Our general goal is to understand the mechanisms of sexual transmission of human pathogens, including the human immunodeficiency virus (HIV), to human genital tract, their tissue pathogenesis, and to develop efficient anti-virals. These goals require a comprehensive understanding of the immunology of human reproduction, one of the missions of the NICHD. Seminal cytokines are key factors in human fertilization. Sexually transmitted pathogens, in particular HIV, use the modulation of the cytokine network for their own benefit. During the last year, we focused on studying seminal cytokines, with a particular interest in their modulation during HIV-1 infection. Our study revealed the importance of coinfecting herepesviruses which, together with HIV-1, alter the immunological landscape of semen. Herpesviruses are known to promote transmission and to facilitate pathogenesis. Our results emphasize the importance of targeting, in addition to HIV-1, coinfecting viruses, in particular herepesviruses, and to prevent viral transmission. Herpesvirus-induced cytokines may serve as another target for the preventive strategy. Recently, tenofovir, applied vaginally as a microbicide, was found to reduce transmission not only of HIV-1 but unexpectedly also of herpes simplex virus. We deciphered the mechanism of this phenomenon using a system of cervico-vaginal tissues infected ex vivo with HIV-1 and herpesviruses. This finding provides a new insight into the development of multi-targeted antivirals. Future studies and ongoing clinical trials will show how efficient the new antivirals are in vivo. However, it is clear that even if HIV-1 is completely suppressed for years, the infected individuals continue to be susceptible to various diseases, most notably atherosclerosis. The current working hypothesis linking HIV-1 disease with atherosclerosis is that the progression of both diseases is fueled by inappropriate activation of the immune system. To test this hypothesis and to identify the aspects of immunoactivation that play a critical role in both pathologies, the activation status of lymphocytes found in atherosclerotic plaques was investigated. This was the focus of our studies on human atherosclerotic plaques.
In general, the system of ex vivo tissues on which we reported earlier is now widely used to study HIV-1 transmission and pathogenesis in lymphoid, cervicovaginal, and rectosigmoid tissues. We further developed and standardized this system to make it reflect various in vivo aspects of intercellular interactions more faithfully than isolated cells in suspension or monolayers cultures and to broaden its application for the scientific community.
HIV sexual transmission from males to their partners occurs through various complex stages (gatekeepers) that control which HIV-1 variant is transmitted to start the infection. During the past year, we focused on one of the earliest of these mechanisms to operate—in semen of HIV-1–infected men. Semen is not merely a passive transporter of virions but plays an active role in HIV-1 transmission through cytokines and other biological factors.
We addressed the relationship between the cytokine milieu and coinfecting viruses in the male genital tract of HIV-1–infected individuals by comparing the levels of 21 cytokines/chemokines as well as the loads of HIV-1 and six coinfecting herpesviruses in seminal and blood plasmas from HIV-1–infected and uninfected men. Semen plasma and blood were collected from 50 HIV-1–infected and thirty-three HIV–uninfected healthy volunteers enrolled at the All India Institute of Medical Sciences in New Delhi, India.
We found that semen and blood are two separate immunological compartments, in which concentrations of cytokines and loads of coinfecting herpesviruses are profoundly different. In particular, we found that semen is enriched in IL-1alpha, IL-7, IL-8, MIP-3alpha, MCP-1, MIG, IP-10, SDF-1alpha, and TGF-beta, while blood is enriched in IL-2, IL-16, MIP-1beta, and eotaxin. Upon HIV infection, the levels of blood and semen cytokines are significantly altered, thus affecting the compartmentalization of the semen and blood cytokine networks. HIV-1 infection changes the seminal cytokine spectrum by upregulating 16 out of the 21 measured cytokines while in blood two cytokines are downregulated and three are upregulated. As a result, HIV-1 infection emphasizes the compartmentalization between blood and semen. Given that the cytokine network and its modulation in semen are different from those in blood, the upregulated seminal cytokines seem to be produced locally in the male genital tract. Thus, changes in cytokine levels upon HIV-1 infection may reflect a profound dysregulation of the functional state of immune cells resident in the male genital tract.
Already altered by HIV-1 infection, the seminal cytokine network is further altered by herpesviruses reactivated in the male genital tract. Our data indicate that such reactivation may occur locally, given that approximately 75% and 54% of the patients, respectively, exhibited cytomegalovirus (CMV) and Epstein Barr virus (EBV) seminal shedding in the absence of blood plasma viremia. Moreover, the median load of CMV in seminal plasma was more than 100 times higher than in blood plasma. Our work indicates that the source of the EBV and CMV seminal shedding is not a systemic reactivation of the infection accompanied by a spill-over of virus from the blood, but rather their specific and compartmentalized reactivation in the male genital tract, further indicating that these two compartments are immunologically distinct.
The seminal cytokine spectra altered by local infection with HIV-1, CMV, EBV, and probably other viruses may favor the selection of particular HIV-1 variants. Co-infecting viruses and cytokines present in semen may alter not only HIV-1 replication and evolution in the male genital tract but also the immunological landscape of the female genital tract when delivered with the ejaculate. These alterations modify the recruitment and activation status of immune cells therein and determine the efficiency of HIV male-to-female and male-to-male HIV transmission and should thus be considered as another target in HIV-1 transmission prevention strategies.
Various new compounds have been tested over the last few years as vaginal microbicides. Recently, instead of testing new compounds as potential microbicides, tenofovir, an efficient nucleotide HIV reverse transcriptase (RT) inhibitor widely used in HIV therapy, was formulated as a 1% gel and became the first example of a microbicide that convincingly diminishes HIV-1 transmission. Surprisingly, a significant reduction of the risk of acquisition of herpes simplex virus type 2 (HSV-2), a common HIV-1 copathogen, which promotes HIV transmission, was also observed. This is important given that herpesviruses facilitate transmission of HIV-1 and adversely affect the clinical course of HIV disease. The effect of tenofovir gel on HSV in vivo was unanticipated, given that tenofovir was not known to exhibit anti-HSV activity.
We resolved this apparent contradiction by providing compelling evidence that, at the concentrations achieved intravaginally by the topical administration of a 1% gel, tenofovir exhibits a direct anti-herpetic activity. We investigated the effect of tenofovir on the replication of both laboratory and clinical HSV-1 and HSV-2 isolates in infected human tissues ex vivo. Tenofovir suppressed replication of HSV-1F, HSV-2G, and HSV-2MS in a dose-dependent manner. At a concentration similar to one achieved in vivo upon local application, tenofovir reduced HSV-1F, HSV-2G, and HSV-2MS replication by 99 %, 87%, and 91.7%, respectively, compared with infected donor–matched untreated tissue. Tenofovir also inhibited replication of HSV in HIV-1–coinfected tissues. Based on the lack of depletion of tonsillar cell subsets and on the unaffected release of 15 cytokines, we found that tenofovir is not toxic. The anti-herpetic effect of tenofovir is not a general property of nucleoside reverse transcriptase inhibitors as there was no effect of lamivudine (3TC) on HSV replication. Furthermore, we deciphered the molecular mechanism of the anti-herpetic activity of tenofovir. Tenofovir diphosphate, to which tenofovir is converted in various human cell types, efficiently inhibits HSV DNA polymerase.
Thus, the antiretroviral drug tenofovir is endowed with a direct anti-herpetic activity at drug concentrations comparable with those achieved in cervico-vaginal fluid following the administration of 1% tenofovir gel. We hypothesize that the discrepancy between the earlier reported lack of significant anti-herpetic activity and the recent clinical data is explained by the striking differences in drug concentrations between systemic and topical applications of tenofovir. Our findings provide a direct explanation for the dual anti-HIV/HSV activity reported in vivo.
In conclusion, tenofovir, which was designed to suppress HIV-1 rather than herpesviruses, at the high concentrations achieved with topical application, suppresses not only HIV-1 but also HSV. Moreover, tenofovir affects HSV directly rather than through complex indirect mechanisms. Antiviral activities of a variety of existing drugs should be revisited as potential microbicides with dual or multiple antiviral properties. These data and the therapeutic principles emerging from our study are important for the development of new drug formulations and administration protocols to design and/or optimize future microbicide trials.
Atherosclerosis is closely related to the activation of immune system. To decipher the immunological mechanisms of plaque maturation and rupture in HIV-1–infected and uninfected individuals, it is necessary to analyze the phenotypes and distribution of individual lymphocytes that migrate to the plaques as well as their activation at different stages of plaque formation. Despite a large volume of clinical and experimental data on the formation, maturation, and rupture of atherosclerotic plaques, the mechanisms of these phenomena are not yet fully understood. A complex process that includes migration of reactive cells, in particular lymphocytes and monocytes, and their activation, followed by the release of various cytokines seems to play an important role in the development of plaques. To decipher the immunological mechanisms of plaque maturation and rupture, it is necessary to analyze the phenotypes and distributions of the lymphocytes in individual plaques. The only current technology that can accomplish these tasks is polychromatic flow cytometry, which earlier modernized other fields of biology and medicine. However, such an analysis was never performed on cells residing within human atherosclerotic plaques. We performed such an analysis by developing an original cell isolation protocol that preserves cell surface markers and uses polychromatic flow cytometry to analyze plaque lymphocytes. We tested various enzyme cocktails on PBMCs, where the surface markers of the treated samples can be compared with those of the untreated control. We chose an optimal enzyme cocktail combination based on collagenase IV, which liberates cells from atherosclerotic plaques while preserving their identity, permitting us to perform a meaningful multi-colored flow-cytometric analysis of these cells.
We found that lymphocytes in plaques and blood differed significantly: plaques are enriched with CD8 T cells. Furthermore, T lymphocytes, especially CD8 T cells in plaques, are more activated than in blood, as evidenced by the expression of the CD25, CD38, and HLA–DR activation markers. The fraction of cells co-expressing CD25 and HLA–DR in plaques was 10 times larger than in blood. Our data indicate that plaque lymphocytes do not penetrate plaques indiscriminately. Analyzing lymphocytes from blood and plaques, we found that blood obtained from patients with plaques contained more CD4 T effector memory, terminally differentiated effector memory CD4 T cells, and CD4+ CD28− T cells, but fewer naive CD4 and CD8 T cells, as well as early differentiated effector memory CD4 T cells. Also, blood from plaque donors contained more activated T cells.
In general, the enrichment of plaques with CD8 T cells and the preferential activation of both these and CD4 T cells compared with activation levels in blood may indicate the presence of foreign antigens in these plaques, as has been suggested. The ability to isolate and characterize these cells may lead to the identification of such antigens.
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