Intrinsic Antiviral Activity of Optineurin Prevents Hyperproliferation of a Primary Herpes Simplex Virus Type 2 Infection.

Very little knowledge exists on virus-specific host cell intrinsic mechanisms that prevent hyperproliferation of primary HSV type 2 (HSV-2) genital infections. In this study, we provide evidence that the Nemo-related protein, optineurin (OPTN), plays a key role in restricting HSV-2 infection both in vitro and in vivo. Contrary to previous reports regarding the proviral role of OPTN during Sendai virus infection, we demonstrate that lack of OPTN in cells causes enhanced virus production. OPTN deficiency negatively affects the host autophagy response and results in a marked reduction of CCL5 induction. OPTN knockout (OPTN-/-) mice display exacerbated genital disease and dysregulated T cell frequencies in infected tissues and lymph nodes. A human transcriptomic profile dataset provides further credence that a strong positive correlation exists between CCL5 upregulation and OPTN expression during HSV-2 genital infection. Our findings underscore a previously unknown OPTN/CCL5 nexus that restricts hyperproliferative spread of primary HSV-2 infection, which may constitute an intrinsic host defense mechanism against herpesviruses in general.

Cellular senescence implication in mustard keratopathy.

Mustard agents are vesicants that were used in warfare multiple times. They are potent alkylating agents that activate cellular pathways of apoptosis, increase oxidative stress, and induce inflammation. Eyes are particularly susceptible to mustard exposure with a wide range of ocular surface damage. Three main categories of mustard-related eye injuries are acute, chronic, and delayed-onset manifestations. Mustard keratopathy (MK) is a known complication characterized by corneal opacification, ulceration, thinning, and neovascularization that can lead to severe vision loss and discomfort. Recently, a few reports demonstrated the role of senescence induction as a new pathological mechanism in mustard-related injuries that could affect wound healing. We ran the first murine model of delayed-onset MK and nitrogen mustard-induced senescence, evaluating the pathological signs of senescence in the cornea using beta-galactosidase staining. Our results suggest that nitrogen mustard exposure causes senescence in the corneal cells, which could be the underlying mechanism for chronic and late-onset ocular surface damage. We also found a significant correlation between the percentage of positive beta-galactosidase staining and the degree of fibrosis in the cornea. This provides valuable insight into the possible role of anti-senescence drugs in the near future for accelerating corneal healing and restricting fibrosis in patients with mustard keratopathy.

Cellular senescence and ophthalmic diseases: narrative review.

PURPOSE: Cellular senescence is a state of permanent growth arrest whereby a cell reaches its replicative limit. However, senescence can also be triggered prematurely in certain stressors including radiation, oxidative stress, and chemotherapy. This stress-induced senescence has been studied in the context of promoting inflammation, tumor development, and several chronic degenerative diseases of aging. Emerging research has elucidated the role of senescence in various ocular diseases. METHODS: The literature search was performed using PubMed with using the query (senescence OR aging) AND (eye disease OR ocular disease OR ophthalmic disease OR cornea OR glaucoma OR cataract OR retina) on October 20th, 2022. No time restriction was proposed. Articles were excluded if they were not referenced in English. RESULTS: Overall, 51 articles regarding senescence and ocular diseases were found and summarized in this study. Several signaling pathways have been implicated in the development of senescence. Currently, senescence has been linked to various corneal and retinal pathologies, as well as cataract and glaucoma. Given the number of pathologies, senolytics, which are small molecules with the ability to selective targeting of senescent cells, can be used as therapeutic or prophylactic agents. CONCLUSIONS: Senescence has been shown to underlie the pathogenesis of numerous ocular diseases. The overall literature on senescence and ocular disease is growing rapidly. There is an ongoing debate whether or not cellular senescence detected in experiments contributes in a significant way to diseases. Research on understanding the mechanism of senescence from ocular cells and tissues is just beginning. Multiple animal models are required to test potential senolytics. Currently, no studies exist to date which have demonstrated the benefits of senolytic therapies in human studies.

Therapeutic Potential of Mesenchymal Stem Cell-Secreted Factors on Delay in Corneal Wound Healing by Nitrogen Mustard.

Ocular surface exposure to nitrogen mustard (NM) leads to severe ocular toxicity which includes the separation of epithelial and stromal layers, loss of endothelial cells, cell death, and severe loss of tissue function. No definitive treatment for mustard gas-induced ocular surface disorders is currently available. The research was conducted to investigate the therapeutic potential of mesenchymal stem cell-conditioned media (MSC-CM) in NM-induced corneal wounds. NM was added to different types of corneal cells, the ocular surface of porcine, and the ocular surface of mice, followed by MSC-CM treatment. NM significantly induced apoptotic cell death, cellular ROS (Reactive oxygen species), and reduced cell viability, metabolic gene expression, and mitochondrial function, and, in turn, delayed wound healing. The application of MSC-CM post NM exposure partially restored mitochondrial function and decreased intracellular ROS generation which promoted cell survival. MSC-CM therapy enhanced wound healing process. MSC-CM inhibited NM-induced apoptotic cell death in murine and porcine corneal tissue. The application of MSC-CM following a chemical insult led to significant improvements in the preservation of corneal structure and wound healing. In vitro, ex vivo, and in vivo results suggest that MSC-CM can potentially provide targeted therapy for the treatment of chemical eye injuries, including mustard gas keratopathy (MGK) which presents with significant loss of vision alongside numerous corneal pathologies.

Heparanase-Regulated Syndecan-1 Shedding Facilitates Herpes Simplex Virus 1 Egress.

Herpes simplex virus 1 (HSV-1) can infect virtually all cell types in vitro An important reason lies in its ability to exploit heparan sulfate (HS) for attachment to cells. HS is a ubiquitous glycosaminoglycan located on the cell surface and tethered to proteoglycans such as syndecan-1. Previously, we have shown that heparanase (HPSE) facilitates the release of viral particles by cleaving HS. Here, we demonstrate that HPSE is a master regulator where, in addition to directly enabling viral release via HS removal, it also facilitates cleavage of HS-containing ectodomains of syndecan-1, thereby further enhancing HSV-1 egress from infected cells. Syndecan-1 cleavage is mediated by upregulation of matrix metalloproteases (MMPs) that accompanies higher HPSE expression in infected cells. By overexpressing HPSE, we have identified MMP-3 and MMP-7 as important sheddases of syndecan-1 shedding in corneal epithelial cells, which are natural targets of HSV-1 infection. MMP-3 and MMP-7 were also naturally upregulated during HSV-1 infection. Altogether, this paper shows a new connection between HSV-1 release and syndecan-1 shedding, a phenomenon that is regulated by HPSE and executed by the MMPs. Our results also identify new molecular markers for HSV-1 infection and new targets for future interventions.IMPORTANCE HSV-1 is a common cause of recurrent viral infections in humans. The virus can cause a range of mucosal pathologies. Efficient viral egress from infected cells is an important step for HSV-1 transmission and virus-associated pathologies. Host mechanisms that contribute to HSV-1 egress from infected cells are poorly understood. Syndecan-1 is a common heparan sulfate proteoglycan expressed by many natural target cells. Despite its known connection with heparanase, a recently identified mediator of HSV-1 release, syndecan-1 has not been previously investigated in HSV-1 release. In this study, we demonstrate that the shedding of syndecan-1 by MMP-3 and MMP-7 supports viral egress. We show that the mechanism behind the activation of these MMPs is mediated by heparanase, which is upregulated upon HSV-1 infection. Our study elucidates a new connection between HSV-1 egress, heparanase, and matrix metallopeptidases; identifies new molecular markers of infection; and provides potential new targets for therapeutic interventions.

Emerging Roles of Heparan Sulfate Proteoglycans in Viral Pathogenesis.

Heparan sulfate is a glycosaminoglycan present in nearly all mammalian tissues. Heparan sulfate moieties are attached to the cell surface via heparan sulfate proteoglycans (HSPGs) which are composed of a protein core bound to multiple heparan sulfate chains. HSPGs contribute to the structural integrity of the extracellular matrix and participate in cell signaling by releasing bound cytokines and chemokines once cleaved by an enzyme, heparanase. HSPGs are often exploited by viruses during infection, particularly during attachment and egress. Loss or inhibition of HSPGs initially during infection can yield significant decreases in viral entry and infectivity. In this review, we provide an overview of HSPGs in the lifecycle of multiple viruses, including herpesviruses, human immunodeficiency virus, dengue virus, human papillomavirus, and coronaviruses.

Pathobiology and treatment of viral keratitis.

Keratitis is one of the most prevalent ocular diseases manifested by partial or total loss of vision. Amongst infectious (viz., microbes including bacteria, fungi, amebae, and viruses) and non-infectious (viz., eye trauma, chemical exposure, and ultraviolet exposure, contact lens) risk factors, viral keratitis has been demonstrated as one of the leading causes of corneal opacity. While many viruses have been shown to cause keratitis (such as rhabdoviruses, coxsackieviruses, etc.), herpesviruses are the predominant etiologic agent of viral keratitis. This chapter will summarize current knowledge on the prevalence, diagnosis, and pathobiology of viral keratitis. Virus-mediated immunomodulation of host innate and adaptive immune components is critical for viral persistence, and dysfunctional immune responses may cause destruction of ocular tissues leading to keratitis. Immunosuppressed or immunocompromised individuals may display recurring disease with pronounced severity. Early diagnosis of viral keratitis is beneficial for disease management and response to treatment. Finally, we have discussed current and emerging therapies to treat viral keratitis.

Heparanase, cell signaling, and viral infections.

Heparanase (HPSE) is a multifunctional protein endowed with many non-enzymatic functions and a unique enzymatic activity as an endo-ß-D-glucuronidase. The latter allows it to serve as a key modulator of extracellular matrix (ECM) via a well-regulated cleavage of heparan sulfate side chains of proteoglycans at cell surfaces. The cleavage and associated changes at the ECM cause release of multiple signaling molecules with important cellular and pathological functions. New and emerging data suggest that both enzymatic as well as non-enzymatic functions of HPSE are important for health and illnesses including viral infections and virally induced cancers. This review summarizes recent findings on the roles of HPSE in activation, inhibition, or bioavailability of key signaling molecules such as AKT, VEGF, MAPK-ERK, and EGFR, which are known regulators of common viral infections in immune and non-immune cell types. Altogether, our review provides a unique overview of HPSE in cell-survival signaling pathways and how they relate to viral infections.

In vitro protocol for the optimal induction of inflammation in human monocyte cell lines.

The study of inflammation is of key interest to biomedical research; however, techniques to induce inflammation in vitro are difficult to implement. Here, we present a protocol that optimizes the induction and measurement of NF-kB-mediated inflammation in vitro using a human macrophage cell line. We describe steps for growing, differentiating, and inducing inflammation in THP-1 cells. We detail the process of staining and grid-based confocal imaging. We also discuss approaches to test anti-inflammatory drug efficacy in inhibiting inflammatory milieu. For complete details on the use and execution of this protocol, please refer to Koganti et al. (2022).1.

Concise Review: Bioengineering of Limbal Stem Cell Niche.

The corneal epithelium is composed of nonkeratinized stratified squamous cells and has a significant turnover rate. Limbal integrity is vital to maintain the clarity and avascularity of the cornea as well as regeneration of the corneal epithelium. Limbal epithelial stem cells (LESCs) are located in the basal epithelial layer of the limbus and preserve this homeostasis. Proper functioning of LESCs is dependent on a specific microenvironment, known as the limbal stem cell niche (LSCN). This structure is made up of various cells, an extracellular matrix (ECM), and signaling molecules. Different etiologies may damage the LSCN, leading to limbal stem cell deficiency (LSCD), which is characterized by conjunctivalization of the cornea. In this review, we first summarize the basics of the LSCN and then focus on current and emerging bioengineering strategies for LSCN restoration to combat LSCD.

Artificial Intelligence and Infectious Keratitis: Where Are We Now?

Infectious keratitis (IK), which is one of the most common and catastrophic ophthalmic emergencies, accounts for the leading cause of corneal blindness worldwide. Different pathogens, including bacteria, viruses, fungi, and parasites, can cause IK. The diagnosis and etiology detection of IK pose specific challenges, and delayed or incorrect diagnosis can significantly worsen the outcome. Currently, this process is mainly performed based on slit-lamp findings, corneal smear and culture, tissue biopsy, PCR, and confocal microscopy. However, these diagnostic methods have their drawbacks, including experience dependency, tissue damage, cost, and time consumption. Diagnosis and etiology detection of IK can be especially challenging in rural areas or in countries with limited resources. In recent years, artificial intelligence (AI) has opened new windows in medical fields such as ophthalmology. An increasing number of studies have utilized AI in the diagnosis of anterior segment diseases such as IK. Several studies have demonstrated that AI algorithms can diagnose and detect the etiology of IK accurately and fast, which can be valuable, especially in remote areas and in countries with limited resources. Herein, we provided a comprehensive update on the utility of AI in IK.

Applications of mesenchymal stem cells in ocular surface diseases: sources and routes of delivery.

INTRODUCTION: Mesenchymal stem cells (MSCs) are novel, promising agents for treating ocular surface disorders. MSCs can be isolated from several tissues and delivered by local or systemic routes. They produce several trophic factors and cytokines, which affect immunomodulatory, transdifferentiating, angiogenic, and pro-survival pathways in their local microenvironment via paracrine secretion. Moreover, they exert their therapeutic effect through a contact-dependent manner. AREAS COVERED: In this review, we discuss the characteristics, sources, delivery methods, and applications of MSCs in ocular surface disorders. We also explore the potential application of MSCs to inhibit senescence at the ocular surface. EXPERT OPINION: Therapeutic application of MSCs in ocular surface disorders are currently under investigation. One major research area is corneal epitheliopathies, including chemical or thermal burns, limbal stem cell deficiency, neurotrophic keratopathy, and infectious keratitis. MSCs can promote corneal epithelial repair and prevent visually devastating sequelae of non-healing wounds. However, the optimal dosages and delivery routes have yet to be determined and further clinical trials are needed to address these fundamental questions.

Ocular graft-versus-host disease (oGVHD): From A to Z.

Allogeneic hematopoietic stem cell transplantation is a definitive therapy for a variety of disorders. One of the complications is acute graft-versus-host disease (aGVHD), which has a high mortality rate. Patients can also develop chronic graft-versus-host disease (cGVHD), a more indolent yet afflicting condition that affects up to 70% of patients. Ocular involvement (oGVHD) is one of the most prevalent presentations of cGVHD and can manifest as dry eye disease, meibomian gland dysfunction, keratitis, and conjunctivitis. Early recognition of ocular involvement using regular clinical assessments as well as robust biomarkers can aid in better management and prevention. Currently, the therapeutic strategies for the management of cGVHD, and oGVHD in particular, have mainly focused on the control of symptoms. There is an unmet need for translating the preclinical and molecular understandings of oGVHD into clinical practice. Herein, we have comprehensively reviewed the pathophysiology, pathologic features, and clinical characteristics of oGVHD and summarized the therapeutic landscape available to combat it. We also discuss the direction of future research regarding a more directed delineation of pathophysiologic underpinnings of oGVHD and the development of preventive interventions.

Pathophysiology of reinfection by exogenous HSV-1 is driven by heparanase dysfunction.

Limited knowledge exists on exogenous DNA virus reinfections. Herpes simplex virus-1 (HSV-1), a prototype DNA virus, causes multiple human diseases including vision-threatening eye infections. While reinfection with an exogenous HSV-1 strain is considered plausible, little is known about the underlying mechanisms governing its pathophysiology in a host. Heparanase (HPSE), a host endoglycosidase, when up-regulated by HSV-1 infection dictates local inflammatory response by destabilizing tissue architecture. Here, we demonstrate that HSV-1 reinfection in mice causes notable pathophysiology in wild-type controls compared to the animals lacking HPSE. The endoglycosidase promotes infected cell survival and supports a pro-disease environment. In contrast, lack of HPSE strengthens intrinsic immunity by promoting cytokine expression, inducing necroptosis of infected cells, and decreasing leukocyte infiltration into the cornea. Collectively, we report that immunity from a recent prior infection fails to abolish disease manifestation during HSV-1 reinfection unless HPSE is rendered inactive.

Wound-Healing Effects of Mesenchymal Stromal Cell Secretome in the Cornea and the Role of Exosomes.

Mesenchymal stromal/stem cells (MSCs) and their secreted factors have been shown to have immunomodulatory and regenerative effects. In this study, we investigated human bone-marrow-derived MSC secretome (MSC-S) for the treatment of corneal epithelial wounds. Specifically, we evaluated the role of MSC extracellular vesicles (EV)/exosomes in mediating the wound-healing effects of the MSC-S. In vitro studies using human corneal epithelial cells showed that MSC-CM increased cell proliferation in HCEC and HCLE cells, while EV-depleted MSC-CM showed lower cell proliferation in both cell lines compared to the MSC-CM group. In vitro and in vivo experiments revealed that 1X MSC-S consistently promoted wound healing more effectively than 0.5X MSC-S, and MSC-CM promoted wound healing in a dose-dependent manner, while exosome deprivation delayed wound healing. We further evaluated the incubation period of MSC-CM on corneal wound healing and showed that MSC-S collected for 72 h is more effective than MSC-S collected for 48 h. Finally, we evaluated the stability of MSC-S under different storage conditions and found that after one cycle of freeze-thawing, MSC-S is stable at 4 °C for up to 4 weeks. Collectively, we identified the following: (i) MSC-EV/Exo as the active ingredient in MSC-S that mediates the wound-healing effects in the corneal epithelium, providing a measure to optimize its dosing for a potential clinical product; (ii) Treatment with EV/Exo-containing MSC-S resulted in an improved corneal barrier and decreased corneal haze/edema relative to EV/Exo-depleted MSC-S; (iii) The stability of MSC-CM for up to 4 weeks showed that the regular storage condition did not significantly impact its stability and therapeutic functions.

Intrastromal versus subconjunctival injection of mesenchymal stem/stromal cells for promoting corneal repair.

PURPOSE: Different approaches to delivery of mesenchymal stem/stromal cells (MSCs) for ameliorating corneal injuries have been investigated. This study was aimed to compare the efficacy of intrastromal and subconjunctival injection of human bone marrow-derived MSCs (hBM-MSCs) in a corneal epithelial injury model. METHODS: Twenty-four C57BL/6J mice underwent total corneal and limbal epithelial debridement. Then, the mice were divided into three different groups: (1) intrastromal hBM-MSCs injection, (2) subconjunctival hBM-MSCs injection, and (3) injection of frozen medium as a control. Mice were monitored by slit lamp and underwent anterior segment optical coherence tomography (ASOCT). Following euthanasia, the corneas were further evaluated by histology and immunostaining. RESULTS: hBM-MSC injection successfully healed epithelial defects regardless of the delivery route (P < 0.001). However, intrastromal injection was superior to subconjunctival injection in reducing defect area (P = 0.001). Intrastromal injection of hBM-MSCs also significantly reduced corneal opacity and neovascularization and improved ASOCT parameters compared to subconjunctival injection or no treatment (P < 0.001, P = 0.003, and P < 0.001, respectively). Although both of the treatment groups were positive for CK12 and had reduced levels of MUC5AC compared to the control, CK12 staining was stronger in the intrastromal group compared to the subconjunctival group. Also, persistency of MSCs was confirmed by in vivo (up to 2 weeks) and in vitro assessments (up to 4 weeks). CONCLUSIONS: Although the injection of hBM-MSC using both intrastromal and subconjunctival methods improve wound healing and reduce neovascularization and opacity, the intrastromal approach is superior in terms of corneal healing.

From the diagnosis of infectious keratitis to discriminating fungal subtypes; a deep learning-based study.

Infectious keratitis (IK) is a major cause of corneal opacity. IK can be caused by a variety of microorganisms. Typically, fungal ulcers carry the worst prognosis. Fungal cases can be subdivided into filamentous and yeasts, which shows fundamental differences. Delays in diagnosis or initiation of treatment increase the risk of ocular complications. Currently, the diagnosis of IK is mainly based on slit-lamp examination and corneal scrapings. Notably, these diagnostic methods have their drawbacks, including experience-dependency, tissue damage, and time consumption. Artificial intelligence (AI) is designed to mimic and enhance human decision-making. An increasing number of studies have utilized AI in the diagnosis of IK. In this paper, we propose to use AI to diagnose IK (model 1), differentiate between bacterial keratitis and fungal keratitis (model 2), and discriminate the filamentous type from the yeast type of fungal cases (model 3). Overall, 9329 slit-lamp photographs gathered from 977 patients were enrolled in the study. The models exhibited remarkable accuracy, with model 1 achieving 99.3%, model 2 at 84%, and model 3 reaching 77.5%. In conclusion, our study offers valuable support in the early identification of potential fungal and bacterial keratitis cases and helps enable timely management.

STAND alert! Prokaryotic immunity for recognition and defense against bacteriophages.

In a recent article, Gao et al. diversify our knowledge of prokaryotic innate immunity by characterizing a novel bacterial defense system that utilizes nucleotide-binding oligomerization-like receptors (NLRs) for recognizing phage proteins.

Topical phenylbutyrate antagonizes NF-κB signaling and resolves corneal inflammation.

Chronic inflammation of the immune privileged cornea originating from viral or nonviral conditions results in significant vision loss. Topical corticosteroids are the common treatments for corneal inflammation, but the drugs cause serious and potentially blinding side effects in the long term. Therefore, new standalone and/or synergistic anti-inflammatory therapies with lower side effects are desperately needed. Here, we show that the aromatic fatty acid phenylbutyrate (PBA) acts as a potent inhibitor of inflammation in preclinical ocular-inflammation models. PBA prevents the transcription as well as translation of pro-inflammatory cytokines by LPS and poly(I:C) via persistent inhibition of NF-κB signaling. PBA quickens the resolution of ocular inflammation in mice by decreasing corneal thickness and immune cell infiltration. More importantly, PBA can synergize with the dexamethasone to antagonize NF-κB signaling at lower drug concentrations. Our results demonstrate that PBA therapy exerts previously unreported anti-inflammatory effects in the eye and facilitates corneal healing during persistent inflammation.

Infection-Induced Porcine Ex Vivo Corneal Wound Model to Study the Efficacy of Herpes Simplex Virus-1 Entry and Replication Inhibitors.

Corneal infections by viruses and bacteria can result in ocular surface defects, ulcers, or wounds. Herpes simplex virus type-1 (HSV-1) is a human virus with global seroprevalence in the range of 60-90%. While the virus more commonly causes mucocutaneous lesions including ulcers on the face and mouth, it is also a leading cause of infection-associated blindness. In this chapter, we discuss an in-depth protocol required to evaluate corneal damage due to HSV-1 infection using porcine models of ex vivo infection. Our methods can be adapted to study similar infections caused by other viruses and bacteria.