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Immunobiology of HPV Infection
Archives of Medical Research 40 (2009) 443e448
REVIEW ARTICLE
Immunobiology of HPV Infection Rodolfo Garcia-Chacon,a Sandra Fabiola Velasco-Ramirez,b Leopoldo Flores-Romo,a and Adria´n Daneri-Navarrob a
b
Departamento de Biologı´a Celular, Cinvestav-IPN, Me´xico D.F., Me´xico, Departamento de Fisiologı´a, Centro Universitario de Ciencias de la Salu´d, Universidad de Guadalajara, Guadalajara, Jalisco, Me´xico Received for publication May 14, 2009; accepted May 18, 2009 (ARCMED-D-09-00201).
Although the high-risk human papillomavirus (HPV) is necessary to cause cervical cancer (CC) and its infectious origin is well recognized, neither the systemic nor the local immune responses to this virus has been well studied or understood. Because the many facets of HPV are excellently described here by others, we will focus on the immune responses of the female genital tract, especially in situ, or in natura, as Casanova argues when studying diseases (1). A general overview is provided where different elements of the innate and adaptive immunity are discussed. We highlight the very successful strategy used by HPV with a protracted and seemingly silent infection, making this virus extremely well adapted to infect humans. The counterpart of this peculiar immunological situation is that whatever immune responses (innate or adaptive, local or systemic) are induced in those chronically infected women, these responses are inefficient to cope with and especially to eradicate the virus, thus resulting in viral persistence. A further follow-up of this line of thought is that among other well-known co-factors some still uncovered complex immune alterations may underlie the enigmatic susceptibility of the minority of women permissive to the chronic HPV infection, i.e., of those who slowly progress from infection to CIN 1e2e3 and then to invasive cervical carcinoma.
Introduction Because the female genital tract has very likely evolved primarily to fulfill reproductive and sexual functions essential to the continuation of the species, immune responses in these anatomic compartments must adapt to protect, but at
Address reprint requests to: Leopoldo Flores-Romo, Departamento de Biologı´a Celular, Cinvestav-IPN, Av. IPN No. 2508, Zacatenco CP 07360, Me´xico D.F., Me´xico; E-mail: [email protected]
the same time to avoid interfering with, these primary functions. This is not a trivial issue especially when considering that this anatomic location faces high and frequent antigenic exposures. Before reproductive age, these antigens should be represented by the corresponding ‘‘own’’ commensal microbiota. Currently, from the trillions of microbes colonizing our bodies, it is not yet known how many species normally inhabit the various subcompartments of the female reproductive tract. However, later on when sexual activity begins, among many other ‘‘exogenous’’ antigens, those derived from the male sexual tract including normal and pathogenic microorganisms such as HPV and sperm components essential for reproduction must also be dealt with. It is thus conceivable that the female genital tract may have evolved an adaptive propensity to low reactivity, a situation that some pathogens may find propitious to the infection. HPV has been referred to as ‘‘non-immunogenic’’ given the complexity, paucity, or non-detectable immune responses in subjects infected naturally. However, in recent years it has been shown in large groups of humans vaccinated intramuscularly with VLPs of HPV that these are not only immunogenic but trigger highly efficient antibodies that prevent the infection with specific HPV genotypes. The protracted infectious strategy used by the HPV is indeed quite successful. There is no viremia, no lytic phase, no viral spread to other distant tissues and it patiently exploits the powerful sexual drives of the species infecting women by HPV-positive men who seldom have a genital cancer but whose sexual impulses are conveniently used by the virus to further spread to human subjects. From another point of view, given that most infected women will effectively eliminate the virus and only a minority of those infected will continue the insidious journey into cancer, it is conceivable that there may be some underlying, yet unknown, complex immune alterations in this small female population permissive for the HPV to thrive.
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2009.05.003
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Immune Responses in the Human Female Genital Tract: Innate and Adaptive Basic Features: Epithelia, Mucus and Antimicrobial Molecules. Basic mechanisms include a variety of properties starting with the sole ‘‘mechanical’’ barrier of the genital epithelia and stroma to the rythmic movements of cilia towards the cervical canal and the vagina, to the pH and the mucus. The latter is an important and unique hydrogel constituent of the cervix composed mainly of water, soluble proteins (albumin, globulin, immunoglobulinseIgA and IgG), mucins with high carbohydrate content, and some lipids such as oleic, linoleic, myristic, palmitic and stearic. It is highly likely that similar to other epithelia such as the skin, small peptides with antimicrobial properties (AMPs) may be present in these female tissues. Cathelicidins (LL37), a and b defensins, are potential candidates. Some may be constitutive like LL37 in the skin, whereas others may be inducible, perhaps preferentially upon certain microbial encounters. Indeed, HD5 mRNA expression has been detected in the vagina, endo- and exocervix, endometrium and fallopian tubes, and the actual protein has been identified as secreted in cervicovaginal lavages (2). Indeed, several other compounds with known antimicrobial properties have been identified particularly in mucus from the cervix, including the neutrophil defensins (HNP) 1e3, secretory leukocyte protease inhibitor (SLP1), lysozyme, lactoferrin, the b defensin 1 (HBD-1) and calprotectin. Trace metals like manganese, iron, zinc and copper have also been found. Basic properties such as the charge and high polysaccharide content likely interfere with adhesion, invasion and perhaps survival of intruding microorganisms; moreover, there may be synergy among these compounds in their anti-pathogen functions. The relative quantities of these various constituents most likely vary along the anatomic subcompartments and also along the menstrual cycle (3). The cellular sources of these antimicrobial (antibacterial, antifungal or antiviral) compounds require identification in situ within these organs. To the best of our knowledge, these various antimicrobial compounds have not been examined regarding their potential effects during the course of the HPV infection. Commensal Microbiota. From the myriad of microbes colonizing our body cavities and contact epithelia, it is unclear yet how many species normally inhabit the various anatomic subcompartments of the female reproductive tract. Apart from the microbial flora typically identified in the vagina (especially lactobacilli), what exactly constitutes the ‘‘own’’ microbiota in the exo- and endocervix, uteri corpus and fallopian tubes still awaits a thorough assessment, not only by means of microbiology tests but with molecular biology and recent genomic tools. The normal commensal microbiota of the various compartments of the female genital tract may function by itself as an important
ecological ‘‘barrier’’ deterring and confining other intruding microbes, non-members of this commensal flora (4). Bacterial vaginosis is an example where the predominant normal vaginal microbiota of predominant lactobacillus is displaced by the overgrowth of anaerobic microorganisms (4). Using genomic probes, a recent study reported the analysis of 13 microbial species of the lower genital tract, finding a correlation between the bacterial species and proinflammatory cytokines (interleukin, IL1e6e8) (4). In another mucosa, for instance, it has been recently found that a bacterial symbiont from the human intestinal tract actually protects from experimental colitis triggered by other intestinal bacteria (5). Conversely, it is unknown whether the sole presence or the long HPV persistence modifies or alters the composition of the genital microbiota. In this regard, it is expected that a thorough analysis of the composition of the normal microbiota of the entire female genital tract compartments will be included in the recent International Human Microbiome Project (6).
Toll-like Receptors. It is likely that some mucosal cells may be armed with (extra- or intracellular) pathogen recognition receptors (PRRs) as microbial sensors or produce themselves putative antimicrobial substances. Regarding the expression of toll-like receptors (TLRs), it is still unknown how the physiological situation is in the female genital tract, whether in basal (non-infectious) conditions TLRs are normally expressed and in which of them or whether may be TLR inducible or preferentially upregulated with defined microorganisms. Again the local cell populations displaying TLRs await identification. At least one study used primary uterine epithelial cells (UECs) and showed the mRNA expression for TLRs 1 9 but not for TLR10. These UECs responded to TLR3 stimulation with Poly I:C by producing several proinflammatory molecules including TNF, GMSCF, chemokines, AMP HBD1e2 and IFNb (7). Another study went further and identified the actual proteins of TLRs 7e10 in endometrial and stromal tissue of healthy uteri (8). A more complex situation is envisaged to ascertain the putative roles of TLRs when the carcinoma is already established because the study of TLRs in cancer is, in general, at its beginning and with contradictory reports. This is because the pro-inflammatory effects of stimulation via TLRs have sometimes been found effective but other times have reportedly promoted tumor growth, survival and metastasis (9). About a century ago, it was already noted that serial administrations of crude microbial extracts could induce anti-tumour responses (9), a phenomenon now presumably exerted via TLRs. If indeed the actual TLR proteins are present in the uterus in basal non-infectious conditions, it needs to be explained why this situation does not trigger a continuous inflammatory response because contrary to expected, the exposure to microorganisms from the cervix to the uteri corpus to the fallopian tubes seems frequent (7).
HPV Immunobiology
Natural Killer Cells. Natural killer (NK) cells represent a first line of defense against viral pathogens killing infected cells directly or via secretion of molecules like certain cytokines (10). There is also accumulating evidence for a crucial role of NK cells in tumor immunosurveillance (11). For instance, NK cell activation and tumor lysis occur through a complex interaction between triggering receptors such as NKp30, NKp44, NKp46 and NKG2D with tumor cell ligands in fine balance with inhibitory receptors and co-receptors (12). Recently, it was reported that activating NK cell receptor ligands MICA (NKG2D ligand) and CD155 (DNAM-1 ligand) are differentially expressed during progression to cervical cancer (13). We found that NK cell-activating receptors NKp30 and NKp46 are significantly downregulated in cervical cancer patients and women with CIN 2e3. NCRs downregulation correlated with low cytolytic activity, HPV16 infection and clinical stage (Garcia T. et al., manuscript submitted). Furthermore, our group recently described that serum levels of soluble MICA (soluble form of this stressinducible activating ligand for NKG2D) increase according to the progression toward cervical cancer. This suggests that soluble MICA may contribute to HPV persistence or tumor progression (14). On the other hand, we described that CEACAM1 significantly increase in high-grade squamous intraepithelial lesions in comparison with low-grade squamous intraepithelial lesions and normal cervical tissues. These findings are relevant because CEACAM1 is an adhesion molecule with immunoregulatory properties and one is NK cell inhibition (15). Indeed, there is still much to do to clarify the intriguing potential role of NK cells, especially in the local tissues affected by the HPV infection and during the progression to carcinoma. Dendritic Cells. Given the constant and varied antigenic exposure in the different subcompartments along the female genital tract (urethra, vulva, vagina, exocervix, endocervix, uterus, fallopian tubes, etc), the presence of dendritic cells (DCs) as sentinel posts of the immune system in these areas is significant, although their precise role has yet to be defined. It is likely, for instance, that conventional myeloid DCs (cDCs) populate these tissues, similar to other peripheral tissues such as the skin, airways, or gastrointestinal mucosa (16e19). Using three DC markers (Langerin, MHC-II and CD1a) and epithelial sheets instead of conventional tissue sections, our group recently identified a dense planar network of DCs in human cervix samples (16). Interestingly, the frequency of these DCs was reduced to about half during high-risk HPV infection (CIN-I) compared to non-infected high-risk HPV-negative samples. We are currently assessing the status of cDCs in situ and whether they are activated/matured, HPV-infected, etc. Likewise, whether the presence of plasmacytoid (p)DCs as the major early producers of natural-type-I interferons is constitutive or inducible under viral infections such as the high-risk HPV still needs to be explored. In this respect, type-I IFNs require assessment in situ, although by using
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keratinocyte cell lines it seems that HPV deregulates this responses at various levels (20). In any case, this line of defense is undoubtedly overcome by the virus because HPV establishes the infection persisting for many years. gdþ T lymphocytes and B1 lymphocytes. As early responder-effector cells, gdþ T lymphocytes also represent another population of innate immune cells whose relevance in these anatomic locations has not been examined during HPV infections but warrants careful scrutiny. Their production of cytokines like interferons and their cytolytic activities may be of importance in the regional defense from HPV and many other potential pathogens. B1 cells are producers of the so-called ‘‘natural’’ antibodies (i.e., those not requiring previous Ag exposure/induction, usually IgM but also IgA) in the different mucosae, and it is highly likely that B1 lymphocytes are important in the innate defense of these tissues. Accordingly, the presence of IgA-producing plasma cells in situ has been reported within the cervix, but their precise lineage remains to be established. HPV and Inflammation? Cell Participants in Early Reactions. The potential role of populations of innate immediate responses such as polymorphonuclear leukocytes (PMN) and mast cells or eosinophils certainly needs to be examined during the course of HPV infection. This is especially true regarding in situ analysis either in human samples or using appropriate animal models for HPV infection where the most early time points could be controlled and assessed, unlike what happens in the natural course of the infection in humans. It is known, for instance, that PMN infiltration is seen mainly in bacterial cervicitis, whereas other cell types such as lymphocytes and monocytes can be found in other diseases such as chlamydia and tuberculosis (the latter being more rare) (21). Although inflammatory infiltrate has been described for other tumor types, this is ill-defined regarding HPV and still remains controversial whether inflammation indeed occurs during the different stages of HPV infection, i.e., from the early changes and NICs to CC. Older studies described infiltration by macrophages, plasma cells and lymphocytes into the epithelium and the stroma in preinvasive and invasive cervix tumors (21e23). However, the controversy about whether there is indeed inflammation and the scarcity of descriptions about it may well indicate a more complex picture of the inflammatory response when facing the HPV. The natural course of the early phase of HPV infection leading to CC is clearly asymptomatic, detected solely by cytology-based tests or by using more elaborate means. Adaptive Immune Responses During Genital HPV Infection: B and T Lymphocytes Adaptive Humoral Immune Responses (B Lymphocytes). Regarding adaptive humoral immunity, it is reported that
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Garcia-Chacon et al./ Archives of Medical Research 40 (2009) 443e448
immunoglobulins (Ig) IgG and IgA are the main Ig classes in female genital secretions such as the mucus from cervix where IgG is found in higher levels than IgA. These IgGs would apparently derive both from locally producing cells and from transudation into the lumen of the genital tract (3). Although the origin of IgAs may be dual, either from B1 or B2 type lymphocytes, IgGs are most likely derived from B2 conventional B cells, i.e., those that have passed through a follicular germinal center reaction experiencing somatic hypermutation and Ig class switching under classical CD4þ T cell help. Because these types of Ab responses normally occur only in highly organized secondary lymphoid tissues, this implies that the exogenous intruding Ags have, by necessity, reached the B cell follicles within regional (iliac) lymph nodes draining these organs. For many obvious limitations, this is an issue not well explored for most pathogens invading these tissues in humans, including HPV. Unlike antibodies from B1 cells, those derived from follicular B2 lymphocytes are Ag-triggered; thus, it is possible that these Abs may be more useful, for example, to interfere with microbial adherence or early interactions (with HPV?) or to clear a given pathogen. Alternatively, if these Abs are triggered and produced only under specific Ag (microbial) exposure but the infection nevertheless progresses—a scenario that seems highly likely during chronic HPV infections—these Abs are thus not effective from the host standpoint. This, in turn, would suggest a rather active strategy from the HPV to deviate the host regional adaptive humoral responses. Regarding systemic responses, it is reported that after transient or even persistent and well-documented HPV infection, some women fail to show seroconversion for HPV, whereas serum IgA has been associated with both HPV persistence or with HPV clearance. Likewise, the extremely long delay of 18 months for IgG and IgA seroconversion to appear after documented HPV infection certainly merits careful investigation (24). In fact, Abs to E7 may not appear even after 10 or 20 years that the protein has been expressed during the infection (25,26). At the regional level, at least one report described the Ab responses in the female genital tract as altered during HPV infection, including the local and serum IgG and IgA, with no apparent changes in IgM. Altered transudation was mentioned as one potential HPV-induced mechanism (26). Another study found that cervical IgA to E7 from HPV 16 was decreased in CC (27). Others have observed fluctuations of VLP-induced Abs during the menstrual cycle (28). Interestingly, anti-VLP 16 IgA Abs in oral fluid of adult women attending dental clinics were found to correlate with cervical HPV positivity. Moreover, the authors suggested that the emergence of these Abs in oral fluids of adolescents correlated with the initiation of sexual activity and depended on the common mucosal immune system (29). The complex situation of the different antibody types (systemic, local, from B1 cells, from memory
responses, etc.) potentially induced during early and chronic HPV infection certainly merits more careful studies. Adaptive Cellular Immune Responses (T cells) Lymphoid aggregates mostly containing CD8þ T lymphocytes surrounding some B cells have long been described by histopathologists in the endometrium, and these aggregates apparently showed variations with the menstrual cycle (30). In pre- and postmenopausal women, the presence of CD3þ, CD4þ, CD8þ T cells, macrophages, and DCs in the endocervix, ectocervix, and vagina was found distributed throughout the lower female reproductive tract in both the epithelium and subepithelial mucosa (31). Our group has also found that CD4þ, CD8þ and some gdþ T lymphocytes are normally present in the basal epithelial layers of the cervix (Jimenez-F, personal communication, 2005). These assessments may provide interesting clues, for example, regarding which T-cell subsets infiltrate (if they do) the actual HPV-infected areas during the progression from early lesions and NIC-I to CC, and whether these T cells are armed or not with pertinent cytokines such as IFN-g or with other relevant molecules like granzyme or granulysin. It is likely that the ultimate host immunity test that HPV must deal with is cytotoxicity, probably by NK or NKTs initially and later on by CD8þ T lymphocytes. However, pertaining functioning, the assessment of cell-mediated immunity during HPV infection has been rather difficult and reports are contradictory (32). For instance, whereas cytotoxic T lymphocytes (CTLs) from an important percentage of chronically (HPV) infected women fail to respond to HPV challenge (33), other studies reported a good correlation between systemic delayed-type hypersensitivity responses (DTH) and CIN resolution (34,35). Discrepancies have also been found regarding the vigorous responses to viral Ags between T cells from peripheral blood and the quite low numbers of tumor-reacting T cells (36). In any case, research is scant regarding the three major T-cell subsets (CD4/CD8/gd T cells), especially in situ in the different subcompartments of the female genital tract. In restricted cases there may be the ethical possibility to obtain the regional draining lymph nodes. Their evaluation may also yield valuable information, for instance, regarding the presence of HPV within the nearest secondary lymphoid tissues or whether the putatively responding T cells are being expanded and activated in the interdigitating dendritic cells (IDC)/T zone area of the nodes. For instance, immune responses are reportedly affected by microenvironment such as extracellular matrix components, growth factors, cytokines and proteases. Our group found that lymphocyte proliferation to PHA was partially inhibited by tissue extracts from cervical carcinoma, mainly by proteases. Experiments with purified proteolytic
HPV Immunobiology
enzymes (trypsin, cathepsin B, uPA and type IV collagenase) reinforce the protease inhibitory role (37). It has also been reported that proteases increase according to the progression towards invasive CC (38). In the search of animal models for HPV infection and carcinogenesis, a transgenic mice expressing E7 from HPV16 was reported with induction of E7-specific immunological tolerance at the CTL level (39). Also in experimental animals, a compartmentalization has been shown of the long-term anamnestic CTL responses into the regional lymphoid tissues draining mucosae (such as the iliac nodes draining the genital tract) or to the systemic lymphoid tissue like the spleen. This depends on the route of immunization (40). The follow-up of the presence or absence of T cells within the genital tract, as well as of pertinent molecules associated with effector T-cell immunity, may certainly yield important cues pertaining to the evolution and prognosis of the HPV infection. In conclusion, a peculiar feature of oncogenic HPVs is the protracted but highly efficient strategy to infect humans and propagate among them. A very enigmatic issue is how, despite facing several multilayer levels of both the innate and the adaptive immunity, apparently the same HPV type is persistent during many years—often during decades—to avoid arising overt and deleterious immune responses, thus revealing HPV as a virus extremely well adapted to humans. In order to efficiently infect the basal layers of the cervix epithelium and transform these cells into cervical carcinoma, it must face and overcome first the primary natural mechanical barriers provided by the epithelium, the mucus, the potential antimicrobial peptides, and the variety of potential alert signals triggered by TLRs. Reports of inflammatory reactions from the female tract to early encounters with HPV are rather scarce, either because they may be difficult to track given the natural course and timing of the infection or because these types of responses are indeed somehow missing. The subsequent phases of immunity must also be dealt with, including the fast reactions from neutrophils, mast cells or eosinophils (if there are any of these reactions during early HPV interactions), then by NKs and plasmacytoid DC with their vigorous type I interferon responses. In a higher complexity level regarding adaptive immunity, HPV probably faces first the so-called natural (‘‘non-inducible’’) antibody responses such as IgMs and IgAs and then the conventionally triggered B2 Ab responses, of which IgA should also be one in this mucosal tissue and then IgGs, this being a chronic infection. It is likely that the ultimate immune test that HPV must deal with is the cytotoxicity against HPV-infected cells, first by NK or NKTs and much later on by CD8þ T lymphocytes. Initially, the cytolytic responses should act upon transformed cervical cells such as koilocytes and, later, over the already carcinogenic cells. However, from the long list provided, it is quite
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evident that HPV manages very well to face, cope with and overcome all the responses that the immune system can produce. Conversely, the ineffectiveness of the several mechanisms of the immune system (innate and adaptive, simple and complex, local and systemic) should also be evident when facing such a virus. Because HPV infection alone seems insufficient to progress from the early infection stages to a carcinoma, another important susceptibility factor may well rely on complex immunodeficiencies not yet described in the minority of women slowly progressing from infection to cancer.
Acknowledgments R. Garcia-Chacon and S.F. Velasco-Ramirez are fellows from the National Council for Science and Technology (Conacyt). L. Flores-Romo and A. Daneri-Navarro are members of the National System of Researchers (SNI). The authors acknowledge the help from the two laboratories, especially Juana Calderon.
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REVIEW ARTICLE
Immunobiology of HPV Infection Rodolfo Garcia-Chacon,a Sandra Fabiola Velasco-Ramirez,b Leopoldo Flores-Romo,a and Adria´n Daneri-Navarrob a
b
Departamento de Biologı´a Celular, Cinvestav-IPN, Me´xico D.F., Me´xico, Departamento de Fisiologı´a, Centro Universitario de Ciencias de la Salu´d, Universidad de Guadalajara, Guadalajara, Jalisco, Me´xico Received for publication May 14, 2009; accepted May 18, 2009 (ARCMED-D-09-00201).
Although the high-risk human papillomavirus (HPV) is necessary to cause cervical cancer (CC) and its infectious origin is well recognized, neither the systemic nor the local immune responses to this virus has been well studied or understood. Because the many facets of HPV are excellently described here by others, we will focus on the immune responses of the female genital tract, especially in situ, or in natura, as Casanova argues when studying diseases (1). A general overview is provided where different elements of the innate and adaptive immunity are discussed. We highlight the very successful strategy used by HPV with a protracted and seemingly silent infection, making this virus extremely well adapted to infect humans. The counterpart of this peculiar immunological situation is that whatever immune responses (innate or adaptive, local or systemic) are induced in those chronically infected women, these responses are inefficient to cope with and especially to eradicate the virus, thus resulting in viral persistence. A further follow-up of this line of thought is that among other well-known co-factors some still uncovered complex immune alterations may underlie the enigmatic susceptibility of the minority of women permissive to the chronic HPV infection, i.e., of those who slowly progress from infection to CIN 1e2e3 and then to invasive cervical carcinoma.
Introduction Because the female genital tract has very likely evolved primarily to fulfill reproductive and sexual functions essential to the continuation of the species, immune responses in these anatomic compartments must adapt to protect, but at
Address reprint requests to: Leopoldo Flores-Romo, Departamento de Biologı´a Celular, Cinvestav-IPN, Av. IPN No. 2508, Zacatenco CP 07360, Me´xico D.F., Me´xico; E-mail: [email protected]
the same time to avoid interfering with, these primary functions. This is not a trivial issue especially when considering that this anatomic location faces high and frequent antigenic exposures. Before reproductive age, these antigens should be represented by the corresponding ‘‘own’’ commensal microbiota. Currently, from the trillions of microbes colonizing our bodies, it is not yet known how many species normally inhabit the various subcompartments of the female reproductive tract. However, later on when sexual activity begins, among many other ‘‘exogenous’’ antigens, those derived from the male sexual tract including normal and pathogenic microorganisms such as HPV and sperm components essential for reproduction must also be dealt with. It is thus conceivable that the female genital tract may have evolved an adaptive propensity to low reactivity, a situation that some pathogens may find propitious to the infection. HPV has been referred to as ‘‘non-immunogenic’’ given the complexity, paucity, or non-detectable immune responses in subjects infected naturally. However, in recent years it has been shown in large groups of humans vaccinated intramuscularly with VLPs of HPV that these are not only immunogenic but trigger highly efficient antibodies that prevent the infection with specific HPV genotypes. The protracted infectious strategy used by the HPV is indeed quite successful. There is no viremia, no lytic phase, no viral spread to other distant tissues and it patiently exploits the powerful sexual drives of the species infecting women by HPV-positive men who seldom have a genital cancer but whose sexual impulses are conveniently used by the virus to further spread to human subjects. From another point of view, given that most infected women will effectively eliminate the virus and only a minority of those infected will continue the insidious journey into cancer, it is conceivable that there may be some underlying, yet unknown, complex immune alterations in this small female population permissive for the HPV to thrive.
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2009.05.003
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Immune Responses in the Human Female Genital Tract: Innate and Adaptive Basic Features: Epithelia, Mucus and Antimicrobial Molecules. Basic mechanisms include a variety of properties starting with the sole ‘‘mechanical’’ barrier of the genital epithelia and stroma to the rythmic movements of cilia towards the cervical canal and the vagina, to the pH and the mucus. The latter is an important and unique hydrogel constituent of the cervix composed mainly of water, soluble proteins (albumin, globulin, immunoglobulinseIgA and IgG), mucins with high carbohydrate content, and some lipids such as oleic, linoleic, myristic, palmitic and stearic. It is highly likely that similar to other epithelia such as the skin, small peptides with antimicrobial properties (AMPs) may be present in these female tissues. Cathelicidins (LL37), a and b defensins, are potential candidates. Some may be constitutive like LL37 in the skin, whereas others may be inducible, perhaps preferentially upon certain microbial encounters. Indeed, HD5 mRNA expression has been detected in the vagina, endo- and exocervix, endometrium and fallopian tubes, and the actual protein has been identified as secreted in cervicovaginal lavages (2). Indeed, several other compounds with known antimicrobial properties have been identified particularly in mucus from the cervix, including the neutrophil defensins (HNP) 1e3, secretory leukocyte protease inhibitor (SLP1), lysozyme, lactoferrin, the b defensin 1 (HBD-1) and calprotectin. Trace metals like manganese, iron, zinc and copper have also been found. Basic properties such as the charge and high polysaccharide content likely interfere with adhesion, invasion and perhaps survival of intruding microorganisms; moreover, there may be synergy among these compounds in their anti-pathogen functions. The relative quantities of these various constituents most likely vary along the anatomic subcompartments and also along the menstrual cycle (3). The cellular sources of these antimicrobial (antibacterial, antifungal or antiviral) compounds require identification in situ within these organs. To the best of our knowledge, these various antimicrobial compounds have not been examined regarding their potential effects during the course of the HPV infection. Commensal Microbiota. From the myriad of microbes colonizing our body cavities and contact epithelia, it is unclear yet how many species normally inhabit the various anatomic subcompartments of the female reproductive tract. Apart from the microbial flora typically identified in the vagina (especially lactobacilli), what exactly constitutes the ‘‘own’’ microbiota in the exo- and endocervix, uteri corpus and fallopian tubes still awaits a thorough assessment, not only by means of microbiology tests but with molecular biology and recent genomic tools. The normal commensal microbiota of the various compartments of the female genital tract may function by itself as an important
ecological ‘‘barrier’’ deterring and confining other intruding microbes, non-members of this commensal flora (4). Bacterial vaginosis is an example where the predominant normal vaginal microbiota of predominant lactobacillus is displaced by the overgrowth of anaerobic microorganisms (4). Using genomic probes, a recent study reported the analysis of 13 microbial species of the lower genital tract, finding a correlation between the bacterial species and proinflammatory cytokines (interleukin, IL1e6e8) (4). In another mucosa, for instance, it has been recently found that a bacterial symbiont from the human intestinal tract actually protects from experimental colitis triggered by other intestinal bacteria (5). Conversely, it is unknown whether the sole presence or the long HPV persistence modifies or alters the composition of the genital microbiota. In this regard, it is expected that a thorough analysis of the composition of the normal microbiota of the entire female genital tract compartments will be included in the recent International Human Microbiome Project (6).
Toll-like Receptors. It is likely that some mucosal cells may be armed with (extra- or intracellular) pathogen recognition receptors (PRRs) as microbial sensors or produce themselves putative antimicrobial substances. Regarding the expression of toll-like receptors (TLRs), it is still unknown how the physiological situation is in the female genital tract, whether in basal (non-infectious) conditions TLRs are normally expressed and in which of them or whether may be TLR inducible or preferentially upregulated with defined microorganisms. Again the local cell populations displaying TLRs await identification. At least one study used primary uterine epithelial cells (UECs) and showed the mRNA expression for TLRs 1 9 but not for TLR10. These UECs responded to TLR3 stimulation with Poly I:C by producing several proinflammatory molecules including TNF, GMSCF, chemokines, AMP HBD1e2 and IFNb (7). Another study went further and identified the actual proteins of TLRs 7e10 in endometrial and stromal tissue of healthy uteri (8). A more complex situation is envisaged to ascertain the putative roles of TLRs when the carcinoma is already established because the study of TLRs in cancer is, in general, at its beginning and with contradictory reports. This is because the pro-inflammatory effects of stimulation via TLRs have sometimes been found effective but other times have reportedly promoted tumor growth, survival and metastasis (9). About a century ago, it was already noted that serial administrations of crude microbial extracts could induce anti-tumour responses (9), a phenomenon now presumably exerted via TLRs. If indeed the actual TLR proteins are present in the uterus in basal non-infectious conditions, it needs to be explained why this situation does not trigger a continuous inflammatory response because contrary to expected, the exposure to microorganisms from the cervix to the uteri corpus to the fallopian tubes seems frequent (7).
HPV Immunobiology
Natural Killer Cells. Natural killer (NK) cells represent a first line of defense against viral pathogens killing infected cells directly or via secretion of molecules like certain cytokines (10). There is also accumulating evidence for a crucial role of NK cells in tumor immunosurveillance (11). For instance, NK cell activation and tumor lysis occur through a complex interaction between triggering receptors such as NKp30, NKp44, NKp46 and NKG2D with tumor cell ligands in fine balance with inhibitory receptors and co-receptors (12). Recently, it was reported that activating NK cell receptor ligands MICA (NKG2D ligand) and CD155 (DNAM-1 ligand) are differentially expressed during progression to cervical cancer (13). We found that NK cell-activating receptors NKp30 and NKp46 are significantly downregulated in cervical cancer patients and women with CIN 2e3. NCRs downregulation correlated with low cytolytic activity, HPV16 infection and clinical stage (Garcia T. et al., manuscript submitted). Furthermore, our group recently described that serum levels of soluble MICA (soluble form of this stressinducible activating ligand for NKG2D) increase according to the progression toward cervical cancer. This suggests that soluble MICA may contribute to HPV persistence or tumor progression (14). On the other hand, we described that CEACAM1 significantly increase in high-grade squamous intraepithelial lesions in comparison with low-grade squamous intraepithelial lesions and normal cervical tissues. These findings are relevant because CEACAM1 is an adhesion molecule with immunoregulatory properties and one is NK cell inhibition (15). Indeed, there is still much to do to clarify the intriguing potential role of NK cells, especially in the local tissues affected by the HPV infection and during the progression to carcinoma. Dendritic Cells. Given the constant and varied antigenic exposure in the different subcompartments along the female genital tract (urethra, vulva, vagina, exocervix, endocervix, uterus, fallopian tubes, etc), the presence of dendritic cells (DCs) as sentinel posts of the immune system in these areas is significant, although their precise role has yet to be defined. It is likely, for instance, that conventional myeloid DCs (cDCs) populate these tissues, similar to other peripheral tissues such as the skin, airways, or gastrointestinal mucosa (16e19). Using three DC markers (Langerin, MHC-II and CD1a) and epithelial sheets instead of conventional tissue sections, our group recently identified a dense planar network of DCs in human cervix samples (16). Interestingly, the frequency of these DCs was reduced to about half during high-risk HPV infection (CIN-I) compared to non-infected high-risk HPV-negative samples. We are currently assessing the status of cDCs in situ and whether they are activated/matured, HPV-infected, etc. Likewise, whether the presence of plasmacytoid (p)DCs as the major early producers of natural-type-I interferons is constitutive or inducible under viral infections such as the high-risk HPV still needs to be explored. In this respect, type-I IFNs require assessment in situ, although by using
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keratinocyte cell lines it seems that HPV deregulates this responses at various levels (20). In any case, this line of defense is undoubtedly overcome by the virus because HPV establishes the infection persisting for many years. gdþ T lymphocytes and B1 lymphocytes. As early responder-effector cells, gdþ T lymphocytes also represent another population of innate immune cells whose relevance in these anatomic locations has not been examined during HPV infections but warrants careful scrutiny. Their production of cytokines like interferons and their cytolytic activities may be of importance in the regional defense from HPV and many other potential pathogens. B1 cells are producers of the so-called ‘‘natural’’ antibodies (i.e., those not requiring previous Ag exposure/induction, usually IgM but also IgA) in the different mucosae, and it is highly likely that B1 lymphocytes are important in the innate defense of these tissues. Accordingly, the presence of IgA-producing plasma cells in situ has been reported within the cervix, but their precise lineage remains to be established. HPV and Inflammation? Cell Participants in Early Reactions. The potential role of populations of innate immediate responses such as polymorphonuclear leukocytes (PMN) and mast cells or eosinophils certainly needs to be examined during the course of HPV infection. This is especially true regarding in situ analysis either in human samples or using appropriate animal models for HPV infection where the most early time points could be controlled and assessed, unlike what happens in the natural course of the infection in humans. It is known, for instance, that PMN infiltration is seen mainly in bacterial cervicitis, whereas other cell types such as lymphocytes and monocytes can be found in other diseases such as chlamydia and tuberculosis (the latter being more rare) (21). Although inflammatory infiltrate has been described for other tumor types, this is ill-defined regarding HPV and still remains controversial whether inflammation indeed occurs during the different stages of HPV infection, i.e., from the early changes and NICs to CC. Older studies described infiltration by macrophages, plasma cells and lymphocytes into the epithelium and the stroma in preinvasive and invasive cervix tumors (21e23). However, the controversy about whether there is indeed inflammation and the scarcity of descriptions about it may well indicate a more complex picture of the inflammatory response when facing the HPV. The natural course of the early phase of HPV infection leading to CC is clearly asymptomatic, detected solely by cytology-based tests or by using more elaborate means. Adaptive Immune Responses During Genital HPV Infection: B and T Lymphocytes Adaptive Humoral Immune Responses (B Lymphocytes). Regarding adaptive humoral immunity, it is reported that
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immunoglobulins (Ig) IgG and IgA are the main Ig classes in female genital secretions such as the mucus from cervix where IgG is found in higher levels than IgA. These IgGs would apparently derive both from locally producing cells and from transudation into the lumen of the genital tract (3). Although the origin of IgAs may be dual, either from B1 or B2 type lymphocytes, IgGs are most likely derived from B2 conventional B cells, i.e., those that have passed through a follicular germinal center reaction experiencing somatic hypermutation and Ig class switching under classical CD4þ T cell help. Because these types of Ab responses normally occur only in highly organized secondary lymphoid tissues, this implies that the exogenous intruding Ags have, by necessity, reached the B cell follicles within regional (iliac) lymph nodes draining these organs. For many obvious limitations, this is an issue not well explored for most pathogens invading these tissues in humans, including HPV. Unlike antibodies from B1 cells, those derived from follicular B2 lymphocytes are Ag-triggered; thus, it is possible that these Abs may be more useful, for example, to interfere with microbial adherence or early interactions (with HPV?) or to clear a given pathogen. Alternatively, if these Abs are triggered and produced only under specific Ag (microbial) exposure but the infection nevertheless progresses—a scenario that seems highly likely during chronic HPV infections—these Abs are thus not effective from the host standpoint. This, in turn, would suggest a rather active strategy from the HPV to deviate the host regional adaptive humoral responses. Regarding systemic responses, it is reported that after transient or even persistent and well-documented HPV infection, some women fail to show seroconversion for HPV, whereas serum IgA has been associated with both HPV persistence or with HPV clearance. Likewise, the extremely long delay of 18 months for IgG and IgA seroconversion to appear after documented HPV infection certainly merits careful investigation (24). In fact, Abs to E7 may not appear even after 10 or 20 years that the protein has been expressed during the infection (25,26). At the regional level, at least one report described the Ab responses in the female genital tract as altered during HPV infection, including the local and serum IgG and IgA, with no apparent changes in IgM. Altered transudation was mentioned as one potential HPV-induced mechanism (26). Another study found that cervical IgA to E7 from HPV 16 was decreased in CC (27). Others have observed fluctuations of VLP-induced Abs during the menstrual cycle (28). Interestingly, anti-VLP 16 IgA Abs in oral fluid of adult women attending dental clinics were found to correlate with cervical HPV positivity. Moreover, the authors suggested that the emergence of these Abs in oral fluids of adolescents correlated with the initiation of sexual activity and depended on the common mucosal immune system (29). The complex situation of the different antibody types (systemic, local, from B1 cells, from memory
responses, etc.) potentially induced during early and chronic HPV infection certainly merits more careful studies. Adaptive Cellular Immune Responses (T cells) Lymphoid aggregates mostly containing CD8þ T lymphocytes surrounding some B cells have long been described by histopathologists in the endometrium, and these aggregates apparently showed variations with the menstrual cycle (30). In pre- and postmenopausal women, the presence of CD3þ, CD4þ, CD8þ T cells, macrophages, and DCs in the endocervix, ectocervix, and vagina was found distributed throughout the lower female reproductive tract in both the epithelium and subepithelial mucosa (31). Our group has also found that CD4þ, CD8þ and some gdþ T lymphocytes are normally present in the basal epithelial layers of the cervix (Jimenez-F, personal communication, 2005). These assessments may provide interesting clues, for example, regarding which T-cell subsets infiltrate (if they do) the actual HPV-infected areas during the progression from early lesions and NIC-I to CC, and whether these T cells are armed or not with pertinent cytokines such as IFN-g or with other relevant molecules like granzyme or granulysin. It is likely that the ultimate host immunity test that HPV must deal with is cytotoxicity, probably by NK or NKTs initially and later on by CD8þ T lymphocytes. However, pertaining functioning, the assessment of cell-mediated immunity during HPV infection has been rather difficult and reports are contradictory (32). For instance, whereas cytotoxic T lymphocytes (CTLs) from an important percentage of chronically (HPV) infected women fail to respond to HPV challenge (33), other studies reported a good correlation between systemic delayed-type hypersensitivity responses (DTH) and CIN resolution (34,35). Discrepancies have also been found regarding the vigorous responses to viral Ags between T cells from peripheral blood and the quite low numbers of tumor-reacting T cells (36). In any case, research is scant regarding the three major T-cell subsets (CD4/CD8/gd T cells), especially in situ in the different subcompartments of the female genital tract. In restricted cases there may be the ethical possibility to obtain the regional draining lymph nodes. Their evaluation may also yield valuable information, for instance, regarding the presence of HPV within the nearest secondary lymphoid tissues or whether the putatively responding T cells are being expanded and activated in the interdigitating dendritic cells (IDC)/T zone area of the nodes. For instance, immune responses are reportedly affected by microenvironment such as extracellular matrix components, growth factors, cytokines and proteases. Our group found that lymphocyte proliferation to PHA was partially inhibited by tissue extracts from cervical carcinoma, mainly by proteases. Experiments with purified proteolytic
HPV Immunobiology
enzymes (trypsin, cathepsin B, uPA and type IV collagenase) reinforce the protease inhibitory role (37). It has also been reported that proteases increase according to the progression towards invasive CC (38). In the search of animal models for HPV infection and carcinogenesis, a transgenic mice expressing E7 from HPV16 was reported with induction of E7-specific immunological tolerance at the CTL level (39). Also in experimental animals, a compartmentalization has been shown of the long-term anamnestic CTL responses into the regional lymphoid tissues draining mucosae (such as the iliac nodes draining the genital tract) or to the systemic lymphoid tissue like the spleen. This depends on the route of immunization (40). The follow-up of the presence or absence of T cells within the genital tract, as well as of pertinent molecules associated with effector T-cell immunity, may certainly yield important cues pertaining to the evolution and prognosis of the HPV infection. In conclusion, a peculiar feature of oncogenic HPVs is the protracted but highly efficient strategy to infect humans and propagate among them. A very enigmatic issue is how, despite facing several multilayer levels of both the innate and the adaptive immunity, apparently the same HPV type is persistent during many years—often during decades—to avoid arising overt and deleterious immune responses, thus revealing HPV as a virus extremely well adapted to humans. In order to efficiently infect the basal layers of the cervix epithelium and transform these cells into cervical carcinoma, it must face and overcome first the primary natural mechanical barriers provided by the epithelium, the mucus, the potential antimicrobial peptides, and the variety of potential alert signals triggered by TLRs. Reports of inflammatory reactions from the female tract to early encounters with HPV are rather scarce, either because they may be difficult to track given the natural course and timing of the infection or because these types of responses are indeed somehow missing. The subsequent phases of immunity must also be dealt with, including the fast reactions from neutrophils, mast cells or eosinophils (if there are any of these reactions during early HPV interactions), then by NKs and plasmacytoid DC with their vigorous type I interferon responses. In a higher complexity level regarding adaptive immunity, HPV probably faces first the so-called natural (‘‘non-inducible’’) antibody responses such as IgMs and IgAs and then the conventionally triggered B2 Ab responses, of which IgA should also be one in this mucosal tissue and then IgGs, this being a chronic infection. It is likely that the ultimate immune test that HPV must deal with is the cytotoxicity against HPV-infected cells, first by NK or NKTs and much later on by CD8þ T lymphocytes. Initially, the cytolytic responses should act upon transformed cervical cells such as koilocytes and, later, over the already carcinogenic cells. However, from the long list provided, it is quite
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evident that HPV manages very well to face, cope with and overcome all the responses that the immune system can produce. Conversely, the ineffectiveness of the several mechanisms of the immune system (innate and adaptive, simple and complex, local and systemic) should also be evident when facing such a virus. Because HPV infection alone seems insufficient to progress from the early infection stages to a carcinoma, another important susceptibility factor may well rely on complex immunodeficiencies not yet described in the minority of women slowly progressing from infection to cancer.
Acknowledgments R. Garcia-Chacon and S.F. Velasco-Ramirez are fellows from the National Council for Science and Technology (Conacyt). L. Flores-Romo and A. Daneri-Navarro are members of the National System of Researchers (SNI). The authors acknowledge the help from the two laboratories, especially Juana Calderon.
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