2019 Volume 44 Issue 2 Pages 83-92
Immunological functions are disturbed in humans who have been chronically exposed to arsenic via contaminated groundwater. Little is known about the specific mechanisms underlying the impairment of immunological defense system caused by arsenic. The activation of macrophage cells upon infection with bacteria and viruses plays important roles in the defense against these pathogens. Here we show that exposure to arsenite (As(III)) suppresses nitric oxide (NO) production in murine RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS) and poly(I:C), the compounds mimicking bacterial and viral infection, respectively. As(III) suppressed the LPS- or poly(I:C)-evoked induction of inducible NO synthase (iNOS) without affecting the transactivation of NF-κB. As the interferon (IFN)-β/STAT1 pathway is also involved in the induction of iNOS in addition to NF-κB, we examined the effects of As(III) on the expression and secretion of IFN-β, the expression of the components of IFN-α/β receptor, the phosphorylation of STAT1, and the levels of cytokines involved in STAT1 activation. The results showed that the expression and secretion of IFN-β were specifically suppressed by As(III) treatment in RAW264.7 cells stimulated with LPS or poly(I:C). These results suggest that As(III) suppresses the expression and secretion of IFN-β, leading to the reduced STAT1 activation and consequently the reduced iNOS induction in macrophage cells. Our data suggest an important role of the arsenic-induced suppression of IFN-β on the disturbances in immunological defense against both bacteria and viruses.
Chronic exposure to inorganic arsenic via drinking tube-well water from arsenic-polluted groundwater in Asian countries has caused various types of disease including cancer of multiple organs, hypertension, and vascular disorders (Engel et al., 1994; Islam et al., 2011, Kadono et al., 2002; Karim et al., 2013; Lagerkvist et al., 1988; Tseng et al., 1968). A variety of genetic, epigenetic, and biochemical events has been shown to be involved in the development of multiple health effects of arsenic. Disturbances in immunological functions have also been observed in humans, animals, and cells exposed to arsenic (Dangleben et al., 2013). Impairment of T-cell activation (Biswas et al., 2008) and decreases in the phagocytic capacity of macrophages have been reported in individuals environmentally exposed to arsenic (Banerjee et al., 2009). A study in Bangladesh demonstrated that the prevalence of diarrhea and that of infectious diseases of the lower respiratory tract were higher in the infants born from mothers chronically exposed to arsenic compared to those from control mothers, suggesting the dysfunction of immunological defense systems against bacterial and viral infection among arsenic-affected infants (Rahman et al., 2011). Although these reports suggest that arsenic affects immunological functions, the mechanisms underlying how arsenic affects the immune system have not been established.
Nitric oxide (NO) plays a variety of roles as a vasodilator, an antiplatelet, and a neurotransmitter. NO also plays an important role in the body’s protection against bacterial and viral infection. Among the three isoforms of NO synthases, inducible NO synthase (iNOS) is known to produce a larger amount of NO than the other two isoforms when cells of the immune system are stimulated. Infection with bacteria such as Escherichia coli and Salmonella typhimurium (Umezawa et al., 1997; Kristof et al., 1998) as well as infection with viruses such as herpes simplex virus type 1 (HSV1), influenza virus, and rabies virus have been shown to produce high amounts of NO via iNOS activation in host animals (Koprowski et al., 1993; Akaike et al., 1996; Akaike et al., 1995). In a clinical study in West Bengal of India, where the groundwater is heavily contaminated by arsenic, the lipopolysaccharide (LPS)-induced production of NO in the macrophages of arsenic-exposed individuals was lower than that in unexposed individuals (Banerjee et al., 2009). In addition, the production of NO induced by interferon (IFN)-γ plus LPS in monocytes was negatively associated with urinary arsenic concentrations in Mexican children exposed to arsenic (Pineda-Zavaleta et al., 2004). The results of these human studies suggest that exposure to arsenic may affect the production of NO, and this may contribute to the depression of the human body’s defense against infection.
The molecular mechanisms underlying the regulation of murine iNOS induction by LPS have been well investigated. The transcription factor nuclear factor (NF)-κB was identified as an important factor in the LPS-induced iNOS expression in murine RAW264.7 macrophage cells (Lowenstein et al., 1993; Xie et al., 1994; Xie et al., 1993). Jacobs and Ignarro (Jacobs and Ignarro, 2001, 2003) showed that iNOS induction by LPS is mediated not only by NF-κB but also by the IFN-β/STAT signaling pathway in RAW264.7 cells. In addition, (Moore and Petro, 2013) showed that poly(I:C), an RNA-virus mimic, stimulates NO production through the activation of a transcription factor, IFN regulatory factor 3 (IRF3). Regarding the effects of arsenic on the pathways of NO production evoked by infection with bacteria, it has been reported that arsenic inhibits LPS-stimulated NO release (Takahashi et al., 2013; Chakravortty et al., 2001). However, no information has been available regarding the effects of arsenic on virus-stimulated NO production.
In this study, we investigated the effects of arsenite (As(III)) exposure on the production of NO by iNOS in RAW264.7 cells treated with LPS or poly(I:C). Our results demonstrate that As(III) suppressed both the LPS-induced and the poly(I:C)-induced production of NO, and that the suppression of the IFN-β/STAT1 pathway is the primary cause of the reduced NO production in RAW264.7 cells exposed to As(III).
Sodium arsenite (As(III)) was purchased from Wako Pure Chemicals (Osaka, Japan). Lipopolysaccharide (LPS) from Escherichia coli, Poly(I:C), and mouse recombinant IFN-β were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti-iNOS (NOS2) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for β-actin, STAT1, and phospho-STAT1 (Tyr701) were purchased from Cell Signaling Technology (Beverly, MA, USA).
Murine RAW264.7 macrophage cells were cultured with RPMI-1640 (189-02025, Wako Pure Chemicals) containing 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere of 5% CO2. RAW264.7 cells were exposed to 1, 5 and 10 μM As(III); the highest concentration of As(III) (10 μM) was confirmed to be non-lethal to RAW264.7 cells.
Nitrite accumulation in the cell culture medium was determined with Griess reagent (1% sulfanilamide, 0.1% naphtylethlenediamine dihydrochloride, and 2% H3PO4). An equal volume of cell culture medium and Griess reagent were mixed, and we measured the absorbance at 550 nm using a microplate reader. Standard curves were constructed with known concentrations of NaNO2.
Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 6.8, 0.5% deoxycholate, 1% NP-40, and 150 mM NaCl) containing protease inhibitor cocktail. The total cell lysates for each analysis were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were transferred to a polyvinylidene difluoride (PVDF) membrane and then placed in a blocking solution consisting of TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween 20) and 5% skim milk for 1 hr. The blotted membranes were incubated with the appropriate antibody, washed with TBST, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. The bound IgG was visualized with Luminata detection reagents (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s protocol.
We used an SV Total RNA Isolation System (Promega, Madison, WI, USA) to extract total RNA from the cells and purify it. For the conversion of total RNA to cDNA, we prepared 20 μL of a reaction mixture containing RT buffer (Fermentas, Burlington, Ontario, Canada), dNTPs, oligo d(T)15 primer, RNase inhibitor (Toyobo, Osaka, Japan), M-MuLV Reverse Transcriptase (Fermentas), and 1 μg of total RNA. The reaction mixture was incubated at 37°C for 90 min followed by inactivation of the enzyme at 65°C for 5 min. For the polymerase chain reaction (PCR) amplification of cDNA, we prepared a 25-μL mixture containing Premix Taq (TaKaRa Bio, Shiga, Japan), 2 μL cDNA, and the specific PCR primers. The PCR reactions were carried out as follows. The PCR amplification profiles for iNOS, IFN-α/β receptor 1 (IFNAR1), IFN-α/β receptor 2 (IFNAR2), IFN-β, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and β-actin each consisted of an initial denaturation at 94°C for 5 min, then 25−35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 1 min.
The primer sequences for iNOS were 5’-CAGAAGCAGAATGTGACCATC-3’ and 5’-CTTCTGGTCGATGTCATGAGC-3’; those for IFNAR1 were 5’-GAACATGTGGGCACTGGAGA-3’ and 5’-ACACAGTACACAGTCAGCGG-3’; for IFNAR2 were 5’-AGCGTTAGGAAGAAGCACGAGCC-3’ and 5’-GGGGCAGCTCAGTGGTGTGCATT-3’; those for IFN-β were 5’-CCATCCAAGAGATGCTCCAG-3’ and 5’-GTGGAGAGCAGTTGAGGACA-3’; those for TNF-α were 5’-TTGCCACTTCATACCAGGAGAA-3’ and 5’-TCACAGAGCAATGACTCCAA-3’; those for IL-6 were 5’-GTTCTCTGGGAAATCGTGGA-3’ and 5’-GGAAATTGGGGTAGGAAGGA-3’, and those for β-actin were 5’-CATGGATGACGATATCGCT-3’ and 5’-CATGAGGTAGTCTGTCAGGT-3’. An automated DNA thermal cycler (Takara Bio) was used. PCR products were analyzed by electrophoresis on a 2% agarose gel. The cycles for each PCR were determined by the quantitative performance.
Nuclear extracts were prepared from RAW264.7 cells with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA). The nuclear extracts were subjected to electrophoresis mobility shift assays (EMSAs) as described below.
Double-stranded oligonucleotides corresponding to the κB elements were end-labeled with [γ-32P]-ATP (Perkin-Elmer, Boston, MA, USA) by using T4 polynucleotide kinase (Toyobo). The sequence of oligonucleotides was a consensus κB element (5’-GGGGACTTTCCC-3’). The binding reaction was carried out in a reaction mixture containing 5 μg of nuclear extract protein, 0.25 μg of poly(dI-dC) (Sigma-Aldrich), 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5 mM dithiothreitol, 1 mM MgCl2, 0.5 mM EDTA, and 0.5 ng of 32P-labeled probe. For the evaluation of the binding competition, 10 ng of unlabeled oligonucleotide was incubated with the reaction mixture prior to the addition of labeled oligonucleotide. After the reaction mixture was incubated with labeled oligonucleotide for 30 min at room temperature, the mixture was electrophoresed on an 8% polyacrylamide gel, fixed and dried. The gel was visualized by FLA2000G (Fuji Photo Film, Kanagawa, Japan).
The IFN-β concentration in the media was measured by a IFN-β ELISA according to the manufacturer’s instructions (eBioscience, San Diego, CA, USA).
Two siRNAs for mouse STAT1 were purchased from Sigma Aldrich. The 19-mer target sequence of siRNAs targeting STAT1 (No. 1 and No. 2) were 5’-CTCAGAACACTCTGATTAA-3’ and 5’-CACAGTATAAACACGAATT -3’, respectively. A 2-nt overhang dTdT was added to 3’ of all siRNAs. Control siRNA was used as a non-silencing siRNA (Sigma Aldrich).
RAW264.7 cells were transfected by using RNAiMAX transfection reagent (ThermoFisher, Waltham, MA, USA) following manufacturer’s instructions.
Data were obtained from three separate experiments. The values are shown as mean ± SD. Statistical significance was assessed with an analysis of variance (ANOVA) followed by Tukey-Kramer post-hoc testing. Differences between groups were considered significant at p < 0.05.
To determine whether exposure to arsenic alters immunological defense reactions against bacterial and viral infection, we examined the effects of As(III) on NO production in murine RAW264.7 macrophage cells treated with LPS or poly(I:C). The cells were treated with LPS or poly(I:C) in the presence of As(III) for 12 hr, and the nitrite accumulation in the cell culture medium was determined by a Griess assay as an indicator of NO release. The results revealed that the concentrations of nitrite in the media were increased by the stimulation of cells with LPS or poly(I:C), but were decreased dose-dependently by the addition of As(III) (Fig. 1A). The stimulation of RAW264.7 cells with LPS or poly(I:C) enhanced the protein levels of iNOS, but the addition of As(III) dose-dependently reduced the iNOS protein levels (Fig. 1B). The enhanced levels of iNOS mRNA were also decreased by the addition of As(III) (Fig. 1C), suggesting that iNOS induction was suppressed at the step of transcription.
Effects of As(III) on the LPS- and poly(I:C)-induced nitrite production in RAW264.7 cells. RAW264.7 cells were exposed to 1, 5 or 10 μM As(III) in the presence of 1 μg/mL LPS or 25 μg/mL poly(I:C). A: After 12-hr incubation, the supernatants were collected and the nitrite concentration was measured with Griess reagent. The values are mean ± SD. *p < 0.05, **p < 0.01 vs. LPS or poly(I:C) alone. B: After 12-hr incubation, western blot analyses were performed for iNOS and β-actin. C: After 6-hr incubation, quantitative RT-PCRs were performed for iNOS and β-actin.
Since iNOS induction is known to be regulated by NF-κB, we examined the effects of As(III) on LPS or poly(I:C)-induced NF-κB activation. The results of the EMSAs showed that the NF-κB activation stimulated by LPS and poly(I:C) was not influenced by As(III) treatment (Fig. 2).
Effects of As(III) on the transactivation of NF-κB in RAW264.7 cells treated with LPS or poly(I:C). A: RAW264.7 cells were exposed to 1, 5 or 10 μM As(III) in the presence of 1 μg/mL LPS or 25 μg/mL poly(I:C). After incubation for 0.5 or 1 hr, nuclear extracts were extracted and 5 μg of nuclear protein was subjected to an EMSA for binding to a consensus κB oligonucleotide. Preincubation with unlabeled, cold oligo (C) significantly attenuated binding. N.S.: Nonspecific band.
We next examined whether the expression of cytokines in RAW264.7 cells stimulated with LPS or poly(I:C) is affected by As(III) treatment. As shown in Fig. 3A, the mRNA levels of TNF-α, IL-6, and IFN-β were all up-regulated by stimulation with LPS or poly(I:C). When the amount of mRNA was quantitated, the mRNA levels of TNF-α and IL-6 stimulated by LPS were not changed by As(III), but those of IFN-β were markedly decreased to less than 0.5-fold by As(III) at all doses. In the case of poly(I:C) stimulation, the mRNA levels of TNF-α and IL-6 showed a 0.6-fold and 0.5-fold reduction only at the dose of 10 μM As(III), while the levels of IFN-β mRNA were decreased to 0.45-fold at 5 μM and 0.26-fold at 10 μM As(III).
Effects of As(III) on the LPS- or poly(I:C)-induced IFN-β production. RAW264.7 cells were exposed to 1, 5 or 10 μM As(III) in the presence of 1 μg/mL LPS or 25 μg/mL poly(I:C). A: After 1-hr incubation, quantitative RT-PCRs were performed for IFN-β, TNF-α, IL-6 and β-actin. B: After 6-hr incubation, the supernatants were collected and the IFN-β concentration was measured with an ELISA. The values are mean ± SD. *p < 0.05, **p < 0.01 vs. LPS or poly(I:C) alone.
To confirm the effects of As(III) on the production of IFN-β, we measured the protein levels of IFN-β in the media of cells. As shown in Fig. 3B, the As(III) treatment dose-dependently decreased the protein levels of IFN-β in the media of RAW264.7 cells stimulated with LPS or poly(I:C).
IFN-β is known to be involved in LPS-induced NO production via the activation of STAT1 (in the IFN-β/STAT1 pathway). We also examined using siRNA technique and found that transfection of siRNA targeting different sites of STAT1 reduced nitrite production stimulated by LPS or poly(I:C) (Fig. 4). Therefore, we next examined the effects of As(III) on STAT1 phosphorylation. Stimulation of RAW264.7 cells with LPS or Poly(I:C) clearly increased the phosphorylation levels of STAT1, but co-treatment with As(III) suppressed the STAT1 phosphorylation (Fig. 5A). These data suggest that the reduced production of IFN-β resulted in the suppression of STAT1 phosphorylation.
Effects of STAT1 siRNA on the LPS- or poly(I:C)-induced nitrite production. A: Mouse STAT1 siRNAs were transfected into RAW264.7 cells. After 48-hr incubation, western blot analyses were performed for STAT1 and β-actin. B and C: Fourty eight hours after siRNA transfection, RAW264.7 cells were exposed to 1 μg/mL LPS (B) or 25 μg/mL poly(I:C) (C). After 12-hr incubation, the supernatants were collected and the nitrite concentration was measured with Griess reagent. *p < 0.05 vs. LPS or poly(I:C).
Effects of As(III) on the activation of the IFN-β/STAT1 signaling pathway in RAW264.7 cells. A: RAW264.7 cells were exposed to 1, 5 or 10 μM As(III) in the presence of 1 μg/mL LPS or 25 μg/mL poly(I:C). After 4-hr incubation, western blot analyses were performed for phospho-STAT1 and STAT1. B: RAW264.7 cells were exposed to 10 μM As(III) for 0.5, 1 or 3 hr. Quantitative RT-PCRs were performed for IFNAR1, IFNAR2, and β-actin. C: RAW264.7 cells were exposed to 1, 5 or 10 μM As(III) in the presence of 100 U/mL mouse recombinant IFN-β for 0.5, 1 or 3 hr. Western blot analyses were performed for phospho-STAT1 and STAT1.
To further identify the factors involved in the As(III)-induced suppression of STAT1 phosphorylation, we examined the effects of As(III) on the expression of the receptor for IFN-α/β and the activity of JAK kinase on STAT1 phosphorylation. As shown in Fig. 5B, the treatment of RAW264.7 cells with As(III) did not alter the mRNA levels of IFNAR1 and IFNAR2, the components of IFN-α/β receptor. The JAK kinase activity for STAT1 phosphorylation were evaluated indirectly by using the recombinant IFN-β as a stimulant for STAT1 phosphorylation. The phosphorylation levels of STAT1 were immediately increased by the addition of recombinant IFN-β in the media, but were not affected by As(III) treatment (Fig. 5C). These results suggest that the suppression of LPS- and poly(I:C)-induced STAT1 phosphorylation by As(III) was caused primarily by the decreases in the production of IFN-β and its subsequent release into the media.
Human studies in arsenic-polluted areas have shown the enhanced prevalence of cancer, infectious diseases, and pulmonary dysfunctions (Tseng et al., 1968; Rahman et al., 2011; Dangleben et al., 2013; Parvez et al., 2013; Smith et al., 2013). In addition to genetic and epigenetic disturbances, immunological dysfunctions may also be involved in the development of these diseases as an underlying mechanism, since disturbances in immune functions are known to weaken tumor immunity, resistance against infection, and anti-inflammatory activities (Schreiber et al., 2011; Vesely et al., 2011). However, little is known about specific mechanisms of how arsenic interferes with immunological defense systems. In the present study, we focused on the effects of As(III) on NO production and the pathways of iNOS activation in RAW264.7 macrophage cells. Since NO production by macrophages plays an important role in the defense against both bacterial and viral infections (Akaike et al., 1998; De Groote et al., 1995; Yoshida et al., 1993), we used both LPS and poly(I:C), which mimic infections of bacteria and viruses, respectively, as stimulants for macrophage cells.
Our results showed that the exposure of RAW264.7 cells to As(III) dose-dependently decreased both the LPS-induced and poly(I:C)-induced release of NO into the culture media. As the protein and mRNA levels of iNOS stimulated by LPS and poly(I:C) were markedly suppressed by As(III), we examined the mechanisms of the suppressed transcriptional activation of iNOS by As(III). The iNOS induction by LPS in RAW264.7 cells is regulated by NF-κB (Xie et al., 1994, 1993), but in the present study, the NF-κB activation by LPS or poly(I:C) was not affected by As(III) in RAW264.7 cells. Several studies have shown enhancing or suppressing effects of As(III) on NF-κB activation. The exposure of porcine aortic endothelial cells to As(III) resulted in an enhanced activation of NF-κB (Barchowsky et al., 1996). In contrast, TNF-α-induced NF-κB activation was suppressed by As(III) in the human lung cell lines A549 and BEAS-2B and the human embryonic kidney cell line HEK293 (Kapahi et al., 2000; Roussel and Barchowsky, 2000; Shumilla et al., 1998). The inconsistent results (including those of our present study) might be caused by the differences in cell lines or the concentrations of As(III) used in the activation of NF-κB.
We next focused on the JAK-STAT signaling pathway, because the LPS-induced and poly(I:C)-induced iNOS production was regulated by not only NF-κB but also the JAK-STAT signaling pathway (Lowenstein et al., 1993; Xie et al., 1994; Xie et al., 1993; Jacobs and Ignarro, 2001, 2003; Moore and Petro, 2013). In addition, Cheng et al. (2004) showed that As(III) inhibited the JAK-STAT pathway activated by IFN-α, IFN-β, IFN-γ and IL-6 via direct interference with the protein kinase activity of JAK. We therefore examined the effects of As(III) on STAT1 phosphorylation induced by LPS or poly(I:C). Our data showed that STAT1 phosphorylation was decreased by As(III) at least at 10 μM. However, the suppression of iNOS induction was observed at 5 μM As(III). These data suggest that the reduction of NO release by As(III) was at least partly dependent on STAT1 activation, but other unknown factors may also be involved.
However, when cells were stimulated by recombinant IFN-β, the STAT1 phosphorylation was not affected by As(III). These data suggest that As(III) does not directly inhibit the kinase activity of JAK in RAW264.7 cells but does affect the release of cytokines that are involved in STAT1 phosphorylation.
Since IFN-β, TNF-α, and IL-6 were shown to activate the JAK-STAT pathway leading to iNOS induction (Jacobs and Ignarro, 2001; Sawada et al., 1997; Kleinert et al., 1998), we examined the effects of As(III) exposure on the mRNA levels of these cytokines. The mRNA levels of IFN-β, TNF-α, and IL-6 were increased in response to stimulation with LPS or poly(I:C). Treatment with As(III), however, decreased the mRNA levels of only IFN-β. Concomitant with the decrease in the levels of IFN-β mRNA, the protein levels of IFN-β in culture media were also decreased by As(III) exposure. Thus, these results suggest that the exposure of RAW264.7 cells to As(III) suppressed the expression of IFN-β, leading to the decrease in the release of IFN-β into the culture media. This, in turn, resulted in the reduced stimulation of IFN- α/β receptor, and finally resulted in the suppression of STAT1 phosphorylation, which plays a critical role in the induction of iNOS gene (Fig. 6).
Schematic description of the effects of As(III) on the IFN-β/STAT1 signaling pathway for iNOS induction in RAW264.7 cells.
It has been shown that the suppression or lack of iNOS-derived NO results in an impairment of bacterial clearance in host animals (Akaike et al., 1998; Yoshida et al., 1993; De Groote et al., 1995), suggesting an antimicrobial role of NO produced in immune cells upon infection with bacteria. The iNOS-derived NO is also produced by infection with viruses such as HSV1, influenza virus, and rabies virus in experimental animals (Akaike et al., 1996, 1995; Koprowski et al., 1993). It was shown that HSV1 replication was suppressed by NO production in RAW24.7 cells (Croen, 1993). Saura et al., (1999) showed that NO inactivates the coxsackie virus protease 3C, an enzyme necessary for the replication of coxsackie virus through the S-nitrosylation. These studies indicate that the suppression of NO production may cause reduced defense activity against both bacterial and viral infection.
Thus, the suppression of iNOS induction by As(III) in macrophage cells stimulated with LPS or with poly(I:C) demonstrated in this study may partly explain the reduced defense activity against infection among individuals chronically exposed to arsenic. Although the reduced ability of macrophages to produce NO in humans exposed to arsenic has been shown (Banerjee et al., 2009), further studies are required for the elucidation of the relationship between the defense against infection and the iNOS inducibility in macrophages in humans.
In this study, we observed that As(III) affected the expression of IFN-β in macrophage cells. In addition to the induction of iNOS, IFN-β is known to play multiple immunomodulatory roles in the control of infection and cancer development (Snell et al., 2017). Future studies are warranted to determine the effects of arsenic on the other immunological events in which IFN-β is deeply involved.
This work was supported by a JSPS KAKENHI Grant, no. JP24310048.
The authors declare that there is no conflict of interest.