Lipid Raft Microdomains of Bladder Epithelial Cells Regulates Apoptosis and Microenvironment Upon Exposure to C. albicans


 INTRODUCTION. Interstitial cystitis/painful bladder syndrome (IC) is characterized by chronic bladder pain and urinary storage symptoms. IC affects more than 3.3 million women in the U.S. alone. Ibis T-5000 assays and next generation sequencing have revealed that the C. albicans fungus is highly abundant in the urine of IC patients, particularly those who report greater pain, urinary urgency, and flares. However, currently, the clinical significance of C. albicans in the urine remains elusive. Here, we report the pathological effects and mechanisms triggered by C. albicans in a healthy normal bladder.
METHODS. Immortalized bladder epithelial cells were infected with C. albicans. Perturbations in gene expression were identified using an Affymetrix gene microarray and subsequently followed with bioinformatic analyses, including gene set enrichment. Inflammatory and apoptotic genes were quantified using RT-PCR and Western blot analyses.Central signal pathways were examined using Western blot analysis. 
RESULTS. DNA microarray analysis showed alterations in the transcriptome of bladder epithelial cells infected with C. albicans over both the short and long terms. Key inflammatory and apoptosis networks were changed, which was consistent with several cellular events. Cellular levels of reactive oxygen species and nitrogen oxide increased after infection. Productions of cyclooxygenase-2 and prostaglandine E2 also increased after C. albicans infection, and their productions were suppressed by blockage of reactive oxygen species-epidermal growth factor receptor-Erk pathway.
CONCLUSIONS. This study provides evidence that C. albicans infection triggers inflammation and cellular damage by dysregulating key regulatory genes, signaling pathways, and bioactive species in normal bladder cells.


IC
Interstitial cystitis/painful bladder syndrome

BACKGROUND Bladder Health in Women
The Prevention of Lower Urinary Tract Symptoms (PLUS) Consortium recently defined the ideal female bladder health as, "a complete state of physical, mental, and social well-being related to bladder function and not merely the absence of lower urinary tract symptoms (LUTS). Healthy bladder function permits daily activities, adapts to short-term physical or environmental stressors, and allows optimal well-being (e.g., travel, exercise, social, occupational, or other activities)." LUTS among women include urinary tract infections (UTIs), overactive bladder (OAB), urinary incontinence (UI), interstitial cystitis/painful bladder syndrome (IC), etc. The Epidemiology of LUTS (EpiLUTS) study compiled the prevalence and associated effects of LUTS in the US, UK, and Sweden (1). This cross-sectional, population-representative survey found that the prevalence of LUTS is more than 75% in women older than 40 years old, and that most patients experienced a reduced quality of life. IC is a chronic bladder syndrome characterized by pain, pressure, and discomfort with urinary symptoms, such as urgency and frequency. The estimated yearly diagnosis of IC among women in the US is more than 3.3 million (2). The burdens of IC on public health and the lack of identified etiology remains a challenging issue in the field of urology.

Role of Urinary Mycobiome in Health
Humans have co-evolved with niche microorganisms that can be normally found in healthy individuals. Understanding the role of the mycobiome has become more and more important in human health and disease. Thanks to recently employed highly sensitive genomics tools and computational analyses, the mycobiome of the bladder has been explored. A series of studies analyzed the mycobiome of urine (voided or catharized) from healthy and diseased individuals.
These recent findings, based on highly sensitive metagenomic technologies using fungal-specific internal transcribed spacer (ITS)-2 amplicon sequencing, revealed consistent patterns that show that the diversity of the mycobiome is reduced in diseased urine samples compared to healthy controls (3,4).
A considerable amount of literature describes the important roles of the mycobiome in human health and disease. Fluctuations in fungal ecosystems may be linked to the abnormalities associated with diseases of the brain(5), heart, gastrointestinal track etc. Type 2 diabetes mellitus (T2DM) is associated dysbiosis in the gut mycobiome. Roles of the gut mycobiome in host immune system and homeostasis have also been suggested (6)(7)(8)(9). Additionally, patients with alzheimer's disease (AD) or mild cognitive impairment (MCI) showed differences in their mycobiome compared to healthy individuals (10).
Although the relationship between the mycobiome and several diseases has been established, the contribution of the mycobiome in bladder homeostasis and alteration of urinary components is a relatively new perspective. Furthermore, the urinary mycobiome may be strongly associated with the bladder ecosystem. Perturbations of the mycobiome community can occur with pathological conditions or they may reflect underlying specific diseases (11)(12)(13)(14). However, it remains uncertain whether specific mycobiome species are diagnostic or predictive of progression in urological diseases.

IC, Mycobiome, and C. albicans
While the etiology of IC remains unknown, much effort has been placed on identifying an infectious cause of IC; however, this has resulted in little to no success (15,16 defined as the presence of greater than 10 5 fungal cfu/ml urine in adults, though as little as 10 3 cfu/ml may also result in pathogenesis among groups at higher risk. When C. albicans is found in urine samples, it is related to the colonization of an indwelling catheter and/or the bladder, symptomatic cystitis, or invasive upper tract infection (21). C. albicans is the most frequently isolated species, but other species are increasingly gaining clinical significance.
The bladder wall has the well-defined layers including the mucosa which is the innermost portion of bladder wall. The mucosa is consisted of urothelium (the transitional epithelium that lines most structures of the urinary tract), basement membrane, lamina propria (22). Mucosal epithelial cells are the first line of barriers defending the invasion of C. albicans. However, the interaction between C. albicans and bladder epithelial cells and the mechanism underlying urinary bladder infection caused by C. albicans are incompletely understood.
Here, we aim to test the hypothesis whether infection with C. albicans causes bladder epithelial cell morphology alteration, cell damage, and production of inflammatory factors, and to characterize the mechanisms of pathogenesis in C. albicans infection of bladder epithelial cells, which may contribute to inflammation, cell apoptosis, and bladder-brain signaling. The findings from this study may further provide valuable information on the mechanisms underlying the infectious etiology related to IC. Elucidation of the potential strategies preventing the C. albicans infection will provide a greater understanding of C. albicans pathogenesis related to bladder health.

Cell lines and Cell Culture
The immortalized human bladder epithelial cells, TRT-HU1, were maintained as described (23).
The TRT-HU1 cell line was constructed and extensively characterized in previously published papers (23)(24)(25)(26)(27)(28). The passage of cell lines was kept below 10, and mycoplasma contamination was incubator with 5% CO2. The culture medium was changed after one day of subculturing and cells were passed again when there was 70%-80% confluence.
For this study, C. albicans (Robin) Berkhout (MYA-2876 ™ , SC5314 (wild type)) were used (ATCC). After thawing the frozen stock of C. albicans, strains were cultured on yeast malt (YM) Agar (BD 271210) as instructed by company (BD, Franklin Lakes, NJ). They were picked from a single colony and grown in yeast malt medium containing amino acids plus 2% glucose at 30°C.
Strains were passaged twice in medium and harvested at the log growing stage. Cells were washed twice in phosphate-buffered saline (PBS) solution before cell counting on a hemacytometer.
To determine the biological effects of C. albicans infection in bladder epithelial cells, they were co-cultured with approximately 85% confluent TRT-HU1 (1x10 6 cells) with multiplicity of infection (MOI) 0.5, 1, 2, 3, or 4 for 2 h. The co-culture plates were incubated in a 37 °C humidified incubator with 5% CO2.

Microarray analysis
After co-incubation of TRT-HU1 cells and C. albicans at MOI 0.5, the TRT-HU1 cells were harvested for sample preparation. First, reverse transcription of total RNA and subsequent steps for sample probe preparation, microarray hybridization, washing, and scanning of microarrays were performed following a standard Affymetrix Human Genome 430 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA) protocol at the UCLA Technology Center for Genomics & Bioinformatics.
The raw data was normalized using the gcrma package (version 2.10.0) in R 2.6.1. The log2 GC-RMA signals were then exported and used for differential expression analysis. To identify differentially expressed genes (DEGs), a two-tailed Welch's t-test was implemented. DEGs were identified as genes with a p-value < 0.05 and fold-change ≥1.5. In order to reduce unreliable detection and false positives, probe sets with average expression levels higher than the average of all probe sets in the data were also considered for further analysis. The DAVID software was used to search for Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were statistically enriched by the DEGs.

SDS-PAGE gel running and Western blotting
Cell lysates were prepared as described previously using a 4% sodium dodecyl sulfate-containing Imaging System was used for visualization of the protein bands and quantification of band intensity was done by using ImageJ.

Cell assay
To test cell growth after exposure to C. albicans, TRT-HU1 cells were seeded onto 10 cm plates with a density of 5 x 10 6 cells/plate. Cells were maintained in standard growth medium for 1 day and then incubated with varying doses of C. albicans for 24, 48, or 72 h. Cell proliferation was measured by manually counting cells using a hemocytometer. The averages of each count were used as the total density of the well after each time point.
After incubating with media containing C. albicans for the indicated times, cells were fixed with 4% paraformaldehyde at room temperature for 5 min. For crystal violet staining, cells were stained with 0.05% crystal violet for 15 min. Tap water was used to wash any extra staining. The cells were dried on filter paper, and the plates were scanned and quantified as described (29). For quantitative analysis, a 10% acetic acid solution was used to dissolve the stained cells, and absorbance at 570-590 nm was measured. All experiments were run in triplicates, and the data are representative of three independent trials. To detect DNA fragmentation related to apoptosis, a TUNEL assay was performed using the TUNEL Assay Kit -FITC (Abcam) after fixating cells with 4% formaldehyde for 15 min on ice.
Fragmented DNA was labeled with fluorescein using the TUNEL reaction mix and then analyzed with flow cytometer or fluorescence microscope (30).

ELISA apoptosis analysis
An ELISA for quantitative apoptosis was used to measure the levels of apoptosis according to the protocol provided by company (Cell Death Detection ELISA; Roche Diagnostics Corp.).

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
Apoptotic cells were also quantified by nuclear fragmentation detection by TUNEL (green) by counting the TUNEL-positive bladder epithelial cells under a microscope.

Measurement of Reactive Oxygen Species (ROS) and Nitric Oxide (NO)
A 2',7'-dichlorofluorescin diacetate (DCFDA) assay to quantitatively assess ROS was performed by using the Intracellular ROS Detection Assay Kit (Abcam), as described in literature (31). Cells were treated with C. albicans under the indicated conditions prior to the assay. Fluorescence spectroscopy with excitation/emission at 490/570 nm was used to detect fluorescence and measure the levels of ROS in response to infection. The control condition (without C. albicans) was equated 100% with the treatment conditions proportionally. Signals were background corrected and adjusted to cell numbers. For NO detection, a commercial NO Assay Kit (colorimetric, Abcam) was used, and total nitrate/nitrite amounts were measured in a simple twostep process (32).

Lipid raft isolation
Lipid raft membranes were isolated using differential extraction with Triton X-100 and octylglucoside (OCG) detergent. Triton-soluble and insoluble (OCG-soluble) membrane components were isolated, as described in previous literature (33). All experimental steps were done on ice or at 4°C.

Statistical analysis
All experiments were repeated at least three times, in most cases six times, with independent biological triplicates. Each of the experiments did not show significantly different results among repetitions. Data was analyzed using an unpaired two-tailed Student's t-test or one-way ANOVA using Prism (GraphPad Software, Inc., La Jolla, CA). Mean values from 6 biological replicates were used for statistical analyses, and all data were presented as the mean ± standard deviation (SD). Students' t test and one-way ANOVA post-hoc Tukey's test were used to compare groups of data. Statistical significance for all analyses was determined at p<0.05.

Apoptotic signatures induced by C. albicans infection
In this study, we sought to investigate the mechanism underlying alterations in the human bladder in response to C. albicans. We first found that infection with C. albicans led to morphological alterations and cellular damage in TRT-HU1 bladder epithelial cells. Co-culture under the conditions of MOI 2 or 3 induced the dramatic morphological changes of TRT-HU1. Under MOI 2 and MOI 3, most of cells were started altering to a rounded morphology after 3 h co-culture. After 12 h co-culture, microscopic observation showed that approximately over 70% of the cells were detached from culture plate both at MOI 2 and MOI 3 (Fig 1A). Because the co-culture condition at MOI 0.5 for 3 h did not show any morphological alterations nor reduced cell viability, we decided to use this condition for further molecular study.
To further investigate the cytotoxic effects of C. albicans on bladder epithelial cells, cell mass was measured using crystal violet staining (Fig 1B). Apoptosis analyses were performed using tryptophan staining and MTT assay (Fig 1C, D). TUNEL staining also supported the increased apoptosis of bladder epithelial cells infected with C. albicans (Fig 1E). An apoptotic biomarker, cleaved PARP, was detected from Western blot analysis (Fig 1F). C-PARP gradually increased in response to C. albicans infection, while protein expression of cyclin D1 was suppressed. This decrease was accompanied by an increase in levels of PARP cleavage form.

DNA microarray analysis shows induced apoptotic signatures after C. albicans infection
To gather molecular evidence of these observations, DNA microarray analyses were performed, which revealed that the global gene expression of normal human bladder epithelial cells were significantly changed after infection with C. albicans.
In total, 5,213 genes were found to be differentially expressed after C. albicans exposure, compared to controls. A heatmap shows 5,213 DEGs, containing 1,144 upregulated and 4,069 downregulated genes 30 min after C. albicans exposure (Fig 2A). A similar analysis identified 1,044 DEGs with 606 upregulated and 438 downregulated genes 3 h after C. albicans exposure (Fig 2B). A z-score transformation was applied to the original gene expression values when generating the heatmaps. There were 547 overlapping DEGs between the two gene lists (Fig   2C). Supplementary Figures 1A and B shows the quality assessment. The numbers of overlapping upregulated and downregulated genes are shown in Figs S2A and S2B. The top 20 DEGs 30-min and 3-h after C. albicans exposure are shown in Fig 2D and 2E, respectively.

Gene enrichment analyses suggested that genes indicative of apoptosis greatly enriched 3 h after
C. albicans infection (Fig 2E). Expression levels of apoptotic proteins were also analyzed. Genes associated with regulation of apoptosis, such as BCL2 Associated Agonist of Cell Death (BAD), BCL2 Associated X, Apoptosis Regulator (BAX), and Transforming Growth Factor Beta-1 (TGFB1) were upregulated in response to C. albicans infection (Fig 2E). Collectively, our results show that C. albicans infection induces transcriptomic alterations, leading to eventual apoptosis in bladder epithelial cells.

Induced apoptosis of human bladder epithelial cells via ROS and NO production by C. albicans infection
Activation of apoptosis includes ROS production, leading to damage of proteins, nucleic acids, lipids, etc. To determine whether C. albicans infection causes ROS and other reactive species, intracellular levels of ROS were measured at different time points (Fig 3A, left). The results were further validated when treatment with antioxidant N-acetyl-L-cisteine (NAC) suppressed ROS production to almost baseline (Fig 3A, right). Western Blot analysis of c-PARP showed upregulation of ROS in response to C. albicans infection was almost completely diminished by NAC pretreatment (Fig 3B). In contrast to ROS, there was a decrease in NO production with C.
albicans infection (Fig 3C). In consistent with this finding, Western blot analyses indicated decreased NOS protein (eNOS and iNOS) production in response to C. albicans (Fig 3D).

Acute infection of C. albicans activates inflammatory networks via activation of EGFR signaling pathways
In the following experiments, we examined the inflammatory responses, including cyclooxygenase-2 (COX-2) protein expression and prostaglandin E2 (PGE2) secretion into conditioned media (Fig 4A-B). In addition, the secretion levels of PGE2 were greatly inhibited when ROS was diminished by NAC treatment (Fig 4B).
Given that ROS production stimulates the activation of key signaling molecules, a series of signaling pathways were tested. Among MAPK pathways, phosphorylation of ERK, p38, and JNK each were determined. Western Blot analysis suggested the temporary deactivation of EGFR and Erk MAPK following C. albicans exposure for 15 min. In contrast, the p38 MAPK was activated, as indicated by increased phosphorylated form of p38 (Fig 4C), while EGFR and Erk MAPK were deactivated. However, these were temporary events that recovered soon after time passed.

These findings are aligned with the previous literature showing that induction of COX-2 gene by
Candida albicans through EGFR, ERK, and p38 pathways in human urinary epithelium (34).
Because ROS stimulated the activation of EGFR signaling pathway, we investigated whether EGFR inhibition can suppress PGE2 secretion, and if NAC can have further effects. Although modest, NAC showed additional inhibition of EGFR inhibitor on extracellular PGE2 secretion (Fig   4D). To further understand the role of the secreted PGE2 in bladder microenvironments, the conditioned medium was collected from the C. albicans-infected bladder cells. After removal of cell debris, RAW264.7, a mouse monocyte/macrophage-like cell line, was challenged by incubation in the collected conditioned medium. We found a significant stimulation of proliferation in Raw 264.7 cells incubated in the conditioned medium (Fig 4E). As a control, the conditioned medium collected in absence of C. albicans was used. albicans-induced cell damage on bladder epithelial cells. We tested this hypothesis and found that MβCD suppresses C. albicans-induced ROS production and apoptosis (Fig 5A). Cell fractionation and enrichment showed that lipid raft microdomains contained EGFR (Fig 5B, top   panel).
Upon C. albicans infection, a portion of EGFR (approximately 50%) translocated into the non-lipid rafts domains. As a result, the EGFR found on lipid rafts decreased with C. albicans infection (Fig   5B, second panel). A-Tubulin and Gia2 were used as marker proteins of non-raft and raft domains, respectively (Fig 5B, 3 rd and 4 th panels). Furthermore, we addressed whether lipid raft disruption by cholesterol-interfering treatments affected EGFR translocation mediated by C.
albicans infection. The decreased levels of EGFR on lipid rafts were recovered by an inhibition of lipid rafts and/or ROS production (Fig 5C). Collectively, these findings suggest that lipid rafts participate in the process of C. albicans infection in bladder epithelial cells.

DISCUSSION
Our present demonstrated what molecular events occur when the bladder is infected with C.
albicans. We aimed at analyzing the role of C. albicans in the bladder because mycobiota communities exist in urine, and the interactions between these communities and the microenvironment are likely to be critical for normal bladder health.
Our hypothesis was inspired by previous studies by colleagues that reported fungal communities from voided urine samples collected from female and male patients with chronic lower urinary track symptoms and healthy controls (4,(35)(36)(37). These findings were based on a cultureindependent next-generation sequencing approach combined with computational analysis. Their able to uptake C. albicans (38). While more data are still needed, these findings suggest that C.
albicans infection could induce cell damage through signaling activation and lipid raft microdomains, as illustrated in Fig 5D. These results also pave the way for future studies to decipher the niche and relationship between the bladder, its microenvironments, and the local mycobiome. Furthermore, these findings also suggest the possible therapeutic strategy of lipid raft disruption on C. albicans-induced immune response and pathological damage on bladder epithelial cells.

CONSENT TO PUBLISH
The authors consent to publish.

AVAILABILITY OF DATA AND MATERIALS
The datasets used during the current study are available from the corresponding author on reasonable request.

COMPETING INTEREST
The authors have nothing to disclose.