HSV-1 and HSV-2 belong to the group of alphaherpesviruses, which have a very broad tropism and replicate lytically in many cell types, most notably cells of the epithelial and fibroblast cell lineages. Importantly, alphaherpesviruses are neurotropic and establish latent infections in these cells after retrograde transport to the ganglia. Due to the neurotropism, alphaherpesviruses can also enter into the CNS, in which they can cause serious diseases. Our data suggest that the Ifnar–/– and Tlr3–/– mice developed myelitis after vaginal HSV-2 infection, and exhibited urinary retention, constipation, and fatty liver more commonly than WT mice.
An important task for the host is thus to restrict the access of HSV to the CNS and spread within the CNS. It has been reported that patients with defects in TLR3 are susceptible to HSV infections in the CNS (24
), thus suggesting a role for TLR3 in immunological control of these viruses in the peripheral tissue prior to neuroinvasion, in the peripheral nervous system, or in the CNS. Our present finding that prevention of viral replication and spread within the CNS is impaired in TLR3-deficient mice, which display unaltered viral loads in peripheral tissue and at early stages after entry into the CNS, suggests that the role of TLR3 in HSV infection is within the CNS. We found higher viral load in the cerebellum of Tlr3–/–
mice compared with that in the Ifnar–/–
mice. These data indicate that TLR3 has antiviral functions in the cerebellum independent of type I IFN. One possibility is that HSV, in addition to spreading to neighboring glial and neuronal cells in the lumbal part of the medulla spinalis, may also spread via long neurons that reach the cerebellum and that this is controlled in a TLR3-dependent but type I IFN–independent way. Moreover, we present in vivo and in vitro data suggesting that Tlr3–/–
astrocytes are more permissive to HSV infection and that this is due to impaired type I IFN production by this cell type in the absence of TLR3. Thus, we propose that astrocytes sense HSV entry into the CNS and mount a type I IFN response, which rapidly restricts the virus after neuronal transport into the CNS.
TLR3 is highly expressed in the CNS (Supplemental Figure 6), and several studies have examined the expression of PRRs in different cell types in the CNS. While microglia cells express most, if not all, TLRs, other glial cells and neurons are much more restricted in their pattern of TLRs (30
). Importantly, TLR3 is selectively expressed as the sole nucleic acid–sensing TLR on astrocytes and oligodendrocytes (Supplemental Figure 6 and refs. 30
), and its expression is further elevated by IFN treatment and virus infections (31
). Thus, the pattern of PRR expression in the CNS further suggests an important role for TLR3 in sensing viruses in the CNS. This is supported by 2 independent studies on TLR3 and West Nile virus infection (20
). While both reports identified key roles for TLR3 in innate recognition of West Nile virus infection, the 2 studies differ with respect to whether TLR3 plays a beneficial or deleterious role for the host during the infection.
In vitro studies with astrocytes have previously shown that HSV-1 and HSV-2 have the capacity to replicate in this cell type and that recombinant IFNs inhibit the viral propagation process (34
). One study has proposed a role for type III IFN in restricting HSV-1 replication in vitro in astrocytes (35
). However, in our model, IFN-λ receptor deficiency did not affect development of disease in the CNS after HSV-2 infection (3
). Collectively, our present work provides in vivo evidence of physiological importance for the previously reported HSV replication in astrocyte cultures, which could be inhibited by type I IFN (34
). In addition, our work demonstrates that the HSV-induced IFN response in astrocytes is mediated through a TLR3-dependent pathway.
Work by Bedoui and associates has recently shown that Tlr3–/–
mice have impaired development of virus-specific CD8+
T cells in a model for cutaneous HSV-1 infection (28
). In our model, we found similar levels of CD8+
T cells in the spleen of WT and Tlr3–/–
mice at day 6 after vaginal HSV-2 infection. Moreover, we did not find recruitment of CD8+
T cells to the lumbar medulla spinalis (Supplemental Figure 5, D–F). These data argue against a role for the adaptive immune system in control of HSV-2 in the CNS in this model. Presently, we do not know whether the findings obtained in the current study on HSV-2 will also apply in a model for HSV-1 encephalitis after initial infection in the periphery (e.g., the ocular infection model) (36
). It is of note, however, that the IFN response to HSV infection in astrocytes in vitro was dependent on TLR3 for both HSV-1 and HSV-2.
We did not find a global defect in activation of innate immune responses in Tlr3–/–
mice, and we present data demonstrating that most PRRs reported to detect HSV are expressed in the CNS during HSV infection. This suggests that other HSV-sensing PRRs are still operative in the CNS and responsible for the strong innate response observed p.i. in the Tlr3–/–
mice. At this stage, we cannot explain why other HSV-sensing PRRs fail to compensate for the lack of TLR3. By contrast, Ifnar–/–
mice fail to induce expression of most PRRs after HSV infection (Supplemental Figure 2B), which could contribute to more severe disease development in these mice. By using an intraperitoneal infection model for HSV-1, Kurt-Jones et al. have previously reported that Tlr2–/–
mice are strongly impaired in inducing an inflammatory response, which renders the mouse resistant to encephalitis in this model (10
). In our model, Tlr2
double-deficient mice were indistinguishable from WT mice with respect to expression of IFNs and inflammatory genes in the medulla spinalis. In an intranasal or corneal HSV-1 infection model, Myd88–/–
mice have previously been reported to develop lethal encephalitis, mainly due to uncontrolled infection. These data suggest a complex role for TLR2 and other TLRs in the immune response to HSV infections, by contributing to both antiviral defense and immunopathology (36
). We have recently reported the identification of IFI16 and its murine ortholog, Ifi204, as intracellular sensors of HSV-1 DNA (17
). It will be interesting to determine whether IFI16/Ifi204 or other intracellular PRRs play a role in immunological control of HSV in the CNS.
Defects in TLR3, TRAF3, and STAT1 function render humans highly susceptible to HSV-1 encephalitis (5
), and a TLR3-deficient patient with repeated HSV-2 Mollaret meningitis has been reported (26
). In this work, we report that TLR3 deficiency in mice, like that in humans, leads to impaired restriction of HSV infection in the CNS. We propose that TLR3 acts in astrocytes to sense HSV-2 infection in the CNS, leading to local autocrine and paracrine IFN-β production, which restricts the virus in the local environment in the lumbar medulla spinalis immediately after entry into the CNS.