In this study we show for the first time that pDC infiltrate the dermis of recurrent genital herpes lesions. Data presented here indicate a role for these cells in the control of HSV spread in such lesions. The pDC were identified unequivocally by dual staining with BDCA-2 and CD123. Their distribution was interesting; although rare in the epidermis, they were found in the dermis and tended to accumulate selectively at the dermo-epidermal junction, where contact with HSV from infected keratinocytes is likely. The presence of pDC at the dermo-epidermal junction may contribute to the inability of HSV to spread into the dermis, by producing IFN-α and protecting dermal fibroblasts from infection with this virus. The production of MxA in cells surrounding pDC in the dermis supports such a role. Perhaps the observed presence of NK cells interacting with pDC contributes by augmenting IFN-α production and cytotoxic elimination of any cells which become infected with HSV. In vivo localization of pDC in herpes lesions provides an explanation for the recent findings in mouse models demonstrating that knockout of TLR9 (and of pDC) leads to enhanced spread of genital infection in mouse models (32
). Our results are also supported by evidence of migration of pDC to the skin during allergy (21
) and more recently by a case report of pDC in lesions of a varicella patient (17
). It is not clear from our data whether the pDC that infiltrate the lesion will migrate to the draining lymph nodes once they have taken up antigen in the skin. Plasmacytoid DC are potent inducers of cutaneous lymphocyte-associated antigen on HSV-reactive CD4 lymphocytes (25
), and their presence in HSV lesions may enhance CD4 T-lymphocyte trafficking to the skin or enable them to remain in the lesion longer. In an elegant study by Zhu et al., HSV-2-specific CD8 T lymphocytes were found to persist at the dermo-epidermal junction even after the lesion had healed, suggesting CD8 T lymphocytes may play a role in local containment of viral replication after subsequent reactivation and shedding from intracutaneous nerve endings (54
). Our data indicate that pDC preferentially induce CD8 T-lymphocyte proliferation and as such pDC may contribute to this local containment. As pDC were found in lesions up to 10 days after onset, we propose that pDC might play an additional role in the local containment of viral replication. Further evidence of a role of pDC in the control of HSV infection comes from a study that showed pDC-depleted mice were less able to control intravaginal HSV infection than wild-type mice (32
). Delivery of CpGs to murine vaginal mucosa results in recruitment of inflammatory cells to the mucosa and protection from subsequent challenge with HSV (5
). In this model, the antiviral effects of CpG were mediated through TLR9. Another study found low levels of pDC in noninflamed vaginal tissue, with elevated numbers following intravaginal treatment with CpG ODN (44
Having found pDC in herpes simplex biopsy lesions, we proceeded to investigate the role that these cells may play during a recurrence. Our laboratory has previously shown that MDDC are infected by HSV and undergo apoptosis and that these apoptotic cells were then taken up by bystander MDDC which presented HSV antigen to CD8 T lymphocytes (7
). In light of this, we investigated whether pDC were infected with HSV in vitro, and we found no viral transcripts or protein expression 16 to 24 h after inoculation. There have been relatively few studies of HSV infection of pDC, with one study mentioning a failure to detect GFP-HSV-1 after 24 h of culture (34
), but to our knowledge no data have been shown. The focus has been more on blood-borne viruses, especially HIV-1. Plasmacytoid DC have been shown to be infected with HIV-1 both in vitro and in vivo (14
), but there is a high viral load in the blood and lymph nodes that provides the opportunity for circulating pDC to encounter and become infected with virus.
Plasmacytoid DC express receptors used by HSV-1 and -2 to infect cells via gD but are not productively infected and do not support viral gene replication, at least as demonstrated by production of early and late viral protein transcripts. Paradoxically, however, these pDC are able to respond to inactivated or live virus through the abundant production of IFN-α (28
) through TLR9, most of which is found in endosomes (27
). TLR9 signaling can be triggered by unmethylated CpG motifs, commonly found in viral and bacterial but not in mammalian DNA. HSV DNA has been shown to contain many CpG motifs (33
). In unstimulated pDC, the TLR9 was shown to be colocalized with the endoplasmic reticulum marker calnexin (27
). However, pDC rapidly internalized purified CpGs into a subcellular compartment and TLR9 was transported from the endoplasmic reticulum to these compartments (27
). Furthermore, TLR9-CpG DNA interactions have been shown to occur in compartments in which the pH is 6.5 to 5, consistent with lysosomes/late endosomes (41
). The intracellular location of TLR9 suggests that HSV-2 does bind and enter the endosomes of pDC but that there is a block of productive viral replication at a stage after binding to the gD receptors. Future studies will investigate the mechanisms of binding and uptake of HSV into endosomes and a possible role for the C-type lectin receptor BDCA-2 in HSV-2 binding, like DC-SIGN concentrating virus on MDDCs. Presumably, the HSV is rapidly destroyed within endosomes producing insufficient viral proteins for detection by our intracellular staining assays. The stage and mechanisms of the block in virus entry and replication will need to be further defined but they do not appear to be IFN-α dependent, as shown by the failure of neutralization of the majority of IFN-α to enhance HSV infection of pDC.
As pDC are mature after exposure to HSV, in terms of upregulating costimulatory molecule expression and cytokine secretion, they may be able to stimulate T-lymphocyte proliferation when skin DC (Langerhans cells and/or dermal dendritic cells) are functionally impaired due to viral infection. Therefore, pDC may provide an auxiliary antigen presentation capacity. In mice, activation of pDC following exposure to HSV is delayed until pDC enter the lymph nodes (53
), but due to constraints of obtaining lymph nodes from humans, there are currently no human data to support this. By visualizing pDC in lesions in close proximity to CD69+
T lymphocytes in recurrent herpes lesions we conclude that in humans, antigen presentation by pDC to T lymphocytes could also be occurring locally in vivo where antigen taken up at the dermo-epidermal junction is presented by pDC to locally infiltrating T lymphocytes, initially CD4 lymphocytes and then CD8 lymphocytes. Infected Langerhans cells are also probably migrating to the draining lymph nodes where HSV antigen is passed on to resident DC in lymph nodes (equivalent to murine CD8α+
DC) or subsequent stimulation of CD8 lymphocytes as described recently in mouse models (3
The ability of pDC to induce CD8 T-lymphocyte proliferation in the absence of any detectable infection indicates a capacity of these cells to cross-present antigens. This function of pDC is somewhat controversial with mouse models suggesting an inability of pDC to cross-present antigen (reviewed in reference 50
). However, our data and another study (20
) in humans clearly indicate that pDC are able to cross-present antigens and stimulate CD8 T-lymphocyte proliferation.
The presence of pDC in recurrent herpetic lesions should lead to further definition of their function and importance in the immune response, particularly their role in the early phases of lesions and whether they play a different role in initial as well as recurrent herpes infection.