Binding of herpesvirus entry mediator (HVEM) and HSV-1 gD affect reactivation but not latency levels.

Previously we reported that the HSV-1 latency associated transcript (LAT) specifically upregulates the cellular herpesvirus entry mediator (HVEM) but no other known HSV-1 receptors. HSV-1 glycoprotein D (gD) binds to HVEM but the effect of this interaction on latency-reactivation is not known. We found that the levels of latent viral genomes were not affected by the absence of gD binding to HVEM. However, reactivation of latent virus in trigeminal ganglia explant cultures was blocked in the absence of gD binding to HVEM. Neither differential HSV-1 replication and spread in the eye nor levels of latency influenced reactivation. Despite similar levels of latency, reactivation in the absence of gD binding to HVEM correlated with reduced T cell exhaustion. Our results indicate that HVEM-gD signaling plays a significant role in HSV-1 reactivation but not in ocular virus replication or levels of latency. The results presented here identify gD binding to HVEM as an important target that influences reactivation and survival of ganglion resident T cells but not levels of latency. This concept may also apply to other herpesviruses that engages HVEM.

Blocking Autophagy in M1 Macrophages Enhances Virus Replication and Eye Disease in Ocularly Infected Transgenic Mice.

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 in vivo and in vitro 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. IMPORTANCE 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.

Herpes Simplex Virus 1 Small Noncoding RNAs 1 and 2 Activate the Herpesvirus Entry Mediator Promoter.

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. IMPORTANCE 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.

Essential role of M1 macrophages in blocking cytokine storm and pathology associated with murine HSV-1 infection.

Ocular HSV-1 infection is a major cause of eye disease and innate and adaptive immunity both play a role in protection and pathology associated with ocular infection. Previously we have shown that M1-type macrophages are the major and earliest infiltrates into the cornea of infected mice. We also showed that HSV-1 infectivity in the presence and absence of M2-macrophages was similar to wild-type (WT) control mice. However, it is not clear whether the absence of M1 macrophages plays a role in protection and disease in HSV-1 infected mice. To explore the role of M1 macrophages in HSV-1 infection, we used mice lacking M1 activation (M1-/- mice). Our results showed that macrophages from M1-/- mice were more susceptible to HSV-1 infection in vitro than were macrophages from WT mice. M1-/- mice were highly susceptible to ocular infection with virulent HSV-1 strain McKrae, while WT mice were refractory to infection. In addition, M1-/- mice had higher virus titers in the eyes than did WT mice. Adoptive transfer of M1 macrophages from WT mice to M1-/- mice reduced death and rescued virus replication in the eyes of infected mice. Infection of M1-/- mice with avirulent HSV-1 strain KOS also increased ocular virus replication and eye disease but did not affect latency-reactivation seen in WT control mice. Severity of virus replication and eye disease correlated with significantly higher inflammatory responses leading to a cytokine storm in the eyes of M1-/- infected mice that was not seen in WT mice. Thus, for the first time, our study illustrates the importance of M1 macrophages specifically in primary HSV-1 infection, eye disease, and survival but not in latency-reactivation.

Restoring Herpesvirus Entry Mediator (HVEM) Immune Function in HVEM-/- Mice Rescues Herpes Simplex Virus 1 Latency and Reactivation Independently of Binding to Glycoprotein D.

The immune modulatory protein herpes virus entry mediator (HVEM) is one of several cellular receptors used by herpes simplex virus 1 (HSV-1) for cell entry. HVEM binds to HSV-1 glycoprotein D (gD) but is not necessary for HSV-1 replication in vitro or in vivo Previously, we showed that although HSV-1 replication was similar in wild-type (WT) control and HVEM-/- mice, HSV-1 does not establish latency or reactivate effectively in mice lacking HVEM, suggesting that HVEM is important for these functions. It is not known whether HVEM immunomodulatory functions contribute to latency and reactivation or whether its binding to gD is necessary. We used HVEM-/- mice to establish three transgenic mouse lines that express either human WT HVEM or human or mouse HVEM with a point mutation that ablates its ability to bind to gD. Here, we show that HVEM immune function, not its ability to bind gD, is required for WT levels of latency and reactivation. We further show that HVEM binding to gD does not affect expression of the HVEM ligands BTLA, CD160, or LIGHT. Interestingly, our results suggest that binding of HVEM to gD may contribute to efficient upregulation of CD8α but not PD1, TIM-3, CTLA4, or interleukin 2 (IL-2). Together, our results establish that HVEM immune function, not binding to gD, mediates establishment of latency and reactivation.IMPORTANCE HSV-1 is a common cause of ocular infections worldwide and a significant cause of preventable blindness. Corneal scarring and blindness are consequences of the immune response induced by repeated reactivation events. Therefore, HSV-1 therapeutic approaches should focus on preventing latency and reactivation. Our data suggest that the immune function of HVEM plays an important role in the HSV-1 latency and reactivation cycle that is independent of HVEM binding to gD.

The Absence of Lymphotoxin-α, a Herpesvirus Entry Mediator (HVEM) Ligand, Affects Herpes Simplex Virus 1 Infection In Vivo Differently than the Absence of Other HVEM Cellular Ligands.

Previously, we reported that the absence of herpesvirus entry mediator (HVEM) decreases latency but not primary infection in ocularly infected mice. Recently, we reported that similar to the absence of HVEM, the absence of HVEM ligands (i.e., LIGHT, CD160, and B and T lymphocyte attenuator [BTLA]) also decreased latency but not primary infection. Similar to LIGHT, CD160, and BTLA, another member of tumor necrosis factor (TNF) superfamily, lymphotoxin-α (LTα), also interacts with HVEM. To determine whether LTα decreases latency in infected mice, we ocularly infected LTα-/- mice with latency-associated transcript-positive [LAT(+)] and LAT(-) viruses using similarly infected wild-type (WT) mice as controls. In contrast to WT C57BL/6 mice, LTα-/- mice were highly susceptible to ocular herpes simplex virus 1 (HSV-1) infection, independent of the presence or absence of LAT. Survival was partially restored by adoptive transfer of CD4+, CD8+, or total T cells. Infected LTα-/- mice had significantly higher corneal scarring than WT mice, and adoptive T cell transfer did not alter the severity of eye disease. In contrast to results in WT mice, the amount of latency was not affected by the absence of LAT. The amount of LAT RNA in LTα-/- mice infected with LAT(+) virus was similar to that in WT mice, and adoptive T cell transfer did not alter LAT RNA levels in LTα-/- infected mice. Increased latency in the absence of LTα correlated with upregulation of HVEM, LIGHT, CD160, and BTLA transcripts as well as with an increase in markers of T cell exhaustion. The results of our study suggest that LTα has antipathogenic and anti-inflammatory functions and may act to protect the host from infection.IMPORTANCE Recently, we evaluated the effects of HVEM and its ligands (LIGHT, CD160, and BTLA) on HSV-1 infectivity. However, the effect of LTα, another member of the TNF superfamily, on HSV-1 latency and eye disease is not known. Here, we demonstrate increased latency and corneal scarring in LTα-/- infected mice, independent of the presence of LAT. In addition, infected mice were highly susceptible to HSV-1 infection, and survival was partially but not significantly restored by adoptive T cell transfer. These results suggest that the absence of LTα affects HSV-1 infectivity differently than the absence of HVEM, LIGHT, CD160, and BTLA.

Roles of Type 1, 2, and 3 Innate Lymphoid Cells in Herpes Simplex Virus 1 Infection In Vitro and In Vivo.

Innate lymphoid cells (ILCs) play important roles in host defense and inflammation. They are classified into three distinct groups based on their cytokine and chemokine secretion patterns and transcriptome profiles. Here, we show that ILCs isolated from mice can be infected with herpes simplex virus 1 (HSV-1) but that subsequent replication of the virus is compromised. After infection, type 2 ILCs expressed significantly higher levels of granulocyte colony-stimulating factor (G-CSF), interleukin 1α (IL-1α), IL-6, IL-9, RANTES, tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, CXCL10, CCL3, and CCL4 than infected type 1 or type 3 ILCs. Transcriptome-sequencing (RNA-seq) analysis of the ILCs 24 h after HSV-1 infection revealed that 77 herpesvirus genes were detected in the infected type 3 ILCs, whereas only 11 herpesvirus genes were detected in infected type 1 ILCs and 27 in infected type 2 ILCs. Compared with uninfected cells, significant upregulation of over 4,000 genes was seen in the HSV-1-infected type 3 ILCs, whereas 414 were upregulated in the infected type 1 ILCs and 128 in the infected type 2 ILCs. In contrast, in all three cell types, only a limited number of genes were significantly downregulated. Type 1, type 2, and type 3 ILC-deficient mice were used to gain insights into the effects of the ILCs on the outcome of ocular HSV-1 infection. No significant differences were found on comparison with similarly infected wild-type mice or on comparison of the three strains of deficient mice in terms of virus replication in the eyes, levels of corneal scarring, latency-reactivation in the trigeminal ganglia, or T-cell exhaustion. Although there were no significant differences in the survival rates of infected ILC-deficient mice and wild-type mice, there was significantly reduced survival of the infected type 1 or type 3 ILC-deficient mice compared with type 2 ILC-deficient mice. Adoptive transfer of wild-type T cells did not alter survival or any other parameters tested in the infected mice. Our results indicate that type 1, 2, and 3 ILCs respond differently to HSV-1 infection in vitro and that the absence of type 1 or type 3, but not type 2, ILCs affects the survival of ocularly infected mice.IMPORTANCE In this study, we investigated for the first time what roles, if any, innate lymphoid cells (ILCs) play in HSV-1 infection. Analysis of isolated ILCs in vitro revealed that all three subtypes could be infected with HSV-1 but that they were resistant to replication. The expression profiles of HSV-1-induced cytokines/chemokines and cellular and viral genes differed among the infected type 1, 2, and 3 ILCs in vitro While ILCs play no role or a redundant role in the outcomes of latency-reactivation in infected mice, absence of type 1 and type 3, but not type 2, ILCs affects the survival of infected mice.

CCR2+ migratory macrophages with M1 status are the early-responders in the cornea of HSV-1 infected mice.

Complex interactions between HSV-1 and infiltrating immune cells play important roles in establishing localized, acute virus replication as well as chronic latent infection. The extent and duration of initial virus replication are the key determinants of subsequent pathologic inflammatory responses and therefore, the accumulation of immune cell populations at this time point is a key target for prevention. Therefore, we evaluated the role of various immune cell infiltrates between 1 h and 28 days post-infection (PI) using mice infected with virulent HSV-1 strain McKrae without corneal scarification. The effect of corneal scarification on immune cell infiltrates was also determined. We first determined the activation status and origin of macrophage infiltrates as early as 1 h PI. We found a sharp increase in the total macrophage population after 12 h PI, that was primarily due to infiltration of CCR2+ migratory macrophages, mostly in M1 status (MHC II+). The number of CCR2- resident macrophages, mostly unpolarized (M0), increased gradually over time and peaked at 48 h PI. Interestingly, some of the resident macrophages gained an M2-like phenotype (CD206Low), which peaked at 12 h PI, concurrent with M1 macrophage infiltration. From 1-7 days PI, infiltration of various immune cells correlated strongly with HSV-1 replication, with neutrophils showing the biggest increase, and NKT cells the biggest decrease, after infection. The presence of geographical ulcer did not correlate with increased infiltration, while mice with corneal scarring had significantly more immune cell infiltration than those without corneal scarring. Overall, we showed time-dependent infiltration of various immune cells in the eye of HSV-1 infected mice. Initial infiltration of macrophages followed by infiltration of T cells at later times PI demonstrates the importance of targeting macrophages rather than other immune cells type, for therapeutic treatment of HSV-1.

Herpes Simplex Virus 1 Latency and the Kinetics of Reactivation Are Regulated by a Complex Network of Interactions between the Herpesvirus Entry Mediator, Its Ligands (gD, BTLA, LIGHT, and CD160), and the Latency-Associated Transcript.

Recently, we reported that the herpesvirus entry mediator (HVEM; also called TNFRSF14 or CD270) is upregulated by the latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1) and that the absence of HVEM affects latency reactivation but not primary infection in ocularly infected mice. gD has been shown to bind to HVEM. LIGHT (TNFSF14), CD160, and BTLA (B- and T-lymphocyte attenuator) also interact with HVEM and can interfere with HSV gD binding. It was not known if LIGHT, CD160, or BTLA affected the level of latency reactivation in the trigeminal ganglia (TG) of latently infected mice. To address this issue, we ocularly infected LIGHT-/-, CD160-/-, and BTLA-/- mice with LAT(+) and LAT(-) viruses, using similarly infected wild-type (WT) and HVEM-/- mice as controls. The amount of latency, as determined by the levels of gB DNA in the TG of the LIGHT-/-, CD160-/-, and BTLA-/- mice infected with either LAT(+) or LAT(-) viruses, was lower than that in WT mice infected with LAT(+) virus and was similar in WT mice infected with LAT(-) virus. The levels of LAT RNA in HVEM-/-, LIGHT-/-, CD160-/-, and BTLA-/- mice infected with LAT(+) virus were similar and were lower than the levels of LAT RNA in WT mice. However, LIGHT-/-, CD160-/-, and BTLA-/- mice, independent of the presence of LAT, had levels of reactivation similar to those of WT mice infected with LAT(+) virus. Faster reactivation correlated with the upregulation of HVEM transcript. The LIGHT-/-, CD160-/-, and BTLA-/- mice had higher levels of HVEM expression, and this, along with the absence of BTLA, LIGHT, or CD160, may contribute to faster reactivation, while the absence of each molecule, independent of LAT, may have contributed to lower latency. This study suggests that, in the absence of competition with gD for binding to HVEM, LAT RNA is important for WT levels of latency but not for WT levels of reactivation.IMPORTANCE The effects of BTLA, LIGHT, and CD160 on latency reactivation are not known. We show here that in BTLA, LIGHT, or CD160 null mice, latency is reduced; however, HVEM expression is upregulated compared to that of WT mice, and this upregulation is associated with higher reactivation that is independent of LAT but dependent on gD expression. Thus, one of the mechanisms by which BTLA, LIGHT, and CD160 null mice enhance reactivation appears to be the increased expression of HVEM in the presence of gD. Thus, our results suggest that blockade of HVEM-LIGHT-BTLA-CD160 contributes to reduced HSV-1 latency and reactivation.

Deficiency of GATA3-Positive Macrophages Improves Cardiac Function Following Myocardial Infarction or Pressure Overload Hypertrophy.

BACKGROUND: Macrophages are highly plastic cells that play an important role in the pathogenesis of cardiovascular disease. OBJECTIVES: This study investigated the role of GATA3-positive macrophages in modulating cardiac function after myocardial infarction (MI) or in response to pressure overload hypertrophy. METHODS: Myeloid-specific GATA3-deficient (mGATA3KO) mice were generated, MI or pressure overload was induced, and cardiac function was determined by echocardiography. GATA3-sufficient Cre mice were used as a control. Immunohistochemical staining, flow cytometry, MILLIPLEX Mouse Cytokine/Chemokine Assay, cultured macrophages, quantitative real-time polymerase chain reaction, and western blot were used to determine the role of GATA3 in macrophages. RESULTS: GATA3-positive macrophages rapidly accumulated in the infarcted region of the myocardium after acute MI. Deficiency of GATA3-positive macrophages led to a significant improvement of cardiac function in response to acute MI or pressure overload hypertrophy compared with the control mice. This improvement was associated with the presence of a large number of proinflammatory Ly6Chi monocytes/macrophages and fewer reparative Ly6Clo macrophages in the myocardium of mGATA3KO mice compared with control mice. Analysis of serum proteins from the 2 mouse genotypes revealed no major changes in the profile of serum growth factors and cytokines between the 2 mice genotypes before and after MI. GATA3 was found to be specifically and transiently induced by interleukin 4 in cultured macrophages through activity of the proximal promoter, whereas the distal promoter remained silent. In addition, the absence of GATA3 in macrophages markedly attenuated arginase-1 expression in cultured macrophages. CONCLUSIONS: We demonstrated that the presence of GATA3-positive macrophages adversely affects remodeling of the myocardium in response to ischemia or pressure overload, whereas the absence of these macrophages led to a significant improvement in cardiac function. Targeting of signaling pathways that lead to the expression of GATA3 in macrophages may have favorable cardiac outcomes.

Binding of Herpes Simplex Virus 1 UL20 to GODZ (DHHC3) Affects Its Palmitoylation and Is Essential for Infectivity and Proper Targeting and Localization of UL20 and Glycoprotein K.

Herpes simplex virus 1 (HSV-1) UL20 plays a crucial role in the envelopment of the cytoplasmic virion and its egress. It is a nonglycosylated envelope protein that is regulated as a γ1 gene. Two-hybrid and pulldown assays demonstrated that UL20, but no other HSV-1 gene-encoded proteins, binds specifically to GODZ (also known as DHHC3), a cellular Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein. A catalytically inactive dominant-negative GODZ construct significantly reduced HSV-1 replication in vitro and affected the localization of UL20 and glycoprotein K (gK) and their interactions but not glycoprotein C (gC). GODZ is involved in palmitoylation, and we found that UL20 is palmitoylated by GODZ using a GODZ dominant-negative plasmid. Blocking of palmitoylation using 2-bromopalmitate (2-BP) affected the virus titer and the interaction of UL20 and gK but did not affect the levels of these proteins. In conclusion, we have shown that binding of UL20 to GODZ in the Golgi apparatus regulates trafficking of UL20 and its subsequent effects on gK localization and virus replication. We also have demonstrated that GODZ-mediated UL20 palmitoylation is critical for UL20 membrane targeting and thus gK cell surface expression, providing new mechanistic insights into how UL20 palmitoylation regulates HSV-1 infectivity.IMPORTANCE HSV-1 UL20 is a nonglycosylated essential envelope protein that is highly conserved among herpesviruses. In this study, we show that (i) HSV-1 UL20 binds to GODZ (also known as DHHC3), a Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein; (ii) a GODZ dominant-negative mutant and an inhibitor of palmitoylation reduced HSV-1 titers and altered the localization of UL20 and glycoprotein K; and (iii) UL20 is palmitoylated by GODZ, and this UL20 palmitoylation is required for HSV-1 infectivity. Thus, blocking of the interaction of UL20 with GODZ, using a GODZ dominant-negative mutant or possibly GODZ shRNA, should be considered a potential alternative therapy in not only HSV-1 but also other conditions in which GODZ processing is an integral component of pathogenesis.

Roles of M1 and M2 Macrophages in Herpes Simplex Virus 1 Infectivity.

Macrophages are the predominant infiltrate in the corneas of mice that have been ocularly infected with herpes simplex virus 1 (HSV-1). However, very little is known about the relative roles of M1 (classically activated or polarized) and M2 (alternatively activated or polarized) macrophages in ocular HSV-1 infection. To better understand these relationships, we assessed the impact of directed M1 or M2 activation of RAW264.7 macrophages and peritoneal macrophages (PM) on subsequent HSV-1 infection. In both the RAW264.7 macrophage and PM in vitro models, HSV-1 replication in M1 macrophages was markedly lower than in M2 macrophages and unstimulated controls. The M1 macrophages expressed significantly higher levels of 28 of the 32 tested cytokines and chemokines than M2 macrophages, with HSV-1 infection significantly increasing the levels of proinflammatory cytokines and chemokines in the M1 versus the M2 macrophages. To examine the effects of shifting the immune response toward either M1 or M2 macrophages in vivo, wild-type mice were injected with gamma interferon (IFN-γ) DNA or colony-stimulating factor 1 (CSF-1) DNA prior to ocular infection with HSV-1. Virus replication in the eye, latency in trigeminal ganglia (TG), and markers of T cell exhaustion in the TG were determined. We found that injection of mice with IFN-γ DNA, which enhances the development of M1 macrophages, increased virus replication in the eye; increased latency; and also increased CD4, CD8, IFN-γ, and PD-1 transcripts in the TG of latently infected mice. Conversely, injection of mice with CSF-1 DNA, which enhances the development of M2 macrophages, was associated with reduced virus replication in the eye and reduced latency and reduced the levels of CD4, CD8, IFN-γ,and PD-1 transcripts in the TG. Collectively, these results suggest that M2 macrophages directly reduce the levels of HSV-1 latency and, thus, T-cell exhaustion in the TG of ocularly infected mice.IMPORTANCE Our findings demonstrate a novel approach to further reducing HSV-1 replication in the eye and latency in the TG by modulating immune components, specifically, by altering the phenotype of macrophages. We suggest that inclusion of CSF-1 as part of any vaccination regimen against HSV infection to coax responses of macrophages toward an M2, rather than an M1, response may further improve vaccine efficacy against ocular HSV-1 replication and latency.

Suppression of IL-12p70 formation by IL-2 or following macrophage depletion causes T-cell autoreactivity leading to CNS demyelination in HSV-1-infected mice.

We have established two mouse models of central nervous system (CNS) demyelination that differ from most other available models of multiple sclerosis (MS) in that they represent a mixture of viral and immune triggers. In the first model, ocular infection of different strains of mice with a recombinant HSV-1 that expresses murine IL-2 constitutively (HSV-IL-2) causes CNS demyelination. In the second model, depletion of macrophages causes CNS demyelination in mice that are ocularly infected with wild-type (WT) HSV-1. In the present study, we found that the demyelination in macrophage-intact mice infected with HSV-IL-2 was blocked by depletion of FoxP3-expressing cells, while concurrent depletion of macrophages restored demyelination. In contrast, demyelination was blocked in the macrophage-depleted mice infected with wild-type HSV-1 following depletion of FoxP3-expressing cells. In macrophage-depleted HSV-IL-2-infected mice, demyelination was associated with the activity of both CD4+ and CD8+ T cells, whereas in macrophage-depleted mice infected with WT HSV-1, demyelination was associated with CD4+ T cells. Macrophage depletion or infection with HSV-IL-2 caused an imbalance of T cells and TH1 responses as well as alterations in IL-12p35 and IL-12p40 but not other members of the IL-12 family or their receptors. Demyelination was blocked by adoptive transfer of macrophages that were infected with HSV-IL-12p70 or HSV-IL-12p40 but not by HSV-IL-12p35. These results indicate that suppression of IL-12p70 formation by IL-2 or following macrophage depletion causes T-cell autoreactivity leading to CNS demyelination in HSV-1-infected mice.

Temozolomide does not impair gene therapy-mediated antitumor immunity in syngeneic brain tumor models.

PURPOSE: Glioblastoma multiforme is the most common primary brain cancer in adults. Chemotherapy with temozolomide (TMZ) significantly prolongs the survival of patients with glioblastoma multiforme. However, the three-year survival is still approximately 5%. Herein, we combined intratumoral administration of an adenoviral vector expressing Flt3L (Ad-Flt3L) with systemic temozolomide to assess its impact on therapeutic efficacy. EXPERIMENTAL DESIGN: Wild-type or immunodeficient mice bearing intracranial glioblastoma multiforme or metastatic melanoma were treated with an intratumoral injection of Ad-Flt3L alone or in combination with the conditionally cytotoxic enzyme thymidine kinase (Ad-TK), followed by systemic administration of ganciclovir and temozolomide. We monitored survival and measured the tumor-infiltrating immune cells. RESULTS: Although treatment with temozolomide alone led to a small improvement in median survival, when used in combination with gene therapy-mediated immunotherapy, it significantly increased the survival of tumor-bearing mice. The antitumor effect was further enhanced by concomitant intratumoral administration of Ad-TK, leading to 50% to 70% long-term survival in all tumor models. Although temozolomide reduced the content of T cells in the tumor, this did not affect the therapeutic efficacy. The antitumor effect of Ad-Flt3L+Ad-TK+TMZ required an intact immune system because the treatment failed when administered to knock out mice that lacked lymphocytes or dendritic cells. CONCLUSIONS: Our results challenge the notion that chemotherapy leads to a state of immune-suppression which impairs the ability of the immune system to mount an effective antitumor response. Our work indicates that temozolomide does not inhibit antitumor immunity and supports its clinical implementation in combination with immune-mediated therapies.

Interactions between herpesvirus entry mediator (TNFRSF14) and latency-associated transcript during herpes simplex virus 1 latency.

Herpesvirus entry mediator (HVEM) is one of several cell surface proteins herpes simplex virus (HSV) uses for attachment/entry. HVEM regulates cellular immune responses and can also increase cell survival. Interestingly, latency-associated transcript (LAT), the only viral gene consistently expressed during neuronal latency, enhances latency and reactivation by promoting cell survival and by helping the virus evade the host immune response. However, the mechanisms of these LAT activities are not well understood. We show here for the first time that one mechanism by which LAT enhances latency and reactivation appears to be by upregulating HVEM expression. HSV-1 latency/reactivation was significantly reduced in Hvem(-/-) mice, indicating that HVEM plays a significant role in HSV-1 latency/reactivation. Furthermore, LAT upregulated HVEM expression during latency in vivo and also when expressed in vitro in the absence of other viral factors. This study suggests a mechanism whereby LAT upregulates HVEM expression potentially through binding of two LAT small noncoding RNAs to the HVEM promoter and that the increased HVEM then leads to downregulation of immune responses in the latent microenvironment and increased survival of latently infected cells. Thus, one of the mechanisms by which LAT enhances latency/reactivation appears to be through increasing expression of HVEM.

A simple alkaline method for decellularizing human amniotic membrane for cell culture.

Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.

The role of LAT in increased CD8+ T cell exhaustion in trigeminal ganglia of mice latently infected with herpes simplex virus 1.

Herpes simplex virus (HSV) infection is a classic example of latent viral infection in humans and experimental animal models. The HSV-1 latency-associated transcript (LAT) plays a major role in the HSV-1 latency reactivation cycle and thus in recurrent disease. Whether the presence of LAT leads to generation of dysfunctional T cell responses in the trigeminal ganglia (TG) of latently infected mice is not known. To address this issue, we used LAT-positive [LAT(+)] and LAT-deficient [LAT(-)] viruses to evaluate the effect of LAT on CD8 T cell exhaustion in TG of latently infected mice. The amount of latency as determined by quantitative reverse transcription-PCR (qRT-PCR) of viral DNA in total TG extracts was 3-fold higher with LAT(+) than with LAT(-) virus. LAT expression and increased latency correlated with increased mRNA levels of CD8, PD-1, and Tim-3. PD-1 is both a marker for exhaustion and a primary factor leading to exhaustion, and Tim-3 can also contribute to exhaustion. These results suggested that LAT(+) TG contain both more CD8(+) T cells and more CD8(+) T cells expressing the exhaustion markers PD-1 and Tim-3. This was confirmed by flow cytometry analyses of expression of CD3/CD8/PD-1/Tim-3, HSV-1, CD8(+) T cell pentamer (specific for a peptide derived from residues 498 to 505 of glycoprotein B [gB(498-505)]), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α). The functional significance of PD-1 and its ligands in HSV-1 latency was demonstrated by the significantly reduced amount of HSV-1 latency in PD-1- and PD-L1-deficient mice. Together, these results may suggest that both PD-1 and Tim-3 are mediators of CD8(+) T cell exhaustion and latency in HSV-1 infection.

Macrophage IL-12p70 signaling prevents HSV-1-induced CNS autoimmunity triggered by autoaggressive CD4+ Tregs.

PURPOSE: CD4(+)CD25(+)FoxP3(+) naturally occurring regulatory T cells (Tregs) maintain self-tolerance and function to suppress overly exuberant immune responses. However, it is unclear whether innate immune cells modulate Treg function. Here the authors examined the role of innate immunity in lymphomyeloid homeostasis. METHODS: The involvement of B cells, dendritic cells (DCs), macrophages, natural killer (NK) cells, and T cells in central nervous system (CNS) demyelination in different strains of mice infected ocularly with herpes simplex virus type 1 (HSV-1) was investigated. RESULTS: The authors found that depletion of macrophages, but not DCs, B cells, NK cells, CD4(+) T cells, or CD8(+) T cells, induced CNS demyelination irrespective of virus or mouse strain. As with macrophage depletion, mice deficient in interleukin (IL)-12p35 or IL-12p40 showed CNS demyelination after HSV-1 infection, whereas demyelination was undetectable in HSV-1-infected, IL-23p19-deficient, or Epstein-Barr virus-induced gene 3-deficient mice. Demyelination could be rescued in macrophage-depleted mice after the injection of IL-12p70 DNA and in IL-12p35(-/-) or IL-12p40(-/-) mice after injection with IL-12p35 or IL-12p40 DNA or with recombinant viruses expressing IL-12p35 or IL-12p40. Using FoxP3-, CD4-, CD8-, or CD25-depletion and gene-deficient mouse approaches, the authors demonstrated that HSV-1-induced demyelination was blocked in the absence of CD4, CD25, or FoxP3 in macrophage-depleted mice. Flow cytometry showed an elevation of CD4(+)CD25(+)FoxP3(+) T cells in the spleens of infected macrophage-depleted mice, and adoptive transfer of CD4(+)CD25(+) T cells to infected macrophage-depleted severe combined immunodeficient mice induced CNS demyelination. CONCLUSIONS: The authors demonstrated that macrophage IL-12p70 signaling plays an important role in maintaining immune homeostasis in the CNS by preventing the development of autoaggressive CD4(+) Tregs.

Ocular infection of mice with an avirulent recombinant HSV-1 expressing IL-4 and an attenuated HSV-1 strain generates virulent recombinants in vivo.

PURPOSE: To assess the relative impact of overexpression of interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN-γ) expressing recombinant herpes simplex virus type 1 (HSV-1) on altering immune responses in ocularly infected mice. METHODS: BALB/c mice were co-infected ocularly with avirulent HSV-1 strain KOS and avirulent recombinant HSV-1 expressing murine IL-4 (HSV-IL-4). Controls mice were co-infected with KOS+HSV-IL-2 or KOS+HSV-IFNγ. Following ocular infection, virus replication in the eye, corneal scarring (CS), and survival were determined. We also isolated recombinant viruses from eye and trigeminal ganglia of KOS+HSV-IL-4 infected mice. RESULTS: In this study we found that ocular infection of BALB/c mice with a mixture of HSV-IL-4 and KOS resulted in increased death and increased eye disease. In contrast, when mice were infected in one eye with KOS and the other eye with HSV-IL-4 no death or eye disease was seen. Intraperitoneal co-infection of mice with KOS and HSV-IL-4 also did not result in HSV-1 induced death. Interestingly, ocular infection of mice with a mixture of HSV-IL-2 and KOS did not have any effect on severity of the disease in infected mice. We isolated recombinant viruses from KOS+HSV-IL-4 infected mice eye and trigeminal ganglia. Some of the isolated viruses were more neurovirulent then either parental virus. Infection of macrophages with IL-4 expressing virus down-regulated IL-12 production by macrophages. CONCLUSIONS: These results suggest a role for IL-4 in suppression of immune response and generation of virulent viruses in vivo.