Despite all efforts and medical progression, worldwide, the HIV-1 infection rate is estimated to be 14,000 per day and more than 40 million people are living with HIV. Most new infections occur in the developing world, and women account for nearly 50% of all people living with HIV.1 In the worst affected area, sub-Saharan Africa, women represent close to 60% of the infected adults; in fact, 76% of young people aged 15 to 24 years living with HIV are female.2 It is suggested that more than 80% of all transmissions are sexual, hence taking place across a mucosal membrane. The mechanisms of viral transmission across mucosal epithelium are not fully understood; however, several pathways have been proposed.3 The virus has been shown to infect epithelial cells directly,4,5 to be transcytosed across the epithelial layer,4,5 and to traverse the mucosal barriers by the use of dendritic cells (DCs).6,7
Knowledge of endogenous factors that mediate resistance to HIV-1 transmission and early dissemination are still largely unclear. A RANTES analogue has been shown to block simian HIV (SHIV) transmucosal infection efficiently, however.8 This antiviral capacity of RANTES was further supported in a recent article by Iqbal et al,9 where the investigators observed significantly higher levels of RANTES in cervical washings of HIV-1-resistant commercial sex workers (CSWs). These findings implicate that RANTES might play a crucial role in preventing HIV-1 infection, and thus become a potent microbicide candidate. The potential value of other protective mediators, including secretory leukocyte protease inhibitor (SLPI), leukemia inhibitory factor (LIF), and defensins that may confine the replication to the portal of entry, still remains to be validated. In this context, interferon-α (IFNα) produced by plasmacytoid dendritic cells (pDCs) is believed to be a key player in linking innate and adaptive immunity. In addition, IFNα production by pDCs exerts a potent antiviral effect on HIV-1.10 It has also been reported that productive infection with HIV-1 correlates with a decrease in IFNα-producing cells in peripheral blood.11
Preventing virus infection at the local mucosal surface with microbicides is an appealing approach, particularly because it would be a prevention method that women can control. Studies on the endogenous expression of innate immune molecules at the single-cell level in the lower female genital tract of women with different HIV-1-encountering risk behaviors may enhance our knowledge about viral and host mechanisms of protective potential.
MATERIALS AND METHODS
Study Population and Sample Collection
Cervical biopsies from the superior portion of the ectocervix were collected from white HIV-1-positive women (n = 6), African HIV-1 IgG-negative CSWs (n = 7), and low-risk healthy African and white HIV-1 IgG-negative women (Neg Ctrls; n = 10). The HIV-1-positive women were selected from a larger study on discordant couples, where the women were defined as nontransmitters of HIV-1 infection. The CSWs were recruited from the Womens Health Center of Reggio Emilia and had been active as sex workers for a median of 8 years; during that time, they had lived and worked a median of 12 months in Italy at the time of biopsy collection (Table 1). Clinical and laboratorial examinations were performed on all study subjects for systemic diseases, including hepatitis B virus (HBV) and hepatitis C virus (HCV), and sexually transmitted diseases (STDs), including Chlamydia, gonorrhea, syphilis, herpes simplex virus type 2 (HSV-2), genital ulcer disease (GUD), Candida, and bacterial vaginosis. Human papillomavirus (HPV) typing was performed in clinically suspected cases. The stage of menstrual cycle, the eventual use of contraceptives, and other relevant clinical information were taken under consideration at time of biopsy collection (see Table 1). The biopsies were immediately snap-frozen in liquid nitrogen.
After enrollment in the study, counseling was offered to all the recruited subjects. Informed consent was obtained from all study subjects, and ethical approval was obtained from the San Raffaele Scientific Institute of Milan Ethical Review Board, the Womens Health Center of Reggio Emilia Ethical Review Board, and the Karolinska Institute Ethical Review Board, as applicable.
In Situ Detection of Innate Immune Molecules by Immunohistochemistry and Confocal Microscopy
The biopsies were analyzed as previously described.12 Cryopreserved biopsy samples were cut 8-μm thick, fixed in 2% formaldehyde, and blocked for endogenous biotin (Biotin/Avidin Blocking Kit; Vector Laboratories, Burlingame, CA). Antihuman IFNα (PBL Biomedical Laboratories, Piscataway, NJ), RANTES (R&D Systems, Abingdon, United Kingdom), β-defensin 2 (Alpha Diagnostic, San Antonio, TX), SLPI (Cell Sciences, Canton, OH), and LIF (Chemicon, Hampshire, United Kingdom) were used to detect the immune markers of interest after extensive evaluation. Negative controls consisted of irrelevant mouse IgG1 (Dako, Denmark). The staining reactions were developed brown using diaminobenzidine tetrahydrochloride (DAB), and counterstaining was performed with hematoxylin. Acquired computerized image analysis13 was performed on the labeled sections, generating digital images that were transferred from a DMR-X microscope (Leica, Wetslar, Germany) into a computerized image analysis system, Quantimet Q 550 IW (Leica Imaging Systems, Cambridge, United Kingdom). Expression of the innate immune molecules of interest was determined as the percentage positive area of the total relevant cell area. Whole-section scans were performed, and each biopsy was assessed twice, with resultant variations always less than 10%. To prevent possible disparities, duplicate biopsies from the same study subject were stained and analyzed when possible. Furthermore, all analyses were performed masked with respect to which risk group the study subject belonged.
For 2-color staining, tissues were stained with antihuman CD8 (mouse IgG1; BD Pharmingen, Sweden) and RANTES (goat IgG; R&D Systems), followed by the appropriate (Alexa 488 or Alexa 594) fluor-conjugated secondary antibody (Molecular Probes; Invitrogen, Sweden). Positive cells were quantified in 10 high-power fields using Qwin 550 software and a filter-free spectral confocal microscope (TCS SP2 AOBS; Leica).
Preliminary tests for normality showed a skewed distribution of values for all parameters we measured. Thus, all analyses were performed with nonparametric tests. Intergroup variations were analyzed in parallel by comparing a single parameter from 1 group of individuals against the corresponding parameter of each other group of individuals using the Kruskall-Wallis test. When significant variations were detected, post hoc comparisons were adjusted with the Bonferroni procedure. The unpaired 2-tailed Mann-Whitney test was used when comparing only 2 groups of individuals. Calculations were performed using STATISTICA System software. A probability value of 0.05 was considered to be statistically significant.
Characteristics of Study Subjects
The median age of the 3 study groups did not vary significantly (see Table 1). The total number of years as a sex worker reported in CSW study group ranged from 3 to 16 years.
One of the 7 CSWs and 2 of the 6 HIV-1-positive women had chronic HCV infection, and 2 CSWs had chronic HBV infection. Furthermore, 3 of the CSWs had histologic signs of inflammation; however, only 1 of these individuals had a clinically suspected and confirmed HPV infection. Three of the 7 CSWs and 1 of the 10 Neg Ctrls were in the preovulatory stage of their menstrual cycle at the time of biopsy collection, whereas the remaining study individuals were in the postovulatory stage. None of the study individuals confirmed condom use or use of any contraceptives at the time of biopsy collection, except for 1 Neg Ctrl, who reported 100% use of condoms, and 2 Neg Ctrls, who reported the use of hormonal contraceptives. Three of the 6 HIV-1-positive individuals were receiving antiretroviral therapy (ART) at the time of biopsy collection, whereas the remaining 3 were treatment naive.
All the previously mentioned intragroup disparities were taken into account when calculating the intergroup variances in expression of the factors of interest. Because of the small number of individuals in the each study group, however, intragroup statistical analysis with the purpose of ruling out significant disparities could not be performed. Nonetheless, no obvious trends were noted. No significant differences were detected in the Neg Ctrl group compared with the African and white subgroups when calculating the expression of the factors of interest; thus, the Neg Ctrl study group remained heterogeneous, consisting of African and white individuals.
Upregulated Expression of Interferon-α and RANTES in HIV-1-Positive and Commercial Sex Worker Individuals
The biopsies were analyzed in situ for innate immune molecules, with proposed involvement in mucosal protection of HIV-1, at the single-cell level.
The HIV-1-positive and CSW study groups had a higher expression of IFNα and RANTES than the Neg Ctrl group (Figs. 1, 2). The total number of years as a sex worker was used as a parameter for HIV-1 risk exposure, and a significant positive correlation between years of sex work and the expression of RANTES was detected (P = 0.028, R2 = 0.65; Fig. 3). Most (>90%) RANTES-expressing cells were CD8+ cells, mainly found in the lamina propria, as determined by 2-color confocal microscopy (Fig. 4). IFNα-expressing cells were predominantly found with subepithelial or intraepithelial localization in large cells with a dendritic appearance (see Fig. 2).
Expressions of Leukemia Inhibitory Factor, Secretory Leukocyte Protease Inhibitor, and β-Defensin 2 in the Study Groups
No difference in the expression of LIF or SLPI was found when comparing the 3 study groups. Lower expression of β-defensin 2 was observed in the CSW subjects compared with HIV-1-infected individuals, however (see Fig. 1).
Correlates of protective immunity against HIV-1 infection have been studied in humans and animals for many years without any definitive conclusion. The most solid evidence of true resistance is probably the homozygous expression of the Δ32 deletion in the HIV-1 coreceptor CCR5.14-16 Studies of HIV-1-exposed uninfected individuals have pointed out a number of HIV-1-specific innate and adaptive immune responses that may confer the resistant status.17 None of these have described the expression of innate immune molecules at the single-cell level in the cervical tissue of women at risk of infection, however.
In the present study, we were able to distinguish a relatively high expression of IFNα and RANTES in HIV-1-infected versus low-risk control subjects. In addition, we were able to identify the same elevated level of expression in a group of CSWs with a reasonably higher HIV-1 encountering risk as compared to low-risk control subjects. It is not possible to distinguish whether the observed higher expressions were attributable to HIV-1 exposure or exposure to other STDs. Although an inflammatory status of the female genital tract is a well-known risk factor for acquiring HIV-1 infection, preexisting IFNα and RANTES may be of clinical importance for protection against HIV-1 at mucosal exposure.
IFNα facilitates major histocompatibility complex (MHC) class I expression of cervical epithelial cells,18 induces a T helper (Th) 1 type of adaptive response, and initiates multiple antiviral mechanisms in neighboring cells. The antiviral effect includes inhibition of HIV-1 replication19 and may be an effective strategy in limiting the initial viral dissemination beyond the critical lamina propria foci of the CD4+ memory T cells in which most initial replication occurs.20 Moreover, local mucosal production of RANTES might be beneficial for resistance to HIV-1 infection by blocking the HIV-1 coreceptor CCR5. In our study, we have shown that the RANTES detected in the cervix mucosa of CSWs was mainly produced by CD8+ cells. Thus, CD8+ cells in the cervix mucosa of exposed seronegative individuals may not only have an HIV-1-specific cytotoxic effect,21,22 but recruitment of such cells to the local site of viral infection may contribute to antiviral activity by the production of RANTES.
It is important to note that the HIV-1-infected women in our study were HIV-1 nontransmitters (ie, their male partners were HIV-1-negative). One could hypothesize that IFNα and RANTES in the cervix mucosa of HIV-1-infected subjects may be correlated to their nontransmitter status.
The CSWs are African women who have worked for a documented 3 to 16 years (median = 36 months), during which they have lived and worked a median of the last 12 months (range: 6-24 months) in Italy. The prevalence of HIV-1 among their clients in Italy is probably lower than in their home country. Although it has not been possible to calculate the number of HIV-1 risk exposures through sexual contacts (as has been done in other highly exposed persistently seronegative [HEPS] cohorts23), and the effects we have observed may be attributable to other concomitant STDs, we believe that the CSWs most probably represent a group at high risk of contracting HIV-1 infection. Interestingly, we observed a positive correlation between the number of years as a CSW and the expression of RANTES. Furthermore, none of the CSWs reported condom use, thus increasing the risk of exposure to multiple viral clades from multiple clients with different human leukocyte antigen (HLA) backgrounds as well as a higher risk of contracting other STDs that may influence the inflammatory status of the genital mucosa. In other studies, reduced numbers of HIV-1 sexual encounters attributable to recess or retirement from sex work resulted in varied HIV-1-specific immunity.24 It is not known whether these CSW subjects had systemic or mucosal HIV-1-specific immunity at the time of biopsy sampling. The innate response pattern found in the CSW group may be an indication of partial protection from HIV acquisition, however.
A number of innate immune molecules have been associated with anti-HIV-1 activity, and most of these were therefore investigated in our study. Induction of LIF has been shown in the initial stages of viral dissemination during primary HIV-1 infection and has thus been suggested to be a part of the virally induced generalized proinflammatory response.25 SLPI has been proven to block HIV-1 replication and is present in mucosa secretions.26 Recently, β-defensins 2 and 3 (but not β-defensin 1) were shown to block HIV-1 infection by modulating the CXCR4 coreceptor and interacting directly with virions.27 Also, a strong constitutive production of α-defensin has been observed in HIV-exposed uninfected individuals, suggesting that these peptides may have a role in the protective immune response that characterizes HIV-1-exposed seronegative individuals.28 In our study, however, the presence of LIF, SLPI, and β-defensin 2 did not differ significantly between the study groups apart from a lower expression of β-defensin 2 in the CSW subjects as compared to HIV-1-infected individuals.
When studying the cervix mucosa, several factors have to be taken into consideration, such as the normal menstrual cycle; the eventual use of oral contraceptives29; and the presence of other STDs, including HPV.30 Alterations during the menstrual cycle have been observed in cervical mucus for LIF,31 SLPI,32 and β-defensins.33 Therefore, we chose to collect the biopsies during the postovulatory stage of the menstrual cycle, with a few exceptions. Studies have shown that the use of oral contraceptives might decrease the expression of β-defensins34 in the cervix mucosa. In our study, we detected a significantly lower expression of β-defensin 2 in the CSW group of individuals. None of the CSWs reported the use of oral contraceptives, however.
Clinical and laboratory examinations were performed on all study subjects for STDs, such as Chlamydia, gonorrhea, syphilis, HPV, HSV-2, GUD, Candida, and bacterial vaginosis. All study subjects were negative for the previously mentioned STDs, except for 3 cases of bacterial vaginosis, 1 case of Candida, 1 case of GUD, and low-grade lesions caused by HPV. Although we could not perform intragroup statistical analysis because of the small number of study individuals, we did not note any obvious differences in the expression of the immunologic factors of interest when comparing the previously mentioned individuals with the other subjects in the same study group concerning the stage of menstrual cycle, use of hormonal contraceptives, or other STDs.
In this study, we have investigated the expression of innate immune molecules at the single-cell level in the cervix mucosa of HIV-1-seronegative individuals with high-risk behavior for contracting HIV-1 infection. Further longitudinal studies of this important compartment for protection from initial viral infection and dissemination in exposed seronegative individuals may generate a better understanding of how the resistance toward HIV-1 infection is mediated. This may ultimately guide us to future preventive interventions, such as the development of improved microbicides, and may assist in the development of vaccines that elicit protective HIV-1 mucosal immunity.
The authors thank C. Pastori for technical help, C. Gemmi and A. Foracchia for management of clinical data and collection of biopsies, and R. Kaul for fruitful discussions.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
cervix; HIV-1; innate immunity