After HSV-1 infection, macrophages infiltrate early into the cornea, where they play an important role in HSV-1 infection. Macrophages are divided into M1 or M2 groups based on their activation. M1 ...macrophages are pro-inflammatory, while M2 macrophages are anti-inflammatory. Macrophage phenotypes can shift between M1 or M2 in vitro and in vivo following treatment with specific cytokines. In this study we looked at the effect of M2 macrophages on HSV-1 infectivity using mice either lacking M2 (M2-/-) or overexpressing M2 (M2-OE) macrophages. While presence or absence of M2 macrophages had no effect on eye disease, we found that over expression of M2 macrophages was associated with increased phagocytosis, increased primary virus replication, increased latency, and increased expression of pro- and anti-inflammatory cytokines. In contrast, in mice lacking M2 macrophages following infection phagocytosis, replication, latency, and cytokine expression were similar to wild type mice. Our results suggest that enhanced M2 responses lead to higher phagocytosis, which affected both primary and latent infection but not reactivation.
Herpes simplex virus-1 (HSV-1) infections are among the most frequent serious viral eye infections in the U.S. and are a major cause of viral-induced blindness. HSV-1 infection is known to induce T ...cell activation, proliferation, and differentiation that play crucial roles in the development of virus-induced inflammatory lesions, leading to eye disease and causing chronic corneal damage. CD80 is a co-stimulatory molecule and plays a leading role in T cell differentiation. Previous efforts to limit lesion severity by controlling inflammation at the cellular level led us to ask whether mice knocked out for CD80 would show attenuated virus replication following reactivation. By evaluating the effects of CD80 activity on primary and latent infection, we found that in the absence of CD80, virus replication in the eyes and virus reactivation in latent trigeminal ganglia were both significantly reduced. However, latency in latently infected CD80
mice did not differ significantly from that in wild-type (WT) control mice. Reduced virus replication in the eyes of CD80
mice correlated with significantly expanded CD11c gene expression as compared to WT mice. Taken together, our results indicate that suppression of CD80 could offer significant beneficial therapeutic effects in the treatment of Herpes Stromal Keratitis (HSK).IMPORTANCEOf the many problems associated with recurrent ocular infection, reducing virus reactivation should be a major goal of controlling ocular herpes simplex virus-1 (HSV-1) infection. In this study, we have shown that the absence of CD80 reduces HSV-1 reactivation, which marks the establishment of a previously undescribed mechanism underlying viral immune evasion that could be exploited to better manage HSV infection.
Herpes simplex virus 1 (HSV-1) latency-associated transcript (LAT) plays a significant role in efficient establishment of latency and reactivation. LAT has antiapoptotic activity and downregulates ...expression of components of the type I interferon pathway. LAT also specifically activates expression of the herpesvirus entry mediator (HVEM), one of seven known receptors used by HSV-1 for cell entry that is crucial for latency and reactivation. However, the mechanism by which LAT regulates HVEM expression is not known. LAT has two small noncoding RNAs (sncRNAs) that are not microRNAs (miRNAs), within its 1.5-kb stable transcript, which also have antiapoptotic activity. These sncRNAs may encode short peptides, but experimental evidence is lacking. Here, we demonstrate that these two sncRNAs control HVEM expression by activating its promoter. Both sncRNAs are required for wild-type (WT) levels of activation of HVEM, and sncRNA1 is more important in HVEM activation than sncRNA2. Disruption of a putative start codon in sncRNA1 and sncRNA2 sequences reduced HVEM promoter activity, suggesting that sncRNAs encode a protein. However, we did not detect peptide binding using two chromatin immunoprecipitation (ChIP) approaches, and a web-based algorithm predicts low probability that the putative peptides bind to DNA. In addition, computational modeling predicts that sncRNA molecules bind with high affinity to the HVEM promoter, and deletion of these binding sites to sncRNA1, sncRNA2, or both reduced HVEM promoter activity. Together, our data suggest that sncRNAs exert their function as RNA molecules, not as proteins, and we provide a model for the predicted binding affinities and binding sites of sncRNA1 and sncRNA2 in the HVEM promoter.
HSV-1 causes recurrent ocular infections, which is the leading cause of corneal scarring and blindness. Corneal scarring is caused by the host immune response to repeated reactivation events. LAT functions by regulating latency and reactivation, in part by inhibiting apoptosis and activating HVEM expression. However, the mechanism used by LAT to control HVEM expression is unclear. Here, we demonstrate that two sncRNAs within the 1.5-kb LAT transcript activate HVEM expression by binding to two regions of its promoter. Interfering with these interactions may reduce latency and thereby eye disease associated with reactivation.
Macrophages are one of the first innate immune infiltrates in the cornea of mice following ocular infection with herpes simplex virus 1 (HSV-1). Using gamma interferon (IFN-γ) and interleukin-4 ...(IL-4) injections to polarize macrophages into M1 and M2, respectively, and in M1 and M2 conditional knockout mice, we have shown that M1 macrophages play an important role in suppressing both virus replication in the eye and eye disease in HSV-1-infected mice. Autophagy is also important in controlling HSV infection and integrity of infected cells. To determine if blocking autophagy in M1 and M2 macrophages affects HSV-1 infectivity and eye disease, we generated two transgenic mouse strains expressing the HSV-1 γ34.5 autophagy gene under the M1 promoter (M1-γ34.5) or the M2 promoter (M2-γ34.5). We found that blocking autophagy in M1 macrophages increased both virus replication in the eyes and eye disease in comparison to blocking autophagy in M2 macrophages or wild-type (WT) control mice, but blocked autophagy did not affect latency-reactivation. However, blocking autophagy affected fertility in both M1 and M2 transgenic mice. Analysis of 62 autophagy genes and 32 cytokines/chemokines from infected bone marrow-derived macrophages from M1-γ34.5, M2-γ34.5, and WT mice suggested that upregulation of autophagy-blocking genes (i.e., Hif1a, Mtmr14, mTOR, Mtmr3, Stk11, and ULK2) and the inflammatory tumor necrosis factor alpha (TNF-α) gene in M1-γ34.5 transgenic mice correlated with increased pathogenicity, while upregulation of proautophagy genes (Nrbf2 and Rb1cc1) in M2-γ34.5 macrophages correlated with reduced pathogenicity. The
and
responses of M1-γ34.5 and M2-γ34.5 transgenic mice to HSV-1 infection were independent of the presence of the γ34.5 gene in wild-type HSV-1. Our results suggest that M1 macrophages, but not M2 macrophages, play an important role in autophagy relative to primary virus replication in the eye and eye disease in infected mice.
Autophagy plays a critical role in clearing, disassembling, and recycling damaged cells, thus limiting inflammation. The HSV-1 γ34.5 gene is involved in neurovirulence and immune evasion by blocking autophagy in infected cells. We found that blocking autophagy in M1 macrophages enhances HSV-1 virus replication in the eye and eye disease in ocularly infected transgenic mice. Our results also show the suppressive effects of γ34.5 on immune responses to infection, suggesting the importance of intact autophagy in M1 but not M2 macrophages in controlling primary infection and eye disease.
Previously, we reported that herpes simplex virus type 1 (HSV-1) ICP22 binds to the CD80 promoter and suppresses its expression
and
. To better understand the impact of ICP22 binding to CD80 on HSV-1 ...infectivity and pathogenicity, we mapped the region of ICP22 required to bind the CD80 promoter to a 40-amino-acid (aa) region of ICP22. We constructed a recombinant HSV-1 expressing a truncated form of ICP22 that lacks these 40 aa, which does not bind to the CD80 promoter (KOS-ICP22Δ40) and retains the ability to replicate efficiently in rabbit skin cells, in contrast to ICP22-null virus. The replication of this recombinant virus
and
was higher than that of the ICP22-null virus, but virus replication kinetics were lower than those of the wild-type (WT) control virus. Similar to ICP22-null virus, the KOS-ICP22Δ40 mutant virus increased CD80 expression in dendritic cells (DCs) and interferon gamma (IFN-γ) expression in CD8
T cells but not CD4
T cells in infected mouse corneas. In contrast to the significantly reduced virus replication in the eyes of ocularly infected mice, the levels of latency reactivation were similar between KOS-ICP22Δ40 virus and WT virus. Thus, blocking ICP22 binding to the CD80 promoter using a recombinant virus expressing a truncated ICP22 that lacks CD80 promoter binding appears to reduce virus replication and enhance CD8
IFN-γ
infiltrates in corneas of infected mice, with no effect on latency reactivation.
Direct binding of HSV-1 ICP22 to the CD80 promoter downregulates the expression of the costimulatory molecule CD80 but not CD86. In this study, we fine mapped the region of ICP22 required for binding to the CD80 promoter and constructed a recombinant virus containing a deletion in ICP22 that failed to bind to the CD80 promoter. This recombinant virus replicated less efficiently
and
than did the WT control virus, although CD80-expressing CD11c
cells and IFN-γ-expressing CD8
T cells were increased. Interestingly, the levels of latency and reactivation in the two viruses were similar despite lower virus replication in the eyes of infected mice. Therefore, blocking the interaction of ICP22 with the CD80 promoter could be used to temper the immune response.
We previously have reported that ICP22, an immediate early gene of herpes simplex virus type 1 (HSV-1), binds to the CD80 promoter to suppress CD80 expression in antigen-presenting cells, leading to ...reduced T-cell function and protection. In contrast, overexpression of CD80 exacerbates corneal scarring (CS) in ocularly infected mice. In this study we tested the hypothesis that the absence of ICP22 could increase disease severity.
To test our hypothesis, BALB/c mice were ocularly infected after corneal scarification with a recombinant HSV-1 lacking the ICP22 gene with its parental wild-type (WT) virus (KOS) as a control. Virus replication in the eye, CS, angiogenesis, latency, and reactivation between ICP22 null virus and WT KOS were determined. In addition, expression of IL-2, IL-4, IFN-γ, IFN-α, granzyme A, granzyme B, and perforin by CD4 and CD8 T cells in corneas of infected mice on days 3, 5, 7, 10, 14, 21, and 28 postinfection were determined by flow cytometry.
We found similar levels of eye disease and angiogenesis in mice following corneal scarification and ocular infection with the ICP22 null virus or parental WT virus despite reduced virus replication in the eye and reduced latency and reactivation in mice ocularly infected with ICP22 null virus. The similar level of eye disease in ICP22 null virus- and WT virus-infected mice correlated with expression of various proinflammatory cytokines that infiltrated the eye after HSV-1 infection.
Our study identified a critical role for ICP22 in HSV-1 pathogenicity and suggests that HSV-1-associated CS is more dependent on host immune responses to infection than to virus replication in the eye. Thus, HSV-1 as means of survival uses ICP22 as a mechanism of immune escape that protects the host from increased pathology.
The HSV-1 latency-associated transcript (LAT) locus contains two small noncoding RNA (sncRNA) sequences (sncRNA1 and sncRNA2) that are not microRNAs (miRNAs). We recently reported that sncRNA1 is ...more important for
activation of the herpesvirus entry mediator than sncRNA2, but its
function is not known. To determine the role, if any, of sncRNA1 during herpes simplex virus 1 (HSV-1) infection
, we deleted the 62-bp sncRNA1 sequence in HSV-1 strain McKrae using dLAT2903 (LAT-minus) virus, creating ΔsncRNA1 recombinant virus. Deletion of the sncRNA1 in ΔsncRNA1 virus was confirmed by complete sequencing of ΔsncRNA1 virus and its parental virus (i.e., McKrae). Replication of ΔsncRNA1 virus in tissue culture or in the eyes of infected mice was similar to that of HSV-1 strain McKrae and dLAT2903 viruses. However, the absence of sncRNA1 significantly reduced the levels of ICP0, ICP4, and gB but not LAT transcripts in infected rabbit skin cells
. In contrast, the absence of sncRNA1 did reduce LAT expression in trigeminal ganglia (TG), but not in corneas, by day 5 postinfection (p.i.) in infected mice. Levels of eye disease in mice infected with ΔsncRNA1 or McKrae virus were similar, and despite reduced LAT levels in TG during acute ΔsncRNA1 infection, McKrae and ΔsncRNA1 viruses did not affect latency or reactivation on day 28 p.i. However, mice infected with ΔsncRNA1 virus were more susceptible to ocular infection than their wild-type (WT) counterparts. Expression of host immune response genes in corneas and TG of infected mice during primary infection showed reduced expression of beta interferon (IFNβ) and IFNγ and altered activation of key innate immune pathways, such as the JAK-STAT pathway in ΔsncRNA1 virus compared with parental WT virus. Our results reveal novel functions for sncRNA1 in upregulating the host immune response and suggest that sncRNA1 has a protective role during primary ocular HSV-1 infection.
HSV-1 latency-associated transcript (LAT) plays a major role in establishing latency and reactivation; however, the mechanism by which LAT controls these processes is largely unknown. In this study, we sought to establish the role of the small noncoding RNA1 (sncRNA1) encoded within LAT during HSV-1 ocular infection. Our results suggest that sncRNA1 has a protective role during acute ocular infection by modulating the innate immune response to infection.
We previously reported that herpes simplex virus 1 (HSV-1) ICP22 binds to CD80 and suppresses CD80 expression
and
. Similar to ICP22, the cellular costimulatory molecules CD28, CTLA4, and PD-L1 also ...bind to CD80. In this study, we asked whether, similar to ICP22-null virus, the absence of these costimulatory molecules will reduce HSV-1 infectivity. To test our hypothesis, CD28
, CD28
CTLA4
, PD-L1
, and wild-type control BALB/c mice were ocularly infected with HSV-1 strain KOS. Levels of virus replication in the eye, corneal scarring (CS), latency, and reactivation in infected mice were determined. Expression of different genes in the trigeminal ganglia (TG) of latently infected mice was also determined by NanoString and quantitative reverse transcription-PCR (qRT-PCR). In the absence of costimulatory molecules, latency levels were higher than those in wild-type control mice, but despite higher latency, a significant number of TG from infected knockout mice did not reactivate. Reduced reactivation correlated with downregulation of 26 similar cellular genes that are associated with inflammatory signaling and innate immune responses. These results suggest that lower reactivation directly correlates with lower expression of interferon signaling. Thus, despite having different modes of actions, we identified a similar function for CD28, CTLA4, and PD-L1 in HSV-1 reactivation that is dependent on their interactions with CD80. Therefore, blocking these interactions could be a therapeutic target for HSV-1-induced reactivation.
Costimulatory molecules play an important role in activation of T cell responses, and T cells contribute to HSV-1-induced eye disease in the host. Similar to HSV-1 ICP22, the cellular costimulatory molecules CD28, CTLA4, and PD-L1 also bind to CD80. In this study, we have shown that the absence of these costimulatory molecules significantly reduced HSV-1
reactivation. Therefore, inhibiting the binding of costimulatory molecules to CD80 could be used to reduce reactivation and, consequently, HSV-1-induced eye disease.
High rates of wild-type (WT) herpes simplex virus 1 (HSV-1) latency reactivation depend on the anti-apoptotic activities of latency-associated transcript (LAT). Replacing LAT with the baculovirus ...inhibitor of apoptosis protein (cpIAP) or cellular FLIP (FLICE-like inhibitory protein) gene restored the WT latency reactivation phenotype to that of a LAT-minus LAT(-) virus, while similar recombinant viruses expressing interleukin-4 (IL-4) or interferon gamma (IFN-γ) did not. However, HSV-1 recombinant virus expressing cpIAP did not restore all LAT functions. Recently, we reported that a similar recombinant virus expressing CD80 in place of LAT had higher latency reactivation than a LAT-null virus. The present study was designed to determine if this CD80-expressing recombinant virus can restore all LAT functions as observed with WT virus. Our results suggest that overexpression of CD80 fully rescues LAT function in latency reactivation, apoptosis, and immune exhaustion, suggesting that LAT and CD80 have multiple overlapping functions.
Recurring ocular infections caused by HSV-1 can cause corneal scarring and blindness. A major function of the HSV-1 latency-associated transcript (LAT) is to establish high levels of latency and reactivation, thus contributing to the development of eye disease. Here, we show that the host CD80 T cell costimulatory molecule functions similarly to LAT and can restore the ability of LAT to establish latency, reactivation, and immune exhaustion as well as induce the expression of caspase 3, caspase 8, caspase 9, and Bcl2. Our results suggest that, in contrast to several other previously tested genes, CD80-expressing virus can completely compensate for all known and tested LAT functions.
Previously we reported that a recombinant HSV-1 expressing murine IL-2 (HSV-IL-2) causes CNS demyelination in different strains of mice and in a T cell-dependent manner. Since T
17 cells have been ...implicated in CNS pathology, in the present study, we looked into the effects of IL-17A
and three of its receptors on HSV-IL-2-induced CNS demyelination. IL-17A
mice did not develop CNS demyelination, while IL-17RA
, IL-17RC
, IL-17RD
and IL-17RA
RC
mice developed CNS demyelination. Adoptive transfer of T cells from wild-type (WT) mice to IL-17A
mice or T cells from IL-17A
mice to Rag
mice induced CNS demyelination in infected mice. Adoptive T cell experiments suggest that both T cells and non-T cells expressing IL-17A contribute to HSV-IL-2-induced CNS demyelination with no difference in the severity of demyelination between the two groups of IL-17A producing cells. IL-6, IL-10, or TGFβ did not contribute to CNS demyelination in infected mice. Transcriptome analysis between IL-17A
brain and spinal cord of infected mice with and without T cell transfer from WT mice revealed that "neuron projection extension involved in neuron projection guidance" and "ensheathment of neurons" pathways were associated with CNS demyelination. Collectively, the results indicate the importance of IL-17A in CNS demyelination and the possible involvement of more than three of IL-17 receptors in CNS demyelination.
PDF
