiments. FZ, YZ, TY, JT, and XL analyzed data and wrote the manuscript. Z-ZW and XL cosupervised the study and revised the paper. All authors read and approved the final manuscript.

### FUNDING

This study was supported by the Chinese National Natural Science Foundation (Grant No. 81771345, 81501062, 31670299), the Natural Science Foundation of Shaanxi Province, China (Grant No. 2018JZ3001, 2018JQ8033, 2019KJXX-022), and the Fundamental Research Funds for the Central Universities (Grant No. GK201903062, GK20182010, GK201701009, 2018CSLZ018, 2018CSLZ019, 2018CSLZ020,<PHONE_NUMBER>52).

### ACKNOWLEDGMENT

We gratefully thank Katherine Regan for editorial assistance.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2019.00863/ full#supplementary-material

experimental autoimmune encephalomyelitis. *Immunol. Cell Biol.* 92, 124–132. doi: 10.1038/icb.2013.73

**Conflict of Interest Statement:** 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.

*Copyright © 2019 Zhang, Zhang, Yang, Ye, Tian, Fang, Han, Wang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Corrigendum: Scopoletin Suppresses Activation of Dendritic Cells and Pathogenesis of Experimental Autoimmune Encephalomyelitis by Inhibiting NF-**κ**B Signaling

#### *Approved by:*

*Frontiers Editorial Office, Frontiers Media SA, Switzerland*

#### *\*Correspondence:*

*Zhe Zhi Wang<EMAIL_ADDRESS>Xing Li<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### *Specialty section:*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology*

*Received: 13 August 2019 Accepted: 14 August 2019 Published: 13 September 2019*

#### *Citation:*

*Zhang F, Zhang Y, Yang T, Ye Z-Q, Tian J, Fang H-R, Han J-J, Wang Z-Z and Li X (2019) Corrigendum: Scopoletin Suppresses Activation of Dendritic Cells and Pathogenesis of Experimental Autoimmune Encephalomyelitis by Inhibiting NF-κB Signaling. Front. Pharmacol. 10:1037. doi: 10.3389/fphar.2019.01037*

*Fei Zhang †, Yuan Zhang †, Ting Yang †, Ze-Qing Ye, Jing Tian, Hai-Rong Fang, Juan-Juan Han, Zhe-Zhi Wang\* and Xing Li\**

*National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest China, The Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, The Ministry of Education, College of Life Sciences, Shaanxi Normal University, Xi'an, China*

Keywords: scopoletin, experimental autoimmune encephalomyelitis, multiple sclerosis, dendritic cells, NF-**κ**B signaling

### **A Corrigendum on**

### **Scopoletin Suppresses Activation of Dendritic Cells and Pathogenesis of Experimental Autoimmune Encephalomyelitis by Inhibiting NF-κB Signaling**

*by Zhang F, Zhang Y, Yang T, Ye Z-Q, Tian J, Fang H-R, Han J-J, Wang Z-Z and Li X (2019). Front. Pharmacol.10:863. doi: 10.3389/fphar.2019.00863*

In the published article, author Zhe-Zhi Wang's identity was incorrect. Instead of "co-author," it should be "co-corresponding author." The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

*Copyright © 2019 Zhang, Zhang, Yang, Ye, Tian, Fang, Han, Wang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Microglial Drug Targets in AD: Opportunities and Challenges in Drug Discovery and Development

*Knut Biber1, Anindya Bhattacharya2, Brian M. Campbell3, Justin R. Piro4, Michael Rohe1, Roland G.W. Staal5, Robert V. Talanian4 and Thomas Möller4\**

*1 AbbVie Deutschland GmbH & Co. KG, Neuroscience Research, Ludwigshafen, Germany, 2 Janssen Research & Development LLC, San Diego, CA, United States, 3 Sage Therapeutics, Cambridge, MA, United States, 4 AbbVie Foundational Neuroscience Center, Cambridge, MA, United States, 5 Paracelsus Neuroscience, Metuchen, NJ, United States*

#### *Edited by:*

*Pietro Giusti, University of Padova, Italy*

#### *Reviewed by:*

*Claes Wahlestedt, Leonard M. Miller School of Medicine, United States Anthony John Hannan, Florey Institute of Neuroscience and Mental Health, Australia*

*\*Correspondence: Thomas Möller<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology*

*Received: 14 March 2019 Accepted: 01 July 2019 Published: 23 August 2019*

#### *Citation:*

*Biber K, Bhattacharya A, Campbell BM, Piro JR, Rohe M, Staal RGW, Talanian RV and Möller T (2019) Microglial Drug Targets in AD: Opportunities and Challenges in Drug Discovery and Development. Front. Pharmacol. 10:840. doi: 10.3389/fphar.2019.00840*

Alzheimer's disease (AD) is a large and increasing unmet medical need with no diseasemodifying treatment currently available. Genetic evidence from genome-wide association studies (GWASs) and gene network analysis has clearly revealed a key role of the innate immune system in the brain, of which microglia are the most important element. Singlenucleotide polymorphisms (SNPs) in genes predominantly expressed in microglia have been associated with altered risk of developing AD. Furthermore, microglia-specific pathways are affected on the messenger RNA (mRNA) expression level in post-mortem AD tissue and in mouse models of AD. Together these findings have increased the interest in microglia biology, and numerous scientific reports have proposed microglial molecules and pathways as drug targets for AD. Target identification and validation are generally the first steps in drug discovery. Both target validation and drug lead identification for central nervous system (CNS) targets and diseases entail additional significant obstacles compared to peripheral targets and diseases. This makes CNS drug discovery, even with well-validated targets, challenging. In this article, we will illustrate the special challenges of AD drug discovery by discussing the viability/practicality of possible microglia drug targets including cluster of differentiation 33 (CD33), KCa3.1, kynurenines, ionotropic P2 receptor 7 (P2X7), programmed death-1 (PD-1), Toll-like receptors (TLRs), and triggering receptor expressed in myeloid cells 2 (TREM2).

Keywords: drug target, microglia, target identification, target validation, screening cascade

### HIGHLIGHTS:

### INTRODUCTION

Microglial reactivity has long been recognized as a pathological hallmark of a wide variety of neurological diseases. While morphological changes of microglia were initially interpreted as a reactive response to neuronal damage, growing evidence suggests that these changes are not merely "reactive" but indicate that glial pathology contributes to disease progression (Raj et al., 2014a; Verkhratsky et al., 2014; Ben Haim et al., 2015; Bergles and Richardson, 2015; Biber et al., 2015; Heneka et al., 2015; Heppner et al., 2015; Jain et al., 2015; Ransohoff and El Khoury, 2015; Robel and Sontheimer, 2015; Zuchero and Barres, 2015; Osborn et al., 2016). Especially with regard to Alzheimer's disease (AD), numerous reviews cover various aspects of microglial biology in the context of disease (see for example: (Guedes et al., 2018; Hansen et al., 2018; Kinney et al., 2018; Shi and Holtzman, 2018; Thei et al., 2018; Ulland and Colonna, 2018; Henstridge et al., 2019). Using the search term "microglia Alzheimer," PubMed lists close to 100 reviews that were published in the last 2 years (Jan 2017–Jan 2019). It is not the purpose of the current review to discuss the potential role of microglia in AD, but to focus on the challenges and practical aspects of microglial proteins or pathways as potential drug targets.

A considerable set of genetic risk factors for AD are predominantly expressed in microglia (Wes et al., 2016; Henstridge et al., 2019). Thus, it is very clear from human genetics that regardless of being "reactive," "active," "dystrophic," "senescent," "dysfunctional," or "disease associated," microglial involvement in AD offers a hitherto unexplored intervention point. Many publications concerning microglia in AD include conclusions like: "The presented data here suggest that protein 'XYZ' can be considered as a drug target for AD." As there are some common misunderstandings between colleagues focused on biology vs. drug discovery regarding the definition of a target, we first aim to clarify what it takes to consider a microglial molecule suitable for a drug discovery program. We will then review some commonly suggested microglial targets and pathways, i.e., cluster of differentiation 33 (CD33), KCa3.1, kynurenines (KYN), ionotropic P2 receptor 7 (P2X7), programmed death-1 (PD-1), Toll-like receptors (TLRs), and triggering receptor expressed in myeloid cells 2 (TREM2), discussing their potential role in AD and their respective challenges in regard to drug discovery.

### TARGET IDENTIFICATION

In general, preclinical drug discovery can be staged as follows: target identification, in which the validity of a target for modulating a disease process and the initial chemical matter along with the means of characterizing them are established; lead optimization, in which target validity is further strengthened while identifying candidate molecules with the full range of necessary drug-like properties; and finally, preclinical testing for safety and efficacy. Apart from target identification, we will not discuss the other stages of preclinical drug development (**Figure 1**). The interested reader is referred to (Möller and Boddeke, 2016) for a detailed description of the whole process.

For potential targets, it is of utmost importance to demonstrate relevancy to the disease and that modulating the target would result in therapeutic benefit with an acceptable safety margin. Evidence for a role of a candidate target in disease might include i) altered expression in disease, ii) integral relationship to disease pathophysiology, iii) genetic association with disease, iv) having a mechanistic link to disease etiology, and v) demonstration of a beneficial effect when modulated in a disease-relevant *in vitro* or (preferably) *in vivo* model.

Unfortunately, generating expression data for the human central nervous system (CNS) is extremely difficult. While for peripheral diseases biopsies, or blood draws are routine, for the CNS, most parameters can only be derived from post-mortem brain tissue that at best represents end-stage disease, or by limited imaging methods, making it difficult to observe changes in earlier and therapeutically more relevant disease states. Non-human models can also provide insight, but with significant translational uncertainties.

A strong genetic association of a microglial molecule with AD, for example, CD33, complement receptor 1 (CR1), phospholipase C gamma 2 (PLCγ2), and TREM2 (Wes et al., 2016; Henstridge et al., 2019), also suggests promise as a drug target. If such evidence can further be strengthened by a mechanistic link to a disease model by *in vitro* or *in vivo* genetic or pharmacologic intervention studies [e.g., knocking out CD33 (Bradshaw et al., 2013)], that would serve to increase confidence in the target.

### WHAT MAKES A GOOD (MICROGLIAL) TARGET?

### Drugability

Besides linking a target to disease, there are other more pragmatic considerations for selecting a target. Admittedly, "drugable" is not a well-defined term; it usually includes chemical tractability with a ligand (small molecule or antibody) but can also refer more broadly to the properties of such ligands, e.g., pharmacokinetics, distribution, metabolism (and the pharmacologic properties of

significant metabolites), pharmaceutical properties including chemical stability, and manufacturability.

Traditionally, a target would be considered chemically tractable if a small molecule or antibody can bind with high affinity and specificity and induce the desired biological effect. For example, G-protein coupled receptors (GPCRs) are generally considered chemically tractable; more than 30% of currently marketed drugs target GPCRs (Ma and Zemmel, 2002; Hauser et al., 2017). Ion channels or enzymes are also considered tractable as those targets often include structural features that permit potent and specific ligand binding, with consequent direct inhibition of conductance or catalytic activity. Blocking protein–protein interactions is generally more difficult; however, recent advances have challenged this view, and modulation of protein–protein interactions by small molecules is now an area of active research and will increase the number of targets considered drugable (Higueruelo et al., 2013). Furthermore, blocking protein–protein interactions can often be accomplished with therapeutic antibodies, and several innovative approaches have been devised to improve antibody penetration into the brain (Watts and Dennis, 2013; Salameh and Banks, 2014; Pardridge, 2015). Despite these recent advancements, developing therapeutics for the CNS remains challenging, and while there are many biologically interesting molecules related to microglia, only a small subset of them might be considered drugable.

### Specificity

It is not merely enough to generate a potent pharmacological agent against a drug target; it is also of utmost importance that the molecule is selectively modulating only this one target to avoid off-target effects that may hamper the interpretation of experimental data. Unfortunately, many so-called "reference compounds," such as commercially available inhibitors, are not as specific as advertised by the suppliers (Frye, 2010). A group of 50 scientists from different life science disciplines have covered this topic in detail in Commentary in *Nature Chemical Biology* (Arrowsmith et al., 2015). This lack of specificity makes interpretation of results gained with many compounds virtually impossible. A prominent example in microglia biology (although not directly related to a single target) is minocycline, which often is referred to as a "microglia inhibitor" (Möller et al., 2016).

### Safety

Arguably the most important criterion for target selection, however, is safety. During clinical development, many drugs fail, not due to the lack of efficacy but due to adverse safety events inadequately predicted by preclinical studies. Toxicity might not only arise from modulating the target itself, meaning that the therapeutic window is small, but that there could be offtarget effects of a drug, or that toxicity may also arise from a metabolite of the drug that itself is toxic. It is therefore important to select targets with the highest likelihood of being safe as early as possible. A common question is how broadly a target is expressed. Localized expression preferentially in the target cell and/or tissue is considered advantageous as there is less likelihood for adverse effects unrelated to the desired mechanism of action. Unfortunately, there are very few potential targets restricted to microglia as most potential targets are also expressed in peripheral myeloid cells, so effects on the peripheral immune system are a persistent concern. Safety concerns, whether based on side effects of a development compound, a target knockout phenotype, or suspected essential physiological roles of the target, need to be addressed as early as possible by experiments.

Taken together, there is a whole range of drugability considerations that must be met to make a target practical for drug discovery. There are few targets in microglia that are often discussed in the literature to be of value in AD. Here we will discuss the potential and challenges around these targets when it comes down to drugability aspects.

### CURRENTLY PROPOSED MICROGLIAL DRUG TARGETS

### Proposed Role of CD33 in AD

In 2011, large-scale genome-wide association studies (GWASs) identified CD33, a member of the sialic acid–binding immunoglobulin-like lectins (siglecs) family, as a genetic locus associated with the risk to develop AD (Hollingworth et al., 2011; Naj et al., 2011). This association with AD risk has been confirmed in a variety of subsequent genetic studies in different ethnic groups, with odds ratios between 0.7 and 0.9; thus, the CD33 rs3865444A allele protects from the development of AD [for recent metaanalysis, see (Jiang et al., 2018)]. It was furthermore described that the protective single-nucleotide polymorphism (SNP) rs3865444A is in linkage disequilibrium with rs12459419T, which causes increased splicing of exon2, and therefore deletes the sialic acid–binding site of CD33. The protective SNP therefore leads to increased expression of a truncated, non-functional version of the receptor, called d2-CD33 or CD33m (Bradshaw et al., 2013; Malik et al., 2013; Raj et al., 2014b). A reduction in full-length CD33 expression was observed in myeloid cells of CD33 rs3865444A allele carriers, resulting in an overall reduced CD33 function (Bradshaw et al., 2013; Raj et al., 2014b).

CD33 signaling in humans depends on two intracellular inhibitory domains, one immunoreceptor tyrosine-based inhibitory motif (ITIM), and one ITIM-like domain. CD33 is therefore considered to be an inhibitory immune receptor. Indeed, when activated CD33 becomes tyrosine-phosphorylated by Src kinases, which subsequently recruit Src homology-2 domain (SH2)–containing tyrosine phosphatases (SHP1/2), this results in the dephosphorylation and inhibition of signaling cascades (see for review Macauley et al., 2014). CD33 is expressed in myeloid cells, and its signaling negatively regulates the function of these cells. Accordingly, knockdown of CD33 in human monocytes leads to spontaneous release of interleukin 1 beta (IL-1β), Tumor necrosis factor alpha (TNFα), and IL-8 (Lajaunias et al., 2005). In monocytes from carriers of the CD33 rs3865444A allele (decreased CD33 function), an increased uptake capacity of amyloid beta(1–42) (Aβ1–42) was described (Bradshaw et al., 2013).

In the brain, CD33 is specifically expressed in microglia (Bradshaw et al., 2013; Griciuc et al., 2013; Malik et al., 2013; Walker et al., 2015), where an inverse relationship between CD33 expression and Aβ uptake capacity was observed (Griciuc et al., 2013). Accordingly, in the cortex of AD patients, a positive correlation of microglial CD33 expression and amyloid pathology was described, indicating that increased CD33 expression in microglia promotes plaque pathology (Griciuc et al., 2013). CD33-deficient Amyloid precursor protein (APP)/ Presenilin 1 (PS1) mice showed reduced amyloid pathology compared to age-matched wildtype (WT) controls, supporting the idea that reduced microglial CD33 function is beneficial with respect to amyloid pathology (Griciuc et al., 2013). Given this variety of data concerning the role of CD33 as regulator of microglial Aβ uptake capacity, this siglec receptor is currently considered as drug target in AD. It is considered that inhibition of CD33 function in microglia would increase their amyloid uptake capacity, which in turn would be beneficial in AD **Figure 2** (Heneka et al., 2015).

### Target Challenges Around CD33

There are many challenges with respect to CD33 as a drug target in AD. In general, finding ligands for siglecs is difficult, as glycan binding often is of relatively low affinity and relies on multivalency to achieve adequate affinity (Varki, 2009). There is evidence that CD33 binds α2.3 sialic acid and α2.6 sialic acid; the corresponding glycosylated proteins that are decorated with these sialic acids, however, have not been identified (Linnartz-Gerlach et al., 2014; Macauley et al., 2014). As CD33 is the shortest of all known siglecs, it most likely is activated in a cis-manner (O'Reilly and Paulson, 2009; Linnartz-Gerlach et al., 2014; Macauley et al., 2014),

activators of microglial phagocytosis, respectively. For TREM2, activating antibodies, and for CD33, inhibitory antibodies, have been proposed as therapeutic interventions. Figure created with Biorender.com

meaning that the sialylated glycoproteins that activate CD33 would also be expressed in microglia. One report provides data that soluble sialyllactosamine in the mM range might activate CD33 in de-sialylated monocytes (Lajaunias et al., 2005). This finding awaits independent confirmation. Apart from our very limited understanding around natural ligands for CD33, functional tool compounds for CD33 have not yet been described. The only published molecule displaying a reasonable binding at CD33 is cpd22, which displaces bead-coupled α2.6 sialic acid with an half maximal effective concentration (EC50) of about 10 µM (Rillahan et al., 2014). It therefore is suggested that cpd22 binds CD33 at the sialic acid–binding domain; whether this binding affects CD33 signaling is not understood. Thus, it is not clear whether cpd22 might function as an agonist or antagonist for CD33. The lack of ligands or tool compounds seriously hampers the development of functional analysis of CD33 in cellular assays, making the development of functional inhibitors for CD33 difficult.

Therapeutic inhibitors of CD33 are hypothesized to increase phagocytic properties in microglia; from a drug development perspective, they might also be envisaged to inhibit the CD33 signaling cascade. It is not yet understood how recruitment of SHP1 leads to inhibition of cellular phagocytosis in CD33 expressing myeloid cells (Linnartz-Gerlach et al., 2014).

Another challenge concerns the large species difference of CD33. Mouse and human CD33 differ not only in the extracellular domain but also in the transmembrane domain as well as intracellularly. In contrast to human CD33, mouse CD33 only has one ITIM-like domain but lacks the ITIM domain (Cao and Crocker, 2011). Moreover, in the transmembrane region of mouse CD33, a positively charged residue is present, which might enable mouse CD33 to recruit immunoreceptor tyrosine-based activation motif (ITAM)-motif–containing adapter proteins like DNAX activating protein of 12 kDa (DAP12) (Linnartz-Gerlach et al., 2014). These data suggest that mouse CD33 can act as an activating receptor instead of being inhibitory, a hypothesis that awaits experimental evidence. In cultured microglia from CD33KO mice, it was shown that CD33 negatively couples to phagocytosis (Griciuc et al., 2013). Whether this is due to ITIM-like signaling or recruitment of ITAM-motif–containing adapter proteins is currently an open question. Potential drugs that inhibit CD33 function, however, need to be tested in animal models. Whether or not the mouse would be an adequate model system to do so is questionable given the species differences and the limited knowledge on CD33 function in mouse cells.

Taken together, despite the genetic association between CD33 and AD development and the straightforward hypothesis of how reduced CD33 function might protect from AD, CD33 remains a target with its unique set of challenges.

## Proposed Role of KCa3.1 in AD

KCa3.1 is an intermediate-conductance potassium channel that is activated when intracellular calcium is increased. The Ca2+ binds to calmodulin, which is constitutively bound to the calmodulin binding domain of the channel, resulting in the opening of the K+ channel (Kd ~ 300 nM) (Xia et al., 1998; Fanger et al., 1999; Bouhy et al., 2011). The KCa3.1-mediated K+ efflux augments the driving force Biber et al. Microglia as Drug Targets in AD

for further Ca2+ influx *via* Ca2+ release-activated calcium (CRAC) channels and subsequent refilling of intracellular calcium stores. Thus, one of the primary functions of KCa3.1 is the augmentation of Ca2+ signaling (Feske et al., 2012). Expression of KCa3.1 in the CNS and its controversies have been reviewed (Dale et al., 2016). Here we will focus on microglia, as the expression of KCa3.1 was established using well-validated antibodies and demonstration of voltage-independent KCa3.1-like Ca2+-activated K+ currents.

Voltage-independent KCa3.1-like Ca2+-activated K+ currents in cultured murine microglia were first reported by Eder et al. (Eder et al., 1997). Khanna et al. subsequently demonstrated the presence of KCa3.1 mRNA in cultured rat and mouse microglia (Khanna et al., 2001). The conclusion that microglia express KCa3.1 was supported by pharmacological studies that demonstrated that charybdotoxin and clotrimazole could inhibit the voltageindependent KCa3.1-like Ca2+-activated K+ currents (Schilling et al., 2002; Schilling and Eder, 2004). There is further evidence that charybdotoxin and clotrimazole prevent the induced microglial oxidative burst (Khanna et al., 2001) and microglial migration (Schilling and Eder, 2004). The KCa3.1 inhibitor TRAM-34 reduced lipopolysaccharide (LPS)-induced microglial neurotoxicity (Kaushal et al., 2007), and inhibition of KCa3.1 using senicapoc (see below) blocked the LPS-induced IL-1β and Nitric oxide (NO) production by microglia (Staal et al., 2017a).

Early *in vivo* studies using antibodies validated in KCa3.1 WT and knock out (KO) mice did not provide any evidence of KCa3.1 expression on any cells in the mouse or human brain (including juvenile, adult, and aged mice) (Schilling and Eder, 2004; Schilling and Eder, 2007). However, studies using antibodies validated in this manner demonstrated KCa3.1 expression on CD68-positive cells (microglia or macrophages) upon injury to the CNS as induced by ischemic injury (Chen et al., 2011), suggesting that KCa3.1 is expressed by microglia only in response to injury. The study by Chen et al. also demonstrated the therapeutic efficacy of inhibiting KCa3.1 (using TRAM-34) in ischemic injury, a finding they validated using KCa3.1-null mice (Chen et al., 2011; Chen et al., 2015). Many other studies have since demonstrated the therapeutic efficacy of KCa3.1 inhibition in other models of neurological injury and disease in which microglia are believed to play a significant role, including experimental autoimmune encephalomyelitis, traumatic brain injury, spinal cord injury, optic nerve transection, glioblastoma multiforme, neuropathic pain, and AD (Dale et al., 2016; Staal et al., 2017b).

Taken together, there is convincing evidence for the expression of KCa3.1 in cultured rodent microglia. While the details of regulation of KCa3.1 await confirmation with more specific tool compounds, the available data suggest a role for KCa3.1 at the very least in microglia reactive oxygen species (ROS) production and signaling.

### Challenges in KCa3.1 Inhibition as Therapeutic Intervention in AD

The presence of "activated" microglia identified by expression of markers of microglial activation [major histocompatibility complex II (MHCII), Iba1] or an activated morphology (enlarged cell bodies and shorter, thicker processes) in brains of AD is well established. The observation that microglial "activation" can occur prior to plaque deposition and neurofibrillary tangle formation, and correlates with cognitive deficits, is consistent with the theory that microglial activation occurs at the earliest stages of disease (perhaps even before disease onset) and is somehow tied to its progression (Okello et al., 2009a; Okello et al., 2009b; Sastre et al., 2011). Maezawa et al. demonstrated that TRAM-34 blocks Aβ-induced proliferation of microglia, p38 Mitogen-activated protein kinase (MAPK) phosphorylation, nuclear factor kappa b (NF-κb) activation, and NO generation in primary microglia cultures (Maezawa et al., 2012). Furthermore, TRAM-34 inhibited neurotoxic effects of Aβ oligomers in mixed microglia–neuron cultures and in organotypic hippocampal slices by decreasing microglial activation and partially preventing synaptic loss (Maezawa et al., 2011).

Senicapoc is a potent and selective inhibitor of KCa3.1 developed for the treatment of sickle cell anemia [half maximal inhibitory concentration (IC50) = 11 nM; screening of 57 targets yielded no hits below 1 µM other than KCa3.1] (Ataga et al., 2006; Ataga et al., 2008; Ataga and Stocker, 2009; Dale et al., 2016; Staal et al., 2017a). In a recent and comprehensive study, Maezawa and colleagues demonstrated that KCa3.1 is functionally upregulated in microglia in brains from 5xFAD mice as well as AD patients (Jin et al., In Press). The authors demonstrate that Aβ oligomers impair long-term potentiation (LTP) and that this effect is reversed by the KCa3.1 inhibitor TRAM-34 as well as senicapoc in hippocampal slices or *in vivo*. It was furthermore demonstrated in this paper that senicapoc can reduce the pro-inflammatory effects as well as rescue the hippocampal LTP affected by Aβ oligomer (Jin et al., In Press). Senicapoc was then tested in the 5xFAD mice, starting at 6 months of age and continuing for three months. Results show that treatment with senicapoc reduced the amyloid load as well as neuroinflammation while enhancing neuronal plasticity compared to vehicle-treated mice (Jin et al., In Press). Thus, the study provides a strong rationale for repurposing senicapoc for the treatment of AD.

In fact, the Alzheimer's Drug Discovery Foundation has provided funding to manufacture clinical-grade senicapoc and conduct the required stability testing in support of an investigational new drug (IND) filing. Upon approval of the IND filing, senicapoc will be tested in a phase II clinical trial in prodromal and mild AD (https://www.alzdiscovery.org/newsroom/announcements/1.8-million-in-new-funding-supportsclinical-stage-treatments).

Senicapoc has been through extensive preclinical safety testing as well as phase I, II, and III clinical trials with few significant side effects noted. Given our knowledge of the pharmacokinetics of senicapoc, including its CNS penetrance (Staal et al., 2017b), it is likely that a dose similar to that used in the previous clinical trials will be used. AD patients, however, are very different from patients with sickle cell disease. It is likely that the AD patients will be older and have more comorbidities than those with sickle cell anemia. The age and comorbidities could reveal underlying susceptibilities to KCa3.1 inhibition that may not have been problematic in younger patients with sickle cell disease (e.g., susceptibility to infections). On the other hand, many diseases or conditions afflicting the elderly have inflammatory components, and a mild immunosuppressant such as senicapoc may benefit those conditions as well (e.g., stroke, chronic and neuropathic pain, atherosclerosis).

Yet, the current AD study is still incredibly important to test the hypothesis that targeting neuroinflammation, specifically KCa3.1, can slow or halt the progression of AD. If the trial results demonstrated significant and convincing slowing of disease progression, pharmaceutical companies could initiate drug discovery projects to develop improved CNS penetrant KCa3.1 inhibitors.

### Targeting the Kynurenine Pathway to Treat AD

Development of drugs that target products of tryptophan metabolism has transformed the treatment of psychiatric disorders, particularly with the advent of serotonin (5-HT) reuptake inhibitors (Wong et al., 1995). However, it is noteworthy that metabolism to 5-HT represents only a small fraction of the fate of free tryptophan, and in fact, the great majority is converted to KYN (Leklem, 1971) and its subsequent products by an array of enzymes located throughout the body and brain. Yet, surprisingly, there are no marketed medications that directly target metabolism of the KYN pathway.

Entry into the KYN pathway begins with metabolism of tryptophan into N-formylkynurenine by indole-2,3-dioxygenase (IDO) or tryptophan-2,3-dioxygenase (TDO), which is then rapidly converted to L-kynurenine (Takikawa et al., 1986; Zhang et al., 2007). Metabolism of KYN produces a host of neuro-active products in the brain that may contribute to CNS diseases (Török et al., 2016; Lovelace et al., 2017; Schwarcz and Stone, 2017). The KYN pathway bifurcates into neurotoxic and neuroprotective metabolic routes. As an example, astrocytes largely express kynurenine aminotransferases (KATs) and so are the primary source of the neuroprotective metabolite, kynurenic acid (KYNA), in the brain (Guidetti et al., 2007). Alternatively, microglia express kynurenine 3-monooxygenase (KMO), which converts KYN to neurotoxic metabolites such as 3-hyroxykynurenine (3-HK) and subsequently quinolinic acid (Quin) (Heyes et al., 1996; Guillemin et al., 2003a) (**Figure 3**). While the topic of this discussion is targeting the disruption in microglial KYN metabolism, one should understand that the various branches of KYN metabolism do not act in isolation, and so a change in one may cause a change in the others by altering the availability of substrate.

Inflammation triggers activation of microglia and dysregulation of KYN metabolism, leading to production of neurotoxic metabolites from the KMO branch, a process that is predicted to be involved in the development of AD pathology (Campbell et al., 2014; Zádori et al., 2018). However, direct evidence of microglial KYN metabolism from human AD subjects suggests that this effect may be limited to localized pathological events rather than global changes in CNS KYN metabolism. As an example, levels of KYN and 3-HK were both surprisingly reduced in CSF of AD patients with dementia (Tohgi et al., 1992). However, tryptophan, 5-HT, and melatonin were all also significantly reduced in the same study. In addition, the same group reported that KYN was reduced in a second cohort of patients with AD dementia but not in ones with vascular dementia (Tohgi et al., 1995). Furthermore, Quin (Mourdian et al., 1989; Heyes et al., 1992) and KYNA (Wennström et al., 2014) were reportedly unchanged in recent studies, though earlier evidence indicated that KYNA could also be decreased (Heyes et al., 1992; Hartai et al., 2007; Gulaj et al., 2010) in AD patients. Overall, there is limited evidence that CSF tryptophan/ KYN metabolites are reproducibly altered in AD, though where it has been assessed, most studies point to either no consistent changes in metabolites or a reduction in KYN metabolism (**Table 1**).

In post-mortem AD brain tissue, early studies reported a lack of change in KYN metabolism. Indeed, Quin levels were shown to be unchanged (Moroni et al., 1986; Mourdian et al., 1989; Sofic et al., 1989; Pearson and Reynolds, 1992), as were KYNA concentrations (Flint Beal et al., 1992). However, more recent analysis revealed