These studies demonstrate that HSV-1 and HSV-2 downregulate SLPI, which may serve as a novel immune evasion strategy. Importantly, downmodulation does not require viral replication, as evidenced by the findings that downmodulation occurs within 6 h following infection and persists in the presence of acyclovir. However, downmodulation requires expression of both ICP4 and wild-type ICP0, as evidenced by results obtained with IE viral variants and UV-inactivated virus.
The observation that both of these IE proteins are required to trigger the downmodulation of SLPI is consistent with their known functional interactions. Both IE proteins play critical roles as transcriptional activators of viral gene expression, and the ability of ICP0 to transactivate promoters is increased synergistically in the presence of ICP4 (34
). Their vital role in regulating HSV infection and reactivation from latency is highlighted by a recent study, which found that the LAT gene encodes several microRNA precursors (miRNAs) and that one of these miRNAs, miR-H2-3p, is transcribed in an antisense orientation relative to ICP0 and inhibits its expression. Notably, a second miRNA (which derives from a transcript distinct from LAT) inhibits expression of ICP4 (48
ICP0 has previously been shown to play a major role in immune evasion by overcoming the antiviral IFN response. The ability of ICP0 to transactivate cellular proteins may contribute to its capacity to overcome the IFN-induced block to viral transcription (37
). ICP0 blocks IFN regulatory factor 3 (IRF3)- and IRF7-mediated activation of IFN-stimulated genes through the activities of the RING finger domain (30
). Additionally, ICP0 stimulates the degradation of a number of host proteins, in part because of its E3 ubiquitin ligase activity as well as its interactions with the cellular ubiquitin-specific protease enzyme USP7 (4
). In the current studies, we found that the primary mechanism by which HSV reduces SLPI is downregulation of mRNA, although virus-induced degradation of SLPI protein may also contribute. Further studies are required to determine whether ICP0 and ICP4 directly or indirectly trigger the downregulation and what the precise mechanism underlying this response is. Notably, the loss of SLPI downmodulation was associated with reduced ability to activate NF-κB at early times p.i. (8 h).
There are several potential consequences of downregulating SLPI. First, we previously demonstrated that recombinant SLPI interacts with human epithelial cells to inhibit HSV infection in vitro (25
). Downregulation could overcome the antiviral activity of SLPI in mucosal secretions, thus providing a mechanism for immune evasion. A clinical study is currently ongoing to examine the concentrations of SLPI in genital tract secretions in women during an acute HSV outbreak. Second, downmodulation of SLPI could promote the activation of NF-κB by releasing a negative regulator, thus further facilitating HSV infection. The ability of HSV to activate NF-κB has been well documented (2
), and interference with NF-κB activation, for example, following expression of a dominant-negative IκBα, resulted in a reduction in virus yield. This has been attributed in part to the role NF-κB may play in preventing virus-induced apoptosis (19
). Thus, it has been proposed that persistent NF-κB activation, rather than being a host response to virus infection, may play a positive role in promoting efficient virus replication.
Additionally, the HSV-induced downregulation of SLPI could also contribute to epidemiological observations of an increased risk of HIV acquisition among HSV-2-seropositive individuals (18
). SLPI inhibits HIV infection of macrophages, and higher concentrations of SLPI in mucosal fluids are associated with a reduced risk of infection (16
). Thus, the reduction in SLPI in the setting of HSV, even in the presence of acyclovir, could promote HIV acquisition. The downregulation of SLPI could also contribute to the increase in HIV viral loads in the genital tract as observed during periods of HSV reactivation in coinfected individuals (20
). We found that exposure of the chronically HIV-infected monocytic cell line U1 to wild-type HSV, but not to the ICP4 deletion virus, resulted in enhanced HIV replication, with a significant increase in p24 production (P. M. M. Mesquita and B. C. Herold, unpublished results). These results could be explained by NF-κB-mediated activation of the HIV long-terminal repeat. Whether SLPI downregulation contributes to this response remains to be determined. It is interesting to speculate whether the downmodulation of SLPI or other changes in the mucosal environment triggered by reactivating HSV, even in the absence of viral replication, played a role in the failure of oral acyclovir suppression to reduce the risk of HIV infection in the recently completed clinical trials among high-risk HSV-infected, HIV-negative individuals (6
; C. L. Celum, presented at the 15th Conference on Retroviruses and Opportunistic Infections, Boston, MA, 2008).
The observation that HSV downmodulates SLPI is unusual, as most other microbes induce SLPI and other antimicrobial peptides. VSV induced no change in SLPI, and consistent with prior studies, we found that HIV exposure resulted in a modest increase in SLPI levels. Notably, the response induced by the cervical cells was less than that previously reported for oral epithelial cells. In the previous study, the authors found that exposure of oral epithelial cells to HIV resulted in a threefold increase in SLPI production and speculated that this response may contribute to the poor oral transmission of HIV (24
). Possibly, the more modest increase in SLPI following exposure of cervical epithelial cells to HIV-1 contributes to the increased risk of genital versus oral mucosal transmission.
Increases in SLPI have also been observed in response to other pathogens. For example, Mycobacterium tuberculosis
has also been shown to increase SLPI production by macrophages. Exposure of murine peritoneal macrophages to M. tuberculosis
or aerosolized infection of mice with M. tuberculosis
triggers an increase in SLPI. Notably, macrophages from TLR2−/−
mice are incapable of inducing this response, suggesting a role for TLR2-dependent pathways in triggering the SLPI response (11
Similarly to results obtained here for HSV, several other pathogens appear to have evolved strategies for escaping the antimicrobial effects of SLPI. Helicobacter pylori
triggers a loss of SLPI in cell cultures, and antral biopsies of H. pylori
-positive subjects show reduced SLPI expression (52
). The H. pylori
-induced decrease in SLPI could not be explained by a transcriptional downmodulation and appeared to be regulated posttranslationally. Additionally, cysteine proteases of Trichomonas vaginalis
have been demonstrated to degrade SLPI, which has been suggested to contribute to the increased risk of HIV in the setting of trichomonas infection (13
In conclusion, these studies describe a novel mechanism by which HSV may interfere with innate mucosal immunity and identify yet another role for HSV in immune evasion. Defining the precise mechanisms by which ICP4 and ICP0 trigger downregulation of this mediator of mucosal host defense may promote the development of strategies for prevention of this mucosal immune response, which could foster prevention of both HSV and HIV.