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1 Department of Diagnostic and Biological Sciences and the Mucosal and Vaccine Research Center, University of Minnesota, 17-164 Moos Tower, 515 Delaware St. SE, Minneapolis, MN 55455, USA
2 Case Western Reserve University, Cleveland, OH 44106, USA; and
3 National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA
Correspondence: * corresponding author, mcherzb{at}umn.edu
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Abstract |
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 TOP Abstract IntroductionList of QuestionsSummary and RecommendationsReferences |
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KEY WORDS: Oral epithelium • squamous keratinocytes • HIV-1 • innate immunity • T-cells
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Introduction |
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TOPAbstractIntroductionList of QuestionsSummary and RecommendationsReferences |
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We considered the oral regional anatomy and modeled how oral keratinocytes and immune cells might be affected by the presence of HIV-1. If the oral mucosae are a portal for systemic infection, mechanisms must exist for the transfer of HIV-1 to the systemic immune cell compartment. To evaluate the evidence, or lack thereof, for an oral route of HIV-1 infection, we considered several related questions.
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List of Questions |
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TOPAbstractIntroductionList of QuestionsSummary and RecommendationsReferences |
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Question 2: Can intrinsic differences between oral and other mucosal epithelial cells explain, in part, why oral mucosae appear to be less-favored routes of infection?
Question 3: Can infectious or inactive virus be found in saliva?
Question 4: Can primary infections be explained by a plausible route of HIV-1 transfer from unbreached oral mucosa to the systemic immune system?
Question 5: What are the factors that minimize infection of target T-cells in the oral mucosa? and
Question 6: What is the fate of HIV-1 upon exposure to oral tissues in animal models?
Question 1: Does HIV-1 translocate and integrate into oral mucosal epithelial cells or other mucosal cells during clinical disease or in vitro?
The epithelium of the oral mucosae is organized into a stratified squamous barrier, separating the intra-oral environment from subepithelial tissues housing viral-susceptible host cells. In the mucosa, the accessibility of susceptible cell targets is critical to launching a productive infection. Epithelial cells lining the mucosa may represent an initial barrier, but may also be a conduit for HIV-1 entry. HIV-1 (phenotypes X4 and R5) appears to interact in vitro with oral keratinocytes. After exposure in vitro, cell-free (using the non-physiological detergent polybrene to promote binding of virus to cells) or cell-associated HIV-1 can be identified within oral mucosal epithelial cells (Pang et al., 2000; Liu et al., 2003, Moore et al., 2003). Viral inocula that are required for internalization or infection, however, are greater than doses that promote infection of epithelial cells from other mucosal sites. Uptake of HIV-1 into CD4-negative squamous epithelial cells in culture appears dependent on expression of GP120 on the virus and may be accompanied by a low level of integration in some cell lines and primary cultures. Whether X4 or R5 type HIV is preferentially captured and integrated into oral keratinocytes is unclear. HIV-1 X4 strains have been reported to preferentially produce low-level integration in a galactosylceramide (gal-cer)- and CXCR4-dependent manner, based largely on the reduction of p24 expression by keratinocytes in the presence of antibody to gal-cer or SDF-1
, respectively (Liu et al., 2003). In other studies, R5 strains have been reported to integrate exclusively (Moore et al., 2003). Earlier work suggests that GP120 promotes uptake in concert with an oligomannosyl co-receptor (Kage et al., 1998). These in vitro studies suggest, therefore, that squamous keratinocytes can potentially harbor HIV-1 and transfer viable virus to targets among peripheral blood mononuclear cells (PBMCs), where virus readily replicates and new virions are actively produced. In vivo corroboration is lacking. Moreover, additional in vitro studies must resolve apparent inconsistencies about the required HIV-1 ligands and the corresponding keratinocyte receptors and co-receptors.
HIV-1 may be identifiable within oral keratinocytes from HIV-positive individuals (Qureshi et al., 1995, 1997), but these reports are as yet unconfirmed. Oral squamous keratinocytes are occasionally positive for nuclear HIV-1 DNA in seropositive patients. Oral epithelium and tissue washings from HIV-seropositive individuals contain fewer Langerhans cells (resident antigen-presenting cells; immature dendritic cells; iDCs) than do those from uninfected controls, suggesting that exposure to the virus mobilizes and depletes these cells from the mucosa (Sporri et al., 1994; Chou et al., 2000). HIV-1 RNA can be detected in saliva, although the virus is reportedly inactive in the saliva of most seropositive patients (Liuzzi et al., 1996). Viral RNA levels are highest before the onset of symptoms (Pilcher et al., 2001), suggesting that the virus could be infectious soon after exposure and before seroconversion. The presence of active virus in the oral cavity before seroconversion has not been investigated, but acquisition of such information is important to our understanding of the oral cavity as a potential route of transmission.
The source of HIV-1 in saliva may depend on the stage of infection. After seroconversion, HIV-1 may appear in saliva from gingival crevicular fluid, which represents contributions from the plasma and cells in the inflamed gingiva (Maticic et al., 2000). HIV-1 may also be released from the palatine tonsils, since tonsillectomy is associated with reduced HIV-1-specific RNA in the secretions (Zuckerman et al., 2003). Once seroconversion occurs, fewer than 1% of patients showed cell-free infectious virus in their saliva samples (Barr et al., 1992, 1996), although the frequency of patients with infectious virus in undiluted salivary supernatants has also been reported to be somewhat higher (six of 28 patients) (Yeung et al., 1993). Although there is little evidence to support the oral mucosal epithelium as a site of primary infection, it cannot be ruled out. Upon shedding of HIV-1, keratinocytes could be a source of transmission to others. If primary infection of the oral mucosae permits carriage of HIV-1 to more permissive cells in the systemic compartment, mechanisms that could enable oral squamous mucosal epithelium to harbor, become infected with, and transfer HIV-1 to lymphoid targets remain important, but open, questions.
Much of our knowledge of mucosal infection by HIV-1 is inferred from studies of the intestinal tissues, which differ in key properties from the oral mucosae. The intestinal epithelium is an established site of mucosal infection by HIV-1. This epithelium is organized in a unicellular layer of columnar cells attached to one another by tight junctions. Based on studies in intestinal epithelial cell lines, R5 and X4 viruses can be translocated from their apical to basolateral surfaces (Bomsel, 1997; Alfsen and Bomsel, 2002; Bomsel and David, 2002), while, in cultured primary intestinal epithelial cells, R5 HIV-1 is selectively captured by interactions with epithelial cell membrane galactosylceramide and CCR5, internalized via microtubule-dependent endocytosis, and transcytosed to PBMCs (Meng et al., 2002). The R5 selectivity of transcytosis by intestinal epithelial cells may explain the greater prevalence of R5 than X4 primary infections. After crossing the intestinal mucosal surface, HIV-1 may encounter myeloid-derived dendritic cells (DCs), which secure HIV-1 through DC-SIGN, a C-type lectin that traps virus for efficient delivery to T-cells (Cameron et al., 1992; Geijtenbeek et al., 2000; Baribaud et al., 2001a; McDonald et al., 2003), and/or other related lectin receptors (Turville et al., 2002; Wu et al., 2002). Upon contact with T-lymphocytes and macrophages, HIV-1 recognition by CD4, together with CCR5 and/or CXCR4 (Berger et al., 1999), initiates fusion, entry, and the replicative viral life cycle. Recent evidence implicates additional co-factors, such as annexin II (Ma et al., 2004), in the macrophage entry/infection process. We continue to compare the intestinal ‘gatekeeper’ model against the plausibility of infection through the squamous epithelia of the oral and oropharyngeal tissues. Yet the structures of the mucosae differ, and it may be unwarranted to extrapolate findings from one site to another.
Contiguous with oral epithelial cells, potential targets of HIV-1 infection also include the lymphoid cells embedded in the epithelium and submucosa, epithelial cells and organized lymphoid tissue of adjacent tonsils, and salivary gland epithelial and proximal lymphoid cells. After incubation with HIV-1, salivary gland epithelial cell lines in vitro will produce infectious virions in a CD4-independent, galactosylceramide-dependent (Han et al., 2000), or CXCR4-dependent (Moore et al., 2002) manner. In seropositive individuals, however, HIV-1 is localized to interstitial mononuclear cells only, and there is no evidence of productive HIV-1 infection of salivary gland epithelium or acinar cells (Wahl et al., 1997a); protection is attributed to secretory leukocyte protease inhibitor (SLPI).
Palatine tonsils can be infected by HIV-1 (Navarrete et al., 2003; Fischer et al., 2004; Moutsopoulos et al., 2006b), which actively replicates in lymphoepithelial crypts (Frankel et al., 1997), even during apparent clinical latency (Pantaleo et al., 1993). The progress of infection can be modeled in tissue explants ex vivo (Maher et al., 2004). In vitro, tonsil epithelial cells reportedly capture and transfer viable type-X4 and R5 HIV-1 to PBMCs in a CD4, CXCR4-, CCR5-, and galactosylceramide receptor-independent manner (Tangjaturonrusamee et al., 2006).
Immature dendritic (iDCs; Langerhans) cells and T-cells can be immunolocalized in the oral squamous epithelium of the gingiva, and their numbers increase with periodontal inflammation (Jotwani et al., 2004). Based largely on studies of skin and vaginal mucosa, HIV-1 will co-localize with iDCs within the epithelium (Dusserre et al., 1992). Once activated, iDCs migrate into the lamina propria to form a synapse with CD4+ T-cells and transfer virus (Blauvelt et al., 1997; Kawamura et al., 2003, 2004). In the connective tissues and regional lymph nodes, DCs presenting HIV-1 show activation markers (Cirrincione et al., 2002). Since DCs present HIV-1 via an immunological synapse (McDonald et al., 2003) to CD4+ T-cells in the connective tissues or regional lymph nodes, HIV-1 becomes poised to enter the circulation and disseminate (Larsson, 2005; Teleshova et al., 2006). Although infection of iDCs is seen in vitro, iDCs in situ appear to capture HIV-1 virions in a DC-SIGN-dependent manner to facilitate transfer and productive infection of CD4+ T-cells (Piguet and Blauvelt, 2002). The roles of iDC CXCR4 and CCR5 in specifying subsequent transfer to T-cells are less clear. Furthermore, it is unclear what role, if any, the oral squamous epithelial cells might play in facilitating HIV-1 interactions with iDCs, or if a life cycle of replication can be supported. If iDCs cannot sample virus on the surface of the oral mucous membranes, interactions between HIV-1 and keratinocytes may specify the progress of transmucosal infection and subsequent involvement of iDCs.
During early stages of HIV-1 infection, most detectable infected cells are T-lymphocytes, and the frequency of infected cells in peripheral lymphoid organs typically reflects plasma viral RNA levels (Schacker et al., 2001). Since HIV-1 replicates preferentially, but not exclusively, in activated CD4+ T-cells (Zack et al., 1990; Bukrinsky et al., 1992), mucosal lymphocytes which are continually exposed to environmental antigens (Schieferdecker et al., 1992) may be especially vulnerable. As mucosal and circulating CD4+ T-cells are depleted, monocytes and macrophages assume an expanding role as viral hosts (Wahl et al., 2003). Furthermore, virus budding from macrophage intracellular membranes (Orenstein et al., 1988; Wahl et al., 2003) may escape immune surveillance, and macrophages may persistently co-exist with virus, whereas infected T-cells are marked for death. Once successful infection is established in these susceptible cell populations, replication occurs, and a cycle of infectious spread to additional target cells is set in motion.
Question 2: Can intrinsic differences between oral and other mucosal epithelial cells explain, in part, why oral mucosae appear to be less-favored routes of infection?
Answers to these key questions are still being pursued. To determine if evidence supports the expression of autonomous cellular resistance to HIV-1, we will compare mucosal epithelial cells from different tissues for (1) physical differences, (2) expression of known HIV-1 co-receptors and infectivity, and (3) expression of oral epithelial cell-derived anti-retroviral peptides.
Physical differences in mucosal epithelial cells
The genital tract mucosa represents a vulnerable exposed mucosal target in HIV-1 transmission. Nonetheless, the actual number of productive infections per encounter is low, suggesting that there are substantial structural, chemical, and/or cellular barriers to virus entry, which may not be invincible. Susceptible mucosal sites, including the female genital tract, express dendritic cells whose extensions can reach the surface, and through which infection can ensue. To date, there is no evidence that the oral mucosa can directly and efficiently sample HIV-1 on its surface through dedicated antigen-presenting cells such as iDCs. Certainly this anatomic difference could explain, in part, the apparently greater sensitivity of the genital mucosa to HIV-1 compared with the oral tissues.
HIV-1 infection of the oral mucosa may also be restricted by the stratified squamous structure of the oral epithelium. Oral epithelium contains differentiated layers of cells that can be 20 or more layers thick. Many squamous mucosal sites show high cell turnover rates, which may encourage the shedding and swallowing of any associated virus. During exposure to HIV-1, the stage of keratinocyte differentiation may affect susceptibility to capture virus or cooperate with iDCs to transfer virus. Cornified keratinocytes cover the so-called ‘attached or masticatory mucosa’ and may be relatively resistant to uptake of virus. Conversely, non-cornified keratinocytes line the buccal tissues, the floor of the mouth, the oropharynx, and the gingival sulcus, which often appears ulcerated during periodontitis. These lining mucosa sites might be more susceptible to infection than the masticatory mucosa. In the neonate, squamous epithelial tissues appear to be somewhat less cornified, and the ontogeny of the developing mucosa and immune system may affect the susceptibility to infection. Thus, the oral mucosa could be a route for mother-to-infant transmission early in post-natal life.
In contrast to the oral mucosa, a single layer of columnar epithelial cells joined by tight junctions generally lines the gut. Consequently, passage of HIV-1 through a single cell layer is likely to be a more efficient route to systemic infection than transcytosis through the multiple layers of oral epithelium. Viral capture and transfer to lymphoid cells may also occur through the lymphoid follicles of the gut, which are arranged periodically along the mucosal wall. The follicles are covered by a follicle-associated epithelium (FAE), which contains M-cells (Kraehenbuhl and Neutra, 2000). M-cells are designed for immunological sampling of the contents of the gut and transport virus (and bacteria) to the follicles for processing (Kraehenbuhl and Neutra, 2000). M-cell transcytosis is considered to be an important route of HIV-1 intestinal infectivity (Kerneis et al., 1997; Fotopoulos et al., 2002). Yet transcytosis of HIV-1 by M-cells may not be the only pathway of infection in the gut. When co-cultured with B-lymphocytes, human colonic epithelial cell monolayers (Caco-2 line) act like M-cells and efficiently transport cell-free and cell-associated HIV-1 to peripheral blood mononuclear cells (Fotopoulos et al., 2002). Whether transcytosis of HIV-1 by oral squamous epithelium is a biological equivalent of B-cell-stimulated Caco-2 cells remains to be determined. While oral epithelium may not express M-cell-like activity, the palatine tonsil epithelium could potentially provide a related function.
Infectivity of HIV-1 may also be affected by the opportunity for the viral inoculum to remain in the presence of the exposed tissues. Increasing the duration of exposure of the tissues to infectious virus would likely increase the opportunity for infection. HIV-1 exposure to intestinal or genital epithelium is likely to be longer than with the oral mucosa, which is actively cleared by saliva and swallowing. During direct exposure to the tissues in the mouth, an oral inoculum would likely be infectious and either highly concentrated (semen) or diluted (mature breast milk). HIV will be diluted many-fold during progression through the alimentary tract, and the infectivity of cell-free and cell-associated HIV-1 in the gut would be challenged by the acidity of the gastric fluids, which show a pH of 4.3 even in the human infant (Crill et al., 2004). In comparison with the gut, the whole salivary fluid shows a pH of about 7.0 in children and adults (Azrak et al., 2003). In this buffered, neutral environment, HIV is exposed to the oral tissues for only a short period (seconds or minutes) before swallowing. Physical characteristics of the mucosal epithelial cells and the proximal environment that may affect susceptibility to HIV-1 infection, therefore, include the time of exposure to HIV-1 before swallowing, the surface area of the mucosal tissues relative to the infectious dose, accessibility to receptive target cells, and the infectivity of the presented form of HIV (cell-free or within macrophage or other cells in saliva, breast milk, or semen).
Expression of HIV-1 co-receptors and infectivity of epithelial cells
Several well-studied receptors and co-receptors can be used by HIV-1 to bind to and promote infection of target cells. HIV-1 receptors and co-receptors reported to be expressed by epithelial cells include CD4 (characteristic of T-cells), CCR5 (the R5 viral phenotype co-receptor), CXCR4 (the X4 viral phenotype co-receptor), and galactosylceramide (GalCer) receptor, a glycosphingolipid receptor that binds gp120 with high affinity and enables HIV-1 to infect CD4-negative cells from neural and intestinal tissues (Yahi et al., 1992). Salivary gland epithelial cell lines, susceptible to HIV-1 infection in vitro, may express GalCer, CCR5, CXCR4, and various levels of CD4 (Han et al., 2000; Moore et al., 2002), albeit this has not been demonstrated on primary cells or in vivo. Human epithelial Langerhans cells (iDCs) express CXCR4 and CCR5 (Blauvelt et al., 2000; Tchou et al., 2001) and DC SIGN, a C-type lectin found on dendritic cells (Geijtenbeek et al., 2000), and can be infected by HIV X4 and R5 strains. GalCer has been detected on cervical and prostate epithelial cells, but not CCR5, CXCR4, or CD4 (Dezzutti et al., 2001). Tonsil epithelial cells in vitro appear to be negative for all of the known purported receptors and co-receptors, including DC-SIGN, gal-cer, CCR5, CXCR4, and CD4, based on preliminary data, yet appear able to transfer virus to activated immune cells (Tangjaturonrusamee et al., 2006). If mucosal keratinocytes take up and transfer HIV to activated immune cells, receptors not common to T-cells and macrophages probably participate.
Alternatively, the expression of typical HIV-1 receptors and co-receptors may depend on interactions with other cells or signaling molecules such as cytokines. Follicle-associated epithelium (FAE), covering human gut lymphoid follicles, expresses CCR5 and GalCer, but not CXCR4, and expression may result from exogenous signaling. When co-cultured with B-lymphocytes, as mentioned above, human colonic epithelial cell monolayers (Caco-2 line) act like M-cells, express GalCer and the appropriate chemokine co-receptor, and efficiently transport cell-free and cell-associated HIV-1 (Fotopoulos et al., 2002). Primary intestinal (jejunal) epithelial cells express gal-cer and CCR5, but not CXCR4, indicating a potential mechanism for the selective transmission of R5 HIV-1 in primary infection acquired through the upper gastrointestinal tract (Meng et al., 2002). Since R5-type HIV-1 is the most frequent cause of primary infections, the female genital mucosa may also transcytose R5-HIV-1 and ‘ignore’ X4-type HIV-1 by a similar ‘gatekeeper’ mechanism. CCR5 is the major HIV co-receptor expressed in the female genital tract (Patterson et al., 1998), but expression of that co-receptor is hormonally regulated on human uterine epithelial cells (Yeaman et al., 2003). Furthermore, the expression of HIV-1 receptors and co-receptors by oral mucosal cells is increased during inflammation associated with periodontitis (Jotwani et al., 2004). It remains to be determined if modulation of HIV-1 receptor and co-receptor expression in vivo enables virus to be taken up by mucosal keratinocytes in a manner that is not easily observed in vitro.
While palatine tonsils exposed to HIV-1 ex vivo can show evidence of incorporated virus and signs of modest progression of infection (Maher et al., 2004), and epithelial cells from various mucosal surfaces (Yahi et al., 1992; Fotopoulos et al., 2002), including salivary glands (Han et al., 2000), capture HIV-1, the evidence remains divided as to whether human oral epithelial cells can be infected. For example, human oral epithelial cells (HOECs) showed low-level replication after high-dose viral challenge in the presence of polybrene (Liu et al., 2003), but frank infection, as evidenced by HIV-1 integration, was not replicated by other workers with either X4- or R5-tropic viruses (Quiñones-Mateu et al., 2003). The discrepancies between these studies may be due to numerous differences in experimental conditions. Quiñones-Mateu et al.(2003) omitted polybrene from their incubation assays, since this detergent is not physiological. Liu et al.(2003) exposed cells using a viral inoculum 8 times higher than that reported by Quiñones-Mateu et al.(2003). Finally, Liu et al.(2003) found HIV-1 co-receptor expression on two-week-cultured HOECs, while flow cytometric analyses by Quiñones-Mateu et al.(2003) did not reveal CD4, CCR5, CXCR4, or GalCer expression on three- to four-day-cultured HOECs. Comprehensive studies are needed to understand if and/or how oral keratinocytes at different stages of differentiation, and populating specific anatomic sites, interact with diverse strains of HIV, and to define the contributory role of epithelial-cell-derived anti-viral peptides in this process.
Epithelial-cell-derived anti-viral peptides
To date, epithelial-cell-associated antimicrobial peptides include calprotectin (Ross and Herzberg, 2001), adrenomedullin (Kapas et al., 2001), secretory leukocyte protease inhibitor (SLPI) (McNeely et al., 1995, 1997; Shugars and Wahl, 1998; Shugars, 1999; Ma et al., 2004), LL37 (Frohm et al., 1997), human beta-defensins (hBDs) (Weinberg et al., 1998; Zasloff, 2002), and a novel host-defense-related ribonuclease, RNase 7 (Harder and Schroder, 2002). Of these, only SLPI (Ma et al., 2004) and hBDs (Quiñones-Mateu et al., 2003) appear to show anti-retroviral activity. Moreover, hBDs (~ 4 kDa cationic, amphipathic peptides) are emerging as important innate immune response agents of human mucosa, showing immunosurveillance properties, and acting as chemo-attractants to modulate the responses of dendritic cells, monocytes, and T-cells (Yang et al., 1999; Biragyn et al., 2002). Since hBD-1 and hBD-2 have recently been shown to be expressed in the basal cell layer and the granular and spinous regions of normal gingival epithelia (Lu et al., 2004) (see Weinberg et al., 2006). Expression of inducible hBDs throughout the oral epithelial mucosa may facilitate ‘cross-talk’ with cells of the adaptive immune system. Furthermore, expression by less-differentiated basally oriented epithelial cells suggests that differentiation is not essential for hBD expression, as previously suggested (Dale et al., 2001), or that hBD-expressing cells, other than epithelial cells, are infiltrating the oral mucosae. With recent findings that hBD-1 and hBD-2 are also expressed in human monocytes, macrophages, and dendritic cells (Duits et al., 2002), it is tempting to speculate that these cells contribute to hBD expression in the basal lamina of the gingival epithelium as cross-talk with the adaptive immune system is facilitated.
Unlike most other epithelia, normal, relatively uninflamed oral epithelium expresses the inducible beta-defensins (hBD-2 and hBD-3) (Dale et al., 2001). In other tissues, including skin, trachea, and gut epithelia, these defensins are expressed in the presence of infection or inflammation (O’Neil et al., 1999). This dichotomy may be due to the exposure of the oral epithelium to specific hBD-inducing commensal bacteria, suggesting that ‘normal’ oral mucosa is subclinically inflamed. Hence, Fusobacterium nucleatum, a ubiquitous Gram-negative organism of the human oral cavity, stimulates hBD-2 and -3 expression in HOECs, conferring protection from P. gingivalis invasion (Weinberg et al., unpublished observations).
Recently, HIV-1 was reported to induce expression of hBD-2 and -3 in human oral epithelial cells. Moreover, hBD-2 and -3 appear to block HIV-1 replication via a direct interaction with virions and through modulation of the CXCR4 co-receptor on immunocompetent cells (Quiñones-Mateu et al., 2003). Constitutively expressed hBD-1 was inactive. When compared with HOECs, vaginal, ectocervical, and endocervical cells do not appear to induce hBD-2 or -3 in response to HIV-1 (Weinberg et al., unpublished observations).
How HIV-1 signals to induce defensin expression is not known. Human epithelial cells also express pattern recognition receptors, known as Toll-like receptors (TLRs) (Rock et al., 1998), which detect pathogen-associated molecular patterns (PAMPs), such as bacterial LPS, peptidoglycan, major outer membrane proteins (MOMPs) (Medzhitov and Janeway, 1997), and viruses and viral particles (Alexopoulou et al., 2001; Haynes et al., 2001; Bieback et al., 2002; Matsumoto et al., 2002; Otten et al., 2002). Whether HIV-1 signals through TLRs is an open question. Signaling through TLR pathways could induce innate immune response genes, including ß-defensins.
The HIV-1-induced hBDs may also act in concert with other HIV-1-inducible oral epithelial cell-derived substances. Hence, agents such as hBD-2 and hBD-3, and SLPI—which is also induced by HIV-1 challenge (Weinberg, unpublished observations), although constitutive levels in the oral cavity (µg) are typically sufficient to inhibit HIV (Greenwell-Wild et al., 2006), and whose mechanism for selectively inhibiting R5 virus was recently elucidated (Ma et al., 2004)—may limit or control HIV-1 infection of the oral epithelium. Given the differences in structures among these molecules or others to be discovered, their mechanisms of action would be expected to differ, and each may function independently or synergistically.
In the oral cavity and oropharynx, where constitutively expressed anti-viral peptides are present, the interval between exposure to infectious HIV-1 and induction of expression of additional anti-viral peptides may represent a window of opportunity for systemic infection by a transmucosal route. We need to determine (1) if and how receptor/co-receptor-independent uptake of HIV-1 by oral keratinocytes results in viral integration, (2) the importance of the inducible anti-HIV-1 molecules and other innate anti-HIV-1 mechanisms such as the APOBEC3 family of proteins (Harris and Liddament, 2004), (3) how coincident commensal and pathogenic microorganisms may modulate anti-retroviral protection or susceptibility to HIV-1 infection, and (4) if differences in epithelial integrity and mode of presentation of the virus in the clinical setting contribute to either facilitating or thwarting HIV-1 at the oral mucosal surface, the site of initial confrontation.
Since rates of viral acquisition across the oral mucosa appear to be infrequent, mechanisms of resistance to HIV-1 are either highly effective or the effective exposure is overestimated or over-reported (McNeely et al., 1995; Vittinghoff et al., 1999; Page-Shafer et al., 2002). Even in vertical transmission, when HIV-1 enters through the oral cavity in infected amniotic fluid, infected blood, and cervical secretions, or through infected breast milk, the virus may access the mucosa through tonsillar and/or upper intestinal mucosa, rather than via the oral mucosa (Spira et al., 1996; Page-Shafer et al., 2002), confounding the interpretation of oral acquisition of HIV-1. Primary exposure of the oral mucosa to HIV-1 may also result solely in transient infection, marked by disappearance of infectious particles, and infected T-cells and DCs from the tissues. If so, viral acquisition across the oral mucosa may be underestimated, although there are, to date, no data to support this.
Question 3: Can infectious or inactive virus be found in saliva?
Primary HIV-1 infections across the oral mucosa would appear to require the passage of infectious virus from external foci through saliva. Available evidence suggests that infectious virions are essentially undetectable in the saliva of seroconverted HIV-1 patients. HIV-1 RNA, proviral DNA, and infected cells appear in salivary secretions of infected persons (Goto et al., 1991; Baron et al., 1999), yet infectious virus is rarely isolated from saliva (Barr et al., 1992; Moore et al., 1993; Coppenhaver et al., 1994; Greenwell-Wild et al., 2006), and transmission through oral secretions is uncommon (Rogers et al., 1990; Moore et al., 1993). Detection of infectious virions ex vivo in salivary or sputum samples may, however, be difficult. Saliva contains several anti-HIV-1 active molecules (Fox et al., 1989; Bolscher et al., 2002), including specific neutralizing secretory IgA antibodies (Lu et al., 1994; Yasuda et al., 1998), mucins (Barr et al., 1996; Nagashunmugam et al., 1998), proline-rich proteins (Robinovitch et al., 2001), lysozyme (Lee-Huang et al., 1999), secretory leukocyte protease inhibitor (SLPI) (McNeely et al., 1995), and anti-viral peptides such as defensins and cystatins, glycoproteins including thrombospondin and lactoferrin, and complement components (Shugars and Wahl, 1998). Indeed, the presence of HIV-1-specific antibodies in saliva is the basis of a clinically useful diagnostic test for seroconversion (O’Connell et al., 2003). Antibodies and mucins can neutralize HIV-1, enabling them to be harmlessly disposed of by swallowing, digestion, and excretion. SLPI has been intensely studied (McNeely et al., 1995; Wahl et al., 1997b; Shugars and Wahl, 1998; Ma et al., 2004), but multiple factors may collectively contribute to viral resistance in the oral cavity (Moutsopoulos et al., 2006a). For example, SLPI may also inhibit infection of macrophages by HIV-1, and may consequently be a novel therapeutic agent (Ma et al., 2004). SLPI can block macrophage uptake of HIV-1 (McNeely et al., 1995) by binding to surface phosphotidylserine-associated annexin II (Ma et al., 2004). While several anti-HIV mechanisms have been identified in saliva, it remains unclear if virions in saliva are infectious before seroconversion, enabling primary infection or transmission by salivary contact to occur. During a plausible window of immunological opportunity, presentation of HIV-1 to the oral tissues in natural vehicles, such as breast milk or semen, may represent a ‘protected flotilla’, maintaining the infectivity of the viral inoculum long enough to transfer the virus to mucosal cells (Fig.
). If within mucosal cells, the virus would likely be shielded from the anti-HIV-1 mechanisms in saliva.
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). The natural host defense mechanisms in the oral cavity are pivotal. Current information supports the potential for virus to infect oral mucosal keratinocytes, but only if salivary and tissue anti-viral mechanisms can be avoided. It is unclear if oral mucosal Langerhans cells (iDCs) in situ can recognize, sample, and process HIV-1 directly, or if keratinocytes can present HIV-1 to professional antigen-presenting cells. In the epidermis (Jakob et al., 1999) and mucosa (Berg et al., 1999), iDC-keratinocyte interactions occur in an E-cadherin-dependent manner. Using mucosal keratinocytes, tumor necrosis factor- (TNF-) and interferon-
(IFN-) synergistically induce MCP-1 mRNA, which is chemotactic for iDCs (Li et al., 2000). Moreover, induced expression of keratinocyte-derived hBDs by HIV-1 (Quiñones-Mateu et al., 2003) could also promote recruitment of iDCs (Yang et al., 1999). Hence, pathways potentially exist to enable keratinocyte-iDC interactions to occur in response to exposure to HIV-1 in the oral mucosa. A DC-SIGN-dependent mechanism appears critical to the formation of an infectious synapse to transfer HIV-1 to CD4+ T-cells (McDonald et al., 2003; Arrighi et al., 2004). If, where, and how the encounter between HIV-1 and DCs occurs, and whether the keratinocyte can contribute as an HIV-1 APC, remains to be determined.
Question 5: What are the factors that minimize infection of target T-cells in the oral mucosa?
First, the host innate and adaptive immune elements appear to provide protection against primary exposure. The protective immune elements differ between mucosal compartments, and epidemiological evidence suggests that protection is most successful in the oral environment. In addition to the distinct oral mucosal architecture and cellular constituents, the complex oral fluid may antagonize HIV-1 infection.
Beyond the presence of inhibitory molecules, additional oral mucosal impediments to HIV-1 have been associated with resistance to infection in the oral cavity. Mucosal integrity, structural configuration, and cellular composition are all contributory. For example, the oral mucosae may contain a paucity of accessible CD4+ Langerhans cells when compared with more susceptible mucosae (Hussain and Lehner, 1995). In addition to the requisite availability of susceptible cells, HIV-1 infection may be dependent upon mucosal integrity. Mucosal trauma such as erosion or ulceration caused by oral co-infections can disrupt the epithelial barrier and provide HIV-1 with direct access to the mucosal microcirculation. Another key location-specific factor is the recruitment of highly HIV-1-susceptible or already-infected inflammatory mononuclear cells to the site of inflammation and/or infection. Thus, primary infection may require susceptible targets to be recruited into the oral mucosa, while secondary infection could be promulgated by HIV-1-infected blood T-lymphocytes and monocytes that could transit to the mucosa. Relevant to these scenarios, seroconversion is followed by local and systemic immunosuppression. Immunosuppression biases mucosal surfaces toward infection by a panoply of opportunistic fungal, viral, parasitic, and bacterial pathogens and may augment HIV-1 replication.
To model a mucosally derived HIV-1 co-infection, Mycobacterium avium complex antigens or viable organisms were exposed to blood monocytes (Wahl et al., 1998). The bacteria were shown to up-regulate monocyte expression of TNF and CCR5, potentially augmenting the pool of CCR5+ mucosal cells at sites of mucosal contact. In addition, mycobacteria induced production of chemokines, such as MIP-1 and MIP-1ß, promoting recruitment of additional target cells to sites of mucosal infection (Hale-Donze et al., 2002). Co-infections of HIV-1 and opportunistic pathogens result in reciprocal activation of virus and bacteria or other viruses (Skolnik et al., 1988; Orenstein et al., 1997; Wahl et al., 1998). Collectively, local infections can enhance the recruitment of infected or infectable cells, viral infection, and replication, thereby promoting enhanced transmission (Wahl et al., 2003) and reflecting the complexity of stimulatory and inhibitory factors in the mucosal micro-environment. In the context of disrupted mucosal integrity, productively infected T-cells can be identified in the oral mucosa (Moutsopoulos et al., 2006a). Even though HIV-1-infected T-cells are infrequently identified in the oral mucosa, circumstances occur in which the local resistance factors are superseded and which may enable HIV-1 penetration to occur either from the outside in or from the inside out.
Question 6: What is the fate of HIV-1 upon exposure to oral tissues in animal models?
For better integration of in vitro data with clinical studies, animal models are useful to establish plausibility of pathogenic mechanisms. Direct inoculation of HIV-1/SIV-1 into the oral cavity of restrained primates results in systemic infection (Baba et al., 1996; Joag et al., 1997; Ruprecht et al., 1998; Herz et al., 2002), but the anatomic site of HIV-1 capture and initiation of a route toward systemic infection has been in doubt. Virus inoculated into the mouth may actually be swallowed and be captured and transmitted across the gut in an R5-selective manner, as suggested by in vitro studies (Meng et al., 2002), where virus replicates and disseminates rapidly in humans. To address the potential for infection across the oral mucosa, large doses of highly infectious SIV were exposed directly and non-traumatically to the lower right cheek and gingiva of neonatal and juvenile primates before they swallowed (Milush et al., 2004). Within a day of inoculation, the oral and esophageal mucosa and tonsils appeared to be the first sites of infection (Milush et al., 2004). At 4 days post-inoculation, virus was first detected in the stomach or intestines and proximal lymphatics, likely secondary to dissemination from oral mucosae to regional and peripheral lymph nodes. Indeed, the acid environment of the stomach was suggested to be a barrier to direct infection by SIV. Furthermore, CD4+ CCR5+ cells were found in the oral squamous mucosa and submucosa at day 7 post-inoculation, although it remains unclear if these were resident or trafficking cells. In primates, however, cells with dendritic cell markers are prominent in the submucosa of the mucous membranes, but not in the epithelia in the presence and absence of SIV-1 (Schwartz et al., 2002). Analysis of these data suggests that non-traumatic oral exposure to SIV at high inocula results in regional dissemination followed by systemic infection. In primates, transmission of SIV-1 from the oral cavity to systemic compartments may not be mediated by intra-epithelial DCs, although submucosal DCs can capture SIV-1 and HIV-1 and transmit the virus to central T-cells (Baribaud et al., 2001b). Whether this pathway of infection by highly infectious SIV is operative in HIV infection of humans remains difficult to confirm.
In humans, primary infection is followed by seroconversion, increasing titers of anti-HIV antibodies, and selective depletion of CD4+ T-cells from effector sites in the gastrointestinal tract (Brenchley et al., 2004; Mehandru et al., 2004). Little is known about depletion of immune cells from the oral tissues during primary infection (before or during seroconversion). Based on in vitro data with human cells and tissues and in primate models, the oral mucosa does appear to be permissive to HIV-1 capture, infection, and spread. Direct demonstration in vivo or ex vivo will be a challenging task. Yet events surrounding natural primary exposure of the oral mucosal tissues to HIV-1 must be characterized.
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