Meredith S. Gregory-Ksander

Staphylococcus aureus Activates the NLRP3 Inflammasome in Human and Rat Conjunctival Goblet Cells

The conjunctiva is a moist mucosal membrane that is constantly exposed to an array of potential pathogens and triggers of inflammation. The NACHT, leucine rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) is a Nod-like receptor that can sense pathogens or other triggers, and is highly expressed in wet mucosal membranes. NLRP3 is a member of the multi-protein complex termed the NLRP3 inflammasome that activates the caspase 1 pathway, inducing the secretion of biologically active IL-1β, a major initiator and promoter of inflammation. The purpose of this study was to: (1) determine whether NLRP3 is expressed in the conjunctiva and (2) determine whether goblet cells specifically contribute to innate mediated inflammation via secretion of IL-1β. We report that the receptors known to be involved in the priming and activation of the NLRP3 inflammasome, the purinergic receptors P2X4 and P2X7 and the bacterial Toll-like receptor 2 are present and functional in conjunctival goblet cells. Toxin-containing Staphylococcus aureus (S. aureus), which activates the NLRP3 inflammasome, increased the expression of the inflammasome proteins NLRP3, ASC and pro- and mature caspase 1 in conjunctival goblet cells. The biologically active form of IL-1β was detected in goblet cell culture supernatants in response to S. aureus, which was reduced when the cells were treated with the caspase 1 inhibitor Z-YVAD. We conclude that the NLRP3 inflammasome components are present in conjunctival goblet cells. The NRLP3 inflammasome appears to be activated in conjunctival goblet cells by toxin-containing S. aureus via the caspase 1 pathway to secrete mature IL1-β. Thus goblet cells contribute to the innate immune response in the conjunctiva by activation of the NLRP3 inflammasome.

Overexpression of Soluble Fas Ligand following Adeno-Associated Virus Gene Therapy Prevents Retinal Ganglion Cell Death in Chronic and Acute Murine Models of Glaucoma

Glaucoma is a multifactorial disease resulting in the death of retinal ganglion cells (RGCs) and irreversible blindness. Glaucoma-associated RGC death depends on the proapoptotic and proinflammatory activity of membrane-bound Fas ligand (mFasL). In contrast to mFasL, the natural cleavage product, soluble Fas ligand (sFasL) inhibits mFasL-mediated apoptosis and inflammation and, therefore, is an mFasL antagonist. DBA/2J mice spontaneously develop glaucoma and, predictably, RGC destruction is exacerbated by expression of a mutated membrane-only FasL gene that lacks the extracellular cleavage site. Remarkably, one-time intraocular adeno-associated virus–mediated gene delivery of sFasL provides complete and sustained neuroprotection in the chronic DBA/2J and acute microbead-induced models of glaucoma, even in the presence of elevated intraocular pressure. This protection correlated with inhibition of glial activation, reduced production of TNF-α, and decreased apoptosis of RGCs and loss of axons. These data indicate that cleavage of FasL under homeostatic conditions, and the ensuing release of sFasL, normally limits the neurodestructive activity of FasL. The data further support the notion that sFasL, and not mFasL, contributes to the immune-privileged status of the eye.

Soluble Fas ligand blocks destructive corneal inflammation in mouse models of corneal epithelial debridement and LPS induced keratitis

ABSTRACT Neutrophil‐mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane‐bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL‐mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil‐mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP‐only, sFasL‐GFP, or mFasL‐GFP were expressed in the corneal stroma of C57BL/6 mice, using intra‐stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS‐induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP‐only, sFasL‐GFP, or mFasL‐GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP‐only and sFasL‐GFP groups and mild corneal inflammation in the mFasL‐GFP group. Similarly, following epithelial debridement, corneas expressing GFP‐only or sFasL‐GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL‐GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL‐GFP plasmid DNA, intrastromal injection of mFasL‐GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL‐GFP AV exacerbated LPS‐induced keratitis, corneal opacity, and neovascularization, while sFasL‐GFP AV expression prevented LPS‐induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS‐induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non‐inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS‐induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil‐mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil‐mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation. HighlightsMembrane‐bound Fas ligand and soluble Fas ligand play opposing roles in corneal inflammation.Membrane‐bound Fas ligand is pro‐inflammatory and exacerbates corneal inflammation.Soluble Fas ligand is non‐inflammatory and antagonizes the activity of membrane‐bound Fas ligand.Soluble Fas ligand specifically inhibits neutrophil‐mediated corneal inflammation and associated tissue damage.

Neither non-toxigenic Staphylococcus aureus nor commensal S. epidermidi activates NLRP3 inflammasomes in human conjunctival goblet cells

Purpose The conjunctiva is a wet mucosal surface surrounding the cornea that is continuously exposed to pathogens. Nevertheless, persistent inflammation is not observed. We examined if the NOD-like receptor pyrin domain 3 (NLRP3) inflammasome functions as a sensor that distinguishes commensal and non-pathogenic bacteria from pathogenic bacteria in human conjunctival goblet cells. Methods Goblet cells were grown from human conjunctiva and co-cultured with commensal Staphylococcus epidermidis, isogenic non-toxigenic S. aureus ACL135 and as a control toxigenic S. aureus RN6390. Activation of the NLRP3 inflammasome was determined by measuring changes in NF-κB activity, expression of pro-interleukin (IL)-1β and NLRP3, activation of caspase-1 and secretion of mature IL-1β. Goblet cell mucin secretion was measured in parallel. Results While all three strains of bacteria were able to bind to goblet cells, neither commensal S. epidermidis nor isogenic non-toxigenic S. aureus ACL135 was able to stimulate an increase in (1) NF-κB activity, (2) pro-IL-1β and NLRP3 expression, (3) caspase-1 activation, (4) mature IL-1β and (5) mucin secretion. Toxigenic S. aureus, the positive control, increased these values: knockdown of NLRP3 with small interfering RNA (siRNA) completely abolished the toxigenic S. aureus-induced expression of pro-IL-1β and secretion of mature IL-1β. Conclusions We conclude that NLRP3 serves as a sensor capable of discriminating commensal and non-pathogenic bacteria from pathogenic bacteria in conjunctival goblet cells, and that activation of the NLRP3 inflammasome induced by pathogenic bacteria mediates secretion of both mature IL-1β and large secretory mucins from these cells.

Soluble guanylate cyclase: an emerging therapeutic target in open angle glaucoma

O peptides and proteins remain a significant challenge for sustained or controlled delivery. Many of these agents have been clinically validated in ophthalmology with great success as exemplified by Macugen®, Eylea® and Lucentis®. However, there remains a critical unmet medical need to sustain or target the delivery of these agents. The clinical application of macromolecule drug substances is limited due to poor bioavailability and disposition. Direct intravitreal administration of these agents mitigates the issues of poor bioavailability, however short half‐lives relative to duration of therapy results in a requirement for frequent high dose administrations.W recently demonstrated that dietary phylloquinone/vitamin K1 releases menadione/vitamin K3 by the cleavage of the side chain in the intestine, followed by delivery of menadione through the mesenteric lymphatic system and blood circulation to tissues, where menadione is converted into MK-4/vitamin K2, and accumulates in the form of MK-4. UBIAD1is a novel MK-4 biosynthetic enzyme screened and identified from the human genome database. Surprisingly, UBIAD1 was recently shown to catalyze the CoQ10 biosynthesis in zebrafish and human cells. Enzymatic analysis using site-directed mutagenesis revealed that UBIAD1 is capable of generating various menaquinones including MK-4, and UBIAD1 missense mutations in Schnyder Corneal Dystrophy (SCD) lower MK-4 biosynthetic activity significantly. To clarify the function of UBIAD1 in vivo, we attempted to generate mice completely lacking ubiad1, a homolog of human UBIAD1by gene targeting. Ubiad1-deficient (ubiad1-/-) mouse embryosfailed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1+/mice developed normally, exhibited normal growth and fertility. Their tissue concentrations and synthetic activity of MK-4 were approximately half of the wild-type levels, whereas their tissue concentrationsand synthetic activity of CoQ9 were similar to the wild-type levels, respectively. Ubiad1-/-mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant ubiad1+/mice. These results suggest that ubiad1plays a critical rolein embryonic development through synthesizing MK-4, but may have additional functions beyond the MK-4 biosynthesis.N anterior ischemic optic neuropathy (NAION) is a sudden ischemic episode that causes permanent visual debilitation. While vision can improve somewhat following NAION, it never returns to baseline function, and many individuals suffer additional visual loss. The reason for these post-ischemic functional changes remains unknown. We hypothesized that these variations result from changes in post-infarct myelination and repair. We obtained human material from a NAION-bilaterally affected human donor who experienced NAION at distant (>20y) and near (~1-1/2y) times. We also generated tissue from the primate NAION (pNAION) and rodent NAION (rAION) models, and evaluated affected ON tissue functionally (visual evoked potentials: VEPs, compound action potentials: CAPs) and histologically using immunohistochemistry and ultrastructural analysis. Considerable post-infarct myelin damage of intact axons was found in both human and animals by confocal analysis, particularly in the human specimen with recent NAION. Ultrastructural analysis revealed myelin damage in both clinical and experimental groups. While in vivo VEPs did not show typical demyelination changes, ex vivo CAPs demonstrated slowing of axonal conduction, consistent with demyelination. Myelin damaged areas with intact axons were associated with significant cellular inflammation. Our results suggest that NAION results not only in isolated retinal ganglion cell and axonal loss, but also post-infarct demyelination and myelin damage that may be responsible for much of the post-infarct variability in visual function, late improvement and decline. Effective NAION treatments may focus on immunomodulation to improve the inflammatory response, reduce long-term, post-infarct demyelination and damage, and improve clinical functional outcomes.Conjunctival papilloma is a benign growth arising from the stratified squamous epithelium of the conjunctiva. It is sometimes described as benign and self limiting and known to occur in both children and adults. The case of a five year old girl who was seen at the University of Benin Teaching Hospital with severe, long standing, bilateral squamous cell papilloma is presented. She had a twelve month history of multiple growths in the conjunctiva of both eyes. Over time, the growths gradually covered most of the palpebral fissure in both eyes. They resulted in visual impairment from obliteration of the gap between the lids when the eyes are open due to the dense, tightly packed fleshy new growth in both eyes, worse in the left eye. The patient’s management involved: Excisional biopsy in both eyes under general anaesthesia, intraoperative use of Mitomycin C and post operative use of Targamet tablets 200 mg BD to prevent recurrence. The definitive histological diagnosis was benign conjunctival papilloma. Various treatment modalities for conjunctival papilloma have been described in the literature. These include conservative management, surgical excision, topical interferon alpha-2b, Carbon dioxide laser, and cryoablation. Mitomycin C has been used as an adjunct therapy after surgical debulking. Excision biopsy was chosen as the treatment modality in this patient due to the level of severity at presentation and the fact that the conjunctiva masses interfered with vision. None of the previously reported complications of use of Mitomycin C occurred in this patient.Interferon alphas are a family of cytokines produced by white blood cells which have antiviral, antiproliferative, and immunomodulatory properties. Systemic interferon alpha-2a has demonstrated efficacy in ocular inflammatory disease of non-infectious cause, most notably Behcet’s disease associated uveitis. Increasingly, the efficacy of systemically administered interferon alphas for uveitic CME, both in the setting of active and inactive uveitis, is being elucidated.I and oxidative stress play an important role in the development and progression of diabetic retinopathy and other retinal vascular diseases. These processes contribute significantly to retinal damage and dysfunction in these conditions, including vascular permeability, neuronal impairment, and capillary degeneration. It is therefore critical to gain further insights into the mechanisms governing oxidative stress and inflammation in the retina, as well as approaches for modulating these processes. The transcription factor nuclear factor erythroid-2-related factor 2 (also known as NFE2L2 or NRF2) is an important regulator of oxidative stress and also has anti-inflammatory effects. It is well-known to play a cytoprotective role in many tissues systemically, but its effects in the retina have been less clear. Our lab has shown that Nrf2 is expressed in the retina in multiple cell types. In order to determine the role of Nrf2, we are examining the effects of Nrf2 deficiency in mouse models of retinal vascular disease, including ischemia-reperfusion injury and diabetic retinopathy. We find that Nrf2 knockout mice (Nrf2 -/-) exhibit significant exacerbation of oxidative stress and inflammation in these models. In addition, Nrf2 knockout mice exhibit increase in pathological endpoints in these models, including retinal vascular permeability, neuronal impairment, and capillary degeneration. With systemic administration of a triterpenoid, we are able to activate Nrf2 in the retina and thereby ameliorate retinal damage. Together, this indicates that Nrf2 plays a protective role in the retina, and pharmacologic modulation of Nrf2 represents a potential therapeutic strategy for retinal vascular disease processes.C is the leading cause of blindness worldwide, and cataract surgery is one of the most common surgical procedures. In developed country settings, cataract surgery is most commonly performed by phacoemulsification (PE). Recently, a new procedure, Femtosecond-laser-assisted cataract surgery (FLACS), has been introduced in which a laser is used to perform certain important steps of cataract surgery. There is the potential for FLACS to reduce complications of cataract surgery, in particular as they relate to corneal endothelial damage and wound architecture. The former is associated with pseudophakic bullous keratopathy (PBK), a complication of cataract surgery in which corneal endothelial cells arelost. An improvement in wound architecture may reduce the rate of post-operative endophthalmitis (POE) and wound leaks. However, actual clinical benefits from use of FLACS have not been documented in controlled studies. Further, the outcome of FLACS has not been studied in an objective and methodical way. Such studies are particularly required given the substantial increase in operative time and expense associated with FLACS. In this talk, we will objectively assess the major studies that have been published to date addressing FLACS.L research represents an explosive field of new discovery in recent years. The cornea provides an ideal tissue for lymphatic research due to its accessible location, transparent nature, and lymphatic-free but-inducible features. Once induced, corneal lymphatic vessels enhance high volume delivery of antigens and immune cells, and accelerate disease progression and transplant rejection. Our research goal is to elucidate the molecular and cellular mechanisms of lymphangiogenesis and to identify new targets for therapeutic intervention. This presentation is to introduce our recent data on corneal lymphatic research and to provide novel insights into corneal lymphangiogenesis, a multi-facet event associated with the pathogenesis of many diseases after an inflammatory, infectious, immunogenic, traumatic, or chemical insult.A (AF) measurement with two-photon microscopy (TPM) and fluorescence lifetime imaging (FLIM) enables the discrimination of different fluorescence molecules located in a different depth of the tissue. We investigated the AF and fluorescence lifetime (FLT) of retinal cells focusing on retinal pigment epithelial (RPE) cells and photoreceptor outer segments (POS). Lipid peroxidation was induced in the RPE and POS of the porcine explants by FeSO4, and the tissues were investigated with TPM and FLIM. Furthermore, the RPE explants were examined with immunofluorescence study for 4-hydroxynonenal (4-HNE)-adducts, adipocyte differentiation related protein (ADFP), and rhodopsin. TPM-AF in RPE cells is mostly originated from the melanosomes under normal conditions, which have a very short fluorescence lifetime (FLT) (mean=117 ps). FeSO4 exposure leads to the appearance of bright granular AF inside and around RPE cells, whose FLT is significantly longer (mean=1388 ps) than melanosome-AF. FeSO4 exposure increases the fluorescence intensity of POS-AF and shortens their FLT. In the immunofluorescence studies, strong 4-HNE staining was observed intraand pericellularly of RPE cells under lipid peroxidation, whose localization was partially consistent with rhodopsin and ADFP, respectively, which suggests that the FeSO4-induced bright AF granules inside/around RPE cells are suggested to be the oxidized POS-membranes and the retinoid-storing inclusions, respectively. From these results, 4-HNE-adducts may be one of the main fluorophores of these AF. Since the amount of 4-HNE-adducts is suggested to reflect cellular oxidative stress status, TPM with FLIM might be a useful tool to detect oxidative stress status of RPE cells and the POS.Methods: A model eye was constructed by incorporating a phase delay map for a diffractive optical element into a reducedeye model incorporating ocular chromatic aberration, pupil apodization, and higher order monochromatic aberrations. The diffractive element was either a monofocal lens of +3.2D diffractive power, or two types of bifocal lenses (unapodized or apodized) of +2.92D ‘add’ power. Polychromatic point-spread functions and image quality for white and monochromatic light were quantified for a series of target vergences, wavelengths and pupil diameters, using modulation transfer functions and a variety of image quality metrics.C epithelial stem cells at the limbus in response to wound are triggered to migrate and suppress proliferation at leading edge. Increased proliferation of basal epithelial cells containing stem cells and concomitant stratification occurs leading to migration of epithelial cell sheet. The extracellular and intracellular signaling pathways that regulate these processes in corneal epithelium by modulating the cell-cell matrix and cell-cell adhesion are less understood. In order to understand the maintenance and functioning of stable cell-cell junctions in corneal epithelium, we studied the protein CDK5 at cell-cell and cell-matrix adhesions during migration. Immuno fluorescence showed co-localization of CDK5, p35, and CDK5(pY15, active form of CDK5)with E-cadherin at cell-cell boundaries indicating that they form an intracellular complex. Inhibiting CDK5 activity with olomoucine (pharmacological inhibitor of CDK5) increased the degradation of surface-biotinylated E-cadherin, generating a degradation product of 29KDa. In contrast, p120 catenin levels were increased two-fold. Similar changes in E-cadherin and p120 expression were seen in cells treated with the CDK5 inhibitor, olomoucine. Reduced E-cadherin immunofluorescence intensity in ShCDK5 was similar to olomoucine treated cells. TIRF analysis of pEGFP-E cadherin in live transfected cells revealed trafficking of cadherin was reduced as opposed to p120. Suppression of CDK5 expression with Sh RNA confirmed previous results obtained with olomoucine and indicating arole for CDK5 instabilizing cell-cell junctions. CDK5 Kinase is therefore required preventing the dissociation, degradation and cadherin–catenins witch, indicating that CDK5 was required for maintaining stable epithelial cell-cell adhesions, homeostasis and tissue repair.W the best animal model for age-related macular degeneration (AMD) should be the nonhuman primate because it has a macula leutea, the cost, availability, and ethical issues have largely limited its usage. Instead, researchers prefer to use the mouse as it can be genetically manipulated. Although the mouse has no maculae and rarely develops drusen, its neuroretina and retinal pigment epithelium (RPE) can develop lesions simulating certain features of AMD. Such lesions include focal RPE and photoreceptor degeneration as well as increased A2E levels. Different genetically engineered AMD mouse models often provide valuable information into AMD pathogenesis and have become useful tools for screening novel preventive and therapeutic compounds for the disease. While using a mouse model can have great benefit on this preclinical research, appropriate controls are critical to assess experiments. In general, there are group control and self control paradigms for the experimental design. In the group control experiment, the genetically manipulated mice with retinal AMD-like lesions and the wild type control mice without retinal AMD-like lesions are randomized by age, size, and weight. The AMD mice and control mice are further divided to treated and non-treated (or placebo-treated) groups (4 total). At the end of the experiment, the group of treated mice is compared to control group of the same mouse strain to determine the efficacy of treatment. In the self control experiment, the two eyes of the same mouse are treated differently; one eye receives a therapeutic agent and the contralateral eye receives a control agent or placebo. At the end of the experiment, the same mouse’s eyes are compared. However, in addition to the lack of macula, the mouse models have other limitations, including small globe size and simple retinal structure, for which some other models have been developed to address. Rat models have the advantage of larger eye size than mouse. Rabbits has a totally dissimilar retinal vasculature compared to human eyes and can be only applied for evaluation of pharmacokinetics and pharmacodynamics. While the pig eye has similar measurements and an area of enriched cone density like humans, more studies are required in this animal to validate its usefulness in AMD studies. In any of these models, findings may not always reflect those in AMD patients. Careful interpretations of animal experiments with clinical and in vitro data will improve our understanding of AMD pathogenesis and our ability to prevent and treat the disease.