Multi-omics analysis of the cervical epithelial integrity of women using depot medroxyprogesterone acetate.

Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA (n = 32) or regularly cycling controls (n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition (Lactobacillus dominant and non-Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4+ cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.

Regular Use of Depot Medroxyprogesterone Acetate Causes Thinning of the Superficial Lining and Apical Distribution of Human Immunodeficiency Virus Target Cells in the Human Ectocervix.

BACKGROUND: The hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may be associated with an increased risk of acquiring human immunodeficiency virus (HIV). We hypothesize that DMPA use influences the ectocervical tissue architecture and HIV target cell localization. METHODS: Quantitative image analysis workflows were developed to assess ectocervical tissue samples collected from DMPA users and control subjects not using hormonal contraception. RESULTS: Compared to controls, the DMPA group exhibited a significantly thinner apical ectocervical epithelial layer and a higher proportion of CD4+CCR5+ cells with a more superficial location. This localization corresponded to an area with a nonintact E-cadherin net structure. CD4+Langerin+ cells were also more superficially located in the DMPA group, although fewer in number compared to the controls. Natural plasma progesterone levels did not correlate with any of these parameters, whereas estradiol levels were positively correlated with E-cadherin expression and a more basal location for HIV target cells of the control group. CONCLUSIONS: DMPA users have a less robust epithelial layer and a more apical distribution of HIV target cells in the human ectocervix, which could confer a higher risk of HIV infection. Our results highlight the importance of assessing intact genital tissue samples to gain insights into HIV susceptibility factors.

Preserved Mucosal-Associated Invariant T Cells in the Cervical Mucosa of HIV-Infected Women with Dominant Expression of the TRAV1-2-TRAJ20 T Cell Receptor α-Chain.

BACKGROUND: Mucosa-associated invariant T (MAIT) cells are innate-like T cells with specialized antimicrobial functions. Circulating MAIT cells are depleted in chronic human immunodeficiency virus (HIV) infection, but studies examining this effect in peripheral tissues, such as the female genital tract, are lacking. METHODS: Flow cytometry was used to investigate circulating MAIT cells in a cohort of HIV-seropositive (HIV+) and HIV-seronegative (HIV-) female sex workers (FSWs), and HIV- lower-risk women (LRW). In situ staining and quantitative polymerase chain reaction were performed to explore the phenotype of MAIT cells residing in paired cervicovaginal tissue. The cervicovaginal microbiome was assessed by means of 16S ribosomal RNA gene sequencing. RESULTS: MAIT cells in the HIV+ FSW group were low in frequency in the circulation but preserved in the ectocervix. MAIT cell T-cell receptor gene segment usage differed between the HIV+ and HIV- FSW groups. The TRAV1-2-TRAJ20 transcript was the most highly expressed MAIT TRAJ gene detected in the ectocervix in the HIV+ FSW group. MAIT TRAVJ usage was not associated with specific genera in the vaginal microbiome. CONCLUSIONS: MAIT cells residing in the ectocervix are numerically preserved irrespective of HIV infection status and displayed dominant expression of TRAV1-2-TRAJ20. These findings have implications for understanding the role of cervical MAIT cells in health and disease.

The healthy female microbiome across body sites: effect of hormonal contraceptives and the menstrual cycle.

STUDY QUESTION: How does hormonal contraceptive use and menstrual cycle phase affect the female microbiome across different body sites? SUMMARY ANSWER: The menstrual cycle phase, but not hormonal contraceptive use, is associated with the vaginal and oral but not the gut microbiome composition in healthy young women. WHAT IS KNOWN ALREADY: Women with low vaginal levels of Lactobacillus crispatus are at increased risk of pre-term birth, fertility treatment failure, sexually transmitted infections and gynaecological cancers. Little is known about the effect of hormonal fluctuations on other body site's microbiomes as well as the interplay between them. STUDY DESIGN, SIZE, DURATION: This study includes a cohort of 160 healthy young Danish women using three different contraceptive regimens: non-hormonal methods (n = 54), combined oral contraceptive (COC, n = 52) or levonorgestrel intrauterine system (LNG-IUS, n = 54). Samples were collected from four body sites during the menstrual cycle (menses, follicular and luteal phases) at Copenhagen University Hospital, Rigshospitalet, Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: The oral, vaginal, rectal and faecal microbiomes were characterized by shotgun sequencing. Microbial diversity and community distance measures were compared between study groups, menstrual phase timepoints and body sites. All participants answered an extensive questionnaire on current health, lifestyle and sex life. Confounding factors such as smoking, BMI and diet were analysed by PERMANOVA. Plasma oestradiol and progesterone levels are correlated with microbiome composition. MAIN RESULTS AND THE ROLE OF CHANCE: The use of COC and LNG-IUS was not associated with the microbiome composition or diversity. However, increased diversity in the vaginal microbiome was observed during menses, followed by a subsequent expansion of Lactobacillus spp. during the follicular and luteal phases which correlated with measured serum oestradiol levels (r = 0.11, P < 0.001). During menses, 89 women (58%) had a dysbiotic vaginal microbiome with <60% Lactobacillus spp. This declined to 49 (32%) in the follicular phase (P < 0.001) and 44 (29%) in the luteal phase (P < 0.001). During menses, bacterial richness and diversity in saliva reached its lowest point while no differences were observed in the faecal microbiome. The microbiome in different body sites was on average more similar within the same individual than between individuals, despite phase or hormonal treatment. Only the vagina presented a clear cluster structure with dominance of either L. crispatus, Lactobacillus iners, Gardnerella vaginalis or Prevotella spp. LARGE SCALE DATA: The microbiome samples analysed in this study were submitted to the European Nucleotide Archive under project number PRJEB37731, samples ERS4421369-ERS4422941. LIMITATIONS, REASONS FOR CAUTION: The cohort is homogenous which limits extrapolation of the effects of ethnicity and socio-economic status on the microbiome. We only present three defined timepoints across the menstrual phase and miss potential important day to day fluctuations. WIDER IMPLICATIONS OF THE FINDINGS: The use of hormonal contraception did not significantly associate with the microbiome composition in the vagina, faeces, rectum or saliva in healthy young women. This is a welcome finding considering the widespread and prolonged use of these highly efficient contraceptive methods. The menstrual cycle is, however, a major confounding factor for the vaginal microbiome. As such, the time point in the menstrual cycle should be considered when analysing the microbiome of women of reproductive age, since stratifying by vaginal dysbiosis status during menstruation could be misleading. This is the first study to confirm by direct measurements of oestradiol, a correlation with the presence of L. crispatus, adding evidence of a possible hormonal mechanism for the maintenance of this desirable microbe. STUDY FUNDING/COMPETING INTEREST(S): This work was partly funded by the Ferring Pharmaceuticals through a research collaboration with The Centre for Translational Microbiome Research (CTMR) at the Karolinska Institutet (L.W.H., E.F., G.E. and I.S.-K.). Ferring Pharmaceuticals also funded the infrastructure to obtain the clinical samples at Copenhagen University Hospital ([#MiHSN01], M.C.K., Z.B., and H.S.N.). This work was also supported by funding from Rigshospitalet's Research Funds ([#E-22614-01 and #E-22614-02] to M.C.K.) and Oda and Hans Svenningsen's Foundation ([#F-22614-08] to H.S.N.). M.C.K., L.W.H., E.F., Z.B., G.E., L.E., I.S.-K. and H.S.N., are partially funded by Ferring Pharmaceuticals, which also provided funds for the collection and processing of the samples analysed in this study. H.S.N.'s research is further supported by Freya Biosciences and the BioInnovation Institute. H.S.N. has received honoraria from Ferring Pharmaceuticals, Merck A/S, Astra-Zeneca, Cook Medical and Ibsa Nordic. A.N.A. reports no competing interests.

Human Immunodeficiency Virus-Infected Women Have High Numbers of CD103-CD8+ T Cells Residing Close to the Basal Membrane of the Ectocervical Epithelium.

Background: Genital mucosa is the main portal of entry for various incoming pathogens, including human immunodeficiency virus (HIV), hence it is an important site for host immune defenses. Tissue-resident memory T (TRM) cells defend tissue barriers against infections and are characterized by expression of CD103 and CD69. In this study, we describe the composition of CD8+ TRM cells in the ectocervix of healthy and HIV-infected women. Methods: Study samples were collected from healthy Swedish and Kenyan HIV-infected and uninfected women. Customized computerized image-based in situ analysis was developed to assess the ectocervical biopsies. Genital mucosa and blood samples were assessed by flow cytometry. Results: Although the ectocervical epithelium of healthy women was populated with bona fide CD8+ TRM cells (CD103+CD69+), women infected with HIV displayed a high frequency of CD103-CD8+ cells residing close to their epithelial basal membrane. Accumulation of CD103-CD8+ cells was associated with chemokine expression in the ectocervix and HIV viral load. CD103+CD8+ and CD103-CD8+ T cells expressed cytotoxic effector molecules in the ectocervical epithelium of healthy and HIV-infected women. In addition, women infected with HIV had decreased frequencies of circulating CD103+CD8+ T cells. Conclusions: Our data provide insight into the distribution of CD8+ TRM cells in human genital mucosa, a critically important location for immune defense against pathogens, including HIV.

A novel multiplexed immunoassay identifies CEA, IL-8 and prolactin as prospective markers for Dukes' stages A-D colorectal cancers.

BACKGROUND: Current methods widely deployed for colorectal cancers (CRC) screening lack the necessary sensitivity and specificity required for population-based early disease detection. Cancer-specific protein biomarkers are thought to be produced either by the tumor itself or other tissues in response to the presence of cancers or associated conditions. Equally, known examples of cancer protein biomarkers (e.g., PSA, CA125, CA19-9, CEA, AFP) are frequently found in plasma at very low concentration (pg/mL-ng/mL). New sensitive and specific assays are therefore urgently required to detect the disease at an early stage when prognosis is good following surgical resection. This study was designed to meet the longstanding unmet clinical need for earlier CRC detection by measuring plasma candidate biomarkers of cancer onset and progression in a clinical stage-specific manner. EDTA plasma samples (1 µL) obtained from 75 patients with Dukes' staged CRC or unaffected controls (age and sex matched with stringent inclusion/exclusion criteria) were assayed for expression of 92 human proteins employing the Proseek® Multiplex Oncology I proximity extension assay. An identical set of plasma samples were analyzed utilizing the Bio-Plex Pro™ human cytokine 27-plex immunoassay. RESULTS: Similar quantitative expression patterns for 13 plasma antigens common to both platforms endorsed the potential efficacy of Proseek as an immune-based multiplex assay for proteomic biomarker research. Proseek found that expression of Carcinoembryonic Antigen (CEA), IL-8 and prolactin are significantly correlated with CRC stage. CONCLUSIONS: CEA, IL-8 and prolactin expression were found to identify between control (unaffected), non-malignant (Dukes' A + B) and malignant (Dukes' C + D) stages.

Compositional and functional differences of the vaginal microbiota of women with and without cervical dysplasia.

Alterations in the vaginal microbiota, including both species composition and functional pathways, have been associated with HPV infection and progression of dysplasia to cervical cancer. To further explore this, shotgun metagenomic sequencing was used to taxonomically and functionally characterize the vaginal microbiota of women with and without cervical dysplasia. Women with histologically verified dysplasia (n = 177; low grade dysplasia (LSIL) n = 81, high-grade dysplasia (HSIL) n = 94, cancer n = 2) were compared with healthy controls recruited from the cervical screening programme (n = 177). Women with dysplasia had a higher vaginal microbial diversity, and higher abundances of Gardnerella vaginalis, Aerococcus christensenii, Peptoniphilus lacrimalis and Fannyhessea vaginae, while healthy controls had higher relative abundance of Lactobacillus crispatus. Genes involved in e.g. nucleotide biosynthesis and peptidoglycan biosynthesis were more abundant in women with dysplasia. Healthy controls showed higher abundance of genes important for e.g. amino acid biosynthesis, (especially L-lysine) and sugar degradation. These findings suggest that the microbiota may have a role in creating a pro-oncogenic environment in women with dysplasia. Its role and potential interactions with other components in the microenvironment deserve further exploration.

Distinct cervical tissue-adherent and luminal microbiome communities correlate with mucosal host gene expression and protein levels in Kenyan sex workers.

BACKGROUND: The majority of studies characterizing female genital tract microbiota have focused on luminal organisms, while the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that these communities may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a cohort of Kenyan female sex workers. RESULTS: We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus, followed by Prevotella, Atopobium, and Sneathia. About half of the L. iners-dominated luminal samples had a corresponding Gardnerella-dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus-dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbial community was associated with epithelial remodeling and pro-inflammatory pathways. Tissue-adherent communities dominated by L. iners and Gardnerella were associated with lower host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, although with a more pro-inflammatory profile for the Gardnerella-dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity. CONCLUSION: We identified ectocervical tissue-adherent bacterial communities in all study participants of a female sex worker cohort. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. We further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community could possibly act as a reservoir that seed the lumen with less optimal, non-Lactobacillus, bacteria. Video Abstract.

HIV-Exposed Seronegative Sex Workers Express Low T-Cell Activation and an Intact Ectocervical Tissue Microenvironment.

Immunological correlates of natural resistance to HIV have been identified in HIV-exposed seronegative (HESN) individuals and include a low-inflammatory genital mucosal status. The cervicovaginal epithelium has not been studied for such correlates despite constituting an important barrier against sexual HIV transmission. To fill this gap in knowledge, we collected samples of blood, cervical mononuclear cells, cervicovaginal lavage, and ectocervical tissue from Kenyan HESN sex workers (n = 29) and controls (n = 33). The samples were analyzed by flow cytometry, protein profiling, 16S rRNA gene sequencing, in situ image analysis, and tissue-based RNA sequencing. A significantly higher relative proportion of regulatory T cells in blood (B7+CD25hiFoxP3+CD127loCD4+ and B7+Helios+FoxP3+CD4+), and a significantly lower proportion of activated cervical T cells (CCR5+CD69+CD4+ and CCR5+CD69+CD8+), were found in the HESN group compared with the controls. In contrast, there were no statistically significant differences between the study groups in cervicovaginal protein and microbiome compositions, ectocervical epithelial thickness, E-cadherin expression, HIV receptor expression, and tissue RNA transcriptional profiles. The identification of an intact ectocervical microenvironment in HESN individuals add new data to current knowledge about natural resistance to sexual transmission of HIV.

Increased Cervical CD4+CCR5+ T Cells Among Kenyan Sex Working Women Using Depot Medroxyprogesterone Acetate.

Depot medroxyprogesterone acetate (DMPA) is the most common hormonal contraceptive used by women in sub-Saharan Africa, however, it has been epidemiologically associated with HIV infections. To assess whether DMPA has an effect on the number and activation of HIV target cells, this study assessed the levels and phenotype of blood- and mucosal-derived HIV target cells among women using DMPA. Thirty-five HIV uninfected women from the Pumwani Sex Worker cohort from Nairobi, Kenya were enrolled in the study (15 using DMPA and 20 not using hormonal contraception). Blood (plasma and peripheral blood mononuclear cells) and cervicovaginal (lavage, cervical cells, and ectocervical biopsies) samples were collected. Cellular phenotype and activation status were determined by flow cytometry, cytokine levels were assessed by bead array and image analysis assessed cell number and phenotype in situ. In blood, the proportion of HIV target cells and activated T cells was lower in DMPA users versus those not using hormonal contraceptives. However, analysis of cervical mononuclear cells showed that DMPA users had elevated levels of activated T cells (CD4+CD69+) and expressed lower levels of the HIV co-receptor CCR5 on a per cell basis, while tissue samples showed that in the ectocervix, DMPA users had a higher proportion of CD4+CCR5+ T cells. This study demonstrates that DMPA users had higher levels of activated T cells and HIV target cells in the genital tract. The increased pool of mucosal HIV target cells provides new biological information about the potential impact of DMPA on HIV susceptibility.

Impact of Q-Griffithsin anti-HIV microbicide gel in non-human primates: In situ analyses of epithelial and immune cell markers in rectal mucosa.

Natural-product derived lectins can function as potent viral inhibitors with minimal toxicity as shown in vitro and in small animal models. We here assessed the effect of rectal application of an anti-HIV lectin-based microbicide Q-Griffithsin (Q-GRFT) in rectal tissue samples from rhesus macaques. E-cadherin+ cells, CD4+ cells and total mucosal cells were assessed using in situ staining combined with a novel customized digital image analysis platform. Variations in cell numbers between baseline, placebo and Q-GRFT treated samples were analyzed using random intercept linear mixed effect models. The frequencies of rectal E-cadherin+ cells remained stable despite multiple tissue samplings and Q-GRFT gel (0.1%, 0.3% and 1%, respectively) treatment. Whereas single dose application of Q-GRFT did not affect the frequencies of rectal CD4+ cells, multi-dose Q-GRFT caused a small, but significant increase of the frequencies of intra-epithelial CD4+ cells (placebo: median 4%; 1% Q-GRFT: median 7%) and of the CD4+ lamina propria cells (placebo: median 30%; 0.1-1% Q-GRFT: median 36-39%). The resting time between sampling points were further associated with minor changes in the total and CD4+ rectal mucosal cell levels. The results add to general knowledge of in vivo evaluation of anti-HIV microbicide application concerning cellular effects in rectal mucosa.

Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

DNA vaccination with a single-plasmid construct coding for viruslike particles protects mice against infection with a highly pathogenic avian influenza A virus.

BACKGROUND: With seasonal outbreaks affecting millions of people each year and devastating pandemics, human influenza is a major health concern. The pandemic threat includes highly pathogenic avian influenza viruses (HPAIVs) that gained the ability to infect humans in Asia and quickly spread throughout the world. Major concerns have been raised regarding today's vaccine production systems against influenza viruses, and new strategies to design efficient vaccines are under intensive investigation. METHODS: We demonstrated elsewhere that viruslike particles (VLPs) incorporating HPAIV hemagglutinin induce strong humoral immune response when injected in mice. In the current study, we evaluated a novel strategy that combines the immunogenicity of influenza VLPs and the advantages of DNA vaccines. RESULTS: We developed minimal expression vectors encoding all genetic information necessary to produce H7N1 influenza VLPs. We showed that mice vaccinated with small DNA amounts developed specific, high-titer neutralizing antibodies against homologous H7N1 strain and were protected against lethal doses of an antigenically distinct H7N7 HPAIV. Moreover, using some of these constructs, we were able to raise cross-neutralizing antibodies against an unrelated H5N1 HPAIV. CONCLUSIONS: DNA vaccination with constructs coding for influenza VLP production is a promising strategy to induce protection against different influenza viruses.