Tripartite-Motif 21 (TRIM21) Deficiency Results in a Modest Loss of Herpes Simplex Virus (HSV)-1 Surveillance in the Trigeminal Ganglia Following Cornea Infection.

Tripartite-motif 21 (TRIM21) is thought to regulate the type I interferon (IFN) response to virus pathogens and serve as a cytosolic Fc receptor for immunoglobulin. Since herpes simplex virus (HSV)-1 is sensitive to type I IFN and neutralizing antibody, we investigated the role of TRIM21 in response to ocular HSV-1 infection in mice. In comparison to wild type (WT) mice, TRIM21 deficient (TRIM21 KO) mice were found to be no more susceptible to ocular HSV-1 infection than WT animals, in terms of infectious virus recovered in the cornea. Similar pathology, in terms of neovascularization, opacity, and loss of peripheral vision function, was observed in both WT and TRIM21 KO mice. However, TRIM21 KO mice did possess a significant increase in infectious virus recovered in the trigeminal ganglia, in comparison to the WT animals. The increased susceptibility was not due to changes in HSV-1-specific CD4+ or CD8+ T cell numbers or functional capabilities, or in changes in type I IFN or IFN-inducible gene expression. In summary, the absence of TRIM21 results in a modest, but significant, increase in HSV-1 titers recovered from the TG of TRIM21 KO mice during acute infection, by a mechanism yet to be determined.

A neutrophil/TGF-ß axis limits the pathogenicity of allergen-specific CD4+ T cells.

The intensity and longevity of inflammatory responses to inhaled allergens is determined largely by the balance between effector and regulatory immune responses, but the mechanisms that determine the relative magnitudes of these opposing forces remain poorly understood. We have found that the type of adjuvant used during allergic sensitization has a profound effect on both the nature and longevity of the pulmonary inflammation triggered by subsequent reexposure to that same provoking allergen. TLR ligand adjuvants and house dust extracts primed immune responses characterized by a mixed neutrophilic and eosinophilic inflammation that was suppressed by multiple daily allergen challenges. During TLR ligand-mediated allergic sensitization, mice displayed transient airway neutrophilia, which triggered the release of TGF-ß into the airway. This neutrophil-dependent production of TGF-ß during sensitization had a delayed, suppressive effect on eosinophilic responses to subsequent allergen challenge. Neutrophil depletion during sensitization did not affect numbers of Foxp3+ Tregs but increased proportions of Gata3+CD4+ T cells, which, upon their transfer to recipient mice, triggered stronger eosinophilic inflammation. Thus, a neutrophil/TGF-ß axis acts during TLR-mediated allergic sensitization to fine-tune the phenotype of developing allergen-specific CD4+ T cells and limit their pathogenicity, suggesting a novel immunotherapeutic approach to control eosinophilia in asthma.

An intact complement system dampens cornea inflammation during acute primary HSV-1 infection.

Corneal transparency is an essential characteristic necessary for normal vision. In response to microbial infection, the integrity of the cornea can become compromised as a result of the inflammatory response and the ensuing tissue pathology including neovascularization (NV) and collagen lamellae destruction. We have previously found complement activation contributes to cornea pathology-specifically, denervation in response to HSV-1 infection. Therefore, we investigated whether the complement system also played a role in HSV-1-mediated neovascularization. Using wild type (WT) and complement component 3 deficient (C3 KO) mice infected with HSV-1, we found corneal NV was accelerated associated with an increase in inflammatory monocytes (CD11b+CCR2+CD115+/-Ly6G-Ly6Chigh), macrophages (CD11b+CCR2+CD115+Ly6G-Ly6Chigh) and a subpopulation of granulocytes/neutrophils (CD11b+CCR2-CD115+Ly6G+Ly6Clow). There were also increases in select pro-inflammatory and pro-angiogenic factors including IL-1α, matrix metalloproteinases (MMP)-2, MMP-3, MMP-8, CXCL1, CCL2, and VEGF-A that coincided with increased inflammation, neovascularization, and corneal opacity in the C3 KO mice. The difference in inflammation between WT and C3 KO mice was not driven by changes in virus titer. However, viral antigen clearance was hindered in C3 KO mouse corneas suggesting the complement system has a dynamic regulatory role within the cornea once an inflammatory cascade is initiated by HSV-1.

Regulation of Immune Responses by Nonhematopoietic Cells in Asthma.

Nonhematopoietic cells are emerging as important contributors to many inflammatory diseases, including allergic asthma. Recent advances have led to a deeper understanding of how these cells interact with traditional immune cells, thereby modulating their activities in both homeostasis and disease. In addition to their well-established roles in gas exchange and barrier function, lung epithelial cells express an armament of innate sensors that can be triggered by various inhaled environmental agents, leading to the production of proinflammatory molecules. Advances in cell lineage tracing and single-cell RNA sequencing have expanded our knowledge of rare, but immunologically important nonhematopoietic cell populations. In parallel with these advances, novel reverse genetic approaches are revealing how individual genes in different lung-resident nonhematopoietic cell populations contribute to the initiation and maintenance of asthma. This knowledge is already revealing new pathways that can be selectively targeted to treat distinct forms of asthma.

Herpes Simplex Virus 1 (HSV-1) 0ΔNLS Live-Attenuated Vaccine Protects against Ocular HSV-1 Infection in the Absence of Neutralizing Antibody in HSV-1 gB T Cell Receptor-Specific Transgenic Mice.

The contribution of T cell and antibody responses following vaccination in resistance to herpes simplex virus 1 (HSV-1) infection continues to be rigorously investigated. In the present article, we explore the contribution of CD8+ T cells specific for the major antigenic epitope for HSV-1 glycoprotein B (gB498-505, gB) in C57BL/6 mice using a transgenic mouse (gBT-I.1) model vaccinated with HSV-1 0ΔNLS. gBT-I.1-vaccinated mice did not generate a robust neutralization antibody titer in comparison to the HSV-1 0ΔNLS-vaccinated wild-type C57BL/6 counterpart. Nevertheless, the vaccinated gBT-I.1 mice were resistant to ocular challenge with HSV-1 compared to vehicle-vaccinated animals based on survival and reduced corneal neovascularization but displayed similar levels of corneal opacity. Whereas there was no difference in the virus titer recovered from the cornea comparing vaccinated mice, HSV-1 0ΔNLS-vaccinated animals possessed significantly less infectious virus during acute infection in the trigeminal ganglia (TG) and brain stem compared to the control-vaccinated group. These results correlated with a significant increase in gB-elicited interferon-γ (IFN-γ), granzyme B, and CD107a and a reduction in lymphocyte activation gene 3 (LAG-3), programmed cell death 1 (PD-1), and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) expressed by TG infiltrating gB-specific CD8+ T cells from the HSV-1 0ΔNLS-vaccinated group. Antibody depletion of CD8+ T cells in HSV-1 0ΔNLS-vaccinated mice rendered animals highly susceptible to virus-mediated mortality similar to control-vaccinated mice. Collectively, the HSV-1 0ΔNLS vaccine is effective against ocular HSV-1 challenge, reducing ocular neovascularization and suppressing peripheral nerve virus replication in the near absence of neutralizing antibody in this unique mouse model.IMPORTANCE The role of CD8+ T cells in antiviral efficacy using a live-attenuated virus as the vaccine is complicated by the humoral immune response. In the case of the herpes simplex virus 1 (HSV-1) 0ΔNLS vaccine, the correlate of protection has been defined to be primarily antibody driven. The current study shows that in the near absence of anti-HSV-1 antibody, vaccinated mice are protected from subsequent challenge with wild-type HSV-1 as measured by survival. The efficacy is lost following depletion of CD8+ T cells. Whereas increased survival and reduction in virus replication were observed in vaccinated mice challenged with HSV-1, cornea pathology was mixed with a reduction in neovascularization but no change in opacity. Collectively, the study suggests CD8+ T cells significantly contribute to the host adaptive immune response to HSV-1 challenge following vaccination with an attenuated virus, but multiple factors are involved in cornea pathology in response to ocular virus challenge.

Complement and CD4+ T cells drive context-specific corneal sensory neuropathy.

Whether complement dysregulation directly contributes to the pathogenesis of peripheral nervous system diseases, including sensory neuropathies, is unclear. We addressed this important question in a mouse model of ocular HSV-1 infection, where sensory nerve damage is a common clinical problem. Through genetic and pharmacologic targeting, we uncovered a central role for C3 in sensory nerve damage at the morphological and functional levels. Interestingly, CD4 T cells were central in facilitating this complement-mediated damage. This same C3/CD4 T cell axis triggered corneal sensory nerve damage in a mouse model of ocular graft-versus-host disease (GVHD). However, this was not the case in a T-dependent allergic eye disease (AED) model, suggesting that this inflammatory neuroimmune pathology is specific to certain disease etiologies. Collectively, these findings uncover a central role for complement in CD4 T cell-dependent corneal nerve damage in multiple disease settings and indicate the possibility for complement-targeted therapeutics to mitigate sensory neuropathies.

Vaccine-induced antibodies target sequestered viral antigens to prevent ocular HSV-1 pathogenesis, preserve vision, and preempt productive neuronal infection.

The cornea is essential for vision yet highly sensitive to immune-mediated damage following infection. Generating vaccines that provide sterile immunity against ocular surface pathogens without evoking vision loss is therefore clinically challenging. Here, we tested a prophylactic live-attenuated vaccine against herpes simplex virus type 1 (HSV-1), a widespread human pathogen that can cause corneal blindness. Parenteral vaccination of mice resulted in sterile immunity to subsequent HSV-1 challenge in the cornea and suppressed productive infection of the nervous system. This protection was unmatched by a relevant glycoprotein subunit vaccine. Efficacy of the live-attenuated vaccine involved a T-dependent humoral immune response and complement C3 but not Fcγ-receptor 3 or interferon-α/ß signaling. Proteomic analysis of viral proteins recognized by antiserum revealed an unexpected repertoire dominated by sequestered antigens rather than surface-exposed envelope glycoproteins. Ocular HSV-1 challenge in naive and subunit-vaccinated mice triggered vision loss and severe ocular pathologies including corneal opacification, scar formation, neovascularization, and sensation loss. However, corneal pathology was absent in mice receiving the live-attenuated vaccine concomitant with complete preservation of visual acuity. Collectively, this is the first comprehensive report of a prophylactic vaccine candidate that elicits resistance to ocular HSV-1 infection while fully preserving the cornea and visual acuity.

Corneal Epithelial Cells Exhibit Myeloid Characteristics and Present Antigen via MHC Class II.

Purpose: To explore the impact of ocular surface insults on the immunomodulatory capacity and phenotype of corneal epithelial cells (CECs) with a focus on epithelial-mesenchymal transition (EMT). Methods: Corneas were harvested from mice 6 days following scratch injury, ragweed pollen-induced allergy, or herpes simplex virus type 1 (HSV-1) infection and compared to healthy tissue controls. Corneas were enzymatically digested and CECs phenotypically characterized using flow cytometry. CECs were defined as epithelial cell adhesion molecule (EpCAM)-positive CD45-negative cells. CECs were assessed by PCR to evaluate EMT-associated transcripts. Recombinant HSV-1 and transgenic mice were utilized to investigate the role of vascular endothelial growth factor A (VEGFA) on the phenotype observed. The immunomodulatory potential of CECs was assessed in coculture assays with ovalbumin-specific CD4 T cells. Results: Ectopic expression of classic "myeloid" antigens Ly6G, CCR2, and CX3CR1 was identified in CEC subsets from all groups with evidence supporting an underlying partial EMT event resulting from loss of cell-cell contacts. Corneal HSV-1 infection induced Ly6C expression and major histocompatibility complex (MHC)-II upregulation in CECs through a VEGFA-linked mechanism. These Ly6C+ MHC-II+ CECs were found to function as amateur antigen-presenting cells and induced CD4 T cell proliferation in vitro. Conclusions: This study characterizes a novel immunomodulatory CEC phenotype with possible implications for immune privilege, chronic inflammation, and tissue fibrosis. Moreover, the identification of CECs masquerading with multiple "myeloid" antigens warrants careful evaluation of flow cytometry data involving corneal digests.

The Neonatal Fc Receptor and Complement Fixation Facilitate Prophylactic Vaccine-Mediated Humoral Protection against Viral Infection in the Ocular Mucosa.

The capacity of licensed vaccines to protect the ocular surface against infection is limited. Common ocular pathogens, such as HSV-1, are increasingly recognized as major contributors to visual morbidity worldwide. Humoral immunity is an essential correlate of protection against HSV-1 pathogenesis and ocular pathology, yet the ability of Ab to protect against HSV-1 is deemed limited due to the slow IgG diffusion rate in the healthy cornea. We show that a live-attenuated HSV-1 vaccine elicits humoral immune responses that are unparalleled by a glycoprotein subunit vaccine vis-à-vis Ab persistence and host protection. The live-attenuated vaccine was used to assess the impact of the immunization route on vaccine efficacy. The hierarchical rankings of primary immunization route with respect to efficacy were s.c. ≥ mucosal > i.m. Prime-boost vaccination via sequential s.c. and i.m. administration yielded greater efficacy than any other primary immunization route alone. Moreover, our data support a role for complement in prophylactic protection, as evidenced by intracellular deposition of C3d in the corneal epithelium of vaccinated animals following challenge and delayed viral clearance in C3-deficient mice. We also identify that the neonatal Fc receptor (FcRn) is upregulated in the cornea following infection or injury concomitant with increased Ab perfusion. Lastly, selective small interfering RNA-mediated knockdown of FcRn in the cornea impeded protection against ocular HSV-1 challenge in vaccinated mice. Collectively, these findings establish a novel mechanism of humoral protection in the eye involving FcRn and may facilitate vaccine and therapeutic development for other ocular surface diseases.

Impact of Type I Interferon on the Safety and Immunogenicity of an Experimental Live-Attenuated Herpes Simplex Virus 1 Vaccine in Mice.

Viral fitness dictates virulence and capacity to evade host immune defenses. Understanding the biological underpinnings of such features is essential for rational vaccine development. We have previously shown that the live-attenuated herpes simplex virus 1 (HSV-1) mutant lacking the nuclear localization signal (NLS) on the ICP0 gene (0ΔNLS) is sensitive to inhibition by interferon beta (IFN-ß) in vitro and functions as a highly efficacious experimental vaccine. Here, we characterize the host immune response and in vivo pathogenesis of HSV-1 0ΔNLS relative to its fully virulent parental strain in C57BL/6 mice. Additionally, we explore the role of type 1 interferon (IFN-α/ß) signaling on virulence and immunogenicity of HSV-1 0ΔNLS and uncover a probable sex bias in the induction of IFN-α/ß in the cornea during HSV-1 infection. Our data show that HSV-1 0ΔNLS lacks neurovirulence even in highly immunocompromised mice lacking the IFN-α/ß receptor. These studies support the translational viability of the HSV-1 0ΔNLS vaccine strain by demonstrating that, while it is comparable to a virulent parental strain in terms of immunogenicity, HSV-1 0ΔNLS does not induce significant tissue pathology.IMPORTANCE HSV-1 is a common human pathogen associated with a variety of clinical presentations ranging in severity from periodic "cold sores" to lethal encephalitis. Despite the consistent failures of HSV subunit vaccines in clinical trials spanning the past 28 years, opposition to live-attenuated HSV vaccines predicated on unfounded safety concerns currently limits their widespread acceptance. Here, we demonstrate that a live-attenuated HSV-1 vaccine has great translational potential.

Herpesvirus-Associated Lymphadenitis Distorts Fibroblastic Reticular Cell Microarchitecture and Attenuates CD8 T Cell Responses to Neurotropic Infection in Mice Lacking the STING-IFNα/ß Defense Pathways.

Type I IFN (IFN-α/ß)-driven immune responses to acute viral infection are critical to counter replication and prevent dissemination. However, the mechanisms underlying host resistance to HSV type 1 (HSV-1) are incompletely understood. In this study, we show that mice with deficiencies in IFN-α/ß signaling or stimulator of IFN genes (STING) exhibit exacerbated neurovirulence and atypical lymphotropic dissemination of HSV-1 following ocular infection. Synergy between IFN-α/ß signaling and efficacy of early adaptive immune responses to HSV-1 were dissected using bone marrow chimeras and adoptive cell transfer approaches to profile clonal expansion, effector function, and recruitment of HSV-specific CD8(+) T cells. Lymphotropic viral dissemination was commensurate with abrogated CD8(+) T cell responses and pathological alterations of fibroblastic reticular cell networks in the draining lymph nodes. Our results show that resistance to HSV-1 in the trigeminal ganglia during acute infection is conferred in part by STING and IFN-α/ß signaling in both bone marrow-derived and -resident cells, which coalesce to support a robust HSV-1-specific CD8(+) T cell response.

A Highly Efficacious Herpes Simplex Virus 1 Vaccine Blocks Viral Pathogenesis and Prevents Corneal Immunopathology via Humoral Immunity.

UNLABELLED: Correlates of immunologic protection requisite for an efficacious herpes simplex virus 1 (HSV-1) vaccine remain unclear with respect to viral pathogenesis and clinical disease. In the present study, mice were vaccinated with a novel avirulent, live attenuated virus (0ΔNLS) or an adjuvanted glycoprotein D subunit (gD-2) similar to that used in several human clinical trials. Mice vaccinated with 0ΔNLS showed superior protection against early viral replication, neuroinvasion, latency, and mortality compared to that of gD-2-vaccinated or naive mice following ocular challenge with a neurovirulent clinical isolate of HSV-1. Moreover, 0ΔNLS-vaccinated mice exhibited protection against ocular immunopathology and maintained corneal mechanosensory function. Vaccinated mice also showed suppressed T cell activation in the draining lymph nodes following challenge. Vaccine efficacy correlated with serum neutralizing antibody titers. Humoral immunity was identified as the correlate of protection against corneal neovascularization, HSV-1 shedding, and latency through passive immunization. Overall, 0ΔNLS affords remarkable protection against HSV-1-associated ocular sequelae by impeding viral replication, dissemination, and establishment of latency. IMPORTANCE: HSV-1 manifests in a variety of clinical presentations ranging from a rather benign "cold sore" to more severe forms of infection, including necrotizing stromal keratitis and herpes simplex encephalitis. The present study was undertaken to evaluate a novel vaccine to ocular HSV-1 infection not only for resistance to viral replication and spread but also for maintenance of the visual axis. The results underscore the necessity to reconsider strategies that utilize attenuated live virus as opposed to subunit vaccines against ocular HSV-1 infection.

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.

Thrombocytopenia as an adverse effect of complementary and alternative medicines, herbal remedies, nutritional supplements, foods, and beverages.

BACKGROUND: Thrombocytopenia is a well-recognized adverse effect of many drugs. However, the association of thrombocytopenia with complementary/alternative medicines, herbal remedies, nutritional supplements, foods, and beverages has been rarely described, except for reports of thrombocytopenia caused by quinine-containing beverages. OBJECTIVES: To systematically identify all published reports of thrombocytopenia associated with these substances and to assess the evidence supporting their causal association with thrombocytopenia. METHODS: Eleven databases were searched to identify relevant published reports. A priori criteria were defined for article selection and assessment. Each selected article was independently assessed by the three authors to document the presence of the criteria and determine the level of evidence for a causal association of the reported substance with thrombocytopenia. RESULTS: Twenty-seven articles were identified that reported the occurrence of thrombocytopenia with 25 substances (other than quinine). However, only six articles describing five substances (cow's milk, cranberry juice, Jui [Chinese herbal tea], Lupinus termis bean, and tahini [pulped sesame seeds]) reported clinical data supporting definite evidence of a causal association with thrombocytopenia. Four articles provided probable evidence for four additional substances, and five articles provided possible evidence for five additional substances. In the remaining articles, the association with thrombocytopenia was unlikely or the articles were excluded from review. CONCLUSIONS: Reports of thrombocytopenia describing definite or probable evidence for an association of a complementary/alternative medicines, herbal remedies, nutritional supplements, foods, and beverages are rare. Whether the occurrence of thrombocytopenia with these substances is uncommon or unrecognized is unknown.