Backgrounds
Macrophages and DCs are bone marrow-derived cells that are involved in antigen capture, processing, and presentation and thus play a key role in triggering the immune system against infectious agents [
1‐
6]. Although both macrophages [
7] and DCs [
8] cross-present antigens, only DCs are capable of stimulating naive CD8
+ T cells [
9,
10]. DCs also play an important role in initiation of NK anti-viral immunity [
11,
12]. Similar to DCs, macrophages also play a variety of roles in immune system-mediated defense, including a central role in innate or natural immunity. Macrophages exhibit a wide variety of functions, including phagocytosis, tumor cytotoxicity, cytokine secretion and antigen presentation [
13‐
15]. A number of factors are known that "activate" or engage macrophages in these activities, including viral infection.
Herpes simplex virus (HSV) infections are among the most frequent serious viral infections in the U.S. and are considered to be a major health issue in developed countries [
16‐
19]. Both macrophages and DCs perform crucial roles in linking innate and adaptive immunity and augmenting the immune response to HSV-1 infection. It was previously shown that human blood monocytes are resistant to HSV-1 infection [
20‐
22], although a more recent study reported that immature monocyte-derived human DCs could be moderately infected with HSV-1, resulting in productive infection [
23]. Bone marrow-derived macrophages are also resistant to HSV-1 infection [
24‐
28].
The factors involved in the resistance of DCs and macrophages to productive HSV-1 infection are not known. The aim of our study was to determine if STAT1 might play a role in DC and macrophage resistance to HSV-1 replication. We found that DCs and macrophages isolated from STAT1-/- mice lost their resistance to HSV-1 infection. Thus, STAT1 seems to be critically important for allowing DCs and macrophages to resist HSV-1 replication.
Discussion
It was previously reported that freshly isolated peripheral blood monocytes and lymphocytes are resistant to HSV infection [
20‐
22]. Similarly, infection of resident peritoneal macrophages with HSV-1 results in an abortive infection in which the viral DNA is not replicated and no infectious virus is produced [
24‐
28]. Another study showed that while both mature and immature monocyte-derived DCs are infected by HSV-1, only immature DCs produce infectious virus, but at ten-fold lower levels than most cell lines, despite the fact that DCs express HSV receptors [
36]. However, the mechanism of DC and macrophage resistance to HSV-1 replication is not known. Mature DCs are also resistant to productive infection with influenza virus [
37] and dengue virus [
38]. We show here that BM-derived DCs and macrophages isolated from wild type mice, including BALB/c or 129SVE strains and C57BL/6 strain (data not shown) do not support replication of HSV-1 as judged by virus yield, viral mRNA transcription, confocal microscopy of a GFP-viral fusion protein, and viral genomic DNA levels. However, in DCs from STAT1
-/- mice, HSV-1 replication approached that seen in RS cells. We report here that HSV-1 attaches as efficiently to DCs as it does to the highly permissive RS cells, suggesting that the DCs non-permissiveness for HSV-1 is not due to a defect in viral attachment. Thus, non-permissiveness of DCs to HSV-1 appears due to either inefficient virus penetration or a block in the virus' replication cycle. Since it seems more likely that STAT1 would affect virus replication, we lean towards this explanation. However, it should be noted that the experiments reported here do not definitively distinguish between a block in virus entry versus a block in virus replication. Since RS cells support very efficient replication of HSV-1, these results suggest that STAT1 plays a key role in the resistance of DCs and macrophages to HSV-1 replication. We would predict that STAT1 may also be involved in resistance of DCs to influenza virus and dengue virus, since
Chlamydia trachomatis also propagates more efficiently in STAT1-null or STAT1 knockdown cells [
39].
STAT1-deficient mice are highly sensitive to infection by microbial pathogens and viruses [
40‐
44] including HSV-1, which replicates to approximately 1000-fold higher titers in the eyes of STAT1
-/- mice compared to wt mice [
45]. We obtained similar results when STAT1
-/- mice were ocularly infected with HSV-1 strain McKrae or KOS (data not shown). The experiments presented here constitute the first report of a virus replicating more efficiently in DCs and macrophages from STAT1 deficient mice. In addition, the increased HSV-1 replication in DCs and macrophages from STAT1
-/- mice was approximately 1000-fold higher than in wild type mice, similar to that reported in the eyes of STAT1
-/- mice [
45,
46]. This raises the possibility that the enhanced sensitivity of STAT1 deficient mice to viruses and particularly the 1000 fold increase in HSV-1 replication in STAT1 deficient mice may be due, at least in part, to increased replication of virus in DCs and macrophages. In this regard, it has been reported that HSV-infected DCs are compromised by the infection process and have reduced T-cell stimulatory capacity [
47‐
49].
The increased replication of HSV-1 in STAT1-/- DCs and macrophages shown here might be an important factor contributing to increased susceptibility of STAT1-/- mice to infection. However, since transfer of BM-derived DCs or macrophages from wild-type mice to STAT1-deficient mice did not reduce the susceptibility of STAT1-deficient mice to HSV-1 infection, even when the avirulent HSV-1 strain KOS was used for ocular challenge (data not shown), additional factors are likely involved. Surprisingly, STAT1-/- and STAT1+/+ mice had similar levels of corneal scarring following ocular HSV-1 infection (data not shown). Thus, the resistance of APCs in STAT1+/+ mice to HSV-1 replication compared to the permissiveness of APCs in STAT1-/- mice to HSV-1 replication, did not appear to play an important role in protecting mice against either death or corneal scarring.
STAT1 is one of the seven members of the mammalian STAT family. STATs participate in gene control and are activated when cells encounter various extracellular polypeptides [
50]. Targeted disruption of the STAT1 gene in mice revealed a role for STAT1 in the JAK-STAT signaling pathway [
40]. The JAK-STAT signaling pathway is involved in mediating biologic responses induced by many cytokines [
51]. STAT1-deficient mice lack responsiveness to IFN-α and IFN-γ [
40‐
44]. Thus, it is possible that the absence of responsiveness to IFN-α or IFN-γ in DCs and macrophages isolated from STAT1-deficient mice contributes to their susceptibility to HSV-1 infection. However, IFN-α production does not appear to correlate with innate protection against HSV-2 [
52] and we previously showed that expression of murine IFN-γ by an HSV-1 recombinant virus does not impair virus replication [
53,
54]. Furthermore, incubation of STAT1
+/+ DCs or macrophages in the presence of anti-IFN-α mAb, anti-IFN-γ mAb, or both mAbs did not increase HSV-1 replication in infected DCs (data not shown).
Conclusion
We have shown here for the first time that STAT1 (likely via the JAK-STAT1 pathway) is involved in suppressing HSV-1 replication in murine DCs and macrophages. In addition, the lack of virus replication in wild-type APCs did not appear to be due to transcriptional blockage of HSV-1 α, β, or γ genes, although viral DNA replication did not occur.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KM was responsible for conducting experiments. DU was responsible for experimental design and conducting experiments. SW was responsible for writing the manuscript. TT was responsible for experimental design and conducting experiments. HG was responsible for experimental design and writing the manuscript