The innate immune defense appears to be particularly adept at protecting the oral cavity against HIV-1 infection, as evidenced by the paucity of HIV-1 transmission through the oral route compared to genital and anorectal routes. Here, we demonstrate that SLPI, an antiviral mucosal protein, is constitutively expressed in immortalized and normal oral keratinocytes. Brief exposure of cells to HIV-1 led to a significant increase in SLPI mRNA and protein secretion that was dose and time dependent, occurred rapidly after virus contact, did not require productive cellular infection, and was elicited specifically by the external envelope glycoproteins of HIV-1 and SIV strains. In this first in-depth characterization of SLPI regulation by a virus, our findings suggest that SLPI stimulation by HIV-1 represents a previously unrecognized mechanism that contributes to the oral host immune defense against this virus.
SLPI has been recognized as an important component of the innate mucosal immunity to microbial pathogens. Previous studies have confirmed its anti-inflammatory, antimicrobial, and wound-healing properties and its role in the maintaining the integrity of mucosal surfaces by inactivating destructive serine proteases during inflammation (reviewed in reference 16
). The natural abundance of SLPI in oral secretions (33
) has led to the speculation that the antiprotease is a major inhibitor of HIV-1 in saliva. Here, we show that oral keratinocytes are a previously unrecognized source of the inhibitor.
In the time course experiment, SLPI stimulation was observed in GMSM-K cells exposed to virus for as little as 5 min. This time frame has biological relevance in that it mirrors the brief duration that oral epithelial cells are likely to be exposed to virus in vivo during receptive oral sex. Oropharyngeal tissues of infants are likely exposed to virus in infected milk for longer, multiple periods during nursing.
In our experiments, the number of SLPI transcripts rose 50-fold within 1 h after exposure to HIV-1 (Fig. ), while a significant increase in SLPI protein secretion were first detected 48 h after virus exposure in most experiments (Fig. , , , and ). The lag period between inhibitor expression and secretion likely reflects the time needed for the activation of SLPI expression and the translation, intracellular transport, secretion and accumulation of the protein in the culture supernatant. Based on the time lag, it is unlikely that virus-mediated SLPI upregulation protects target cells and tissues against virus acquisition during initial infection. Rather, antiviral inhibitors (including SLPI) already present in salivary fluids likely serve this purpose. When initial protective oral defenses are overwhelmed by a bolus of virus (e.g., during breastfeeding, receptive oral sex, and experimentally infected rhesus macaques; reviewed in reference 49
), virus can penetrate the mucosa via transcytosis and possibly direct infection (29
), and create an initial focus of viral replication. Over time, subsequent rounds of replication spread the virus through the tissues and draining lymph nodes, and systemic infection occurs. We propose that HIV-1-induced SLPI from oral keratinocytes may help to limit virus spread through tissues rather than prevent initial infection. In addition, distal regions of the oral mucosa may be protected by the elevated SLPI level in oral fluids that ensue after virus stimulation. In support of this latter hypothesis, Lin et al. (28
) recently reported higher mean SLPI concentrations in saliva from submandibular/sublingual glands of HIV-1-infected individuals compared to uninfected individuals. The anti-inflammatory property of the inhibitor may also help to control and/or limit viral spread by dampening the inflammatory response and suppressing the activation of susceptible and/or infected cells.
Our data suggest that interactions between HIV-1 gp120 and component(s) of the cellular membrane activate a signaling pathway that ultimately “turns on” SLPI expression. The mechanism of action may include NF-κβ, activated protein 1 (AP-1), and CAAT enhancer binding protein (C/EBP), nuclear transcription factors that activate an array of cytokines and chemokines in response to inflammation, microbial infections, and stress. The human SLPI promoter contains binding sites for NF-κβ, AP-1, and C/EBP (3
). HIV-1 binding to cell surface CD4 during infection activates the binding of these transcription factors to the promoters of HIV-1, inflammatory cytokines and chemokines (41
), thus creating an inflammatory milieu conducive to viral replication. SLPI appears to be a previously unrecognized cellular gene that is also activated by gp120-CD4 interactions. Given its anti-inflammatory property, SLPI upregulation in virus-exposed keratinocytes may serve as a mechanism to dampen the local inflammatory response to infection, as exogenous SLPI attenuated NF-κβ-dependent inflammatory responses in human endothelial cells and macrophages after atherogenic stimuli (15
Another possibility is that SLPI stimulation is induced by virus interactions with other cell surface molecules such as Toll-like receptors (TLRs), evolutionarily conserved pattern recognition receptors engaged by specific pathogens on the surfaces of immune and epithelial cells (20
). Pathogen-TLR interactions also lead to activated inflammatory gene expression through AP-1 and NF-κβ-mediated pathways. By RT-PCR, we have detected mRNAs for TLRs 1 to 9 in unstimulated GMSM-K cells (unpublished data). Preincubation of GMSM-K cells with antibodies to TLRs 2 and 4 blocked SLPI induction by BaL in GMSM-K cells (unpublished data), suggesting a role for the TLR pathway in the stimulatory effect. Many viruses, including respiratory syncytial virus, measles virus, cytomegalovirus, and influenza virus, modulate host immune responses through TLR activation by viral envelope glycoproteins, core proteins, and replicative nucleic acid intermediates (e.g., single- or double-stranded RNAs) (56
). Thus, engagement of TLR-mediated pathways may represent a common mechanism by which cells detect and respond to viruses. Alternatively, interactions between HIV-1 gp120 and a non-CD4 cell surface protein involved in viral entry, such as sulfatide, heparans, galactosyl ceramide (GalCer), and intercellular adhesion molecule 1 (6
), may initiate the effect. Future investigations will clarify the mechanism of HIV-1-induced SLPI upregulation and identify the signaling pathway involved in the effect.
Our finding of virus-mediated induction of SLPI mirrors a recent report (42
) describing HIV-1 induction of hBD-2 and hBD-3, cationic antimicrobial peptides also produced in epithelial cells. In that study, normal oral epithelial cells exposed to X4 and R5 viruses expressed higher levels of hBD-2 and hBD-3 (but not hBD-1) mRNA compared to controls. The active peptides were specific for X4 viruses, had little effect against R5 viruses, and blocked viral replication through two mechanisms: direct binding to virions and down-modulation of cell surface CXCR4 expression. In contrast, SLPI antiviral activity involves a cellular rather than a viral target (30
) and is active against both R5 and X4 viruses (33
), although activity is diminished slightly with HIV-1 isolates having broad coreceptor usage patterns (e.g., CCR5, CXCR4, and either CCR2 or CCR3) (51
). Thus, in its role as “gatekeeper” of the body, the oral cavity has evolved complementary strategies involving at least two distinct antimicrobial molecules in its quest to protect the body against HIV-1.
Under our experimental conditions, no productive HIV-1 infection was detected in oral keratinocytes, as evaluated by p24 secretion or PCR for newly generated proviral DNA (unpublished data). This result is in agreement with that of Quiñones-Mateu et al. (42
), who found no productive infection in normal oral keratinocytes inoculated with either X4 or R5 viruses. The findings, however, contrast with those of Liu et al. (29
), who showed infection of oral keratinocytes using high doses of X4 and R5/X4 viruses but not an R5 virus through a CD4-independent mechanism that may involve GalCer and/or CXCR4 as cellular receptors. In another study, Moore et al. (35
) showed infection of oral keratinocytes with cell-free R5 viruses but not X4 viruses. The discrepancies may be due to variations in experimental conditions, such as the use of Polybrene during virus exposure by Liu et al. (29
) and differences in virus dosages and/or levels of cell surface GalCer, CCR5, and/or CXCR4 expression in the cell models.
It is not known whether HIV-1 stimulates SLPI production at nonoral mucosal sites and what effects enhanced inhibitor production has on the susceptibility of mucosal tissues to virus infection. Oral and genital mucosae differ in several aspects that may alter their susceptibility to this virus, including the architecture of the epithelium, composition and viscosity of the fluids bathing the sites, and local immune responses. In addition, tissue-specific differences in SLPI regulation by HIV-1 may contribute to tissue susceptibility, as treatment with tumor growth factor β1 suppressed SLPI mRNA levels in respiratory epithelial cells (21
) but had the opposite effect in endometrial epithelial cells (50
). Characterization of the SLPI response to HIV-1 in nonoral mucosal tissues will shed light on this important issue.
In summary, our study reveals that oral epithelial cells constitutively express SLPI. Furthermore, SLPI expression can be manipulated by HIV-1 through infection-independent interactions initiated at the cell surface. Given the anti-inflammatory and antiviral properties of SLPI, the induction of SLPI in virus-stimulated cells represents a tug-of-war between the virus and the host immune response, as the virus attempts to stimulate the local inflammatory response while the inhibitor tries to dampen the response and/or protect neighboring cells against infection. An imbalance between the opposing responses may dictate whether virus exposure ultimately results in productive infection or protection. It will be important to identify other immune response genes whose expression is modified after contact of virus with the oral epithelium. These studies will lead to a greater understanding of the mechanisms through which the host immune response protects against mucosal HIV-1 infection.