The potential association of HSV2 infection status in HIV-negative FSWs with changes in genital CD4/CCR5+ T-cell or CD1a/DC-SIGN+ iDC populations was assessed (Fig. 2a; representative FACS plot of cervical cell populations). There were no differences in the absolute number of cervical CD4+ T cells, but there was a 0.3 log10 increase (three-fold higher absolute cell numbers) of CD4/CCR5+ T cells in the cervix of HSV2-infected FSWs (P < 0.05; Fig. 2b). Cervical CD4+ T cells were also more activated, with a greater absolute number and proportion (46% in HSV2+ versus 16% in HSV2) expressing CD69 (P < 0.001 for both; Fig. 2b). Strikingly, there was an entire log10 increase (ten-fold higher absolute cell numbers) in DC-SIGN+ iDCs (P < 0.001; Fig. 2c), and an increased proportion of cervical iDCs expressed DC-SIGN (11.9% in HSV2+ versus 4.6% in HSV2–, P < 0.05; Fig. 2c). Further characterization demonstrated that over 90% of cervical CD1a+ iDCs co-expressed CD11c, suggesting that these cells were of myeloid origin (data not shown).
Overall, HSV2 infection was associated with increases in genital HIV target cell populations, even in the absence of genital ulceration or HSV2 reactivation. We did not find any significant association between HSV2 infection and cervical cytokine or chemokine levels in HIV-uninfected FSWs. Since HSV2 shedding was not detected in HIV-negative FSWs, changes in the genital immune milieu related to HSV2 reactivation could not be examined.
In comparison with HIV-negative women, there was a profound depletion of iDC populations in the genital mucosa of HIV–HSV2 co-infected FSWs. Cervical CD1a+ iDCs were reduced (P < 0.05; Fig. 3a), particularly iDC subsets expressing either DC-SIGN (P < 0.05) or Toll-like receptor 9 (TLR9; P = 0.05), an innate immune receptor that binds CpG motifs on HSV2 DNA to trigger signaling cascades that culminate in type 1 interferon and inflammatory cytokine release, and a potentially important mediator of HSV2 immune control [36,37]. This iDC depletion was independent of systemic immune status (blood CD4+ T-cell counts or CD4/CD8 ratio; data not shown). HIV-infected FSWs had increases in cervical CD3+ T cells (P < 0.05) and CD8+ T cells (P = 0.001; Fig. 3a), without differences in activation levels. Cervical CD4+ T-cell numbers did not vary with HIV status (P = 0.7), but the CD4/CD8+ T-cell ratio was decreased in both the blood (P < 0.001) and cervix (P < 0.001) of HIV-infected FSWs, and to a similar degree at both sites (ratio 0.5 in blood versus 0.6 in the cervix; P = 0.5). There were no significant differences in CCR5+CD4+ cervical cell populations (absolute numbers, log10 transformed or % CCR5 expression by CD4 cells) between HIV-uninfected and infected women, although there was a trend (P = 0.07) for a decrease in the proportion of CCR5 expressing CD4+ T cells in HIV-infected FSWs. Although there were no significant differences in cervical cytokines/chemokines levels between HIV-infected and uninfected women, there were some trends to increased cytokines/chemokines in HIV-infected FSWs (MCP, IP-10, IL-10, IL-5, IL-2; P < 0.1 for all). HIV infection status was not associated with overall differences in the expression of TLRs 1–10 or FoxP3.
Cervical HIV shedding was detected in 10 of 36 FSWs (28%), and was strongly associated with immune activation in the genital tract. Levels of chemokines (IP-10, MCP, RANTES; all P < 0.001 and MIG; P < 0.05) and inflammatory cytokines (IL-1β, IL-8, IL-6 and IFN-γ; all P < 0.05; Fig. 3b) were elevated in the cervico-vaginal secretions of the 10 HIV shedders. In this subgroup, absolute HIV levels were inversely correlated with TLR9+ iDC numbers (r2 = −0.7; P < 0.05) and with mRNA expression of TLR8 (r2 = −0.9; P = 0.001) and TLR9 (r2 = −0.7; P < 0.05). Cervical HIV load correlated with both the number and proportion of activated cervical CD4+ T cells expressing CD69 (r2 = 0.7; P < 0.05; and r2 = 0.7; P < 0.05, respectively). Neither blood CD4 cell counts, blood HIV viral load, nor cervical immune cell populations varied significantly with HIV RNA shedding status.
Cervical HSV2 shedding was only detected in HIV-infected sex workers, and the level of cervical HIV RNA was strongly correlated with HSV2 DNA levels (r2 = 0.9; P < 0.001: Fig. 3c). Activated cervical CD4+ CD69+ T cells were increased in the genital mucosa of FSWs shedding HSV2 (202 versus 68 cells/cytobrush; P < 0.05, Fig. 4a), as were cervical levels of chemokines (P < 0.05 for all; Fig. 4b), but not pro-inflammatory cytokines. HSV2 levels correlated with the number and proportion of CD4 T cells expressing CCR5 (r2 = 0.7; P < 0.05; and r2 = 0.7; P = 0.02; respectively) (Fig. 4c), and with levels of the chemokines IP-10, MCP, MIG and RANTES (all r2 > 0.6; all P ≤ 0.05). Cervical iDC numbers were unchanged in FSWs shedding HSV2 (13 588 versus 10 696 cells/cytobrush; P = 0.2), although fewer iDCs expressed TLR9 (3.1 versus 11.6%; P = 0.001). HSV2 DNA levels correlated inversely with the number of DC-SIGN+ iDCs (r2 = −0.8; P < 0.05) and TLR9+ iDCs (r2 = −0.8; P < 0.05) (Fig. 3d, e). This suggests that TLR9+ and DC-SIGN+ iDCs may mediate local immune control of HSV2 reactivation at a genital level.
This study confirms the substantial epidemiological synergy reported between HIV and HSV2, and is the first to demonstrate that this may be underpinned by a profound negative ‘mucosal synergy’ between these viruses in the female genital tract. In HIV-negative at-risk women, HSV2 infection was associated with increases in genital mucosal target cell populations that would be expected to increase susceptibility to HIV infection. These mucosal immune changes were evident even in the absence of genital ulceration or HSV2 reactivation, implying that HSV2 induces a persistent state of increased mucosal HIV susceptibility. The ten-fold increase in cervical iDCs expressing the DC-SIGN lectin, and the three-fold increase in cervical CD4+ T cells expressing the HIV co-receptor CCR5, provides a putative biological explanation for the observation that HSV2 seropositivity is a strong independent risk factor for HIV acquisition [6–9] even in the absence of symptomatic ulceration.
Both DC-SIGN and CD1a are important in mucosal immune responses. Upon antigen encounter, immature dendritic cells mount innate immune responses and undergo a coordinated series of dynamic cellular events that lead to maturation, antigen peptide loading onto MHC class II molecules, migration to lymph nodes and antigen presentation to T cells to induce adaptive immune responses. DC-SIGN binding of various viral and microbial pathogens triggers DC activation and/or maturation, as well as signaling via Toll-like receptors [25,39]. In addition, CD1a molecules can bind and efficiently present antigen to T cells in a maturation-independent manner . Intravaginal inoculation of mice with HSV2 led to rapid recruitment of submucosal DCs to the infected epithelium, followed by local stimulation of IFNγ production from HSV-specific CD4+ T cells  and submucosal DCs were the primary cells responsible for priming and mounting protective T-cell helper 1 responses during HSV2 infection . HSV2 infection of rhesus macaques impaired iDC maturation and promoted the release of chemokines RANTES and MIP1α, offering another possible explanation for the association of increased mucosal iDCs with HSV2 infection, and for increased genital chemokine levels during HSV2 reactivation. Together with these studies, our in vivo demonstration that HSV2 infection was associated with increased cervical DC-SIGN+ CD1a+ iDCs suggests that these cells may be important in mediating local immune control of HSV2.
However, DC-SIGN has also been demonstrated to permit the propagation of productive HIV infection in CD4+ T cells at low viral titers , such as are present in the genital mucosa soon after sexual acquisition of HIV . Therefore, HIV may exploit the local increases in DC-SIGN+ iDCs mediating HSV2 immune control in the genital mucosa, to enhance host susceptibility to HIV infection after sexual exposure.
HIV infection was associated with an overall depletion of cervical iDCs, as well as depletion of iDC subpopulations expressing DC-SIGN+ and/or TLR9+, regardless of HIV disease stage. HIV nef has been shown to reduce surface expression of CD1a on iDCs through redistribution into late endosomal/lysosomal compartments . Therefore, we cannot determine whether HIV infection was associated with a true depletion of iDCs, or a downregulation of CD1a surface expression by iDCs. Regardless, the reduced numbers of cervical iDCs expressing CD1a was strongly associated with local HSV2 reactivation in HIV-infected FSWs, again implying that these iDCs mediate local HSV2 immune control, and that their depletion in the context of HIV infection may further enhance the sexual transmission of both HSV2 and HIV.
The association of HSV2 and HIV shedding levels in FSWs co-infected by both HIV and HSV2 suggests that HSV2 infection may increase HIV transmission from this core group to their male clients. During reactivation, HSV2-encoded proteins may directly increase transcription from HIV-LTR [17,19–21]. HSV2 shedding was also associated with increased chemokine levels and the amount of HSV2 viral DNA was positively correlated with CCR5+ CD4+ cells. CCR5 receptor engagement by HIV envelope glycoproteins activates signalling that culminates in the release of various chemokines that generate a chemokine gradient that recruits T cells to sites of inflammation [45–47]. The increase in CCR5+ cells and chemokines during episodes of HSV2 shedding may recruit activated CD4+ T cells to the genital tract, and therefore enhance local replication of HIV  and consequently increase HIV transmission during sexual intercourse.
Overall, we observed strong immune synergy between HSV2 and HIV specifically in the mucosal genital compartment that may have direct implications for HIV prevention strategies. Our ex vivo approach allowed direct characterization of the female genital immune milieu, and elucidation of the immediate HSV2-associated mucosal changes that may underlie increased sexual HIV transmission. The resulting static ‘snap shot’ of the FGT immune milieu does not permit study of the extremely dynamic processes of immune cell migration to and from the genital mucosa, but does allow precise definition of the cell populations present in the mucosa at the time of sexual exposure to HIV, which is likely to be a critical determinant of transmission.
In sub-Saharan Africa, over half of new HIV infections occur in women, with young women from 15–24 years of age being at particularly high risk . Given the expected lack of a protective HIV vaccine in the near future, these statistics highlight the urgent need for an accessible alternative to prevent HIV infection . Our finding of increased DC-SIGN+ iDCs and CCR5+CD4+ T cells in the cervix of HSV2-infected women implies that microbicides targeting these molecules may hold promise, and such compounds are in early clinical testing [50,51]. Since DC-SIGN+ dendritic cells in the rectal mucosa also bind HIV avidly, an effective microbicide targeting this molecule might be important for both men and women practicing anal intercourse . Our results strongly suggest that HSV2 suppression, both in HIV-infected and uninfected individuals, holds promise as an HIV prevention strategy, and supports the rationale for large-scale clinical trials that are currently testing this hypothesis .
We thank Dr. Mario Ostrowski for helpful discussions and critical reading of the manuscript, Jane Kamene and the Pumwani clinic nurses for study recruitment and providing treatment, Ann Miangi, Nyakio Chinga and the laboratory staff at the University of Nairobi Microbiology Annex for specimen processing and performing diagnostic assays. Above all, we thank the women of the Pumwani ML cohort for their continued participation and support of our studies.
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