Modulation of dysbiotic vaginal complications by cervical mucus revealed in linked human vagina and cervix chips

Modulation of dysbiotic vaginal complications by cervical mucus revealed in linked human vagina and cervix chips | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Modulation of dysbiotic vaginal complications by cervical mucus revealed in linked human vagina and cervix chips Ola Gutzeit, Aakanksha GULATI, Zohreh IZADIFAR, Anna STEJSKALOVA, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3898191/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background The cervicovaginal mucus which coats the upper surface of the vaginal epithelium is thought to serve as a selective barrier that helps to clear pathogens, however, its role in modulating the physiology and pathophysiology of the human vagina is poorly understood. Bacterial vaginosis (BV), a common disease of the female reproductive tract that increases susceptibility to sexually transmitted infections, pelvic inflammatory disease, infertility, preterm birth, and both maternal and neonatal infections is characterized by the presence of a wide array of strict and facultative anaerobes, often including Gardnerella vaginalis . Objective To assess the role of cervical mucus in preventing dysbiosis-associated complications and preserving vaginal health. Study Design: To better understand the role of cervicovaginal mucus in vaginal health, we used human organ-on-a-chip (Organ Chip) microfluidic culture technology to analyze the effects of cervical mucus produced in a human Cervix Chip and then transferred to a human Vagina Chip BV model. Both chips are lined by primary human organ-specific (cervical or vaginal) epithelium interfaced with organ-specific stromal fibroblasts. Results Our data show that mucus-containing effluents from Cervix Chips protect Vagina Chips from inflammation and epithelial cell injury caused by co-culture with a dysbiotic microbiome containing G. vaginalis . Proteomic analysis of proteins produced by the Vagina Chip following treatment with the Cervix Chip mucus also revealed a collection of differentially abundant proteins that may contribute to the vaginal response to a dysbiotic microbiome, which could represent potential diagnostic biomarkers or therapeutic targets for the management of BV. Conclusions This study highlights the importance of cervical mucus in controlling human vaginal physiology and pathophysiology, and demonstrates the potential value of Organ Chip technology for studies focused on the health and diseases of the female reproductive tract. Biological sciences/Physiology Biological sciences/Physiology/Reproductive biology cervical mucus vaginal microbiota dysbiosis organ-on-a-chip bacterial vaginosis inflammation diagnostic biomarkers therapeutic targets Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The cervicovaginal fluid that covers the surface of the vaginal epithelium is essential to women's health and reproductive functions because it serves as a selective barrier that protects against environmental pathogens. 1 , 2 This fluid contains mucus that is mainly produced by the cervical epithelium along with vaginal secretions, and it contains a range of cytokines, chemokines, immunoglobulins, and other immune mediators that help to prevent pathogens from crossing this critical mucosal interface. However, the role of cervical mucus in regulating vaginal microbiome composition and its effects on health outcomes has largely remained unexplored. 3 This is important because dysbiotic changes in the composition of the female genital tract microbiome, as observed for example in patients with bacterial vaginosis (BV) whose microbiome often contains strict and facultative anaerobes including Garderenella vaginalis 4 , 5 have been linked to increased susceptibility to sexually transmitted infections, pelvic inflammatory disease, infertility, preterm birth, and maternal and neonatal infections. 6 , 7 BV is also the most prevalent cause of vaginal symptoms in women with a more than 50% recurrence rate, yet the underlying factors contributing to these conditions remain elusive. 8 In this study, we leveraged human organ-on-a-chip (Organ Chip) microfluidic culture technology to directly explore how cervical mucus secretions influence human vaginal epithelium under both healthy and dysbiotic conditions. To acheive this, we connected two recently described human Organ Chip models of female reproductive organs. The first is a human vagina-on-a-chip (Vagina Chip) that is lined by primary, hormone-sensitive, vaginal epithelium interfaced with underlying stromal fibroblasts that we have shown recapitulates the pathophysiology of a dysbiotic vaginal epithelium when co-cultured with a G. vaginalis -containing microbiome and that enables analysis of human host-microbiome interactions in vitro . 9 The second is a human cervix-on-a-chip (Cervix Chip) lined by primary cervical epithelium interfaced with cervical fibroblasts 10 that produces abundant cervical mucus with compositional, biophysical, and hormone-responsive properties similar to those observed in vivo . To model and study the effect of cervical mucus on vaginal responses in vitro , we co-cultured a dysbiotic microbiome in the human Vagina Chip in the presence or absence of mucus-containing effluents that were transferred from the epithelial channel of human Cervix Chips. These studies revealed that human cervical epithelial secretions exert immunomodulatory effects and protect the vaginal epithelium against a dysbiotic microbiome by reducing innate inflammatory responses and inhibiting the growth of G. vaginalis bacteria, thereby reducing vaginal cell injury. MATERIALS AND METHODS Human Vagina Chip culture The Human Vagina Chip was cultured as previously described (36434666). Breifly, microfluidic two-channel co-culture Organ Chip devices (CHIP-S1TM) were obtained from Emulate Inc. (Boston, MA). The Polydimethylsiloxane )PDMS( membrane was coated with collagen IV (30 µg/mL) (Sigma, cat. no. C7521) and collagen I (200 µg/mL) (Corning, cat. no. 354236) in Dulbecco's Modified Eagle Medium )DMEM, ThermoFisher, cat. no. 12320-032) in the apical channel. The basal channel was coated with collagen I (200 µg/mL) (Corning, USA) and poly-L-lysine (15 µg/mL) (ScienCell, Cat# 0403(. Primary human uterine fibroblasts (ScienCell Research Laboratories, cat. no. 7040) were then seeded at a density of 1 × 10 6 cells/mL in the basal channel and human vaginal epithelial cells (Lifeline Cell Technology, cat. no. FC-0083; donors 05328) were seeded at a density of 3 × 10 6 cells/mL in the apical channel. Chips were incubated at 37°C with 5% CO2 under static conditions until the cells formed a uniform monolayer. Chips were then connected to the culture module instrument (ZOË™ CULTURE MODULE, Emulate Inc., USA) and placed under flow conditions. Vagina Chips were cultured using a periodic flow regimen in which vaginal epithelium growth medium (Lifeline, Cat# LL-0068 ) was flowed through the apical channel for 4 h/day at 15 µL/h. The basal channel was flowed continuously with fibroblast growth medium (ScienCell, Cat# 2301) at 30 µL/h. After 5–6 days, the basal medium was replaced with an in-house differentiation medium 9 for eight days following the same intermittent and continuous perfusion regime in the apical and basal channels, respectively. The apical medium was replaced with customized HBSS Low Buffer/+Glucose (HBSS (LB/+G)) and the basal medium was replaced with antibiotic-free differentiation medium for one day followed by three days of microbial co-culture as described below. Human Cervix Chip culture Human Cervix Chips were cultured as previously described (Izadifar et al., 2023, BioRxiv). Breifly, microfluidic two-channel co-culture Organ Chip devices (CHIP-S1™) were obtained from Emulate Inc. (Boston, MA). The PDMS membrane was coated with 500 µg/mL collagen IV (Sigma-Aldrich, Cat. no. C7521) in the apical channel and with 200 µg/mL Collagen I (Advanced BioMatrix, Cat. no. 5005) and 30 µg/mL fibronectin (Corning, Cat. no. 356008) in the basal channel. Primary cervical fibroblasts (0.65 x10 6 cells/mL, P5)(isolated from hysterectomy cervical tissues) were seeded on the basal side followed by seeding primary cervical epithelial cells (1.5 x 10 6 cells/mL, P5) (LifeLine Cell Technology Cat# FC-0080) on the apical side. The respective chip media were refreshed after seedingfor each channel and the chips were incubated at 37°C, 5% CO2 under static conditions overnight. Chips were then connected to the culture module instrument (ZOË™ CULTURE MODULE, Emulate Inc.,USA). Cervix Chips were cultured using a periodic flow regimen in which cervical growth medium was flowed through the apical channel for four hours per day at 30 µL/h while fibroblast growth medium was continuously perfused basally at 40 µL/h. After five days the apical medium was replaced by Hank’s Buffer Saline Solution (HBSS) (Thermo Fisher, 14025076) while being fed through the basal channel by differentiation medium consisting of cervical epithelial medium (LifeLine Cell Technology, Cat. no. LL-0072) supplemented with 5 nM estradiol-17β (E2) (Sigma, Cat. no. E2257) and 50 µg/mL ascorbic acid (ATCC, Cat. no. PCS-201-040). On day 2 of differentiation the apical medium was replaced by HBSS with low buffering salts and no glucose (HBSS (LB/-G) at pH ~ 5.4 and cultured for five additional days. Mucus collection from Cervix Chips Cervix Chip mucus was collected every day starting at day 4 of differentiation for 10 days. During the collection period, the basal channel was continuously perfused with antibiotic- free differentiation medium at a volumetric flow rate of 40 µL/h. The apical channel was perfused with HBSS (LB/-G) for 4 h/day at 40 µL/h and the chip effluents were collected and stored at -80°C until the end of the experiment. Before introduction to the Vagina Chip, 5.56 mM D-glucose (Sigma, cat. no. G7021) was added to the Cervix Chip mucus. Culture of a non-optimal Gardnerella vaginalis- containing consortium in Vagina Chips In non-optimal vaginal microbiota, Gardnerella species are typically found as dominant bacteria 7 accompanied by other frequent taxa such as Prevotella species and Atopobium species. 11 To mimic the ecology of non-optimal vaginal microbiota, we used a synthetic dysbiotic consortia (BVC1: Gardnerella vaginalis E2, Gardnerella vaginalis E4, Prevotella bivia BHK8, and Atopobium vaginae ). The Gardnerella isolates used in this study were selected because they represent distinct genomic groups, exhibit phenotypic diversity in vitro , and were co-resident, meaning that they were co-isolated from a single participant in the UMB-HMP study. 12 P. bivia and A. vaginae are prevalent species in Lactobacillus -deficient vaginal microbiota. The two strains used in this study were co-resident, isolated from a single participant in the Females Rising Through Education Support and Health study. 13 The apical channel of each Vagina Chip was inoculated with ~ 10 5 CFU of prepared BVC1 consortia and then chips were incubated statically at 37°C and 5% CO2 for 20 h before starting flow using the Zöe culture module. The basal channel was continuously perfused with in-house antibiotic-free differentiation medium and the apical channel was perfused for 4 h/day with customized HBSS (LB/+G) medium at a volumetric flow rate of 40 µL/h. Study design The study was carried out under five conditions: control, BVC1, pre-treatment, post-treatment and pre + post treatment. Vaginal epithelium cultured on-chip for 72 h in the absence (Control( or presence of BVC1 consortium, in the absence or presence of cervical mucus. In the Mucus Pre-treatment group, cervical mucus was used as the apical medium for 24 h before BVC1 infection, followed by the use of customized HBSS Low Buffer/+Glucose (HBSS (LB/+G)) as the apical medium. In the Mucus Post-Treatment group, cervical mucus was used as the apical medium for the duration of the experiment, beginning 24 h after BVC1 infection. In the Mucus pre + post treatment group, cervical mucus was used as the apical medium for 24 h prior to BVC1 infection, and for 72 h during BVC1 co-culture. Bacterial enumeration from Vagina Chip co-culture To enumerate all cultivable bacteria in the effluents, effluent samples (50 µL) were collected at 24, 48, and 72 h. Effluent samples from Vagina Chips containing BVC1 consortia were plated on Brucella blood agar (with hemin and vitamin K1) (Hardy, cat. no. A30) at 37°C under completely anaerobic conditions. After 48 h incubation, CFU/chip was calculated for each sample. To enumerate all cultivable bacteria engrafted in the Vagina Chip, the whole epithelial cell layer was digested for 1 h with 1 mg/mL of collagenase IV (Gibco, cat. no. 17104019) in TrypLE (ThermoFisher, cat. no. 12605010). Cell layer digests were diluted and processed in the same way as effluent samples and CFU/chip was calculated for each chip digest. Analysis of cytokines and chemokines Samples (100 µL) of apical effluents from Vagina Chips were collected and analyzed for a panel of cytokines and chemokines, including TNF-α, IFN-y, IL-1α, IL-1β, IL-10, IL-8, IL-6, MIP-1α, MIP-1β, IP-10, and RANTES using custom ProcartaPlex assay kits (ThermoFisher Scientific). Analyte concentrations were determined using a Luminex 100/200 Flexmap3D instrument coupled with Luminex XPONENT software. Protein extraction and mass spectrometry Sample digestion: Samples were run through a 50 kDa filter (Amicron Ultracel, Merck Millipore, Ireland) and digested according to the manafacturer’s protocol for 1 h at 50 °C by Trypsin Platinum (Promega, WV), and digested material was dried in a Speedvac (Eppendorf, Germany). Mass spectrometry: Each sample was resolubilized in 10 µL of 0.1% formic acid. Single LC-MS/MS was performed on a 240 Exploris Orbitrap (ThermoScientific, Germany) equipped with a NEO nano-HPLC pump (ThermoScientific, Germany). Peptides were separated onto a micropac 5 cm trapping column (Thermo, Belgium) followed by a 50 cm micropac analytical column (ThermoScientific, Belgium). Separation was achieved by applying a 5–24% ACN gradient in 0.1% formic acid over 90 min at 250 nL min − 1. Electrospray ionization was achieved at 1.8 kV with an electrode junction (PepSep, Denmark) at the end of a microcapillary column with a stainless-steel 4 cm needle (ThermoScientific, Denmark). The Exploris Orbitrap was operated in data-dependent mode, and the mass spectrometry survey scan was performed at 450 − 1,200 m/z and a resolution of 1.2 × 10 5 , followed by selection of the ten most intense ions (TOP10) for HCD-MS2 fragmentation. FFor each HCD MS2 scan, the fragment ion isolation width was 0.8 m/z, AGC was 50,000, maximum ion time was 150 ms, normalized collision energy was 32V and an activation time of 1 ms. Data analysis: Raw data were submitted for analysis in Proteome Discoverer 3.0 (Thermo Scientific, CA) software. Assignment of MS/MS spectra was performed using the Sequest HT algorithm by searching the data against a protein sequence database including all entries from the Human Uniprot database (SwissProt, 2019) and full Uniprot bacteria database (SwissProt, 2022) as well as known contaminants such as human keratins and common lab contaminants. Sequest HT searches were performed using a 15 ppm precursor ion tolerance requiring each peptide N-/C terminiusto adhere with Trypsin protease specificity, while allowing up to two missed cleavages. For searches, methionine oxidation (+ 15.99492 Da) and asparagine and glutamine deamidations (+ 0.984016 Da) were set as variable modifications as well as N-terminal acetylation of protein termini. A MS2 spectra assignment false discovery rate (FDR) of 1% on the protein level was achieved by applying the target-decoy database search. Filtering was performed using a Percolator (64bit version). 14 For quantification analysis between samples, label-free quantitation mode using Minora detection features of the Proteome Discoverer platform was used. Statistical analysis All results presented are from at least two independent experiments and all data points shown indicate the mean ± standard deviation (s.d.) from n ≥ 3 Organ Chips unless otherwise mentioned. Statistically significant differences between groups were determined using unpaired t-test, statistical analyses were performed using GraphPad Prism 9.0.2. RESULTS Modulation of innate immunity in Vagina Chips by mucus-containing effluents from Cervix Chips We recently described a human Vagina Chip lined by primary human vaginal epithelium interfaced across an extracellular matrix (ECM)-coated porous membrane with underlying stromal fibroblasts that enables analysis of human host-microbiome interactions in the vaginal microenvironment, 9 as well as a human Cervix Chip containing primary cervical epithelium interfaced with stromal cervical fibroblasts that produces cervical mucus with physical and chemical properties similar to those observed in vivo . 10 Here, we collected mucus-containing effluents from the epithelial channel of the Cervix Chip ('cervical chip mucus' containing 4.01 ± 3.04 mg/mL of mucus glycoproteins) for 7 days and then perfused it through the epithelial channel of a Vagina Chip to simulate the natural flow of mucus in the reproductive tract in vivo ( Fig. 1A ). Presence of this mucus in the Vagina Chip induced statistically significant decreases in secretion of multiple relevant proinflammatory cytokines, including interleukin-1α (IL-1α), IL-1β, and macrophage inflammatory protein-1β (MIP-1β), accompanied by a concomitant increase in anti-inflammatory IL-10 protein production after 24 h of exposure compared to control chips without mucus ( Fig. 1B ). These results demonstrate that the mucus-containing fluids produced by human cervical epithelium in Cervix Chips in vitro can directly influence the vaginal epithelium and result in suppression of inflammatory cytokine production, even in the absence of immune cells. Modulation of the dysbiotic vaginal microbiome by introducing cervical mucus into the Vagina Chip We next studied the effects of cervical mucus on a dysbiotic (non-optimal) vaginal microbiome in Vagina Chips by inoculating them with a consortium containing G. vaginalis E2 and E4 combined with P. bivia BHK8 and A. vaginae (BVC1; ~10 5 CFU/chip) on day 14 of Vagina Chip culture in the presence or absence of mucus-containing effluents from the Cervix Chip. Interestingly, the presence of human cervical mucus inhibited the consortium's ability to colonize the epithelium and thrive on the Vagina Chip. The total number of CFU of live non-adherent bacteria collected in effluents from the epithelial channel during 72 h of infection ( Fig. 2A ), as well as the number of live adherent bacteria in tissue digests at the end of the 72 h culture (Fig. 2B), were significantly reduced whether Vagina Chips were pretreated with mucus effluents for 1 day before microbiome introduction, 1 day after BVC 1 addition, or continuously for the entire 3-day culture starting 1 day before the addition of bacteria. Consistent with these data, when we quantified vaginal epithelial cells with a Clue Cell-like appearance (i.e., covered with bound bacteria) 15 in digests of Vagina Chip epithelium with the dysbiotic BVC1 consortium, we observed a decrease in the number of these cells in the presence of cervical mucus ( Fig. 2C,D ). Not surprisingly, this reduction in bacterial cell number induced by the presence of cervical mucus was also accompanied by a concomitant increase in vaginal epithelial cell viability (retained cell number) ( Fig. 2E ), as well as significant downregulation of the proinflammatory cytokines, IL-8, IL-10, Rantes (CCL5), TNF-α, MIP-1β, and IL-1α after 72 h of co-culture ( Fig. 3 ). These results demonstrate that Cervix Chip mucus can directly influence the Vaginal Chip epithelium to dampen production of inflammatory cytokines and this correlates with protection of the vaginal epithelium against injury. Suppression of G. vaginalis growth in Vagina Chip effluents To explore whether cervical mucus acts directly to suppress bacterial cell growth or indirectly by altering vaginal cell physiology, we next compared the growth of G. vaginalis in mucus-containing effluent samples collected from the epithelial channel of control Cervix Chips (perfused with HBSS) versus effluents from Vagina Chips that were perfused with Cervix Chip-derived mucus-containing effluent for 1 day, with the bacteria cultured directly in HBSS that was used to perfuse the apical channels of our Cervix and Vagina chips as a control. Our results demonstrate that G. vaginalis grew well in the Cervix Chip mucus in 2D culture, but growth was suppressed when cultured in effluents from the Vagina Chip perfused with similar Cervix Chip-derived mucus-containing effluent or in HBSS that lacks critical nutrients ( Fig. 4A,B ). Importantly, when similar studies were carried out after the addition of 50% bacterial broth to provide optimal nutrient conditions, bacterial growth was restored in the control HBSS sample, but not in the sample from the Vagina Chip exposed to Cervix Chip-derived mucus effluent ( Fig. 4C, D ). These findings suggest that mucus components produced by the Cervix Chip induce the cells lining the Vagina Chip to express factors that suppress G. vaginalis growth. Cervix Chip mucus alters the vaginal secretome To further explore the effects of cervical mucus on the vaginal epithelium, we conducted mass spectrometry analysis to compare the proteome composition of the Cervix Chip effluent before and after exposure to the Vagina Chip versus the untreated Vagina Chip effluent. Of the 1752 proteins identified ( Supplementary Table 1 ), 103 were found to be differentially abundant as determined by fold change (|log2 fc| >= 1), p adj ≤ 0.05) in Cervix Chip effluents that had passed through the Vagina Chip versus either the Cervix Chip effluent or Vagina Chip effluent alone. Significant changes in the expression of multiple proteins were observed, with 64 proteins showing increased expression ( Fig. 5A ) and 39 proteins showing decreased expression ( Fig. 5B ), with the most prominent alterations highlighted in a volcano plot ( Fig. 5C ). PCA analysis of the proteomics data also revealed distinct segregation among these sample groups, indicating notable changes in protein expression in effluents from Vagina Chips exposed to Cervix Chip mucus compared to those from untreated Cervix or Vagina Chips alone ( Fig. 5D ). Interestingly, using STRING analysis, which incorporates both physical protein-protein interactions and functional associations from various sources (e.g., automated text mining, computational interaction predictions from co-expression, conserved genomic context, databases of interaction experiments, and curated sources of known complexes/pathways), 16 we found that 3 of the 37 down-regulated proteins exhibit calcium channel inhibitor activity (PHPT1, AMBP, SLC30A1). Previous research has shown that G. vaginalis strongly induces epithelial calcium influx and contraction. 17 In addition, 6 of the down-regulated proteins are ECM molecules (LGALS3BP, GPC1, AMBP, SERPING1, VASN, FBLN1, FBLN2), which may play a role in G. vaginalis adhesion and biofilm formation. 18 Finally, 3 down-regulated proteins are members of the Lipocalin family (AMBP, APOD, RBP4), which is known for its role in regulating inflammation and antioxidant responses. 19 The STRING analysis additionally showed that 17 of the up-regulated proteins are RNA binding proteins (RBPs). Previous studies have highlighted the vital role of RBPs in bacterial replication by binding to and regulating their RNAs. 20 These proteins also play a crucial role in immune system response to viral infections by regulating viral RNA stability and translation. 21 Considering that BV increases susceptibility to sexually transmitted infections, including viral infections, these findings further support the potential involvement of RBPs in the immune response within the reproductive tract. Twenty-five proteins related to the male reproductive system were also found to be upregulated. This finding is significant because past studies have established a notable link between BV and infertility. 6 For instance, one of the proteins identified, CSTF2T, has the potential to contribute to sperm adhesion to the zona pellucida 22 while the also identified TMED10 protein may be involved in in sperm capacitation and the acrosome reaction. 23 Importantly, exposure of the Vagina Chip to cervical mucus also resulted in enhanced production of potential antimicrobial proteins PLAU and WASF2. PLAU is a serine protease with immunomodulatory functions 24 and WASF2 is a member of the Wiskott-Aldrich syndrome protein family that regulates autophagy and inflammasome activity. 25 One of the prominent down-regulated proteins, GNS, is an N-acetylglucosamine-6-sulfatase. This is interesting because BV is often associated with the breakdown of mucins, which are necessary for these dysbiotic bacteria to colonize the vagina. 26 Thus, downregulation of GNS could contribute to the inhibition of dysbiotic bacterial growth we observed by increasing glycoprotein sulfation and thereby prevent mucin degradation. Potential role of exosomes as mediators of the effects of cervical mucus on the Vagina Chip Exosomes, which are small extracellular vesicles containing nucleic acids, lipids, and proteins, play a significant role in intercellular communication in the female reproductive tract by modulating the immune system and promoting tissue repair. 27 This is accomplished by presenting antigenic peptides, regulating gene expression through exosomal miRNA, and inducing differential signaling through exosomal surface ligands. Importantly, when we carried out STRING analysis of the proteins differentially expressed in Vagina Chip effluents exposed to Cervix Chip mucus, we found that a significant proportion of the differentially expressed proteins were associated with exosomes. Specifically, 23 out of 37 down-regulated proteins and 17 out of 64 upregulated proteins were found to be linked to extracellular exosomes ( Supplementary Table 2 ). Notable among the upregulated proteins were DDR1 28 and COMP, 29 which regulate cellular adhesion to the ECM and its remodeling, subsequently influencing bacterial adhesion. 30 Conversely, among the down-regulated proteins, 5 ECM proteins (AMBP, FBLN1, GPC1, LGALS3BP, and SERPING1) were identified, which may also influence bacterial adhesion. Interestingly, SERPING1 functions as a regulator of the complement system 31 , and three of the down-regulated proteins (AMBP, SERPING1, and SPINT1) belong to the Kunitz family of serine protease inhibitors that are involved in coordinating inflammation. 32 Cervicovaginal antimicrobial peptides Additionally, we identified 12 antimicrobial peptides in the Cervix Chip and Vagina Chip effluents ( Table 1 ). Of these, 6 (Dermcidin, Ubiquicidin, Chemerin, Acipensin 6, hSAA1, and Psoriasin) were present in both Vagina and Cervix Chip effluents, 1 was solely produced by the Vagina Chip (KAMP-19), and 5 were exclusively produced by the Cervix Chip. No antimicrobial peptides were specifically induced in Vagina Chips exposed to Cervix Chip effluents. The Cervix Chip-derived antimicrobial peptides include Histone H4, Histone H3, CXCL1, BHP, and Chromacin. Histones and their fragments have a variety of antimicrobial actions and functions, including bacterial cell membrane permeabilization, penetration into the membrane followed by binding to bacterial DNA and/or RNA, binding to bacterial lipopolysaccharide (LPS) and neutralizing its toxicity, and entrapping pathogens as a component of neutrophil extracellular traps. 33 , 34 It is noteworthy that P. bivia , which is included in our bacterial consortium, has been shown to produce high concentrations of LPS. 35 CXCL1 also inhibits the growth of E. coli and S. aureus in vitro 36 and BHP impedes growth of M. luteus, S. epidermidis , and several fungi (e.g., C. albicans, S. cerevisiae, and A. nidulans) 37 , while Chromacin suppresses the growth of Bacillus megaterium and Micrococcus luteus . 38 Comment Principal Findings : These data show that mucus-containing effluents from human Cervix Chips suppress the growth of dysbiotic microbiota, associated inflammation, and epithelial cell injury in human Vagina Chips. By analyzing the differentially abundant proteins in the secretome of Vagina Chips following treatment with Cervix Chip effluents, we identified multiple proteins that may contribute to this protective response and that potentially could be used as clinical biomarkers for monitoring female reproductive tract health. Results in the Context of What is Known : Maintaining homeostasis is crucial for the health of epithelial barriers, which can be disrupted during infection or injury. Inflammation plays a vital role in supporting the body's defense against pathogens, promoting tissue healing, and restoring homeostasis. 39 However, chronically high levels of proinflammatory cytokines that undermine normal protective immune signals have been linked to an imbalanced microbiome and compromised epithelial cell stability. 40 This study presents evidence that communication between cervical and vaginal tissues in the lower reproductive tract via transfer of cervical mucus-containing secretions helps to suppress vaginal inflammation in the presence of a dysbiotic microbiome. There is a growing body of clinical evidence suggesting that medical cervical procedures may disrupt this crucial communication between cervical and vaginal epithelium, and lead to changes in composition of the vaginal microbiome. 41 , 42 Our results support this observation and suggest that it is a direct effect of reducing cervical mucus transfer to the vagina, which could only be studied directly using this type of engineered in vitro model. The recurrence of abnormal vaginal flora after treatment of BV (e.g., with metronidazole) is commonly detected in most women, 8 however, the underlying factors contributing to these recurrences remain elusive. Our findings suggest that alterations in cervical mucus levels may influence the susceptibility of the vaginal epithelium to BV infection. Therefore, an imbalance in the cervicovaginal mucus may be a possible contributing factor to the high rate of BV recurrence. In this context, it is important to note that we identified five cervical antimicrobial peptides that appear to play a role in the antimicrobial effects we observed on-chip. These findings suggest that interactions between antimicrobial peptides and the host vaginal epithelium can enhance innate immune protection against dysbiotic flora. Immune effectors and specialized stromal cells at epithelial surfaces produce cytokines and antimicrobial defenses to orchestrate tissue repair and minimize opportunistic infections. Exosomes can act as mediators for this form of inter-tissue communication. We identified 40 exosomal proteins produced by vaginal epithelium that were modulated by exposure to cervical mucus produced in the human Cervix Chip. Human cervicovaginal exosomes have been previously shown to be part of the female innate defense system and to protect against HIV-1 infection 43 as well as bacterial toxins. 44 Exosomes are also currently being explored as potential therapeutic agents and drug delivery vehicles. Thus, the ability to study the role of exosomes in host-microbiome interactions in the female reproductive tract in vitro using the human Organ Chip models described here may facilitate the development of novel treatments for vaginal dysbiosis as well as other diseases of the female reproductive tract. Clinical Implications : Our study has important clinical implications as it has the potential to identify new targets for diagnosis and treatment of vaginal diseases. Identifying patients with a high likelihood of recurrent vaginal dysbiosis can help to customize their treatment plan and prevent complications. In this study, we identified multiple proteins and antimicrobial peptides that may contribute to the protective response against dysbiotic microbiota and associated inflammation and injury to the vaginal epithelium. These proteins and peptides could potentially be used as clinical biomarkers for monitoring the health of the female reproductive tract in the future. Several proteins we identified (e.g., TPM3, PLAU, ALDH3A2, GAS6, DTYMK, SERPING, STAT6, CMPK1) are known to be targeted by existing approved drugs (Progesterone, Urokinase, Disulfiram, Warfarin, Zidovudine, Rhucin, Indomethacin, and Gemcitabine, respectively). Thus, if these molecules actively contribute to the BV disease phenotype, one or more of these therapeutics could be added to current clinical regimens. Research Implications : Our results show the value of human Organ Chip technology for studying vaginal health and diseases of the female reproductive tract. However, further research is needed to evaluate the effects of these compounds as well as modulators of the other putative targets we identified for maintaining vaginal homeostasis and a healthy microbiome. Strengths and Limitations : While the human Vagina and Cervix Chips used in this study replicate many physiological and pathophysiological features of the female reproductive tract, we did not incorporate immune cells. As these cells play a crucial role in mounting antibacterial immune responses, the model would be strengthened by incorporating them in the future. Additionally, it should be noted that the Organ Chips we used were created with epithelial cells from a single human donor and thus, these studies should be extended to include chips lined by cells from multiple donors from different ethnic groups as well. Conclusions This study highlights the crucial role that cervical mucus plays in maintaining vaginal health and preventing dysbiosis-related complications. Our results directly demonstrate that cervical mucus-containing secretions can suppress the growth of dysbiotic microbiota as well as associated inflammation and epithelial cell injury in the human Vagina Chip. We also identified several proteins and antimicrobial peptides that could serve as clinical biomarkers for monitoring the health of the female reproductive tract and potentially be targeted for the treatment of vaginal dysbiosis. This study also sheds light on the potential role of exosomes in inter-tissue communication and immune protection against dysbiotic flora in the female reproductive tract. In addition, these findings could have important clinical implications, particularly for identifying patients with a high risk of recurrent dysbiosis and customizing their treatment plans. Further research is needed to evaluate the effects of modulating the potential molecular mediators we identified on maintaining healthy vaginal microbial homeostasis. However, these findings provide further evidence showing that human Organ Chip models can provide a valuable tool for studying host-microbiome interactions in the female reproductive tract as well as for identifying potential clinical biomarkers and therapeutic targets for patients with vaginal dysbiosis and other related diseases. Declarations ACKNOWLEDGMENTS This research was sponsored by funding from the Bill and Melinda Gates Foundation (OPP1173198 & INV-035977 to D.E.I., OPP1189217 to J.R. and INV-031642 to S.R-N.) and the Wyss Institute for Biologically Inspired Engineering (D.E.I.). Gwenn Merry (Wyss Institute) provided technical editing. AUTHORS CONTRIBUTIONS O.G.: conceptualization, data curation, formal analysis, investigation, writing - original draft. A.G.: investigation, writing - review & editing. Z.I.: conceptualization, writing - review & editing. A.S.: conceptualization. H.R.: investigation.J.C.: investigation. B.B.: investigation, proteomics methodology. S.S.: software. G.G.: supervision, writing - review & editing. A.J.: supervision, writing - review & editing. D.E.I.: conceptualization, supervision, funding acquisition, writing - review & editing. POTENTIAL CONFLICTING INTERESTS D.E.I. is a founder, board member, and chairs the SAB of Emulate Inc., in which he also holds equity. The author O.G, A.G, Z.I, A.S, H.R, J.C, B.B, S.S, G.G and A.J report no conflict of interest. References Lacroix G, Gouyer V, Gottrand F, Desseyn JL. The Cervicovaginal Mucus Barrier. Int J Mol Sci 2020;21. Vagios S, Mitchell CM. Mutual Preservation: A Review of Interactions Between Cervicovaginal Mucus and Microbiota. Front Cell Infect Microbiol 2021;11:676114. McLoughlin K, Schluter J, Rakoff-Nahoum S, Smith AL, Foster KR. Host Selection of Microbiota via Differential Adhesion. Cell Host Microbe 2016;19:550–9. Juliana NCA, Suiters MJM, Al-Nasiry S, Morre SA, Peters RPH, Ambrosino E. The Association Between Vaginal Microbiota Dysbiosis, Bacterial Vaginosis, and Aerobic Vaginitis, and Adverse Pregnancy Outcomes of Women Living in Sub-Saharan Africa: A Systematic Review. Front Public Health 2020;8:567885. Janulaitiene M, Paliulyte V, Grinceviciene S, et al. Prevalence and distribution of Gardnerella vaginalis subgroups in women with and without bacterial vaginosis. BMC Infect Dis 2017;17:394. Ravel J, Moreno I, Simon C. Bacterial vaginosis and its association with infertility, endometritis, and pelvic inflammatory disease. Am J Obstet Gynecol 2021;224:251–57. van de Wijgert J, Jespers V. The global health impact of vaginal dysbiosis. Res Microbiol 2017;168:859–64. Neal CM, Kus LH, Eckert LO, Peipert JF. Noncandidal vaginitis: a comprehensive approach to diagnosis and management. Am J Obstet Gynecol 2020;222:114–22. Mahajan G, Doherty E, To T, et al. Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip. Microbiome 2022;10:201. Izadifar Z, Cotton J, Chen C, et al. Mucus production, host-microbiome interactions, hormone sensitivity, and innate immune responses modeled in human endo- and ecto-cervix chips. Nature Communications, ‘in press’. Earlier preprint version for Reviewers can be found at bioRxiv 2023:2023.02.22.529436. Ravel J, Gajer P, Abdo Z, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 2011;108 Suppl 1:4680–7. Ravel J, Brotman RM, Gajer P, et al. Daily temporal dynamics of vaginal microbiota before, during and after episodes of bacterial vaginosis. Microbiome 2013;1:29. Bloom SM, Mafunda NA, Woolston BM, et al. Cysteine dependence of Lactobacillus iners is a potential therapeutic target for vaginal microbiota modulation. Nat Microbiol 2022;7:434–50. Kall L, Storey JD, Noble WS. Non-parametric estimation of posterior error probabilities associated with peptides identified by tandem mass spectrometry. Bioinformatics 2008;24:i42-8. Gardner HL, Dukes CD. Haemophilus vaginalis vaginitis: a newly defined specific infection previously classified non-specific vaginitis. Am J Obstet Gynecol 1955;69:962–76. Szklarczyk D, Kirsch R, Koutrouli M, et al. The STRING database in 2023: protein-protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res 2023;51:D638-D46. Abbasian B, Shair A, O'Gorman DB, et al. Potential Role of Extracellular ATP Released by Bacteria in Bladder Infection and Contractility. mSphere 2019;4. Hardy L, Cerca N, Jespers V, Vaneechoutte M, Crucitti T. Bacterial biofilms in the vagina. Res Microbiol 2017;168:865–74. Rassart E, Desmarais F, Najyb O, Bergeron KF, Mounier C. Apolipoprotein D. Gene 2020;756:144874. Van Assche E, Van Puyvelde S, Vanderleyden J, Steenackers HP. RNA-binding proteins involved in post-transcriptional regulation in bacteria. Front Microbiol 2015;6:141. Gao Q, Jiang M, Zhao Y, et al. eIF4A3 Promotes RNA Viruses' Replication by Inhibiting Innate Immune Responses. J Virol 2022;96:e0151322. Tardif S, Akrofi AS, Dass B, Hardy DM, MacDonald CC. Infertility with impaired zona pellucida adhesion of spermatozoa from mice lacking TauCstF-64. Biol Reprod 2010;83:464–72. Castillo J, Bogle OA, Jodar M, et al. Proteomic Changes in Human Sperm During Sequential in vitro Capacitation and Acrosome Reaction. Front Cell Dev Biol 2019;7:295. Jin T, Bokarewa M, Tarkowski A. Urokinase-type plasminogen activator, an endogenous antibiotic. J Infect Dis 2005;192:429–37. Lee PP, Lobato-Marquez D, Pramanik N, et al. Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells. Nat Commun 2017;8:1576. Roberton AM, Wiggins R, Horner PJ, et al. A novel bacterial mucinase, glycosulfatase, is associated with bacterial vaginosis. J Clin Microbiol 2005;43:5504–8. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science 2020;367. Duan X, Xu X, Zhang Y, Gao Y, Zhou J, Li J. DDR1 functions as an immune negative factor in colorectal cancer by regulating tumor-infiltrating T cells through IL-18. Cancer Sci 2022;113:3672–85. Posey KL, Coustry F, Hecht JT. Cartilage oligomeric matrix protein: COMPopathies and beyond. Matrix Biol 2018;71–72:161–73. Marrs CN, Knobel SM, Zhu WQ, Sweet SD, Chaudhry AR, Alcendor DJ. Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis. Microbes Infect 2012;14:500–8. Zeerleder S. C1-inhibitor: more than a serine protease inhibitor. Semin Thromb Hemost 2011;37:362–74. Shigetomi H, Onogi A, Kajiwara H, et al. Anti-inflammatory actions of serine protease inhibitors containing the Kunitz domain. Inflamm Res 2010;59:679–87. Wira CR, Patel MV, Ghosh M, Mukura L, Fahey JV. Innate immunity in the human female reproductive tract: endocrine regulation of endogenous antimicrobial protection against HIV and other sexually transmitted infections. Am J Reprod Immunol 2011;65:196–211. Drab T, Kracmerova J, Hanzlikova E, et al. The antimicrobial action of histones in the reproductive tract of cow. Biochem Biophys Res Commun 2014;443:987–90. Aroutcheva A, Ling Z, Faro S. Prevotella bivia as a source of lipopolysaccharide in the vagina. Anaerobe 2008;14:256–60. Yang D, Chen Q, Hoover DM, et al. Many chemokines including CCL20/MIP-3alpha display antimicrobial activity. J Leukoc Biol 2003;74:448–55. Conlon JM, Kolodziejek J, Nowotny N. Antimicrobial peptides from the skins of North American frogs. Biochim Biophys Acta 2009;1788:1556–63. Strub JM, Goumon Y, Lugardon K, et al. Antibacterial activity of glycosylated and phosphorylated chromogranin A-derived peptide 173–194 from bovine adrenal medullary chromaffin granules. J Biol Chem 1996;271:28533–40. Rathinam VAK, Chan FK. Inflammasome, Inflammation, and Tissue Homeostasis. Trends Mol Med 2018;24:304–18. Clavel T, Haller D. Bacteria- and host-derived mechanisms to control intestinal epithelial cell homeostasis: implications for chronic inflammation. Inflamm Bowel Dis 2007;13:1153–64. Kawahara R, Fujii T, Kukimoto I, et al. Changes to the cervicovaginal microbiota and cervical cytokine profile following surgery for cervical intraepithelial neoplasia. Sci Rep 2021;11:2156. Wiik J, Sengpiel V, Kyrgiou M, et al. Cervical microbiota in women with cervical intra-epithelial neoplasia, prior to and after local excisional treatment, a Norwegian cohort study. BMC Womens Health 2019;19:30. Smith JA, Daniel R. Human vaginal fluid contains exosomes that have an inhibitory effect on an early step of the HIV-1 life cycle. AIDS 2016;30:2611–16. Keller MD, Ching KL, Liang FX, et al. Decoy exosomes provide protection against bacterial toxins. Nature 2020;579:260–64. Tables Table 1 is available in the Supplementary Files section. Supplementary Files Supplementary Tables is not available with this version. Additional Declarations Competing interest reported. D.E.I. is a founder, board member, and chairs the SAB of Emulate Inc., in which he also holds equity. The author O.G, A.G, Z.I, A.S, H.R, J.C, B.B, S.S, G.G and A.J report no conflict of interest. Supplementary Files Table1.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 17 Jun, 2024 Reviews received at journal 07 Jun, 2024 Reviews received at journal 02 Jun, 2024 Reviewers agreed at journal 22 May, 2024 Reviewers agreed at journal 26 Apr, 2024 Reviewers agreed at journal 23 Apr, 2024 Reviews received at journal 17 Apr, 2024 Reviewers agreed at journal 03 Mar, 2024 Reviewers agreed at journal 18 Feb, 2024 Reviewers invited by journal 05 Feb, 2024 Editor assigned by journal 31 Jan, 2024 Submission checks completed at journal 29 Jan, 2024 First submitted to journal 25 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3898191","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":269882963,"identity":"b65412d7-9527-4fad-916f-36cecff0849e","order_by":0,"name":"Ola Gutzeit","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ola","middleName":"","lastName":"Gutzeit","suffix":""},{"id":269882964,"identity":"5b8f5167-e516-46bf-b539-291fc9df882c","order_by":1,"name":"Aakanksha GULATI","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aakanksha","middleName":"","lastName":"GULATI","suffix":""},{"id":269882965,"identity":"46de80bf-aff3-4b03-a095-8e624db4dbf6","order_by":2,"name":"Zohreh IZADIFAR","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zohreh","middleName":"","lastName":"IZADIFAR","suffix":""},{"id":269882966,"identity":"9e34d9e7-d49b-4773-ad50-c11be278fb15","order_by":3,"name":"Anna STEJSKALOVA","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"STEJSKALOVA","suffix":""},{"id":269882967,"identity":"4094777e-5ff7-4081-bdfb-8fe7f25b8eb3","order_by":4,"name":"Hassan RHBINY","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hassan","middleName":"","lastName":"RHBINY","suffix":""},{"id":269882968,"identity":"08381300-b678-4b5b-b61a-4f6087b6bfd5","order_by":5,"name":"Justin COTTON","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Justin","middleName":"","lastName":"COTTON","suffix":""},{"id":269882969,"identity":"ab167e6d-4d19-498d-80be-5fca54b1839e","order_by":6,"name":"Bogdan BUDNIK","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bogdan","middleName":"","lastName":"BUDNIK","suffix":""},{"id":269882970,"identity":"74c4f1b4-4917-4782-9908-a9f5f7dcb0d9","order_by":7,"name":"Sanjid SHAHRIAR","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sanjid","middleName":"","lastName":"SHAHRIAR","suffix":""},{"id":269882971,"identity":"cbd88f13-ca67-44fa-a5fa-c017391cab27","order_by":8,"name":"Girija GOYAL","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Girija","middleName":"","lastName":"GOYAL","suffix":""},{"id":269882972,"identity":"82f29486-5af5-4449-b892-80017353f617","order_by":9,"name":"Abidemi JUNAID","email":"","orcid":"","institution":"Harvard University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abidemi","middleName":"","lastName":"JUNAID","suffix":""},{"id":269882973,"identity":"a34ec088-e6df-4968-ad8c-88106100e340","order_by":10,"name":"Donald E. INGBER","email":"data:image/png;base64,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","orcid":"","institution":"Harvard University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Donald","middleName":"E.","lastName":"INGBER","suffix":""}],"badges":[],"createdAt":"2024-01-25 19:44:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3898191/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3898191/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50460537,"identity":"a055d63f-ed78-4a52-862f-32b07ee2141d","added_by":"auto","created_at":"2024-01-31 20:36:07","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of cervical mucus on cytokine production by the Vagina Chip.\u003c/strong\u003e A) Schematic diagrams of Cervix and Vagina Chips and the transfer of cervical mucus between chips. B) Cytokine protein levels for IL-1α, IL-1β, MIP-1β, and IL-10 measured in effluents of Vagina Chips cultured with (light gray bars) without (dark gray bars) cervical mucus for 1 day. Each data point indicates one chip; data shown are from 3 different experiments and are presented as mean ± sd; significance was calculated by unpaired t-test; ***, P\u0026lt; 0.0001; **, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898191/v1/071c99949d7d07c599ee5afb.jpg"},{"id":50459809,"identity":"6dd453d6-33a6-4721-ac7d-7eb3da8e4592","added_by":"auto","created_at":"2024-01-31 20:28:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87606,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of dysbiotic vaginal microbiome by cervical mucus introduced to the Vagina Chip.\u003c/strong\u003e Vaginal epithelium cultured on-chip for 72 h in the absence (Control: grey with stripes bars) or presence of BVC1 consortium (black bars) perfused either with media alone or with mucus-containing effluents from Cervix Chips that were added 1 day prior to the addition of bacteria (Pre; dark grey bar), 1 day after BVC 1 addition (Post; light grey bar), or continuously for the entire 3-day culture starting 1 day prior to addition of bacteria (Pre+Post; white bar). \u003cstrong\u003eA\u003c/strong\u003e) Total non-adherent bacterial cell number (CFU) per chip determined by quantification of bacteria collected in effluents from the apical epithelial channel during 72 h of co-culture with BVC1 in Vagina Chips. \u003cstrong\u003eB\u003c/strong\u003e) Total adherent CFU/chip determined by quantification of bacteria retained within epithelial tissue digests after 72 h of culture. \u003cstrong\u003eC\u003c/strong\u003e) Quantification of vaginal epithelial cell injury (percent cell viability) assessed by calculating the number of live cells relative to control using Trypan blue exclusion assay. \u003cstrong\u003eD\u003c/strong\u003e) Bright field microsopic image showing Clue-like cells and live epithelial cells using Trypan blue stain. \u003cstrong\u003eE\u003c/strong\u003e) Ratio of Clue-like cells to live cells detected as described in \u003cstrong\u003eD\u003c/strong\u003e. In all graphs, results were obtained from at least 2 different experiments; each data point indicates one chip. Data are presented as mean ± sd; significance was calculated by unpaired t-test; ***, P\u0026lt; 0.0001; **, P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898191/v1/ab92f9f42ea41a7f7fd13a05.jpg"},{"id":50459807,"identity":"415d71a0-cfd0-45cd-904b-454f73701355","added_by":"auto","created_at":"2024-01-31 20:28:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51442,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokine production by the Vagina Chip exposed to a dysbiotic consortium\u003c/strong\u003e. Heat map showing the innate immune response of vaginal epithelium cultured on-chip for 72 h in the absence (Control) or presence of BVC1 consortium perfused either without or with mucus-containing effluents from Cervix Chips that were added 1 day prior to the addition of bacteria (Pre), 1 day after BVC 1 addition (Post), or continuously for the entire 3-day culture starting 1 day prior to the addition of bacteria (Pre+Post). \u0026nbsp;IL-10, RANTES(CCL5), TNF-α, MIP-1β, and IL-1α protein levels in the epithelial channel effluents were normalized for cell number. The gray scale represents fold-change in cytokine levels relative to levels in control chips. n=4-10 individual chips for each group from 4 independent experiments; significance was calculated by unpaired t-test ; *P\u0026lt;0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001 compared to BVC1.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898191/v1/06d923a966d3cda972c28b46.jpg"},{"id":50459804,"identity":"2d9df174-4e2b-4f8e-8c45-ce7e0510655a","added_by":"auto","created_at":"2024-01-31 20:28:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppression of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e G. vaginalis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egrowth in Vagina Chip effluents\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein 2D culture. \u0026nbsp;A) \u003c/strong\u003e\u003cem\u003eG. vaginalis\u003c/em\u003e growth in 2D culture wells by optical density (OD) measurement every 30 min for 24 h; (M) within mucus-containing Cervix Chip effluent; (V+M) similar effluent perfused through a Vagina Chip for 1 day; and (HBSS) control.\u003cstrong\u003e B) \u003c/strong\u003eLogistic growth rate constant (k) for bacterial growth in panel A. \u003cstrong\u003eC)\u003c/strong\u003e Bacterial growth in permissive conditions in which the effluents shown in A were supplemented with 50% bacterial broth (PYT). While permissive conditions allowed for growth in the HBSS control group, \u003cem\u003eG. vaginalis\u003c/em\u003e growth remained suppressed in the V+M group. \u003cstrong\u003eD) \u003c/strong\u003eLogistic growth rate constant (k) for bacterial growth in panel C. \u0026nbsp;n=5; *P\u0026lt;0.05, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898191/v1/f8558697eaba39ad15d81cd3.jpg"},{"id":50459808,"identity":"0fd8a39b-7a67-40a2-a4cb-ccb24c3b3fc7","added_by":"auto","created_at":"2024-01-31 20:28:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCervical mucus alters the vaginal secretome.\u003c/strong\u003e Mass spectrometry analysis of Vagina Chip effluents pre-exposed to Cervix Chip mucus-containing effluent for 1 day (V+M) compared to Vagina Chip effluent alone (V) and Cervix Chip mucus-containing effluent (M).\u0026nbsp; \u003cstrong\u003eA)\u003c/strong\u003e Upregulated and \u003cstrong\u003eB)\u003c/strong\u003e Downregulated proteins in (V+M) compared (V) and (M), determined by fold change (|log2 fc| \u0026gt;= 1, p\u003csub\u003eadj \u003c/sub\u003e£ 0.05). \u003cstrong\u003eC\u003c/strong\u003e) Volcano plot showing differentially expressed proteins in (V+M) compared to (V) and (M). The plot was constructed using the normalized protein expression data with the negative logarithm of the adjusted p-value represented on the y-axis and the log2 fold change represented on the x-axis. Each dot on the plot corresponds to a protein, with color coding used to indicate the statistical significance of differential expression. Proteins with a \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05 and fold change \u0026gt; 2 are colored red, while proteins with a \u003cem\u003ep\u003c/em\u003e value \u0026lt; 0.05, and fold change \u0026lt; -2 are colored blue (Gray, proteins with \u003cem\u003ep \u003c/em\u003evalue \u0026gt; \u0026nbsp;0.05).\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898191/v1/86e6094e74b8baf1b49d6d58.jpg"},{"id":50460834,"identity":"f5171ce7-839c-46e0-b8bc-121d159c55c1","added_by":"auto","created_at":"2024-01-31 20:44:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":835836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3898191/v1/053f4922-eba0-4eb6-aa90-b4d4b10be64e.pdf"},{"id":50459806,"identity":"22b69422-bbd9-440a-83d6-8bcf24d7cb22","added_by":"auto","created_at":"2024-01-31 20:28:07","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":102477,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3898191/v1/800b99a7051e0005233be88b.jpg"}],"financialInterests":"Competing interest reported. D.E.I. is a founder, board member, and chairs the SAB of Emulate Inc., in which he also holds equity. The author O.G, A.G, Z.I, A.S, H.R, J.C, B.B, S.S, G.G and A.J report no conflict of interest.","formattedTitle":"Modulation of dysbiotic vaginal complications by cervical mucus revealed in linked human vagina and cervix chips","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe cervicovaginal fluid that covers the surface of the vaginal epithelium is essential to women's health and reproductive functions because it serves as a selective barrier that protects against environmental pathogens.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e This fluid contains mucus that is mainly produced by the cervical epithelium along with vaginal secretions, and it contains a range of cytokines, chemokines, immunoglobulins, and other immune mediators that help to prevent pathogens from crossing this critical mucosal interface. However, the role of cervical mucus in regulating vaginal microbiome composition and its effects on health outcomes has largely remained unexplored.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e This is important because dysbiotic changes in the composition of the female genital tract microbiome, as observed for example in patients with bacterial vaginosis (BV) whose microbiome often contains strict and facultative anaerobes including \u003cem\u003eGarderenella vaginalis\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e have been linked to increased susceptibility to sexually transmitted infections, pelvic inflammatory disease, infertility, preterm birth, and maternal and neonatal infections.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e BV is also the most prevalent cause of vaginal symptoms in women with a more than 50% recurrence rate, yet the underlying factors contributing to these conditions remain elusive.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we leveraged human organ-on-a-chip (Organ Chip) microfluidic culture technology to directly explore how cervical mucus secretions influence human vaginal epithelium under both healthy and dysbiotic conditions. To acheive this, we connected two recently described human Organ Chip models of female reproductive organs. The first is a human vagina-on-a-chip (Vagina Chip) that is lined by primary, hormone-sensitive, vaginal epithelium interfaced with underlying stromal fibroblasts that we have shown recapitulates the pathophysiology of a dysbiotic vaginal epithelium when co-cultured with a \u003cem\u003eG. vaginalis\u003c/em\u003e-containing microbiome and that enables analysis of human host-microbiome interactions \u003cem\u003ein vitro\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e The second is a human cervix-on-a-chip (Cervix Chip) lined by primary cervical epithelium interfaced with cervical fibroblasts\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e that produces abundant cervical mucus with compositional, biophysical, and hormone-responsive properties similar to those observed \u003cem\u003ein vivo\u003c/em\u003e. To model and study the effect of cervical mucus on vaginal responses \u003cem\u003ein vitro\u003c/em\u003e, we co-cultured a dysbiotic microbiome in the human Vagina Chip in the presence or absence of mucus-containing effluents that were transferred from the epithelial channel of human Cervix Chips. These studies revealed that human cervical epithelial secretions exert immunomodulatory effects and protect the vaginal epithelium against a dysbiotic microbiome by reducing innate inflammatory responses and inhibiting the growth of \u003cem\u003eG. vaginalis\u003c/em\u003e bacteria, thereby reducing vaginal cell injury.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHuman Vagina Chip culture\u003c/h2\u003e \u003cp\u003eThe Human Vagina Chip was cultured as previously described (36434666). Breifly, microfluidic two-channel co-culture Organ Chip devices (CHIP-S1TM) were obtained from Emulate Inc. (Boston, MA). The Polydimethylsiloxane )PDMS( membrane was coated with collagen IV (30 \u0026micro;g/mL) (Sigma, cat. no. C7521) and collagen I (200 \u0026micro;g/mL) (Corning, cat. no. 354236) in Dulbecco's Modified Eagle Medium )DMEM, ThermoFisher, cat. no. 12320-032) in the apical channel. The basal channel was coated with collagen I (200 \u0026micro;g/mL) (Corning, USA) and poly-L-lysine (15 \u0026micro;g/mL) (ScienCell, Cat# 0403(. Primary human uterine fibroblasts (ScienCell Research Laboratories, cat. no. 7040) were then seeded at a density of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL in the basal channel and human vaginal epithelial cells (Lifeline Cell Technology, cat. no. FC-0083; donors 05328) were seeded at a density of 3 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/mL in the apical channel. Chips were incubated at 37\u0026deg;C with 5% CO2 under static conditions until the cells formed a uniform monolayer. Chips were then connected to the culture module instrument (ZO\u0026Euml;\u0026trade; CULTURE MODULE, Emulate Inc., USA) and placed under flow conditions. Vagina Chips were cultured using a periodic flow regimen in which vaginal epithelium growth medium (Lifeline, Cat# LL-0068 ) was flowed through the apical channel for 4 h/day at 15 \u0026micro;L/h. The basal channel was flowed continuously with fibroblast growth medium (ScienCell, Cat# 2301) at 30 \u0026micro;L/h. After 5\u0026ndash;6 days, the basal medium was replaced with an in-house differentiation medium\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e for eight days following the same intermittent and continuous perfusion regime in the apical and basal channels, respectively. The apical medium was replaced with customized HBSS Low Buffer/+Glucose (HBSS (LB/+G)) and the basal medium was replaced with antibiotic-free differentiation medium for one day followed by three days of microbial co-culture as described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eHuman Cervix Chip culture\u003c/h2\u003e \u003cp\u003eHuman Cervix Chips were cultured as previously described (Izadifar et al., 2023, BioRxiv). Breifly, microfluidic two-channel co-culture Organ Chip devices (CHIP-S1\u0026trade;) were obtained from Emulate Inc. (Boston, MA). The PDMS membrane was coated with 500 \u0026micro;g/mL collagen IV (Sigma-Aldrich, Cat. no. C7521) in the apical channel and with 200 \u0026micro;g/mL Collagen I (Advanced BioMatrix, Cat. no. 5005) and 30 \u0026micro;g/mL fibronectin (Corning, Cat. no. 356008) in the basal channel. Primary cervical fibroblasts (0.65 x10\u003csup\u003e6\u003c/sup\u003e cells/mL, P5)(isolated from hysterectomy cervical tissues) were seeded on the basal side followed by seeding primary cervical epithelial cells (1.5 x 10\u003csup\u003e6\u003c/sup\u003e cells/mL, P5) (LifeLine Cell Technology Cat# FC-0080) on the apical side. The respective chip media were refreshed after seedingfor each channel and the chips were incubated at 37\u0026deg;C, 5% CO2 under static conditions overnight. Chips were then connected to the culture module instrument (ZO\u0026Euml;\u0026trade; CULTURE MODULE, Emulate Inc.,USA). Cervix Chips were cultured using a periodic flow regimen in which cervical growth medium was flowed through the apical channel for four hours per day at 30 \u0026micro;L/h while fibroblast growth medium was continuously perfused basally at 40 \u0026micro;L/h. After five days the apical medium was replaced by Hank\u0026rsquo;s Buffer Saline Solution (HBSS) (Thermo Fisher, 14025076) while being fed through the basal channel by differentiation medium consisting of cervical epithelial medium (LifeLine Cell Technology, Cat. no. LL-0072) supplemented with 5 nM estradiol-17β (E2) (Sigma, Cat. no. E2257) and 50 \u0026micro;g/mL ascorbic acid (ATCC, Cat. no. PCS-201-040). On day 2 of differentiation the apical medium was replaced by HBSS with low buffering salts and no glucose (HBSS (LB/-G) at pH\u0026thinsp;~\u0026thinsp;5.4 and cultured for five additional days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMucus collection from Cervix Chips\u003c/h2\u003e \u003cp\u003eCervix Chip mucus was collected every day starting at day 4 of differentiation for 10 days. During the collection period, the basal channel was continuously perfused with antibiotic- free differentiation medium at a volumetric flow rate of 40 \u0026micro;L/h. The apical channel was perfused with HBSS (LB/-G) for 4 h/day at 40 \u0026micro;L/h and the chip effluents were collected and stored at -80\u0026deg;C until the end of the experiment. Before introduction to the Vagina Chip, 5.56 mM D-glucose (Sigma, cat. no. G7021) was added to the Cervix Chip mucus.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCulture of a non-optimal\u003c/b\u003e \u003cb\u003eGardnerella vaginalis-\u003c/b\u003e\u003cb\u003econtaining consortium in Vagina Chips\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn non-optimal vaginal microbiota, \u003cem\u003eGardnerella\u003c/em\u003e species are typically found as dominant bacteria\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e accompanied by other frequent taxa such as \u003cem\u003ePrevotella\u003c/em\u003e species and \u003cem\u003eAtopobium\u003c/em\u003e species.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e To mimic the ecology of non-optimal vaginal microbiota, we used a synthetic dysbiotic consortia (BVC1: \u003cem\u003eGardnerella vaginalis E2, Gardnerella vaginalis E4, Prevotella bivia BHK8, and Atopobium vaginae\u003c/em\u003e). The \u003cem\u003eGardnerella\u003c/em\u003e isolates used in this study were selected because they represent distinct genomic groups, exhibit phenotypic diversity \u003cem\u003ein vitro\u003c/em\u003e, and were co-resident, meaning that they were co-isolated from a single participant in the UMB-HMP study.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eP. bivia\u003c/em\u003e and \u003cem\u003eA. vaginae\u003c/em\u003e are prevalent species in \u003cem\u003eLactobacillus\u003c/em\u003e-deficient vaginal microbiota. The two strains used in this study were co-resident, isolated from a single participant in the Females Rising Through Education Support and Health study.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The apical channel of each Vagina Chip was inoculated with ~\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e CFU of prepared BVC1 consortia and then chips were incubated statically at 37\u0026deg;C and 5% CO2 for 20 h before starting flow using the Z\u0026ouml;e culture module. The basal channel was continuously perfused with in-house antibiotic-free differentiation medium and the apical channel was perfused for 4 h/day with customized HBSS (LB/+G) medium at a volumetric flow rate of 40 \u0026micro;L/h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThe study was carried out under five conditions: control, BVC1, pre-treatment, post-treatment and pre\u0026thinsp;+\u0026thinsp;post treatment. Vaginal epithelium cultured on-chip for 72 h in the absence (Control( or presence of BVC1 consortium, in the absence or presence of cervical mucus. In the Mucus Pre-treatment group, cervical mucus was used as the apical medium for 24 h before BVC1 infection, followed by the use of customized HBSS Low Buffer/+Glucose (HBSS (LB/+G)) as the apical medium. In the Mucus Post-Treatment group, cervical mucus was used as the apical medium for the duration of the experiment, beginning 24 h after BVC1 infection. In the Mucus pre\u0026thinsp;+\u0026thinsp;post treatment group, cervical mucus was used as the apical medium for 24 h prior to BVC1 infection, and for 72 h during BVC1 co-culture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBacterial enumeration from Vagina Chip co-culture\u003c/h2\u003e \u003cp\u003eTo enumerate all cultivable bacteria in the effluents, effluent samples (50 \u0026micro;L) were collected at 24, 48, and 72 h. Effluent samples from Vagina Chips containing BVC1 consortia were plated on Brucella blood agar (with hemin and vitamin K1) (Hardy, cat. no. A30) at 37\u0026deg;C under completely anaerobic conditions. After 48 h incubation, CFU/chip was calculated for each sample. To enumerate all cultivable bacteria engrafted in the Vagina Chip, the whole epithelial cell layer was digested for 1 h with 1 mg/mL of collagenase IV (Gibco, cat. no. 17104019) in TrypLE (ThermoFisher, cat. no. 12605010). Cell layer digests were diluted and processed in the same way as effluent samples and CFU/chip was calculated for each chip digest.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of cytokines and chemokines\u003c/h2\u003e \u003cp\u003eSamples (100 \u0026micro;L) of apical effluents from Vagina Chips were collected and analyzed for a panel of cytokines and chemokines, including TNF-α, IFN-y, IL-1α, IL-1β, IL-10, IL-8, IL-6, MIP-1α, MIP-1β, IP-10, and RANTES using custom ProcartaPlex assay kits (ThermoFisher Scientific). Analyte concentrations were determined using a Luminex 100/200 Flexmap3D instrument coupled with Luminex XPONENT software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and mass spectrometry\u003c/h2\u003e \u003cp\u003eSample digestion: Samples were run through a 50 kDa filter (Amicron Ultracel, Merck Millipore, Ireland) and digested according to the manafacturer\u0026rsquo;s protocol for 1 h at 50 \u0026deg;C by Trypsin Platinum (Promega, WV), and digested material was dried in a Speedvac (Eppendorf, Germany). Mass spectrometry: Each sample was resolubilized in 10 \u0026micro;L of 0.1% formic acid. Single LC-MS/MS was performed on a 240 Exploris Orbitrap (ThermoScientific, Germany) equipped with a NEO nano-HPLC pump (ThermoScientific, Germany). Peptides were separated onto a micropac 5 cm trapping column (Thermo, Belgium) followed by a 50 cm micropac analytical column (ThermoScientific, Belgium). Separation was achieved by applying a 5\u0026ndash;24% ACN gradient in 0.1% formic acid over 90 min at 250 nL min\u0026thinsp;\u0026minus;\u0026thinsp;1. Electrospray ionization was achieved at 1.8 kV with an electrode junction (PepSep, Denmark) at the end of a microcapillary column with a stainless-steel 4 cm needle (ThermoScientific, Denmark). The Exploris Orbitrap was operated in data-dependent mode, and the mass spectrometry survey scan was performed at 450 \u0026minus;\u0026thinsp;1,200 m/z and a resolution of 1.2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, followed by selection of the ten most intense ions (TOP10) for HCD-MS2 fragmentation. FFor each HCD MS2 scan, the fragment ion isolation width was 0.8 m/z, AGC was 50,000, maximum ion time was 150 ms, normalized collision energy was 32V and an activation time of 1 ms.\u003c/p\u003e \u003cp\u003eData analysis: Raw data were submitted for analysis in Proteome Discoverer 3.0 (Thermo Scientific, CA) software. Assignment of MS/MS spectra was performed using the Sequest HT algorithm by searching the data against a protein sequence database including all entries from the Human Uniprot database (SwissProt, 2019) and full Uniprot bacteria database (SwissProt, 2022) as well as known contaminants such as human keratins and common lab contaminants. Sequest HT searches were performed using a 15 ppm precursor ion tolerance requiring each peptide N-/C terminiusto adhere with Trypsin protease specificity, while allowing up to two missed cleavages. For searches, methionine oxidation (+\u0026thinsp;15.99492 Da) and asparagine and glutamine deamidations (+\u0026thinsp;0.984016 Da) were set as variable modifications as well as N-terminal acetylation of protein termini. A MS2 spectra assignment false discovery rate (FDR) of 1% on the protein level was achieved by applying the target-decoy database search. Filtering was performed using a Percolator (64bit version).\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e For quantification analysis between samples, label-free quantitation mode using Minora detection features of the Proteome Discoverer platform was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results presented are from at least two independent experiments and all data points shown indicate the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (s.d.) from n\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;3 Organ Chips unless otherwise mentioned. Statistically significant differences between groups were determined using unpaired t-test, statistical analyses were performed using GraphPad Prism 9.0.2.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eModulation of innate immunity in Vagina Chips by mucus-containing effluents from Cervix Chips\u003c/h2\u003e \u003cp\u003eWe recently described a human Vagina Chip lined by primary human vaginal epithelium interfaced across an extracellular matrix (ECM)-coated porous membrane with underlying stromal fibroblasts that enables analysis of human host-microbiome interactions in the vaginal microenvironment,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e as well as a human Cervix Chip containing primary cervical epithelium interfaced with stromal cervical fibroblasts that produces cervical mucus with physical and chemical properties similar to those observed \u003cem\u003ein vivo\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Here, we collected mucus-containing effluents from the epithelial channel of the Cervix Chip ('cervical chip mucus' containing 4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;3.04 mg/mL of mucus glycoproteins) for 7 days and then perfused it through the epithelial channel of a Vagina Chip to simulate the natural flow of mucus in the reproductive tract \u003cem\u003ein vivo\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;1A\u003c/b\u003e). Presence of this mucus in the Vagina Chip induced statistically significant decreases in secretion of multiple relevant proinflammatory cytokines, including interleukin-1α (IL-1α), IL-1β, and macrophage inflammatory protein-1β (MIP-1β), accompanied by a concomitant increase in anti-inflammatory IL-10 protein production after 24 h of exposure compared to control chips without mucus (\u003cb\u003eFig.\u0026nbsp;1B\u003c/b\u003e). These results demonstrate that the mucus-containing fluids produced by human cervical epithelium in Cervix Chips \u003cem\u003ein vitro\u003c/em\u003e can directly influence the vaginal epithelium and result in suppression of inflammatory cytokine production, even in the absence of immune cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eModulation of the dysbiotic vaginal microbiome by introducing cervical mucus into the Vagina Chip\u003c/h2\u003e \u003cp\u003eWe next studied the effects of cervical mucus on a dysbiotic (non-optimal) vaginal microbiome in Vagina Chips by inoculating them with a consortium containing G. \u003cem\u003evaginalis E2\u003c/em\u003e and \u003cem\u003eE4\u003c/em\u003e combined with \u003cem\u003eP. bivia BHK8\u003c/em\u003e and \u003cem\u003eA. vaginae\u003c/em\u003e (BVC1; ~10\u003csup\u003e5\u003c/sup\u003e CFU/chip) on day 14 of Vagina Chip culture in the presence or absence of mucus-containing effluents from the Cervix Chip. Interestingly, the presence of human cervical mucus inhibited the consortium's ability to colonize the epithelium and thrive on the Vagina Chip. The total number of CFU of live non-adherent bacteria collected in effluents from the epithelial channel during 72 h of infection (\u003cb\u003eFig.\u0026nbsp;2A\u003c/b\u003e), as well as the number of live adherent bacteria in tissue digests at the end of the 72 h culture (Fig.\u0026nbsp;2B), were significantly reduced whether Vagina Chips were pretreated with mucus effluents for 1 day before microbiome introduction, 1 day after BVC 1 addition, or continuously for the entire 3-day culture starting 1 day before the addition of bacteria.\u003c/p\u003e \u003cp\u003eConsistent with these data, when we quantified vaginal epithelial cells with a Clue Cell-like appearance (i.e., covered with bound bacteria)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e in digests of Vagina Chip epithelium with the dysbiotic BVC1 consortium, we observed a decrease in the number of these cells in the presence of cervical mucus (\u003cb\u003eFig.\u0026nbsp;2C,D\u003c/b\u003e). Not surprisingly, this reduction in bacterial cell number induced by the presence of cervical mucus was also accompanied by a concomitant increase in vaginal epithelial cell viability (retained cell number) (\u003cb\u003eFig.\u0026nbsp;2E\u003c/b\u003e), as well as significant downregulation of the proinflammatory cytokines, IL-8, IL-10, Rantes (CCL5), TNF-α, MIP-1β, and IL-1α after 72 h of co-culture (\u003cb\u003eFig.\u0026nbsp;3\u003c/b\u003e). These results demonstrate that Cervix Chip mucus can directly influence the Vaginal Chip epithelium to dampen production of inflammatory cytokines and this correlates with protection of the vaginal epithelium against injury.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSuppression of\u003c/b\u003e \u003cb\u003eG. vaginalis\u003c/b\u003e \u003cb\u003egrowth in Vagina Chip effluents\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore whether cervical mucus acts directly to suppress bacterial cell growth or indirectly by altering vaginal cell physiology, we next compared the growth of \u003cem\u003eG. vaginalis\u003c/em\u003e in mucus-containing effluent samples collected from the epithelial channel of control Cervix Chips (perfused with HBSS) versus effluents from Vagina Chips that were perfused with Cervix Chip-derived mucus-containing effluent for 1 day, with the bacteria cultured directly in HBSS that was used to perfuse the apical channels of our Cervix and Vagina chips as a control. Our results demonstrate that \u003cem\u003eG. vaginalis\u003c/em\u003e grew well in the Cervix Chip mucus in 2D culture, but growth was suppressed when cultured in effluents from the Vagina Chip perfused with similar Cervix Chip-derived mucus-containing effluent or in HBSS that lacks critical nutrients (\u003cb\u003eFig.\u0026nbsp;4A,B\u003c/b\u003e). Importantly, when similar studies were carried out after the addition of 50% bacterial broth to provide optimal nutrient conditions, bacterial growth was restored in the control HBSS sample, but not in the sample from the Vagina Chip exposed to Cervix Chip-derived mucus effluent (\u003cb\u003eFig.\u0026nbsp;4C, D\u003c/b\u003e). These findings suggest that mucus components produced by the Cervix Chip induce the cells lining the Vagina Chip to express factors that suppress \u003cem\u003eG. vaginalis\u003c/em\u003e growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCervix Chip mucus alters the vaginal secretome\u003c/h2\u003e \u003cp\u003eTo further explore the effects of cervical mucus on the vaginal epithelium, we conducted mass spectrometry analysis to compare the proteome composition of the Cervix Chip effluent before and after exposure to the Vagina Chip versus the untreated Vagina Chip effluent. Of the 1752 proteins identified (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e), 103 were found to be differentially abundant as determined by fold change (|log2 fc| \u0026gt;= 1), p\u003csub\u003eadj\u003c/sub\u003e \u0026le; 0.05) in Cervix Chip effluents that had passed through the Vagina Chip versus either the Cervix Chip effluent or Vagina Chip effluent alone. Significant changes in the expression of multiple proteins were observed, with 64 proteins showing increased expression (\u003cb\u003eFig.\u0026nbsp;5A\u003c/b\u003e) and 39 proteins showing decreased expression (\u003cb\u003eFig.\u0026nbsp;5B\u003c/b\u003e), with the most prominent alterations highlighted in a volcano plot (\u003cb\u003eFig.\u0026nbsp;5C\u003c/b\u003e). PCA analysis of the proteomics data also revealed distinct segregation among these sample groups, indicating notable changes in protein expression in effluents from Vagina Chips exposed to Cervix Chip mucus compared to those from untreated Cervix or Vagina Chips alone (\u003cb\u003eFig.\u0026nbsp;5D\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, using STRING analysis, which incorporates both physical protein-protein interactions and functional associations from various sources (e.g., automated text mining, computational interaction predictions from co-expression, conserved genomic context, databases of interaction experiments, and curated sources of known complexes/pathways),\u003csup\u003e16\u003c/sup\u003e we found that 3 of the 37 down-regulated proteins exhibit calcium channel inhibitor activity (PHPT1, AMBP, SLC30A1). Previous research has shown that \u003cem\u003eG. vaginalis\u003c/em\u003e strongly induces epithelial calcium influx and contraction.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e In addition, 6 of the down-regulated proteins are ECM molecules (LGALS3BP, GPC1, AMBP, SERPING1, VASN, FBLN1, FBLN2), which may play a role in \u003cem\u003eG. vaginalis\u003c/em\u003e adhesion and biofilm formation.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Finally, 3 down-regulated proteins are members of the Lipocalin family (AMBP, APOD, RBP4), which is known for its role in regulating inflammation and antioxidant responses.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe STRING analysis additionally showed that 17 of the up-regulated proteins are RNA binding proteins (RBPs). Previous studies have highlighted the vital role of RBPs in bacterial replication by binding to and regulating their RNAs.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e These proteins also play a crucial role in immune system response to viral infections by regulating viral RNA stability and translation.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Considering that BV increases susceptibility to sexually transmitted infections, including viral infections, these findings further support the potential involvement of RBPs in the immune response within the reproductive tract. Twenty-five proteins related to the male reproductive system were also found to be upregulated. This finding is significant because past studies have established a notable link between BV and infertility.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e For instance, one of the proteins identified, CSTF2T, has the potential to contribute to sperm adhesion to the zona pellucida\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e while the also identified TMED10 protein may be involved in in sperm capacitation and the acrosome reaction.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eImportantly, exposure of the Vagina Chip to cervical mucus also resulted in enhanced production of potential antimicrobial proteins PLAU and WASF2. PLAU is a serine protease with immunomodulatory functions\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and WASF2 is a member of the Wiskott-Aldrich syndrome protein family that regulates autophagy and inflammasome activity.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e One of the prominent down-regulated proteins, GNS, is an N-acetylglucosamine-6-sulfatase. This is interesting because BV is often associated with the breakdown of mucins, which are necessary for these dysbiotic bacteria to colonize the vagina.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Thus, downregulation of GNS could contribute to the inhibition of dysbiotic bacterial growth we observed by increasing glycoprotein sulfation and thereby prevent mucin degradation.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePotential role of exosomes as mediators of the effects of cervical mucus on the Vagina Chip\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExosomes, which are small extracellular vesicles containing nucleic acids, lipids, and proteins, play a significant role in intercellular communication in the female reproductive tract by modulating the immune system and promoting tissue repair.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e This is accomplished by presenting antigenic peptides, regulating gene expression through exosomal miRNA, and inducing differential signaling through exosomal surface ligands. Importantly, when we carried out STRING analysis of the proteins differentially expressed in Vagina Chip effluents exposed to Cervix Chip mucus, we found that a significant proportion of the differentially expressed proteins were associated with exosomes. Specifically, 23 out of 37 down-regulated proteins and 17 out of 64 upregulated proteins were found to be linked to extracellular exosomes (\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e). Notable among the upregulated proteins were DDR1\u003csup\u003e28\u003c/sup\u003e and COMP,\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e which regulate cellular adhesion to the ECM and its remodeling, subsequently influencing bacterial adhesion.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Conversely, among the down-regulated proteins, 5 ECM proteins (AMBP, FBLN1, GPC1, LGALS3BP, and SERPING1) were identified, which may also influence bacterial adhesion. Interestingly, SERPING1 functions as a regulator of the complement system\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, and three of the down-regulated proteins (AMBP, SERPING1, and SPINT1) belong to the Kunitz family of serine protease inhibitors that are involved in coordinating inflammation.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCervicovaginal antimicrobial peptides\u003c/h2\u003e \u003cp\u003eAdditionally, we identified 12 antimicrobial peptides in the Cervix Chip and Vagina Chip effluents (\u003cb\u003eTable\u0026nbsp;1\u003c/b\u003e). Of these, 6 (Dermcidin, Ubiquicidin, Chemerin, Acipensin 6, hSAA1, and Psoriasin) were present in both Vagina and Cervix Chip effluents, 1 was solely produced by the Vagina Chip (KAMP-19), and 5 were exclusively produced by the Cervix Chip. No antimicrobial peptides were specifically induced in Vagina Chips exposed to Cervix Chip effluents. The Cervix Chip-derived antimicrobial peptides include Histone H4, Histone H3, CXCL1, BHP, and Chromacin. Histones and their fragments have a variety of antimicrobial actions and functions, including bacterial cell membrane permeabilization, penetration into the membrane followed by binding to bacterial DNA and/or RNA, binding to bacterial lipopolysaccharide (LPS) and neutralizing its toxicity, and entrapping pathogens as a component of neutrophil extracellular traps.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e It is noteworthy that \u003cem\u003eP. bivia\u003c/em\u003e, which is included in our bacterial consortium, has been shown to produce high concentrations of LPS.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e CXCL1 also inhibits the growth of \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus in vitro\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and BHP impedes growth of \u003cem\u003eM. luteus, S. epidermidis\u003c/em\u003e, and several fungi (e.g., \u003cem\u003eC. albicans, S. cerevisiae, and A. nidulans)\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, while Chromacin suppresses the growth of \u003cem\u003eBacillus megaterium\u003c/em\u003e and \u003cem\u003eMicrococcus luteus\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eComment\u003c/h2\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePrincipal Findings\u003c/span\u003e: These data show that mucus-containing effluents from human Cervix Chips suppress the growth of dysbiotic microbiota, associated inflammation, and epithelial cell injury in human Vagina Chips. By analyzing the differentially abundant proteins in the secretome of Vagina Chips following treatment with Cervix Chip effluents, we identified multiple proteins that may contribute to this protective response and that potentially could be used as clinical biomarkers for monitoring female reproductive tract health.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eResults in the Context of What is Known\u003c/span\u003e: Maintaining homeostasis is crucial for the health of epithelial barriers, which can be disrupted during infection or injury. Inflammation plays a vital role in supporting the body's defense against pathogens, promoting tissue healing, and restoring homeostasis.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e However, chronically high levels of proinflammatory cytokines that undermine normal protective immune signals have been linked to an imbalanced microbiome and compromised epithelial cell stability.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e This study presents evidence that communication between cervical and vaginal tissues in the lower reproductive tract via transfer of cervical mucus-containing secretions helps to suppress vaginal inflammation in the presence of a dysbiotic microbiome. There is a growing body of clinical evidence suggesting that medical cervical procedures may disrupt this crucial communication between cervical and vaginal epithelium, and lead to changes in composition of the vaginal microbiome.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Our results support this observation and suggest that it is a direct effect of reducing cervical mucus transfer to the vagina, which could only be studied directly using this type of engineered \u003cem\u003ein vitro\u003c/em\u003e model.\u003c/p\u003e \u003cp\u003eThe recurrence of abnormal vaginal flora after treatment of BV (e.g., with metronidazole) is commonly detected in most women,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e however, the underlying factors contributing to these recurrences remain elusive. Our findings suggest that alterations in cervical mucus levels may influence the susceptibility of the vaginal epithelium to BV infection. Therefore, an imbalance in the cervicovaginal mucus may be a possible contributing factor to the high rate of BV recurrence. In this context, it is important to note that we identified five cervical antimicrobial peptides that appear to play a role in the antimicrobial effects we observed on-chip. These findings suggest that interactions between antimicrobial peptides and the host vaginal epithelium can enhance innate immune protection against dysbiotic flora.\u003c/p\u003e \u003cp\u003eImmune effectors and specialized stromal cells at epithelial surfaces produce cytokines and antimicrobial defenses to orchestrate tissue repair and minimize opportunistic infections. Exosomes can act as mediators for this form of inter-tissue communication. We identified 40 exosomal proteins produced by vaginal epithelium that were modulated by exposure to cervical mucus produced in the human Cervix Chip. Human cervicovaginal exosomes have been previously shown to be part of the female innate defense system and to protect against HIV-1 infection\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e as well as bacterial toxins.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Exosomes are also currently being explored as potential therapeutic agents and drug delivery vehicles. Thus, the ability to study the role of exosomes in host-microbiome interactions in the female reproductive tract \u003cem\u003ein vitro\u003c/em\u003e using the human Organ Chip models described here may facilitate the development of novel treatments for vaginal dysbiosis as well as other diseases of the female reproductive tract.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eClinical Implications\u003c/span\u003e: Our study has important clinical implications as it has the potential to identify new targets for diagnosis and treatment of vaginal diseases. Identifying patients with a high likelihood of recurrent vaginal dysbiosis can help to customize their treatment plan and prevent complications. In this study, we identified multiple proteins and antimicrobial peptides that may contribute to the protective response against dysbiotic microbiota and associated inflammation and injury to the vaginal epithelium. These proteins and peptides could potentially be used as clinical biomarkers for monitoring the health of the female reproductive tract in the future. Several proteins we identified (e.g., TPM3, PLAU, ALDH3A2, GAS6, DTYMK, SERPING, STAT6, CMPK1) are known to be targeted by existing approved drugs (Progesterone, Urokinase, Disulfiram, Warfarin, Zidovudine, Rhucin, Indomethacin, and Gemcitabine, respectively). Thus, if these molecules actively contribute to the BV disease phenotype, one or more of these therapeutics could be added to current clinical regimens.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eResearch Implications\u003c/span\u003e: Our results show the value of human Organ Chip technology for studying vaginal health and diseases of the female reproductive tract. However, further research is needed to evaluate the effects of these compounds as well as modulators of the other putative targets we identified for maintaining vaginal homeostasis and a healthy microbiome.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStrengths and Limitations\u003c/span\u003e: While the human Vagina and Cervix Chips used in this study replicate many physiological and pathophysiological features of the female reproductive tract, we did not incorporate immune cells. As these cells play a crucial role in mounting antibacterial immune responses, the model would be strengthened by incorporating them in the future. Additionally, it should be noted that the Organ Chips we used were created with epithelial cells from a single human donor and thus, these studies should be extended to include chips lined by cells from multiple donors from different ethnic groups as well.\u003c/p\u003e "},{"header":"Conclusions","content":"\u003cp\u003e This study highlights the crucial role that cervical mucus plays in maintaining vaginal health and preventing dysbiosis-related complications. Our results directly demonstrate that cervical mucus-containing secretions can suppress the growth of dysbiotic microbiota as well as associated inflammation and epithelial cell injury in the human Vagina Chip. We also identified several proteins and antimicrobial peptides that could serve as clinical biomarkers for monitoring the health of the female reproductive tract and potentially be targeted for the treatment of vaginal dysbiosis. This study also sheds light on the potential role of exosomes in inter-tissue communication and immune protection against dysbiotic flora in the female reproductive tract. In addition, these findings could have important clinical implications, particularly for identifying patients with a high risk of recurrent dysbiosis and customizing their treatment plans. Further research is needed to evaluate the effects of modulating the potential molecular mediators we identified on maintaining healthy vaginal microbial homeostasis. However, these findings provide further evidence showing that human Organ Chip models can provide a valuable tool for studying host-microbiome interactions in the female reproductive tract as well as for identifying potential clinical biomarkers and therapeutic targets for patients with vaginal dysbiosis and other related diseases.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eACKNOWLEDGMENTS\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was sponsored by funding from the Bill and Melinda Gates Foundation (OPP1173198 \u0026amp; INV-035977 to D.E.I., OPP1189217 to J.R. and INV-031642 to S.R-N.) and the Wyss Institute for Biologically Inspired Engineering (D.E.I.). Gwenn Merry (Wyss Institute) provided technical editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHORS CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eO.G.: conceptualization, data curation, formal analysis, investigation, writing - original draft.\u0026nbsp;A.G.: investigation, writing - review \u0026amp; editing. Z.I.: conceptualization, writing - review \u0026amp; editing. A.S.: conceptualization. H.R.: investigation.J.C.: investigation. B.B.: investigation, proteomics methodology. S.S.: software. G.G.: supervision, writing - review \u0026amp; editing. \u0026nbsp; A.J.: supervision, writing - review \u0026amp; editing. D.E.I.: conceptualization, supervision, funding acquisition, writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePOTENTIAL CONFLICTING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.E.I. is a founder, board member, and chairs the SAB of Emulate Inc., in which he also holds equity. The author \u0026nbsp;O.G, A.G, Z.I, \u0026nbsp;A.S, H.R, J.C, B.B, S.S, G.G and A.J report no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLacroix G, Gouyer V, Gottrand F, Desseyn JL. The Cervicovaginal Mucus Barrier. Int J Mol Sci 2020;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVagios S, Mitchell CM. Mutual Preservation: A Review of Interactions Between Cervicovaginal Mucus and Microbiota. Front Cell Infect Microbiol 2021;11:676114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcLoughlin K, Schluter J, Rakoff-Nahoum S, Smith AL, Foster KR. Host Selection of Microbiota via Differential Adhesion. Cell Host Microbe 2016;19:550\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJuliana NCA, Suiters MJM, Al-Nasiry S, Morre SA, Peters RPH, Ambrosino E. 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AIDS 2016;30:2611\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeller MD, Ching KL, Liang FX, et al. Decoy exosomes provide protection against bacterial toxins. Nature 2020;579:260\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"},{"header":"Supplementary Files","content":"Supplementary Tables is not available with this version."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Women's Health](https://www.nature.com/npjwomenshealth/)","snPcode":"44294","submissionUrl":"https://submission.springernature.com/new-submission/44294/3","title":"npj Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"cervical mucus, vaginal microbiota, dysbiosis, organ-on-a-chip, bacterial vaginosis, inflammation, diagnostic biomarkers, therapeutic targets","lastPublishedDoi":"10.21203/rs.3.rs-3898191/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3898191/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe cervicovaginal mucus which coats the upper surface of the vaginal epithelium is thought to serve as a selective barrier that helps to clear pathogens, however, its role in modulating the physiology and pathophysiology of the human vagina is poorly understood. Bacterial vaginosis (BV), a common disease of the female reproductive tract that increases susceptibility to sexually transmitted infections, pelvic inflammatory disease, infertility, preterm birth, and both maternal and neonatal infections is characterized by the presence of a wide array of strict and facultative anaerobes, often including \u003cem\u003eGardnerella vaginalis\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo assess the role of cervical mucus in preventing dysbiosis-associated complications and preserving vaginal health.\u003c/p\u003e\u003ch2\u003eStudy Design:\u003c/h2\u003e \u003cp\u003eTo better understand the role of cervicovaginal mucus in vaginal health, we used human organ-on-a-chip (Organ Chip) microfluidic culture technology to analyze the effects of cervical mucus produced in a human Cervix Chip and then transferred to a human Vagina Chip BV model. Both chips are lined by primary human organ-specific (cervical or vaginal) epithelium interfaced with organ-specific stromal fibroblasts.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur data show that mucus-containing effluents from Cervix Chips protect Vagina Chips from inflammation and epithelial cell injury caused by co-culture with a dysbiotic microbiome containing \u003cem\u003eG. vaginalis\u003c/em\u003e. Proteomic analysis of proteins produced by the Vagina Chip following treatment with the Cervix Chip mucus also revealed a collection of differentially abundant proteins that may contribute to the vaginal response to a dysbiotic microbiome, which could represent potential diagnostic biomarkers or therapeutic targets for the management of BV.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study highlights the importance of cervical mucus in controlling human vaginal physiology and pathophysiology, and demonstrates the potential value of Organ Chip technology for studies focused on the health and diseases of the female reproductive tract.\u003c/p\u003e","manuscriptTitle":"Modulation of dysbiotic vaginal complications by cervical mucus revealed in linked human vagina and cervix chips","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-31 20:28:02","doi":"10.21203/rs.3.rs-3898191/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-18T00:02:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-07T23:40:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-02T13:48:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121245079034472013369130281958373202296","date":"2024-05-22T16:03:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248223070533701359375580370058220128626","date":"2024-04-26T14:36:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157e0469-3dbe-4368-a3ff-befca003a699","date":"2024-04-23T07:11:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-17T17:00:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61767097-6943-4ea0-90ca-4e4191170f72","date":"2024-03-03T18:02:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"56d270d8-f058-49e3-a05e-b98151f376e5","date":"2024-02-18T11:49:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-06T00:28:57+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-01-31T23:50:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-01-29T12:54:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Women's Health","date":"2024-01-25T19:42:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-womens-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Women's Health](https://www.nature.com/npjwomenshealth/)","snPcode":"44294","submissionUrl":"https://submission.springernature.com/new-submission/44294/3","title":"npj Women's Health","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"82f6075c-51d5-40b1-ac9f-c37a947318b0","owner":[],"postedDate":"January 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":28429395,"name":"Biological sciences/Physiology"},{"id":28429396,"name":"Biological sciences/Physiology/Reproductive biology"}],"tags":[],"updatedAt":"2025-01-21T03:53:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-31 20:28:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3898191","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3898191","identity":"rs-3898191","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}