To our knowledge, this is the first in vivo
study showing an increase in TLR expression in cervical samples with incident HPV 16 infection that is subsequently associated with viral clearance and lack of an observed increase was associated with persistence. TLRs whose change in expression was significantly different between women with HPV clearance and those with persistence on follow-up included TLR2, TLR3, TLR7, TLR8, and TLR9. Notably, with the exception of TLR2, these are exclusively localized in the endosomal membrane and are known to recognize viral nucleic acids. The difference in expression of multiple nucleic acid-sensing TLRs is particularly interesting in light of synergistic co-activation which has been described for TLR3 and TLR7 31
as well as TLR3 and TLR9.32
The difference in expression of TLR2 is interesting in that this is a cell surface-expressed receptor; TLR2 has been found to be activated by various viral protein components. 33, 34
However, human macrophage recognition of hepatitis C virus through herterodimers of TLR2 with TLR1 or TLR6 has also been recently demonstrated. 35
The true role of surface-expressed TLRs in anti-viral host responses, however, is not clear. 19
For example, activation of TLR2 by measles virus or herpes simplex virus proteins may be detrimental to the host through upregulation of viral receptors or induction of proinflammatory cytokines, respectively. 33, 36
In contrast, TLR2-mediated activation of natural killer cells has been shown to be important in early control of murine cytomegalovirus. 37
In some cases, particularly viruses with low structural complexity, the viral protein responsible for TLR engagement has been identified, such as measles virus hemagglutinin protein and TLR2 33
and respiratory syncytial virus fusion protein and TLR4. 38
In other cases, the protein ligand is unknown. The association shown here between TLR2 and clearance of HPV16 suggests that it may play a beneficial role in the host in infection with this virus. Further work, particularly in vitro
studies, will be needed to identify HPV proteins that engage TLRs.
Examination of changes within groups revealed that the difference between women exhibiting clearance on follow-up and those with persistence was primarily due to significant increases in TLR expression upon HPV 16 incidence in the former group, rather than decreases in expression in the latter group. These results suggest that increases in TLR expression are necessary for clearance. This observation is in line with studies that show that activation of TLRs is necessary for a successful innate immune response. In addition, the increase in IFN-α2 associated with the TLR increase in women who cleared HPV 16 gives further evidence that this TLR response assisted in HPV clearance. 16-18
Our findings are consistent, however, with in vitro
and animal studies that have shown that HPV 16 is able to evade immune recognition and this evasion results in its persistence, 20, 21
as indicated by the lack of TLR expression increase in the persistence group and even trends, albeit non-significant, in the downward direction. Interestingly, IFN-α2 was not significantly associated with this decrease. Likely, in the absence of TLR activation there is no correlation between TLR expression and cytokine levels. Taken together, we believe these data suggest that HPV may evade the immune system by avoiding TLR recognition. Certainly, studies have shown that HPV is associated with several mechanisms that may assist in avoiding immune recognition including diminishing Langerhans cell populations, dampening natural killer cell activity and MHC expression. 11, 39
We also investigated whether the changes in TLR expression in the women with incident HPV 16 infections occurred in parallel or opposite directions. Generally, there was good agreement in the direction of change in the women with persistence on follow-up, except between TLR2 and TLR9. In contrast, there was less agreement among women with clearance on follow-up. In this group, pairings involving the nucleic acid-sensing TLRs tended to be those showing moderate or better agreement, while TLR2 showed poor agreement in all pairings. The cases of moderate or better agreement may suggest either coordinated regulation or migration of cells bearing multiple TLRs in or out of the mucosa. That TLR2 showed relatively poor agreement with other TLRs in both groups is consistent with its presumed less central role in response to viruses.
Our findings appear to be specific for HPV 16 and were not paralleled by findings for the other high-risk type, HPV 51. Hasan et al. reported that, in vitro
, HPV 16 is much more efficient than HPV 18 in downregulating TLR9
suggesting that modulation of TLR expression may be part of a mechanism by which HPV 16 evades innate immunity, and that this is not common to all high-risk HPV types. Unfortunately, we were unable to identify enough cases of HPV 18 in our cohort to study. However, HPV 51, which was the second most prevalent HPV type in our cohort, did not show similar differences between women with clearance on followup and those without. The reason for this remains unclear and underscore the difficulties of interpreting in vivo
studies which are potentially affected by numerous environmental factors. Certainly, HPV 16 is 50 times more common in cervical cancers than HPV 51. 40
Our different findings for types 16 and 51 support the hypothesis that immune mechanisms involved in clearance, as well as in viral immune evasion, may differ by HPV types.
The approach presented here, evaluating change in TLR expression within each subject, recognizes the fact that there is no information regarding what constitutes a “normal” level of expression, and that this may be different in each woman owing to the myriad immunogenic factors that make the cervical mucosa such a complex environment. While our findings support our hypothesis that HPV 16 uniquely affects TLR expression in a way that helps predict the subsequent course of infection, we were surprised that baseline TLR expression appeared to be markedly different between the persistence and clearance groups. This observation may suggest some level of pre-existing non-specific immune activation in the former group. Because women were excluded from this study if they had, at baseline (or incident), abnormal cytology, yeast, C. trachomatis, N. gonorrhoeae
, or T. vaginalis
infection, or were pregnant, none of these covariates appears to explain the difference. However, the cervical vaginal microbiologic and immunologic milieu is extremely complicated and is affected by a host of environmental and genetic factors. 41
We speculate that there may be some genetic component that affected baseline TLR expression and resulted in increasing these women's vulnerability to HPV persistence.
For the purposes of this study, follow-up was limited to one four-month visit after the incident HPV infection and clearance or persistence defined based on that follow-up interval. As HPV, and in particular, HPV 16, may sometimes persist for extended periods with eventual clearance occurring months to years later or not at all 3
, much longer prospective follow-up will be needed to determine the role of TLR expression in regression of these more prolonged infections. Extended follow-up, while beyond the scope of this substudy, remains ongoing in our cohort.
In summary, we have shown that incident HPV 16 infection that results in clearance is associated with increased expression of several TLRs compared to women who do not clear, including the 4 TLRs known to recognize viral nucleic acids. Imiquimod, a TLR7 agonist, is approved for treatment of genital warts and has been suggested for treatment of other HPV-associated disease. 42, 43
If TLR agonists are capable of mitigating the diminished TLR expression shown here, their use in the treatment of cervical HPV persistence may be promising. Understanding the mechanisms involved might lay the foundation for new therapies for HPV-associated diseases.