REVIEW ARTICLE
Secreted Mucosal Antimicrobials in the Female Reproductive Tract that are Important to Consider for HIV Prevention Mimi Ghosh Department of Epidemiology and Biostatistics, Milken Institute School of Public Health, The George Washington University, Washington, DC, USA
Keywords Antimicrobials, cervical-vaginal lavage, female reproductive tract, HIV, hormones, sexually transmitted infections Correspondence Mimi Ghosh, Epidemiology and Biostatistics, The George Washington University, 2300 Eye Street, Washington, DC 20037, USA. E-mail: [email protected]
The mucosal microenvironment of the female reproductive tract (FRT) is rich in secreted endogenous antimicrobials that provide the first line of defense against pathogens. This review focuses on the spectrum of secreted antimicrobials found in the FRT that have anti-HIV functions and are regulated by the natural hormonal changes in women’s life cycle. Understanding the complex nature of FRT, mucosal microenvironment will enable us to better design therapeutic interventions for women against sexually transmitted pathogens.
Submission January 2, 2014; accepted March 14, 2014. Citation Ghosh M. Secreted mucosal antimicrobials in the female reproductive tract that are important to consider for HIV prevention. Am J Reprod Immunol 2014; 71: 575–588 doi:10.1111/aji.12250
Introduction As of 2012, the HIV pandemic claimed an estimated 36 million lives globally, and 35.3 million people are currently living with HIV/AIDS. HIV is the leading cause of death among women of reproductive age with heterosexual transmission, accounting for >80% of infections in women. The biological risks of transmission are further enhanced when coupled with high prevalence of non-consensual sex, unprotected sex, and high-risk behavior.1,2 In spite of the staggering global statistics, the rate of male-to-female transmission per act of vaginal intercourse is low, 1-200 to 1-2000, as determined by studies in sero-discordant couples.3,4 In reality, the transmission rates are often higher and dictated by a multitude of factors, including high donor semen viral load.5 However, multiple layers of protection do exist in the mucosa of the female reproductive tract (FRT) that can protect against sexually American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
transmitted infections including HIV (reviewed6). For the purpose of this review, we will focus on molecules with antimicrobial properties that are secreted by epithelial and immune cells of the FRT into the fluid that bathes the luminal surfaces of both the upper (Fallopian tube, uterus, endocervix) and the lower (ectocervix, vagina) tract. Layers of protection in the female reproductive tract Immune responses in the FRT have to be critically balanced as the system is geared toward successful reproduction and propagation of species. Therefore, the immune system must allow the entry of allogeneic sperm and the implantation of semi-allogeneic fetus, at the same time protecting the system against sexually transmitted pathogens. Multiple layers of immune protection exist in the FRT (reviewed6). The mucosal lining of the FRT is 575
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made up of epithelial cells and bathed in mucus, thus providing an effective physical and immunological barrier that can protect against pathogenic invasion. The lower FRT, consisting of the vagina and ectocervix, is lined with multiple layers of non-keratinized stratified squamous epithelium attached to a basement membrane. The upper FRT (Fallopian tube, endometrium, endocervix) is lined with a single layer of columnar epithelium with tight junctions between them. The integrity of this barrier can be disrupted if the tight junction proteins are disrupted, for example, by exposure to pathogens such as HIV or exposure to inflammatory cytokines such as TNF-a.7 A barrier breach caused by tight junction disruption can allow easy entry and passage of pathogens. The barrier is regulated by estradiol8,9 as well as by underlying stromal fibroblasts that secrete growth factors and cytokines and are also under hormonal control. Mucus that bathes the FRT also protects against pathogens by physically retarding movement of the virus into tissue.6,10 The texture of the mucus is hormonally regulated, being thin and watery at the estrogenic proliferative and ovulation stages to allow sperm entry and thick and viscous at progestrogenic secretory phase to prevent sperm entry. Acidic vaginal pH is another deterrent for pathogens such as HIV. In healthy women, vaginal pH is between 4 and 5 depending on ethnicity,11 which can destabilize bacterial cell wall and viral envelopes, including HIV envelope. However, during sexual transmission, virus enters along with sperm and seminal fluid, which raises the pH to 7–7.8 and thereby facilitates diffusion of the virus into the host.12 Semen or seminal fluid can also directly interact with HIV. Studies have shown that amyloid fibrils in semen known as SEVI can enhance HIV infection.13,14 Others have shown a protective effect of seminal plasma on HIV infection of CD4+ target cells.15 HIV can also directly bind to sperm cells using mannose receptor or heparin sulfate binding and potentially piggyback all the way up to the upper FRT.16–18 Beneficial commensals such as lactobacillus species, especially those producing hydrogen peroxide and lactic acid,19 are primarily responsible for maintaining the acidic pH of the vagina that is hostile to pathogens including HIV. An alteration in the microbiome balance of the FRT, such as due to bacterial vaginosis, can raise the pH and enhance the risk of HIV transmission.20,21 576
Innate (epithelial, NK, neutrophils, macrophages, dendritic/Langerhans’ cells) and adaptive (CD4+ T cells, CD8+ T cells, B cells) immune cells are present throughout the FRT (reviewed22,6) and fluctuate through natural hormonal changes (menstrual cycle, pregnancy, menopause) during women’s life cycle as well as due to inflammation resulting from infection. The FRT immune cells express functional pattern recognition receptors (PRR), such as toll-like receptors (TLR), NOD-like receptor (NOD), and RIG-like helicases.23 Stimulation of PRR with synthetic ligands or actual virus results in an immune response as denoted by the production of cytokines, chemokines or antimicrobials.23,24 Finally, the FRT mucosa consists of a host of antimicrobial peptides, which are discussed in more detail in the following paragraphs. Cervical-vaginal lavage Many studies have demonstrated the presence of immune mediators in FRT secretions. However, it is important to appreciate that the different sampling and processing methods in these studies might have resulted in variability in reported findings. A critical publication by Dezzutti et al.25 compared the levels of FRT soluble immune mediators collected by different methods and reported significant variability in findings depending upon the method used. Recently, Birse et al.26 have used a proteomic approach to demonstrate significant variability in FRT immune factors depending upon site as well as method of sample collection. Swabs and cervical-vaginal lavage (CVL), collected from normal saline, are considered to be the most optimal method for the recovery of the soluble immune mediators that are commonly measured for anti-HIV activity.25 CVL is a complex biological fluid consisting of not only antimicrobials but also water, electrolytes, organic compounds (glucose, amino acids, and lipids), cells (leukocytes, lymphocytes, and epithelial cells), and a multitude of proteins (antimicrobials, immunoglobulins), proteolytic enzymes, and secretion from commensals.27–29 The critical function of anti-HIV FRT antimicrobials was first demonstrated by Venkataraman et al.,30 where the authors showed that whole CVL had HIV inhibitory functions. They observed loss of this function if the whole CVL was depleted of cationic peptides, the fraction containing most known antimicrobials. Since then, other groups have confirmed intrinsic anti-HIV activity of CVL.31–33 American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
FEMALE REPRODUCTIVE TRACT ANTIMICROBIALS
Antimicrobials in FRT secretions At least 22 distinct endogenous antimicrobials in the FRT have been described to date, but it is likely that there are others that have not been characterized yet. Endogenous antimicrobials are primarily small cationic peptides produced by epithelial and immune cells of the FRT and secreted in the fluids that bathe the FRT mucosa. Endogenous antimicrobials are broad-spectrum in nature, showing activity against a range of bacterial, viral, and fungal pathogens including HIV.34,35 Antimicrobials prevent and/or reduce infection by killing or preventing the growth of microorganisms by direct or indirect mechanisms (reviewed36). They can interact directly with cell membranes and destabilize them by forming pores that abolish pH and ionic concentration gradients.35,37 Indirectly, they can modulate the immune system by inducing chemotaxis, cell proliferation, cytokine induction, and regulation of antigen uptake. For example, human b-defensin 2 (hBD2) can directly kill bacteria through membrane pore formation, whereas the chemokines CCL3/MIP-1a, CCL4/MIP-1b, CCL5/RANTES, and CXCL12/SDF-1a block HIV-1 binding to coreceptors CCR5 and CXCR4 on host cells (see reviews36,38). In fact, it is an underappreciated fact that a majority of chemokines have antimicrobial properties and several antimicrobials have chemotactic properties.39 For example, defensins have chemotactic functions40 in addition to their antimicrobial activity, and these activities are located in distinct domains of the molecule. Other examples of multifunctional antimicrobials are SLPI and trappin-2/elafin. They belong to the family of whey acidic protein (WAP) and share a 40% homology.41 Both are broad-spectrum antimicrobials found in mucosal sites and inhibit bacteria, fungi, and viruses including HIV. In addition, they are both potent anti-inflammatory molecules that mediate their activity by inhibiting endogenous proteases and reducing secretion of proinflammatory mediators.41,42 As with defensins, the antiprotease and antimicrobial activities are distinct and located within distinct domains of the molecules. In addition, both SLPI and elafin have been shown to be hormonally regulated both in vitro and in vivo.8,43,44 Recent studies have identified other ‘non-traditional’ antimicrobials in the sense that they are not cationic peptides, yet play potentially important roles in mucosal HIV inhibition. Antiproteases, such as serpins and cystatins, have recently been linked to American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
reduced susceptibility to HIV infection in highly exposed sero-negative women.45 Although most antiproteases have been demonstrated to indirectly affect HIV (through interference with host proteases), serpins A1 and C1, and cystatins A and B have also been shown to directly inhibit HIV.13,45 Some endogenous antimicrobials, such as surfactants, are lectin-like molecules46–48 or glycoproteins (e.g., thrombospondin).49 Lectins are a class of pattern recognition molecules that are chemotactic, and several surfactants, for example, surfactants A and D, are present in the FRT and have been shown to have anti-HIV functions.46,47,50,51 Another class of FRT molecules that can have pathogen-inhibiting functions is mucin. Mucins are the predominant component of mucus, a thick viscous fluid that covers all mucosal surfaces and acts as a protective barrier against invading pathogens.52 Mucin proteins are specific to a given mucosal compartment. Mucin proteins MUC 1, 4, 5AC, 5B, and 6 have been described in the FRT.53–55 Recent studies have demonstrated interactions between FRT mucus and IgG and IgA, which would potentially trap and immobilize pathogens in the mucus and cleared as the mucus is shed.52 Indeed, cervical-vaginal mucus has been shown to have an activity that hinders HIV-1 movement.56 Radtke et al.57 showed that MUC 1, 4, and 16 were upregulated in 3D human ectocervical epithelial cell model upon stimulation with synthetic viral agonist Poly(I:C), indicating the dynamic nature of the MUC proteins in the FRT. In summary, the molecules described here are able to impact HIV replication/infection in multiple ways. They can have a direct effect on the virus, direct effect on target cells, and/or effects on downregulating inflammatory markers and/or proteases resulting from HIV exposure (reviewed58). Whereas most studies focus on antimicrobials one at a time, it is important to consider that the mucosal microenvironment is a complex milieu where antimicrobials (and other molecules) interact with each other synergistically or antagonistically. Venkataraman et al.30 showed that anti-HIV functions were almost completely abolished when the cationic fraction was depleted from CVL. Singh et al.59 showed additive and synergistic antimicrobial interactions between SLPI and lysozyme as well as lactoferrin and lysozyme in airway surface liquid, another rich mucosal microenvironment. Another study by Ong et al.60 also demonstrated synergistic interactions between the antimicrobials HBD2 and LL-37. These 577
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data stress the importance of considering interactions between antimicrobials in vivo when evaluating their effectiveness. The complete antimicrobial repertoire in FRT secretions is unknown. While the best-studied antimicrobials present in the FRT are shown in Table I (modified from70), this list is incomplete and should be considered a ‘work in progress’. As proteomic studies of vaginal secretions have identified more than 600 unique proteins,28,71 it is likely that many molecules are yet to be characterized as anti-HIV factors. Hormonal regulation of antimicrobials Estradiol can influence both innate and adaptive immune systems locally and systemically by binding
to two main forms of the estradiol receptor (ER), ERa and ERb, which mediate transcription and translation of hundreds of genes.72 Within the upper and lower FRT, ERa and ERb, as well as progesterone receptors (PR), are expressed in a cell-specific manner locally by stromal and epithelial cells and fluctuate through the menstrual cycle (reviewed58,72). In vivo, antimicrobial peptides have been found to be distributed differentially throughout the FRT73 and expression patterns are altered through women’s life cycle, indicating hormonal regulation. Recent studies using cervical explants and nonhuman primate models show evidence of a window of vulnerability during the late ovulatory to secretory phase of the menstrual cycle, where women might be more susceptible to HIV acquisition.74–76 Endogenous
Table I Antimicrobials in the FRT Antimicrobials
Location
Source
References
a–defensins (HNP1–3, 5, 6)
U/L FRT, CVF, CM
34,37,80,106,108,137–145
b-Defensins (HBD1–5)
U/L FRT, CVF, CM
MIP3a/CCL20
U/L FRT, CVF
Elafin
U/L FRT, CM, CVF
SLPI
U/L FRT, CM, CVF
Cathelicidin (LL37)
U/L FRT, CVF, CM.
Calprotectin (MRP8/14)
L FRT, CVF, CM.
Lysozyme Lactoferrin Surfactant D
L FRT, CVF, CM. L FRT, CVF, CM U/L FRT
Surfactant A
U/L FRT
Thrombospondin
U/L FRT
Serpins A1, A3, B, C1 Cystatins A, B
CVF CVF
Neutrophils Macrophages Monocytes UEC LEC Macrophages Monocytes Dendritic cells UEC VEC UEC VEC Neutrophils UEC LEC Neutrophils Basal LEC Neutrophils Monocytes Superficial LEC Neutrophils Neutrophils UEC LEC UEC LEC Fibroblasts Monocytes/macrophages LEC LEC
8,34,37,80,137
104,146,147 32,43,81,148–152
34,73,80,82,106,137,138,152–156,156,157 34,106,158,159 34,100,138,160,161
34,137,138,160 46,137,138,154,162,163 46,50 47,51,79 49,164 45,133,165 45,133
U, upper; L, lower; FRT, female reproductive tract; CVF, cervical-vaginal fluid; CM, cervical mucus; UEC, upper FRT epithelial cells; LEC, lower FRT epithelial cells; STC, stromal fibroblasts; NK, natural killer; ND, not determined.
578
American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
FEMALE REPRODUCTIVE TRACT ANTIMICROBIALS
antimicrobials are likely to account in part for this observation as reports indicate that they can change through puberty77,78 menstrual cycle,34,50,79–81 pregnancy,81–84 and menopause.44 Unpublished data from our laboratory have also shown lower levels of SLPI in postmenopausal women compared with pre-menopausal women (Ghosh, in preparation). Rollenhagen et al. showed85 higher levels of inflammatory markers and enhanced HIV replication in cervical explant cultures obtained from postmenopausal women when compared to pre-menopausal women. These data point toward a more inflammatory, less protective microenvironment in postmenopausal women, which might support enhanced HIV replication. Overall, current data demonstrate that natural hormonal fluctuations through a woman’s life cycle can alter her immune functions in the FRT, thereby affecting her susceptibility to HIV and other sexually transmitted pathogens. Table II (adapted and updated from70) summarizes effects of hormones on antimicrobials. Exogenous hormones such as usage of hormonal contraceptives can also affect immune system in the FRT and hence alter susceptibility to HIV acquisition and transmission. Recent studies have demonstrated significantly higher risks of HIV acquisition and transmission in women on hormonal contraceptives, especially those using injectable DMPA.86,87 In HIV-
positive women, contraceptives have been shown to interact with some antiretrovirals, thereby reducing their efficiency.88,89 There are not too many studies that show effects of hormonal contraceptives on endogenous antimicrobials. Fleming et al.90 have shown reduction in beta defensins 1 and 2 in women using oral contraceptive or levonorgestrel intrauterine system. Others have shown inhibition of SLPI in DMPA users, thereby potentially altering their susceptibility to HIV acquisition/transmission.91 This is certainly an area that needs further investigation. ‘Real’ effects of antimicrobials: considerations regarding bioactivity Studies are sometimes in disagreement about the extent of ‘real’ effects of antimicrobials in vivo. One reason is that inherent caveats exist in the methodology of the in vitro studies designed to demonstrate antimicrobial functions. For example, it is important to consider that antimicrobials are susceptible to the effects of pH, ion concentration (e.g., Na+, Mg2+), serum proteins, and protease inhibitors, many of which can be antagonistic toward antimicrobial activity.37,61–66 Another major caveat is that when testing antimicrobial activity in vitro, the physiologically
Table II Antimicrobial Changes in the Female Reproductive Tract due to Menstrual Status, Pregnancy, and Contraceptive Use Antimicrobial
Proliferative
Midcycle
Secretory
Pregnancy
Postmenopausal
Oral Contraceptive
References
a-Defensins (HNP1–3) SLPI
++++ ++++
+ ++
+++ +++
Present Present
ND Decrease
NC ND
Lysozyme Lactoferrin b-Defensin 1 b-Defensin 2
+++ ++++ ND ++++
+ ++
+++ +++
++
+++
ND ND Present Decrease
ND ND ND Decrease
NC NC Decrease NC
b-Defensin 3 b-Defensin 4 MIP3a/CCL20
ND ND ND
Present Present Decrease
ND ND Decrease
Decrease ND ND
Elafin Surfactant A Surfactant D Serpin A1 Serpin A3
ND +++ – +++ NC
Decrease
ND
ND
Present
ND Decrease Decrease
ND Decrease Decrease
80,137,166–168 44,80,90,91,137,155,169,170 Ghosh, in preparation 80,137 80,137,163 90,170 80,84,90,137,166,170 Ghosh, in preparation 81,90,137,166,170 81 84 Ghosh, in preparation 43,84,170 50,79 50 132 132
++
+ ++ ++
+ symbols indicate level of each antimicrobial determined in CVL relative to the proliferative phase with increasing numbers of + symbols indicating higher levels of antimicrobial. (NC, no change; ND, not determined).
American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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relevant concentrations of these molecules are often not taken into consideration. As most in vitro studies use commercially available recombinant antimicrobials, the method of construction and accuracy of the recombinant peptides tested should also be considered. How a recombinant is made can affect its bioactivity leading to controversial results. For example, although SLPI is now widely regarded as a wellcharacterized anti-HIV molecule, 15 years ago there was compelling doubt shed on this premise.67 Possible explanations for variability in findings may have been in the quality (incomplete linkage of eight disulfide bridges) of different recombinant SLPIs that were used, variability in the expression of a cell surface receptor for SLPI on HIV target cells,68,69 or the selection of assay for HIV activity.58 Levels and activity of antimicrobials can vary based on local hormonal levels and the presence of proteases and antiproteases that can act as regulators by activating and deactivating these molecules. Whereas most studies focus on measuring levels of antimicrobials or other soluble immune mediators using standard ELISA or Luminex-type assays, the bioactivity of these molecules is often not considered. It is conceivable that an antimicrobial might be present in high levels (as measured by ELISA or Luminex) in a given CVL, yet not correlate with anti-HIV activity.31 It is the bioactivity of individual molecules in a CVL sample that will determine the overall antimicrobial functionality of the whole CVL. We have previously shown that conditioned media from epithelial cell cultures from both the upper and lower FRT could inhibit a variety of pathogens including HIV while not affecting beneficial lactobacillus species.92 Studies with CVL have also shown anti-HIV activity in HIV-negative as well as HIVpositive women with high CD4 counts.31 However, in HIV-positive women with lower CD4 counts, this activity is almost completely abolished.32 This loss of activity in HIV-positive women with CD4 counts of
Secreted Mucosal Antimicrobials in the Female Reproductive Tract that are Important to Consider for HIV Prevention Mimi Ghosh Department of Epidemiology and Biostatistics, Milken Institute School of Public Health, The George Washington University, Washington, DC, USA
Keywords Antimicrobials, cervical-vaginal lavage, female reproductive tract, HIV, hormones, sexually transmitted infections Correspondence Mimi Ghosh, Epidemiology and Biostatistics, The George Washington University, 2300 Eye Street, Washington, DC 20037, USA. E-mail: [email protected]
The mucosal microenvironment of the female reproductive tract (FRT) is rich in secreted endogenous antimicrobials that provide the first line of defense against pathogens. This review focuses on the spectrum of secreted antimicrobials found in the FRT that have anti-HIV functions and are regulated by the natural hormonal changes in women’s life cycle. Understanding the complex nature of FRT, mucosal microenvironment will enable us to better design therapeutic interventions for women against sexually transmitted pathogens.
Submission January 2, 2014; accepted March 14, 2014. Citation Ghosh M. Secreted mucosal antimicrobials in the female reproductive tract that are important to consider for HIV prevention. Am J Reprod Immunol 2014; 71: 575–588 doi:10.1111/aji.12250
Introduction As of 2012, the HIV pandemic claimed an estimated 36 million lives globally, and 35.3 million people are currently living with HIV/AIDS. HIV is the leading cause of death among women of reproductive age with heterosexual transmission, accounting for >80% of infections in women. The biological risks of transmission are further enhanced when coupled with high prevalence of non-consensual sex, unprotected sex, and high-risk behavior.1,2 In spite of the staggering global statistics, the rate of male-to-female transmission per act of vaginal intercourse is low, 1-200 to 1-2000, as determined by studies in sero-discordant couples.3,4 In reality, the transmission rates are often higher and dictated by a multitude of factors, including high donor semen viral load.5 However, multiple layers of protection do exist in the mucosa of the female reproductive tract (FRT) that can protect against sexually American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
transmitted infections including HIV (reviewed6). For the purpose of this review, we will focus on molecules with antimicrobial properties that are secreted by epithelial and immune cells of the FRT into the fluid that bathes the luminal surfaces of both the upper (Fallopian tube, uterus, endocervix) and the lower (ectocervix, vagina) tract. Layers of protection in the female reproductive tract Immune responses in the FRT have to be critically balanced as the system is geared toward successful reproduction and propagation of species. Therefore, the immune system must allow the entry of allogeneic sperm and the implantation of semi-allogeneic fetus, at the same time protecting the system against sexually transmitted pathogens. Multiple layers of immune protection exist in the FRT (reviewed6). The mucosal lining of the FRT is 575
GHOSH
made up of epithelial cells and bathed in mucus, thus providing an effective physical and immunological barrier that can protect against pathogenic invasion. The lower FRT, consisting of the vagina and ectocervix, is lined with multiple layers of non-keratinized stratified squamous epithelium attached to a basement membrane. The upper FRT (Fallopian tube, endometrium, endocervix) is lined with a single layer of columnar epithelium with tight junctions between them. The integrity of this barrier can be disrupted if the tight junction proteins are disrupted, for example, by exposure to pathogens such as HIV or exposure to inflammatory cytokines such as TNF-a.7 A barrier breach caused by tight junction disruption can allow easy entry and passage of pathogens. The barrier is regulated by estradiol8,9 as well as by underlying stromal fibroblasts that secrete growth factors and cytokines and are also under hormonal control. Mucus that bathes the FRT also protects against pathogens by physically retarding movement of the virus into tissue.6,10 The texture of the mucus is hormonally regulated, being thin and watery at the estrogenic proliferative and ovulation stages to allow sperm entry and thick and viscous at progestrogenic secretory phase to prevent sperm entry. Acidic vaginal pH is another deterrent for pathogens such as HIV. In healthy women, vaginal pH is between 4 and 5 depending on ethnicity,11 which can destabilize bacterial cell wall and viral envelopes, including HIV envelope. However, during sexual transmission, virus enters along with sperm and seminal fluid, which raises the pH to 7–7.8 and thereby facilitates diffusion of the virus into the host.12 Semen or seminal fluid can also directly interact with HIV. Studies have shown that amyloid fibrils in semen known as SEVI can enhance HIV infection.13,14 Others have shown a protective effect of seminal plasma on HIV infection of CD4+ target cells.15 HIV can also directly bind to sperm cells using mannose receptor or heparin sulfate binding and potentially piggyback all the way up to the upper FRT.16–18 Beneficial commensals such as lactobacillus species, especially those producing hydrogen peroxide and lactic acid,19 are primarily responsible for maintaining the acidic pH of the vagina that is hostile to pathogens including HIV. An alteration in the microbiome balance of the FRT, such as due to bacterial vaginosis, can raise the pH and enhance the risk of HIV transmission.20,21 576
Innate (epithelial, NK, neutrophils, macrophages, dendritic/Langerhans’ cells) and adaptive (CD4+ T cells, CD8+ T cells, B cells) immune cells are present throughout the FRT (reviewed22,6) and fluctuate through natural hormonal changes (menstrual cycle, pregnancy, menopause) during women’s life cycle as well as due to inflammation resulting from infection. The FRT immune cells express functional pattern recognition receptors (PRR), such as toll-like receptors (TLR), NOD-like receptor (NOD), and RIG-like helicases.23 Stimulation of PRR with synthetic ligands or actual virus results in an immune response as denoted by the production of cytokines, chemokines or antimicrobials.23,24 Finally, the FRT mucosa consists of a host of antimicrobial peptides, which are discussed in more detail in the following paragraphs. Cervical-vaginal lavage Many studies have demonstrated the presence of immune mediators in FRT secretions. However, it is important to appreciate that the different sampling and processing methods in these studies might have resulted in variability in reported findings. A critical publication by Dezzutti et al.25 compared the levels of FRT soluble immune mediators collected by different methods and reported significant variability in findings depending upon the method used. Recently, Birse et al.26 have used a proteomic approach to demonstrate significant variability in FRT immune factors depending upon site as well as method of sample collection. Swabs and cervical-vaginal lavage (CVL), collected from normal saline, are considered to be the most optimal method for the recovery of the soluble immune mediators that are commonly measured for anti-HIV activity.25 CVL is a complex biological fluid consisting of not only antimicrobials but also water, electrolytes, organic compounds (glucose, amino acids, and lipids), cells (leukocytes, lymphocytes, and epithelial cells), and a multitude of proteins (antimicrobials, immunoglobulins), proteolytic enzymes, and secretion from commensals.27–29 The critical function of anti-HIV FRT antimicrobials was first demonstrated by Venkataraman et al.,30 where the authors showed that whole CVL had HIV inhibitory functions. They observed loss of this function if the whole CVL was depleted of cationic peptides, the fraction containing most known antimicrobials. Since then, other groups have confirmed intrinsic anti-HIV activity of CVL.31–33 American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
FEMALE REPRODUCTIVE TRACT ANTIMICROBIALS
Antimicrobials in FRT secretions At least 22 distinct endogenous antimicrobials in the FRT have been described to date, but it is likely that there are others that have not been characterized yet. Endogenous antimicrobials are primarily small cationic peptides produced by epithelial and immune cells of the FRT and secreted in the fluids that bathe the FRT mucosa. Endogenous antimicrobials are broad-spectrum in nature, showing activity against a range of bacterial, viral, and fungal pathogens including HIV.34,35 Antimicrobials prevent and/or reduce infection by killing or preventing the growth of microorganisms by direct or indirect mechanisms (reviewed36). They can interact directly with cell membranes and destabilize them by forming pores that abolish pH and ionic concentration gradients.35,37 Indirectly, they can modulate the immune system by inducing chemotaxis, cell proliferation, cytokine induction, and regulation of antigen uptake. For example, human b-defensin 2 (hBD2) can directly kill bacteria through membrane pore formation, whereas the chemokines CCL3/MIP-1a, CCL4/MIP-1b, CCL5/RANTES, and CXCL12/SDF-1a block HIV-1 binding to coreceptors CCR5 and CXCR4 on host cells (see reviews36,38). In fact, it is an underappreciated fact that a majority of chemokines have antimicrobial properties and several antimicrobials have chemotactic properties.39 For example, defensins have chemotactic functions40 in addition to their antimicrobial activity, and these activities are located in distinct domains of the molecule. Other examples of multifunctional antimicrobials are SLPI and trappin-2/elafin. They belong to the family of whey acidic protein (WAP) and share a 40% homology.41 Both are broad-spectrum antimicrobials found in mucosal sites and inhibit bacteria, fungi, and viruses including HIV. In addition, they are both potent anti-inflammatory molecules that mediate their activity by inhibiting endogenous proteases and reducing secretion of proinflammatory mediators.41,42 As with defensins, the antiprotease and antimicrobial activities are distinct and located within distinct domains of the molecules. In addition, both SLPI and elafin have been shown to be hormonally regulated both in vitro and in vivo.8,43,44 Recent studies have identified other ‘non-traditional’ antimicrobials in the sense that they are not cationic peptides, yet play potentially important roles in mucosal HIV inhibition. Antiproteases, such as serpins and cystatins, have recently been linked to American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
reduced susceptibility to HIV infection in highly exposed sero-negative women.45 Although most antiproteases have been demonstrated to indirectly affect HIV (through interference with host proteases), serpins A1 and C1, and cystatins A and B have also been shown to directly inhibit HIV.13,45 Some endogenous antimicrobials, such as surfactants, are lectin-like molecules46–48 or glycoproteins (e.g., thrombospondin).49 Lectins are a class of pattern recognition molecules that are chemotactic, and several surfactants, for example, surfactants A and D, are present in the FRT and have been shown to have anti-HIV functions.46,47,50,51 Another class of FRT molecules that can have pathogen-inhibiting functions is mucin. Mucins are the predominant component of mucus, a thick viscous fluid that covers all mucosal surfaces and acts as a protective barrier against invading pathogens.52 Mucin proteins are specific to a given mucosal compartment. Mucin proteins MUC 1, 4, 5AC, 5B, and 6 have been described in the FRT.53–55 Recent studies have demonstrated interactions between FRT mucus and IgG and IgA, which would potentially trap and immobilize pathogens in the mucus and cleared as the mucus is shed.52 Indeed, cervical-vaginal mucus has been shown to have an activity that hinders HIV-1 movement.56 Radtke et al.57 showed that MUC 1, 4, and 16 were upregulated in 3D human ectocervical epithelial cell model upon stimulation with synthetic viral agonist Poly(I:C), indicating the dynamic nature of the MUC proteins in the FRT. In summary, the molecules described here are able to impact HIV replication/infection in multiple ways. They can have a direct effect on the virus, direct effect on target cells, and/or effects on downregulating inflammatory markers and/or proteases resulting from HIV exposure (reviewed58). Whereas most studies focus on antimicrobials one at a time, it is important to consider that the mucosal microenvironment is a complex milieu where antimicrobials (and other molecules) interact with each other synergistically or antagonistically. Venkataraman et al.30 showed that anti-HIV functions were almost completely abolished when the cationic fraction was depleted from CVL. Singh et al.59 showed additive and synergistic antimicrobial interactions between SLPI and lysozyme as well as lactoferrin and lysozyme in airway surface liquid, another rich mucosal microenvironment. Another study by Ong et al.60 also demonstrated synergistic interactions between the antimicrobials HBD2 and LL-37. These 577
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data stress the importance of considering interactions between antimicrobials in vivo when evaluating their effectiveness. The complete antimicrobial repertoire in FRT secretions is unknown. While the best-studied antimicrobials present in the FRT are shown in Table I (modified from70), this list is incomplete and should be considered a ‘work in progress’. As proteomic studies of vaginal secretions have identified more than 600 unique proteins,28,71 it is likely that many molecules are yet to be characterized as anti-HIV factors. Hormonal regulation of antimicrobials Estradiol can influence both innate and adaptive immune systems locally and systemically by binding
to two main forms of the estradiol receptor (ER), ERa and ERb, which mediate transcription and translation of hundreds of genes.72 Within the upper and lower FRT, ERa and ERb, as well as progesterone receptors (PR), are expressed in a cell-specific manner locally by stromal and epithelial cells and fluctuate through the menstrual cycle (reviewed58,72). In vivo, antimicrobial peptides have been found to be distributed differentially throughout the FRT73 and expression patterns are altered through women’s life cycle, indicating hormonal regulation. Recent studies using cervical explants and nonhuman primate models show evidence of a window of vulnerability during the late ovulatory to secretory phase of the menstrual cycle, where women might be more susceptible to HIV acquisition.74–76 Endogenous
Table I Antimicrobials in the FRT Antimicrobials
Location
Source
References
a–defensins (HNP1–3, 5, 6)
U/L FRT, CVF, CM
34,37,80,106,108,137–145
b-Defensins (HBD1–5)
U/L FRT, CVF, CM
MIP3a/CCL20
U/L FRT, CVF
Elafin
U/L FRT, CM, CVF
SLPI
U/L FRT, CM, CVF
Cathelicidin (LL37)
U/L FRT, CVF, CM.
Calprotectin (MRP8/14)
L FRT, CVF, CM.
Lysozyme Lactoferrin Surfactant D
L FRT, CVF, CM. L FRT, CVF, CM U/L FRT
Surfactant A
U/L FRT
Thrombospondin
U/L FRT
Serpins A1, A3, B, C1 Cystatins A, B
CVF CVF
Neutrophils Macrophages Monocytes UEC LEC Macrophages Monocytes Dendritic cells UEC VEC UEC VEC Neutrophils UEC LEC Neutrophils Basal LEC Neutrophils Monocytes Superficial LEC Neutrophils Neutrophils UEC LEC UEC LEC Fibroblasts Monocytes/macrophages LEC LEC
8,34,37,80,137
104,146,147 32,43,81,148–152
34,73,80,82,106,137,138,152–156,156,157 34,106,158,159 34,100,138,160,161
34,137,138,160 46,137,138,154,162,163 46,50 47,51,79 49,164 45,133,165 45,133
U, upper; L, lower; FRT, female reproductive tract; CVF, cervical-vaginal fluid; CM, cervical mucus; UEC, upper FRT epithelial cells; LEC, lower FRT epithelial cells; STC, stromal fibroblasts; NK, natural killer; ND, not determined.
578
American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
FEMALE REPRODUCTIVE TRACT ANTIMICROBIALS
antimicrobials are likely to account in part for this observation as reports indicate that they can change through puberty77,78 menstrual cycle,34,50,79–81 pregnancy,81–84 and menopause.44 Unpublished data from our laboratory have also shown lower levels of SLPI in postmenopausal women compared with pre-menopausal women (Ghosh, in preparation). Rollenhagen et al. showed85 higher levels of inflammatory markers and enhanced HIV replication in cervical explant cultures obtained from postmenopausal women when compared to pre-menopausal women. These data point toward a more inflammatory, less protective microenvironment in postmenopausal women, which might support enhanced HIV replication. Overall, current data demonstrate that natural hormonal fluctuations through a woman’s life cycle can alter her immune functions in the FRT, thereby affecting her susceptibility to HIV and other sexually transmitted pathogens. Table II (adapted and updated from70) summarizes effects of hormones on antimicrobials. Exogenous hormones such as usage of hormonal contraceptives can also affect immune system in the FRT and hence alter susceptibility to HIV acquisition and transmission. Recent studies have demonstrated significantly higher risks of HIV acquisition and transmission in women on hormonal contraceptives, especially those using injectable DMPA.86,87 In HIV-
positive women, contraceptives have been shown to interact with some antiretrovirals, thereby reducing their efficiency.88,89 There are not too many studies that show effects of hormonal contraceptives on endogenous antimicrobials. Fleming et al.90 have shown reduction in beta defensins 1 and 2 in women using oral contraceptive or levonorgestrel intrauterine system. Others have shown inhibition of SLPI in DMPA users, thereby potentially altering their susceptibility to HIV acquisition/transmission.91 This is certainly an area that needs further investigation. ‘Real’ effects of antimicrobials: considerations regarding bioactivity Studies are sometimes in disagreement about the extent of ‘real’ effects of antimicrobials in vivo. One reason is that inherent caveats exist in the methodology of the in vitro studies designed to demonstrate antimicrobial functions. For example, it is important to consider that antimicrobials are susceptible to the effects of pH, ion concentration (e.g., Na+, Mg2+), serum proteins, and protease inhibitors, many of which can be antagonistic toward antimicrobial activity.37,61–66 Another major caveat is that when testing antimicrobial activity in vitro, the physiologically
Table II Antimicrobial Changes in the Female Reproductive Tract due to Menstrual Status, Pregnancy, and Contraceptive Use Antimicrobial
Proliferative
Midcycle
Secretory
Pregnancy
Postmenopausal
Oral Contraceptive
References
a-Defensins (HNP1–3) SLPI
++++ ++++
+ ++
+++ +++
Present Present
ND Decrease
NC ND
Lysozyme Lactoferrin b-Defensin 1 b-Defensin 2
+++ ++++ ND ++++
+ ++
+++ +++
++
+++
ND ND Present Decrease
ND ND ND Decrease
NC NC Decrease NC
b-Defensin 3 b-Defensin 4 MIP3a/CCL20
ND ND ND
Present Present Decrease
ND ND Decrease
Decrease ND ND
Elafin Surfactant A Surfactant D Serpin A1 Serpin A3
ND +++ – +++ NC
Decrease
ND
ND
Present
ND Decrease Decrease
ND Decrease Decrease
80,137,166–168 44,80,90,91,137,155,169,170 Ghosh, in preparation 80,137 80,137,163 90,170 80,84,90,137,166,170 Ghosh, in preparation 81,90,137,166,170 81 84 Ghosh, in preparation 43,84,170 50,79 50 132 132
++
+ ++ ++
+ symbols indicate level of each antimicrobial determined in CVL relative to the proliferative phase with increasing numbers of + symbols indicating higher levels of antimicrobial. (NC, no change; ND, not determined).
American Journal of Reproductive Immunology 71 (2014) 575–588 ª 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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relevant concentrations of these molecules are often not taken into consideration. As most in vitro studies use commercially available recombinant antimicrobials, the method of construction and accuracy of the recombinant peptides tested should also be considered. How a recombinant is made can affect its bioactivity leading to controversial results. For example, although SLPI is now widely regarded as a wellcharacterized anti-HIV molecule, 15 years ago there was compelling doubt shed on this premise.67 Possible explanations for variability in findings may have been in the quality (incomplete linkage of eight disulfide bridges) of different recombinant SLPIs that were used, variability in the expression of a cell surface receptor for SLPI on HIV target cells,68,69 or the selection of assay for HIV activity.58 Levels and activity of antimicrobials can vary based on local hormonal levels and the presence of proteases and antiproteases that can act as regulators by activating and deactivating these molecules. Whereas most studies focus on measuring levels of antimicrobials or other soluble immune mediators using standard ELISA or Luminex-type assays, the bioactivity of these molecules is often not considered. It is conceivable that an antimicrobial might be present in high levels (as measured by ELISA or Luminex) in a given CVL, yet not correlate with anti-HIV activity.31 It is the bioactivity of individual molecules in a CVL sample that will determine the overall antimicrobial functionality of the whole CVL. We have previously shown that conditioned media from epithelial cell cultures from both the upper and lower FRT could inhibit a variety of pathogens including HIV while not affecting beneficial lactobacillus species.92 Studies with CVL have also shown anti-HIV activity in HIV-negative as well as HIVpositive women with high CD4 counts.31 However, in HIV-positive women with lower CD4 counts, this activity is almost completely abolished.32 This loss of activity in HIV-positive women with CD4 counts of
