S100‐A9 protein in exosomes derived from follicular fluid promotes inflammation via activation of NF‐κB pathway in polycystic ovary syndrome

Abstract Exosomes have recently emerged as key mediators of different physiological and pathological processes. However, there has been few report about proteomic analysis of exosomes derived from human follicular fluid and their association with the occurrence of PCOS. Herein, we used TMT‐tagged quantitative proteomic approach to identify proteomic profiles in exosomes derived from follicular fluid of PCOS patients and healthy controls. We identified 662 proteins in exosomes derived from human ovarian follicular fluid. Eighty‐six differently expressed proteins (P < .05) were found between PCOS and healthy women. The alterations in the proteomic profile were related to the inflammation process, reactive oxygen species metabolic process, cell migration and proliferation. Importantly, we observed that follicular fluid exosomes contain S100 calcium‐binding protein A9 (S100‐A9) protein. Exosome‐enriched S100‐A9 significantly enhanced inflammation and disrupted steroidogenesis via activation of nuclear factor kappa B (NF‐κB) signalling pathway. These data demonstrate that exosomal proteins are differentially expressed in follicular fluid during disease process, and some proteins may play important roles in the regulation of granulosa cell function. These results highlight the importance of exosomes as extracellular communicators in ovarian follicular development.

diseases. 10,11 However, the pathogenesis and molecular defects in PCOS are not fully understood. 12 Follicular fluid (FF) provides an important microenvironment for follicular development and oocyte maturation. The accumulation of follicular fluid is mainly formed by the secretion of granulosa cells, theca cells and oocytes as well as by diffusion of plasma components from capillaries to the antrum. 13,14 The major components of follicular fluid are proteins, steroids, metabolites and polysaccharides. 15,16 Follicular fluid is the medium for bi-directional communication between oocytes and the surrounding somatic cells. 17 These cells can produce growth factors and cytokines that contribute to the regulation of ovarian function through paracrine/autocrine systems. 18 Exosomes are small membrane-enclosed vesicles (30-100 nm in diameter) secreted by a wide range of living cells under normal or pathophysiological circumstances. 19,20 Exosomes contain several regulatory molecules, such as mRNAs, microRNAs (miRNAs), proteins and lipids. 21 These compositions can be transferred between different types of cells and influence biological activities. [21][22][23] Recently, some studies uncovered the potential function of exosomes derived from FF as carriers of miRNAs in steroidogenesis, follicular development and other pathological conditions. [24][25][26][27] Although proteome and peptidome were identificated in human FF, [28][29][30][31][32][33] little was known about the proteomic analysis of exosomes in follicular fluid and their potential roles in follicular development and reproduction-related disorders. Therefore, we used an TMT-tagged quantitative proteomic approach to compare the proteomic profile of exosomes derived from FF of PCOS and healthy women. Then, we focused on differently expressed proteins and several important biological pathways altered during PCOS progression. We found exosomes carrying S100 calcium-binding protein A9 (S100-A9) were able to activate the nuclear factor kappa B (NF-κB) pathway, increase inflammation and disrupt steroidogenesis, which probably involved in the occurrence of PCOS.

| Study population and sample collection
All the participants included in the study were women undergoing IVF or ICSI at Shanghai First Maternity and Infant Hospital between February 2017 and December 2017. Patients for this study were divided into two groups: PCOS (n = 8) and matching control (n = 8). PCOS was defined according to the Rotterdam consensus criteria. 1 The control group contained patients undergoing IVF due to male factor infertility or tubal factors. The exclusion criteria for both groups included women with endometriosis, cancer, premature ovarian insufficiency (POI) or other medical disorders that could affect follicular development. The information of involved patients is shown in Table 1

| Exosome isolation
The follicular fluid exosomes were collected by ExoQuick-TC Exosome Precipitation Solution Kit (System Biosciences). In brief, 250 µL ExoQuick Solution was added to the 1 mL follicular fluid (1:4), mixed well and incubated overnight at 4°C. Then, the mixture was centrifuged at 1500 g for 30 minutes at 4°C, and the supernatant was removed. The exosome pellet was resuspended in 500 µL PBS and passed through a 0.22-µm filter.
Exosomes from cell culture supernatants were isolated by ultracentrifugation. In brief, 293T cells were starved in medium containing 1% BSA for 48 hours. Then, cell supernatant was centrifuged at 300 g for 10 minutes to remove cells. The supernatant fluid was then centrifuged at 2000 g for 10 minutes at 4°C to remove dead cells. The resultant supernatant fluid was transferred to an ultracentrifuge tube and centrifuged at 100 000 g for 2 hours. The pellet was suspended in PBS and filtered through a 0.22-μm filter, and then centrifuged at 100 000 g for 2 hours. The pellet was resuspended in 200 μL PBS and stored at −80°C.

| Transmission electron microscopy
Exosomes were analysed by transmission electron microscopy (TEM) as previously described. 34 and air-dried. The samples were observed by FEI Tecnai G2 spirit transmission electron microscope (FEITM) at an acceleration voltage of 120 kV.

| Nanoparticle tracking analysis
Nanoparticle tracking analysis (NTA) measurements were performed using a NanoSight NS300 instrument (Malvern Panalytical) with a 488-nm laser and sCMOS camera module (Malvern Panalytical). Measurements in flow mode were performed with a flow rate of 50, these flow measurements consisted of 3 measurements of 60 seconds, and the captured data were analysed using NTA 3.2 software.

| Western blot analysis
The concentration of proteins was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Proteins were separated by 12% SDS-PAGE gels and transferred to PVDF membranes by gel electrophoresis and electroblotting, respectively. After blocking with 5% BSA, blots were probed with primary antibodies at 4°C overnight. Then, membranes were washed and incubated with second antibodies. Ultimately, proteins were visualized using the enhanced chemiluminescence reagents (Thermo Fisher Scientific). The antibodies we used are listed in Table S2. The relative protein expression levels were analysed by densitometry using the ImageJ imaging analysis software (NIH).

| ELISA
Concentration of S100-A9 protein in 100 μL follicular fluid, as well as in exosomes derived from 100 μL follicular fluid (FF-Exos), was measured by commercial ELISA kits (Boster Biological Technology) according to the manufacturer's instruction. In order to prepare exosome lysis solution, 25 µL ExoQuick Exosome Precipitation Solution was added to the 100 μL follicular fluid, mixed well and incubated overnight at 4°C. Then, the mixture was centrifuged at 1500 g for 30 minutes at 4°C. The exosome pellet was resuspended in 100 µL PBS, repeated freeze-thaw cycles three times to gain exosome lysis solution.

| Construction of S100-A9-enriched exosomes
The expression vector S100-A9 (pBABE-puro-S100-A9) and its con- and culture expansion, the stable cell clones overexpressing S100-A9 were attained. And the infected cells with an empty pBABE-puro vector were used as control. Quantitative RT-PCR was used to detected S100-A9 expression level in constructed 293T cells. Western blot was used to analyse the S100-A9 protein level in constructed 293T cells and their exosomes. To explore the potential effects of S100-A9 protein in exosomes, KGN cells were incubated with 10 µg/mL S100A9 protein (Sino Biological) or 100 µg/mL exosomes secreted by 293T cell which enriched S100-A9. To inhibit the NF-κB pathway, the cells were pre-treated for 1 hour with BAY-117082 (NF-κB inhibitor, 10 µM; Beyotime).

| Exosome uptake assay
For exosome uptake analysis, exosomes isolated from engineered for 24 hours at 37°C, 5% CO 2 . After incubation, the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature.
Nuclei were stained with DAPI. The signals were examined by confocal microscopy (TCS SP8; Leica).

| Cell proliferation assay
To assess the effect of exosomal protein S100-A9 on cell proliferation, about 3000 KGN cells/well were seeded in a 96-well plate with complete DMEM/F12. After 24-hour attachment, the cells were starved for 12 hours and then treated with different dose of exosomes which overexpressed S100-A9. After 48-hour incubation, cell proliferation was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (Promega) according to the manufacturer's instructions.

| Luminex array assay of cytokine expression
KGN cells were seeded in 6-well plates at 1 × 10 5 per well, and after 24-hour attachment, the cells were starved for 12 hours and then treated with 100 µg/mL exo-S100-A9. After 24-hour incubation, the culture medium was centrifuged at 3000 g for 5 minutes. The

| Statistical analyses
Data were expressed as the mean ± standard error of the mean (SEM) of at least three independent times. All statistics were performed with SigmaStat, v.3.5 (Jandel Co.). Statistical significance was analysed by unpaired Student's t tests or one-way ANOVA. A P-value < .05 was considered statistically significant. were consistent with previously reported characteristics of exosomes. 26,36 TEM image of microvesicles (MVs) from FF (particles >100 nm) as negative control is shown in Figure S2A.

| Proteomic analysis of differentially expressed exosomal proteins
Proteomic analysis using TMT technology was used to detect the proteomic profiles in exosomes from normal and PCOS patients.
The proteins which were detectable in at least one samples were included as quantifiable proteins in the analysis. In total, 662 proteins were identified, among which 546 proteins were quantified in control group, 547 proteins were quantified in PCOS. The quantifiable protein expression profiles of two groups were essentially the same.
Differentially expressed proteins were identified with a cut-off of absolute fold change ≥1.2 and P-value < .05. The results showed that 86 proteins were differentially expressed between PCOS and normal control (Figure 2A). Among them, 27 exosomal proteins were significantly higher and 59 proteins were lower in PCOS patients than controls. Most differentially expressed proteins are listed in Table 2.
To select the proteins involved in PCOS disease progression, we  down-regulated, such as neuropilin-1 and dopamine beta-hydroxy- Our results confirmed the presence of these proteins in exosomes derived from follicular fluid (FF-Exos) of PCOS patients, whereas low presence was detected in healthy controls ( Figure 3A).

| S100-A9 expression level in follicular fluid and FF-Exos
S100-A9 is an activator of the NF-κB pathway, and its function is associated with inflammation. 37 We measured the expression level of S100-A9 in follicular fluid and FF-Exos lysis solution by ELISA.
As shown in Figure 3B, concentration of S100-A9 was significantly higher in FF-Exos of PCOS patients compared with controls, whereas no significant difference was found in the supernatant of follicular fluid between PCOS and control group.
F I G U R E 3 Validation of expression of several differentially expressed exosomal proteins. A, The representative image of expression of up-regulated exosomal proteins S100-A9, peroxiredoxin 6, APMAP and angiotensinogen by Western blot in exosomes from normal and PCOS follicular fluid (n = 6 per group). B, Concentration of S100-A9 in follicular fluid supernatant (100 µL) and exosome lysis solution (exosomes were isolated from 100 µL FF, repeated freeze-thaw cycles three times to gain lysis solution) measured by ELISA (n = 18 per group), *P < .05. All data were presented as means ± SEM. Exo-lysis, exosome lysis solution; FF, follicular fluid; NC, normal control F I G U R E 4 Construction of S100-A9-enriched exosomes. A, S100-A9 mRNA expression level in 293T cells which stably transfected with empty pBABE-puro vector (named 293T-pBABE) or pBABE-puro-S100-A9 vector (named 293T-S100-A9). ***P < .001. B, Western blot analysis of S100-A9 expression level in 293T whole cell lysate (WCL) and exosomes from 293T cells culture supernatants. C, The detection of constructed exosome uptake by KGNs in vitro. KGNs that had been incubated for 24 h with CM-Dil labelled exosomes are shown (CM-Dil in red, PKH67 in green, DAPI in blue). Scale bar, 25 µm. D, Cell viability when treated with varying concentrations of control or S100-A9enriched exosomes (0, 25, 50, 100 and 150 µg/mL) for 48 h. All values were presented as the means ± SEM of three pairs of independent experiments performed 3.4 | Expression levels of S100-A9 receptors S100-A9 can bind to the receptor of advanced glycation end products (RAGE), 38 Toll-like receptor 4 (TLR4) 38 as well as extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), 39 which might play roles in mediating inflammatory effects of S100-A9. Our western blot results showed S100-A9 receptors (TLR4, EMMPRIN, RAGE) were expressed in granulosa cells of normal control and PCOS women and KGN cell line ( Figure S2B).
Western blot analysis showed S100-A9 protein expression level was significantly increased in 293T whole cell lysate and 293T cell-secreted exosomes ( Figure 4B). Since appropriate function of granulosa cells is critical for normal follicular development, we selected the most widely used granulosa-like tumour cell lines, KGN cells, to explore the effect of S100-A9 on its functions. The exosome uptake experiment confirmed this kind of exosome could be uptook by KGNs ( Figure 4C). Cell viability assay indicated that control exosome (exo-pBABE) and S100-A9-enriched exosome (exo-S100-A9) did not significantly impact cell viability ( Figure 4D).

| S100-A9 affects expression of genes associated with steroidogenesis
CYP19A1 and CYP17 are key enzymes in androgen metabolic pathways. Increased activity of CYP17 has been hypothesized to enhance androgen biosynthesis and secretion in PCOS. 41 To investigate the role of S100-A9 in steroidogenesis, the expression levels of CYP17 and CYP19A1 mRNA in KGNs were determined by qRT-PCR.
The effect of S100-A9 on expression of these genes was suppressed by NF-κB pathway inhibitor. These results suggested S100-A9 disturbed several genes expression and hormone signalling mainly through a NF-κB pathway-dependent manner. To identify the role of exosomal proteins during PCOS progression, we focused on the S100-A9 protein, which was up-regulated in exosomes of PCOS patients. S100-A9 belongs to a family of 25 homologous low-molecular-weight intracellular calcium-binding proteins. 49 It is well known that S100-A8/S100-A9 are mainly released by activated granulocytes, can trigger signalling pathways involved in inflammation and play important roles in a number of cellular processes, such as cell cycle progression, cell survival, proliferation and migration. 50 S100-A9 can exist as a homodimer, with its own functions. 51,52 Meanwhile, we did not observe differential expression of S100-A8 between the two groups in our proteomic profile results. Our data showed the S100-A9 concentration was significantly higher in FF-Exos of PCOS patients compared with normal control, but in supernatant of follicular fluid with no significant difference, which probably suggested that S100-A9 might exercise its function through exosomes in follicles during PCOS progression. The S100-A9-enriched exosomes might be secreted by granulosa cells, ovarian inflammatory cells, peripheral leucocytes or other cell types during PCOS process, affect the crosstalk between granulosa cells and local/ distant environment.

| D ISCUSS I ON
Indeed, we observed that S100-A9-enriched exosomes could significantly up-regulate expression of several pro-inflammatory factors and chemokines in KGN cell lines, as well as promote NF-κB pathway activation. Importantly, the specific inhibitor of NF-κB pathway, BAY-117082, significantly blocked S100-A9-induced inflammation. Our results were consistent with previous study which found S100-A9 protein in exosomes from chronic lymphocytic leukaemia cells promotes NF-kB activity. 53 We also found exosomeenriched S100-A9 disturbed steroidogenesis by deregulation of CYP17 mRNA expression via NF-κB pathway-dependent manner.
Previous studies reported S100-A9 protein could induce specific cytokine secretion, such as IL-1, IL-6 and IL-8, which in turn enhance the expression of S100-A9. 51,54-56 So we supposed S100-A9 and inflammatory cytokines might also form part of a feedback loop in follicles. Our results also confirmed S100-A9 mRNA expression was up-regulated after exosomal S100-A9 treatment ( Figure 6B).
This positive feedback loop can promote a persistent inflammatory response and cause an adverse effect on reproductive functions or other organ systems.
We also used exosomes from follicular fluid of normal and PCOS patients to treat KGN cells. We found TNF-a, IL-1 and IL-8 mRNA expression level was increased in PCOS-treated group ( Figure S2C).
But we did not observe significant activation (a little increase trend) in NF-κB pathway ( Figure S2D), which probably attribute to lower concentration of S100-A9 in mixed protein cargo of follicular fluid exosome when compared with engineered S100-A9-enriched exosome. That's why we chose engineered exosomes to study the functional role of S100-A9. As too many proteins mixed in the follicular fluid, the inflammation effect might be caused by many proteins and different pathways. Our results found exosomes might be one of the mechanisms of inflammatory process regulation. Future studies about specific exosomal or non-exosomal proteins and their roles in PCOS are needed to further investigate.
In conclusions, we reported the different proteomic profiles of follicular fluid exosomes among healthy and PCOS patients, some of which are important in reproductive signalling pathways. We found for the first time that S100-A9 protein in exosomes could activate NF-κB signalling pathways in granulosa cells, increase the production of inflammatory cytokines and disturb steroidogenesis.
Our findings suggest the importance of exosomes as extracellular mediators in the pathophysiology of PCOS. Furthermore, this study contributes to the better understanding of exosomal proteins as potential therapeutic target.

ACK N OWLED G EM ENTS
This work was supported by National Key Research and Development Program of China (2017YFA01046003; to K. W.) and the grant from the National Natural Science Foundation of China (81873832; to Y. J.).

CO N FLI C T O F I NTE R E S T
All authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Kai Wang and Yazhong Ji contributed to conception and design. University. Written informed consent to participate was obtained from all participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.