Sexually transmitted microbial diseases or bacterial vaginosis expose genital tract cells to TLR ligands. In this study we performed experiments to determine if exposure to defined TLR ligands affects HCMV infection and found that TLR ligands inhibit HCMV infection of both HFF and ectocervical explant tissue through induction of IFNβ. While no previous studies directly investigated the effect of TLR ligand stimulation of cells in vitro on HCMV infection, Sainz et al [25
] showed that the pretreatment of HFF with either IFN-α, IFNβ, or IFN-γ inhibited HCMV infection. Several previous studies showed induction of IFNβ in HFF, HEK fibroblasts, and human lung fibroblasts in response to stimulation with Poly I:C [26
While the effect of genital microbial infections on initial HCMV infection of women has not been reported, Ross et al. [4
] recently reported that HCMV shedding was found at a higher rate in women with BV than in women with normal flora. Infection with T. vaginalis
, gonorrhea, and BV were independently associated with intrauterine transmission of HCMV [7
]. Thus, these clinical studies show that under some in vivo conditions, HCMV infection can be enhanced by infections with other infectious agents. This suggests that TLR ligands may enhance HCMV infection in vivo since GC, T. vaginalis
and BV all have TLR ligands (TLR2, TLR4 and TLR2 respectively) associated with their infections [8
]. The clinical studies contrast with the findings of our in vitro and ex vivo studies where inhibition by defined TLR ligands was observed. A possible explanation for the differences could be that many of the clinical infections are chronic infections that in vitro 24 and 48 hour treatments with TLR ligands fail to accurately model. Also, in vivo adaptive immune responses or other stimuli may be present that affect HCMV that are lacking in vitro. Further studies are needed to understand these apparent differences.
A recent study showed that during infection with murine CMV, virus replicates to higher levels in mice lacking TLR2 [29
]. Depletion of Natural Killer (NK) cells eliminated the difference between TLR2-positive and TLR2-negative mice suggesting NK cells were involved in virus suppression in TLR2-positive mice. Also, type 1 interferon was lower in the TLR2 negative mice suggesting a role in virus suppression. The CMV inhibition in mice is different than the in vitro HCMV inhibition described in our study since in the mice no exogenous TLR ligands were given before infection. Intact HCMV virions have been reported to activate TLR2, possibly via glycoproteins B and H [30
], although murine CMV is not known to have this activity. Iverson et al. [32
] showed that human NK cells can suppress HCMV through secretion of IFNβ, and NK cells can be stimulated through certain TLR including TLR2 [33
]. In our in vitro studies, no NK cells were present in HFF cultures showing that TLR3- and TLR4-ligands had a direct effect on the HCMV infection targets. However, in ectocervical tissue, it is possible that targets of HCMV infection as well as non-targets, such as immune cells, could have produced interferons. In mice, murine HCMV replicates to higher levels in mice deficient in TLR9 or MyD88 [34
]. This higher replication is again associated with lower levels of type 1 IFN and decreased NK cell activity. However, mouse embryonic fibroblasts, dendritic cells and macrophges, and human fibroblasts have all been shown to secrete IFNβ in response to stimulation with LPS [22
]. Thus, it is likely that multiple cell types in ectocervical tissues secrete IFNβ and contribute to the anti-HCMV effect.
Another interesting observation made in the current study was that the response pattern to the TLR ligands was different between HFF and ectocervical explants. Anti-HCMV responses in HFF were only found with TLR4 and TLR3 ligands while significant HCMV inhibition was induced by ligands to TLR2, TLR3, TLR4 and TLR9 in ectocervical explants. In our studies, IL-8 was measured to determine the responsiveness of HFF and explants to the TLR ligands. The IL-8 response pattern to the TLR ligands was also different between HFF and ectocervical explants with only TLR3 and TLR4 ligands inducing IL-8 in HFF but TLR3, TLR4 and TLR9 ligands inducing IL-8 in the tissue. Analysis of mRNA indicated that the ectocervical tissue expressed all four of the TLR while HFF only expressed TLR3 and TLR4. Many cell types express restricted repertoires of TLR receptors. For example, many epithelial cells have been observed to lack expression of TLR4 but to respond to TLR2 ligands [39
]. This highlights the importance of using models to study HCMV infection that most closely mirror the types of cells that are present in vivo. Cultures of ectocervical tissue have been used to study factors that affect HIV-infection [20
] and to assess the interactions of HIV with HCMV [18
], but this is the first study to investigate how TLR ligands affect HCMV infection in this tissue.
The inability of a TLR9 ligand to inhibit HCMV in HFF may be due to a lack of expression of TLR9 in these cells. TLR2 is not generally recognized to activate signaling pathways that lead to IFN production and may explain the lack of anti-HCMV effect in HFF due to this TLR ligand [40
]. However, TLR2 induced an anti-HCMV effect in ectocervical tissue and this appeared to be dependent on IFNβ. The mechanism for induction of IFNβ by TLR2 in tissues is not known although as mentioned above, some cells may produce IFN in response to TLR2 ligands. Also, stimulation through TLR2 can upregulate a number of molecules involved in anti-viral responses such as TRIF [41
] possibly leading to enhanced IFN production by cells due to other stimuli.
In conclusion this study shows that defined TLR ligands inhibit HCMV replication via IFNβ which suggests that different types of flora in the female genital tract can influence HCMV infection. This further suggests that reactivation and shedding of HCMV in the genital tract may be determined by alterations in the normal flora, which results from underlying conditions such as bacterial vaginosis or sexually transmitted diseases.