Molecules and Cells

The Effect of A1 as a Potential New Therapeutic Target on Neuronal Damage by

Activated Microglia Ji-Eun You, Se-Hwa Jung, and Pyung-Hwan Kim*

Department of Biomedical Laboratory Science, Konyang University, Daejeon 35365, Korea *Correspondence: [email protected] https://doi.org/10.14348/molcells.2021.0020 www.molcells.org

Brain disease is known to cause irrevocable and fatal loss of INTRODUCTION biological function once damaged. One of various causes of its development is damage to neuron cells caused by Recent trends in the study of neurodegenerative diseases hyperactivated microglia, which function as immune cells in such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) brain. Among the expressed in microglia stimulated focus on the role and function of microglia and the relation by various antigens, annexin A1 (ANXA1) is expressed in with neuron (Arends et al., 2000; Perry et al., 2010). The the early phase of the inflammatory response and plays an main causes of this diseases are the high susceptibility of neu- important role in controlling the immune response. In this rons to inflammatory damage, and the main role of microglia study, we assessed whether ANXA1 can be a therapeutic as mediators of the inflammatory process (Chan et al., 2001; target for the initial reduction of the immune response Magnus et al., 2001). Various omics data suggest about the induced by microglia to minimize neuronal damage. To key role of microglia in the pathogenesis of brain diseases address this, mouse-origin microglial cells were stimulated such as AD (Lewcock et al., 2020). There are evidences that to mimic an immune response by lipopolysaccharide (LPS) neurodegenerative diseases, which are caused by misfolding treatment. The LPS treatment caused activation of ANXA1 and aggregation of such as β-amyloid and tau tan- gene and expression of inflammatory cytokines. To assess gles, could cause dementia (Ashrafian et al., 2020; Binder the biological function in microglia by the downregulation et al., 2005). Among brain disorders, neuroinflammation is of ANXA1 gene, cells were treated with short hairpin RNA- a defense mechanism that initially protects the brain by re- ANXA1. Downregulated ANXA1 affected the function of moving or inhibiting diverse pathogens (Picca et al., 2020). mitochondria in the microglia and showed reduced neuronal This inflammatory response can have beneficial effects that damage when compared to the control group in the co- activated microglia by pathogens involve immune regula- culture system. Taken together, our results showed that tion or injury resolution by acquiring phenotypes as well as ANXA1 could be used as a potential therapeutic target for participate in cell cytotoxic. However, when inflammatory re- -related neurodegenerative diseases. sponses continue, it become chronic inflammatory which are detrimental, and they inhibit regeneration as well as involve Keywords: annexin A1, brain diseases, immune response, microglia and astrocytes and can lead to neurodegenerative microglia, neural damage diseases (Scheiblich et al., 2020). Microglia, which are immune cells in the central nervous

Received 29 January, 2021; revised 25 February, 2021; accepted 15 March, 2021; published online 22 April, 2021 eISSN: 0219-1032 ©The Korean Society for Molecular and Cellular Biology. All rights reserved. cc This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.

Mol. Cells 2021; 44(4): 195-206 195 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al. system (CNS) (Fan et al., 2017), responds to endogenous stimuli generated following infection or injury. Upon appro- priate stimulation, classically activated microglia trigger the LPS treatment Cytokine expression :IL-6, TNF-α first line of host defense against pathogens, leading to a neu- roprotective role (Graeber, 2010; Nimmerjahn et al., 2005).

However, chronic activation of microglia leads to potential Damaged neuron cytotoxicity (Franco and Fernandez-Suarez, 2015; Pascual et al., 2012), such as pro-inflammatory cytokines and reactive Microglia ActivatedActivated mimicrogliacroglia oxygen intermediates, which can cause various CNS disor- Knock-down of ders such as pain, neurodegeneration, and stroke that carry ANXA1 actual or potential risks (Dai et al., 2015). Consequently, the activated microglia in the brain attack the neurons, leading Healthy neuron to dementia due to the damaged neurons (Eikelenboom and Fig. 1. Schematic representation of the proposed role in the Veerhuis, 1996; Shaji et al., 2018). control of microglial . Microglia is activated by LPS Currently, active research in various fields has been con- treatment, and in the CNS, the activated microglia damage the ducted to find effective treatments, including gene therapy neuron. (Nilsson et al., 2010), stem cell-based cell therapy (Duncan and Valenzuela, 2017), and drug development. Among them, risperidone is the only treatment currently approved ride (LPS)-stimulated microglia. We evaluated whether the to treat agitation and aggression in patients with AD. Risper- neuronal damage was reduced when ANXA1, expressed in idone is a clinically preferred drug, because it has fewer side the microglia activated by antigens, was substantially down- effects as compared to other drugs, and it effectively reduces regulated (Fig. 1). the symptoms and works against the negative symptoms by blocking dopamine receptors (Katz et al., 1999). Although MATERIALS AND METHODS several other drugs such as citalopram, aripiprazole, and quetiapine are used, they are not recommended because of Cell cultures the greater risk of side effects than positive effects (Dai et BV2 microglial cells and SH-SY5Y neuron cell lines were pro- al., 2015). Despite various treatments, including β-amyloid- vided by professor Seung-Ju Yang of Konyang University. Two inhibiting drugs, and studies conducted for dementia, no cell lines were incubated at 37°C and 5% CO2 in Dulbecco’s fundamental treatment or significant therapeutic effect has modified Eagle medium with high glucose (HyClone, USA) been reported. Therefore, in addition to the existing treat- containing 10% fetal bovine serum (HyClone) and 1% peni- ment methods, the development and approaches of new cillin G-streptomycin solution (Gibco, USA). treatments that can cure the fundamental causes is required. To address this issue, we focused on genetic material RNA extraction and relative quantitative real-time poly- related to immune response as a way to treat the disease merase chain reaction or conventional polymerase chain at its source in order to slow it down. Among the genes ex- reaction pressed in microglia stimulated by various antigens, annexin Total RNA was isolated from BV2 microglial cells using Trizol A1 (ANXA1) was considered as a candidate genetic material reagent (Invitrogen, USA) by following the manufacturer’s involved in inflammatory reactions that causes microglia to instructions. It was quantified using NanoDropTM One (Ther- damage nerve cells. ANXA1 is a that is expressed and mo Fisher Scientific, USA), and the reverse transcription-poly- activated in the early phase of the inflammatory response and merase chain reaction (RT-PCR) was performed with plays an important role in controlling the immune response DiaStar 2X RT Pre-Mix (Solgent, Korea) on T100 (Bio-Rad, (McArthur et al., 2010). It exists in the plasma membrane, USA). The RT-PCR experiment using primers for the detec- extracellular region, and of cells (Han et al., 2020). tion of each gene was performed as per the manufacturer’s Moreover, several studies have reported that the expression PCR conditions. The products were electrophoresed in 2% of ANXA1 is found in glial cells (specifically in microglia) of agarose gels (Agarose, Molecular Biology Grade; Vivantis the normal adult human and rodent brain (Buckingham et Technologies, Malaysia) prepared in 1X tris-acetate-ethylene- al., 2006; Dreier et al., 1998). Thus, ANXA1 is observed to diaminetetraacetic acid buffer (Biosesang, Korea). Gel images be constitutively expressed in cells related with the innate were obtained using a Fusion SL (Vilber Lourmat, France). immune stage of the normal brain (Solito et al., 2008). Al- Real-time PCR was conducted in a CFX96TM real-time system though ANXA1 is controversial because it is responsible for employing the SolgTM 2X RT-PCR Smart mix (Solgent). Table 1 the functions of pro-inflammatory and anti-inflammatory shows the primer sequences used in this study. in the CNS through various studies, enhanced ANXA1 ex- pression has been found in brain lesions of patients with PD Transfection of annexin A1 short hairpin RNA to BV2 mi- (Solito et al., 2008), and AD, which may specifically act in the croglial cells inflammatory phase before dementia occurs. ANXA1 short hairpin RNA (shRNA) was constructed using Using this characteristic of ANXA1, we hypothesized that the same sequences (NM_010730) (Supplementary Fig. S1). ANXA1 would engage in early immune responses and affect To establish the conditions of ANXA1 knockdown (Kd) and neuronal damage in the immune response by lipopolysaccha- scrambles (Scr), a total of 1 × 105 BV2 cells were transfected

196 Mol. Cells 2021; 44(4): 195-206 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al.

Table 1. List and sequences of primers used Gene Forward Reverse

GAPDH TCACCACCATGGAGAAGG GCTAGGCAGTTGGTGGTGCA IL-6 ACAAGTCCGGAGAGGAGACT GGTCTTGGTCCTTAGCCACTC TNF-α CAAGGGACAAGGCTGCCCCG GCAGGGGCTCTTGACGGCAG ICAM1 AGCACCTCCCCACCTACTTT AGCTTGCACGACCCTTCTAA ANXA1 ACGTGAACGTCTTCACCACA TCGGCAAAGAAAGCTGGAGT

with pGPU6/GFP/Neo vector with shRNA-ANXA1 (OriGene, β-actin (1:500 dilution, 8432; Santa Cruz Biotechnology). USA) by using TransIT-X2 (Takara Bio, Japan) in serum-free After five washes with 0.1% phosphate-buffered saline with medium according to the manufacturer’s instructions. The Tween®, the cells were stained with a goat anti-mouse IgG same plasmid without any insert and a plasmid with a Scr IRDyeTM 680 antibody (1:800 dilution; LI-COR Biosciences) vector were used as negative controls. After the transfection, at RT for 1 h. The microplates were scanned using the Od- green fluorescent protein (GFP)-expressing BV2 cells were yssey CLx Infrared Imaging System (LI-COR Biosciences). The observed after 48 h of incubation, and then RNA was extract- integrated fluorescence intensities representing the protein ed and subjected to conventional PCR to examine ANXA1 expression levels were acquired using the software provided expression level. with the imager station (Odyssey Software Version 3.0; LI- COR Biosciences) (Ma et al., 2017). The relative amount of Enzyme-linked immunosorbent assay for interleukin-6 protein was obtained by normalizing to endogenous β-actin In order to confirm the cytokine expression pattern of microg- in all experiments. lia activated by LPS and the Kd by shRNA, BV2 microglial cells were cultured in 48-well plates at 5 × 104 cells/ml for 24 h. Measurements of oxygen consumption rate (OCR) The cells were transfected with ANXA1 shRNA or Scr shRNA BV2 microglial cell were assayed as described in the manufac- using Mirus (Takara Bio) in a serum-free medium according to turer’s instructions in XF DMEM (containing 2 mM glutamine, the manufacturer’s instructions. In brief, culture supernatants Agilent Seahorse; Agilent Technologies, USA) supplemented were collected after transfection. Pro-inflammatory cytokine with 10 mM pyruvate and 25 mM glucose. BV2 microglial interleukin-6 (IL-6) secreted from microglia stimulated by cell were plated at 1 × 104 cells per well in 8-well Seahorse LPS was confirmed using the Mouse IL-6 DuoSet ELISA (R&D culture plates (Agilent Seahorse) in 200 μl of the appropriate Systems, USA) as per the manufacturer’s instructions and ex- growth medium. One day after plating, BV2 microglial cell pressed as pg/ml. All assays were performed thrice, and the was treated LPS 2 h. After then, we measured OCR on a absorbance of the solution was measured at 450 nm using Seahorse FXp as described by the manufacturer, with modi- VersaMaxTM (Molecular Device, LLC, USA). This was calculat- fications (Shin et al., 2019). The concentrations of the inhibi- ed using the standard value obtained from a linear regression tors used in this study were the following: 1 mM oligomycin, equation. 1 mM FCCP, and 0.5 mM each of antimycin A and rotenone.

Cell viability test assay Based on a 24-well plate, 2 mg/ml solution of 3-(4,5-di- To clarify whether ANXA1 was downregulated in microglia methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; treated with shRNA cell death (the apoptotic effect was Sigma, UK) was added to each well for 300 μl and incubated caused by ANXA1 in microglial cells), apoptosis was detected at 37°C for 4 h and 30 min. After the formation of formazan using the FITC-Annexin V Apoptosis Detection Kit 1 (BD Bio- salt, the supernatant was removed and treated with dimethyl sciences) according to the manufacturer’s protocol. Samples sulfoxide at 37°C for 15 min in the same amount as the MTT were assessed using a co-culture system. First, the microglia reagent, and the absorbance was measured at 570 nm using prepared in the upper insert were treated with LPS for 2 h. VerxaMax. The SH-SY5Y neuron cells that had been seeded on the bot- tom plate were then harvested and washed twice with cold In-cell Western assay PBS and re-suspended in 1X binding buffer at a concentra- The in-cell Western (ICW) assay was performed using the Od- tion of 1 × 106 cells/ml. The cells were transferred to 100 μl yssey Imaging System (LI-COR Biosciences, USA) according to of the solution (1 × 105 cells/ml), 5 µl of FITC-Annexin V, and the manufacturer’s instructions. Cells were seeded at 1 × 104 5 µl propidium iodide (PI) for 15 min in the dark at RT. Next, cells/ml for 24 h in 96-well plates (FalconTM, Cat. #161093; 400 μl of binding buffer was added to each sample. Finally, BD Biosciences, USA). The cells were then fixed and per- apoptotic levels were analyzed by flow cytometry (Novo Cyte meabilized with 3.7% formaldehyde for 20 min at room flow cytometer; ACEA Bioscience, USA). Data were analyzed temperature (RT) and blocked with LI-COR Odyssey Blocking using Novoexpress software (ACEA Biosciences). Solution (LI-COR Biosciences) for 90 min. The cells were incubated at RT overnight with nuclear factor-kappa-B p65 Statistical analysis (NF-κB) (SC-514451; Santa Cruz Biotechnology, USA) pre- All data are presented as mean ± SEM. The values were ob- mixed with a mouse immunoglobulin (Ig) G antibody against tained from at least three repetitive experiments. The exper-

Mol. Cells 2021; 44(4): 195-206 197 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al. iment results were determined by applying the paired t-test innate immune system, which occurs within the first few and one-way ANOVA. Significance of analysis is indicated in hours or days (Tang and Le, 2016). The microglia secrete the figures (*P < 0.05, **P < 0.02, and ***P < 0.01). pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1 beta (IL-1β), and IL-6, and produce re- RESULTS active oxygen species (ROS) and nitric oxide (NO). Therefore, we evaluated whether the LPS-stimulated microglia convert- The expression of annexin A1 in microglia stimulated by ed to M1 type and secreted pro-inflammatory cytokines in lipopolysaccharides response. As shown in Figs. 3A and 3B, the expression of First, we assessed the optimal conditions for checking wheth- intercellular adhesion molecule-1 (ICAM1), which is an in- er the expression of ANXA1 was induced in the microglia by flammatory protein that acts as a biomarker of the M1 type, pathogenic stimuli. ANXA1 expression in BV2 microglia was was increased in the LPS-treated microglia when compared examined according to the treatment concentration and time to the untreated microglia. This indicated that the LPS-stimu- of LPS treatment. RNA extraction and cDNA synthesis by RT- lated microglia converted to the M1 type to function as mac- PCR were used to confirm the expression of the ANXA1 gene rophages in the brain. In addition, cytokine secretion, one in BV2 microglia. The total RNA isolated from these cells was of the major functions of , was expressed at normalized to glyceraldehyde 3-phosphate dehydrogenase. high levels at 2 h, similar to the expression of ANXA1 in the As shown in Fig. 2A, the treatment concentration of LPS for microglia treated with LPS (Figs. 3C and 3D). These results the induction of ANXA1 expression was significantly higher indicate that microglia stimulated by LPS present ANXA1 as in the microglia group treated with 1,000 ng/ml LPS than in soon as they switch to the M1 type. the untreated microglial cells. The quantitative data of the ANXA1 band showed a similar tendency (Fig. 2B). Using the Decreased cell viability of neuron by activated microglia optimal concentration conditions for LPS treatment from the In previous experiments, we confirmed that microglia dif- results of Fig. 2A, the expression of ANXA1 was evaluated ferentiate into cells that function as M1 phenotype by LPS over time (Fig. 2C). The highest expression was observed stimulation (Fig. 3). Next, we assessed how the activated after 2 h of LPS treatment (Figs. 2C and 2D). These data in- microglia affect neurons. To address this, co-culture was per- dicate that LPS is a potent inducer of ANXA1 expression in formed and stabilized by seeding microglia (BV2) cells in the microglial cells. upper well and neuronal (SH-SY5Y) cells in the down well. The wells were combined for 1 day after the activation of mi- Cytokine expression and M1 phenotype polarization of croglial cells by LPS for 4 h (Fig. 4A). As shown in the results microglia by lipopolysaccharide stimulation of Fig. 4B, co-cultured neurons with microglia may be slightly Microglia in the brain contribute to the immune response by affected by the unstimulated microglia, although some cyto- switching to M1 or M2 types to function as macrophages, toxicity was observed in neuron cells treated with LPS. How- depending on the stimulation. Activated pro-inflammatory ever, neuronal cell viability was significantly reduced when (M1) phenotype often act as the first line of defense of the co-cultured with LPS-activated microglia (Fig. 4B). This result

A B 13 *** ANXA1 Fig. 2. Optimal conditions for the 10 expression induction of ANXA1 in microglial cells by LPS. When GAPDH 7 LPS was treated to BV2 according

0 10 100 1000 to various concentration, ANXA1

(ANXA1/folds) 4 LPS concentration (ng/ml) was significantly increased at 1,000 Relative expression levels expression Relative 1 ng/ml. (A and B) The expression 0 10 100 1000 of ANXA1 according to various LPS concentration (ng/ml) LPS concentration (10-1,000 ng/ ml) and its quantitative data. (C and D) The expression of ANXA1 C D 2.5 according to the time of LPS treatment (1,000 ng/ml) and its ANXA1 * quantitative data. Quantifications 2 of ANXA1 of conventional PCR GAPDH intensities normalized to their

0 1 2 3 4 1.5 respective controls (defined as (ANXA1/folds) Time of LPS treatment (h) 1.0). Original gel images showed

Relative expression levels expression Relative in Supplementary Fig. S2. ***P < 1 0.01, as compared with control 0 1 2 3 4 group. *P < 0.05, as compared Time of LPS treatment (h) with control group.

198 Mol. Cells 2021; 44(4): 195-206 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al.

A B 2 * LPS - + 1.5 ICAM1

GAPDH 1

0.5

Relative expression (ICAM1/folds) Relative expression 0 NC LPS Fig. 3. The assessment of BV2 Treatment groups microglia stimulated by LPS to confirm the function as C D 6 . (A and B) The confirmation of ICAM1 biomarker IL-6 5 for the conversation of pro- inflammatory M1 macrophage GAPDH 4 of microglia stimulated with LPS. 0 1 2 3 4 3 (C and D) The expression of pro- Time of LPS treatment (h) 2 inflammatory cytokine IL-6 in activated microglial cells. Data 1 are expressed as mean ± SEM. Relative expression (IL-6/folds) (IL-6/folds) expression Relative 0 Original gel images showed in 0 1 2 3 4 Supplementary Fig. S3. *P < 0.05, Time of LPS treatment (h) as compared with control group.

A

SH-SY5Y, BV2 LPS treatment for 2 h seeding 2 5 ସ 1 Day 1 Day ൈ ͳͲ ସ ൈ ͳͲ Co-culture of Neuron Co-culture of neuron with neuron with Co-culture of microglia (upper) microglia activated microglia and neurons (lower) Fig. 4. The evaluation of bio­ Co-culture Microglia Activated Neuron started (BV2) microglia (SH-SY5Y) logical linkage between activa­ ted BV2 microglia and neuron cells. (A) Co-culture was stabilized by seeding microglia (BV2) in the upper well and neuron (SH- B 100 SY5Y) in the down well, and then the wells were combined after 80 24 h. (B) Although co-culture of neurons may be affected by 60 unstimulated microglia to some extent, neuron viability was 40 ** significantly reduced by activated

Cell viability (%) (%) viability Cell microglia. This confirmed that 20 activated microglia can damage neurons. Data are presented 0 as mean ± SEM for three inde­ Neuron only LPS-treated neuron Neuron with Neuron with pendent experiments. **P < 0.02, microglia activated microglia by LPS as compared with neuron with Treatment groups microglia.

Mol. Cells 2021; 44(4): 195-206 199 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al. demonstrated that microglia activated by foreign stimulation Downregulated nuclear factor-kappa B activity of microg- have a serious effect on neurons. lia in protein level by annexin A1 knockdown suppression It is well known that many pro-inflammatory cytokines such Characterization change of activated BV2 microglia by an- as IL-6 and TNF-α induction mainly depend on the NF-κB ac- nexin A1 silencing tivity with the onset of an immune response. Among various To investigate the role of ANXA1 in the regulation of M1 cytokines, NF-κB is a protein complex that controls transcrip- phenotype polarization and function in activated microglia, tion of DNA, cytokine production, and cell survival. NF-κB not various shRNA were generated to induce the knock-down only plays a key role in regulating the immune response to (Kd) of ANXA1 at the transcriptome level. Successful cell infection, but is also a major transcription factor that regu- transfection efficiency of shRNA series was confirmed by the lates the genes responsible for innate and adaptive immune expression of GFP protein inserted into the shRNA expres- responses. When NF-κB is activated, various cytokines are se- sion vector in Fig. 5A. Among the shRNA used, ANXA1 gene creted, causing damage to neurons. Thus, we evaluated how expression in BV2 microglia treated with shB successfully the Kd of ANXA1 in microglia affects NF-κB activity using the showed the best gene silencing efficiency through markedly ICW assay. As shown in Fig. 6, the expression of phosphory- decreased mRNA expression levels of ANXA1 (Fig. 5B). As lated NF-κB increased with cytokine secretion after LPS treat- shown in Fig. 5C, the mRNA levels of ICAM1, a good indi- ment. The shScr-treated group also showed similar results to cator of the pro-inflammatory state, were decreased as low those of the LPS-treated group. However, microglia treated as that of microglia not activated by the ANXA1 Kd. These with shB-ANXA1 showed significantly decreased NF-κB results demonstrate that ANXA1 Kd can reduce the immune expression after LPS treatment as compared to the LPS- or reaction to foreign pathogens in microglia. shScr-treated cells (Fig. 6A). The quantitative data of NF-κB in the Odyssey image results showed a similar tendency (Fig.

A X100

shScr shA shB shC shD

B 1.2

1.0

0.8

0.6 Fig. 5. Knock-down of ANXA1

(ANXA1/folds) 0.4 and deactivation in BV2 microglia. ** Short hairpin RNA (shRNA) series Relative expression ratios expression Relative 0.2 were constructed in pGPI6/GFP/ Neo vector to effectively knock- 0 shScr shA shB shC shD down ANXA1 gene in the activated Treatment groups microglia. (A and B) Knock-down efficiency of ANXA1 by const­ ructed shRNAs through GFP

C LPS - + + expression and qPCR experiment. **P < 0.02, as compared with ICAM1 shScr group. (C) The assessment of M1 type depolarization of activated microglial cells treated GAPDH with shRNA. Original gel images NC shScr shB-ANXA1 showed in Supplementary Fig. S4.

200 Mol. Cells 2021; 44(4): 195-206 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al.

6B). The decreased activity of NF-κB by ANXA1 Kd could also flammatory M1 phenotype display a metabolic transition be caused by the downregulation of cytokines. To address towards aerobic glycolysis. Since mitochondria play a key role this, we examined the expression level of pro-inflammatory in this transition, we assessed whether the downregulation cytokine IL-6 following shRNA treatment. In BV2 microglial of ANXA1 affected mitochondrial function. To address this, cells stimulated by LPS, the secretory levels of IL-6 in the su- a cellular respiratory assay using the Seahorse XF analyzer pernatant detected by ELISA were significantly increased as was used to assess mitochondrial function in ANXA1 Kd. compared to the unstimulated cells (NC group). Conversely, Microglia were exposed to glycolytic stress compounds in the shB-ANXA1 treated BV2 microglia cells showed reduced IL-6 following order: oligomycin, FCCP, and rotenone & antimycin expression as compared to the LPS- or shScr-treated groups A during the real-time OCR measurement. The OCR of cells, (Fig. 6C). In conjunction with the results seen in Fig. 6C, we an important indicator of normal cellular function, is used examined whether ANXA1 Kd could affect cytokine expres- as a parameter to study mitochondrial function. As shown sion at the transcriptome level. Consistent with the protein in Fig. 7, ANXA1 Kd showed a significantly decreased OCR level results, the increase in TNF-α and IL-6 mRNA levels in- than that of the LPS-treated microglial cells. However, OCR duced by LPS was significantly alleviated by ANXA1 Kd (Figs. was restored in normal conditions like in the negative control 6D and 6E). These data indicate that ANXA1 Kd could de- (NC) group, or when BV2 microglia were treated with shB- crease LPS-induced inflammatory cytokine expression in BV2 ANXA1 (Fig. 7A). These results indicate that the activation of microglial cells, leading to neuronal cell survival. ANXA1 impairs mitochondrial function in microglia. More- over, the baseline respiratory rate and energy demand of Inhibition of mitochondria dysfunction in annexin the cell under baseline conditions did not differ between the A1-downregulated microglia NC and ANXA1 Kd groups after 2 h of LPS stimulation (Fig. Metabolic reprogramming has been tightly correlated with 7B). Impairment in mitochondrial respiration was more pro- macrophage polarization and function. For instance, pro-in- nounced in LPS-induced microglial cell, as demonstrated by

A

NF-κB

β-actin

NC LPS shScr shB-ANXA1

B 1.8 C 140

1.5 120

) * 100 κB 1.2 80 * 0.9 60 Fig. 6. The effect of on the 0.6 40 decreased NF-κB activity in LPS-

Intensity ratios (NF- 0.3 stimulated microglial cells via IL-6 expression level (pg/ml) level (pg/ml) IL-6 expression 20 knock-down of ANXA1. BV2 0 0 NC LPS shScr shB-ANXA1 NC LPS shScr shB-ANXA1 cells were transfected with Treatment groups Treatment groups ANXA1 shRNA for 48 h and then incubated with LPS (1 μg/ml) for 2 h. (A) The scanned imaging results D 12 E 120 and (B) quantitative intensity ratio

/folds) /folds) (ANXA1/β-action) by ICW analysis 10 100 for NF-κB activation. (C) The 8 80 secretory levels of IL-6 in activated BV2 microglial cells treated with 6 * 60 shRNA-B using ELISA. (D and E) 4 40 The expression of TNF-α and IL-6 ** in the transcriptomic levels. Data 2 20 were presented as mean ± SEM of Relative expression level expression (IL-6/folds) Relative 0 level expression (TNF- Relative 0 three independent experiments. NC LPS shScr shB-ANXA1 NC LPS shScr shB-ANXA1 *P < 0.05 and **P < 0.02, as Treatment groups Treatment groups compared with LPS group.

Mol. Cells 2021; 44(4): 195-206 201 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al. a significant reduction in maximal respiration, spare respira- Kd (Figs. 7F and 7G). These results suggest that microglial cell tory capacity, and coupling efficiency (Fig. 7C-7E). While no activated by LPS may have impaired mitochondrial function. significant difference in proton leak was observed between Thus, these results elicit the downregulation of ANXA1 in the NC and BV2 cells treated with shB-ANXA1 after LPS microglia under the stimulatory conditions induced by LPS. stimulation, adenosine triphosphate (ATP) production was The data suggested that in BV2 cells, a switch to glycolysis significantly lower in LPS-treated BV2 cells than in the ANXA1 appears to serve as a survival response to maintain ATP levels

A Rotenone& 180 oligo FCCP antimycin A

150 NC LPS 120 shB-ANXA1

90

60

30 OCR (pmol/min/Proteins) (pmol/min/Proteins) OCR 0 0 10 20 30 40 50 60 70 80 Time (min)

B 100 Basal C 150 Maximal Respiration

80 120

60 90

40 60

20 30

OCR (pmol/min/Proteins) (pmol/min/Proteins) OCR Fig. 7. Effect of ANXA1 down­ OCR level (pmol/min/Proteins) 0 0 regulation on mitochondrial NC LPS shB-ANXA1 NC LPS shB-ANXA1 function.­ BV2 microglial Treatment groups Treatment groups cells were seeded in 8-well Seahorse plate. Representative D E example of bioenergetics profile 60 Spare Respiratory Capacity 120 Coupling Efficiency (Supplementary Fig. S5) shows a normal response pattern 50 100 for control BV2 cells for basal 40 80 respiration, and following addition of the mitochondrial stressors 30 60 oligomycin (oligo; 1.0 μM),

20 40 FCCP (1.0 μM), and Rotenone & OCR (pmol/min/Proteins) (pmol/min/Proteins) OCR antimycin A (0.5 μM). (A) Calcu­ 20 OCR (pmol/min/Proteins) (pmol/min/Proteins) OCR 10 lation of OCR as a percentage of control demonstrated a significant 0 0 NC LPS shB-ANXA1 NC LPS shB-ANXA1 difference between controls and Treatment groups Treatment groups LPS-exposed cells suggesting an increased extracellular acidifi­ cation of the media. (G) While F ATP Production G Protein Leak 50 45 no respectable reduction in proton leak, (B-F) LPS-exposed 40 cells showed a decrease in basal 30 30 respiration (B), maximal respiration (C), spare respiratory capacity 20 (D), coupling efficiency (E), and 15 ATP production (F) and were 10 OCR (pmol/min/Proteins) (pmol/min/Proteins) OCR OCR (pmol/min/Proteins) OCR (pmol/min/Proteins) unresponsive to the different

0 0 mitochondrial stressors, suggesting NC LPS shB-ANXA1 NC LPS shB-ANXA1 an impairment of mitochondrial Treatment groups Treatment groups function.

202 Mol. Cells 2021; 44(4): 195-206 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al. after inhibition of oxidative phosphorylation by NO. upon LPS stimulation, and this effect was significantly sup- pressed by the shRNA Kd of ANXA1 in BV2 cells. SH-SY5Y Increased survival effects of neuron on annexin A1 down- neuron cells were evaluated by double-staining fluores- regulation in BV2 microglia cence-activated cell sorting using FITC-annexin-V and PI. The We examined how the decrease in ANXA1 expression in cell death population was lower in neurons treated with shB- microglia affects the cell viability of neurons. As shown in the ANXA1 (annexin-V positive and PI negative, 0.18%; annex- results of Fig. 8A, the lowest cell survival rate was observed in-V positive and PI positive, 0.95%) as compared to those via MTT assay in the neurons co-cultured with activated mi- treated with LPS (annexin-V positive and PI negative, 0.92%; croglia when compared to untreated neurons. The viability annexin-V positive and PI positive, 4.15%) or those of other of neurons co-cultured with activated microglia reduced to groups (Figs. 8B and 8C). These results demonstrate that twice as low as that of neurons co-cultured with non-activat- ANXA1 Kd expression by LPS stimulation in microglia elicits ed microglia. In this result, as shown in Fig. 4, LPS treatment enhanced cell survival. showed some cytotoxicity to neuron cells, but, not as shown in activated microglia by LPS group. On the other hand, in DISCUSSION the case of neurons co-cultured with shB-ANXA1-treated microglia, the cell survival of neurons significantly improved With the increase in life expectancy, the neurodegenerative when compared to that of neurons co-cultured with LPS-acti- diseases, such as AD (Jeong, 2017; Masters et al., 2015), PD vated microglia. (Davie, 2008), and Tay-Sachs disease (Ramani and Sankaran, Flow cytometry experiments to clarify the improved cell 2020), is increasing considerably. They are involved by several viability and cell death mechanism showed results similar to factors including genetic, environmental, and endogenous those of MTT experiments. It showed that BV2 cells signifi- factors, however, the exact mechanism underlying neurode- cantly enhanced SH-SY5Y apoptosis (annexin V-positive) generative diseases are not fully known. Among the various

A 120

100

80 *

60

40 Cell viability (%) (%) viability Cell

20

0 Neuron LPS Microglia Microglia shScr-treated shB-GBP2-treated activated microglia microglia by LPS activated by LPS activated by LPS Treatment groups

B C Fig. 8. Enhanced cell survival of 8 neuron by down-regulation of ANXA1 in microglial cell. BV2 6 microglial cells were transfected with shB-ANXA1 or scramble vector for 48 h in co-culture 4 system followed by LPS incubated for 2 h. (A) SH-SY5Y neuron cell NC LPS viability by the MTT assay. *P

Neueonal cell death (%) 2 < 0.05, as compared with LPS group. (B and C) FACS analysis *** 0 of Annexin-V and PI double NC LPS shScr shB-ANXA1 staining in SH-SY5Y neuron cell co-cultured with microglial cells. Treatment groups Data are representative of at least three independent experiments. PI shScr shB-ANAX1 ***P < 0.01, as compared with Annexin-V LPS group.

Mol. Cells 2021; 44(4): 195-206 203 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al. researches regarding treatment of dementia (Summers et al., Stimulation like LPS activates NF-κB pathway, which leads 1986), the fundamental treatment using genetic material is in to further production of pro-inflammatory cytokines (McAr- the spotlight (Duncan and Valenzuela, 2017). Among them, thur et al., 2010). It is well known that pro-inflammatory M1 ANXA1 play a potential anti-inflammatory functions such as phenotype undergo metabolic re-programming typically char- action, removal of apoptotic leukocytes in pe- acterized by increased glycolysis (Choi et al., 2020) and de- ripheral nervous system (Buckingham et al., 2006). However, creased oxidative phosphorylation (Ransohoff, 2016; Wang the exact mechanism and action of microglia in controlling et al., 2014). Interestingly, oxidative phosphorylation was immune response in CNS remains poorly understood. significantly decreased in ANXA1 Kd. This speculation is sup- Here, we elucidated that ANXA1 Kd plays a protective role ported by previous reports that increased glucose uptake and against inflammation induced by LPS in BV2 microglia to enhanced glycogen accumulation were seen in M1 polarized maintain mitochondrial function and prevent neuronal dam- phenotype (Qiu et al., 2018). As shown in the results of the age. BV2 microglial cells activated by LPS showed a remark- energy map (Fig. 7), shRNA-ANXA1-treated microglial cell able up-regulation in the ANXA1 expression (Figs. 2 and 3). tended to return the OCR of reduced mitochondria to their These characteristics are important results indicating that the original state (Fig. 7), suggesting that these cells may favor expression of ANXA1 is induced in relation to the immune entering the G0 cell cycle and transform into quiescent status. response in microglial cell. Microglia, as macrophage-like Quiescent microglial cell are usually deactivated and have a resident immune cell in the CNS (Block et al., 2007), are steady-state phenotype. Further research may be needed, but considered the first line of defense in the CNS and provide the downregulation of ANXA1 showed a mitochondrial func- tissue defense and protection (Ransohoff and Perry, 2009). tion similar to that of non-activated microglia. Eventually, the However, severe neuronal damage could cause by excessive ANXA1 Kd can inhibit intracellular energy production, and acute or chronic microglial activation via the release of cyto- microglia can differentiate into M1 type based on the pattern toxic factors such as NO, ROS, TNF-α, and IL series (Colonna of oxygen consumption in the mitochondria. and Butovsky, 2017). Thus, microglia may work as a dou- These results indicate that activated microglia revert to the ble-edged sword because it is responsible for neurotoxic and deactivated state and eventually reduce neuronal cell dam- neuroprotective effects (Dai et al., 2015). age. The ANXA1 Kd suggests that the damage and viability of In the injured brain, the activated microglia participate in neuronal cells are affected. To address this, we also assessed the course of inflammation (Liu and Hong, 2003), a process the cell viability of neurons in the co-culture system when the that includes actions of various cytokines by foreign stim- ANXA1 gene was downregulated. As a result, the cell surviv- ulation (Gordon and Taylor, 2005), leading to particularly al of neurons by ANXA1 Kd microglia showed decreased cell harmful effects on the neurons. Various materials secreted death with reduced apoptotic cell population (Fig. 8). Over- from activated microglia, such as IL-6 and TNF-α, can form a all, our study suggests that downregulation of ANXA1 can vicious cycle of pro-inflammatory responses that continuously improve neuronal survival to clear damaged microglia caused damage neurons and other important nervous system struc- by pro-inflammatory cytokine stimulation. Thus, this indicates tures (Hanisch and Kettenmann, 2007; Henkel et al., 2009). the possibility that ANXA1 could be the new regulator of As shown in the results of Figs. 3C and 4, neuronal damage neuroinflammation by external stimulation for activating the was observed to be induced by the activated microglia. The microglia. release and accumulation of pro-inflammatory factors secret- In the CNS, microglia act as macrophages when foreign ed by activated microglia are thought to induce neurotoxicity, pathogens invade, causing elimination of foreign substances especially in neurodegenerative diseases (Colton and Wilcock, by the induction of immune responses as the first step in 2010). Additionally, the damaged neurons secrete neurotox- defense. However, hyperactive microglia in the brain have a ic soluble factors that in turn induce microglial reactivation. negative effect on neurons, which can also cause dementia. Thus, a vicious cycle causing prolonged neuroinflammation In this study, we used ANXA1 genetic material to reduce the and progressive neurodegeneration is created. Therefore, it is side effects of activated BV2 microglia on neurons and pre- important to clarify the cellular crosstalk signaling pathways vent neuronal damage. When the “resting microglia” become between microglia and neurons to identify future therapeutic “activated microglia” by pathogens such as LPS, the NF-κB targets of CNS disorders, including pain, infection, neurode- signal is activated in them. Therefore, we conducted a study generation, and stroke. on the effects of Kd ANXA1 genes overexpressed in the For this, ANXA1 is a good genetic material that can control activated microglia. Collectively, our data indicated that the the crosstalk between microglia and neurons. The increase of downregulation of ANXA1 resulted in decreased secretion ANXA1 in LPS-activated microglia indicates that external stim- of cytokines from the LPS-activated microglia. In a co-culture uli act as inducers of ANXA1 expression (Fig. 2). Although system between activated microglia and SH-SY5Y neurons, ANXA1 works in the early stages of external stimuli, it induc- microglia treated with shRNA-ANXA1 induced decreased es the differentiation of microglia into M1 type to function as damage and apoptosis in neurons. Taken together, our study a macrophage (Fig. 3), i.e., conversion into cells that damage suggest that ANXA1 could be used as a new potential target neurons. Hence, the upregulation of ANXA1 could damage on neuronal damage by activated microglia. the adaptive response of microglia during inflammatory con- ditions. It is an attempt of the system to reduce inflammation Note: Supplementary information is available on the Mole- in the early stages of the disease, since downregulation of cules and Cells website (www.molcells.org). ANXA1 effectively resolves the inflammatory phase.

204 Mol. Cells 2021; 44(4): 195-206 Biological Function of ANXA1 in Microglia Activated by LPS Ji-Eun You et al.

ACKNOWLEDGMENTS Aging 17, 673-680. The research leading to these results received from the Kon- Fan, Y., Xie, L., and Chung, C.Y. (2017). Signaling pathways controlling yang University Research Fund in 2019. microglia . Mol. Cells 40, 163. Franco, R. and Fernandez-Suarez, D. (2015). Alternatively activated AUTHOR CONTRIBUTIONS microglia and macrophages in the central nervous system. Prog. P.H.K. designed experiment, coordinated the project and Neurobiol. 131, 65-86. helped in manuscript preparation and writing. J.E.Y. and Gordon, S. and Taylor, P.R. (2005). and macrophage S.H.J. performed the experiments and contributed to the heterogeneity. Nat. Rev. Immunol. 5, 953-964. data collection and analysis. J.E.Y. wrote the manuscript. Graeber, M.B. (2010). Changing face of microglia. Science 330, 783-788. Han, P.F., Che, X.D., Li, H.Z., Gao, Y.Y., Wei, X.C., and Li, P.C. (2020). Annexin CONFLICT OF INTEREST A1 involved in the regulation of inflammation and pathways. 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