RESUMO
PROBLEM: Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide and known to increase the risk for HIV acquisition. Few studies have investigated how infection of epithelial cells compromises barrier integrity and antimicrobial response. METHOD OF STUDY: ECC-1 cells, a human uterine epithelial cell line, were treated with live and heat-killed C. trachomatis. Epithelial barrier integrity measured as transepithelial resistance (TER), chemokines antimicrobial levels, and antimicrobial mRNA expression was measured by ELISA and Real-time RT-PCR. RESULTS: Epithelial barrier integrity was compromised when cells were infected with live, but not with heat-killed, C. trachomatis. IL-8 secretion by ECC-1 cells increased in response to live and heat-killed C. trachomatis, while MCP-1, HBD2 and trappin2/elafin secretion decreased with live C. trachomatis. CONCLUSION: Live C. trachomatis suppresses ECC-1 innate immune responses by compromising the barrier integrity, inhibiting secretion of MCP-1, HBD2, and trappin-2/elafin. Differential responses between live and heat-killed Chlamydia indicate which immune responses are dependent on ECC-1 infection rather than the extracellular presence of Chlamydia.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Células Epiteliais/fisiologia , Anti-Infecciosos/metabolismo , Linhagem Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Elafina/genética , Elafina/metabolismo , Feminino , Regulação da Expressão Gênica , Temperatura Alta , Humanos , Imunidade Inata , Imunomodulação , Interleucina-8/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Útero/patologia , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
Tenofovir (TFV) is a reverse transcriptase inhibitor used in microbicide preexposure prophylaxis trials to prevent HIV infection. Recognizing that changes in cytokine/chemokine secretion and nucleotidase biological activity can influence female reproductive tract (FRT) immune protection against HIV infection, we tested the hypothesis that TFV regulates immune protection in the FRT. Epithelial cells, fibroblasts, CD4(+) T cells, and CD14(+) cells were isolated from the endometrium (Em), endocervix (Cx), and ectocervix (Ecx) following hysterectomy. The levels of proinflammatory cytokines (macrophage inflammatory protein 3α [MIP-3α], interleukin 8 [IL-8], and tumor necrosis factor alpha [TNF-α]), the expression levels of specific nucleotidases, and nucleotidase biological activities were analyzed in the presence or absence of TFV. TFV influenced mRNA and/or protein cytokines and nucleotidases in a cell- and site-specific manner. TFV significantly enhanced IL-8 and TNF-α secretion by epithelial cells from the Em and Ecx but not from the Cx. In contrast, in response to TFV, IL-8 secretion was significantly decreased in Em and Cx fibroblasts but increased with fibroblasts from the Ecx. When incubated with CD4(+) T cells from the FRT, TFV increased IL-8 (Em and Ecx) and TNF-α (Cx and Ecx) secretion levels. Moreover, when incubated with Em CD14(+) cells, TFV significantly increased MIP-3α, IL-8, and TNF-α secretion levels relative to those of the controls. In contrast, nucleotidase biological activities were significantly decreased by TFV in epithelial (Cx) and CD4(+) T cells (Em) but increased in fibroblasts (Em). Our findings indicate that TFV modulates proinflammatory cytokines, nucleotidase gene expression, and nucleotidase biological activity in epithelial cells, fibroblasts, CD4(+) T cells, and CD14(+) cells at distinct sites within the FRT.
Assuntos
5'-Nucleotidase/biossíntese , Adenina/análogos & derivados , Fármacos Anti-HIV/farmacologia , Citocinas/biossíntese , HIV-1/efeitos dos fármacos , Organofosfonatos/farmacologia , 5'-Nucleotidase/genética , Adenina/farmacologia , Adulto , Idoso , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colo do Útero/citologia , Colo do Útero/efeitos dos fármacos , Quimiocina CCL20/metabolismo , Endométrio/citologia , Endométrio/efeitos dos fármacos , Células Epiteliais/imunologia , Feminino , Fibroblastos/imunologia , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Pessoa de Meia-Idade , Mucosa/citologia , Mucosa/efeitos dos fármacos , Inibidores da Transcriptase Reversa/farmacologia , Tenofovir , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The conflicting results of recent pre-exposure prophylaxis (PrEP) trials utilizing tenofovir (TFV) to prevent HIV infection in women led us to evaluate the accumulation of intracellular TFV-diphosphate (TFV-DP) in cells from the female reproductive tract (FRT) and whether sex hormones influence the presence of TFV-DP in these cells. Following incubation with TFV, isolated epithelial cells, fibroblasts, CD4+ T cells and CD14+ cells from the FRT as well as blood CD4+ T cells and monocyte-derived macrophages convert TFV to TFV-DP. Unexpectedly, we found that TFV-DP concentrations (fmol/million cells) vary significantly with the cell type analyzed and the site within the FRT. Epithelial cells had 5-fold higher TFV-DP concentrations than fibroblasts; endometrial epithelial cells had higher TFV-DP concentrations than cells from the ectocervix. Epithelial cells had 125-fold higher TFV-DP concentrations than FRT CD4+ T cells, which were comparable to that measured in peripheral blood CD4+ T cells. These findings suggest the existence of a TFV-DP gradient in the FRT where epithelial cells > fibroblasts > CD4+ T cells and macrophages. In other studies, estradiol increased TFV-DP concentrations in endometrial and endocervical/ectocervical epithelial cells, but had no effect on fibroblasts or CD4+ T cells from FRT tissues. In contrast, progesterone alone and in combination with estradiol decreased TFV-DP concentrations in FRT CD4+ T cells. Our results suggest that epithelial cells and fibroblasts are a repository of TFV-DP that is under hormonal control. These cells might act either as a sink to decrease TFV availability to CD4+ T cells and macrophages in the FRT, or upon conversion of TFV-DP to TFV increase TFV availability to HIV-target cells. In summary, these results indicate that intracellular TFV-DP varies with cell type and location in the FRT and demonstrate that estradiol and/or progesterone regulate the intracellular concentrations of TFV-DP in FRT epithelial cells and CD4+ T cells.
Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/farmacologia , Estradiol/farmacologia , Organofosfatos/farmacologia , Progesterona/farmacologia , Adenina/farmacologia , Adulto , Idoso , Transporte Biológico/efeitos dos fármacos , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Colo do Útero/citologia , Colo do Útero/metabolismo , Colo do Útero/cirurgia , Endométrio/citologia , Endométrio/metabolismo , Endométrio/cirurgia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Pessoa de Meia-Idade , Especificidade de Órgãos , Cultura Primária de CélulasRESUMO
PROBLEM: Whether the concentrations of antiviral proteins, and anti-HIV activity, within human vaginal secretions change across the menstrual cycle is unknown. METHOD OF STUDY: Using a menstrual cup, vaginal secretions from pre-menopausal women were recovered at the proliferative (d6-8), mid-cycle (d13-15), and secretory (d21-23) stages of the menstrual cycle. Antiviral protein concentration was determined by ELISA, and anti-HIV activity assessed using the TZM-bl reporter cell line. RESULTS: CCL20, RANTES, elafin, HBD2, SDF-1α, and IL-8 levels were detectable in the secretions. Vaginal secretions had anti-HIV activity against specific clade B strains of HIV, with significant inhibition of IIIB and increased infectivity of transmitted/founder CH077.t. No significant differences in either antiviral protein concentration or anti-HIV activity with respect to menstrual cycle stage were measured, but marked differences were observed in both parameters over the course of the cycle between different women and in consecutive cycles from the same woman. CONCLUSION: The vagina contains a complement of antiviral proteins. The variation in anti-HIV activity demonstrates that immune protection in the vagina is not constant. Intra- and interindividual variations suggest that factors in addition to sex hormones influence antiviral protection. Lastly, the menstrual cup is a new model for recovering undiluted vaginal secretions from women throughout their reproductive life.
Assuntos
Secreções Corporais/imunologia , HIV/imunologia , Imunidade Inata/imunologia , Vagina/imunologia , Vagina/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Ciclo Menstrual , Pré-MenopausaRESUMO
The immune system in the female reproductive tract (FRT) does not mount an attack against human immunodeficiency virus (HIV) or other sexually transmitted infections (STI) with a single endogenously produced microbicide or with a single arm of the immune system. Instead, the body deploys dozens of innate antimicrobials to the secretions of the FRT. Working together, these antimicrobials along with mucosal antibodies attack viral, bacterial, and fungal targets. Within the FRT, the unique challenges of protection against sexually transmitted pathogens coupled with the need to sustain the development of an allogeneic fetus, has evolved in such a way that sex hormones precisely regulate immune function to accomplish both tasks. The studies presented in this review demonstrate that estradiol (E2 ) and progesterone secreted during the menstrual cycle act both directly and indirectly on epithelial cells, fibroblasts and immune cells in the reproductive tract to modify immune function in a way that is unique to specific sites throughout the FRT. As presented in this review, studies from our laboratory and others demonstrate that the innate and adaptive immune systems are under hormonal control, that protection varies with the stage of the menstrual cycle and as such, is dampened during the secretory stage of the cycle to optimize conditions for fertilization and pregnancy. In doing so, a window of STI vulnerability is created during which potential pathogens including HIV enter the reproductive tract to infect host targets.
Assuntos
Estradiol/imunologia , Genitália Feminina/imunologia , Infecções por HIV/imunologia , Imunidade nas Mucosas , Mucosa/imunologia , Progesterona/imunologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Genitália Feminina/virologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Tolerância Imunológica , Linfócitos/imunologia , Linfócitos/virologia , Macrófagos/imunologia , Macrófagos/virologia , Ciclo Menstrual/imunologia , Mucosa/patologia , Mucosa/virologia , GravidezRESUMO
Despite extensive studies of the mucosal immune system in the female reproductive tract (FRT) and its regulation by sex hormones, relatively little attention has been paid to the tissue environment in the FRT that regulates immune cell function. Consisting of secretions from epithelial cells (EC), stromal fibroblasts, and immune cells in tissues from the upper (Fallopian tubes, uterus, and endocervix) and lower (ectocervix and vagina) tracts, each tissue compartment is unique and precisely regulates immune cells to optimize conditions for successful pregnancy and protection against sexually transmitted diseases including HIV. Our goal in this review is to focus on the mucosal (tissue) environment in the upper and lower FRT. Specifically, this review will identify the contributions of EC and fibroblasts to the tissue environment and examine the impact of this environment on HIV-target cells. Much remains to be learned about the complex interactions with the tissue environment at different sites in the FRT and the ways in which they are regulated by sex hormones and chemical contraceptives. Awareness of the involvement of the tissue environment in determining immune cell function and HIV acquisition is crucial for understanding the mechanisms that lead to HIV prevention, acquisition, and the development of new therapeutic modalities of immune protection.
Assuntos
Microambiente Celular/imunologia , Genitália Feminina/imunologia , Hormônios Esteroides Gonadais/imunologia , Infecções por HIV/imunologia , Imunidade nas Mucosas , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Receptor 1 de Quimiocina CX3C , Células Epiteliais/imunologia , Células Epiteliais/virologia , Feminino , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Genitália Feminina/virologia , Hormônios Esteroides Gonadais/genética , HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Humanos , Gravidez , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/imunologiaRESUMO
Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4⺠T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24 hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5'-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5' nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4⺠T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4⺠T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.
Assuntos
5'-Nucleotidase/metabolismo , Adenina/análogos & derivados , Citocinas/metabolismo , HIV-1/fisiologia , Fatores Imunológicos/farmacologia , Organofosfonatos/farmacologia , Adenina/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunidade Inata/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Tenofovir , Fatores de TempoRESUMO
Mucosal tissues represent the front line in defense against potential pathogens, and one means by which mucosa provide protection is via the secretion of antimicrobials which can interfere with potential pathogens as well as recruit and modify the responses of immune cells. Here we describe adaptation of ELISA assays to microsphere format, facilitating simultaneous quantification of antimicrobial peptides including elafin, MIP3α, HBD2, HBD3, SLPI, RANTES, SDF1, lactoferrin, LL-37, and HNP1-3. The multiplexed assay exhibits excellent reproducibility, shows linearity over a two order of magnitude concentration range for most analytes, is compatible with biological fluids such as cervicovaginal lavage fluid, and presents significant cost and sample savings relative to traditional ELISA assays.
Assuntos
Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/metabolismo , Líquidos Corporais/química , Líquidos Corporais/citologia , Ensaio de Imunoadsorção Enzimática , Líquidos Corporais/imunologia , Feminino , HumanosRESUMO
The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5'-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5'-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.
Assuntos
5'-Nucleotidase/metabolismo , Células Epiteliais/efeitos dos fármacos , Estradiol/farmacologia , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , 5'-Nucleotidase/genética , Separação Celular , Células Cultivadas , Colo do Útero/citologia , Colo do Útero/efeitos dos fármacos , Colo do Útero/enzimologia , Citosol/efeitos dos fármacos , Citosol/enzimologia , Endométrio/citologia , Endométrio/efeitos dos fármacos , Endométrio/enzimologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Especificidade de Órgãos , Vagina/citologia , Vagina/efeitos dos fármacos , Vagina/enzimologiaRESUMO
The magnitude of the HIV epidemic in women requires urgent efforts to find effective preventive methods. Even though sex hormones have been described to influence HIV infection in epidemiological studies and regulate different immune responses that may affect HIV infection, the direct role that female sex hormones play in altering the susceptibility of target cells to HIV-infection is largely unknown. Here we evaluated the direct effect of 17-ß-estradiol (E2) and ethinyl estradiol (EE) in HIV-infection of CD4(+) T-cells and macrophages. Purified CD4(+) T-cells and monocyte-derived macrophages were generated in vitro from peripheral blood and infected with R5 and X4 viruses. Treatment of CD4(+) T-cells and macrophages with E2 prior to viral challenge reduced their susceptibility to HIV infection in a dose-dependent manner. Addition of E2 2 h after viral challenge however did not result in reduced infection. In contrast, EE reduced infection in macrophages to a lesser extent than E2 and had no effect on CD4(+) T-cell infection. Reduction of HIV-infection induced by E2 in CD4(+) T-cells was not due to CCR5 down-regulation, but was an entry-mediated mechanism since infection with VSV-G pseudotyped HIV was not modified by E2. In macrophages, despite the lack of an effect of E2 on CCR5 expression, E2-treatment reduced viral entry 2 h after challenge and increased MIP-1ß secretion. These results demonstrate the direct effect of E2 on susceptibility of HIV-target cells to infection and indicate that inhibition of target cell infection involves cell-entry related mechanisms.
Assuntos
Linfócitos T CD4-Positivos/virologia , Estradiol/farmacologia , Estradiol/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Macrófagos/virologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Suscetibilidade a Doenças/imunologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Etinilestradiol/farmacologia , Etinilestradiol/uso terapêutico , Feminino , Humanos , Ligantes , Macrófagos/efeitos dos fármacos , Receptores CCR5/metabolismo , Receptores de Estrogênio/metabolismo , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
PROBLEM: Vaginal epithelial cells (VEC) are the first line of defense against incoming pathogens in the female reproductive tract. Their ability to produce the anti-HIV molecules elafin and HBD2 under hormonal stimulation is unknown. METHOD OF STUDY: Vaginal epithelial cells were recovered using a menstrual cup and cultured overnight prior to treatment with estradiol (E2), progesterone (P4) or a panel of selective estrogen response modulators (SERMs). Conditioned media were recovered and analyzed for protein concentration and anti-HIV activity. RESULTS: E2 significantly decreased the secretion of HBD2 and elafin by VEC over 48 hrs, while P4 and the SERMs (tamoxifen, PHTTP, ICI or Y134) had no effect. VEC conditioned media from E2 -treated cells had no anti-HIV activity, while that from E2 /P4 -treated cells significantly inhibited HIV-BaL infection. CONCLUSION: The menstrual cup allows for effective recovery of primary VEC. Their production of HBD2 and elafin is sensitive to E2, suggesting that innate immune protection varies in the vagina across the menstrual cycle.
Assuntos
Elafina/metabolismo , Células Epiteliais/efeitos dos fármacos , Estradiol/farmacologia , Vagina/imunologia , beta-Defensinas/metabolismo , Adulto , Fármacos Anti-HIV/análise , Células Cultivadas , Meios de Cultivo Condicionados/química , Elafina/análise , Células Epiteliais/imunologia , Feminino , Humanos , Imunidade Inata , Pessoa de Meia-Idade , Progesterona/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Vagina/citologia , beta-Defensinas/análiseRESUMO
PROBLEM: Estradiol regulates chemokine secretion from uterine epithelial cells, but little is known about estradiol regulation in vivo or the role of estrogen receptors (ERs). METHOD: CCL20 and CXCL1 present in reproductive washes following treatment with selective estrogen receptor modulators (SERMs) were compared with that during estrous and following estradiol-treated ovariectomized BALB/c mice. Cellular regulation was determined using isolated vaginal and uterine epithelial/stromal cells in vitro. RESULTS: Uterine and vaginal chemokine secretion is cyclically regulated with CCL20 at low levels but CXCL1 at high levels during high estradiol, generally mimicking estradiol effect in vivo. ERα but not ERß regulated CCL20/CXCL1 secretion by uterine epithelial cells in vitro and vaginal CCL20 in vivo. Estradiol/SERMs failed to alter uterine CCL20 secretion in ovariectomized mice. Diminished uterine epithelial ERα staining following ovariectomy corresponded with estradiol unresponsiveness of uterine tissue. CONCLUSION: Estrogen receptors α regulates CCL20/CXCL1 secretion in the female reproductive tract, and ERα antagonists directly oppose the regulation by estradiol. Understanding ER-mediated antimicrobial chemokine expression is important to elucidate cyclic susceptibility to sexually transmitted pathogens.
Assuntos
Quimiocina CCL20/metabolismo , Quimiocina CXCL1/metabolismo , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Útero/efeitos dos fármacos , Vagina/efeitos dos fármacos , Animais , Células Epiteliais/efeitos dos fármacos , Receptor alfa de Estrogênio/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Relação Estrutura-Atividade , Útero/citologia , Útero/metabolismo , Vagina/citologia , Vagina/metabolismoRESUMO
PROBLEM: Expression patterns and regulation of cytosolic pattern recognition receptors (PRR) NOD-1, NOD-2, RIG-1, and MDA5 have not been elucidated in the human female reproductive tract (FRT). METHOD OF STUDY: Primary epithelial cells (EC) isolated from Fallopian tube (FT), endometrium (EM), cervix (Cx), and ectocervix (Ecx) were treated with estradiol, poly(I:C), Neisseria gonorrhea (GC), and HIV-1. PRR mRNA expressions were analyzed by Real-time RT-PCR. Conditioned media were analyzed for IL-8 by ELISA. RESULTS: EC from all FRT compartments constitutively expressed NOD1, NOD2, RIG-1, and MDA5 with highest levels expressed by FT. Stimulation with poly(I:C) resulted in upregulation of NOD2, RIG-1, and MDA5 in all FRT compartments and correlated with increased secretion of IL-8, whereas estradiol treatment had no effects. Exposure to GC and HIV-1 IIIB but not BaL resulted in selective upregulation of NOD2 and MDA5. CONCLUSION: PRR are expressed throughout the FRT and differentially regulated by poly(I:C), GC and HIV-1.
Assuntos
Células Epiteliais/imunologia , Genitália Feminina/imunologia , Gonorreia/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Neisseria gonorrhoeae/imunologia , Infecções do Sistema Genital/imunologia , Células Cultivadas , Citosol/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células Epiteliais/microbiologia , Estradiol/imunologia , Feminino , Regulação da Expressão Gênica/imunologia , Genitália Feminina/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Helicase IFIH1 Induzida por Interferon , Interleucina-8/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Poli I-C/imunologia , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismoRESUMO
This study investigates the cyclic changes in innate immunity in the female reproductive tract (FRT) of mice during the estrous cycle. By examining uterine and vaginal tissues and secretions we show that innate immunity varies with the stage of the estrous cycle and site in the FRT. Secretions from the uterine lumen contained cytokines and chemokines that were significantly higher at proestrus and estrus relative to that measured at diestrus. In contrast, analysis of vaginal secretions indicated that only IL-1ß and CXCL1/mouse KC changed during the cycle, with highest levels measured at diestrus and estrus. In contrast, vaginal α-defensin 2 and ß-defensins 1-4 mRNA levels peaked at proestrus and estrus and are expressed 1-4 logs greater than that seen in the uterus. These studies further indicate that TLR5 and TLR12 in the uterus, and TLR1, TLR2, TLR5 and TLR13 in the vagina varies with stage of the estrous cycle, with some peaking at proestrus/estrus and others at diestrus. Overall, these studies indicate that innate immune parameters in the uterus and vagina are separate and discrete, and regulated precisely during the estrous cycle.
Assuntos
Quimiocina CXCL1/metabolismo , Ciclo Estral/imunologia , Útero/metabolismo , Vagina/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica/imunologia , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Útero/imunologia , Vagina/imunologia , alfa-Defensinas/genética , alfa-Defensinas/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
BACKGROUND: We have previously demonstrated intrinsic anti-HIV activity in cervicovaginal lavage (CVL) from HIV-infected women with high CD4 counts and not on antiretroviral therapy. However, the impact of HIV disease progression on CVL innate immune responses has not been delineated. METHODS: CVL from 57 HIV-infected women not on antiretroviral therapy were collected by washing the cervicovaginal area with 10 ml of sterile normal saline. We characterized subject HIV disease progression by CD4 count strata: >500 cells/µl, 200-500 cells/µl, or <200 cells/µl of blood. To assess CVL anti-HIV activity, we incubated TZM-bl cells with HIV plus or minus CVL. Antimicrobials, cytokines, chemokines and anti-gp160 HIV IgG antibodies were measured by ELISA and Luminex. RESULTS: CVL exhibited broad anti-HIV activity against multiple laboratory-adapted and transmitted/founder (T/F) viruses, with anti-HIV activity ranging from 0 to 100% showing wide variation between viral strains. Although there was broad CVL inhibition of most both laboratory-adapted and T/F virus strains, there was practically no inhibition of T/F strain RHPA.c, which was isolated from a woman newly infected via heterosexual intercourse. HIV disease progression, measured by declining CD4 T cell counts, resulted in a selective reduction in intrinsic anti-HIV activity in CVL that paralleled CVL decreases in human beta-defensin 2 and increases in Elafin and secretory leukocyte protease inhibitor. HIV disease progress predicted decreased CVL anti-HIV activity against both laboratory-adapted and T/F strains of HIV. Anti-HIV activity exhibited close associations with CVL levels of fourteen cytokines and chemokines. CONCLUSIONS: Amid a multifaceted immune defense against HIV-1 and other sexually transmitted pathogens, HIV disease progression is associated with selective disturbances in both CVL anti-HIV activity and specific innate immune defenses in the human female reproductive tract (FRT). Overall, these studies indicate that innate immune protection in the FRT is compromised as women progress to AIDS.
Assuntos
Progressão da Doença , Genitália Feminina/imunologia , Genitália Feminina/virologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Sistema Imunitário/imunologia , Imunidade Inata/imunologia , Adulto , Fármacos Anti-HIV/farmacologia , Contagem de Linfócito CD4 , Quimiocinas/imunologia , Coito , Demografia , Feminino , Genitália Feminina/efeitos dos fármacos , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Heterossexualidade/efeitos dos fármacos , Humanos , Sistema Imunitário/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Pessoa de Meia-Idade , Fatores de Risco , Tropismo/efeitos dos fármacos , Ducha Vaginal , Carga Viral/efeitos dos fármacos , Adulto JovemRESUMO
Interferon ß (IFNß) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNß and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I:C) induced the mRNA expression of IFNß, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNß or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNß upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I:C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I:C), had no effect on IFNß or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNß partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I:C) had no effect on IFNß and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.
Assuntos
Células Epiteliais/efeitos dos fármacos , Estradiol/farmacologia , Interferon beta/antagonistas & inibidores , Poli I-C/farmacologia , Receptores de Interferon/antagonistas & inibidores , Útero/efeitos dos fármacos , Anticorpos Neutralizantes/farmacologia , Linhagem Celular , Células Epiteliais/citologia , Células Epiteliais/imunologia , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Interferon beta/genética , Interferon beta/imunologia , Proteínas de Resistência a Myxovirus , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Cultura Primária de Células , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Útero/citologia , Útero/imunologia , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
As the AIDS epidemic continues with women being disproportionately affected, it is crucial to understand factors that predict the risk of heterosexual HIV-1 transmission. We investigated whether genital tract viral load (GTVL) in cervical-vaginal lavages (CVL) from HIV-1-positive women with moderately low CD4 T cell counts correlates with cytokine levels, antimicrobial concentrations, and intrinsic anti-HIV activity. CVL were collected from 19 HIV-1-positive women with moderately low CD4 T cell counts [mean 381 cells/mm(3) (227-536 cells/mm(3))]. None of the women was on antiretroviral therapy. The women were categorized into those with detectable GTVL or those with undetectable GTVL (detectable GTVL RNA levels > 400 copies/ml). Women were also categorized according to bacterial vaginosis (BV) status irrespective of GTVL. The TZM-bl assay was used to determine the presence of infectious virus and anti-HIV activity. Significantly higher levels of RANTES, Eotaxin, Fractalkine, IL-1α, IL-6, MCP-1, MIP1ß, MIP1α, TNF-α, and GM-CSF were observed in women with detectable GTVL compared to women with undetectable GTVL. No significant differences were observed in the following cytokines and chemokines: G-CSF, IL-1RA, IL-8, and IP-10. GTVL did not correlate either with antimicrobials known to have anti-HIV activity or with the presence of infectious virus. BV status did not have a significant effect on anti-HIV activity. These findings further our understanding of the role of GTVL in determining the cytokine and chemokine milieu in the female reproductive tract.
Assuntos
Síndrome da Imunodeficiência Adquirida/virologia , Colo do Útero/virologia , Citocinas/metabolismo , HIV-1 , Vagina/virologia , Síndrome da Imunodeficiência Adquirida/patologia , Síndrome da Imunodeficiência Adquirida/transmissão , Adulto , Contagem de Linfócito CD4 , Colo do Útero/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , RNA Viral , Vagina/metabolismo , Ducha Vaginal , Carga ViralRESUMO
PROBLEM: Hepatocyte Growth Factor (HGF) secretion facilitates epithelial cell growth and development in the female reproductive tract (FRT) and may contribute to pathological conditions such as cancer and endometriosis. We hypothesized that estradiol and poly (I:C), a synthetic RNA mimic, may have a regulatory effect on HGF secretion by stromal fibroblasts from FRT tissues. METHOD OF STUDY: Following hysterectomies, normal tissue from the uterus, endocervix, and ectocervix were dispersed into stromal cell fractions by enzymatic digestion and differential filtering. Stromal fibroblasts were cultured and treated with estradiol and/or poly (I:C), and conditioned media were analyzed for HGF via enzyme-linked immunosorbent assay. RESULTS: Treating uterine fibroblasts with estradiol or poly (I:C) significantly increased HGF secretion. When uterine fibroblasts were co-treated with estradiol and poly (I:C), the effect on HGF secretion was additive. In contrast, stromal fibroblasts from endo- and ecto-cervix were unresponsive to estradiol, but were stimulated to secrete HGF by poly (I:C). CONCLUSION: HGF secretion is uniquely regulated in the uterus, but not in ecto- and endo-cervix, by estradiol. Moreover, potential viral pathogens further induce HGF. These findings have potential applications in understanding both hormonal regulation of normal tissue as well as the role of HGF in tumorogenesis, endometriosis, and human immunodeficiency virus infection.
Assuntos
Colo do Útero/imunologia , Endometriose/imunologia , Estradiol/farmacologia , Fibroblastos/imunologia , Fator de Crescimento de Hepatócito/imunologia , Poli I-C/farmacologia , Células Estromais/imunologia , Separação Celular , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/patologia , Células Cultivadas , Colo do Útero/citologia , Colo do Útero/efeitos dos fármacos , Colo do Útero/metabolismo , Colagenases/metabolismo , Meios de Cultivo Condicionados/farmacologia , Endometriose/patologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Estradiol/imunologia , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , HIV/imunologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Fator de Crescimento de Hepatócito/biossíntese , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Especificidade de Órgãos , Poli I-C/imunologia , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
Mucosal surfaces of the female reproductive tract (FRT) contain a spectrum of antimicrobials that provide the first line of defense against viruses, bacteria, and fungi that enter the lower FRT. Once thought to be a sterile compartment, the upper FRT is periodically exposed to pathogens throughout the menstrual cycle. More recently, secretions from the upper FRT have been shown to contribute to downstream protection in the lower FRT. In this review, we examine the antimicrobials in FRT secretions made by immune cells and epithelial cells in the upper and lower FRT that contribute to innate protection. Because each site is hormonally regulated to maintain fertility, this review focuses on the contributions of hormone balance during the menstrual cycle to innate immune protection. As presented in this review, studies from our laboratory and others demonstrate that sex hormones regulate antimicrobials produced by innate immune cells throughout the FRT. The goal of this review is to examine the spectrum of antimicrobials in the FRT and the ways in which they are regulated to provide protection against pathogens that compromise reproductive health and threaten the lives of women.
Assuntos
Glândulas Endócrinas/fisiologia , Genitália Feminina/imunologia , Imunidade Inata , Infecções Sexualmente Transmissíveis/imunologia , Anti-Infecciosos/metabolismo , Feminino , Hormônios Esteroides Gonadais/fisiologia , Infecções por HIV/imunologia , HIV-1 , Humanos , Ciclo MenstrualRESUMO
The studies presented in this review explore three distinct areas with potential for inhibiting HIV infection in women. Based on emerging information from the physiology, endocrinology and immunology of the female reproductive tract (FRT), we propose unique 'works in progress' for protecting women from HIV. Various aspects of FRT immunity are suppressed by estradiol during the menstrual cycle, making women more susceptible to HIV infection. By engineering commensal Lactobacillus to secrete the anti-HIV molecule Elafin as estradiol levels increase, women could be protected from HIV infection. Selective estrogen response modifiers enhance barrier integrity and enhance secretion of protective anti-HIV molecules. Finally, understanding the interactions and regulation of FRT endogenous antimicrobials, proteases, antiproteases, etc., all of which are under hormonal control, will open new avenues to therapeutic manipulation of the FRT mucosal microenvironment. By seeking new alternatives to preventing HIV infection in women, we may finally disrupt the HIV pandemic.