Christopher D. Conrady

Resistance to HSV-1 infection in the epithelium resides with the novel innate sensor, IFI-16.

Toll-like receptors (TLRs) are innate sentinels required for clearance of bacterial and fungal infections of the cornea, but their role in viral immunity is currently unknown. We report that TLR signaling is expendable in herpes simplex virus (HSV)-1 containment as depicted by plaque assays of knockout mice (MyD88−/−, Trif−/− and MyD88−/− Trif−/− double knockout) resembling wild-type controls. To identify the key sentinel in viral recognition of the cornea, in vivo knockdown of the DNA sensor IFI-16/p204 in the corneal epithelium was performed and resulted in a loss of IFN-regulatory factor-3 (IRF-3) nuclear translocation, interferon-α production, and viral containment. The sensor seems to have a similar function in other HSV clinically relevant sites such as the vaginal mucosa in which a loss of p204/IFI-16 results in significantly more HSV-2 shedding. Thus, we have identified an IRF-3-dependent, IRF-7- and TLR-independent innate sensor responsible for HSV containment at the site of acute infection.

Microglia and a Functional Type I IFN Pathway Are Required To Counter HSV-1–Driven Brain Lateral Ventricle Enlargement and Encephalitis

HSV-1 is the leading cause of sporadic viral encephalitis, with mortality rates approaching 30% despite treatment with the antiviral drug of choice, acyclovir. Permanent neurologic deficits are common in patients that survive, but the mechanism leading to this pathology is poorly understood, impeding clinical advancements in treatment to reduce CNS morbidity. Using magnetic resonance imaging and type I IFN receptor–deficient mouse chimeras, we demonstrate HSV-1 gains access to the murine brain stem and subsequently brain ependymal cells, leading to enlargement of the cerebral lateral ventricle and infection of the brain parenchyma. A similar enlargement in the lateral ventricles is found in a subpopulation of herpes simplex encephalitic patients. Associated with encephalitis is an increase in CXCL1 and CXCL10 levels in the cerebral spinal fluid, TNF-α expression in the ependymal region, and the influx of neutrophils of encephalitic mouse brains. Reduction in lateral ventricle enlargement using anti-secretory factor peptide 16 reduces mortality significantly in HSV-1–infected mice without any effect on expression of inflammatory mediators, infiltration of leukocytes, or changes in viral titer. Microglial cells but not infiltrating leukocytes or other resident glial cells or neurons are the principal source of resistance in the CNS during the first 5 d postinfection through a Toll/IL-1R domain-containing adapter inducing IFN-β–dependent, type I IFN pathway. Our results implicate lateral ventricle enlargement as a major cause of mortality in mice and speculate such an event transpires in a subpopulation of human HSV encephalitic patients.

Microglia-induced IL-6 protects against neuronal loss following HSV-1 infection of neural progenitor cells

Herpes virus type 1 (HSV‐1) is one of the most widespread human pathogens and accounts for more than 90% of cases of herpes simplex encephalitis (HSE) causing severe and permanent neurologic sequelae among surviving patients. We hypothesize such CNS deficits are due to HSV‐1 infection of neural progenitor cells (NPCs). In vivo, HSV‐1 infection was found to diminish NPC numbers in the subventricular zone. Upon culture of NPCs in conditions that stimulate their differentiation, we found HSV‐1 infection of NPCs resulted in the loss of neuronal precursors with no significant change in the percentage of astrocytes or oligodendrocytes. We propose this is due a direct effect of HSV‐1 on neuronal survival without alteration of the differentiation process. The neuronal loss was prevented by the addition of microglia or conditioned media from NPC/microglia co‐cultures. Using neutralizing antibodies and recombinant cytokines, we identified interleukin‐6 (IL‐6) as responsible for the protective effect by microglia, likely through its downstream Signal Transducer and Activator of Transcription 3 (STAT3) cascade. GLIA 2014;62:1418–1434

CD8+ T Cells Suppress Viral Replication in the Cornea but Contribute to VEGF-C–Induced Lymphatic Vessel Genesis

HSV-1 is the leading cause of infectious corneal blindness in the industrialized world. CD4+ T cells are thought to be the major leukocyte population mediating immunity to HSV-1 in the cornea as well as the likely source of immunopathology that reduces visual acuity. However, the role of CD8+ T cells in immune surveillance of the cornea is unclear. Thus, we sought to evaluate the role of CD8+ T cells in ocular immunity using transgenic mice in which >98% of CD8+ T cells are specific for the immunodominant HSV-1 epitope (gBT-I.1). We found a significant reduction in virus, elevation in HSV-specific CD8+ T cell influx, and more CD8+ T cells expressing CXCR3 in the cornea of transgenic mice compared with those in the cornea of wild-type controls yet similar acute corneal pathology. However, by day 30 postinfection, wild-type mice had drastically more blood and lymphatic vessel projections into the cornea compared with gBT-I.1 mice, in which only lymphatic vessel growth in response to VEGF-C could be appreciated. Taken together, these results show that CD8+ T cells are required to eliminate virus more efficiently from the cornea but play a minimal role in immunopathology as a source of VEGF-C.

Granulocytes in Ocular HSV-1 Infection: Opposing Roles of Mast Cells and Neutrophils

PURPOSE The contributions of mast cells (MCs) to immunologic defense against pathogens in the eye are unknown. We have characterized pericorneal MCs as tissue-resident innate sentinels and determined their impact on the immune response to herpes simplex virus type-1 (HSV-1), a common ocular pathogen. METHODS The impact of mast cells on the immune response to HSV-1 infection was investigated using MC-deficient Kit(W-sh) mice. Virus titers, inflammatory cytokine production, eicosanoid profiles, cellular immune responses, and ocular pathology were evaluated and compared with C57BL/6J mice during an acute corneal HSV-1 infection. RESULTS Corneas of Kit(W-sh) mice have higher viral titers, increased edema, and greater leukocyte infiltration following HSV-1 infection. Following infection, cytokine profiles were slightly elevated overall in Kit(W-sh) mice. Eicosanoid profiles were remarkably different only when comparing uninfected corneas from both groups. Neutrophils within infected corneas expressed HSV-1 antigen, lytic genes, and served as a disease-causing vector when adoptively transferred into immunocompromised animals. Myeloid-derived suppressor cells did not infiltrate into the cornea or suppress the expansion, recruitment, or cytokine production by CD8+ T cells following acute HSV-1 infection. CONCLUSIONS Collectively, these findings provide new insight into host defense in the cornea and the pathogenesis of HSV-1 infection by identifying previously unacknowledged MCs as protective innate sentinels for infection of the ocular surface and reinforcing that neutrophils are detrimental to corneal infection.

A Functional Type I Interferon Pathway Drives Resistance to Cornea Herpes Simplex Virus Type 1 Infection by Recruitment of Leukocytes.

Type I interferons are critical antiviral cytokines produced following herpes simplex virus type-1 (HSV-1) infection that act to inhibit viral spread. In the present study, we identify HSV-infected and adjacent uninfected corneal epithelial cells as the source of interferon-α. We also report mice deficient in the A1 chain of the type I IFN receptor (CD118−/−) are extremely sensitive to ocular infection with low doses (100 PFU) of HSV-1 as seen by significantly elevated viral titers in the cornea compared to wild type (WT) controls. The enhanced susceptibility correlated with a loss of CD4+ and CD8+ T cell recruitment and aberrant chemokine production in the cornea despite mounting an adaptive immune response in the draining mandibular lymph node of CD118−/− mice. Taken together, these results highlight the importance of IFN production in both the innate immune response as well as eliciting chemokine production required to facilitate adaptive immune cell trafficking.

Loss of Mandibular Lymph Node Integrity Is Associated with an Increase in Sensitivity to HSV-1 Infection in CD118-Deficient Mice

Type I IFNs are potent antiviral cytokines that contribute to the development of the adaptive immune response. To determine the role of type I IFNs in this process in an infectious disease model, mice deficient in the type I IFN receptor (CD118−/−) were ocularly infected with HSV-1 and surveyed at times post infection in the nervous system and lymph node for virus and the host immune response. Virus titers were elevated in the trigeminal ganglia and brain stem with virus disseminating rapidly to the draining lymph node of CD118−/− mice. T cell and plasmacytoid dendritic cell infiltration into the brain stem was reduced in CD118−/− mice following infection, which correlated with a reduction in CXCL10 but not CXCL9 expression. In contrast, CXCL1 and CCL2 levels were up-regulated in the brainstem of CD118−/− mice associated with an increase in F4/80+ macrophages. By day 5 post infection, there was a significant loss in T, NK, and plasmacytoid dendritic cell numbers in the draining lymph nodes associated with an increase in apoptotic/necrotic T cells and an appreciable lack of HSV-specific CD8+ T cells. The adoptive transfer of HSV-specific TCR transgenic CD8+ T cells into CD118−/− mice at the time of infection modestly reduced viral titers in the nervous system suggesting in addition to the generation of HSV-specific CD8+ T cells, other type I IFN-activated pathways are instrumental in controlling acute infection.

HSV-1 Targets Lymphatic Vessels in the Eye and Draining Lymph Node of Mice Leading to Edema in the Absence of a Functional Type I Interferon Response

Herpes simplex virus type-1 (HSV-1) induces new lymphatic vessel growth (lymphangiogenesis) in the cornea via expression of vascular endothelial growth factor by virally infected epithelial cells. Here, we extend this observation to demonstrate the selective targeting of corneal lymphatics by HSV-1 in the absence of functional type I interferon (IFN) pathway. Specifically, we examined the impact of HSV-1 replication on angiogenesis using type I IFN receptor deficient (CD118(-/-)) mice. HSV-1-induced lymphatic and blood vessel growth into the cornea proper was time-dependent in immunocompetent animals. In contrast, there was an initial robust growth of lymphatic vessels into the cornea of HSV-1-infected CD118(-/-)mice, but such vessels disappeared by day 5 postinfection. The loss was selective as blood vessel integrity remained intact. Magnetic resonance imaging and confocal microscopy analysis of the draining lymph nodes of CD118(-/-) mice revealed extensive edema and loss of lymphatics compared with wild-type mice. In addition to a loss of lymphatic vessels in CD118(-/-) mice, HSV-1 infection resulted in epithelial thinning associated with geographic lesions and edema within the cornea, which is consistent with a loss of lymphatic vasculature. These results underscore the key role functional type I IFN pathway plays in the maintenance of structural integrity within the cornea in addition to the anti-viral characteristics often ascribed to the type I IFN cytokine family.

Comparison of the Host Immune Response to Herpes Simplex Virus 1 (HSV-1) and HSV-2 at Two Different Mucosal Sites

ABSTRACT A study was undertaken to compare the host immune responses to herpes simplex virus 1 (HSV-1) and HSV-2 infection by the ocular or genital route in mice. Titers of HSV-2 from tissue samples were elevated regardless of the route of infection. The elevation in titers of HSV-2, including cell infiltration and cytokine/chemokine levels in the central nervous system relative to those found following HSV-1 infection, was correlative with inflammation. These results underscore a dichotomy between the host immune responses to closely related alphaherpesviruses.

Type I interferon and lymphangiogenesis in the HSV-1 infected cornea – Are they beneficial to the host?

Herpes simplex virus type 1 (HSV-1) is a highly successful pathogen that can result in significant human morbidity. Within the cornea, it was thought the initial recognition of the pathogen was through Toll-like receptors expressed on/in resident cells that then elicit pro-inflammatory cytokine production, activation of anti-viral pathways, and recruitment of leukocytes. However, our lab has uncovered a novel, TLR-independent innate sensor that supersedes TLR induction of anti-viral pathways following HSV-1 infection. In addition, we have also found HSV-1 induces the genesis of lymphatic vessels into the cornea proper by a mechanism independent of TLRs and unique in the field of neovascularization. This review will focus on these two innate immune events during acute HSV-1 infection of the cornea.