In addition to previously-described mechanisms, our results show that estrogen-induced vaginal mucus inhibits antigen penetration and hence immune reactivity against antigens delivered to the vaginal lumen by vaccination, and the same mechanism potentially contributes to the prevention of maternal responses to paternal antigens introduced during insemination.
Using this model, we can extrapolate that estradiol-induced mucus may contribute to the failed initiation of anti-paternal responses during insemination. Due to the extensive degradation of paternally-derived cells after mating, it is likely that maternal exposure to paternal antigens includes live cells, cellular debris and degradation products including peptides. Although mucus is known to block penetration of large particles, such as bacteria, into the vaginal wall, it is more surprising that, given its high water content, mucus also prevents penetration of antigens as small as peptides. Thus vaginal mucus may cause substantial variability in responses to intravaginal vaccination at different stages of the hormonal cycle, and may provides effective barrier for the spectrum of antigens likely to be introduced during insemination.
Our results showing that APC removed from the hormone-controlled environment of the vagina can stimulate mouse CD8+
(not shown) T cells ex vivo
is in contrast to the inhibition of T cell proliferation stimulated by rat vaginal cells presenting antigen ex vivo
]. In addition to the potential differences between species, this discrepancy may also be influenced by the short-term cytokine secretion for antigen presentation used in our studies, compared to the longer-term proliferation assay used in previous work. In addition, proliferation in response to protein antigens may be mainly mediated by CD4+
T cells, whereas most of our experiments focused on CD8+
Our demonstration of the role for mucus in inhibiting presentation of even a peptide antigen adds to several other mechanisms that help to reduce infections by pathogens such as HIV transferred during intercourse [23
]. Mucus forms a barrier that impedes the penetration of microorganisms, macromolecules, and toxins from entering host tissues. The major components of mucus are mucins, a family of large, heavily glycosylated proteins. The vaginal mucus barrier is maximally developed during the ovulatory and luteal phases, and is initiated by the rising estrogen levels during proliferative and follicular phases. Mucus also contains immunomodulatory properties. Mid-cycle cervical mucus adversely affected the bioactive properties of IL-4 and the immunoglobulins IgA and IgG by decreasing their recovery from aqueous media after contact [24
]. Cervical mucus contains antagonists of two key inflammatory cytokines, IL-8 and IL-1, both involved in the recruitment of phagocytic and inflammatory cell types [25
]. Thus mucus may regulate immune parameters in the female reproductive tract by both physical and biochemical mechanisms.
Additional functions of estradiol may account for the partial inhibition of antigen presentation and CD8+
T cell activation that remains even after mucinase treatment. For example, estradiol induces the proliferation of vaginal epithelial cells [20
] resulting in thickening of the epithelial layer which may reduce antigen penetration to the underlying APC. Estradiol also inhibits APC maturation [26
] and APC function [10
]; expands T-regulatory cell populations [27
]; upregulates the suppressive ligand, PD-1, on several APCs; and reduces expression of both chemokine receptors and T cell migratory activity [28
]. In view of all these mechanisms, it is not surprising that there is residual estradiol-mediated inhibition of CD8+
T cell immunization in mucinase-treated animals.
Our studies using vaginal immunization and analysis of vaginal APC are relevant to models of IVAG vaccination, and may also provide information on immune responses against vaginal pathogens at different stages of the hormonal cycle. However, these results are only partially relevant to the regulation of maternal immune responses against paternal antigens delivered during insemination, because in mice the majority of the ejaculate is delivered to the uterine lumen, although a percentage does contact the vaginal wall via the coital plug [29
]. Also, semen contains several potent immunomodulators that further modify the maternal response.
Mice normally mate only during estrus, so exposure to paternal antigens normally occurs only during estrogen-induced inhibition of immune priming. Unlike estrus cycling mammals (e.g., rodents), humans can be sexually active throughout the menstrual cycle, although there is a strong preference for mating when fertile [30
]. As human semen deposition is therefore not as strongly restricted to the high-estrogen phase of the cycle, additional mechanisms probably prevent anti-paternal responses. Both human and mouse semen contain large quantities of the immunosuppressive cytokine TGFβ, and human semen also contains PGE2
which also suppresses T cell responses [31
]. If anti-paternal T cell responses are generated, several mechanisms may prevent anti-fetal attack later during pregnancy [32
], including restricted antigen presentation [35
]. Although unrestricted mating in humans may reduce the importance of antigen blockade by estradiol-induced mucus as a mechanism for lack of responsiveness to paternal antigens, inhibition of antigen penetration by mucus may still contribute to the reduced efficacy of human vaccination during the proliferative phase [5
Although the levels of estradiol used in our studies result in higher circulating hormone levels than occur in natural estrous cycles, higher levels of systemically administered exogenous estradiol may be required to achieve the levels of hormone in the FRT that are provided by local production. The exogenous levels in our study are similar to those used in other studies of the effect of reproductive hormones on the immune system [16
]. Furthermore, the experiments in also show a reduction in vaginal wall APC numbers in mice selected for being in the estrous phase after hormone induction, thus reinforcing our hypothesis that estradiol can reduce antigen presentation during normal estrous cycles.
We propose that access to antigen deposited in the vaginal tract is partially restricted by an estradiol-induced mucus barrier that lines the vaginal lumen during the estrous phase in mice. This physical barrier may contribute to immunological ignorance to paternal antigens, complementing the immunoregulatory components in the seminal fluid [1
] and the female reproductive tract [36
] that further enhance maternal tolerance or ignorance to paternal antigens during insemination [5
]. These data may have implications for the design of intravaginal immunization strategies attempting to induce efficient cell mediated and humoral immunity in the reproductive tract, such as vaccines for sexually transmitted pathogens.