Person:

Gregory-Ksander, Meredith

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNF (\alpha) triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the (α_1) subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase (α_1)–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the (α_1) and (β_1) subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

Purpose: CD36 is a Class B scavenger receptor that is constitutively expressed in the corneal epithelium and has been implicated in many homeostatic functions, including the homeostasis of the epidermal barrier. The aim of this study is to determine (1) whether CD36 is required for the maintenance of the corneal epithelial barrier to infection, and (2) whether CD36-deficient mice present with an increased susceptibility to bacterial keratitis. Methods: The corneas of CD36−/−, TSP1−/−, TLR2−/−, and C57BL/6 WT mice were screened via slit lamp microscopy or ex vivo analysis. The epithelial tight junctions and mucin layer were assessed via LC-biotin and Rose Bengal staining, respectively. Bacterial quantification was performed on corneal buttons and GFP-expressing Staphylococcus aureus was used to study bacterial binding. Results: CD36−/− mice develop spontaneous corneal defects that increased in frequency and severity with age. The mild corneal defects were characterized by a disruption in epithelial tight junctions and the mucin layer, an infiltrate of macrophages, and increased bacterial binding. Bacterial quantification revealed high levels of Staphylococcus xylosus in the corneas of CD36−/− mice with severe defects, but not in wild-type controls. Conclusions: CD36−/− mice develop spontaneous bacterial keratitis independent of TLR2 and TSP1. The authors conclude that CD36 is a critical component of the corneal epithelial barrier, and in the absence of CD36 the barrier breaks down, allowing bacteria to bind to the corneal epithelium and resulting in spontaneous keratitis. This is the first report of spontaneous bacterial keratitis in mice.

A fully automated and robust method was developed to quantify b-III-tubulin-stained retinal ganglion cells, combining computational recognition of individual cells by CellProfiler and a machine-learning tool to teach phenotypic classification of the retinal ganglion cells by CellProfiler Analyst. In animal models of glaucoma, quantification of immunolabeled retinal ganglion cells is currently performed manually and remains time-consuming. Using this automated method, quantifications of retinal ganglion cell images were accelerated tenfold: 1800 images were counted in 3 h using our automated method, while manual counting of the same images took 72 h. This new method was validated in an established murine model of microbead-induced optic neuropathy. The use of the publicly available software and the method's user-friendly design allows this technique to be easily implemented in any laboratory.

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.