This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Damalie Nakanjako
Makerere University College of Health Sciences
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0935-2112
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Women with diverse genital anaerobic bacterial communities, are at over 4-fold higher risk of HIV acquisition than women with a Lactobacillus-rich vaginal microbiome. The mechanisms underlying this are poorly understood. We set out to examine how vaginal microbiota diversity affects epithelial integrity in HIV sero-negative pregnant women. We also investigated how HIV infection alters mucosal integrity within the prevalent genital microbiome diversity.
Methods
We assessed epithelial permeability by measuring the concentrations of tight junction proteins, claudin-1 and zonula occludens- (ZO)-1, in cervico-vaginal lavages (CVL) by enzyme-linked immunosorbent assay (ELISA). Cytokines in the vaginal fluids were measured by multiplex magnetic bead assay to establish the inflammatory state.
Bacterial cell-free supernatants were used to treat vaginal epithelial cells and tissues, mimicking the in-vivo vaginal milieu. Gene and protein expression levels of tight junctions of vaginal epithelial cells and tissues in response to treatment were quantified by QuantiGene™ Plex Gene Expression Assay and by western blot respectively. The cytokine response of vaginal epithelial cells, VK2 (E6/E7, ATCC® CRL-2616™), to bacterial cell-free supernatants was measured by ELISA method.
Results
Among women with CT3 cervicotype, HIV sero-negative pregnant women had significantly higher claudin-1 in their CVL than the HIV-infected pregnant women (p=0.0011). IL-8 (p=0.0028), IL-1beta (p<0.0001) and TNF-alpha (p=0.0283) were significantly higher among HIV-negative pregnant women with a non-Lactobacillus dominant vaginal microbiota than those with a Lactobacillus-dominant vaginal microbiota. Bacterial cell-free supernatants from Lactobacillus elicited low levels of pro-inflammatory cytokines IL-1alpha, IL8, IL-6 and IL-1 beta in comparison to media and G. vaginalis. Treatment with G. vaginalis supernatant lowers claudin-1 and claudin-4 expression yet presence of either L. crispatus or L. iners mitigates this effect of G. vaginalis as observed by immuno-staining of treated vaginal cells.
Conclusions
Pregnant women with a non-Lactobacillus dominant microbiome had a disrupted epithelial barrier and elevated pro-inflammatory cytokines, making them at a higher risk of HIV acquisition than women with a Lactobacillus-dominant microbiome. Targeting vaginal microbiota and/or its effects on the vaginal epithelium and cervico-vaginal milieu can potentially lower rates of HIV acquisition and transmission.
Keywords
vaginal, microbiota, epithelial, tight junctions, HIV, susceptibility
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Damalie Nakanjako
Makerere University College of Health Sciences
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0935-2112
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 22 Feb, 2021
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Microbiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
Women with diverse genital anaerobic bacterial communities, are at over 4-fold higher risk of HIV acquisition than women with a Lactobacillus-rich vaginal microbiome. The mechanisms underlying this are poorly understood. We set out to examine how vaginal microbiota diversity affects epithelial integrity in HIV sero-negative pregnant women. We also investigated how HIV infection alters mucosal integrity within the prevalent genital microbiome diversity.
Methods
We assessed epithelial permeability by measuring the concentrations of tight junction proteins, claudin-1 and zonula occludens- (ZO)-1, in cervico-vaginal lavages (CVL) by enzyme-linked immunosorbent assay (ELISA). Cytokines in the vaginal fluids were measured by multiplex magnetic bead assay to establish the inflammatory state.
Bacterial cell-free supernatants were used to treat vaginal epithelial cells and tissues, mimicking the in-vivo vaginal milieu. Gene and protein expression levels of tight junctions of vaginal epithelial cells and tissues in response to treatment were quantified by QuantiGene™ Plex Gene Expression Assay and by western blot respectively. The cytokine response of vaginal epithelial cells, VK2 (E6/E7, ATCC® CRL-2616™), to bacterial cell-free supernatants was measured by ELISA method.
Results
Among women with CT3 cervicotype, HIV sero-negative pregnant women had significantly higher claudin-1 in their CVL than the HIV-infected pregnant women (p=0.0011). IL-8 (p=0.0028), IL-1beta (p<0.0001) and TNF-alpha (p=0.0283) were significantly higher among HIV-negative pregnant women with a non-Lactobacillus dominant vaginal microbiota than those with a Lactobacillus-dominant vaginal microbiota. Bacterial cell-free supernatants from Lactobacillus elicited low levels of pro-inflammatory cytokines IL-1alpha, IL8, IL-6 and IL-1 beta in comparison to media and G. vaginalis. Treatment with G. vaginalis supernatant lowers claudin-1 and claudin-4 expression yet presence of either L. crispatus or L. iners mitigates this effect of G. vaginalis as observed by immuno-staining of treated vaginal cells.
Conclusions
Pregnant women with a non-Lactobacillus dominant microbiome had a disrupted epithelial barrier and elevated pro-inflammatory cytokines, making them at a higher risk of HIV acquisition than women with a Lactobacillus-dominant microbiome. Targeting vaginal microbiota and/or its effects on the vaginal epithelium and cervico-vaginal milieu can potentially lower rates of HIV acquisition and transmission.
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
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