The Impact of the Vaginal Microbiome on Vaginal Epithelial Wound Healing

doi:10.21203/rs.3.rs-3282980/v1

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The Impact of the Vaginal Microbiome on Vaginal Epithelial Wound Healing

https://doi.org/10.21203/rs.3.rs-3282980/v1

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The human microbiome has been shown to be an important player in a variety of health outcomes. Within the female genital tract specifically, clinical data has shown an association between bacterial vaginosis-associated anaerobes and postoperative infections after pelvic surgery. This study investigates vaginal epithelial healing when exposed to conditioned media from commensal Lactobacillus spp. and BV-associated spp. using an in vitro model. We demonstrated improved wound healing when exposed to Lactobacillus conditioned media when compared to that of pathogenic bacteria. These differences in wound healing corresponded to increased levels of CM-CSF, IL-6, IL-7, IL-8, TNF-a, and VEGF as well as decreased levels of IL-1B and IFN-γ. These findings suggest an important role for the vaginal microbiome in tissue healing potentially mediated by modifiable immunologic markers.

Biological sciences/Physiology/Reproductive biology

Biological sciences/Microbiology/Communities

Biological sciences/Immunology/Mucosal immunology

Wound healing involves a complex interplay of multiple intrinsic and extrinsic factors. One important, but understudied factor is the skin microbiome; commensal species are essential for the regulation of the cutaneous immune system and provide protection from intracellular pathogens¹. Studies have shown interactions between commensal microbiota and innate immune responses to be important to normal healing^2,3. In contrast, pathogenic microorganisms are thought to play a role in delayed wound healing in non-healings skin ulcers⁴. In diabetic wounds skin microbes are associated with outcomes and can even be used as an indicator of healing prognosis⁵. Identification of interactions between the skin microbiome and wound healing has led to recent advances targeting the microbiome to improve healing outcomes⁶. Within the oral microbiome alterations in microbial communities overall are associated with dental caries⁷, peri-implantitis and periodontitis⁸, and severity of bone loss around non-submerged implants⁹. Oral mucosal healing is impacted by the composition of the oral microbiome and delayed healing can result from both the bacterial load and composition¹⁰. In mice, altered oral microbiomes are associated with a deficiency of oral mesenchymal stem cells, which contributes to impaired wound healing¹¹. These studies further suggest that throughout mucosal surfaces in the body, the microbiome plays an important role in clinical healing outcomes.

The restoration of the vaginal epithelial barrier is an important step in wound healing after gynecologic surgery. The vaginal microbial community associated with optimal surgical and reproductive health outcomes is low diversity and dominated by Lactobacillus species. These microbes produce lactic acid, which is anti-inflammatory in vitro, and may promote epithelial barrier integrity. Bacterial vaginosis is a syndrome characterized by a high diversity vaginal microbial community comprised of a mixture of anaerobic and facultative bacteria, such as Gardnerella vaginalis¹². Clinically, BV is associated with a variety of poor outcomes including surgical site infection after pelvic surgery, specifically vaginal cuff cellulitis¹³. The mechanism behind this clinical finding is not well understood. In vitro, BV-associated microbes decrease epithelial barrier integrity and increase inflammation^14,15, suggesting that wound complications may be linked to impaired healing in the presence of BV-associated bacterial species.

We hypothesized that BV-associated bacterial species would be associated with decreased healing in a vaginal epithelial injury model.

Lactobacillus crispatus, L. iners, Gardnerella vaginalis and Atobobium vaginae (now known as Fannyhessea vaginae) were grown in NYCIII media to stationary phase, centrifuged and filtered to create bacterial cell free supernatants (CFS). This was added at 5% and 20% concentration to confluent culture vaginal epithelial cells (VK2) which had undergone scratch injury, and closure of the scratch visualized at 4, 8, 12 and 24 hours. At 5% concentration, there were no observed differences in wound healing between CFS from different species at any timepoint [Supp Fig. 1]. However, cells exposed to CFS did demonstrate more healing than NYCIII media controls at 4 and 8 hours of healing. With 20% concentration of CFS, pairwise comparisons demonstrated significant differences between L. crispatus and A. vaginae CFS exposure at 12 [Supp Fig. 1] and 24 hours [Fig. 1] Overall, wound healing occurred more slowly with 20% CFS compared to 5% CFS.

After 24 hours of exposure, supernatant was collected and immune markers were measured using Luminex and ELISA. At 24 hours, higher levels of GM-CSF, IL-6, IL-8, and TNF-a were seen after exposure to 5% L. iners and A. vaginae CFS compared with L. crispatus. Additionally, higher levels of IL-6 and VEGF were seen after exposure to L. iners CFS compared with G. vaginalis [Fig. 2]. With 20% CFS, we found higher levels of IL-6 and IL-7 after exposure to the L. crispatus and L. iners CFS when compared to G. vaginalis and A. vaginae. Higher levels of IL-1β were seen with exposure to L. iners CFS when compared to A. vaginae. Finally higher levels of VEGF were seen with L. crispatus CFS when compared to G. vaginalis [Fig. 3].

When comparing mean soluble cytokine levels for all bacterial CFS conditions between the 5% concentration and 20% concentration assays, significantly higher levels of GM-CSF, IL-6, IL-7, IL-8, VEGF, and TNF-α were observed in the 20% CFS concentration, while levels of IFN-γ and IL-1B were significantly lower [Fig. 4].

We demonstrated that vaginal epithelial wound healing is slower in the presence of soluble metabolites from the BV-associated species G. vaginalis and A. vaginae when compared to commensal L. crispatus.

Our findings are consistent with prior data, including a study using MS74 immortalized vaginal cells which demonstrated that exposure to both live L crispatus and CFS accelerated epithelial cell repair in a scratch assay, accompanied by an increase in VEGF particularly along the scratch edge¹⁶. A second study using HeLa cells noted that diverse bacterial communities dominated by G. vaginalis are associated with epithelial barrier disruption and enhanced immune activation, while low diversity communities dominated by Lactobacillus species were associated with increased expression of host mucosal proteins that are protective of the mucosal barrier¹⁷. The same study also performed a scratch healing assay and found that soluble products from G. vaginalis inhibited wound healing while those derived from L. iners did not. Overall these studies share some similarities with our findings in that commensal species are beneficial when compared to BV associated species for vaginal epithelial healing. However, differences in the precise findings may be due to variations in experimental design such as the usage of HeLa cells which are derived from cervical cancer cells and thus may behave differently than VK2 immortalized vaginal epithelial cells.

Clinical studies have suggested that patients with BV are at increased risk for cuff cellulitis following hysterectomy^13,18. Notably, in those older studies¹⁹ prophylactic antibiotics were not routine, and newer large-scale studies in the era of preoperative antibiotic prophylaxis have shown a decreased incidence of surgical site infection in patients receiving a B-lactam antibiotics with metronidazole compared to B-lactam antibiotics alone²⁰. Another study found that preoperative administration of vaginal metronidazole significantly decreased both pelvic and genitourinary infections following radical hysterectomy²¹. These findings together suggest that BV associated organisms, which are targeted by medications like metronidazole but not by B-lactams, may indeed play a role in surgical healing and postoperative outcomes like surgical site infection.

While it may seem obvious that having more anaerobic bacteria would lead to more infection, these microbes alone do not usually cause abscesses or even clinical vaginal erythema or induration. After surgery, restoration of the epithelial barrier is important to prevention of tissue invasion by pathogens. BV associated organisms like G. vaginalis have been shown to negatively impact the vaginal mucosa as a barrier by degrading mucin which functions as a dense physical barrier in the vaginal wall²² and may prevent infiltration by other pathogens. The vaginal mucosal barrier is also negatively impacted by the secretion of the pore forming toxin vaginolysin by G. vaginalis which results in the formation of microscopic defects in the vaginal epithelium²³ and further compromises the integrity of the mucosal barrier. Conversely, a L. crispatus dominated microbiota has been shown to reinforce the cervicovaginal mucus barrier and protect against HIV penetration²⁴. Lactic acid has been shown to promote production of anti-inflammatory cytokines like IL-1RA and inhibit production of inflammatory cytokines and chemokines like IL-6, TNFa, IL-8, RANTES, and MIP-3a in the presence of toll like receptors^25,26.

In our data, low concentrations of cell free supernatant did not induce significant differences in vaginal epithelial wound healing. However, at higher concentrations conditioned media from commensal Lactobacilli species resulted in faster healing than CFS from BV associated species. Exposure to CFS at either concentration was associated with differences in the soluble cytokines and chemokines found at the conclusion of the scratch healing assay. The biochemistry of wound healing is a complex process that is still under investigation and there are limited data about the mechanisms of vaginal mucosal healing. More data regarding the roles of certain cytokines and chemokines exists in other mucosal or dermal healing literature.

In our study, GM-CSF was found in higher quantities after exposure to the lower concentration of L. iners and A. vaginae CFS compared to the L. crispatus, although this difference was not statistically significant at higher concentrations. Overall we observed a dose response increase in GM-CSF: at higher concentrations of L. crispatus CFS we saw higher concentrations of GM-CSF, as well as improved scratch healing in comparison to BV associated species. These findings suggesting a role for GM-CSF in vaginal epithelial cell wound healing. A study of gut mucosal healing reported that GM-CSF drives the maturation and polarization of inflammatory intestinal macrophages which promotes anti-microbial functions while also inhibiting pro-repair functions associated with fibrosis and stricturing²⁷ and these findings may suggest a similar positive role in vaginal mucosal healing. Similarly, for IL-8 and TNF-α, levels were higher after exposure to L. iners and A. vaginae compared with L. crispatus at low concentrations. Levels of IL-8 and TNF-α seen in all bacterial CFS conditions increased without significant differences between them at higher concentrations of conditioned media where differences in wound healing were observed. These findings suggest a role for increased levels of these molecules in moderating vaginal mucosal healing.

Many studies in wound healing have demonstrated a complex process that includes macrophages, neutrophils, fibroblasts and platelets and molecules including interleukins (IL), tumor necrosis factor alpha (TNF-α), and growth factors. These studies have also shown that high levels of cytokines like IL-1 and IL-6 are associated with non-healing wounds²⁸. On the other hand, deletion of IL-1 receptor signaling impairs oral wound healing but has relatively little impact on dermal healing²⁹. Another study found that IL-6 deficient mice had significantly delayed wound healing³⁰. Higher levels of pro-inflammatory IL-1α, IL-2, IL-4, and IL-5, IL-6, GM-CSF, and TNF-α as well as lower levels of anti-inflammatory IL-10 were found in patients with chronic non-healing diabetic foot ulcers compared with non-diabetic healing wounds³¹. These findings, when combined with our own observations suggest that inflammatory cytokines such as IL-1B, IL-6, IL-7, IL-8, and TNF- α, could play a critical role in facilitating wound healing in the vagina, and when affected by the vaginal microbiome may lead to abnormal wound healing.

Finally, we noted that levels of VEGF were significantly higher in L. crispatus conditions compared with G. vaginalis conditions at the higher concentrations of cell free supernatant where differences in wound healing were observed. Other studies have shown an important role in wound healing for mast cells that release inflammatory mediators like IL-6, IL-8, and VEGF, which increase endothelial permeability and vasodilation, and facilitate migration of inflammatory cells, mainly monocytes and neutrophils to the site of injury³². The same study also noted that mast cells are capable of activating fibroblasts and keratinocytes via IL-4 and VEGF. Our findings suggest that VEGF may play a similar mediator role within vaginal wound healing.

We demonstrated slower vaginal epithelial wound healing after exposure to conditioned media from BV associated bacterial species compared with commensal Lactobacillus species. This difference in healing phenotype can be correlated with higher levels of regulatory molecules including CM-CSF, IL-6, IL-7, IL-8, TNF-a, and VEGF and lower levels of IL-1B and IFN-γ. Further study is needed to better elucidate the physiology of vaginal wound healing and future studies may consider the addition of fibroblasts to better represent the full thickness of vaginal wall tissues, investigation into the interaction with polypropylene mesh as used in pelvic reconstructive surgery or include interventions such as vaginal estrogen to study its effect on wound healing in the context of the microbiome.

Vaginal Epithelial Cells

Immortalized vaginal epithelial cells (VK2, ATCC) were grown under aerobic conditions at 37°C in complete keratinocyte serum free (KSF) media (i.e. supplemented with 0.4 mM calcium chloride, 0.1 ng/mL epidermal growth factor and 0.05 mg/mL bovine pituitary extract per manufacturer specifications) (ThermoFisher) with 100ug/ml streptomycin and 100U/ml penicillin.

Cell Free Supernatant

Cell free supernatants, or conditioned media, were prepared for Lactobacillus crispatus (JV-V01), Lactobacillus iners (ATCC 55195), Gardnerella vaginalis (ATCC 49145), and Atopobium vaginae (BAA-55) by inoculating 20 ml of fresh NYC III media with a single colony from an agar plate and incubating the broth for 48 hours until turbid, indicating stationary phase of growth.. The bacterial culture was then centrifuged at 10,000 RCF for 10 minutes and the supernatant collected and filtered through 0.2 µM surfactant free cellulose acetate filter to create the final cell free supernatant. Lactic acid concentrations in the supernatants were measured using EnzyChrom L-Lactate and D-Lactate Assay Kits (BioAssay Systems) [Supp Table 1]

Scratch Healing Assay

For the scratch assay, 6-well cell culture plates were marked at 5 mm intervals on the bottom of each well. Two million VK2 cells were inoculated into each well in 8 mL complete KSF media. Cell cultures were incubated at 37°C for 48 hours to achieve complete confluence.

A scratch was made through the center of each well using a 200 µL sterile pipette tip traveling perpendicularly to the lines drawn on the bottom of the plate. The supernatant was then aspirated and replaced with either 8 ml of KSF or 8 ml of a premade solution of 5% or 20% NYCIII or cell free supernatant in KSF. Additional controls were prepared using 1 M L-lactic acid to achieve a final concentration of 2, 4, 8, or 16 mM in NYCIII. These were used to create lactic acid control solutions at 5% or 20% in KSF.

Images were captured at 4, 8, 12, and 24 hours using the EVOS M5000 imaging system (ThermoFisher) and analyzed using ImageJ (NIH). The width of the scratch was measured at each 5mm interval/marking for all time points captured. Immediately following the 24-hour time point, supernatant was aspirated for further biochemical analysis of soluble cytokines and chemokines. Epithelial cell viability was tested using an LDH Assay (Pierce) and maintained above 70% for all conditions at the end of the scratch healing assay [Supp Fig. 2].

Cell viability assay

Cell viability at the end of the assay with various concentrations of the analytes was evaluated using either trypan blue staining and cell counting in a hemocytometer or an LDH cell viability assay (Pierce LDH Cytotoxicity Assay Kit).

Cytokine and Chemokine Analysis

Cytokine and chemokine analysis was performed using the MILLIPLEX Human High Sensitivity T Cell Magnetic Bead Panel (HSTCMAG28SPMX13, Millipore) using manufacturer specified protocols. This panel measures GM-CSF IFNγ IL-1β IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 IL-10 IL-12 (p70) IL-13, and TNF-α. The range of detection for each analyte is as follows: GM-CSF: 1.22–5,000 pg/mL, IFNγ: 0.61–2,500 pg/mL, IL-1β: 0.49–2,000 pg/mL, IL-2: 0.49–2,000 pg/mL, IL-4: 1.83–7,500 pg/mL, IL-5: 0.49–2,000 pg/mL, IL-6: 0.18–750 pg/mL, IL-7: 0.37–1,500 pg/mL, IL-8: 0.31–1,250 pg/mL, IL-10: 1.46–6,000 pg/mL, IL-12 (p70): 0.49–2,000 pg/mL, IL-13: 0.24–1,000 pg/mL, TNFα: 0.45–1,750 pg/mL.

VEGF ELISA was performed using the Human VEGF DuoSet ELISA Kit (R&D Systems) and results measured at OD 570 nm using a BioTek Eon Microplate Spectrophotometer per manufacturer guidelines. Analysis was performed using a 1:2 dilution of the human cell culture supernatant following the manufacturer specified protocol. The range of detection for this assay is 31.2–2,000 pg/mL.

Data analysis

Measurements were imported into Prism Graphpad 9 for analysis. Values were compared across conditions using Kruskal-Wallis one way ANOVA with Dunn correction for multiple comparisons or Mann-Whitney test as described.

AUTHOR CONTRIBUTIONS

Joe Y Shi: conceptualization, methodology, formal analysis, investigation, writing, visualization

Agnes M Bergerat-Thompson: conceptualization, methodology, resources

Caroline M Mitchell: conceptualization, validation, writing, visualization, supervision

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author, JS, upon reasonable request.

ADDITIONAL INFORMATION

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported

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No competing interests reported.

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The Impact of the Vaginal Microbiome on Vaginal Epithelial Wound Healing

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