Citrus flavonoids suppress IL-5 and ROS through distinct pathways in PMA/ionomycin-induced EL-4 cells

Abstract

Interleukin-5 (IL-5) strongly initiates the asthmatic inflammatory response, which affects 300 million patients with asthma annually worldwide, through oxidative stress generation. Citrus flavonoids have beneficial properties, such as anti-inflammatory and antioxidant properties, but the precise molecular mechanism of the inhibition of the asthmatic inflammatory response is still unclear. This study aimed to investigate the underlying mechanisms of ROS and IL-5 reduction with citrus flavonoid treatment in PMA/ionomycin-induced EL-4 cells. Our results showed that hesperetin and gardenin A dramatically suppressed ROS and IL-5 production through distinct pathways. Interestingly, hesperidin induced HO-1 expression through the transcription factor Nrf2 coupled with the PI3K/AKT or ERK/JNK signaling pathway, consequently downregulating NFAT activity and IL-5 secretion. Likewise, gardenin A induced HO-1 expression and subsequently suppressed IL-5 production by reducing NFAT activity and upregulating PPARγ in EL-4 cells, suggesting that inducing HO-1 expression may inhibit asthmatic inflammation. Altogether, hesperidin and gardenin A have great potential for regulating the asthma-associated immune responses through antioxidant properties.

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1. Introduction

It is well known that air pollution from electricity, fuel, and transportation is a permanent physical risk and contributes to the global burden of disease (GBD), such as asthma, which has over 300 million patients annually in the world.^1–4 In addition to ambient air pollution, indoor endotoxin exposure is also linked to allergic rhinitis and asthma.^5,6 Although investigators and clinicians are striving to find personalized prevention strategies through epidemiological studies on air pollution and asthma, the high incidence and prevalence of asthma still cannot be mitigated.^7–9

How air pollution affects asthma and allergic rhinitis is unclear. Despite intensive research, scientists still cannot completely understand why some individuals develop antigen-specific cell-mediated immune responses. Nevertheless, it is certain that individual genetic susceptibility may affect an especially proinflammatory subtype of antigen-specific T helper 2 (Th2) cells, which are dominant in initiating and orchestrating the asthmatic inflammatory response pathways through the generation of oxidative stress.^10–12 An overactive Th2 immune response is stimulated by allergens and typically produces characteristic effector cytokines such as interleukin-5 (IL-5).¹¹ Th2 cytokine expression is posttranscriptionally regulated by Th2-related transcription factors, including nuclear factor of activated T cells (NFAT), signal transducer and activator of transcription 6 (STAT6), and activator protein-1 (AP-1).^13,14

IL-5, a Th2 cell-derived cytokine, is involved in eosinophil development and differentiation from myeloid precursor cells in bone marrow.¹⁵ Dysregulation of IL-5 secretion by Th2 cells has been linked to eosinophilic inflammation in asthma.¹⁶ In addition, reactive oxygen species (ROS), such as nitric oxide (NO) and eosinophil extracellular traps (EETs), a cytotoxic granule protein, have been found to be involved in severe eosinophilic asthma.^17,18 Mepolizumab, an anti-IL 5 humanized monoclonal antibody (trade name Nucala), has been documented as having therapeutic benefits and is a standard care for severe eosinophilic asthma patients.¹⁹ However, anti-IL-5 therapy with mepolizumab induces a sustained decrease in blood eosinophils with an increase in lymphocyte IL-5 and IL-5 receptor α production, suggesting that the endogenous IL-5 autoregulatory mechanism or IL-5-independent pathway exacerbates eosinophilic asthma processes and should be considered before asthma treatment.^20–22 Therefore, finding a safe, effective, and economical alternative treatment strategy is important for allergic rhinitis and asthma control.

Flavonoids are the largest group of phytonutrients in the human diet and are ubiquitously found in foods, including tea, cocoa, vegetables, red wine, and fruits.²³ Several epidemiological studies have examined the relationship between flavonoids in dietary intake and asthma.²⁴ A previous study demonstrated that flavonoids from apples and red wine consumption show a protective effect on asthma through a powerful antioxidant capacity.²⁵ A recent review summarized that flavonoids may be potential therapeutic agents for asthma treatment by targeting oxidizing molecules that conjugate immune mediators and sequence-specific DNA-binding factors.²⁶ Moreover, a previous report demonstrated that fisetin, a flavonol, inhibits IL-5 cytokine production in the human KU812 basophil cell line.²⁷ Likewise, hesperidin effectively treats asthma by reducing IL-5 cytokine levels.²⁸ Citrus flavonoids, including nobiletin, tangeretin, 5-demethylnobiletin (5-DN), gardenin A, and hesperidin, have been shown to have powerful antioxidant activity and anti-inflammatory properties in promoting health.^29–32 However, the underlying mechanisms by which citrus flavonoids suppress Th2 cell-derived IL-5 and ROS remain unclear. In this study, we aimed to uncover the signaling related to the downregulation of IL-5 and ROS expression by citrus flavonoid treatment in EL-4 lymphocyte cells.

2. Materials and methods

2.1. Chemicals

Dulbecco's modified Eagle's medium (DMEM), horse serum (HS), and penicillin−streptomycin (PS) antibiotic solution were obtained from Invitrogen (Carlsbad, CA, USA). Citrus flavonoids (nobiletin, 5-demethylnobiletin, tangeretin, and gardenin A) were kindly provided by Professor Chi-Tang Ho (Rutgers University, USA). Hesperidin, phorbol 12-myristate 13-acetate (PMA), ionomycin, dimethyl fumarate, retinoic acid, GW9662, and rosiglitazone were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). AS1517499 was purchased from Axon Medchem (Axon Medchem (Groningen, The Netherlands). SR 11302 was purchased from Tocris Bioscience (Ellisville, MO, USA). NFAT inhibitor was obtained from Cayman Chemical (Ann Arbor, MI, USA). Anti-Nrf2 was purchased from Novus Biologicals (Littleton, CO, USA). Anti-HO-1 and anti-lamin B1 antibodies were obtained from BioVision (Milpitas, CA, USA). Anti-NFAT, anti-STAT6, anti-β-actin, anti-c-Jun, anti-c-Fos, anti-PI3k, anti-phospho-PI3k, anti-Akt, anti-phospho-Akt, anti-ERK, anti-phospho-ERK, anti-p38, anti-phospho-p38, anti-JNK, and anti-phospho-JNK were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-PPARγ was obtained from Merck Millipore (Billerica, MA, USA). Anti-PCNA was purchased from GeneTex (Irvine, CA, USA). PD98059, wortmannin, and SP600125 were obtained from Biosource (Camarillo, CA, USA).

2.2. Cell culture

The EL-4 murine T-lymphoma cell line (BCRC 60179) was obtained from the Bioresource Collection and Research Center (BCRC, Food Industry Research and Development Institute, Hsinchu, Taiwan). Cells were grown in DMEM and supplemented with 10% HS. Confluent cells were subcultured at a ratio of 1 : 3, and the medium was changed twice a week. Cells were cultured at 37 °C under a humidified atmosphere of 5% CO₂.

2.3. Cell viability analysis

Cell viability analysis was performed according to our previous report.³³ Briefly, after treatment, cells were then suspended in trypan blue solution for viability measurement.

2.4. Enzyme-linked immunosorbent assay (ELISA)

Quantification of IL-5 secretion by PMA/ionomycin-induced EL-4 cells was performed after treatment with or without citrus flavonoids (10 μM) for 24 h. The level of IL-5 was evaluated according to the ELISA kit instructions (R&D system Inc., Minneapolis, MN, USA).

2.5. Intracellular ROS determination

The 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) method was used to measure intracellular ROS production according to the method described by Cheng et al. (2019).³⁴

2.6. Nuclear and cytosolic protein extraction

After citrus flavonoid (10 μM) or inhibitor stimulation, the nuclear and cytosolic proteins were extracted using a nuclear/cytosol fractionation kit (BioVision, Mountain View, CA, USA) and according to the method described by Ho et al. (2012).³⁵

2.7. Western blotting

Western blotting was performed according to the report by Chang et al. (2019).³⁶ The primary antibodies used in this study were NFAT, c-Jun, c-Fos, STAT6, Nrf2, PPARγ, HO-1, phospho-PI3k, PI3k, phospho-Akt, Akt, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, lamin B1, β-actin, and PCNA.

2.8. Statistical analysis

All results are presented as mean ± SD. Significant differences were evaluated by ANOVA followed by Duncan's test between multiple groups. A p value <0.05 was considered to be significant.

3. Results

3.1. Regulating IL-5 and ROS production through the transcription factors NFAT, STAT6, and AP-1 in PMA/ionomycin-induced EL-4 cells

Several articles have shown that PMA plus ionomycin enhances Th2 cytokine production in EL-4 cells.^37–39 In the present study, we found that the maximum IL-5 and ROS production by EL-4 cells occurred with 0.1 μg mL⁻¹ PMA combined with 400 nM ionomycin treatment for 24 h (Fig. 1A and B). The cell viability was not affected by 0.1 μg mL⁻¹ PMA plus 400 nM ionomycin treatment compared to untreated cells (Fig. 1C). To validate the potent transcription factors involved in IL-5 and ROS production, nuclear protein expression of the Th2-associated NFAT, STAT6, and AP-1 family members c-Fos and c-Jun transcription factors were evaluated in PMA/ionomycin-induced EL-4 cells. As shown in Fig. 1D and E (quantification data), all the nuclear protein expression levels of these transcription factors were upregulated by PMA/ionomycin treatment in EL-4 cells. To confirm this result, PMA/ionomycin-induced EL-4 cells were pretreated with an NFAT inhibitor, STAT6 inhibitor (AS1517499), or AP1 inhibitor (SR11302) for 1 h. After stimulation, IL-5 secretion was significantly reduced by inhibitor treatment, particularly in NFAT inhibitor-treated cells (Fig. 1F). Likewise, ROS production was significantly inhibited by inhibitor treatment (Fig. 1G), indicating that not only IL-5 but also ROS production was regulated by NFAT, STAT6, and AP-1 transcription factors in PMA/ionomycin-treated EL-4 cells. In addition, cell viability was not affected in inhibitor-treated cells (Fig. 1H).

Fig. 1 Regulating IL-5 and ROS production in PMA/ionomycin-induced EL-4 cells. (A) IL-5 and (B) ROS were induced by 0–10 μg mL⁻¹ PMA combined with 400 nM ionomycin treatment for 24 h. (C) Cell viability was determined after treatment with 0.1 μg mL⁻¹ PMA combined with 400 nM ionomycin for 24 h. (D) Th2-associated NFAT, c-FOS, c-Jun, and STAT6 transcription factor protein expression. (E) Quantification data of transcription factors were assessed. The relative level of protein is expressed as the fold change over the control to normalize against PCNA (loading control). The effects of transcription factor inhibitors on (F) IL-5, (G) ROS, and (H) cell viability were measured in PMA/ionomycin-induced EL-4 cells. The results are expressed as mean ± SD (n = 3). * indicates p < 0.05 compared with the control cells. Different letters indicate p < 0.05 compared with every other group.

3.2. Citrus flavonoids suppressed IL-5 and ROS production in PMA/ionomycin-treated EL-4 cells

It is well known that citrus flavonoids have excellent antioxidant activity and anti-inflammatory properties that promote health.^29–31 Here, citrus flavonoids, including nobiletin, tangeretin, 5-DN, gardenin A, and hesperidin, were used to explore their effects on IL-5 and ROS production in EL-4 cells treated with PMA/ionomycin. As shown in Fig. 2A, gardenin A or hesperidin displayed an intensely suppressive effect on IL-5 secretion by PMA/ionomycin-treated EL-4 cells, respectively. Additionally, ROS production was inhibited by gardenin A or hesperidin treatment in PMA/ionomycin-incubated EL-4 cells (Fig. 2B). Surprisingly, 5-DN treatment dramatically increased IL-5 production and significantly decreased ROS expression in PMA/ionomycin-treated EL-4 cells (Fig. 2A and B). In addition, no cytotoxicity was found in 10 μM gardenin A- or hesperidin-treated cells compared with untreated cells (Fig. 2C). NFAT was the major transcription factor in regulating IL-5 secretion by PMA/ionomycin-induced EL-4 cells. NFAT protein expression was inhibited by gardenin A or hesperidin treatment in a dose-dependent manner (5 to 10 μM) in PMA/ionomycin-treated EL-4 cells (Fig. 2D and E). For the reasons given above, we chose those doses of citrus flavonoids to perform the study.

Fig. 2 Citrus flavonoids inhibited IL-5 and ROS production in PMA/ionomycin-induced EL-4 cells. Cells were co-treated with citrus flavonoids and PMA/ionomycin for 24 h. The level of (A) IL-5 in the medium was evaluated by ELISA. (B) ROS level in cells was determined using the fluorescent probe DCFH-DA (20 μM). The cells were incubated with gardenin A or hesperidin for 24 h. (C) Cell viability was measured. (D and E) NFAT protein expression was regulated by hesperidin or gardenin A combined with PMA/ionomycin treatment in EL-4 cells for 24 h. The results are expressed as mean ± SD (n = 3). Different letters indicate p < 0.05 compared with each group.

3.3. Hesperidin induced HO-1 expression through the transcription factor Nrf2 coupled with the PI3K/AKT or ERK/JNK signaling pathway in EL-4 cells

Heme oxygenase-1 (HO-1) is upregulated in an ROS-dependent manner through the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) or peroxisome proliferator-activated receptor γ (PPARγ) in rosiglitazone-treated human pulmonary alveolar epithelial cells.⁴⁰ As shown in Fig. 3A and B, upregulated Nrf2 nuclear protein expression and HO-1 antioxidant cytosolic protein levels were observed in EL-4 cells with hesperidin incubation for 2 h. Further investigation of the related kinases that modulate HO-1 expression found an increase in the phosphorylation of PI3K/Akt, ERK, and c-JNK in EL-4 cells with hesperidin treatment (Fig. 3C). However, kinase phosphorylation was impeded by PI3K inhibitor (Wortmannin), MAP kinase inhibitor (PD98059) or JNK inhibitor (SP600125) pretreatment in hesperidin-treated EL-4 cells (Fig. 3D).

Fig. 3 Hesperidin induced HO-1 expression via the transcription factor Nrf2 coupled with the PI3K/AKT or ERK/JNK signaling pathway in EL-4 cells. Cells were incubated with or without 10 μM hesperidin for 0, 2, 4, and 6 h. Protein expression of (A) transcription factors Nrf2 and PPARγ and (B) HO-1 was measured by western blot analysis. The cells were incubated with or without 10 μM hesperidin for 2 h. (C) PI3K, Akt, ERK, p38, and JNK protein expression was assessed. Cells were pretreated with or without wortmannin (20 μM), PD98059 (20 μM) or SP600125 (20 μM) for 1 h and then incubated with hesperidin (10 μM) for another 2 h. (D) Nrf2 protein expression was analyzed by western blot analysis. The results are expressed as mean ± SD (n = 3). The relative level of protein is expressed as the fold change over the control to normalize against the loading control (Lamin B1, β-actin or PCNA).

3.4. Hesperidin suppressed IL-5 production through the transcription factor NFAT in PMA/ionomycin-induced EL-4 cells

Previous evidence demonstrated the negative regulation of NFAT protein expression by the transcription factor Nrf2.⁴¹ We therefore assessed NFAT nuclear protein expression by pretreating EL-4 cells with or without an Nrf2 agonist (dimethyl fumarate, DMF) and an Nrf2 antagonist (retinoic acid, RA), followed by hesperidin coupled with PMA/ionomycin treatment. As shown in Fig. 4A, pretreatment with DMF slightly altered NFAT protein expression in hesperidin- and PMA/ionomycin-treated cells compared with PMA/ionomycin-treated cells. Interestingly, NFAT protein expression increased with RA pretreatment in PMA/ionomycin-treated EL-4 cells; however, this increase was abolished by hesperidin treatment in a dose-dependent manner (Fig. 4A). Furthermore, IL-5 levels were evaluated under DMF or RA pretreatment conditions in hesperidin- and PMA/ionomycin-treated EL-4 cells. Pretreatment with DMF significantly inhibited IL-5 secretion in hesperidin- and PMA/ionomycin-treated cells compared with non-DMF-stimulated cells (Fig. 4B). Additionally, elevated IL-5 production by RA pretreatment in PMA/ionomycin-treated EL-4 cells was significantly blocked by 5 or 10 μM hesperidin treatment (Fig. 4B). These results suggest that hesperidin and the signaling it initiates strongly affected NFAT and IL-5 and had suppressive effects even under Nrf2 antagonist stimulation (Fig. 4A and B). Hesperidin-initiated signaling in PMA/ionomycin-treated EL-4 cells was explored. As shown in Fig. 4C and D, the phosphorylation of PI3K, PKC, ERK, JNK, and p38 was promoted by PMA/ionomycin treatment, whereas hesperidin treatment reduced phosphorylation in a dose-dependent manner in PMA/ionomycin-treated EL-4 cells.

Fig. 4 Hesperidin suppressed IL-5 production through the transcription factor NFAT in PMA/ionomycin-induced EL-4 cells. The cells were pretreated with or without DMF (25 μM) or RA (2 μM) for 1 h and then incubated with or without hesperidin (10 μM) and exposed to PMA/ionomycin for 24 h. After stimulation, the effect of DMF and RA on (A) NFAT protein expression and (B) IL-5 levels were assessed. Effect of hesperidin on (C) PI3K/PKC and (D) ERK/JNK/p38 protein expression in PMA/ionomycin-induced EL-4 cells after 24 h of incubation. The results are expressed as mean ± SD (n = 3). The relative level of protein is expressed as the fold change over the control to normalize against the loading control (Lamin B1 or β-actin). Different letters indicate p < 0.05 compared with each group.

3.5. Gardenin A induced HO-1 expression and suppressed IL-5 production through the transcription factor PPARγ in EL-4 cells

As previously mentioned, HO-1 may be regulated by the transcription factors Nrf2 and PPARγ.⁴⁰ We found that the maximum PPARγ nuclear protein level and HO-1 protein expression were upregulated in EL-4 cells by 10 μM gardenin A treatment for 4 h (Fig. 5A and B). Phosphorylation of the related kinase ERK was increased in gardenin A-treated EL-4 cells (Fig. 5C). Moreover, IL-5 production by PMA/ionomycin-treated EL-4 cells was significantly reduced by gardenin A treatment in a dose-dependent manner (Fig. 5D). Interestingly, a decrease in IL-5 expression by PMA/ionomycin-incubated EL-4 cells that were treated with a PPARγ agonist (Rosiglitazone) coupled with gardenin A was observed (Fig. 5D). In contrast, the opposite result was observed with PPARγ antagonist (GW9662) treatment in PMA/ionomycin-treated EL-4 cells (Fig. 5D).

Fig. 5 Gardenin A reduced HO-1 expression through the transcription factor PPARγ in EL-4 cells. Cells were incubated with or without 10 μM gardenin A for 0, 2, 4, and 6 h. Protein expression of (A) transcription factors Nrf2 and PPARγ and (B) HO-1 were measured by western blot analysis. The cells were incubated with or without 10 μM gardenin A for 4 h. (C) PI3K, Akt, ERK, p38, and JNK protein expression was assessed. The cells were pretreated with or without rosiglitazone (10 μM) and GW9662 (2 μM) for 1 h and then incubated with or without gardenin A (10 μM) and exposed to PMA/ionomycin for 24 h. (D) IL-5 levels were assessed. The results are expressed as mean ± SD (n = 3). The relative level of protein is expressed as the fold change over the control to normalize against the loading control (Lamin B1 or β-actin).

4. Discussion

The asthmatic-Th2 immune response typically produces the characteristic effector cytokine IL-5 and generates oxidative stress.^10–12 Citrus flavonoids have been reported to have a variety of beneficial qualities, including impairing dengue virus replication, ameliorating cardiac hypertrophy, and preventing hepatic steatosis, improving hepatic ischemia-reperfusion injury, as well as antimicrobial activities and antiendotoxemic, anti-inflammatory and antioxidant properties.^42–48 However, the effect of citrus flavonoids on the asthmatic inflammatory response is still unclear. Here, we found that citrus flavonoids, including hesperetin and gardenin A, dramatically prohibited ROS and IL-5 production in PMA/ionomycin-induced EL-4 cells.

In this study, citrus flavonoids, including nobiletin, tangeretin, 5-DN, hesperetin, and gardenin A, were used to investigate the suppressive effect on ROS and IL-5 production. IL-5 secretion was increased by nobiletin, tangeretin, and especially 5-DN treatment in PMA/ionomycin-incubated EL-4 cells (Fig. 2A). However, IL-5 has been demonstrated to repress the Th1 response and induction of antigen-specific CD4⁺CD25⁺ T regulatory cells,^49,50 indicating that 5-DN could be a potential natural compound for preventing autoimmune disease.

NFAT, STAT6, and AP-1 family transcription factors c-Fos and c-Jun are involved in balancing the Th1 and Th2 immune responses.¹⁴ NFAT has been shown to be a master transcription factor in enhancing the Th2 response for choreographing the cytokine profile.^51,52 In addition, a previous study demonstrated that the NFAT/AP-1 binding element plays an important role in IL-5 synthesis in peripheral T cells from patients with asthma.⁵³ Consistently, our study found that IL-5 production was strongly regulated by the transcription factor NFAT in PMA/ionomycin-treated EL-4 cells (Fig. 1). Treatment with 10 μM hesperetin dramatically suppressed IL-5 secretion by downregulating NFAT nuclear protein expression (Fig. 2).

Increasing HO-1 expression suppressed the asthmatic immune response through MAPK, Nrf2, and PPARγ transcriptional signaling pathways for the therapeutic modulation of inflammation.⁵⁴ Convincingly, our results demonstrated that hesperetin and gardenin A upregulated HO-1 production in PMA/ionomycin-treated EL-4 cells. Interestingly, hesperidin induced HO-1 expression via the transcription factor Nrf2 coupled with the PI3K/AKT or ERK/JNK signaling pathway in EL-4 cells (Fig. 3). Moreover, gardenin A induced HO-1 expression and suppressed IL-5 production through the transcription factor PPARγ in EL-4 cells (Fig. 5). Hesperetin and gardenin A upregulated HO-1 production through distinct pathways. However, both hesperetin and gardenin A significantly downregulated IL-5 secretion by the transcription factor NFAT (Fig. 2, 3 and 5), indicating that increasing HO-1 expression may inhibit asthmatic inflammation by suppressing IL-5 secretion.

Our study demonstrated that hesperidin induced the activation of Nrf2 and HO-1 via increasing the phosphorylation of the PI3K/AKT or ERK/JNK signaling pathway. These results played an important role in the inhibition of NFAT expression and consequently suppressed IL-5 secretion in PMA/ionomycin-treated EL-4 cells. In addition, gardenin A treatment showed a similar effect in inducing HO-1 expression and suppressing IL-5 production through the transcription factor PPARγ in EL-4 cells. Taken together, downregulating NFAT via the activation of Nrf2 and PPARγ contributed to the hesperidin- and gardenin A-mediated inhibitory effects on IL-5 and ROS production by PMA/ionomycin-incubated EL-4 cells, suggesting that stimulating HO-1 expression may inhibit asthmatic inflammatory effects by suppressing IL-5 secretion and ROS production. Our study showed that citrus flavonoid consumption may protect against asthma pathogenesis through antioxidant properties.

Abbreviations

5-DN	5-Demethylnobiletin
AP-1	Activator protein-1
AP1 inhibitor	SR11302
DMF	Dimethyl fumarate (Nrf2 agonist)
GBD	Global burden of disease
HO-1	Heme oxygenase-1
IL-5	Interleukin-5
NFAT	Nuclear factor of activated T cells
NO	Nitric oxide
Nrf2	Nuclear factor-erythroid 2 related factor 2
PMA	Phorbol 12-myristate 13-acetate
PPARγ	Peroxisome proliferator activated receptor γ
PPARγ agonist	Rosiglitazone
PPARγ antagonist	GW9662
ROS	Reactive oxygen species
RA	Retinoic acid (Nrf2 antagonist)
STAT6	Signal transducer and activator of transcription 6
STAT6 inhibitor	AS1517499
Th2	T helper 2

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

This research work was supported in part by the grant MOST 108-2320-B-005-001- from the Ministry of Science and Technology, Taiwan.

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Footnote

† These authors contributed equally to this work.

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