Naveen K. Rajasagi

In Vivo Ablation of CD11c-Positive Dendritic Cells Increases Susceptibility to Herpes Simplex Virus Type 1 Infection and Diminishes NK and T-Cell Responses

ABSTRACT The precise role of each of the seven individual CD11c+ dendritic cell subsets (DCs) identified to date in the response to viral infections is not known. DCs serve as critical links between the innate and adaptive immune responses against many pathogens, including herpes simplex virus type 1 (HSV-1). The role of DCs as mediators of resistance to HSV-1 infection was investigated using CD11c-diphtheria toxin (DT) receptor-green fluorescent protein transgenic mice, in which DCs can be transiently depleted in vivo by treatment with low doses of DT. We show that ablation of DCs led to enhanced susceptibility to HSV-1 infection in the highly resistant C57BL/6 mouse strain. Specifically, we showed that the depletion of DCs led to increased viral spread into the nervous system, resulting in an increased rate of morbidity and mortality. Furthermore, we showed that ablation of DCs impaired the optimal activation of NK cells and CD4+ and CD8+ T cells in response to HSV-1. These data demonstrated that DCs were essential not only in the optimal activation of the acquired T-cell response to HSV-1 but also that DCs were crucial for innate resistance to HSV-1 infection.

Galectin-9/TIM-3 Interaction Regulates Virus-Specific Primary and Memory CD8+ T Cell Response

In this communication, we demonstrate that galectin (Gal)-9 acts to constrain CD8+ T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with α-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8+ T cell response to HSV particularly in the acute phase. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8+ T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved direct inhibitory effects on TIM-3+ CD8+ T effector cells as well as the promotion of Foxp3+ regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

Controlling Herpes Simplex Virus-Induced Ocular Inflammatory Lesions with the Lipid-Derived Mediator Resolvin E1

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye caused by HSV-1 infection and a common cause of blindness in humans. The inflammatory lesions are primarily perpetuated by neutrophils with the active participation of CD4+ T cells. Therefore, targeting these immune cell types represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin E1 (RvE1), an endogenous lipid mediator, was shown to promote resolution in several inflammatory disease models. In the current report, we determined whether RvE1 administration begun at different times after ocular infection of mice with HSV could influence the severity of SK lesions. Treatment with RvE1 significantly reduced the extent of angiogenesis and SK lesions that occurred. RvE1-treated mice had fewer numbers of inflammatory cells that included Th1 and Th17 cells as well as neutrophils in the cornea. The mechanisms by which RvE1 acts appear to be multiple. These included reducing the influx of neutrophils and pathogenic CD4+ T cells, increasing production of the anti-inflammatory cytokine IL-10, and inhibitory effects on the production of proinflammatory mediators and molecules, such as IL-6, IFN-γ, IL-17, KC, VEGF-A, MMP-2, and MMP-9, that are involved in corneal neovascularization and SK pathogenesis. These findings are, to our knowledge, the first to show that RvE1 treatment could represent a novel approach to control lesion severity in a virally induced immunopathological disease.

Role of IL-17 and Th17 Cells in Herpes Simplex Virus-Induced Corneal Immunopathology

HSV-1 infection of the cornea leads to a blinding immunoinflammatory lesion of the eye termed stromal keratitis (SK). Recently, IL-17–producing CD4+ T cells (Th17 cells) were shown to play a prominent role in many autoimmune conditions, but the role of IL-17 and/or of Th17 cells in virus immunopathology is unclear. In this study, we show that, after HSV infection of the cornea, IL-17 is upregulated in a biphasic manner with an initial peak production around day 2 postinfection and a second wave starting from day 7 postinfection with a steady increase until day 21 postinfection, a time point when clinical lesions are fully evident. Further studies demonstrated that innate cells, particularly γδ T cells, were major producers of IL-17 early after HSV infection. However, during the clinical phase of SK, the predominant source of IL-17 was Th17 cells that infiltrated the cornea only after the entry of Th1 cells. By ex vivo stimulation, the half fraction of IFN-γ–producing CD4+ T cells (Th1 cells) were HSV specific, whereas very few Th17 cells responded to HSV stimulation. The delayed influx of Th17 cells in the cornea was attributed to the local chemokine and cytokine milieu. Finally, HSV infection of IL-17R knockout mice as well as IL-17 neutralization in wild-type mice showed diminished SK severity. In conclusion, our results show that IL-17 and Th17 cells contribute to the pathogenesis of SK, the most common cause of infectious blindness in the Western world.

Role of miR-132 in Angiogenesis after Ocular Infection with Herpes Simplex Virus

MicroRNAs (miRNAs) are small regulatory molecules that control diverse biological processes that include angiogenesis. Herpes simplex virus (HSV) causes a chronic immuno-inflammatory response in the eye that may result in corneal neovascularization during blinding immunopathological lesion stromal keratitis (SK). miR-132 is a highly conserved miRNA that is induced in endothelial cells in response to growth factors, such as vascular endothelial growth factor (VEGF). In this study, we show that miR-132 expression was up-regulated (10- to 20-fold) after ocular infection with HSV, an event that involved the production of both VEGF-A and IL-17. Consequently, blockade of VEGF-A activity using soluble VEGF receptor 1 resulted in significantly lower levels of corneal miR-132 after HSV infection. In addition, low levels of corneal miR-132 were detected in IL-17 receptor knockout mice after HSV infection. In vivo silencing of miR-132 by the provision of anti-miR-132 (antagomir-132) nanoparticles to HSV-infected mice led to reduced corneal neovascularization and diminished SK lesions. The anti-angiogenic effect of antagomir-132 was reflected by a reduction in angiogenic Ras activity in corneal CD31-enriched cells (presumably blood vessel endothelial cells) during SK. To our knowledge, this is one of the first reports of miRNA involvement in an infectious ocular disease. Manipulating miRNA expression holds promise as a therapeutic approach to control an ocular lesion that is an important cause of human blindness.

IL-17A Differentially Regulates Corneal Vascular Endothelial Growth Factor (VEGF)-A and Soluble VEGF Receptor 1 Expression and Promotes Corneal Angiogenesis after Herpes Simplex Virus Infection

Ocular infection with HSV causes corneal neovascularization (CV), an essential step in the pathogenesis of the blinding immunoinflammatory lesion stromal keratitis. The infection results in IL-17A production, which contributes to CV in ways that together serve to shift the balance between corneal concentrations of vascular endothelial growth factor A (VEGF-A) and the soluble vascular endothelial growth factor receptor 1 molecule, which binds to VEGF-A and blocks its function (a so-called VEGF trap). Accordingly, animals lacking responses to IL-17A signaling, either because of IL-17 receptor A knockout or wild-type animals that received neutralizing mAb to IL-17A, had diminished CV, compared with controls. The procedures reduced VEGF-A protein levels but had no effect on the levels of soluble vascular endothelial growth factor receptor 1. Hence the VEGF trap was strengthened. IL-17A also caused increased CXCL1/KC synthesis, which attracts neutrophils to the inflammatory site. Neutrophils further influenced the extent of CV by acting as an additional source of VEGF-A, as did metalloproteinase enzymes that degrade the soluble receptor, inhibiting its VEGF-blocking activity. Our results indicate that suppressing the expression of IL-17A, or increasing the activity of the VEGF trap, represents a useful approach to inhibiting CV and the control of an ocular lesion that is an important cause of human blindness.

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

ABSTRACT The role of CD4+ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4+ T cells are needed for the generation of the protective HSV-1-specific CD8+-T-cell response. This study examined the contribution of CD4+ T cells in the generation of the primary CD8+-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8+-T-cell response generated in the draining lymph nodes of CD4+-T-cell-depleted C57BL/6 mice and B6-MHC-II−/− mice is quantitatively and qualitatively distinct from the CD8+ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8+ T cells express comparable levels of the activation marker CD44 in mice lacking CD4+ T cells and normal mice. In contrast, CD8+ T cells generated in the absence of CD4+ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8+ T cells in the CD4+-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8+ T cells is diminished in the absence of CD4+-T-cell help. These results suggest that CD4+-T-cell help is essential for the generation of fully functional CD8+ T cells during the primary response to HSV-1 infection.

Galectin-1 Reduces the Severity of Herpes Simplex Virus-Induced Ocular Immunopathological Lesions

Stromal keratitis is a chronic immunopathological lesion of the eye caused by HSV-1 infection that can result in blindness. Because the inflammatory lesions are primarily orchestrated by Th1 cells, and to a lesser extent by Th17 cells, inhibiting their activity represents a useful form of therapy. In this study we evaluated the therapeutic potential of galectin-1 (gal-1), an endogenous lectin that in some autoimmune diseases was shown to suppress the functions of Th1 and Th17 cells. Treatment was begun at different times after ocular infection with HSV and the outcome was assessed clinically as well as for effects on various immune parameters. Treatment with recombinant gal-1 significantly diminished stromal keratitis lesion severity and the extent of corneal neovascularization. Treated mice had reduced numbers of IFN-γ– and IL-17–producing CD4+ T cells, as well as neutrophil infiltration in the cornea. Furthermore, disease severity was greater in gal-1 knockout mice compared with their wild-type counterparts. The many effects of gal-1 treatment include reduction in the production of proinflammatory cytokines and chemokines, increased production of IL-10, and inhibitory effects on molecules involved in neovascularization. To our knowledge, our findings are the first to show that gal-1 treatment represents a useful approach to control lesion severity in a virally induced immunopathological disease.

Influence of Galectin-9/Tim-3 Interaction on Herpes Simplex Virus-1 Latency

After HSV-1 infection, CD8+ T cells accumulate in the trigeminal ganglion (TG) and participate in the maintenance of latency. However, the mechanisms underlying intermittent virus reactivation are poorly understood. In this study, we demonstrate the role of an inhibitory interaction between T cell Ig and mucin domain-containing molecule 3 (Tim-3)–expressing CD8+ T cells and galectin 9 (Gal-9) that could influence HSV-1 latency and reactivation. Accordingly, we show that most Kb-gB tetramer-specific CD8+ T cells in the TG of HSV-1–infected mice express Tim-3, a molecule that delivers negative signals to CD8+ T cells upon engagement of its ligand Gal-9. Gal-9 was also upregulated in the TG when replicating virus was present as well during latency. This could set the stage for Gal-9/Tim-3 interaction, and this inhibitory interaction was responsible for reduced CD8+ T cell effector function in wild-type mice. Additionally, TG cell cultures exposed to recombinant Gal-9 in the latent phase caused apoptosis of most CD8+ T cells. Furthermore, Gal-9 knockout TG cultures showed delayed and reduced viral reactivation as compared with wild-type cultures, demonstrating the greater efficiency of CD8+ T cells to inhibit virus reactivation in the absence of Gal-9. Moreover, the addition of recombinant Gal-9 to ex vivo TG cultures induced enhanced viral reactivation compared with untreated controls. Our results demonstrate that the host homeostatic mechanism mediated by Gal-9/Tim-3 interaction on CD8+ T cells can influence the outcome of HSV-1 latent infection, and manipulating Gal-9 signals might represent therapeutic means to inhibit HSV-1 reactivation from latency.

Dendritic Cells Are Required for Optimal Activation of Natural Killer Functions following Primary Infection with Herpes Simplex Virus Type 1

ABSTRACT Natural killer (NK) cells play an important role in the optimal clearance of herpes simplex virus type 1 (HSV-1) infection in mice. Activated NK cells function via cytokine secretion or direct cytolysis of target cells; dendritic cells (DCs) are thought to make critical contributions in the activation of both of these functions. Yet, the magnitude and physiological relevance of DC-mediated NK cell activation in vivo is not completely understood. To examine the contribution of DC help in regulating NK cell functions after infection with HSV-1, we utilized a transgenic mouse model that allows the transient ablation of DCs. Using this approach, it was found that the gamma interferon (IFN-γ) expression potential of NK cells is quantitatively and qualitatively impaired in the absence of DCs. With regard to priming of NK cytolytic functions, the ablation of DCs did not significantly affect cytotoxic protein expression by NK cells. An in vivo cytolytic assay did, however, reveal impairments in the magnitude of NK cell cytotoxicity. Overall, this study provides direct evidence that functional DCs are required for optimal IFN-γ expression and cytolytic function by NK cells following infection with HSV-1.