Ectocervical Epithelial Cells Research Articles

Chlamydia-driven ISG15 expression dampens the immune response of epithelial cells independently of ISGylation.

Excessive inflammation upon Chlamydia trachomatis infection can cause severe damages in the female genital tract. This obligate intracellular bacterium develops mainly in epithelial cells, whose innate response contributes to the overall inflammatory response to infection. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) stimulates interferon γ (IFNγ) production and is required for bacterial clearance in several infectious contexts. Here, we describe and investigate the consequences of the increase in ISG15 expression by epithelial cells infected with C. trachomatis. Infection of HeLa cells and primary ecto-cervical epithelial cells resulted in a transcriptional upregulation of ISG15 expression. This did not involve the canonical type I interferon (IFN-I) signaling pathway and depended instead on the activation of the STING/TBK1/IRF3 pathway. The absence or reduction of ISG15 synthesis led to increased production of several cytokines and chemokines, including interleukin (IL) 6 and IL8. This implicates that ISG15 normally dampens the immune response induced by C. trachomatis infection in epithelial cells. ISG15 exerted its control from an intracellular location, but without involving ISGylation. Finally, higher levels of inflammation and delayed bacterial clearance were observed in the genital tracts of ISG15-KO mice infected by C. trachomatis compared with wild-type animals; however, IFNγ production was unchanged. Altogether, our data show that ISG15 expression acts as a brake on the immune response to C. trachomatis infection in epithelial cells and limits bacterial burden and inflammation in mice.IMPORTANCEInfection of epithelial cells by Chlamydia trachomatis elicits an innate immune response by these cells. The signaling pathways involved, and their outcomes, are still very poorly understood. In this paper, we described how Chlamydia infection triggered the expression of ISG15, a small molecule normally associated to type I interferon (IFN-I) signaling and control of INF-γ production. ISG15 synthesis by epithelial cells attenuated their immune response to Chlamydia infection. In mice, we observed that ISG15 displayed a marginal role in modulating the production of IFN-γ, a key component of the host immune response to infection, but facilitated bacterial clearance. Overall, our study strengthens the importance of ISG15 not only in the resolution of viral but also of bacterial infection and document its role of "immune brake" in the context of Chlamydia infection.

Effects of Ureaplasma parvum infection in the exosome biogenesis-related proteins in ectocervical epithelial cells.

Ureaplasma parvum is a mycoplasma commonly associated with female reproductive pathologies, such as preterm birth and infertility. It can survive intracellularly and utilize exosomes to propagate infection and its virulence factors. This study explored the differential protein composition of exosomes derived from normal and U. parvum-infected cells. We also investigated the impact of U. parvum on exosome biogenesis in ectocervical epithelial cells. Ectocervical epithelial (ECTO) cells were infected with U. parvum, and immunocytochemical staining was performed using U. parvum-specific marker multiple banded antigen (mba) and exosome marker CD9. NanoLC-MS/MS analysis was conducted to identify differentially expressed proteins in exosomes. Ingenuity Pathway Analysis (IPA) was performed to identify affected canonical pathways and biological functions associated with the protein cargo of exosomes. Western blot analysis of ECTO cells validated the proteomic findings in ECTO cells. U. parvum exhibited colonization of ECTO cells and colocalization with CD9-positive intraluminal vesicles. Proteomic analysis revealed decreased protein abundance and distinct protein profiles in exosomes derived from U. parvum-infected ECTO cells. Differentially expressed proteins were associated with clathrin-mediated endocytosis and various signaling pathways indicative of infection, inflammation, and cell death processes. Additionally, U. parvum infection altered proteins involved in exosome biogenesis. In ECTO cells, U. parvum infection significantly decreased clathrin, ALIX, CD9, and CD63 and significantly increased TSG101, Rab5, Rab35, and UGCG. These findings contribute to our understanding of the infection mechanism and shed light on the importance of exosome-mediated communication in the pathophysiology of diseases affecting the cervix, such as cervicitis and preterm birth.

Impact of bisphenol A on cell viability and inflammatory cytokine production in human cervical epithelial cells.

An intact cervix is a barrier that prevents pathogenic bacteria from invading the uterine and amniotic cavity during pregnancy. Its disruption is associated with ascending infection and adverse pregnancy outcomes. This study analyzed the effects of bisphenol A (BPA), a chemical used in plastics manufacturing, on cell death and inflammation in cervical epithelial cells. Ectocervical epithelial (ecto) and endocervical epithelial (endo) cells were treated with 100ng/mL and 300ng/mL of BPA for 48h. The cells were subjected to flow cytometry using annexin V and propidium iodide to determine apoptosis and necrosis, cell cycle analysis, and ELISA to determine the levels of inflammatory cytokines (IL-6, IL-8, and IL-10). Low-dose and high-dose BPA significantly increased the live ecto cell population dose-dependently. BPA did not have any noticeable effect on cell cycle progression in either cell type. BPA treatment also decreased the apoptotic ecto and endo cell population dose-dependently. Lastly, high dose BPA significantly increased IL-6 in ecto and endo cells. However, IL-8 and IL-10 were not affected by BPA treatments. Chemical exposure damage to the cervix can lead to adverse pregnancy outcomes. Our study showed that the BPA concentrations reported in pregnant subjects do not induce cervical cell toxicity . The decrease in apoptosis and increase in live cells may be a compensatory mechanism to preserve the integrity of the cervical epithelial layer.

Xenobiotic metabolites modify immune responses of the cervicovaginal epithelium: potential mechanisms underlying barrier disruption.

Xenobiotic metabolites are exogenous biochemicals that can adversely impact reproductive health. We previously identified xenobiotics in cervicovaginal fluid during pregnancy in association with short cervix. In other organ systems, xenobiotics can modify epithelial barrier function. We hypothesise that xenobiotics dysregulate epithelial cell and macrophage immune responses as a mechanism to disrupt the cervicovaginal barrier. In vitro cell culture system. Laboratory within academic institution. Vaginal, ectocervical and endocervical epithelial cell lines and primary macrophages. Cells were treated with diethanolamine (2.5 mM), ethyl glucoside (5 mM) or tartrate (2.5 mM) for 24 h. Cytokines and matrix metalloproteinases were measured in cell supernatants (n = 3 per condition). One-way analysis of variance (ANOVA) with Dunnett's test for multiple comparisons was performed. Diethanolamine induces inflammatory cytokines, whereas ethyl glucoside and tartrate generally exert anti-inflammatory effects across all cells. Diethanolamine increases interleukin 6 (IL-6), IL-8, interferon γ-induced protein 10 kDa (IP-10), growth-regulated oncogene (GRO), fractalkine, matrix metalloproteinase 1 (MMP-1), MMP-9 and MMP-10 (p < 0.05 for all), factors involved in acute inflammation and recruitment of monocytes, neutrophils and lymphocytes. Ethyl glucoside and tartrate decrease multiple cytokines, including RANTES and MCP-1 (p < 0.05 for all), which serve as chemotactic factors. Vaginal cells exhibit heightened inflammatory tone compared with cervical cells and macrophages, with a greater number of differentially expressed analytes after xenobiotic exposure. Xenobiotic metabolites present in the cervicovaginal space during pregnancy modify immune responses, unveiling potential pathways through which environmental exposures may contribute to the pathogenesis of cervical remodelling preceding preterm birth. Future work identifying xenobiotic sources and routes of exposure offers the potential to modify environmental risks to improve pregnancy outcomes.

Exosomes from Ureaplasma parvum-infected ectocervical epithelial cells promote feto-maternal interface inflammation but are insufficient to cause preterm delivery.

This study determined if exosomes from ectocervical epithelial (ECTO) cells infected with Ureaplasma parvum (U. parvum) can carry bacterial antigens and cause inflammation at the feto-maternal interface using two organ-on-chip devices, one representing the vagina-cervix-decidua and another one mimicking the feto-maternal interface, and whether such inflammation can lead to preterm birth (PTB). Exosomes from U. parvum-infected ECTO cells were characterized using cryo-electron microscopy, nanoparticle tracking analysis, Western blot, and Exoview analysis. The antigenicity of the exosomes from U. parvum-infected ECTO cells was also tested using THP-1 cells and our newly developed vagina-cervix-decidua organ-on-a-chip (VCD-OOC) having six microchannel-interconnected cell culture chambers containing cells from the vagina, ectocervical, endocervical, transformation zone epithelia, cervical stroma, and decidua. The VCD-OOC was linked to the maternal side of our previously developed feto-maternal interface organ-on-a-chip (FMi-OOC). Cell culture media were collected after 48 h to determine the cytokine levels from each cell line via ELISA. For physiological validation of our in vitro data, high-dose exosomes from U. parvum-infected ECTO cells were delivered to the vagina of pregnant CD-1 mice on E15. Mice were monitored for preterm birth (PTB, < E18.5 days). Exosomes from ECTO cells infected with U. parvum (UP ECTO) showed significant downregulation of exosome markers CD9, CD63, and CD81, but contained multiple banded antigen (MBA), a U. parvum virulence factor. Monoculture experiments showed that exosomes from UP ECTO cells delivered MBA from the host cell to uninfected endocervical epithelial cells (ENDO). Moreover, exposure of THP-1 cells to exosomes from UP ECTO cells resulted in increased IL-8 and TNFα and reduced IL-10. The OOC experiments showed that low and high doses of exosomes from UP ECTO cells produced a cell type-specific inflammatory response in the VCD-OOC and FMi-OOC. Specifically, exosomes from UP ECTO cells increased pro-inflammatory cytokines such as GM-CSF, IL-6, and IL-8 in cervical, decidual, chorion trophoblast, and amnion mesenchymal cells. The results from our OOC models were validated in our in vivo mice model. The inflammatory response was insufficient to promote PTB. These results showed the potential use of the VCD-OOC and FMi-OOC in simulating the pathophysiological processes in vivo.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human β-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin.

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Expanded Histopathology and Tropism of Ebola Virus in the Rhesus Macaque Model: Potential for Sexual Transmission, Altered Adrenomedullary Hormone Production, and Early Viral Replication in Liver

The pathogenesis of Ebola virus disease (EVD) is still incomplete, in spite of the availability of a nonhuman primate modelfor more than 4 decades. To further investigate EVD pathogenesis, a natural history study was conducted using 27 Chinese-origin rhesus macaques. Of these, 24 macaques were exposed intramuscularly to Kikwit Ebola virus and euthanized at predetermined time points or when end-stage clinical disease criteria were met, and 3 sham-exposed macaques were euthanized on study day 0. This study showed for the first time that Ebola virus causes uterine cervicitis, vaginitis, posthitis, and medullary adrenalitis. Not only was Ebola virus detected in the interstitial stromal cells of the genital tract, but it was also present in the epididymal and seminal vesicular tubular epithelial cells, ectocervical and vaginal squamous epithelial cells, and seminal fluid. Furthermore, as early as day 3 after exposure, Ebola virus replicative intermediate RNA was detected in Kupffer cells and hepatocytes. These findings in the nonhuman model provide additional insight into potential sexual transmission, possible disruption of sympathetic hormone production, and early virus replication sites in human EVD patients.

Cross talk: trafficking and functional impact of maternal exosomes at the feto-maternal interface under normal and pathologic states†.

Fetal cell-derived exosomes promote inflammation in uterine and cervical cells to promote labor and delivery. However, the effect of maternal exosomes on fetal cells is still not known. We tested the hypothesis that cervical cells exposed to infectious and oxidative stress (OS) signals produce exosomes that can induce inflammation at the feto-maternal interface (FMi). Exosomes isolated from medium samples from human ectocervical epithelial cells (ecto), endocervical epithelial cells (endo), and cervical stromal cells (stroma) in normal cell culture (control) or exposed to infection or OS conditions were characterized based on morphology, size, quantity, expression of tetraspanin markers, and cargo proteins. Human decidual cells, chorion trophoblast cells (CTC), chorion mesenchymal cells (CMC), amnion mesenchymal cells (AMC), and amnion epithelial cells (AEC) were treated with control, LPS-, or OS-treated cervical exosomes. Enzyme-linked immunosorbent assay for pro-inflammatory cytokines and progesterone was done to determine the recipient cells' inflammatory status. Ecto, endo, and stroma released ∼110nm, cup-shaped exosomes. LPS and OS treatments did not affect exosome size; however, OS significantly increased the number of exosomes released by all cervical cells. Cervical exosomes were detected by fluorescence microscopy in each target cell after treatment. Exosomes from LPS- and CSE-treated cervical cells increased the inflammatory cytokine levels in the decidual cells, CMC, AMC, and AEC. LPS-treated stromal cell exosomes increased IL-6, IL-8, and progesterone in CTC. In conclusion, infection and OS can produce inflammatory cargo-enriched cervical exosomes that can destabilize FMi cells. However, the refractoriness of CTC to exosome treatments suggests a barrier function of the chorion at the FMi.

Oxidative stress promotes cellular damages in the cervix: implications for normal and pathologic cervical function in human pregnancy†.

A physiologic increase in reactive oxygen species throughout pregnancy is required to remodel the cervix. Oxidative stress can cause cellular damage that contributes to dysfunctional tissue. This study determined the oxidative stress-induced cell fate of human cervical epithelial and cervical stromal cells. We treated the ectocervical and endocervical epithelial cells and cervical stromal cells with cigarette smoke extract, an oxidative stress inducer, for 48 h. Cell viability (crystal violet assay); cell cycle, apoptosis, and necrosis (flow cytometry); senescence (senescence-associated β-galactosidase staining); autophagy (staining for autophagosome protein, microtubule-associated protein 1 light chain 3B); stress signaler p38 mitogen-activated protein kinases pathway activation (western blot analyses); and inflammation by measuring interleukin-6 (enzyme-linked immunosorbent assay) were conducted after 48 h of cigarette smoke extract treatment. Oxidative stress induced reactive oxygen species production in cervical cells, which was inhibited by N-acetylcysteine. Oxidative stress promoted cell cycle arrest and induced necrosis in cervical cells. High senescence and low autophagy were observed in cervical stromal cells under oxidative stress. Conversely, senescence was low and autophagy was high in endocervical epithelial cells. Oxidative stress induced p38 mitogen-activated protein kinases (p38MAPK) activation in all cervical cells but only increased interleukin-6 production by the ectocervical epithelial cells. Inhibition of interleukin-6 production by a p38 mitogen-activated protein kinases inhibitor confirmed the activation of an oxidative stress-induced pathway. In conclusion, oxidative stress can promote cell death and sterile inflammation that is mediated by p38 mitogen-activated protein kinases activation in the cellular components of the cervix. These cellular damages may contribute to the normal and premature cervical ripening, which can promote preterm birth.

Primary ectocervical epithelial cells display lower permissivity to Chlamydia trachomatis than HeLa cells and a globally higher pro-inflammatory profile

The tumoral origin and extensive passaging of HeLa cells, a most commonly used cervical epithelial cell line, raise concerns on their suitability to study the cell responses to infection. The present study was designed to isolate primary epithelial cells from human ectocervix explants and characterize their susceptibility to C. trachomatis infection. We achieved a high purity of isolation, assessed by the expression of E-cadherin and cytokeratin 14. The infectious progeny in these primary epithelial cells was lower than in HeLa cells. We showed that the difference in culture medium, and the addition of serum in HeLa cultures, accounted for a large part of these differences. However, all things considered the primary ectocervical epithelial cells remained less permissive than HeLa cells to C. trachomatis serovar L2 or D development. Finally, the basal level of transcription of genes coding for pro-inflammatory cytokines was globally higher in primary epithelial cells than in HeLa cells. Transcription of several pro-inflammatory genes was further induced by infection with C. trachomatis serovar L2 or serovar D. In conclusion, primary epithelial cells have a strong capacity to mount an inflammatory response to Chlamydia infection. Our simplified purification protocol from human explants should facilitate future studies to understand the contribution of this response to limiting the spread of the pathogen to the upper female genital tract.

649 Cervical epithelial organ-on-chip: a platform to study cellular remodeling of the cervix

To develop an organ-on-chip for the cervical epithelial layer (CE-OOC) for testing the intercellular interactions between the epithelial cells from distinct regions of the cervix and their contributions in maintaining the integrity of the cervix during infection and inflammation often associated with spontaneous preterm birth. The CE-OOC is composed of two chambers connected by microchannels to culture ectocervical (Ecto) and endocervical (Endo) epithelial cells and recreated the cervical epithelial layer (Fig 1A-B). Using CE-OOC, we tested the effect of lipopolysaccharide (LPS; a proxy for infection), and tumor necrosis factor-alpha (TNFα; a proxy for inflammation), on cell fate (apoptosis and necrosis [flow cytometry] and senescence [senescence associated β-galactosidase staining], cellular migration, and epithelial-to-mesenchymal transition [EMT] using cytokeratin-18 [epithelial marker] and vimentin [mesenchymal marker] immunostaining) as well as on the secretion of inflammatory cytokines (IL-6 and IL-8 multiplex assay) inside the CE-OOC for 96 h. The ectocervical and the endocervical epithelial cells were successfully cultured in a CE-OOC for 96 h (Fig 1C). Using this OOC, we determined that LPS and TNFα induced irreversible EMT but only LPS increased the migration of Ecto and Endo. Co-culture allowed bidirectional cellular migration through microchannels that led to the development of the transformation zone. This mimicked, endocervical cells migrating towards the ectocervix, as seen in vivo. Apoptosis, necrosis, and senescence were significantly increased by LPS and TNFα treatments in both cell types (Fig 2A-B). LPS and TNFα increased the IL6- and IL-8 production by Ecto and Endo. CE-OOC established an in vitro model that recapitulated the ectocervical, the transformation zone, and the endocervical epithelial region of the cervix. This model allowed us to study the pathological effects of infection and inflammation in the distinct zones of the cervix. This CE-OOC can be used to study the epithelial layer in cervical remodeling during pregnancy and parturition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

673 Oxidative stress promotes pathologic cellular remodeling of the cervix: implications for pregnancy and parturition

A physiologic increase in reactive oxygen species (ROS) throughout pregnancy is required to remodel uterine tissues including the cervix. Oxidative stress (OS) and excessive ROS can cause cellular damages resulting in a dysfunctional tissue. This study determined the effects of OS and OS induced cell fate of cervical epithelial and stromal cells. Cervical cells were isolated and immortalized from biopsies after hysterectomy from women with benign gynecologic conditions. Ectocervical (Ecto) and endocervical epithelial cells (Endo) and cervical stromal cells (CSC) were treated with cigarette smoke extract (CSE; 1:50), an OS inducer, for 48 h. Cell viability (crystal violet assay), cell cycle, apoptosis and necrosis and (flow cytometry), senescence (senescence-associated b-galactosidase staining), autophagy (staining for autophagosome protein LC3B), stress signaler p38MAPK pathway activation (western blot analyses), and sterile inflammation by measuring IL-6 (ELISA) were conducted after 48 h of CSE treatment. Our main findings are as follows: (1) OS induced ROS production in cervical cells that was inhibited by the antioxidant N-acetyl cysteine (NAC). Distinct forms of cell fate induced by OS in different cell types are listed in Table 1. 2) OS induced apoptosis and necrosis in Ecto but only caused necrosis in Endo and CSC. 3) High senescence and low autophagy were observed in CSC under OS. Conversely, senescence was lower and autophagy was higher in Endo; 4) OS decreased cell cycle progressions in G1G0 phase and increased cells in G2 phase indicating a cell cycle arrest; 5) OS induced p38MAPK activation in all three cervical cells (Fig.1A), and increased IL-6 production. Inhibition of IL-6 production by a p38MAPK inhibitor confirmed activation of OS induced stress signaling pathway (Fig.1B). OS can cause cell cycle arrest, promote different forms of cell deaths, and sterile inflammation mediated by p38MAPK activation in the cellular components of the cervix. OS may contribute to accelerating the cervical ripening process which can predispose patients to preterm birth.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Sperm can act as vectors for HIV-1 transmission into vaginal and cervical epithelial cells.

Sperm are the major cells in semen. Human sperm possess a number of HIV-1 gp120 binding ligands including sulfogalactosylglycerolipid (SGG). However, the mechanisms of how sperm capture HIV-1 onto their surface are unclear. Furthermore, the ability of sperm to deliver HIV-1 to vaginal/cervical epithelial cells lining the lower female reproductive tract, as a first step in HIV-1 transmission, needs to be determined. Sperm from healthy donors were incubated with dual-tropic HIV-1CS204 (clinical isolate), and virus capture was determined by p24 antigen ELISA. The involvement of SGG in HIV-1 capture was assessed by determining Kd values of HIV-1 gp120-SGG binding as well as computational docking of SGG to the gp120 V3 loop. The ability of sperm-associated HIV-1 to infect peripheral blood mononuclear cells (PBMCs) and TZM-bl indicator cells was determined. Lastly, infection of vaginal (Vk2/E6E7), ectocervical (Ect1/E6E7), and endocervical (End1/E6E7) epithelial cells mediated by HIV-1-associated sperm was evaluated. Sperm were able to capture HIV-1 in a dose-dependent manner, and the capture reached a maximum within 5minutes. Captured HIV-1, however, could be removed from sperm by Percoll-gradient centrifugation. Affinity of gp120 for SGG was substantial, implicating sperm SGG in HIV-1 capture. Sperm-associated HIV-1 could productively infect PBMCs and TZM-bl cells, and was capable of being transmitted into vaginal/cervical epithelial cells. Sperm are able to capture HIV-1, which remains infectious and is able to be transmitted into vaginal/cervical epithelial cells, a result indicating the importance of sperm in HIV transmission.

Epithelial Cells and Fibroblasts from the Human Female Reproductive Tract Accumulate and Release TFV and TAF to Sustain Inhibition of HIV Infection of CD4+ T cells

Tenofovir (TFV) treatment of female reproductive tract (FRT) cells results in differential accumulation of intracellular Tenofovir diphosphate (TFV-DP) in different cell types, with greater concentrations in epithelial cells (100-fold) and fibroblasts (10-fold) than in CD4+ T cells. The possibility that TFV-DP accumulation and retention in epithelial cells and fibroblasts may alter TFV availability and protection of CD4+ T cells against HIV infection, prompted us to evaluate TFV and/or Tenofovir alafenamide (TAF) release from FRT cells. Endometrial, endocervical and ectocervical polarized epithelial cells and fibroblasts were pre-loaded with TFV or TAF, and secretions tested for their ability to inhibit HIV infection of activated blood CD4+ T cells. Epithelial cell basolateral secretions (1, 2 and 3 days post-loading), but not apical secretions, suppressed HIV infection of CD4+ T cells, as did secretions from pre-loaded fibroblasts from each site. Intracellular TFV-DP levels in epithelial cells following preloading with TFV or TAF correlated directly with ARV protection of CD4+ T cells from HIV infection. When added apically to epithelial cells, TFV/TAF was released basolaterally, in part through Multidrug Resistant Protein transporters, taken up by fibroblasts and released into secretions to partially protect CD4+ T cells. These findings demonstrate that epithelial cells and fibroblasts release TFV/TAF for use by CD4+ T cells and suggest that the tissue environment plays a major role in the sustained protection against HIV infection.

Overexpression of LINC00037 represses cervical cancer progression by activating mTOR signaling pathway.

Long non-coding RNAs have been reported to play crucial roles in tumorigenesis including cervical cancer. LINC00037 has been identified as a significant regulator in several cancers. Our study was aimed to investigate the function of LINC00037 in cervical cancer progression. LINC00037 was significantly downregulated in human cervical cancer cells (HeLa, HCC94, HT-3, Caski, and SiHa cells) compared with the ectocervical epithelial cells (End1/E6E7 cells). Overexpression of LINC00037 was able to inhibit cervical cancer cell proliferation, induce cell apoptosis, and restrain the cell migration/invasion capacity. Reversely, knockdown of LINC00037 exhibited an opposite process in vitro. mTOR has been recognized as an atypical serine/threonine kinase that is involved in regulating significant cellular functions. In our present study, we observed that the mTOR signaling pathway was strongly activated in human cervical cancer cells. Meanwhile, upregulation of LINC00037 contributed to the inactivation of mTOR signaling whereas downregulation of LINC00037 activated the pathway. Subsequently, in vivo animal models using SiHa cells were established and we proved that LINC00037 repressed cervical cancer progression via targeting the mTOR signaling pathway. All these findings implied that LINC00037 could regulate cervical cancer pathogenesis via mTOR signaling. In conclusion, a novel role of LINC00037 was manifested in cervical cancer progression.

Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR-429.

Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells. Silencing of MALAT1 obviously reduced cervical cell viability, induced cell apoptosis, and repressed cell invasion capacity. Conversely, overexpression of MALAT1 exhibited an opposite phenomenon. Furthermore, miR-429 was predicted as a direct target of MALAT1, and it was dramatically decreased in cervical cancer cells. It has been shown that miR-429 plays a crucial role in cervical cancer progression. In our current study, the targeting correlation between MALAT1 and miR-429 was confirmed by luciferase reporter assays and RIP experiments. Finally, in vivo animal models were established, and we indicated that MALAT1 inhibited cervical cancer progression via targeting miR-429. These findings revealed that MALAT1 can sponge miR-429 and regulate cervical cancer pathogenesis in vivo and in vitro. In conclusion, we indicated that the MALAT1/miR-429 axis was involved in cervical cancer development.

Long non-coding RNA BCYRN1 promotes the proliferation and metastasis of cervical cancer via targeting microRNA-138 in vitro and in vivo.

Cervical cancer is one of the most malignant types of tumor and the fourth leading cause of cancer-associated mortality in females worldwide. High expression of brain cytoplasmic RNA 1 (BCYRN1) has been detected in various tumors. The present study aimed to investigate the effect of BCYRN1 in the viability and motility of cervical cancer, and the relevant mechanism. The results demonstrated that BCYRN1 was upregulated in cervical cancer tissues compared with normal tissues. Elevated levels of BCYRN1 were also detected in three human cervical cancer cell lines (SiHa, HeLa and CaSki) compared with non-cancerous ectocervical epithelial cell line (Ect1/E6E7). The expression of BCYRN1 was suppressed following transfection with small interfering RNA (siRNA) in HeLa cells. The silence of BCYRN1 significantly reduced cell viability and motility. Furthermore, microRNA (miR)-138 was predicted as a direct target of BCYRN1 and the expression of miR-138 was elevated in HeLa cells transfected with BCYRN1 siRNA. Subsequently, elevated levels of miR-138 were suppressed by transfection with miR-138 inhibitor in HeLa cells pretreated with BCYRN1 siRNA. The targeting association between BCYRN1 and miR-138 was supported by luciferase reporter assays. Additionally, BCYRN1 siRNA partially counteracted the effect of miR-138 inhibitor on promoting cell viability and mobility in HeLa cells. Finally, the in vivo experiment verified that BCYRN1 siRNA was able to prevent tumor growth, and reduced the expression of migration marker proteins metalloproteinase 2 and vascular endothelial cell growth factor, with enhanced expression levels of miR-138. These results suggest that lncRNA BCYRN1 promotes the proliferation and invasion of cervical cancer via targeting miR-138.

Conditional cell reprogramming involves non-canonical β-catenin activation and mTOR-mediated inactivation of Akt.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-267362, converts primary epithelial cells growing in vitro into an undifferentiated adult stem cell-like state that is characterized by long-term proliferation. This cell culture method also maintains the proliferation of adult epithelial stem cells from various tissues. Both primary and adult stem cells retain their tissue-specific differentiation potential upon removal of the culture conditions. Due to the ability to modulate the proliferation and differentiation of the cells, this method is referred to as conditional reprogramming and it is increasingly being used in studies of tumor heterogeneity, personalized medicine and regenerative medicine. However, little is known about the biology of these conditionally reprogrammed (CR) cells. Previously we showed that β-catenin activation, a hallmark of stem cells in vivo, occurs in CR human ectocervical cells (HECs). Here we show that β-catenin-dependent transcription is necessary for the induction of epithelial stem cell markers, and that β-catenin is activated via a non-canonical pathway that is independent of Wnt and Akt/GSK-3. Active Akt actually decreases due to increased mTOR signaling, with a consequent increase in dephosphorylated, active GSK-3. Despite the increase in active GSK-3, β-catenin associates with protein phosphatase 2A (PP2A) and is activated. Inhibition of PP2A catalytic activity reduces both the level of active β-catenin and the acute induction of stem cell markers, suggesting an important role for PP2A in the activation of β-catenin. Moreover, we demonstrate similar results using human prostate and breast cells, indicating that these changes are not restricted to ectocervical epithelial cells and may represent a more fundamental property of conditional reprogramming.

Estradiol alters the immune-responsiveness of cervical epithelial cells stimulated with ligands of Toll-like receptors 2 and 4.

The mucosa of the female reproductive tract plays a pivotal role in host defence. Pregnancy must alter immunological mechanisms at this interface to protect the conceptus. We sought to determine how estradiol (E2) alters the immune-responsiveness of cervical epithelial cells to ligand stimulation of Toll-like receptor (TLR)-2 and -4. Human ectocervical epithelial cells (HECECs) were cultured and co-incubated with two concentrations of E2 and peptidoglycan (PGN) or lipopolysaccharide (LPS) over durations that ranged between 10 minutes and 18 hours. Cytometric Bead Array was performed to quantify eight cytokines in the supernatant fluid. In response to PGN, HECECs co-incubated with E2 released lesser quantities of IL-1ß and IFNγ, higher levels of RANTES, and variable levels of IL-6 and IL-8 than those not exposed to E2. In contrast, HECECs co-incubated with LPS and E2 secreted increased levels of IL-1ß, IL-6, IL-8, and IFNγ at 2 and 18 hours than HECECs not exposed to E2, and reduced levels of RANTES at same study time-points. Estradiol alters the immune-responsiveness of cultured HECECs to TLR2 and TLR4 ligands in a complex fashion that appears to vary with bacterial ligand, TLR subtype, and duration of exposure. Our observations are consistent with the functional complexity that this mucosal interface requires for its immunological roles.