HSV-2 enhances HIV-1 acquisition and transmission during symptomatic and asymptomatic stages of HSV-2 infection 
. However, due to the lack of a suitable animal model for HSV-2 infection that closely relates to humans, the underling mechanism(s) that leads to enhanced risk of HIV-1 infection remains unknown. One potential explanation holds that increased presence and persistence of HSV-2-reactive CD4+
T cells facilitate HIV-1 transmission 
Herein, we provide in-vivo
data collected in a novel non-human primate model of HSV-2 infection and we describe in-vitro
experiments that add new insights into HSV-2/HIV-1 interplay. In-vivo
we observed an increase in the percentages of α4
T cells both locally and systemically a few days after rectal HSV-2 challenge. We show that CD11c+
DCs from peripheral blood are susceptible to HSV-2 infection in-vitro
and that HSV-2 infection of immature moDCs amplifies the α4
T subset in autologous DC-T co-cultures. We show that HSV-2 infection increases ALDH1A1 expression in DCs, a phenomenon that enhances their potential to produce RA. The latter mediates the HSV-2-driven up-regulation of α4
in our DC-T co-cultures and has a plethora of immunomodulatory effects, including influencing HIV-1 replication 
Indeed, we found that blocking the RARα in T cells inhibits HIV-1 replication in HSV-2-infected DC-T cell cultures.
Localization, retention, function and survival of antigen-experienced T cells that infiltrate mucosal sites following pathogen invasion, are influenced by the expression of adhesion molecules, substantially modulated by microenvironmental factors 
. Among such adhesion molecules, the integrin receptor α4
mediates lymphocyte migration to the gastrointestinal tract. However, recent findings indicate that STIs can modulate the expression and migration of α4
lymphocytes also in other tissues, such as the endocervix of human females infected with Clamydia trachomatis 
. We developed a macaque HSV-2 rectal infection model and show that in macaques the mucosal site of HSV-2 infection, its draining LNs, and blood are enriched in α4
T cells within 6 days of HSV-2 exposure. The increased percentages of α4
T cells were not observed in animals treated with UV-HSV-2, suggesting that HSV-2 replication is important to this phenomenon.
Several factors could explain the enrichment in α4
T cells at the site of HSV-2 infection. Among them is the ability of α4
T cells to specifically target mucosal sites, a possible inflammation-driven induction of the α4
receptor MadCam 
, and specific responses of CM T cells to inflammatory soluble factors. However, DCs are present and persist at the site of HSV-2 infection 
, they are critical to the immunological response to HSV-2 
and are able, in determinate circumstances, to induce α4
on T cells. Therefore, we explored the possibility of their contribution to the increased percentage of α4
T cells in HSV-2-infected macaques.
HSV-2 is able to skew DC immunological responses 
. While moDCs and langerhans cells are highly susceptible to HSV in-vitro 
, plasmacytoid DCs, critical players in the innate response to HSV 
, seem to be resistant to infection 
myeloid DCs are important in antigen presentation and adaptive response to HSV 
. Mimicking the mixed leukocyte populations potentially encountering HSV-2 in-vivo
using blood, we confirmed that (macaque and human) myeloid CD11c+
DCs are the primary leukocyte target for HSV-2 infection in-vitro
. Due to the variety of CD11c+
DC subsets implicated at different stages of the immune response 
future studies will need to investigate the precise phenotype of the susceptible population, the differences between HSV-2-infected in-vitro
generated moDCs, and infected CD11c+
DCs in their interaction with T cells. Since the primary goal of this study was to explore whether modulation of myeloid DC function by HSV-2 infection was involved in the enrichment of α4
T cells observed in-vivo
, we were able to use the established moDC-HSV-2 model to dissect this biology.
The effect of HSV-2 infection on moDCs has been extensively studied, typically using relatively large amounts of virus 
. Our work reveals that even a much smaller viral inoculum significantly influences DC biology. We confirmed that low dose HSV-2 infection caused a down-regulation of the maturation receptors HLA-DR, CD86 and CD54, as seen with higher HSV-2 doses 
. We also demonstrated a down-regulation of CD209, which would be expected in a maturing DC. The latter can be explained by the ability of HSV to bind this receptor 
, although it could be also the result of a skewed maturation process. We demonstrated that DCs infected with a low HSV-2 inoculum down-modulated CD69 expression on T cells, supporting an earlier report that HSV-infected moDCs inhibit T cell activation. Notably, we found that HSV-2-infected DCs up-regulate the expression of α4
and, by blocking the binding of RA to its receptors on the CD4+
T cells, we showed that RA was directly involved in the HSV-2 driven increase in α4
expression. RA impacts several immunological mechanisms, in particular it is known to induce a mucosal-type phenotype in DCs 
, playing an important role in inducing and sustaining the tolerogenic microenvironment of the gut 
We provide the first evidence that human immature moDCs express ALDH1A1 (and ALDH1A2), have the potential to convert serum retinol into RA, and that HSV-2 infection significantly increases this capability. This supports earlier work in mice showing that GM-CSF and IL-4 induce ALDH1A2 expression in BM-DCs 
. The same study also reported that this gene was up-regulated by TLR ligands in DCs cultured with GM-CSF and IL-4 and matured with LPS. However, the up-regulation of ALDH1A1 expression by HSV-2 infection in human moDCs did not appear to be due a ligand effect of HSV-2 proteins or DNA (triggering through TLRs), since even 25-times more UV-HSV-2 was unable to reproduce these responses. Additional studies are needed in order to ascertain whether other TLR ligands or other pathogens can stimulate human DCs (like HSV-2 infection) to up-regulate ALDH1A1 expression and subsequently increase α4
expression on CD4+
T cells. The specific mechanism through which HSV-2 infection increases ALDH1A1 expression in moDCs was not a major focus of this work and might be a direct effect of newly synthesized HSV-2 components and/or an indirect effect of CCs/CKs secreted by DCs in response to HSV-2 replication. Given the potentially important role that RA holds in immune responses to pathogens, this subject is worthy of further research. That HSV-2 is able to mediate the up-regulation of an enzyme that serves as a key metabolic checkpoint in the conversion of retinol to RA is noteworthy, because RA has the capacity to modulate immune responses and replication of many pathogens including HIV-1 
These studies also revealed that significantly elevated amounts of other soluble factors are released by HSV-2-infected moDCs. In particular, we detected a notable increase in IL-7, which is known to induce HIV-1 reactivation and replication in T cells 
and, as previously reported 
, of CXCL10 which is responsible of recruiting activated T cells, therefore contributing to viral replication in inflamed tissues 
. All these factors could cooperate in enhancing HIV-1 infection. However, an RAR antagonist ablated the HSV-2-mediated enhancement of HIV-1 amplification, suggesting that RA is one of the major factors driving this biology.
HIV-1 infection of moDCs could also be affected by HSV-2 infection. Though, the apoptotic nature of the HSV-2 infection, suggests little contribution of HIV-1 replication in moDCs to the enhanced HIV-1 replication in the co-cultures.
We previously reported that α4
T cells are the most susceptible HIV-1 target in T cells cultures supplemented with RA and that blocking α4
binding to HIV-1 inhibits HIV-1 replication 
. Herein, we show that the α4
T cells also constitute the most susceptible HIV-1 target in the DC-T cell co-cultures and that this is independent of the effect of HSV-2 on the DCs. Therefore, being particularly susceptible to HIV seems an intrinsic characteristic of α4
T and an expansion of this cell-subset likely has a greater impact than the expansion of less susceptible subsets, contributing to fuel infection.
This work gives us new insights into HSV-2 modulation of the mucosal microenvironment. A low- level HSV-2 infection of immature myeloid DCs could play a role in increasing the susceptibility to HIV-1 by influencing its surroundings in a way favorable to HIV-1 infection. In we try to integrate our findings in a bigger picture with the new different actors that HSV-2 infected DCs add to the scene. Further studies will have to dissect how these mechanisms interplay in-vivo, the respective role of factors such as RA and α4β7 and their relative importance in transmission across the rectal and genital mucosa.
Potential mechanism of HSV-2-infected DCs enhancement of HIV-1 infection.