Innate and Adaptive Anti-HIV Immune Responses in the Female Reproductive Tract

Abstract

The mucosal surface of the female reproductive tract (FRT) is the primary site of transmission for a plethora of sexually transmitted infections, including human immunodeficiency virus (HIV), that represent a significant burden upon womens' health worldwide. However, fundamental aspects of innate and adaptive immune protection against HIV infection in the FRT are poorly understood. The FRT immune system is regulated by the cyclical changes of the sex hormones estradiol and progesterone across the menstrual cycle, which as we have hypothesized, leads to the creation of a window of vulnerability during the secretory stage of the menstrual cycle, when the risk of HIV transmission is increased. The goal of this review is to summarize the multiple levels of protection against HIV infection in the FRT, the contribution of different cell types including epithelial cells, macrophages, T cells, and dendritic cells to this, and their regulation by estradiol and progesterone. Understanding the unique immune environment in the FRT will allow for the potential development of novel therapeutic interventions such as vaccines and microbicides that may reduce or prevent HIV transmission in women.

1. Introduction: the window of vulnerability

Women represent half of the 34 million people currently living with human immunodeficiency virus (HIV) according to the latest World Health Organization report (WHO 2011). Young women (aged 15–24) are a particularly susceptible group, accounting for 22% of all new infections, almost twice as high as young men. In sub-Saharan Africa, women represent 59% of infected individuals; a new young woman is infected every minute and the likelihood of their living with HIV is eight times higher than in men. Consequently, HIV is the main cause of death in women of reproductive age (UNAIDS 2010).

Sexual transmission of HIV is the main route of HIV acquisition in women (NIAID 2010). While the risk of transmission with sexual intercourse is low, ranging from 1/200 to 1/2000 per sexual act, once infection occurs, HIV rapidly replicates in the mucosal tissues and CD4+ T cells are depleted from the gut within weeks of infection (Haase 2011). There is a short period of time when early immune responses at mucosal surfaces could effectively reduce susceptibility to HIV infection or abort the infection before it spreads. Even though the female reproductive tract (FRT) is the main portal of entry for HIV, early HIV infection at this mucosal surface has not been extensively studied and most of our knowledge about mucosal HIV infection and antiviral responses in humans comes from studies performed in the gastrointestinal tract.

The mucosal FRT environment needs to be evaluated in the context of its main function – reproduction. The FRT immune system is tightly regulated by cyclic changes in the sex hormones estradiol and progesterone to optimize conditions for implantation; thus, an adequate balance between immune protection against pathogens and tolerance to allogeneic sperm and semi-allogeneic fetus results in successful reproduction (Hickey et al. 2011). As discussed elsewhere, changes in hormone levels during the menstrual cycle result in cyclical changes in FRT innate and adaptive immune responses, as well as the immune cell populations within the upper and lower tract, which in turn modulate conditions for the opportunistic establishment of HIV infection (Wira and Fahey 2008). Based on these changes, we developed the hypothesis of a window of vulnerability that lasts about 7–10 days after ovulation during the secretory phase when HIV infection is more likely to occur because of the dampening of protective immune responses (Wira and Fahey 2008). This review summarizes the current literature on innate and adaptive immune responses in the FRT with emphasis on how modulation of these responses by sex hormones influences susceptibility to HIV infection.

2. HIV Tropism

The primary cellular receptor for HIV entry is CD4 (Sattentau et al. 1986). HIV also uses either CCR5 (Deng et al. 1996) or CXCR4 (Feng et al. 1996) as coreceptors alongside CD4. There are two main strains of HIV:

• (1)CCR5-tropic (R5) strains that interact with the CCR5 coreceptor, and
• (2)CXCR4-tropic (X4) strains that utilize the CXCR4 coreceptor.

In addition, there are alternative cell surface receptors, such as the C-type lectins DC-SIGN (Turville et al. 2001), mannose receptor (MR) (Turville et al. 2001), heparan sulfate proteoglycans (HSPG) (Patel et al. 1993) and syndecans, as well as gal ceramide (GalC) (Bhat 1992), gp340 (Stoddard et al. 2007) and α4β7 integrin (Arthos et al. 2008) amongst others that HIV also employs to enter the cell. The majority of heterosexual transmission events are due to CCR5-tropic strains of HIV. Viral infection is characterized by a genetic bottleneck as approximately 80% of new cases of sexually transmitted HIV are due to a single infectious CCR5-tropic virion. The reasons behind this phenomenon are unclear, but are currently an active area of research.

3. HIV and Cervico-Vaginal Secretions

Bathing the lining of the lower FRT are cervico-vaginal fluids (CVF), composed of vaginal transudate, mucus, and epithelial cell secretions (Owen and Katz 1999) from the cervix, uterus, and Fallopian tubes. CVF is known to restrict the infection of target cells by multiple pathogenic organisms including HIV and HSV-2 (Ghosh et al. 2010a; Shust et al. 2010). Both HIV-positive and HIV-negative women possess anti-HIV activity in their CVF. Anti-HIV activity in the CVF of HIV-positive women varies with disease progression (Lahey et al. 2012), as women with lower CD4+ T cell counts have diminished protection in their CVF (Ghosh et al. 2010a). CVF antiviral activity against different viral strains is not uniform, with some strains of reporter cells being highly infectious and others having very low infectivity in the presence of CVF (Ghosh et al. 2010b). This suggests that the constituents of CVF have differential activity against HIV, potentially creating a situation where some strains of virus are more easily transmitted than others.

The CVF contains a rich cocktail of more than 20 antimicrobials, chemokines, and cytokines including CCL20, RANTES, human beta-defensin 2 (HBD2), elafin, and secretory leukocyte protease inhibitor (SLPI) (Valore et al. 2002; Keller et al. 2007; Ghosh et al. 2010a), which can potently reduce HIV infection of target cells in vitro and are associated with greater protective activity against HIV in CVF (Lahey et al. 2012). They exert their effects via several mechanisms:

• (a)By preventing binding of HIV to coreceptors expressed on HIV target cells;
• (b)By directly interacting with the viral membrane and subsequently destabilizing it; and
• (c)By altering HIV target cell signaling pathways that alter gene expression.

Epithelial cells produce several of the anti-HIV proteins that inhibit HIV infection in vitro. The protein complement of cellular secretions varies with cell type. For example, the anti-HIV cytokine CCL20 is present in secretions from uterine (Ghosh et al. 2009), but not superficial vaginal epithelial cells (Wira et al. 2010) suggesting that antiviral activity is not equal at each site in the FRT. Apical secretions collected from polarized endometrial epithelial cells contain potent, but variable anti-HIV activity that reduces infection of TZM-bl cells (Wira et al. 2011a). In contrast, there is no detectable anti-HIV activity in superficial vaginal epithelial cell secretions (Patel and Wira, unpublished observation). Unfortunately, no one has compared the protein complement of epithelial secretions, and thus their potential antiviral activity, between the different anatomical regions of the FRT epithelium.

Several of the antiviral proteins are differentially regulated by hormonal status. For example, HBD2 and SLPI concentration decreases in CVF recovered at ovulation (Keller et al. 2007). Both HBD2 and elafin are increased by estradiol in uterine epithelial cells (Fahey et al. 2008), but decreased in superficial vaginal epithelial cells (Patel and Wira, unpublished observation). Contraceptive use is associated with decreased SLPI production in uterine tissues (Li et al. 2008) and in the vaginal epithelium (Kumar et al. 2011). Whether hormonal status modulates anti-HIV activity in CVF is unknown, but urgently needs to be addressed, especially in light of recent publications suggesting that contraceptive use might be linked to increased risk of HIV acquisition (Heffron et al. 2012).

4. HIV and FRT Epithelial Cells

Despite being the first cells exposed to incoming pathogens, the responses of epithelial cells from the lower and upper FRT to HIV have not been adequately studied. Much remains to be determined about the initial interactions between HIV and epithelial cells that occur in the hours following deposition of infected semen in the vagina. Given their role as the primary sentinel cells of the FRT, the epithelial cell response to HIV will be a key event in determining whether viral transmission occurs.

Epithelial cells form a physical barrier that protects the underlying FRT tissue and its resident immune cells against potential viral and bacterial pathogens. The stratified squamous epithelium of the ectocervix and vagina is 25–50 layers thick and consists of the superficial, parabasal and basal layers (Patton et al. 2000). The squamo-columnar junction or transformation zone is where the stratified epithelium of the lower FRT meets the single layer columnar epithelium of the upper FRT and is considered to be uniquely vulnerable to HIV infection because of the abundance of immune target cells (CD4+ T cells, macrophages, dendritic cells) (Pudney et al. 2005) and the transition in epithelial phenotype (Hladik and McElrath 2008). Given their structural differences, the lower FRT epithelium is thought to provide a more robust mechanical barrier to incoming pathogens than the upper FRT epithelium. Whether increased thickness correlates with increased protection against HIV is not definitively known. However, cervical ectopy, where the columnar epithelium of the endocervix extrudes onto the surface of the ectocervix, is associated with increased transmission risk of HIV (Moss et al. 1991), human papillomavirus (HPV) (Rocha-Zavaleta et al. 2004), Chlamydia trachomatis (Lee et al. 2006), and cytomegalovirus (CMV) (Critchlow et al. 1995). Intriguingly, occurrence of cervical ectopy varies with stage of reproductive development and is more prevalent in adolescent women (Rahm et al. 1991) as well as pregnant women (Goldacre et al. 1978) and women taking oral/hormonal contraceptives (Critchlow et al. 1995); each of these populations of women is thought to be at increased risk of HIV infection.

Epithelial thickness varies across the menstrual cycle and is under the control of estradiol. In the vagina epithelial thickness peaks at ovulation, following the surge in blood estradiol concentration (Galand et al. 1971). In post-menopausal women, the lack of ovarian estradiol secretion is associated with long-term thinning of the epithelium, which may be partially responsible for the increased risk of HIV infection in this population (Nilsson et al. 1995, Aaby et al. 1996). The administration of systemic or topical estrogens alleviates these symptoms. Indeed, the topical administration of estradiol in the primate vagina is associated with decreased risk of simian immunodeficiency virus (SIV) infection (Smith et al. 2004). In the endometrium, the thickness of the stratum functionalis increases through the proliferative phase along with the appearance of tortuous glands lined with columnar epithelial cells that penetrate into the tissue. The thickened endometrium is naturally shed during menstruation because of decreased levels of progesterone and estradiol. Whether these natural changes in turn degrade epithelial barrier protection and lead to increased rates of HIV transmission is unknown. The effect of hormonal contraceptive use on FRT epithelial cells is controversial. For example, administration of Depo-Provera (DMPA), a progestin-containing contraceptive in rhesus macaques leads to dramatic thinning of the vaginal epithelium that is associated with increased SIV transmission (Marx et al. 1996). However, the thinning effect of DMPA on the vaginal epithelium is considerably less pronounced in women (Bahamondes et al. 2000; Miller et al. 2000). Given the large numbers of women who use oral/hormonal contraceptives to regulate their reproductive function, further studies are needed to understand how these compounds alter the epithelial barrier function and innate immune protection in vivo.

5. Crossing the Epithelial Barrier

HIV must cross the epithelial barrier to infect the immune target cells present in the reproductive stromal tissues. As mentioned previously, the nature of this barrier changes between the upper and lower FRT. There are at least three potential mechanisms by which HIV and other pathogens including bacteria can access the FRT stroma:

• (1)Via existing gaps or breaches in the epithelial barrier;
• (2)Transcytosis through epithelial cells, and
• (3)Paracellular movement between epithelial cells.

Breaches in the epithelium can arise in several ways:

• (a)The effects of repeated penile entry into the vagina can result in microabrasions in the vaginal epithelium, allowing direct viral access to resident intraepithelial lymphocytes and Langerhans cells present in the basal and parabasal layers (Patton et al. 2000). Because of the mechanics of vaginal intercourse, however, it is unlikely that penile entry will directly disrupt the single-layer epithelium in the upper FRT.
• (b)The introduction of foreign compounds into the FRT can sometimes disrupt the integrity of the epithelial barrier. For example, the spermicide/microbicide Nonoxynol-9, which leads to increased HIV infection, causes rapid exfoliation and loss of uterine, vaginal, and rectal epithelial cell sheets, thus exposing the underlying stroma (Krebs et al. 2000; Phillips et al. 2000; Jain et al. 2005; Cone et al. 2006). Nonoxynol-9 further alters the phenotype of regenerating uterine epithelial cells, causing them to transition from columnar to cuboidal epithelial cells (Dayal et al. 2003).
• (c)Sexually transmitted pathogens such as HSV2 infect FRT epithelial cells and, as part of their lytic cycle, cause cell death and ulcerations through which viruses can penetrate into the FRT stroma (Horbul et al. 2011).

A second potential mechanism by which HIV can cross the epithelial barrier is transcytosis, where virus is taken up at the apical surface of epithelial cells, transported to the basolateral membrane and secreted into the stromal compartment (Bomsel 1997). Several studies using both cell lines and primary epithelial cells have demonstrated that transcytosis is exceedingly inefficient with an average of less than 0.5% of the initial cell-free viral inoculum crossing polarized epithelial monolayers in vitro and entering the basolateral compartment (Hocini et al. 2001; Bobardt et al. 2007). Viral uptake is an active, temperature-dependent process that relies on the expression of HIV receptors (Bobardt et al. 2007). However, depending on the system employed, HSPG (Bobardt et al. 2007), gp340 (Stoddard et al. 2007, 2009), MR (Hocini et al. 2001) and the co-receptors CCR5 and CXCR4 (Hocini et al. 2001) have been implicated in transcytosis demonstrating a fundamental difference between experimental systems. The question of whether cell-free or cell-associated HIV transcytose with greater efficiency is unresolved, with one study showing no difference between the two (Hocini et al. 2001), and another demonstrating preferential movement of cell-associated HIV (Lawrence et al. 2012). The virus that passes through cells is significantly less infectious than the initial viral inoculum suggesting that the intracellular environment is detrimental to viral integrity (Saidi et al. 2007). Intriguingly, some investigators have reported that R5 viruses are preferentially transcytosed over X4 viruses (Bobardt et al. 2007; Lawrence et al. 2012) representing a potential mechanism explaining why R5 viruses are the dominant sexually-transmitted strain in vivo.

The third potential mechanism by which HIV can cross the epithelium is paracellular movement. This is controlled by tight junctions that link adjacent columnar epithelial cells in the upper FRT. Tight junctions are protein complexes located at the apical membrane of epithelial cells and are composed of occludin, ZO-1, and multiple claudins amongst others, which regulate the paracellular movement of solutes, and thus create a polarized environment between the apical and basolateral surface (Matter and Balda 2003). Both R5 and X4 strains of HIV decrease transepithelial resistance (TER) in polarized monolayers of primary human endometrial epithelial cells in vitro by approximately 60% over 6–48 h (Nazli et al. 2010). Gp120 binding to host cell receptors leads to the induction of TNFα, which in turn decreases the expression of tight junction proteins. This is associated with increased translocation of bacterial and viral components across the epithelium (Nazli et al. 2010). However, TER is unaffected in polarized monolayers of HEC-1A cells after R5 exposure (Lawrence et al. 2012). Furthermore, recombinant TNFα and IL-1β are unable to alter TER in HEC-1A cells (Lawrence et al. 2012), suggesting that there might be a fundamental difference between primary cells and cell lines in their response to HIV.

In the stratified epithelium of the lower FRT, superficial epithelial cells are not bound together by tight junctions (Blaskewicz et al. 2011) and have gaps or pores between adjacent cells that are filled with interstitial fluid and vaginal secretions. It is believed that HIV diffuses deeper into the epithelium layer via these fluid phase channels to potentially access lymphocytes in the stratum corneum. Parabasal and basal epithelial cells express claudins and occludins and are linked by tight junctions (Blaskewicz et al. 2011). Whether HIV can induce the relaxation of these protein complexes and thus reduce barrier function, as it does in the upper FRT, remains to be determined.

6. HIV Receptor Expression on FRT Epithelial Cells

The primary viral receptor CD4, the coreceptors CCR5 and CXCR4, and the alternative receptor GalC are expressed on fresh human endometrial epithelial cells. In contrast, CD4 and CCR5 vary in freshly isolated ectocervical epithelial cells with expression absent in superficial cells and low in parabasal and basal cells (Yeaman et al. 2003, 2004). CXCR4 is absent from all the epithelial layers in the ectocervix. GalC displays a different expression profile from other receptors and is highest in superficial and parabasal epithelial cells and undetectable in the basal epithelium (Yeaman et al. 2003, 2004). Expression of the receptors varies with the stage of the menstrual cycle (Table 1). However, whether this predisposes epithelial cells to greater interactions with HIV is unknown.

Table 1.

Effect of menstrual cycle stage on receptor expression in freshly isolated human endometrial and ectocervical epithelial cells

	Endometrial		Ectocervical
	Proliferative	Secretory	Proliferative	Secretory
CD4	Increase	Decrease	Increase	Decrease
CCR5	Increase	Decrease	No Change	No Change
CXCR4	Decrease	Increase	Absent	Absent
GalC	No Change	No Change	No Change	No Change

The expression profile of most alternative HIV receptors on FRT epithelial cells is relatively understudied. β1 integrin is expressed by endo- and ectocervical epithelial cells in vivo (Maher et al. 2005). Mannose receptor (MR) and DC-SIGN are absent from endometrial, endocervical, and vaginal epithelial cells (Hirbod et al. 2011; Kaldensjö et al. 2011). gp340 is present on multiple cell lines and primary epithelial cells from the FRT (Stoddard et al. 2007, 2009). Recent studies suggest that alternative receptors may play a pivotal role in HIV translocation and infection in epithelial cells. As noted above, receptor expression can vary dramatically between anatomical sites in vivo. Thus, in order to understand the potential contribution of these receptors to HIV transmission one must first elucidate their spatial and temporal expression patterns.

7. Epithelial Cell Infection and Transmission

The question of whether FRT epithelial cells in vivo are permissive to HIV infection is controversial. Given that FRT epithelial cells express known HIV receptors, it is possible for HIV to enter and infect them. However, examination of human and non-human primate FRT tissue exposed to HIV has demonstrated the presence of viral particles in small populations of leukocytes, but their total absence in epithelial cells. Several groups have tried to infect upper FRT epithelial cells in vitro using either primary human cells or cell lines. Unfortunately, the data from these studies are contradictory, with a spectrum of results ranging from no detectable sign of viral entry to de novo production of infectious virions (Tan et al. 1993; Furuta et al. 1994; Howell et al. 1997; Dezzutti et al. 2001; Asin et al. 2003; Wu et al. 2003; Asin et al. 2004; Berlier et al. 2005; Saidi et al. 2007). Epithelial cells also sequester virus and release it slowly over time (Wu et al. 2003; Saidi et al. 2007). This observation may explain the appearance of infectious virions in basolateral secretions in the absence of detectable proviral integration.

Primary epithelial cells and multiple cell lines (HEC-1A, CaSKi, ECC-1, RL95-2, ME-180, and SiHa) have been used as models for HIV infection. The variation in infectivity experiments probably results from the use of cell preparations that are phenotypically different from each other. For example, Asin et al. (2003) reported that 20, 0, 6, and 5% of HEC-1A cells expressed CD4, CCR5, CXCR4, and GalC respectively. In contrast, Berlier et al. (2005) reported that 0, 0, 59, and 14% of their HEC-1A cells expressed CD4, CCR5, CXCR4, and GalC. In addition, the use of polarized versus nonpolarized epithelial cells may be a confounding factor given their different responses to identical stimuli. Another source of variation may arise from the use of different HIV strains used to infect epithelial cells. Unfortunately, most studies have only used two or three different viral strains in their protocols. Given that some strains of HIV are more infectious than others, it will be essential to expand the panel of viruses used in infection studies, and standardize the cell preparations used in order to derive clear and consistent results.

8. Innate Immune Responses to HIV

The majority of studies investigating the interactions between HIV and FRT epithelial cells have focused on whether epithelial cells are permissive with regard to infection, and if they can subsequently infect immune target cells. The direct innate immune response of epithelial cells to HIV, in terms of altered gene expression and protein secretion, has not been addressed in significant detail. Epithelial cells from throughout the FRT are able to respond to pathogens and pathogen ligands by modulating the expression of potentially hundreds of genes. Determining the immune response to HIV is essential to understanding the early interactions between host and pathogen and should provide valuable insight into how HIV is transmitted.

HEC-1A cells differentially respond to R5 and X4 strains of HIV. R5 HIV induces the secretion of IL-8, GRO, TNFβ, and IL-1α while X4 HIV decreases the secretion of TGFβ, IL-1α, RANTES, IL-8, and GRO (Saidi et al. 2007). Primary human uterine epithelial cells also upregulate apical secretion of a panel of cytokines including TNFα, IL-6, IL-10, and IL-1β in response to R5 HIV (ADA) (Nazli et al. 2010). In studies from our laboratory using primary human uterine epithelial cells and the ECC-1 cell line, we observed the preferential induction of the cytokines IP-10, IL-6, and TNFα in the presence of X4 HIV (IIIB), while MIP1β was selectively induced after R5 HIV (BaL) treatment (Patel and Wira, unpublished observation). In general, IIIB (X4) induces a more proinflammatory environment than BaL (R5). Intriguingly, we also observed the selective induction of the intracellular interferon-stimulated genes MxA, OAS2, PKR, and A3G in the presence of X4 (IIIB) but not R5 (BaL) virus. The expression and secretion of anti-HIV molecules such as HBD2, elafin, and CCL20 are unaltered by either virus (Patel and Wira, unpublished observation). Together, these studies demonstrate the selective induction of a unique panel of genes by different strains of HIV in primary epithelial cells. Whether this expression profile is responsible for the preferential transmission of R5 strains over X4 strains remains a tantalizing question. Unfortunately, there has been no systematic evaluation of epithelial cell responses to different strains of HIV or to identifying the underlying mechanisms regulating the differential response.

Recognizing that epithelial cell secretions can alter the phenotype of resident immune cells, and that R5 and X4 viruses induce altered protein secretion profiles by epithelial cells, it is quite likely that the epithelial response to HIV can affect the phenotype of FRT immune cells and thus their susceptibility to HIV infection. Incubation of macrophages with basolateral secretions from polarized HEC-1A monolayers apically-exposed to R5 HIV leads to their decreased secretion of MCP1, MCP2, MIP1α, MIP1β, MIP3α, and RANTES (Saidi et al. 2009). In contrast, secretions from X4-treated HEC-1A cells induce higher levels of MIP1β, RANTES, IP-10, I-TAC, MIG, MCP1, eotaxin 2, and IL-8 secretion by macrophages (compared with R5 HIV) (Saidi et al. 2009). These phenotypic changes extend to the ability of macrophages to recruit CD4+ T cells. Macrophages cultured in the presence of R5 HIV HEC-1A secretions recruited lower numbers of CD4+ T cells than macrophages cultured in the presence of X4 HEC-1A secretions.

Given the dearth of information regarding epithelial responses to HIV, three fundamental questions remain to be answered:

• (1)Do R5 strains in general induce epithelial responses that are different from those seen with X4 strains or are the responses unique to individual viruses in each strain?
• (2)Do epithelial cells from throughout the FRT respond in the same manner to individual strains of HIV or are there fundamental differences that are site-specific in the FRT?
• (3)If there are differential responses, can these alter the susceptibility of target cells in the stroma, thus increasing or decreasing the chances of successful transmission?

9. Immune cells susceptible to HIV infection in the lower and upper FRT

After successfully surviving the secretions, mucus, and epithelial barriers, HIV virions will encounter immune cells that are both ideal targets for viral replication and active participants in restricting viral infection. Susceptible target cells in the FRT mucosa include CD4+ T cells, macrophages, dendritic cells (DC), and Langerhans cells (LC), which can be found all along the upper and lower FRT.

CD4+ T cells

Many studies concur that CD4+ T cells may represent the initial and main target cell for HIV replication (McKinnon and Kaul 2012). However, not all CD4+ T cells subsets are equally susceptible to HIV infection and the location and phenotype of these initial CD4+ T cell targets in the FRT remain unknown. T cells are present in the vagina, cervix, and endometrium, localized in the sub-epithelial stroma, and also located as intraepithelial lymphocytes between epithelial cells that line the lumen (Hickey et al. 2011). T cells constitute a main proportion of all leukocytes present in the FRT, representing about 40% of leukocytes in the upper tract and about 50% in the lower tract (Givan et al. 1997). Non-human primate models with rhesus macaques showed that SIV infection begins with a small focus of infected CD4+ T cells, with productive SIV infection detected in both activated and resting cells (Zhang et al. 1999; Li et al. 2005). Studies in humans using explants from vagina, ectocervix, and endocervix indicate that HIV productively infects CD4+ T cells in the genital mucosa very efficiently. Infected cells can be found in the stroma one day after viral exposure, but analysis of the distribution of virions in the initial hours after vaginal challenge demonstrated viral fusion with intraepithelial CD4+ T cells (Hladik et al. 2007). Since sexual transmission of HIV is established by CCR5-tropic viral strains (Keele et al. 2008; Salazar-Gonzalez et al. 2009), characterization of CD4+ T cells subsets expressing CCR5 has become a priority. Using immune cells obtained from cytobrush, a recent report found a subset of CD4+ T cells expressing CCR5 in the cervix (McKinnon et al. 2011). Interestingly, CCR5 expression was enhanced in endocervical CD4+ T cells from postmenopausal women relative to premenopausal controls (Meditz et al. 2012), suggesting a possible hormonal control of CCR5 expression. Nevertheless, molecules and markers other than CCR5 most likely influence infection of CD4+ T cells since expression of CXCR4 is high in CD4+ T cells at mucosal surfaces (McKinnon et al. 2011); paradoxically, initial infection has not been described to involve CXCR4-tropic strains.

Macrophages

Approximately 10% of the leukocytes present in the FRT are macrophages (Givan et al. 1997). These cells are irregularly distributed among FRT tissues, with numbers in the endometrial stroma and myometrial connective tissue (Wira et al. 2005) higher than those seen in the endo- or ectocervix. Influx of macrophages into the endometrium is under the influence of estradiol and progesterone with their numbers increasing just prior to menstruation (Starkey et al. 1991). In contrast, vaginal macrophage numbers are not affected by sex hormones and remain constant throughout the menstrual cycle (Wira et al. 2005). Compared with gastrointestinal macrophages, vaginal macrophages display greater susceptibility to HIV infection, most likely because of higher levels of the HIV-1 receptor CD4 and co-receptors CCR5 and CXCR4 (Shen et al. 2009; Cassol et al. 2010), and resemble the monocytic phenotype found in blood with high CD14 expression. The involvement of macrophages as initial targets for HIV infection in the FRT mucosa is controversial. While some in vitro HIV infection studies in women demonstrated that initial infection was supported by macrophages (Collins et al. 2000; Greenhead et al. 2000; Cummins et al. 2007), SIV infection studies in primate models did not support these findings. Whether macrophages are involved in the initial steps of HIV infection in women as primary cells that support initial replication, or as cells that capture the virus and transmit it to T cells, remains to be elucidated (Sharova et al. 2005).

Dendritic cells

Dendritic cells are potent antigen-presenting cells that generate and regulate adaptive immune T-cell responses (Banchereau and Steinman 1998; Steinman and Hemmi 2006). Hematopoietic stem cells in the bone marrow continuously produce DCs that migrate to the different mucosal surfaces of the human body, where they reside in an immature state. In the upper FRT, DCs can be found in the sub-epithelial stroma in the endometrium, while in the vagina DCs mostly reside within the epithelial layer (Iijima et al. 2007, 2008). In the uterine endometrium, immature CD1a+ DCs outnumber mature CD83+ DCs throughout the menstrual cycle (Schulke et al. 2008), while CD83+ DCs remain constant. The movement of CD1a+ DCs into the FRT is regulated by hormonal changes during the menstrual cycle with numbers increasing from the proliferative to the secretory phases and peaking at menses (Schulke et al. 2008).

As sentinel cells, DCs can sample antigens at the mucosal surface, which potentially makes them one of the first cells that encounters HIV (Wu and Kewalramani 2006; Randolph et al. 2008; Liu et al. 2009; Altfeld et al. 2011). HIV can exploit the biological properties of DCs (Cunningham et al. 2010; Lambotin et al. 2010) to spread virus from mucosal surfaces to lymph nodes, the most important site for HIV replication. Thus, interaction between DCs and HIV can be either advantageous to the host if the final outcome is induction of efficient immune responses able to control infection, or beneficial to the virus if it exploits DC function to facilitate the spread of infection. DC-SIGN expression has been demonstrated to mediate HIV transmission from DCs to CD4+ T cells in vitro (Chehimi et al. 2003; Gringhuis et al. 2010). However, the role of DC-SIGN+ DCs in sexual transmission in vivo is not so well characterized. DC-SIGN+ DCs remain relatively constant in the human endometrium throughout the proliferative and secretory stages of the menstrual cycle (Rieger et al. 2004), although possible changes during menses have not been explored. Previous studies from our laboratory indicate that uterine epithelial cells through secreted soluble factors modulate DC phenotype. This phenotype is characterized by decreased sensitivity to Toll-like receptor (TLR) 3 and TLR4 stimulation and reduced expression of co-stimulatory molecules and DC-SIGN (Ochiel et al. 2010a, 2010b). Importantly, this down-regulation in DC-SIGN was associated with a reduction in HIV trans-infection by immature DCs (Ochiel et al. 2010b). These results highlight the complexity of the FRT, where not only the influence of sex hormones, but also the local microenvironment needs to be taken into account when evaluating immune responses.

In addition to their major role in triggering adaptive immune responses, DCs exert antiviral activity, which can also be modulated by sex hormones. For example, production of α-defensins by immature DC is inhibited by high doses of estradiol (Rodriguez-Garcia et al. 2007; Escribese et al. 2011). Additionally, higher production of α-defensins by immature DCs is associated with slower disease progression in HIV-infected subjects (Rodriguez-Garcia et al. 2010).

Plasmacytoid DCs (pDC) represent a DC subset of a different origin. pDCs are also present in the FRT and rapidly respond to viral infections secreting large amounts of Type I and Type II interferons (IFN) (Kaldensjö et al. 2011; Southern et al. 2011). Differences between men and women have been described in the responses of blood-derived pDC to HIV stimulation. For example, HIV-encoded TLR7 ligands induce a stronger IFNα response in women than in men (Meier et al. 2009), resulting in enhanced CD8+T cell activation. In non-primate human models, pDCs were found very early after SIV infection to produce both Type I IFN and chemokines within the infected foci of cells in the cervical mucosa (Li et al. 2009).

Whether these early innate immune responses effectively inhibit HIV infection or favor infection by attracting new target cells to the infected foci remains to be determined. While in vitro studies in humans have demonstrated potent anti-HIV activity of innate immune molecules and correlations with protection (Ghosh et al. 2010a; Rodriguez-Garcia et al. 2010; Wira et al. 2011b), studies of acute SIV infection in rhesus macaques show that inhibition of proinflammatory cytokine and chemokine production by 5% glycerol monolaurate prior to SIV high-dose intravaginal challenge protects from systemic viral infection (Li et al. 2009). The general biology and immunology of stromal DCs in the FRT constitute a major gap in our knowledge, and a better understanding is needed regarding which DC subsets are present in the FRT and what their role is in HIV infection.

Langerhans cells

Distinct from DCs, LCs are characterized by the expression of Langerin and their location at the mucosal surface. LCs are target cells for HIV that can be productively infected in vitro, although it appears likely that their main role is to capture and transfer virus to susceptible cells (Wu and Kewalramani 2006). Studies using epidermal cells showed that viral up-take through Langerin, a C-type lectin, mediates internalization and degradation of the virus in Birbeck granules (de Witte et al. 2007). Analysis of vaginal LCs, however, indicated that viral internalization occurs shortly after HIV exposure primarily by endocytosis with intact virions persisting in the cytoplasm for days (Hladik et al. 2007). Furthermore, non-productively infected LCs containing virus migrate from the exposed vaginal epithelium and transfer HIV to CD4+ T cells (Ballweber et al. 2011). In the upper FRT, LCs coexpressing CD1a, CD11c, and Langerin are mainly located in the luminal or glandular epithelium in the endometrium, where they represent potential targets for HIV (Kaldensjö et al. 2011). The role that LCs from the upper FRT play in HIV infection has not yet been determined and further studies are required to elucidate their contribution to HIV transmission.

10. Induction of HIV-specific cellular immune responses in the FRT

Although in vitro studies found that adaptive immune responses can control HIV infection, in vivo correlates of protection against HIV infection remain to be determined (Peretz et al. 2012). The role of the adaptive immune response in HIV infection is thought to be too little and too late (Reynolds et al. 2005), since by the time an adaptive immune response is mounted against HIV, it is too late to clear infection. A better understanding of the uniqueness of the FRT physiology and immunology is necessary to achieve induction of stronger immune responses at the site of entry that can rapidly act in response to the viral challenge.

The role that humoral immune responses play in protecting mucosal surfaces against HIV infection and the differences between these responses in the FRT and other mucosal surfaces constitute an area of active research and have been reviewed elsewhere (Mestecky et al. 2009,; Ackerman et al. 2012). Something to consider for the induction of effective cellular immune responses in the FRT is that immune control is more effectively achieved by localized memory T cells than circulating memory T cells (Iwasaki 2010), suggesting that immunization against HIV should induce memory T cell populations in the genital mucosa. Certain tissues, such as the FRT mucosa, skin or central nervous system have restricted access of circulating memory CD8+ T cells in response to localized infections (Gebhardt et al. 2009). Entry into these tissues is only facilitated after a subset of CD4+ T cells access the tissue, first in response to local signals, and induce the secretion of chemokines, which in turn allows the migration of other CD4+ and CD8+ memory T cells (Nakanishi et al. 2009).

Within one to two weeks following viral infection, effector cells (CD4+ and CD8+ T cells) die, leaving a residual population of quiescent antigen-specific memory T cells in tissues to provide a rapid response against subsequent infections (Pepper and Jenkins 2011). This retention process is partially mediated by TGF-β in non-lymphoid tissues by downregulation of CCR7 and upregulation of E-cadherin receptor CD103 (Sheridan and Lefrancois 2011). In the lower FRT, memory CD4+ or CD8+ T cell and B cell clusters appear in the submucosa and below the epithelium in the vagina after infection clearance. These cell clusters are absent in uninfected individuals, but can be detected in infected subjects for months following infection resolution (Iwasaki 2010). In the upper FRT, lymphoid aggregate structures consisting of a B cell core surrounded by CD8+ T cells and a halo of macrophages are located in the endometrium (Yeaman et al. 1997). CD8+ T cells are found in these lymphoid aggregates located between glands in the functionalis region, while CD4+ T cells can be found outside the aggregates, usually located in the stroma. In contrast to the lower FRT CTL whose activity remains high and independent of hormonal changes throughout the menstrual cycle (White et al. 1997a, 1997b), aggregates in the upper FRT are regulated by cyclical hormonal fluctuations. A reduced size of the lymphocyte aggregates is accompanied by high CTL activity during the proliferative phase of the menstrual cycle while maximal size of the lymphoid aggregates during the secretory phase is accompanied by suppression of the CTL activity (Yeaman et al. 1997, 2001). The demonstration that uterine lymphocyte aggregates are absent in postmenopausal women, when FRT CTL activity is significantly higher than in pre-menopausal women at any stage of the menstrual cycle (White et al. 1997a, 1997b), further proves the implication of sex hormones in both processes.

A major gap in our knowledge remains the characterization of HIV-specific immune responses and the T-cell repertoire in the FRT. HIV-specific T-cell responses have been described in the lower and upper FRT of chronically infected women (Musey et al. 2003; Shacklett and Greenblatt 2011) and also in the cervix of exposed uninfected individuals (Kaul et al. 2000). Several reports demonstrate a lack of correlation between peripheral blood and the mucosal tissue (White et al. 2001; Gumbi et al. 2008) highlighting the necessity of characterizing local responses since HIV-specific T-cell responses in blood are not necessarily predictive of mucosal protection.

11. Current state of vaginal microbicides and vaccine trials

Recent partial but hopeful success has been achieved with vaginal microbicides and vaccine trials (Opoku-Anane et al. 2012). Since women are primarily infected via heterosexual intercourse (70–90%), an effective vaginal/rectal microbicide represents a female-controlled protective method that will have a great impact on the prevention of HIV acquisition in women. Microbicides act by reducing susceptibility of target cells at the mucosal surface. The CAPRISA 004 trial showed that the use of 1% tenofovir gel (a reverse-transcriptase inhibitor) applied before and after sex was 39% effective in preventing HIV acquisition overall, with 54% efficacy in highly adherent users (Abdool Karim et al. 2010). However, a second trial testing the daily application of 1% tenofovir gel (VOICE) was stopped owing to futility and a lack of protection (van der Straten et al. 2012). Reasons for this disparity in tenofovir effectiveness are under investigation and include the different treatment regimens (continuous versus intermittent), user adherence to the daily application, and how sustained use of 1% tenofovir gel alters the immunobiology of the FRT. Further trials are being conducted to determine the effectiveness of tenofovir, including one with the application of 1% tenofovir gel before and after sex (FACTS 001) to try to reproduce the success of the CAPRISA 004 trial. New microbicides are also being developed and a new trial with maraviroc, which blocks CCR5, will soon be initiated (Shattock and Rosenberg 2012).

The RV144 Thai vaccine trial showed a 31% efficacy in preventing HIV acquisition (Rerks-Ngarm et al. 2009). The exact mechanisms of how the vaccine worked remain under investigation. Current results suggest that IgG antibodies directed against envelope proteins protect against HIV infection while IgA antibodies mitigate the effects of protective antibodies (Haynes et al. 2012). Protective cellular responses in peripheral blood were not found in this trial. Unfortunately, mucosal samples are unavailable since they were not part of the study design; thus, the state of the mucosal innate and adaptive immune responses as well as the characteristics of the potential HIV target cells cannot be determined at present.

Successful preventive strategies in women require a better understanding of the FRT and how immune responses are induced. Animal model studies using vaccination followed by infection demonstrate that cyclical hormonal changes influence the induction of adaptive immune responses. Strong immune responses can be generated when antigen challenge is performed at diestrus (low estradiol levels) while challenge at estrus (high estradiol levels) results in induction of tolerance (Black et al. 2000; Kaushic et al. 2000; Gockel et al. 2003). New vaccine trials need to take into account the biology of women and plan genital cells and secretions sampling in their design for evaluation of anti-HIV responses at the portal of entry.

12. Conclusions

The continued expansion and magnitude of the HIV epidemic in women require urgent efforts to find effective preventive methods. The characteristics of the HIV target cells and innate and adaptive anti-HIV responses in the FRT remain largely unknown; thus, a considerable increase in knowledge is necessary to achieve this goal. Given the complexity of interactions between HIV and the FRT, it appears likely that a combination of strategies that act at the portal of entry to protect the HIV target cells induces an effective local innate and adaptive immune response, and control of hormonal changes to reduce local HIV susceptibility will be necessary for successful prevention.