Study population: The HIV and Microbiome (HM) study enrolled a total of 360 pregnant women with a gestation age >12 weeks, attending routine antenatal clinic at Mulago National Referral Hospital. All study participants were aged 18 years and older, with the average age of 26 years (SD+ 5.14 years). The average gestational age of all women enrolled in the study was 27.9 weeks (SD+6.4 weeks). Of the 360 pregnant women enrolled, we included only the 179 who had had their vaginal microbiome sequenced. We included 79 (44%) HIV-infected (all receiving antiretroviral therapy) and 100 (56%) were HIV-negative pregnant women. Participants gave written informed consent to participate in the study and their vaginal microbiome was determined by 16S rRNA sequencing. Four cervicotypes (CT) were identified; CT1 (the least diverse) which was predominantly non-iners Lactobacillus, CT2 which was dominated by L. iners, CT3 that was Gardnerella dominant and CT4 (most diverse), a mixed CT co-dominated by L. iners, Gardnerella and Atopobium. Approval for this study was obtained from the Institutional Review Board of School of Biomedical Sciences, Makerere University College of Health Sciences and Uganda National Council for Science and Technology (study reference number: HS 2257).
ELISAs for claudin-1 and ZO-1
We used the human tight junction protein ZO-1 and claudin 1 ELISA kits (abbexaÒ, Cambridge, UK) to measure the concentrations of the afore-mentioned proteins in CVL. Briefly, standards and samples were added to 96-well plate pre-coated with antibodies to either ZO-1 or claudin-1, incubated and thereafter washed as per the manufacturer’s instructions. A detection antibody that is biotin labelled and specific to either ZO-1 or claudin-1 was added, incubated then followed by incubation with avidin-conjugated horse radish peroxidase (HRP). TMB was catalyzed by HRP to give a blue product that changed to yellow upon addition of an acidic stop solution. The absorbance was read using a spectrophotometer set at 450nm.
Preparation of bacterial cell-free supernatants
Based on the cervicotypes previously identified among pregnant mothers in a Ugandan study, we chose the bacterial species of interest; L. crispatus (L. crispatus, ATCC 33820) representing CT1, L. iners (L. iners, ATCC 55195) representing CT2, G. vaginalis (G. vaginalis, ATCC 14018) representing CT3 and combinations of G. vaginalis with either L. crispatus or L. iners representing CT4.L. crispatus was grown in Lactobacilli MRS broth (BD 288130) while L. iners and G. vaginalis were grown in NYC III media with 10% v/v heat inactivated horse serum at 37°C in a 5% CO2 incubator for an initial 48 hours. Subsequently, 1 ml of each bacterial culture was inoculated into 100ml of NYC III media and grown for a further 20 hours to OD600 0.7 at 37°C in a 5% CO2 incubator. The cultures were centrifuged two times for 10 min each at 2,500 rpm at 4°C to remove the bacteria. The resulting supernatants were filter-sterilized through a 0.22 μM membrane filter (EMD Millipore, Darmstadt, Germany) to remove any remaining bacterial components or debris. Cell-free supernatants and NYC III media (negative control to determine the baseline measurements of background growth media) were then used for in vitro cell and tissue culture experiments. For each treatment, experiment was done in duplicate or triplicate.
Cell culture
Vaginal epithelial (VK2/E6E7, ATCC® CRL-2616™) cell lines (American Type Culture Collection, Bethesda, MD, United States) were cultured in keratinocyte serum-free medium (K-SFM) supplemented with 0.1 ng/ml human recombinant epidermal growth factor (EGF) and 50 μg/ml bovine pituitary extract (ScienCell Research Laboratories, Carlsbad, CA, United States), 44.4mg/L calcium chloride100 U/mL penicillin, and 100 μg/mL of streptomycin at 37°C in a 5% CO2 humidified incubator. The cells were grown to an 80% confluence before use in subsequent experiments.
In vitro tissue model (VEC tissue)
A human organotypic vaginal tissue model, EpiVaginal™ VEC-100-FT, was used to study the effects of bacterial cell-free supernatants on gene expression of tight junctions of fully differentiated vaginal epithelium. This model is reconstructed from primary human vaginal epithelial cells and fibroblasts and produced by the MatTek Corporation, Ashland, Massachusetts, USA. The EpiVaginal™ VEC-100-FT tissue is a three-dimensional tissue model reconstructed using normal vaginal ectocervical epithelial cells and is well stratified, containing differentiated basal, suprabasal, intermediate, and superficial cell layers similar to in vivo tissue 24.
Treatment of vaginal epithelial cells and tissues
To model the vaginal epithelium, vaginal epithelial cells (VK2/E6E7) and epithelial tissues (MatTek VEC-100-FT Epivaginal™, (VEC)) were used. VEC was continuously treated on the apical surface with 50uL (50% v/v) of bacterial cell-free supernatants for 18 hours at 37°C, 5% carbon dioxide. VK2/E6E7 cells were plated at 1.0 × 106 cells/ml into culture plates and treated with 1mL (50%v/v) of bacterial cell-free supernatants for 18 hours at 37°C, 5% carbon dioxide. To mimic a diverse microbiota, we used combinations of G.vaginalis and L. crispatus or G.vaginalis and L.iners supernatants at final concentrations of 25% (v/v) of each supernatant. At the end of each experiment, cell culture media were collected for multiplex cytokine assays and/or the cells and tissues were collected in RNAlater for RNA extraction. Experiments were performed in triplicate or duplicate.
Multiplex cytokine assay
To establish the inflammatory state within the vaginal micro-environment, we measured cytokines in CVL and within the supernatants from the in-vitro experiments. For the CVLs, the cytokines IL-1β, IL-1RA, IL-6, IL-8, IL-10, and TNF-α were measured. All samples were run in duplicate as per the manufacturer’s instructions. Vaginal epithelial cells and tissue were treated with bacterial cell-free supernatant as described above. A 6-plex human cytokine magnetic bead panel (PPX-06-MXKA3DC, ProcartaPlex, ThermoFisher Scientific, Vienna, Austria) was run on apical and basal tissue culture supernatants and cell culture supernatants after 18 hours of treatment. We measured the concentrations of IL-1α, IL-1β, IL-1RA, IL-6, IL-8 and MCP-1.
Multiplex gene expression assay
A multiplex gene expression assay, QuantiGene™ Plex Gene Expression Assay (Affymetrix), was used to determine the changes in expression of cell junction proteins of the epithelial tissue namely; claudin-1 and 4, occludin, JAM-A, ZO-1 in response to treatment with the bacterial supernatants. The QuantiGene™ Plex assay is a probe hybridization-based method of target-specific RNA capture and quantification that utilizes branched DNA signal amplification and Luminex multi-analyte profiling technology. MatTek™ tissues were lysed using the QuantiGene™ sample processing kit for cultured cells (QS0100) as per manufacturer’s instructions. 10ul of Proteinase K was added per 1ml of lysis buffer. One part of this mixture was further diluted by adding it to two parts RNAse-free water. MatTek™ tissues were removed from their inserts and cut up before adding to the lysis buffer. Lysis of the tissue was achieved by vortexing for 1 minute and then incubating the tissue in lysis buffer at 50-55ºC for 30 minutes with gentle agitation. The lysed samples were kept on ice and used immediately for the QuantiGene™ Plex Gene Expression Assay (Affymetrix Inc., Santa Clara, USA) or stored at -80°C for later use, as per the manufacturer’s instructions. Output was initially analyzed using QuantiGene Plex Data Analysis application available from ThermoFisher.
Western blots for claudin-1 and e-cadherin
To quantify the changes in barrier permeability due to bacterial cell-free treatment, tight junction protein expression by VK2 cells was quantified by western blot. VK2 cells were plated in a 6-well tissue-culture treated plate at a concentration of 0.7x106 cells/mL in 1mL volume. The next day, the media was aspirated, and cell-free bacterial supernatants were added, diluted 50% (v/v) in K-SFM. After an 18-hour incubation, supernatants were collected and cells were lysed using 0.5mL of RIPA Lysis Buffer (Santa Cruz Biotechnology Inc, Santa Cruz, California, USA). Lysates were quantified by BCA and 15ug of total protein per sample was loaded and run on a 4-15% polyacrylamide gel (Bio-Rad, Hercules, California, USA). Proteins were transferred to a PVDF membrane and stained with antibodies at the following concentrations: rabbit anti-human claudin-4 at 1ug/mL (Thermo Fisher, Rockford, IL, United States, cat#51-9000), mouse anti-human e-cadherin at 1ug/mL (ThermoFisher cat#33-400), and rabbit anti-human beta-tubulin (Novus Biologicals, Colorado, USA, cat#NB600-936) at 1:1000. Anti-rabbit or anti-mouse HRP antibodies were used as secondary antibodies and the membrane was visualized using Pierce ECL Western Blotting Substrate (ThermoFisher cat#32106) on a luminescent imager (FujiFilm LAS-4000). Restore Western Blot Stripping Buffer (ThermoFisher cat#21059) was used to strip the membrane before re-staining with a different antibody. Protein expression was quantified using the gel analyzer tool in the Fiji distribution of ImageJ (version 1.52p) 25.
Staining for tight junctions
To assess effect of bacterial soluble factors on barrier integrity, we stained VK2 (E6/E7) cells treated with bacterial cell-free supernatants to localize tight junctions claudin-1 and claudin-4. Slides with VK2 (E6/E7) cells were fixed in acetone and rehydrated in Tris-Buffered saline (TBS, Biocare Medical Immunocare, Pacheco, California, USA, diluted to 1x with distilled water) prior to staining. Vaginal epithelial tissues were embedded in wax and sliced into 5-micron thick sections, thereafter rehydrated in decreasing concentrations of ethanol and subjected to heat-mediated antigen retrieval prior to staining. Slides were then washed liberally and subsequently blocked with 10% normal donkey serum in TBS (Jackson ImmunoResearch, West Grove, Pennsylvania, USA, cat#017-000-001) for tissues sections and DAKO serum-free protein block (Agilent Technologies Inc, Santa Clara, California, USA, cat#X0909) for cells for 30 minutes. Rabbit anti-human monoclonal antibodies for claudin-1 and claudin-4 were diluted to 5ug/mL ThermoFisher catalog# 51-9000 and cat#PA5-16875) in DAKO antibody diluent with background reducing components (Agilent Technologies Inc, Santa Clara, California, USA, DAKO cat#S3022) were added to each slide and incubated for one hour in a wet box with gentle shaking. Slides were then washed in TBS and thereafter, secondary antibody, Cy3 diluted 1:1000 or FITC diluted 1:200 in TBS was added to each tissue. Stained cells were incubated at room temperature for one hour in a wet box under darkness and with gentle shaking. Vectashield anti-fade mounting medium with DAPI (Novus Biologicals, Colorado, USA, cat#H-1200) were added to stained cells after washing with TBS.
Statistical analysis
Statistical analysis and visualization of data was done in GraphPad Prism version 8.2.1. Comparisons between cytokine or tight junction proteins levels between two unpaired groups were made using Mann-Whitney test and statistical significance was set at p<0.005.