In order to examine the effect of reproductive hormones on susceptibility and immune responses to genital HSV-2 infection, we used a mouse model where the endogenous source of hormones was removed. We then reconstituted the mice with physiological doses of either E2 or P4 or a combination of both. These mice were infected IVAG with HSV-2 to examine how the hormonal environment altered susceptibility. We found that in the presence of E2, mice were not susceptible to genital HSV-2 infection, as shown by the absence of pathology and viral shedding in their vaginal secretions. In the absence of any hormones, mice were highly susceptible to vaginal infection with HSV-2. P4 treatment in these mice did not appear to alter their susceptibility significantly at a high inoculation dose (105 PFU). At lower inoculation doses of HSV-2, the infection in P4-treated mice progressed faster than in the non-hormone-treated group, indicating that P4 was possibly exacerbating the infection. At the lowest inoculation dose (102 PFU), 100% of P4-treated mice died, while some survival was seen in the control saline group. The P4-treated mice had extensive inflammatory response, characterized by a persistent and heavy leukocytic infiltration into the vaginal tissue and lumen. This persistent inflammatory response was absent in animals infected under other hormone conditions and in non-hormone-treated controls. The presence of inflammation coincided with the induction of a number of chemokines and receptors in the vaginal tracts of P4-treated, infected mice. The E+P group had intermediate susceptibility at the high inoculation doses, characterized by focal infection that spread slowly. At lower inoculation doses, increased protection was seen in this group, indicating the dominating protective effect of estradiol.
A salient result from the present study is that E2
, in the absence of any other hormonal influence, made mice resistant to vaginal infection with HSV-2. Previous studies of intact mice show that medroxyprogesterone acetate treatment increased susceptibility to genital HSV-2 infection, while mice were refractory to infection following Depo-estradiol (2 μg/mous; The Upjohn Co., Kalamazoo, Mich.) treatment (18
). However, in these studies the exogenous hormones were injected in non-OVX mice, superimposing their effect on the circulating hormone levels and making it difficult to determine the effect of individual hormones. Both estradiol and progesterone regulate the other's receptors and antagonize the biological effects of each other (7
). It is therefore critical to examine the outcome of each hormone directly before combining or superimposing their effects. In the present study, we examined the effect of estradiol and progesterone on their own and in combination by using physiological doses of the hormones. The results showed that when it was administered alone, estradiol made the mice resistant to genital infection with HSV-2. With the combination of estradiol and progesterone used in this study, the estradiol effect was dominant on susceptibility when virus was present in low numbers. However, in the presence of large amounts of virus, this protective effect was overcome.
The mechanism by which E2
made mice nonsusceptible is not clear. One well-accepted mechanism is that during estrus and under the influence of estradiol, the vaginal epithelium is several layers thick and keratinized in superficial layers, making it impermeable to viral entry (18
). While this is a plausible explanation that may be true when mice are solely under the influence of estradiol, there may be additional factors that affect susceptibility. So far, the presence of HSV-2 has not been examined in the absence of “productive” infection. It is possible that the virus does enter the vaginal epithelium in estradiol-treated mice. In this case, estradiol may influence factors present in the epithelium or the surrounding tissue to exert antiviral effects that could limit or even terminate infection in the epithelium. The second possibility is that the entry of HSV-2 into the genital epithelium could be modified by the expression of viral receptors that may be hormonally regulated. Support for this possibility comes from a recent study, where nectin-1-δ, one of the HSV-2 receptors, was not expressed in mouse vaginae at estrus, when estradiol levels are high and mice are known to be resistant to genital HSV-2 (13
). These other possibilities need to be explored to fully understand the mechanism by which E2
In this study, progesterone by itself did not appear to have a significant role in modulating susceptibility. Only at lower inoculation doses was there an indication that P4
-treated mice may be more susceptible than non-hormone-treated controls. However, P4
-treated mice did have significantly increased inflammation following infection at all challenge doses. Previously, we have seen similar proinflammatory effects of progesterone in genital infection with Chlamydia trachomatis
in a rat model (8
). Progesterone therefore appears to have a role in inducing inflammation and possibly immune responses following infection in the genital tract. Neutrophils were the predominant cell type constituting the inflammatory infiltrate following infection in P4
-treated mice. Neutrophils are known to play an important role in inflammatory and innate immune responses (17
). They have been shown to be involved in protection against HSV challenge in the vaginal mucosa (16
). In the present study, however, the presence of neutrophils did not correlate with protection. This result indicates that they may be playing a different role. Neutrophils have been shown to be a cellular source of chemokine production that can orchestrate sequential recruitment of other immune cells (19
). There is also evidence that in respiratory infections, they augment epithelial damage induced by viral infection and contribute to pathophysiology (24
). Indeed, the main consequence of progesterone treatment in our studies appeared to be persistent inflammation, signifying that neutrophils may be contributing to epithelial damage. However, given the wide array of chemokines seen postinfection in P4
-treated mice, it is likely that they also participate in coincident induction of innate and adaptive immune responses. In fact, this is supported by our more recent experiments, where we immunized mice under P4
influence with an attenuated HSV-2 strain and saw excellent protection against wild-type challenge (6a
). Interestingly, although these mice were protected, they did exhibit symptoms of chronic pathology.
The present model should provide valuable insights into the mechanism of hormone regulation of immune responses to sexually transmitted viral infections in general and HSV-2 in particular. Clinical studies have clearly documented the effect of hormones on susceptibility to viral sexually transmitted infections, including HSV-2 and HIV, in women. Experimental work in monkey models provided clear evidence that hormones influence infection by SIV. Progesterone implants enhanced SIV vaginal transmission and viral loads, while estradiol provided protection from infection (15
). More recent studies have shown that HIV-infected women showed enhanced viral shedding in their cervicovaginal secretions following hormonal contraceptive treatments (12
). These studies emphasize the importance of understanding the role of hormones in susceptibility to sexually transmitted viral agents. This knowledge is critical for developing better prophylactic and therapeutic strategies against these infections in women.