Charu Kaushic, Department of Pathology & Molecular Medicine, Michael DeGroote Center for Learning & Discovery Room 4014, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada. E-mail: email@example.com
Citation Kaushic C. HIV-1 infection in the female reproductive tract: role of interactions between HIV-1 and genital epithelial cells. Am J Reprod Immunol 2011; 65: 253–260
Despite recent progress in understanding the mucosal transmission of human immunodeficiency virus (HIV)-1, the immediate events following transmission in the female genital tract are incompletely understood. Recent in vivo studies in primate models indicate that HIV-1 transmission may occur in the upper or lower genital tract and the initial HIV-1 replication occurs primarily in the target T cells and in some subsets of DCs localized in the genital tract. However, the principal mechanism(s) that allow the virus to cross the primary barrier of genital epithelial cells (GECs) are still unclear. A number of pathways have been proposed as possible ways that HIV-1 could use to cross the epithelium. However, little attention has been paid to the response of GECs to HIV-1. We recently demonstrated that exposure to HIV-1 rapidly upregulates a wide array of pro-inflammatory cytokine production by GECs. Among these cytokines, tumour necrosis factor (TNF)-α impaired the tight junction barrier allowing HIV-1 and luminal bacteria to translocate across the epithelium. This study illustrated that GECs are dynamically active cells that mount rapid host responses to HIV-1, independent of viral replication. Cytokine responses of GECs could play a critical role in HIV transmission and replication. Further understanding of GEC responses to HIV-1 and their regulation could be critical to understanding HIV-1 transmission dynamics during heterosexual transmission.
Human immunodeficiency virus-1 (HIV-1) is primarily a mucosal pathogen since >85% of HIV-1 infections occur through genital and intestinal mucosal exposure. Recent estimates show that despite the low transmission probability per exposure, female reproductive tract accounts for about 40% of HIV-1 transmission.1 Contrary to the picture portrayed by the overall stabilization in number of HIV-1 infected people worldwide in last few years, the pandemic is still growing in women. According to UNAIDS, women now constitute ∼50% of the 40 million people who are currently living with HIV worldwide.2 In order to address the issue of HIV-1 prevention in women, it is critical to understand heterosexual transmission of HIV-1 since this is the primary route of HIV-1 transmission in women. Unfortunately, despite recent efforts and progress in understanding mucosal transmission of HIV-1 via the intestinal and genital tract, the acute events following HIV-1 exposure in the female genital tract that result in either successful infection or protection against HIV-1 remain unclear.
Recent observations from studies conducted on in vitro cell cultures, ex vivo cervico-vaginal tissues and non-human primates have provided some insights into the acute transmission events in the female genital tract.3,4 These studies indicate that HIV-1 transmission can occur both in the upper or lower female genital tract. This has helped in overcoming the previous dogma that the primary, and perhaps the only, route of HIV-1 transmission in the female genital tract is through microtears in the vaginal mucosa, during sexual intercourse. Perhaps the most convincing confirmation of this comes from recent studies examining acute simian immunodeficiency virus (SIV) infection in non-human primates.4,5 These studies clearly indicate that when inoculated intravaginally with high doses, SIV crossed the epithelial barrier preferentially in the endocervix, within hours, and established infection in small focal “hot spots” within 48–72 hr. The formation of a few small foci of productive infection is consistent with the recent observations that HIV infection in infected hosts can be traced back to small (between 1 and 5) founder virus populations. In the macaque SIV infection study, the small foci underwent a local amplification in the genital mucosa in the first few days prior to systemic dissemination.4,5 Based on the elegant studies by Hladik et al.6 that have been confirmed by others, there is general consensus that HIV-1 replication occurs primarily in the target T cells. While some subsets of DCs can be both productively infected by HIV-1 as well as play a role in trans-infection to T cells, their contribution to local amplification of HIV-1 in the genital tract is likely limited.3,4 The subset of T cells that are most susceptible to HIV-1 and the role played by various subsets of DCs in trans-infection versus viral replication is still being investigated and progress in these areas has been previously reviewed.7,8
Despite these insights, the mechanism(s) that allow, and the factors that regulate, the crossing of the primary barrier of genital epithelial cells (GECs) that line the genital mucosa of the female reproductive tract by HIV-1 remains incompletely understood. This review will focus on this area, summarizing the current information and outlining some of the challenges that remain.
Epithelial cells lining upper and lower reproductive tract
One of the important considerations for understanding the heterosexual HIV-1 transmission is the morphological and functional distinctions between the epithelial cells that line the different compartments of the female reproductive tract. These epithelial cells are the first cells that HIV-1 or any other pathogen encounters therefore their physical and functional characteristics are important determinants in the outcome of exposure to HIV-1. The lower reproductive tract in women is composed of exocervix and the vaginal tract. The mucosal lining in these compartments consists of stratified squamous epithelium that can be more than 25-cell layers thick.9 In contrast, the upper reproductive tract, made up of the endocervix and endometrium, is composed of a single layer of columnar epithelium that rests on a continuous, thin basement membrane.10 The columnar epithelium is characterized by the presence of tight junctions between cells that makes it impermeable to entry of any large molecules and particulate matter, including pathogens. The thick stratified epithelium of the lower reproductive tract, although not impermeable, is robust and provides a substantive physical barrier compared to the delicate single layer of columnar epithelium of the upper reproductive tract. In addition, the continuous sloughing of the superficial layers of the stratified epithelium of the vagina and ectocervix prevents many pathogens from colonizing and establishing infections, thus providing a better mechanical protection against HIV invasion than the single layer columnar epithelium that lines the upper reproductive tract. However, the greater surface area of the vaginal wall and ectocervix arguably allows greater access for HIV entry, particularly when breaches occur in the epithelium, such as during sexual intercourse.1,11,12
In addition to the differences in the morphology, the upper and lower reproductive tract epithelium are quite distinct in terms of the local microenvironment and functions. The lower reproductive tract, particularly the vaginal mucosa is continuously exposed to the external environment and is rich in resident flora. Functionally, this requires the lower tract to continually differentiate between commensal bacteria and pathogens. Normal, healthy vaginal mucosa has commensal microflora rich in lactobacilli that serves a number of important and beneficial functions including lowering the local pH in the vagina, making it an inhospitable environment for pathogens to survive.13 The epithelium of the endometrium, on the other hand, lacks direct contact with the external environment. This does not imply that the upper reproductive tract is a sterile environment. Rather, its function is primarily reproductive, serving as a site for implantation and development of the foetus. These functional differences are reflected in the ability of the upper and lower reproductive tract epithelial cells to respond to various stimulations. These will be discussed in more detail in the next section. One important similarity between both the columnar and squamous epithelium is that they are exquisitely responsive to, and functionally regulated by reproductive hormones, estradiol and progesterone.14
The squamo-columnar junction between the exocervix and the endocervix where the squamous epithelium abruptly changes to the single layer of columnar epithelium is called the transitional or transformation zone. In addition to the morphological changes, the transformation zone is the most immunologically active site in the reproductive tract; lymphocytes and antigen presenting cells are present in abundance in normal women and further increased during inflammatory conditions.15 The increased physical vulnerability and the presence of an enriched target population could easily make the transformation zone the most vulnerable point in the genital epithelium and an easy target as an entry point for HIV-1. Indeed clinical studies have shown that presence of ectopy (the protrusion of the cervical transformation zone outside the external os) of the cervix has been associated with increased risk of heterosexual transmission of HIV-1.16
Immune functions of genital epithelial cells
Genital epithelial cells are dynamically active cells. As the primary cells that form the barrier between the external environment and the female genital tract, they play a critical role as the first responders to any incoming pathogen. They perform the dual function of responding directly to the pathogen as well as relaying signals to activate other innate and adaptive cells of the immune system. GECs perform both these tasks very efficiently. They secrete a variety of anti-microbial factors constitutively and upon induction. They also express a complete repertoire of Toll-like receptors (TLRs), which allows them to recognize a wide array of pathogens and respond by production of cytokines and chemokines, which in turn attract other immune cells including neutrophils and DCs to the genital tract. There is also evidence that GECs condition DCs to initiate adaptive immune responses.
Among the anti-microbial peptides secreted by GECs, many are produced constitutively and others are induced or upregulated upon exposure to stimuli. Studies carried out in in vitro culture systems indicate that some of these, including secretory leukocyte protease inhibitor (SLPI), lactoferrin, β defensin and trappin-2/elafin have anti-HIV properties. The anti-leukoprotease SLPI has been shown to be secreted by ECs both in the human cervix and endometrium and regulated by female sex hormones oestrogen and progesterone. Based on in vitro studies, it has been suggested that SLPI inhibits HIV-1 prior to replication.17–19 Similarly, lactoferrin, a protein found in breast milk and the genital tract, has also been shown to inhibit HIV at the early stages of viral infection in vitro, by blocking viral adsorption and uptake.20,21 Defensins, a family of small cationic proteins produced in the genital tract have demonstrated significant antimicrobial effects in studies.22,23 Epithelial cells of the genital mucosa produce mainly β-defensins.24 Human β-defensin-1 (HBD-1) is expressed constitutively while HBD-2 and HBD-3 are inducible and have been shown to inhibit HIV-1, particularly X4 tropic strains.25–27 More recently, the serine protease inhibitor, trappin-2/elafin has been shown to be secreted by GECs and implicated in anti-HIV activity at mucosal surfaces.28,29
Similar to the epithelial cells that line the gastrointestinal and lung mucosa, GECs express a rich array of pattern recognition receptors. In particular, expression of and activation by TLRs in GECs have been well described. Vaginal and cervical epithelial cells and cell lines express TLRs 1, 2, 3, 5 and 6, while primary endocervical ECs express TLRs 1, 2, 3 and 6 allowing them to sense both bacterial and viral pathogenic motifs and rapidly relay messages to other innate and adaptive cells should a pathogenic breach occur (reviewed in Ref. 30). Primary human endometrial ECs express an even broader array of TLRs 1–9, indicating the potential of upper reproductive tract to respond to a wide range of pathogens. TLR-mediated activation leads to production of chemokines and cytokines, including IL-6, IL-8, SDF-1 and β-chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES.31–35 Using an ex-vivo cell culture system of GECs, we found that the antiviral responses induced in genital ECs following treatment with eight different TLR ligands correlated with differential decrease in HSV-2 replication in GEC cultures.36 TLR3 [poly (I:C)], TLR9 [cytosine–phosphate–guanosine (CpG A)] and TLR5 ligands (flagellin) showed the greatest ability to reduce HSV-2 replication. Poly (I:C) treatment induced maximum anti-viral effects, but also enhanced production of inflammatory cytokines IL-1α, IL-6 and tumour necrosis factor (TNF)-α. Similar effects on the inhibition of human cytomegalovirus (CMV) replication following activation by TLR ligands in human genital tissues have also been reported.37
In addition to the pro-inflammatory cytokines and chemokines, GECs are also capable of producing Type I Interferon, mainly IFN-β. Type I interferons (IFN-α, -β) impede the HIV replication cycle through numerous mechanisms, including induction of the antiviral molecule apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G).38,39 Type I IFNs also induce the production of inactive 2′,5′-oligoadenylate synthetase (2′,5′-OAS), which activates a latent endo-ribonuclease, RNase L that degrades viral and cellular RNAs resulting in inhibition of protein synthesis.40,41 The 2`,5`-OAS/RNase L pathway has been shown to inhibit the replication of HIV-1.42,43 Protein kinase R (PKR), inducible nitric oxide synthase (iNOS), myxovirus (Mx)-family proteins and 9–27 proteins are other interferon-inducible proteins shown to have anti-HIV properties.44 Schaefer et al.45 reported the upregulation of the message levels for IFN-β, MyxA and OAS in cultures of primary endometrial ECs following exposure to TLR 3 ligand Poly I:C. Since then, other studies have measured and reported production of IFN-β in cervical and cervicovaginal cells following activation by TLR ligands or virus exposure.37,46 In our studies, we found TLR3, 5, 9 ligands including Poly I:C, Flagellin and CpG oligos upregulated biologically active Type I IFN and NO production by GEC cultures resulting in significant protection of GECs against HSV-2 infection.36 Neutralization of IFN-β in the culture supernatants abrogated protection against HSV-2 infection in the GEC cultures.
The ability of GECs to respond so exquisitely to environmental stimuli with an array of immune factors could potentially have a profound effect on the local milieu of the genital tract and on other immune cells that are present or infiltrate into the genital tract as a consequence of chemotactic influences. Chemokines including IL-8, MIP-1β, monocyte chemotactic protein-1 (MCP-1) produced by GECs would play an important role in attracting other immune cells such as neutrophils, monocytes, DCs and T cells. In addition, local production of cytokines by epithelial cells including TNF-α, GM-CSF, IL-6 could influence differentiation and activation state of these immune cells. To this effect, a recent study utilizing an in vitro co-culture model of endometrial epithelial cell cultures with DCs demonstrated that soluble mediators produced by ECs do indeed modify DC differentiation and functions. Thus, GECs play a key role as initiators of both innate and adaptive responses in the genital tract.47
Crossing of epithelial cell barrier by HIV-1
Earlier in vitro studies indicated that genital tract epithelial cell lines could be infected by HIV.48 Subsequent studies have shown that HIV-1 can infect both ECs from the lower49,50 and upper female genital tract in vitro.51,52 In one study, X4-tropic strain of HIV (T cell-tropic) was shown to replicate in cultured human primary uterine epithelial cells; however, R5-tropic strain (macrophage-tropic) was taken up and released from the cells, unmodified.53 These and other studies have raised the possibility that the virus could directly enter GECs using receptors. In support of this likelihood, canonical HIV-1 receptors including CCR5, CXCR4 and CD4 have been demonstrated in the female genital tract and shown to be regulated during the menstrual cycle.54,55 Alternative cellular receptors, such as Gal-Cer, C-type lectins such as DC-SIGN, mannose receptors, proteoglycans such as heparin sulphate and syndecan that bind to HIV have also been demonstrated.48,56–59 More recently, the organ culture models of intestine, tonsil and cervix have been able to add relevant information regarding HIV transmission across epithelium.6,60,61 These studies indicate that HIV-1 could possibly bind to epithelial cells via β-1 integrin and penetrate the ectocervical epithelial cell surface. A variant of salivary agglutinin named gp340, which is expressed on cervical and vaginal ECs has also been implicated in the passage of HIV-1 through the epithelium. The relative contribution of these receptors to HIV-1 entry and infection in genital ECs is unclear. In addition to the reported diversity in expression of these alternate receptors, there is also ambiguity regarding which cellular processes may play a primary role in allowing HIV-1 to penetrate the GEC barrier. Direct infection,52 transcytosis,51,57,62 sequestration, transmigration, penetration of the virus through breaches in the epithelium11,12 have all been implicated as possible ways in which HIV crosses the genital epithelium. Despite a significant body of information regarding HIV-1 interactions with GECs, the exact mechanism involved in crossing of the genital epithelium by HIV-1 remains inconclusive.
Direct interactions of genital epithelial cells with HIV-1
As outlined above, much of the attention on genital epithelial interactions with HIV-1 has focused on examining the mechanism by which the virus crosses the epithelium. We recently reported a novel mechanism demonstrating that GECs interact directly with HIV-1, resulting in a rapid cellular response from the GECs (Fig. 1).63 Interaction of viral surface glycoprotein gp120 leads to production of an array of pro-inflammatory cytokines from GECs. Among these, TNF-α production led to breaching of the epithelial barrier by reduction of trans-epithelial resistance (TER), a measure of epithelial monolayer integrity, by 30–60% in primary genital epithelium. The disruption in barrier functions was associated with viral and bacterial translocation across the epithelial monolayers. A mutant of HIV-1 lacking viral surface glycoprotein as well as replication incompetent HIV-1 were unable to induce any response from GECs, indicating that the interaction was mediated by viral surface glycoprotein, gp120. Given the rapid timing of the response (within 4 hr), one of the conclusions from this was that GECs can recognize HIV-1 and initiate cytokine production independent of viral replication. While in this study we focused on the effect of pro-inflammatory cytokines on the barrier function of GECs, the overall implications of this phenomenon could be more extensive. Presence of pro-inflammatory cytokines in the genital tract within such a short time of HIV-1 exposure could directly affect HIV-1 transmission and replication dynamics in the genital tract. Currently, we are examining both HIV-1 replication dynamics as well as activation of other pathways in this system. These results show that while HIV-1 replication is not productive, HIV-1 pro-viral DNA can be detected in GEC cultures (Kafka et al., unpublished). Interestingly, NF-kB activation was observed GECs within 1 hr post-HIV-1 exposure indicating rapid signal transduction following HIV-1 exposure (Nazli et al., unpublished).
Conclusions and challenges
Genital epithelial cells play a critical role as the primary defenders of genital mucosa and as initiators of innate and adaptive immune responses. The female reproductive tract mucosa consists of epithelial lining of multiple organs, each with distinct morphological and functional characteristics. These cells are dynamically active, equipped to recognize a wide range of pathogens, produce an array of anti-microbial factors, cytokines and chemokines constitutively and upon stimulation by pathogenic motifs. There is significant evidence that a number of alternate receptors are expressed on GECs that could lead to a range of interactions between GECs and HIV-1. Suggested pathways leading to HIV-1 crossing the epithelial barrier include among others, direct infection, transcytosis and sequestration. However, because of the differences in biological properties of human cell lines and primary cells and unique complexities of ex-vivo cell or organ culture systems, significant challenges remain in interpreting the physiological relevance of these pathways to HIV-1 transmission. In vivo studies in non-human primate models that focus on early events of HIV-1 interaction with genital epithelium could provide useful insight on the primary mechanism/s involved in viral crossing of epithelium and relative contribution of various pathways. There is also emerging evidence that the GECs respond to HIV-1 exposure very rapidly by production of an array of cytokines and chemokines and this in turn could influence HIV-1 transmission and possibly replication. Altogether, these studies emphasize the importance of examining the GEC-HIV-1 interactions more closely since GEC responses could directly influence the genital microenvironment and consequently, HIV-1 transmission and replication.
CK’s research is supported by research grants from Canadian Institutes of Health Research (CIHR), Ontario HIV Treatment Network (OHTN) and Canadian Foundation of AIDS Research (CANFAR) to C.K. C.K. is a recipient of an OHTN Scholarship Award and a New Investigator’s Award from CIHR. The author would like to thank Varun Anipindi and Kristen Mueller for help with the figure.