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Environmentally Relevant Concentrations of Bisphenol A Interact with Doxorubicin Transcriptional Effects in Human Cell Lines

The worldwide production of synthetic chemicals, including endocrine disruptor chemicals (EDCs), such as Bisphenol A (BPA) has increased significantly in the last two decades. Human exposure to BPA, particularly through ingestion, is continuous and ubiquitous. Although, considered a weak environmental estrogen, BPA can induce divergent biological responses through several signaling pathways, including carcinogenesis in hormone-responsive organs. However, and despite the continuous increase of tumor cell-resistance to therapeutic drugs, such as doxorubicin (DOX), information regarding BPA drug interactions is still scarce, although its potential role in chemo-resistance has been suggested. This study aims to assess the potential interactions between environmentally relevant levels of BPA and DOX at a therapeutic dosage on Hep-2 and MRC-5 cell lines transciptome. Transcriptional effects in key-player genes for cancer biology, namely c-fos, p21, and bcl-xl, were evaluated through qRT-PCR. The cellular response was analyzed after exposure to BPA, DOX, or co-exposure to both chemicals. Transcriptional analysis showed that BPA exposure induces upregulation of bcl-xl and endorses an antagonistic non-monotonic response on DOX transcriptional effects. Moreover, the BPA interaction with DOX on c-fos and p21 expression emphasize its cellular specificity and divergent effects. Overall, Hep-2 was more susceptible to BPA effects in a dose-dependent manner while MRC-5 transcriptional levels endorsed a non-monotonic response. Our data indicate that BPA environmental exposure may influence chemotherapy outcomes, which emphasize the urgency for a better understanding of BPA interactions with chemotherapeutic agents, in the context of risk assessment.

Introduction
The worldwide production of synthetic chemicals, such as endocrine disruptor chemicals (EDCs), has grown exponentially in the last two decades. Among EDCs, which are classified by the World Health Organization (WHO) as "an exogenous substance or mixture that alters function(s) of the endocrine system, and consequently causes adverse health effects in an intact organism, or its progeny, or (sub) populations" (World Health Organization 2011) [1,2], Bisphenol A (BPA) appears as a paradigmatic case. Currently, human exposure to this compound is persistent and continuous, particularly through ingestion of contaminated food and beverages. BPA also appears to have bioaccumulation capacity, which raises public and scientific concerns regarding its possible health-related effects [3]. Although, BPA is still the main identified bisphenol in environmental monitoring studies worldwide, diverse bisphenol analogues, such as Bisphenol F (BPF) and Bisphenol S (BPS), have been developed and produced, with the aim of replacing BPA in multiple applications [4]. Nevertheless, all these compounds have the molecular scaffold of bisphenols (BPA, bisphenol analogues, and bisphenol derivates) which result in the ability to interact with specific protein families, such as GTPases, including H-Ras, N-Ras, and K-Ras isoforms, crucial for cancer etiology [5,6].
Relevantly, the associations between human exposure to these compounds and hazardous effects on fertility, fetal development, and several types of cancer have been reported [7,8]. Even though BPA is considered a weak xenoestrogen, due to its low binding affinity to classical estrogen receptors (ERα and ERβ), this compound is capable of triggering diverse and divergent estrogen-signaling pathways [9,10], and to endorse tissue-specific, as well as developmental effects, including cellular proliferation and gene expression [11][12][13][14]. BPA-induced alterations on transcriptional patterns have been correlated to epigenetic effects, which were first demonstrated in mice after maternal exposure to BPA [15]. For the past two decades, BPA exposure effects have been exceedingly studied in carcinogenesis in hormone-responsive organs [16]. However, and despite the worldwide increase in cancer chemo-resistance, ref [17] information regarding potential interactions with chemotherapeutic drugs, such as Doxorubicin (DOX), has been scarce. DOX is one of the most utilized antineoplastic drugs for cancer treatments worldwide, ref [18] which at therapeutic concentrations, induces cancer cells apoptosis as a result of DNA damage [19]. Previous studies have demonstrated BPA's counteracting effects in breast cancer cells [20,21] and HT29 cells [22] pre-exposed to DOX, which suggests an effective interaction and raises awareness regarding BPA's role in chemo-resistance.
Moreover, considering that the ingestion of contaminated food and beverages are responsible for more than 90% of overall human environmental exposure to BPA at all age groups [23], the cells from the upper gastrointestinal tract, such as laryngeal cells, are particularly exposed to non-conjugated BPA (active form).
Here, we aimed to evaluate the effects of environmentally relevant concentrations of BPA, commonly found in human biological samples [24,25], and in co-exposure with a therapeutic concentration of DOX in human epithelial Type 2 (Hep-2) cells (originating from a human laryngeal carcinoma) and MRC-5 cells (DNA repair-proficient cell line). Our group's previously published results, regarding BPA/DOX interactions in cytotoxicity and genotoxicity, as well as cytological evaluation of mitotic disruption, demonstrated its genotoxic potential, as well as antagonistic effect to DOX [26].
Here, the effects resulting from the combined exposures to BPA and DOX, were evaluated regarding the transcriptional disruption of genes, known as key players in cancer biology. The transcriptional effects were assessed for the anti-apoptotic gene bcl-xl, for which upregulation has been previously observed in BPA/DOX interaction [20]. While, p21 gene, which encodes for a cyclin-dependent kinase inhibitor, is particularly related to cell cycle progression and cancer outcome [27] and c-fos gene a relevant component of the activator protein-1 (AP-1) transcription factor, is critical for accurate regulation of numerous genes implicated in cell proliferation, differentiation, apoptosis, and oncogenic transformation [28]. Data presented in this work increases the knowledge regarding BPA-induced effects in the transcriptional response to a frequently utilized therapeutic DOX concentration with potential consequences for chemotherapy outcome and risk assessment.

Cell Cultures and Reagents
In this work, Hep-2 and MRC5 cell lines were kindly offered by Centro Hospitalar Lisboa Ocidental; Hospital Egas Moniz, Microbiology and Molecular Biology Laboratory, and cultivated in 75 cm 2 flasks with RPMI media containing GlutaMAX™ I, 25 mM HEPES (Invitrogen, Carlsbad, CA, USA). Culture media was supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM L-glutamine. All cell cultures were maintained in a 5% (v/v) CO 2 humidified atmosphere at 37 • C. In the subcultures, the cells stabilized for 24 h before treatments.

Drugs and Treatments
Here, BPA (Sigma, St. Louis, MI, USA) was newly diluted in vehicle ethanol and supplemented in the culture media at final concentrations of 4.4 µM (1 µg/mL), 4.4 nM (1 ng/mL) and 0.44 nM (0.1 ng/mL). Relevantly, 0.44 nM dosages, in the range of BPA-detected levels in human biological samples, was associated with environmental exposure, whereas 4.4 µM was selected based on the reference value of 50 µg/kg body weight/day, considering an average body weight of 70 kg and 3 L of water intake per day. Considering that the U.S. Environmental Protection Reference Dose for Chronic Oral BPA Exposure (RfD) is 50 µg/kg body weight/day [29], which is in agreement with the BPA Tolerable Daily Intake (TDI), which is the value set until January 2015 by the European Food Safety Authority, and reduced to 4 µg/kg body weight/day [30] thereafter. For interactions assessment, DOX (AppliChem, Darmstadt, Germany) was diluted in water and supplemented the culture medium at 4 µM (2.5 µg/mL), in final concentration, which correspond to free DOX levels in blood in cancer chemotherapy patients [31]. Regarding, BPA/DOX co-exposures, the cells were previously exposed to BPA for 24 h. Afterwards, 24 h of co-exposure to BPA and DOX followed. Additionally, cell cultures were exposed to BPA for 48 h after the 24 h stabilization period, and DOX standard medium was substituted by the medium with DOX 48 h after subculture for single-drug treatments. In all experiments, the controls were utilized in cells grown in standard culture medium or in medium supplemented with ethanol 170 µM (vehicle concentration for BPA).

RNA Isolation and Real-Time PCR
The total RNA was extracted from cell lysates using the GF-1 Total RNA Extraction Kit (Vivantis technologies, Subang Jaya, Malaysia), according to the manufacturer's protocol. The concentrations of all RNA samples were determined by a fluorescence-based assay, the Qubit™ RNA HS Assay Kit in Qubit™ 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Then, 1 µg of total RNA was reverse transcribed to cDNA using random hexamers from the First-Strand cDNA Synthesis Kit (GE Healthcare, Little Chalfont, UK). Quantitative real-time PCR (qRT-PCR) was conducted on a CFX Connect™ Real-Time PCR Detection System (Bio-rad) for the genes c-fos, p21, bcl-xl and for both reference genes GAPDH and β-actin, which were used for data normalization. Each reaction was performed in triplicate using the 5x HOT FIREPol ® EvaGreen ® qPCR Supermix (Solis BioDyne, Tartu, Estonia) in a final volume of 20 µl. Control PCRs were also performed for all primer combinations without a template. The primers used are listed in Table 1 and the cycling conditions were as follows: Initial activation of 95 • C for 12 min, followed by 40 cycles of 95 • C for 30 s, 55 • C for 30 s, and 72 • C for 40 s. Then, those cycles were followed by the acquisition of a melting curve, in order to check for primer-dimer formation and contaminations. The relative quantification was undertaken by normalizing threshold cycles (Ct) of the target genes with the mean Ct of GAPDH and β-actin, since no significant differences were detected between these two reference genes. Transcript levels were analyzed by calculating ∆∆Ct (∆∆Ct = ∆Ct treatment − mean ∆Ct control) and the control samples always had a value of 0.

Statistical Analysis
All statistical analysis was performed by using IBM SPSS statistics 22 software (Armonk, NY, USA). Significant differences between different groups were assessed through Student's t-test (comparison for two groups) and p < 0.05 was considered statistically significant. Results are presented as mean ± standard deviation.

BPA Alters bcl-xl and p21 Transcription Levels but Does Not Significantly Affect c-fos Expression
Relative expression of cancer-related genes, namely bcl-xl, c-fos and p21, was analyzed in Hep-2 and MRC-5 cells, through qRT-PCR, after 48 h of exposure to different BPA environmentally relevant concentrations (0.44 nM, 4.4 nM, and 4.4 µM). We observed a pronounced divergence in cellular response between the two tested cell lines. In Hep-2 cells, the higher BPA concentration assessed induced an upregulation (p = 0.003) of bcl-xl mRNA levels ( Figure 1A). No significant changes were observed for c-fos mRNA levels after BPA exposure ( Figure 1B) and p21 mRNA expression was upregulated after exposure to 4.4 nM and 4.4 µM BPA concentrations (p = 0.018) ( Figure 1C). Regarding MRC-5 cell line, no significant differences were observed in bcl-xl mRNA expression ( Figure 1D) or c-fos mRNA expression ( Figure 1E). On the other hand, we observed an upregulation of p21 mRNA levels after exposure to the lowest BPA concentration (p = 0.035) ( Figure 1F). All statistical analysis was performed by using IBM SPSS statistics 22 software (Armonk, NY, USA). Significant differences between different groups were assessed through Student's t-test (comparison for two groups) and p < 0.05 was considered statistically significant. Results are presented as mean ± standard deviation.

BPA Alters bcl-xl and p21 Transcription Levels but Does Not Significantly Affect c-fos Expression
Relative expression of cancer-related genes, namely bcl-xl, c-fos and p21, was analyzed in Hep-2 and MRC-5 cells, through qRT-PCR, after 48 h of exposure to different BPA environmentally relevant concentrations (0.44 nM, 4.4 nM, and 4.4 μM). We observed a pronounced divergence in cellular response between the two tested cell lines. In Hep-2 cells, the higher BPA concentration assessed induced an upregulation (p = 0.003) of bcl-xl mRNA levels ( Figure 1A). No significant changes were observed for c-fos mRNA levels after BPA exposure ( Figure 1B) and p21 mRNA expression was upregulated after exposure to 4.4 nM and 4.4 μM BPA concentrations (p = 0.018) ( Figure 1C). Regarding MRC-5 cell line, no significant differences were observed in bcl-xl mRNA expression ( Figure 1D) or c-fos mRNA expression ( Figure 1E). On the other hand, we observed an upregulation of p21 mRNA levels after exposure to the lowest BPA concentration (p = 0.035) ( Figure 1F).  F). GAPDH and β-actin were used for normalization. The error bars represent the standard deviation between two independent treatments and three qRT-PCR replicates. Significant statistical values, which were compared with the vehicle (EtOH) and calculated with Student's t-test, are illustrated as: * p < 0.05 and ** p < 0.01.

BPA Interacts with DOX Transcriptional Effects in Hep-2 and MRC-5 Cell Lines with Cell Type Specificity
The DOX effects alone, and combined with BPA, were assessed using the same primers described above (Figure 2). Ours results demonstrate that DOX severely decreases bcl-xl, c-fos and p21 expression levels in Hep-2 cells, with higher significance when compared to vehicle (p < 0.001 for all the three genes). However, the combined exposure with BPA antagonized DOX transcriptional effects with the exception of DOX/BPA 0.44 nM combination in bcl-xl and p21 expression ( Figure  2A,C). Regarding MRC-5 cells effects, DOX alone did not significantly affect c-fos and p21 expression GAPDH and β-actin were used for normalization. The error bars represent the standard deviation between two independent treatments and three qRT-PCR replicates. Significant statistical values, which were compared with the vehicle (EtOH) and calculated with Student's t-test, are illustrated as: * p < 0.05 and ** p < 0.01.

BPA Interacts with DOX Transcriptional Effects in Hep-2 and MRC-5 Cell Lines with Cell Type Specificity
The DOX effects alone, and combined with BPA, were assessed using the same primers described above (Figure 2). Ours results demonstrate that DOX severely decreases bcl-xl, c-fos and p21 expression levels in Hep-2 cells, with higher significance when compared to vehicle (p < 0.001 for all the three genes). However, the combined exposure with BPA antagonized DOX transcriptional effects with the exception of DOX/BPA 0.44 nM combination in bcl-xl and p21 expression (Figure 2A,C). Regarding MRC-5 cells effects, DOX alone did not significantly affect c-fos and p21 expression levels. However, the combined exposure of DOX/BPA resulted in a significant downregulation, and upregulation, respectively of these genes. The anti-apoptotic bcl-xl, BPA interaction with DOX were not observed. ( Figure 2D)

Discussion
The continuous growth of chemo-resistance and the concurrent worldwide increase of manmade products to which human are continuously and ubiquitous exposed has led to the question: Are humans exposed to environmental contaminants that may enhance and/or endorse cellular resistance to chemotherapy?
One of the most utilized drugs in chemotherapy worldwide is DOX, with distinct concentrationdependent cellular effects. DOX often used therapeutic dosages to induce cellular death through cell cycle arrest and apoptosis [32]. Previous studies, in HT29 cell line, demonstrated that the endocrine disruptor, BPA, was able to interfere with DOX transcriptional effects on genes related to cell cycle progression, mitotic regulation and apoptosis [22]. Moreover, considering that, several studies indicated that BPA effects are cell type and dose dependent, here we aimed to evaluate in Hep-2 and MRC-5 cell lines. Environmentally-relevant concentrations of BPA were able to interfere and/or antagonize DOX cellular effects in transcription levels of c-fos, p21 and bcl-xl, as previously reported in HT29 cell line.
Cellular cytotoxicity and genotoxicity of BPA tested concentrations, namely, 4.4 μM (1 μg/mL), 4.4 nM (1 ng/mL), and 0.44 nM (0.1 ng/mL), which correspond to the range of BPA detected levels in human biological samples due to environmental exposure, have already been evaluated in Hep-2 and MRC-5 cell lines in a previously published study [26]. We have demonstrated that these BPA concentrations do endorse genotoxic effects, such as micronuclei formation at 4.4 nM and 0.44 nM  E) and p21 (C,F). GAPDH and β-actin were used for normalization. Error bars represent the standard deviation between two independent treatments and three qRT-PCR replicates. Significant statistical values, which were compared with the vehicle (EtOH) and calculated with Student's t-test, are illustrated as: * p < 0.05, ** p < 0.01 and *** p < 0.001.

Discussion
The continuous growth of chemo-resistance and the concurrent worldwide increase of man-made products to which human are continuously and ubiquitous exposed has led to the question: Are humans exposed to environmental contaminants that may enhance and/or endorse cellular resistance to chemotherapy?
One of the most utilized drugs in chemotherapy worldwide is DOX, with distinct concentrationdependent cellular effects. DOX often used therapeutic dosages to induce cellular death through cell cycle arrest and apoptosis [32]. Previous studies, in HT29 cell line, demonstrated that the endocrine disruptor, BPA, was able to interfere with DOX transcriptional effects on genes related to cell cycle progression, mitotic regulation and apoptosis [22]. Moreover, considering that, several studies indicated that BPA effects are cell type and dose dependent, here we aimed to evaluate in Hep-2 and MRC-5 cell lines. Environmentally-relevant concentrations of BPA were able to interfere and/or antagonize DOX cellular effects in transcription levels of c-fos, p21 and bcl-xl, as previously reported in HT29 cell line.
Cellular cytotoxicity and genotoxicity of BPA tested concentrations, namely, 4.4 µM (1 µg/mL), 4.4 nM (1 ng/mL), and 0.44 nM (0.1 ng/mL), which correspond to the range of BPA detected levels in human biological samples due to environmental exposure, have already been evaluated in Hep-2 and MRC-5 cell lines in a previously published study [26]. We have demonstrated that these BPA concentrations do endorse genotoxic effects, such as micronuclei formation at 4.4 nM and 0.44 nM concentrations and DNA, as well as oxidative damage with no detected effects in cellular viability, with associated non-monotonic responses [26].
One of the generally recognized genes acting as a key player in cancer biology is the anti-apoptotic bcl-xl [33]. We have previously demonstrated that in HT29 cell line BPA at 4 uM induced a significant transcriptional upregulation [22], as was also observed in this study in Hep-2 cell line. Moreover, La Pensee and coworkers have correlated BPA antagonistic effect to DOX with increased expression of anti-apoptotic proteins [20]. Here we also demonstrate that BPA induces an antagonistic effect of DOX transcriptional effect in Hep-2 cells, and more importantly, this effect is particularly observed within DOX interactions, with the lowest tested BPA concentration, which indicates a non-monotonic response effect. On the other hand, in MCR-5 cells, no significant effects were observed after BPA exposure alone, or in co-exposure with DOX, which may indicate that transcriptional alterations in the expression levels of this anti-apoptotic gene are cell type-specific and are more likely to appear in tumors cells. Considering that overexpression of bcl-xl impairs apoptosis, induced by DOX [33] and that DOX therapeutic concentrations induce apoptosis [32], BPA antagonizing effect may negatively influence DOX therapeutic outcomes.
Moreover, another key gene in cancer biology is c-fos, which encodes a crucial component of the activator protein-1 (AP-1) transcription factor, required for accurate regulation of several genes associated with cell proliferation, differentiation, apoptosis, and oncogenic transformation [28].
In agreement with previous studies [22], c-fos transcriptional levels were not affected by BPA exposure; however, the interaction with DOX was obvious for both cell lines. Remarkably, although BPA interaction occurs in both cells the effects are divergent. While, in Hep-2 cells BPA decreases the downregulation induced by DOX exposure, which is concordant with previous results in HT29 cancer cell line [22], in MRC-5 cells BPA enhances c-fos downregulation with more pronounced effects in the lowest tested concentrations.
The third analyzed gene, p21 encodes a cyclin-dependent kinase inhibitor is tightly connected with cell cycle progression and cancer outcome [27]. BPA effect in the transcriptional expression of this gene emphasizes once more its cellular specificity. In Hep-2 cells, we observed an upregulation of transcriptional levels at the highest tested concentrations, which indicates a dose-dependent effect, while in MRC-5 cells, p21 upregulation was observed at the lowest BPA concentration, which demonstrates a non-monotonic effect. Interestingly, the BPA interaction with DOX is evident and results in striking divergent effects. Whereas, in Hep-2, BPA antagonizes DOX induced downregulation of p21 in a dose-dependent manner, while in MRC-5 cells BPA co-exposure with DOX results in a very significant upregulation of gene expression. Interestingly, previous studies in colon epithelial cells have also demonstrated an inversed correlation between c-fos and p21 expression associated with oxidative stress, ref [34] which we have demonstrated occurs in these cells (Hep-2 and MRC-5) after BPA and DOX co-exposure [26].
Overall our data demonstrate that Hep-2 cells were more susceptible to BPA effects in a dose-dependent manner, while MRC-5 transcriptional levels present non-monotonic response. Interestingly, previous studies demonstrate that the tested BPA concentrations induced higher levels of DNA damage and oxidative stress in Hep-2 cells, rather than MRC-5 also in a dose-dependent manner [26].

Conclusions
In this study, we provide new evidence regarding BPA cell type-specific effects and non-monotonic responses. Furthermore, we demonstrate that relevant environmental doses of BPA directly affect transcriptional levels of key player genes in cancer biology and interact with DOX-induced effects, that might be crucial for cancer patients undergoing this treatment. Our data emphasize the necessity of a better understanding of BPA interactions with chemotherapeutic agents in the context of risk assessment.
Author Contributions: E.R. designed the study; E.R. and M.D. performed the experiments; E.R. and M.B. supervised the project. All authors prepared, read, and approved the final manuscript.


s2fieldsofstudy: Biology Medicine

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Neutrophils in Leprosy

Leprosy is an infectious disease caused by the intracellular bacillus Mycobacterium leprae that mainly affects the skin and peripheral nerves. One of the most intriguing aspects of leprosy is the diversity of its clinical forms. Paucibacillary patients are characterized as having less than five skin lesions and rare bacilli while the lesions in multibacillary patients are disseminated with voluminous bacilli. The chronic course of leprosy is often interrupted by acute episodes of an inflammatory immunological response classified as either reversal reaction or erythema nodosum leprosum (ENL). Although ENL is considered a neutrophilic immune-complex mediated condition, little is known about the direct role of neutrophils in ENL and leprosy disease overall. Recent studies have shown a renewed interest in neutrophilic biology. One of the most interesting recent discoveries was that the neutrophilic population is not homogeneous. Neutrophilic polarization leads to divergent phenotypes (e.g., a pro- and antitumor profile) that are dynamic subpopulations with distinct phenotypical and functional abilities. Moreover, there is emerging evidence indicating that neutrophils expressing CD64 favor systemic inflammation during ENL. In the present review, neutrophilic involvement in leprosy is discussed with a particular focus on ENL and the potential of neutrophils as clinical biomarkers and therapeutic targets.

INTRODUCTION
Leprosy is a millennial disease that continues to adversely impact the public health systems of endemic countries. The most commonly affected sites are the dermis and the peripheral nerves. Permanent disabilities are the direct consequence of the neurological damage caused by the Mycobacterium leprae infection, especially when the damage is left untreated in its early stages. During 2017, 150 countries reported 210,671 new cases of leprosy at a detection rate of 2.77/100,000 (1).
Leprosy severity is determined by the regulation of cell-mediated immunity, ranging anywhere from mild, presenting with a single, well-demarcated lesion (termed tuberculoid: TT), to severe, involving widespread, poorly-demarcated, raised, or nodular lesions (termed lepromatous lepromatous: LL). Biopsies of TT lesions reveal well-developed granulomatous inflammation associated with the marked presence of Langerhans cells (CD1a + ) and rare acid-fast bacilli. LL dermal lesions are characterized by the presence of numerous heavily-infected foamy macrophages, a sparse infiltrate of lymphoid cells, and the number of Langerhans cells is consistently low (2)(3)(4). The so-called borderline patients (BT, BB, and BL) are situated between the extremes of the TT and LL poles. These patients display a mixed unstable immune response whose characteristics are in accordance with their proximity to one pole or the other (5).
During disease evolution, 50% of LL and 5-10% of BL patients present a variety of dermatological inflammatory phenomena with systemic symptoms (6,7), referred to as erythema nodosum leprosum (ENL, or Type 2 reaction). ENL together with reversal reaction are core aspects of leprosy that profoundly impact both the course of the disease and the development of nerve damage (8). Clinically, ENL patients demonstrate painful subcutaneous nodules on the apparently normal skin (Figure 1). More severe cases display systemic inflammation accompanied by neutrophilic leukocytosis, fever, and malaise similar to sepsis (9).
Currently, ENL is often designed as a neutrophilic immune complex-mediated disease (23). The cause of ENL is eminently complex. Immune complex deposits have been implicated in the cutaneous lesions of ENL (13,24). It is primarily driven by an aberrant dermal immune response that is modified by genetic susceptibility (25) and various environmental stimuli (e.g., pregnancy, lactation, puberty, intercurrent infections, vaccination, and psychological stress) (26). Elevated levels of tumor necrosis factor (TNF)-α and other pro-inflammatory  Frontiers in Immunology | www.frontiersin.org cytokines have been associated with ENL episodes while, in the opposite direction, TNF suppression leads to clinical improvement (27,28). Learning more about the factors that ultimately trigger and/or sustain ENL could lead to the identification of strategies to control and, most importantly, prevent the associated inflammation.
A case can be made that the role played by neutrophils in leprosy has been largely overshadowed by several studies dedicated to the macrophages and Schwann cells targeted by M. leprae (29). In the blood of multibacillary leprosy patients (LL and BL), neutrophils and monocytes are loaded with the bacilli (30) and their clearance will only effectively occurs after 2-3 months of multidrug therapy (31). Novel aspects of neutrophilic biology reported in recent papers strongly indicate that, in ENL, neutrophils are active and not neutral, thus providing new insights into their participation in the disease. In the present review, we tried to highlight some of the potential gaps in knowledge among neutrophils in leprosy. Our focus was on attempting to identify the possible ways neutrophils might contribute to ENL-linked systemic inflammation. As a final concern, the potential of these cells as clinical biomarkers and therapeutic targets was highlighted.

SOME OLD AND NEW FINDINGS ON NEUTROPHIL BIOLOGY
Neutrophils have always been considered effector cells of innate immunity with a limited biosynthetic capacity. The primary role ascribed to these cells was as warriors against extracellular pathogens and in acute inflammation. These cells were classically characterized by their phagocytic ability, the release of lytic enzymes from their granules, and the production of reactive oxygen intermediates with a microbicidal potential. In the 1990s, however, this limited view was challenged by evidence that neutrophils actually survive much longer than initially believed (32) and have added ability to express genes encoding proinflammatory key mediators as components of the complement system, Fc receptors, chemokines, and cytokines (33).
Neutrophils are continuously generated in the bone marrow from its myeloid precursor. Daily production approximates 2 × 10 11 cells. In humans, 50-70% of circulating leukocytes are neutrophils whereas, in mice, they range from 10 to 25%. This process is largely controlled by the granulocytic colony stimulating factor (G-CSF), produced in response to interleukin 17A (IL-17A). IL-17A is primarily synthesized by Th17 cells. But, innate immune cells, including γδ T cells, neutrophils, macrophages, innate lymphocyte cells (ILC), mast cells, and keratinocytes, have recently been found to be involved in IL-17 secretion (34). Other molecules-such as granulocytic-macrophage-colony stimulating factor (GM-CSF), IL-6, and KIT ligand (KITL, also known as KITLG)likewise induce granulopoiesis. The production of this cytokine storm during the inflammatory responses results in overactive granulopoiesis and neutrophilia. During maturation, neutrophils undergo a number of stages referred to as either myeloblasts, pro-myelocytes, myelocytes, metamielocytes, band neutrophil, or, lastly, polymorphonuclear cells (segmented). Neutrophilic granules are formed sequentially during maturation of the promyeloid stage (35).
Neutrophils have long been considered short, half-life cells in the circulation that normally survive approximately 1.5 h in mice and 8 h in humans (37,38). Pillay et al. demonstrated that, under baseline conditions, the average life span of neutrophils in the circulation is 12.5 h in mice and 5.4 days in humans (37). During inflammation, neutrophils become activated and longevity increases, ensuring the presence of these cells at the inflammation site (32,39). Endogenous products such as cytokines and growth factors together with bacterial products activate neutrophils. This increased half-life may allow neutrophils to perform more complex activities in the tissue. Examples may include: resolution of inflammation through the production of lipid mediators, modulation of the adaptive response, and reverse transmigration, which could involve the ability to exit the initial injury site and migrate to other tissues such as bone marrow (40).
Neutrophils eliminate pathogens through various intraand extracellular mechanisms. When neutrophils encounter microorganisms, phagocytosis occurs followed by the formation of phagosomes. Microorganisms could be killed by NADPH oxygenase-dependent pathways (reactive oxygen species, ROS) or antimicrobial proteins (cathepsins, defensins, lactoferrin, and lysozyme) (35). These microbial proteins are released into either the phagosomes or the extracellular environment, acting against intra-or extracellular pathogens, respectively.
ROS production, i.e., oxidative burst, is considered a key component of the innate immune defense against bacterial and fungal infections (41). To the best of our knowledge the literature hasn't shown yet oxidative burst in leprosy neutrophils. However, oxidative stress was evaluated by measuring serum levels of malondialdehyde (MDA) and superoxide dismutase (SOD) activity and the results showed that leprosy patients present increased serum levels of MDA, MDA/SOD ratio together with a decreased SOD activity when compared to healthy controls suggesting oxidative stress in leprosy (42). In addition, it was demonstrated that the oxidative stress gradually increased along the spectrum from TT to LL (43).
An elegant strategy was used to measure the antimicrobial capacity of neutrophils to contain methicillin-resistant Staphylococcus aureus (MRSA). Leliefeld et al. (44) used a longterm neutrophil bacterial interaction in a 3D scaffold reminiscent of the in vivo environment. In such condition it was possible to evaluate the capacity for long-term intracellular containment of live bacteria. Neutrophils from chronic granulomatous disease (CGD) patients who lack a functional NADPH oxidase and healthy neutrophils under hypoxic conditions did not exhibit impaired bacterial containment and in that way this containment was independent of ROS (44). Conversely, failure of the phagosomal acidification led to impaired intracellular containment of MRSA (44).
Activated neutrophils have the capacity to eliminate extracellular microorganisms after releasing neutrophilic extracellular traps (NETs) (45). NETs are composed of nuclear DNA in association with histones and granular proteins such as antimicrobial proteins and enzymes (MPO and neutrophil elastase). The functions of NETs include immobilizing pathogens to prevent them from disseminating and facilitating the subsequent phagocytosis of trapped microorganisms. Many pathogens, namely viruses, bacteria, parasites, and fungi can induce NETs. For this reason, the mechanisms for both the initiation and evasion of NETs by pathogens have been intensively studied (46). To our knowledge, no report exists in the literature regarding the ability or inability of M. leprae to induce NETs. In addition, there are two open questions: (1) Is there a connection between NET formation, neutrophil infiltration and ENL systemic inflammation? (2) Does the massive presence of neutrophils in the ENL lesions could generate necrotic areas? It is possible to observe a collection of neutrophils in ENL lesions forming a microabscess (Figure 2). The NETosis role in limiting the spread of necrotic tissues was demonstrated in acute abdominal inflammation where netting neutrophils create a barrier between necrotic and viables areas (47).
Although ROS production by neutrophils during infections is an important antimicrobial mechanism, the exacerbated production of ROS due to the massive involvement of neutrophils can lead to oxidative stress accompanied of cell death and necrosis (47). The large amounts of neutrophil-released enzymes could degrade cytokines but also modify glycan on the surrounding tissues (48). Alteration of glycan residues on IgG molecules is associated with high lupus disease activity (49). Moreover, the incomplete clearance of DNA-released material leads to systemic inflammation and autoantibody production (50).
Dias et al. (51) demonstrated that ENL patients displayed higher levels of human DNA-histone complexes than either BL/LL patients or healthy individuals. The increased levels of TLR-9 ligands and the TLR-9 per se in peripheral mononuclear cells was considered by the authors as a major innate immunity pathway activated during ENL (51). Meanwhile, the source of the DNA-histone complex has yet to be identified.
There is much evidence in support of the existence of neutrophilic subpopulations and their role in inflammation, infection, and tumor immunology. The neutrophils subsets have been characterized according to their phenotypic, functional, morphological, and physical characteristics under both homeostatic and pathophysiologic conditions ( Table 1). A detailed description of all neutrophils subsets is beyond the scope of this work, but additional reviews can be found elsewhere (58). Nonetheless, it is not yet known whether these subpopulations are distinct subsets or rather represents the plastic development of their neutrophilic precursor.
Recruitment of leukocytes at a site of blood vessel growth is a crucial event for proper angiogenesis and subsequent Frontiers in Immunology | www.frontiersin.org tissue perfusion. Pro-angiogenic neutrophils CD11b + /GR-1 + /CXCR4 high producing high levels of MMP9 were recruited into the tissue in response to VEGFA in a mouse model of nonvascularized transplant (59). These pro-angiogenic neutrophils have been shown to be essential in promoting neovascularization of transplanted pancreatic islands (59) and may be the same cells that are known to promote cancer cell survival (60). Neutrophils with regulatory function were identified in several models. Lung neutrophils isolated from both bronchoalveolar lavage fluid and parenchyma of infected mice produced IL-10 and negatively regulating local lung inflammation during chronic phase of M. tuberculosis infection (61). Secreting IL-10 neutrophils reported in Trypanosoma cruzi-infected mice showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production (62). However, these anti-inflammatory neutrophils may change into pro-inflammatory phenotypes (IL-10 low /IL-12 high ) after interacting with Natural Killer T cells in a CD1d-dependent manner (63). It was showed that neutrophils (G-neutrophils) from G-CSF-treated human and mice donors could inhibit T cell activation both in vitro and in vivo in a model of experimental acute graft-versus-host disease (64,65). The disease inhibition induced by G-Neutrophils is dependent on neutrophil IL-10 competence (66).
During infection with MRSA, distinct types of neutrophils have been identified in association with resistance and susceptibility to MRSA. Neutrophilic populations isolated from both resistant and susceptible MRSA, PMN-I, and PMN-II, respectively-were distinct from neutrophils isolated from healthy mice, PMN-N ( Table 1). It is possible that these proand anti-inflammatory neutrophils may alter the course of the adaptive response by inducing M1 or M2 macrophages, respectively. It cannot be ruled out that these neutrophils may change their phenotypes during the course of inflammation to fit a particular aggressor and do not necessarily represent distinct lineages (52). To date, no work in the literature has identified any neutrophilic subpopulations in leprosy that might correlate with the TT leprosy-resistant or LL susceptible leprosy polar forms, TT vs. LL.
A myriad of neutrophilic subpopulations (CD16 bright / CD62L dim , CD16 dim / CD62L bright , CD16 bright /CD62L bright ) has been identified in the circulation of human volunteers receiving lipopolysaccharide in contrast to the number in untreated individuals (53, 54) ( Table 1). The subset CD16 bright /CD62L dim hypersegmented neutrophils displayed normal phagocytosis associated with a remarkably poor capacity to contain bacteria intracellularly. This defect in bacterial containment was associated with failure of acidification in the phagosomal compartment. On the other hand, CD16 dim /CD62L bright banded neutrophils were the only neutrophil subset that adequately contained MRSA (44).
Fridlender et al. (55) were the first to describe the existence of additional neutrophilic subsets nominated N1 e N2. The former is pro-inflammatory and the latter, anti-inflammatory (55). Via a murine model of cancer, the authors demonstrated the presence of tumor associated neutrophils (TANs), characterized by differential activation and phenotypical states. Neutrophils with an N1 phenotype possess a hypersegmented nucleus with proinflammatory and antitumor properties due to increased tumoral cytotoxicity, high NETs production, high ICAM1 expression, and production of inflammatory cytokines and chemokines like TNF. On the other hand, the N2 phenotype plays an opposite role and is classified as immunosuppressive and pro-tumoral mostly due to the elevated production of arginase like G-MDCs. These neutrophils usually possess an immature nucleus although some works have described them as being segmented. Another interesting fact about N2 neutrophils is their proangiogenic profile, which is driven by their capacity to produce elevated levels of MMP-9 and VEGF (57). Furthermore, it has been shown that TGF-β plays a critical role in neutrophilic polarization as a result of its ability to induce plasticity between a N1 subset into a N2 profile (55,56). Other factors, namely angiotensin-II and type I IFNs, have recently been to shown to promote N1/N2 polarization too (58). Beyond phenotypic differences, it became clear by way of the transcriptomic approach that the N1 and 2 subsets represent distinct populations with diverse transcriptional signatures (56).
Interestingly, a new neutrophil subset with different densities has been the focus of several research projects. Associated with disease severity in some inflammatory disorders, a subset of low-density neutrophils (LDN) that co-localizes with peripheral blood mononuclear cells (PBMC) after density gradient separation has been reported (67). It is also noteworthy that, in cancer, this population was found to increase within tumor growth and be characterized by a morphologically homogeneous population that may contain band and segmented neutrophils (68,69). Even though the origin and role of this subpopulation remain somewhat nebulous, some works have reported that LDNs display diverse profiles. The analyses of PBMC preparations from patients with Systemic Lupus Erythematous reveals that LDNs have an activated phenotype. In this scenario, LDN produce higher levels of such proinflammatory mediators as type I IFNs, IFN-γ, and TNF and are capable of modulating endothelial cell functions and increasing vascular damage (70). In addition, they are more disposed to form NETs that favor the chronic inflammation and disease severity (71,72). While LDN have also been detected in many other pathologies like sepsis, HIV infection, malaria and also in tuberculosis (73)(74)(75)(76), increasing our understanding of their surface marker patterns, cytokine expression, transcription factor regulators, and other trademarks of activation is of prime importance. Despite the uptick of studies describing the diversity of neutrophilic subpopulations, their distinct origins and plastic capacity remain unknown. New data need to be put forward that corroborate the existence of neutrophilic subpopulation in leprosy.
Under certain physiological conditions, the death of circulating neutrophils takes place in the liver, spleen, and bone marrow. Observed in old neutrophils, increased CXCR4 expression helps to direct them back to the bone marrow and subsequent elimination. CXCR4 is also involved in the down regulation of the newly-formed neutrophilic release into the marrow (39). Some studies suggest that terminal neutrophilic trafficking inside the intestinal tract also takes place to help FIGURE 3 | ENL skin lesions present neutrophils expressing CD64. The protein expression of CD64 on neutrophil (MPO + cells) was evaluated by immunofluorescence. Skin lesion was stained for CD64 (red), myeloperoxidase (MPO; green), and nuclei (DAPI; blue). Images are representative of three independent samples of ENL patients. Co-localized areas of MPO + CD64 + cells were identified with arrows. The regulation of neutrophil CD64 expression has the potential to be useful in the ENL treatment, as well as to prevent ENL reactional episodes. Scale bars: 10 µm. Images from Leprosy Laboratory collection.
regulate the commensal flora (77). Both senescent neutrophils and those that die after fighting infection may also expire within the vasculature and be removed by Kupffer cells (resident liver macrophages) (78). Removal of neutrophils by both Kupffer and dendritic cells is mainly regulated by the IL-23/IL17/G-CSF axis. This cytokinetic axis stimulates neutrophilic production in the bone marrow and is down regulated by liver X receptor (LXR) (79). The mechanisms involved in the clearance of NETotic cells are not yet known. On the other hand, clearance of apoptotic cells is well-studied (50). Different cell types participates in the uptake of apoptotic bodies by employing different types of receptors. This process can be amplified or suppressed by different types of plasma proteins (80). The complement system, pentraxins, and collectins have been implicated in apoptotic cell clearance in circulation or in injured tissue (81,82).

NEUTROPHILS IN LEPROSY
A number of studies in the 1970's addressed the neutrophilic functions in the different forms of leprosy (83)(84)(85)(86). These studies used the nitro blue tetrazolium that measures neutrophilic activation through reduction of in vitro. Goihman-Yahr et al. (83,85) found that, during reaction, there is spontaneous neutrophilic activation not witnessed in LL leprosy (83,85). Moreover, neutrophils are equally well-activated by endotoxin and M. leprae in vitro (87) in all forms of leprosy. In sharp contrast, Sher et al. (86) found no spontaneous activation rate in neutrophils of TT, LL, or ENL patients. Even so, ENL sera activated neutrophils of healthy donors in vitro, suggesting that ENL sera contain a neutrophilic activation inductor (86). Another parameter assessed of neutrophilic activation was cell motility that was measured by three different assays (random migration, chemotaxis, and chemokinesis), all of which were defective in LL neutrophils whether in the absence or presence of ENL (86). Drutz et al. (88) reported no important differences among TT and LL patients and normal control subjects in the bactericidal and fungicidal functions of their phagocytic cells, including monocytes, macrophages, and neutrophils (88).
Circulating neutrophils from leprosy patients are loaded with M. leprae (30,31) and there is apparently no sign of systemic inflammation. It remains unclear whether neutrophils are capable of killing the bacilli. Our group has reported that neutrophils isolated from LL patients with or without ENL released TNF and IL-8 subsequent to stimulation with M. leprae ex vivo (89). Besides, the apoptotic rate of ENL neutrophils is higher in comparation to that found in BL/LL patients and healthy volunteers (89). It has been previously demonstrated that apoptotic neutrophils infected or not with M. tuberculosis trigger a proinflammatory response in M. tuberculosis-infected macrophages through a caspase-1-and IL-1β-dependent mechanism (90). The biological responses of macrophages included an enhanced production of proinflammatory cytokines as well as an enhanced capacity to control the intracellular growth of M. tuberculosis. The interaction of apoptotic neutrophils and macrophages in leprosy has yet to be determined with certainty.
Our group has demonstrated that, during ENL, circulating and lesional neutrophils exclusively expresses CD64 (FcγRI) while leprosy patients without reaction, such as BL/LL, BT, and RR individuals, do not (9) (Figure 3). Besides, the higher CD64 levels on circulating neutrophils have been correlated with disease severity, pointing to CD64 as an early biomarker for ENL as well as a marker of severity (9). Since neutrophils function as biosensors, the proinflammatory microenvironment and/or fragments of the bacillus could induce the expression of CD64 on the neutrophil surface. The biological impact of surface expression of CD64 in neutrophils needs to be better evaluated. Upregulation of CD64 in vivo has likewise been associated with enhanced neutrophilic functionality (91)(92)(93). At the onset of sepsis or septic shock, the CD64 expression rate in neutrophils is augmented (94). Fadlon et al. showed that the CD64 + neutrophils bound to endothelial monolayers and CD64 + enriched neutrophils were 7 times more strongly adherent to endothelial monolayers than were CD64-depleted neutrophils (95).
Pentraxin-3 (PTX3), a protein present in the secondary neutrophilic granule, was originally identified as being induced by such primary inflammatory signals as TNF and interleukin 1β (IL-1β) (96,97). Our group has shown evidence that PTX3 is released systemically and at the site of ENL lesions (98). Our research has also demonstrated that PTX3 serum levels correlated to the surface expression of CD64 in circulating neutrophils and that thalidomide treatment of ENL down regulated PTX3 levels (98). Interestingly, PTX3 serum levels were high in MB patients without reaction yet persistent in patients who developed ENL. In contrast, MB patients who developed RR had low levels of PTX3 prior to and at the onset of the event. These results indicate that high and persistent levels of PTX3 in MB patients may be associated with the occurrence of ENL while also identifying PTX3 as a potentially predictive ENL biomarker capable of differentiating it from an RR episode.
In recent years, several large-scale gene expression studies have been conducted to monitor the host response to pathogen. These study results could potentially serve as diagnostic tool to either distinguish disease-afflicted patients from healthy individuals or classify different forms of the same disease. Via microarray analyses, Lee et al. (99) compared LL -reaction free skin lesions to those of ENL patients. Their global gene expression profiles revealed the up-regulation of genes involved in cell-movement, including E-selectin and its ligands, both key molecules in mediating neutrophilic recruitment to inflammatory sites (99). Transcriptome profiles derived from ENL skin lesions have also recently detailed the participation of neutrophilic and endothelial cell-gene networks in the vasculitis resulting in tissue damage (100).
To date, no study has yet reported a gene expression signature based on leprosy whole blood. However, via the microarray, the global transcriptional profiles of PBMC revealed that there were 275 genes differentially expressed in RR and 517 differentially expressed in ENL (101). In addition, a granulocytic gene signature was identified in gene-expression arrays derived from ENL PBMCs (101). These data suggest that PBMC fractions of ENL patients may be contaminated with LDN subpopulation, as has been similarly described in autoimmune diseases. Nonetheless, the presence of LDN in light of their functional capacity and potential to contribute to the clinical manifestations of ENL remain basically unexplored.
Naranbhai et al. have recently mapped the quantitative trait loci (eQTL) expression in peripheral blood CD16 + neutrophils from 101 healthy Europeans (102). The analyses found that leprosy and Crohn's disease, an autoimmune inflammatory bowel illness, showed a profound overlap in genetic architecture. The ancestral T allele of rs1981760 was associated with an increased susceptibility to MB leprosy. The authors observed a strong link between rs1981760-T and a reduced NOD2 expression in neutrophils in conjunction with a conversely elevated expression in monocytes. In addition, neutrophils stimulated with a NOD2 ligand, muramyl dipeptide supplemented with Pam3-CSK4, a synergistic agonist, express significantly higher levels of mRNA for IFNβ (102). These data demonstrate that rs1981760 affects NOD2 expression and the subsequent IFNβ responses to its ligand. Interestingly, eQTL in neutrophils are enriched for genes in the IFNβ network. These data suggest that type-1 interferons and neutrophils may be involved in leprosy such as has been previously shown in tuberculosis (103).

CONCLUSION
There are still large gaps in our understanding of the role of neutrophils in ENL and leprosy disease despite the large number of studies examining their immunological functions. Future works should aim to further determine the roles of neutrophils in host-mycobacterial interactions, particularly as relates to their early defensive posture and possible contribution to disease progression. The identification of subpopulations of neutrophils associated with the clinical forms of leprosy could provide novel insights of neutrophil function and reveal new targets in leprosy. The present review suggests the roles performed by neutrophils as both migratory and, for the first time, effector cells following chemo-attractants in the context of leprosy.

AUTHOR CONTRIBUTIONS
VS wrote the original draft of manuscript. IT, FP, JdS, and CdS helped with literature collating and referencing. IT, PP, AM, and ES revised and edited the manuscript.


text:
Plasma MicroRNA-200c as A Prognostic Biomarker for Epithelial Ovarian Cancer

1Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No.6, Jakarta, Indonesia 2Department of Community Medicine, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No.6, Jakarta, Indonesia 3Department of Biochemistry and Molecular Biology, Division of Oral Biology, Faculty of Dentistry, Universitas Trisakti, Jl. Kyai Tapa No.260, Jakarta, Indonesia

Introduction
Ovarian cancer is the third most prevalent cancer in women in Indonesia. Usually diagnosed in stage III-IV, ovarian cancer patient survival is generally low.(1) In a study performed at Dr. Cipto Mangunkusumo Central General Hospital, the 5-year survival rate of epithelial ovarian cancer patients was 54.8%. (2) There is barely any difference between incidence and mortality rate of ovarian cancer in developed and developing countries due to the fact that in all countries, 70% of patients were diagnosed in advanced stage.(3) Although new developments in detection and treatment of ovarian cancer are continually appearing, ovarian cancer is still the deadliest cancer in women. Determination of prognosis in ovarian cancer patients is an essential part of evaluation and therapy. Efforts to identify prognostic factors in epithelial ovarian cancer (EOC) continue to be sought in further research areas, including tumor characteristics, surgery, genetic patterns, immunological actors, and proteomic patterns.
Over the past ten years, scientific developments have changed the traditional concept of ovarian cancer. Ovarian cancer is currently not identified as a single disease, but rather diagnosed as a group of diseases with distinct morphologies and behaviors. About 80% of patients who have tumor residual disease after surgery and receive platinumbased combination chemotherapy, will have a median of 18-month disease-free survival rate.(4) Identification of factors related to disease aggressiveness can direct the development of new biomarkers and identification of targeted therapies that would greatly ameliorate the burden of this disease. (5) MicroRNAs (miRs) are short RNA sequences which do not encode proteins but are the main expression regulators of various genes by acting on base pairs of 3'UTR mRNA targets that determine mRNA degradation or translation inhibition. The miRs are involved in the process of carcinogenesis, cell cycle, apoptosis, proliferation, invasion, metastasis, and chemoresistance. Their dysregulation is related with initiation and development of cancer in human, including ovarian cancer. miRs can act as onco-miR or as a tumor suppressor-miR. miRs are stable in blood circulation so they have potential as non-invasive biomarkers. Research on the role of miRs in tumorigenesis can provide a new paradigm for understanding ovarian cancer. The pattern of miR expression in primary tissue, circulation, and effusion fluid of ovarian cancer patients is not the only a potential marker in the detection and screening of non-invasive ovarian cancer, but can also be associated with survival, prognosis, and predictions of recurrence. (6) miR-200c, a member of the miR-200 family, influences various types of cancer, including ovarian cancer. The miR-200c has been reported to play a role in epithelialmesenchymal transition (EMT), cancer stem cells (CSC), differentiation, modulation of cell division, apoptosis, and chemoresistance. (7) In this study, miR-200c in blood plasma is studied as a potential biomarker in determining prognosis in EOC.

Subject Recruitment
A prospective cohort study was conducted at Dr. Sardjito Central General Hospital in Yogyakarta from September 2015 to July 2018. Ethical clearance was granted from the Faculty of Medicine Universitas Gadjah Mada (KE/ FK/1085/EC/2015). Sixty-two subjects were recruited with consecutive sampling method. Subjects who suffer EOC, confirmed with clinical and histopathological examinations, were included, whereas subjects who had history of benign tumor and other cancer, were excluded. The EOC stage was classified based on The International Federation of Gynecology and Obstetrics/Fédération Internationale de Gynécologie et d'Obstétrique (FIGO).

Sample Collection
Pre-operative 5 mL peripheral venous blood was drawn in an ethylenediaminetetraacetic acid (EDTA) tube. Half of the blood was used to measure CA-125 level at Laboratory of Clinical Pathology, Dr. Sardjito Central General Hospital. And the other half was processed to get plasma for further analysis using Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR). After surgery, tumor was collected, prepared for hematoxylin-eosin staining and examined by pathologists.

RT-qPCR
RNA was isolated from plasma using miRCURY RNA Isolation Kit-Biofluids (Exiqon, Vedbaek, Denmark). Then total cDNA was prepared immediately from RNA using a Universal cDNA Synthesis kit (Exiqon). The qPCR was performed using miRCURY LNA Universal RT microRNA (Exiqon) with hsa-miR-16-5p LNA (Exiqon) and hsa-miR-200c LNA primer sets (Exiqon). PCR was set for 40 cycles. miR-16 was used as the internal control. Analysis was carried out using GenEx 6 software to get results in the form of fold change and to measure the expressions of miR-200c. Data was processed using the CFX Manager 96 Biorad program to obtain the quantification cycle (Cq).

Survival Rate and Cox Regression
Analysis of survival rate and Cox regression was done using SPSS version 23.0 (IBM Corp, New York, USA). Determination of high and low miR expressions was conducted using software R2: Genomic Analysis and Visualization Platform.

The miR-200c Expression of EOC Subjects
The expression of the miR-200c was shown in fold change compared to miR-16. The miR-200c fold changes ( Figure   Results

miR-200c and Overall Survival of EOC Subjects
The overall survival was recorded for 3 months starting from the date of surgery. Among 40 subjects who were included in the study, 15 subjects died (35%) and 25 people survived (65%). Using R2 Genomic Analysis and Visualization Platform, low and high miR-200c groups were classified and then analyzed using the Kaplan-Meier method for overall survival. Using log-rank test, low expression of miR-200c was shown to be associated with higher overall survival, although not statistically significant (Table 3, Figure 2).

Discussion
Most of the EOC subjects in this study were in the stage I-II (57.5%), which is different from the majority of studies whose subjects were 60-70% in the stage III-IV.(1) This might be related with the fact that the majority of the subjects of this study were having histopathology type I (60%). The results of this study are not much different from studies which found that the most common histopathology type was serous carcinoma, followed by mucinous and endometrioid carcinoma. (8,9) The EOC type I grows slowly, usually in stage I-II, rarely has a p53 mutation and tends to be resistant to platinum-based chemotherapy. (10) The miR-200c expression of the group with preoperative CA-125 level >500 U/mL was significantly higher (p=0.009) than the group with <500 U/mL. Although not significant, miR-200c expression was higher in the EOC histopathology type II than in the type I. miR-200c expressions was also higher in EOC Stage I-II than Stage III-IV. The increase of miR-200c expression in this study showed a tendency towards severity of EOC. This is in agreement with the previous study, which found a significant difference in the miR-200c expression between EOC stage III-IV and healthy controls. (11) Some studies stated that higher expression level of the miR-200 family was associated with decrease in patient's survival, while in other studies the opposite effect of high miR-200 expression has been reported. The exact reason underlying these contradictory findings is still not clear. One report shows that miR-200c can act as a suppressor or trigger for aggressive phenotypes, depending on the location   of the Human antigen R (HuR). (12) In current study, after bivariate analysis using log-rank test and Kaplan-Meier curve, it was found that high miR-200c expression had lower overall EOC patient survival, but not statistically significant. This result suggests a possible oncomiR role of miR-200c in EOC. The finding of miR-200c as an oncomiR has been reported in serous ovarian cancer, the high expression of miR-200c had a worse median overall survival compared to the one of low miR-200c expression. (13) The relationship between the high expression of the miR-200 family and the prognosis of cancer is still controversial. It has been shown in a meta-analysis report that high expression of the miR-200 family in various tumors was associated with a low overall survival. The expression of the miR-200 family is associated with a poor prognosis of various types of cancer, implying the potential of the family miR-200 family for cancer prognosis in clinical practice. (14) Survival Rate Duration (months)

Conclusion
The miR-200c expression is correlated with the severity in EOC. Higher expression of miR-200c correlate with worse overall survival, although not statistically significant. miR-200c could be a promising biomarker for EOC. Further studies with larger sample sizes are needed to clarify the prognostic value of miR-200c.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Medicine Chemistry

text:
NADPH oxidase 4 mediates TGF-β1/Smad signaling pathway induced acute kidney injury in hypoxia

Hypoxia is an important cause of acute kidney injury (AKI) in various conditions because kidneys are one of the most susceptible organs to hypoxia. In this study, we investigated whether nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase 4 (Nox4) plays a role in hypoxia induced AKI in a cellular and animal model. Expression of Nox4 in cultured human renal proximal tubular epithelial cells (HK-2) was significantly increased by hypoxic stimulation. TGF-β1 was endogenously secreted by hypoxic HK-2 cells. SB4315432 (a TGF-β1 receptor I inhibitor) significantly inhibited Nox4 expression in HK-2 cells through the Smad-dependent cell signaling pathway. Silencing of Nox4 using Nox4 siRNA and pharmacologic inhibition with GKT137831 (a specific Nox1/4 inhibitor) reduced the production of ROS and attenuated the apoptotic pathway. In addition, knockdown of Nox4 increased cell survival in hypoxic HK-2 cells and pretreatment with GKT137831 reproduce these results. This study demonstrates that hypoxia induces HK-2 cell apoptosis through a signaling pathway involving TGF-β1 via Smad pathway induction of Nox4-dependent ROS generation. In an ischemia/reperfusion rat model, pretreatment of GKT137831 attenuated ischemia/reperfusion induced acute kidney injury as indicated by preserved kidney function, attenuated renal structural damage and reduced apoptotic cells. Therapies targeting Nox4 may be effective against hypoxia-induced AKI.

Introduction
Acute kidney injury (AKI) is defined as "an abrupt (within 48 hours) reduction in kidney function" [1]. The incidence of AKI gradually increases, reaching 8-16% of hospitalized patients a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 [2]. AKI can progress to chronic kidney disease (CKD) and increase in-hospital mortality four-fold [2]. Despite the clinical importance of AKI, there is no absolute prevention or treatment has yet been found. Among the many causes of AKI, ischemic or non-ischemic kidney tissue hypoxia is the leading cause of AKI. Hypoxia not only leads to energy shortages in tissues, but also induces changes in intracellular signaling systems and gene expression. Kidney tubular epithelial cells are more vulnerable to hypoxia because these are less energy producing in anaerobic condition than other cells and consume more oxygen to maintain active transtubular absorption and excretion [3,4]. Various mechanisms have been introduced as mediators of the AKI caused by hypoxia, including calcium overload, endoplasmic reticulum stress, complement system activation, and reactive oxygen species (ROS) [5][6][7][8][9]. ROS play an important role in maintaining a normal intracellular signaling system in a stable state under normal conditions but the amount of ROS increases markedly in the pathologic state, acting as a crucial cause of AKI. Excessive production of ROS promotes hypoxia induced AKI by affecting the function of cellular DNA, proteins, and lipids [10,11]. Among various sources of ROS, nicotinamide adenine dinucleotide 3-phosphate (NADPH) oxidase (Nox) is the major intracellular non-mitochondrial ROS source. Because among seven Nox families, Nox4 is the most abundant in the human kidney, changes of Nox4 expression in hypoxia are predicted to affect the progression of AKI by altering the intracellular ROS level in the kidney. But few studies have investigated the role of Nox4 in hypoxia induced AKI [12][13][14].
Based on these observations, we investigated the role of Nox4 and the benefits of Nox4 inhibition in hypoxia induced AKI.

Cell viability
Cell viability was measured with the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as reported previously [15][16][17]. Briefly, after hypoxia exposure, HK-2 cells were cultured in a 24 well plate. After 24 h incubation 5 mg/ml MTT solution (Sigma) was added to the wells and we incubated the cells for another 4 h. The supernatant was then removed and 1 ml of dimethyl sulfoxide (DMSO) was added to each well. Immediately after purple formazan crystals formed and dissolved, the solution was collected and pipetted into a 96-well plate. The optical density was measured at 590 nm (VICTOR X3; PerkinElmer, Waltham, MA, USA).

Immunoblotting
HK-2 cells exposed to hypoxia for 24 h were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in a radioimmunoprecipitation assay (RIPA) buffer on ice. Whole cell lysates (50 μg) were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane. The membrane were blocked with 5% nonfat dried milk for 2 h at room temperature and incubated overnight with 0.2 μg/ml of primary antibody in PBS (pH 7.2) at 4˚C. After two washes, the membrane were incubated with secondary antibodies (horseradish peroxidase-conjugated antibodies) for 2 h at room temperature. Signals were visualized using enhanced chemiluminescence (Thermo Fisher Scientific Inc., Rockford, IL) using Image Quant 400 (GE Healthcare, Buckinghamshire, UK). Following antibodies were used for this study. Nox4 antibody was from Abcam (Cambridge, MA). Antibodies for pp38 and p-c-Jun N-terminal kinase (JNK) were obtained from Cell signaling Technology (Denver, MA, USA). Anti-p38, JNK, p-extracellular signal-regulated kinase (ERK), ERK, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Reactive oxygen species (ROS) measurements
Hydrogen peroxide (H 2 O 2 , end product of Nox4) was measured with Amplex red assays using Amplex red hydrogen peroxide/peroxidase assay kits (Invitrogen) according to manufacturer recommendations as described in author's previous studies [15,16]. Briefly reactions containing 50 μM Amplex Red reagents and 0.1 U/mL catalase in 50 mM sodium phosphate buffer (pH 7.4) were prepared in a darkroom under a red light. White enzyme-linked immunosorbent assay (ELISA) plates containing the samples (100 μL/well) were kept in the dark for 30 or 60 min. Fluorescence (excitation, 535 nm; emission, 595 nm) was measured on an HTS Multi-Label Reader (Perkin Elmer).
For the measurement of intracellular superoxide, the oxidative fluorescent dye dihydroethidium (DHE) was obtained from Thermo Fisher Scientific Inc. Cells were grown on glass slides in 12-well plates. Cells were fixed with cold methanol for 10 min and permeabilization for 30 min with 1% bovine serum albumin, cellular immunofluorescence was evaluated by staining with DHE for 40 min. The nuclei were counterstained with 4 0 ,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Carlsbad, CA, USA), and the cells were then washed with ice-cold PBS three times for 5 min and examined by confocal microscopy as previously described (LSM710; Carl Zeiss, Jena, Germany) [15,16].

Quantification of transforming growth factor-β levels
HK-2 cells were incubated in the hypoxia chamber (APM-30D, Astec., Tokyo) for 48 h. Transforming growth factor (TGF)-β1 was measured by an ELISA kit for human TGF-β1 (R&D Systems Inc.) in the culture media at different times according to the manufacturer's instructions. After the sampling steps were completed, the optical density of each well was measured at 450 nm (VICTOR X3; PerkinElmer, Waltham, MA, USA).

Animal models
All procedure in this study complied with the regulations of the Institutional Animal and Use Committee of the Konyang University. The animal care protocol for the experiments performed in our study was approved by the Institutional Animal Care and Use Committee of Konyang University. Experiments were carried out using twenty Sprague-Dawley female rats, 7 weeks old and weighing 180-200g. The rats were maintained in cages on a 12 h light: dark cycle at 22˚C and received water ad libitum and food. After 1 week acclimation period, rats were randomized into four groups and placed into separate cages: Group 1(control, normal saline pretreatment plus sham operation; n = 5), Group 2 (GKT137831 pretreatment plus sham operation; n = 5), Group 3 [normal saline pretreatment plus ischemia/reperfusion (I/R); n = 5] and Group 4 (GKT137831 pretreatment plus I/R; n = 5) (S2 Fig). Renal I/R injury progressed as previously described [22]. Briefly, two incisions were placed on both flank area and the arteries and veins were clipped with a clip for 45 minutes and then reperfused for 24 hours. Sham operation was performed in the same way as renal I/R operation, except for arterial and vein ligation. Group 1 and Group 3 were given 5ml/kg of normal saline by gavage daily for 5 days while Group 2 and Group 4 received 10mg/kg (5ml/kg) of GKT137831.

Biochemical and histological analysis
The blood samples were collected for biochemical analysis and kidney tissues were harvested for histological examination from separate group of rats after 24 hour of ischemic injury under anesthesia with ketamine/xylazine (0.5 ml of 100 mg/ml ketamine combined with 0.05ml of 20 mg/ml xylazine) at a dosage of 0.55ml/100g body weight. Serum blood urea nitrogen (BUN) and creatinine concentrations were measured using a Fufi Dri Chem 3500 (FUJIFILM, Tokyo, Japan). Kidney tissues were dehydrated with ethanol and fixed in 10% phosphate buffered formalin. After embedding in paraffin, it was cut into 4 um thickness and then stained with hematoxylin-eosin (H&E) or periodic acid-Shiff staining (PAS) to observe the structural changes. In order to examine apoptosis of kidney cells, we performed a Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay according to manufacturer's instructions as previously described (HRP kit DBA; Apotag, Milan, Italy). [15,16].

Statistical analysis
All graphed data are presented as the means ± standard deviation. The results were analyzed using analysis of variance (ANOVA) or a Student's t-test. P-values of <0.05 and <0.01 were considered to indicate statistically significant and highly statistically significant differences, respectively.

Hypoxia induces Nox4 expression
We explored the use of the HK-2 cell line as an in vitro model of hypoxia-induced kidney injury. We first investigated the effect of hypoxia on Nox homologues (Nox1, Nox2, Nox3, Nox4, Nox5) mRNA expression in HK-2 cells. Expression of mRNA was measured by real time-PCR after 0, 3, 6 and 12 h exposure to 1% oxygen, 5% CO 2 and 94% nitrogen using the hypoxia chamber (APM-30D, Astec., Tokyo). As shown in Fig 1A, Nox2 and Nox4 mRNA expression were significantly increased after exposure 3 h of hypoxia. Expression of Nox5 mRNA also increased after 6 h of exposure to hypoxia but was significantly less than that of Nox2 and Nox4. Little Nox1 and Nox3 mRNA expression was detected in HK-2 cells.
We further examined the effect of hypoxia on Nox4 protein levels in cultured HK-2 cells for 72 h. Nox4 protein levels peaked 12 h after hypoxia exposure and maintained high for 72 h (Fig 1B). We also examined the effects of CoCl 2 (300 μM) on Nox4 protein and mRNA transcript levels in cultured HK-2 cells. We found that Nox4 mRNA expression was significantly increased after CoCl 2 exposure as measured by real-time-PCR ( Fig 1C). CoCl 2 also increased the Nox4 protein level as measured by western blot (Fig 1D).

Role of Nox4 in hypoxia-induced apoptosis in HK-2 cells
To determine the role of Nox4 in hypoxia-induced apoptosis, cells were infected with Nox4targeting or control small-interfering RNA (siRNA). The efficiency of Nox4 knockdown showed in S3 Fig. Nox4 gene silencing blunted hypoxia-induced apoptosis (Fig 2A) as determined by caspase 3/7 activity. To reaffirm the results, we treated the cells with the specific Nox1/4 inhibitor GKT137831, finding that GKT137831 also attenuated caspase 3/7 activation (Fig 2B). In addition, Nox4 knockdown increased the survival of HK-2 cells in response to hypoxia as assessed using the MTT assay ( Fig 2C). Pretreatment with GKT137831 also showed the renoprotective effects observed with Nox4 silencing (Fig 2D).

Effects of Nox4 inhibition on hypoxia-induced cellular ROS levels
We evaluated superoxide production after hypoxia exposure in HK-2 cells with and without Nox4 knockdown or treated with and without GKT137831 using dihydroethidium (DHE) staining (Fig 3A and 3B). We also assessed hydrogen peroxide generation using an Amplex red assay and DCF-DA assay in cells with and without Nox4 silencing or treated with and without GKT137831 after hypoxia exposure (Fig 3C-3F). Hypoxia induced a significant increase in ROS production 24 h after hypoxia exposure. Intriguingly, the extent of the effect of hypoxia on ROS levels was significantly suppressed by Nox4 knockdown or GKT137831 pretreatment. The similar results were obtained when the hypoxia chamber was replace with

Effect of Nox4 inhibition on mitochondria in hypoxic HK-2 cells
We observed the effect of Nox4 inhibition on mitochondrial changes after hypoxia exposure using Mitotracker (Mitotracker Orange CMTMRos, Thermo Fisher Scientific, Inc., USA). Compared with the control group, the number of mitochondria was significantly reduced in the group exposed to hypoxia and recovered when Nox4 was blocked through Nox4 knockdown ( Fig 4A) or GKT137831 pretreatment (Fig 4B). The mitochondrial ROS measured by MitoSOX (Invitrogen) significantly increased in hypoxia but decreased when exposed to hypoxia after Nox4 knockdown (Fig 3C) or GKT137831 pretreatment (Fig 3D). To compare mitochondrial and cytoplasmic ROS changes, cytoplasmic ROS were measured again using DHR 123. Similar to the results of mitochondrial ROS, it increased with exposure to hypoxia and decreased with Nox4 knockdown (Fig 3E) or GKT137831 pretreatment (Fig 3F). When the ratio of mitochondrial ROS and cytoplasmic ROS were checked, the ratio of mitochondria ROS was higher after Nox4 inhibition. As a result, we could guess that the degree of decreased of cytoplasmic ROS was higher than that of mitochondrial ROS through inhibition of Nox4.

TGF-β1 induces Nox4 expression in hypoxic HK-2 cells
Culture media were collected from HK-2 cells that were exposed to hypoxia for 24 h and then added to normoxic HK-2 cells to examine whether hypoxia directly promote Nox4 expression in HK-2 cells or influences through a mediator as previously reported [23]. The Nox4 protein expression level was estimated 24 h later. Nox4 protein of normoxic HK-2 cells grown in hypoxia-conditioned culture media significantly increased compared with HK-2 cells grown in normoxic culture media in a volume-dependent manner (Fig 5A). This means that hypoxic HK-2 cells release soluble mediators that induce Nox4 expression [23]. Hypoxia is well known  to cause the upregulation of TGF-β1 [23,24]. The TGF-β1 levels in hypoxic HK-2 cells were measured by ELISA to know whether HK-2 cells exposed to hypoxia secrete TGF-β1 [23]. There was a significant increase in active TGF-β1 within 12 h under hypoxic conditions. TGF-β1 levels peaked 36 h after the hypoxia and then decreased; however, their levels remained high for 48 h compared with media from HK-2 cells cultured under normoxic conditions (Fig 5B).
To further confirm whether TGF-β1 induces Nox4 expression in hypoxia, HK-2 cells were grown with and without the TGF-β1 type 1 tyrosine kinase inhibitor, SB431542. Treatment with SB431542 significantly decreased Nox4 expression with real-time PCR and western blotting (Fig 5C and 5D).
To examination the effect of TGF-β1 on cellular apoptosis and survival in hypoxia, HK-2 cells were pretreated with and without SB431542 and then incubated under normoxia and hypoxia. Caspase 3/7 activity was measured and an MTT assay was performed. The caspase 3/ 7 activity was significantly attenuated and cellular survival significantly increased by pretreatment with SB431542 under hypoxia (Fig 5E and 5F).

The Smad pathway mediates TGF-β1-induced Nox4 expression in hypoxic cell injury
To examine whether Smad-dependent signaling pathway is involved in Nox4 expression in TGF-β1-induced hypoxic injury, HK-2 cells were infected with Smad4 siRNA or control siRNA. The gene silencing efficacy of the Smad4 siRNA was confirmed by western blot, with Nox4 in hypoxia induced acute kidney injury Smad4 gene silencing significantly decreasing TGF-β1-induced Nox4 expression measured by mRNA transcripts and western blot (Fig 6A and 6B).

Mitogen-activated protein kinases (MAPKs) pathways mediate redoxsensitive, hypoxia-induced apoptosis in HK-2 cells
We analyzed activation of the redox sensitive MAPK pathways (p38, JNK, and ERK) to identify the signaling mechanisms underlying hypoxia and Nox4 medicated apoptosis. Hypoxia (300uM of CoCl 2 ) led to increased phosphorylation of p38, JNK and ERK after 24 h. To determine the role of Nox4 in MAPK activation, cells were infected with Nox4-targeting or control Nox4 in hypoxia induced acute kidney injury siRNA. Nox4 gene silencing significantly reduced hypoxia-induced phosphorylation of JNK and p38. The levels of Bax decreased and Bcl-2 increased (Fig 7A). We further confirmed the relation between hypoxia and JNK, p38 and ERK pathways by inhibition of them with SP600125, SB203580 and PD98059, respectively. Notably, inhibition of JNK and p38 improved cellular survival (Fig 7B).

Pretreatment of GKT137831 attenuated hypoxia induced acute kidney injury and oxidative stress in rats
To assay changes in renal function after ischemic injury, we measured blood urea nitrogen (BUN) and creatinine in serum of ischemic / reperfusion (I/R) injured rats. Serum BUN and creatinine levels were not changed in the Group 2 (GKT treatment + sham operation) compared to Group 1. In Group 3 (I/R operation) serum BUN and creatinine levels were significantly increased when compared to Group 1 (control, sham operation). However, in Group 4 (GKT pretreatment + I/R operation), the damage of renal function due to ischemia was improved (Table 1).
Histologic changes were analyzed in control and experimental rats. There was no significant difference between Group1 and Group 2, but tubular dilatation, cellular casts, loss of tubular brush borders, vacuolar degeneration and tubular epithelial cell shedding were observed in Group 3. In Group 4, tubular damage was restored when compared to Group 3 (Fig 8A-8F).

Fig 7. Mitogen-activated protein kinases (MAPKs) pathways involved in Nox4 -induced HK-2 cell apoptosis in hypoxia.
Immunoblotting of Nox4-dependent intracellular signaling in CoCl 2 treated HK-2 cells with and without Nox4 knockdown (A). HK-2 cell viability was assayed using the MTT assay with and without pharmacologic inhibition of JNK, p38 and ERK with SP600125, SB203580 and PD98059, respectively under hypoxic condition (B). To detect apoptotic changes in in kidney cells, we performed TUNEL assay in control and experimental rats. In Group 3, there was a significant number of positive cells in the TUNEL staining, whereas in Group 4, the number of TUNEL positive cells was significantly decreased (Fig 9)

Discussion
Although it is located ubiquitously in cells, Nox4 is considered a renox agent because it is predominantly present in the kidney. The primary role of Nox4 lies in the generation of free radicals, especially hydrogen peroxide. Given the importance of ROS in hypoxia induced AKI, we hypothesized that Nox4 inhibition could prevent hypoxia induced AKI.
HK-2 cell apoptosis driven by hypoxia-induced oxidative stress involves the TGF-β/Smad/ Nox4 signaling pathway in this study. Our results show that HK-2 cell apoptosis, characterized by caspase 3/7 activity, is regulated through Nox4-generated ROS. By using a pharmacological inhibitor of TGF-β1 signaling, we demonstrated that TGF-β1-induced HK-2 cell apoptosis acts through the TGF-β receptor 1/Smad/Nox4 signaling cascade. TGF-β1 enhanced the expression of Nox4 and the TGF-β1 receptor blocker SB431542 decreased Nox4 expression.
Although Nox4 generated ROS, its function in the cell is not entirely clear and it may be regulated by multiple factors according to the cell type and variant of Nox homolog(s) present in the cell [25]. Previous studies have reported that in pathologic conditions, overexpression of Nox4 in podocytes and mesangial cells leads to renal fibrosis and chronic kidney disease through increasing cellular ROS level. [13,26,27]. Even though renal tubular epithelial cell injury is a key feature of the initiation of AKI and overexpression of Nox4 in kidney epithelial cells has been noted, its role in AKI has not been well characterized in these cells. A few studies including previous author's studies reported Nox4 involvement in apoptosis in human proximal tubule cells in several AKI models [14-16, 28,29]. Song et al. reported before that diphenylene iodonium (DPI) abolish hypoxia induced apoptosis of HK-2 cells [14]. But DPI is known to nonspecifically reduce Nox activity by inhibition of flavoprotein. So this is the first study in our knowledge to describe the role of Nox4 in HK-2 cells in hypoxic condition. There is a study that reported the contradictory results of the author's findings on the role of Nox4 in hypoxia. Nlandu-Khodo et al [30], reported that Nox4 knockout mice showed increased tubular cell death during acute ischemia/reperfusion injury with decreased Nrf2 protein expression. We performed in-vivo study with wild type rats and GKT/137831 for Nox4 inhibition instead of Nox4 knockout mouse. In our study, in contrast to Nlandu-Khodo et al, kidney damage due to I/R was alleviated by GKT137831 pretreatment. ROS are supposed to have dual roles in biological systems. Even though maintaining a basal level of ROS is crucial for life, immoderate concentrations of ROS can destroy balance and cause adverse effects. Thus, whether Nox4  plays a good or bad role in body might be determined by how much of the appropriate ROS is present in various cells and different condition [31]. Different levels of oxidative stress could lead to different effects on kidney injury in hypoxia. AKI due to hypoxia not present only in the ischemia/reperfusion injury but in other ischemic or non-ischemic injury can occur, further in-vivo study in various settings is needed. In our study, not only cytoplasmic but also mitochondrial ROS were reduced by Nox4 inhibition. It is known that Nox4 directly or indirectly affects mitochondrial ROS. With direct influence, Nox4 is located not only cytoplasmic membrane but also mitochondrial membrane. Mitochondrial Nox4 is known as one of the important source of mitochondrial ROS in diabetes [32] and aging associated cardiovascular disease [33]. On the other hand, indirectly H 2 O 2 produced by cytoplasmic Nox4 activates Nox2, which increases the ROS of mitochondria and activates the VEGF signaling in endothelial cells [34]. Crosstalk between mitochondria and NADPH oxidases has been studied as an interesting subject [35]. Even though further studies are needed to clarify the relationship, the results of this study may be a good basis for explaining the relationship between Nox4 and mitochondria in hypoxia.
Among several reported pathways that related in TGF-β1 induced the expression of Nox4 (Smad pathway, PI3K pathway, MAPK pathways and RhoA/ROCK pathway) [36], TGF-β1/ Smad signaling pathway was involved in this study. The Smads accumulate in the nucleus as the Smad2-3/Smad4 complex induce Nox4 expression by binding in the Nox4 gene promoter region [37]. It is well known that TGF-β induced Nox4 associated fibrotic response in many Nox4 in hypoxia induced acute kidney injury cells [36] but a few studies including author's previous studies reported the role of TGF-β/ Nox4 signaling in cellular apoptosis [15,38].
In this study, the JNK and p38 pathways were critical in mediating hypoxia-induced HK-2 cell apoptosis. Although phosphorylation of JNK, ERK and p38 increased after hypoxia exposure, only phosphorylation of JNK and p38 decreased after knockdown of Nox4. Our results are consistent with previous studies that suggest that p38 and JNK are involved in apoptosis and ERK is involved in cellular survival in ischemia/reperfusion kidney injury [39]. Because MAPK pathway has various effects on different cells and even in the same cells shows different changes depending on the situation, it is necessary to study the change pattern of MAPKs according to the change of amount of ROS.
Our findings show that Nox4 participates in the pathogenesis of HK-2 cell apoptosis in hypoxia. Our present study suggest that AKI driven by hypoxia-induced oxidative stress involves the TGF-β/Smad/Nox4/MAPK signaling pathway to drive redox signaling in HK-2 cells (Fig  10). Our data also strongly suggest that the renoprotective effects of Nox4 inhibition involve Nox4-dependent reductions in oxidative damage and apoptosis. Thus, we propose that hypoxia upregulates the expression of Nox4 via the TGF-β/Smad pathway and increases ROS production and therapies targeting Nox4 may be effective against hypoxia-induced acute kidney injury. ischemic reperfusion injury model GKT137831 (10mg/kg) or normal saline (5ml/kg) was administered orally once a day during four days prior to the ischemic injury (45min) and once after ischemic injury. The rats were scarified 24 hours after ischemic injury. (TIF)

S4 Fig. Effects of Nox4 inhibition on hypoxia-induced ROS generation.
HK-2 cells were exposed to CoCl 2 . Confocal microscopy images of cells subjected to dihydroethidium (DHE) staining with and without Nox4 knockdown (A) or treated with and without GKT137831 (B). Levels of H 2 O 2 , a product of Nox4, were measured by the Amplex red assay with and without Nox4 knockdown (C) or treated with and without GKT137831 (D). Levels of ROS measured by DCF-DA with and without Nox4 knockdown (E) or treated with and without GKT137831 (F). Data represent means ± SD; � p< 0.01, �� p < 0.05, ��� p < 0.001 vs. control and # p < 0.05, Validation: Se-Hee Yoon.