ABCB5 is a limbal stem cell gene required for corneal development and repair.

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

An open-source computational tool to automatically quantify immunolabeled retinal ganglion cells.

A fully automated and robust method was developed to quantify ß-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.

Increased resistance to Staphylococcus aureus endophthalmitis in BALB/c mice: Fas ligand is required for resolution of inflammation but not for bacterial clearance.

FasL was recently shown be required for bacterial clearance in C57BL/6 mice that express the FasL.1 allotype. The FasL.2 allotype is expressed in BALB/c mice and exhibits increased binding affinity to and increased cytotoxic activity against Fas(+) target cells. Therefore, we hypothesized that BALB/c mice would be more resistant to Staphylococcus aureus-induced endophthalmitis. To test this hypothesis, C57BL/6, BALB/c, and BALB(gld) mice received intravitreal injections of 2,500 CFU of S. aureus (RN6390). Clinical examinations, electroretinography (ERG), histology, and bacterial quantification were performed at 24, 48, 72, and 96 h postinjection. The myeloperoxidase (MPO) assay was used to quantitate neutrophil infiltration. At 96 h postinfection, 86% of C57BL/6 mice presented with complete destruction of the eye, compared to only 29% of BALB/c mice with complete destruction. To our surprise, in the absence of Fas ligand, BALB(gld) mice showed no difference in bacterial clearance compared to BALB/c mice. However, histology and ERG analysis revealed increased retinal damage and significant loss of retinal function. MPO analysis revealed equal numbers of neutrophils in BALB(gld) and BALB/c mice at 24 h postinfection. However, at 48 h, the neutrophil numbers remained significantly elevated in BALB(gld) mice, correlating with the increased retinal damage observed in BALB(gld) mice. We conclude that the increased resistance to S. aureus induced endophthalmitis in BALB/c mice is not dependent upon the FasL. However, in contrast to C57BL/6 mice, FasL is required for resolution of inflammation and protecting host tissue from nonspecific damage in BALB/c mice.

alphaB-crystallin protects retinal tissue during Staphylococcus aureus-induced endophthalmitis.

Bacterial infections of the eye highlight a dilemma that is central to all immune-privileged sites. On the one hand, immune privilege limits inflammation to prevent bystander destruction of normal tissue and loss of vision. On the other hand, bacterial infections require a robust inflammatory response for rapid clearance of the pathogen. We demonstrate that the retina handles this dilemma, in part, by activation of a protective heat shock protein. During Staphylococcus aureus-induced endophthalmitis, the small heat shock protein alphaB-crystallin is upregulated in the retina and prevents apoptosis during immune clearance of the bacteria. In the absence of alphaB-crystallin, mice display increased retinal apoptosis and retinal damage. We found that S. aureus produces a protease capable of cleaving alphaB-crystallin to a form that coincides with increased retinal apoptosis and tissue destruction. We conclude that alphaB-crystallin is important in protecting sensitive retinal tissue during destructive inflammation that occurs during bacterial endophthalmitis.

The Role of the Immune System in Glaucoma: Bridging the Divide Between Immune Mechanisms in Experimental Glaucoma and the Human Disease.

Glaucoma is one of the leading causes of visual impairment worldwide. Classically, clinicians have evaluated patients through a full ophthalmological examination including gonioscopy, measurement of intraocular pressure (IOP), and assessment of the optic nerve. New imaging modalities have further enhanced our ability to evaluate glaucoma; however, our treatments have not evolved as much. Whether one uses medical treatment with topical ocular antihypertensives, laser trabeculoplasty, or filtering surgery, the mainstay of treatment is to lower IOP. However, as our understanding of the disease evolves, mechanisms other than elevated IOP have been implicated in glaucoma pathogenesis. Recent animal model studies have shown a possible role of the immune system in the pathophysiology of glaucoma. This article explores the current understanding of immune reactions in glaucoma, which could lead to a new paradigm of treatment for human disease.

A novel treatment for ocular tumors using membrane FasL vesicles to activate innate immunity and terminate immune privilege.

PURPOSE: Ocular immune privilege promotes tumor growth by hindering the development of innate and adaptive immunity. A prior study showed that ocular tumors expressing the membrane-only form of Fas ligand (FasL) terminate immune privilege, induce vigorous inflammation, undergo rejection, and induce systemic protective immunity. In these previous experiments the tumor cells used were genetically engineered to express membrane FasL. As an initial step toward developing an immunotherapy for intraocular tumors, the present study was conducted to examine whether injection of microvesicles expressing membrane FasL into ocular tumors (that are FasL negative) would have a similar effect. METHODS: Microvesicles expressing either no FasL or membrane-only Fas ligand were coinjected with L5178Y-R lymphoma cells into the anterior chambers (AC) of DBA/2 mice. RESULTS: Tumor cells coinjected with control vesicles grew progressively in the AC, and all mice died of metastatic disease by day 15. By contrast, a single injection of membrane FasL vesicles induced a potent inflammatory response characterized by GR1+ neutrophils and F4/80+ macrophages and significantly improved survival from 0% in untreated mice to 58% in mFasL-treated mice. Among the surviving mice, the ocular tumor was eliminated in 55%, and the mice exhibited systemic protection from a second tumor challenge. In the remaining 45%, the ocular tumor was not eliminated, but the mice were protected from liver metastases. CONCLUSIONS: Bioactive membrane FasL microvesicles coinjected with tumor cells induce a potent inflammatory response that terminates immune privilege, eliminates ocular tumors, and prevents metastatic disease.

Effects of High Estrogen Levels on Monocyte Chemoattractant Protein-1 and Wound Healing.

Objective: Herein, we tested the effects of high levels of supplemental estrogen treatment on cutaneous wound healing. Approach: Female mice were implanted with a 17ß-estradiol (E2) secreting pellet or placebo before receiving a full-thickness dermal excisional wound. Mice receiving the E2 pellet attained hormone levels that are comparable to those achieved during pregnancy. At 1, 3, and 5 days after injury, the dermal excision wound was examined for their histologic appearance, rate of closure, and chemokine levels. Results: Wound closure, assessed by percent reepithelialization, was slower in E2-treated mice relative to placebo (42.6%±6.6% vs. 70.0%±5.3%, respectively, 3 days after injury). In addition, there was a marked reduction in the subepithelial inflammatory infiltrate and granulation tissue in E2-treated mice relative to placebo. Wound levels of monocyte chemoattractant protein-1 (MCP-1) were increased by 3 days after injury and continued to rise at 5 days after injury in placebo-treated mice (p<0.01). By contrast, MCP-1 levels were significantly reduced at 3 and 5 days after injury in E2-treated mice relative to placebo-treated controls (p<0.01). This attenuation could be reversed by treatment with an estrogen receptor antagonist. Innovation: High levels of estrogen are able to suppress normal wound closure. Conclusion: Dermal wound healing can be altered by manipulating the gonadal steroid hormone levels. In particular, high levels of estrogen can be utilized to slow down the rate of wound healing through a reduction in the inflammatory response.

Estrogen regulation of immune responses after injury.

There is a naturally occurring gender difference in immune responses which persists after traumatic injury. Physiological levels of 17beta-estradiol (E(2)) are immunostimulatory, whereas high pregnancy and superphysiological levels are immunosuppressive. In contrast, at all concentrations, testosterone suppresses immune responses. Evidence from this laboratory and others suggest that the gender difference in immune responses after injury is mediated in part by alterations in the circulating levels of gonadal steroid hormones through modulation of production of inflammatory and immunoregulatory cytokines, including interleukin-6 (IL-6). Aberrant production of IL-6 is known to be an important mediator of immunity after injury. Since E(2) is a critical regulator of IL-6 production and overall immune function, it suggests gender specific therapies should be considered for the treatment of patients.

NLRP3 inflammasome activation in retinal pigment epithelial cells by lysosomal destabilization: implications for age-related macular degeneration.

PURPOSE: To evaluate the effect of lysosomal destabilization on NLRP3 inflammasome activation in RPE cells and to investigate the mechanisms by which inflammasome activation may contribute to the pathogenesis of age-related macular degeneration (AMD). METHODS: Human ocular tissue sections from patients with geographic atrophy or neovascular AMD were stained for NLRP3 and compared to tissues from age-matched controls. Expression of the IL-1ß precursor, pro-IL-1ß, was induced in ARPE-19 cells by IL-1α treatment. Immunoblotting was performed to assess expression of NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1ß in ARPE-19 cells. Lysosomes were destabilized using the lysosomotropic agent L-leucyl-L-leucine methyl ester (Leu-Leu-OMe). Active caspase-1 was detected using FAM-YVAD-FMK, a fluorescent-labeled inhibitor of caspases (FLICA) specific for caspase-1. IL-1ß was detected by immunoblotting and ELISA, and cytotoxicity was evaluated by LDH quantification. RESULTS: RPE of eyes affected by geographic atrophy or neovascular AMD exhibited NLRP3 staining at lesion sites. ARPE-19 cells were found to express NLRP3, ASC, and procaspase-1. IL-1α dose-dependently induced pro-IL-1ß expression in ARPE-19 cells. Lysosomal destabilization induced by Leu-Leu-OMe triggered caspase-1 activation, IL-1ß secretion, and ARPE-19 cell death. Blocking Leu-Leu-OMe-induced lysosomal disruption with the compound Gly-Phe-CHN(2) or inhibiting caspase-1 with Z-YVAD-FMK abrogated IL-1ß release and ARPE-19 cytotoxicity. CONCLUSIONS: NLRP3 upregulation occurs in the RPE during the pathogenesis of advanced AMD, in both geographic atrophy and neovascular AMD. Destabilization of RPE lysosomes induces NLRP3 inflammasome activation, which may contribute to AMD pathology through the release of the proinflammatory cytokine IL-1ß and through caspase-1-mediated cell death, known as "pyroptosis."

Soluble guanylate cyclase α1-deficient mice: a novel murine model for primary open angle 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 α1 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 α1-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 α1 and ß1 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.

Spontaneous bacterial keratitis in CD36 knockout mice.

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.

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death.

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 TNFα 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.

Control of ocular tumor growth and metastatic spread by soluble and membrane Fas ligand.

Fas ligand (FasL) can be either membrane bound, or cleaved by metalloproteinases (MMP) to produce a soluble protein. The two different forms of FasL are reported to have opposite functions-membrane-bound FasL (mFasL) is proinflammatory and soluble FasL (sFasL) is antiinflammatory. We previously showed that, within the immune-privileged eye, tumors expressing high levels of mFasL overcame the suppressive ocular environment, triggered an inflammatory response, and were subsequently rejected. By contrast, eye tumors expressing low levels of mFasL grew progressively. To evaluate the effect of sFasL on the tumor growth and metastatic potential of ocular FasL-expressing tumors, we compared tumor cell clones that expressed equal amounts of (low) mFasL in the presence or absence of sFasL. Tumor cells transfected with a modified FasL gene expressed only mFasL (noncleavable), grew progressively within the eye, and induced systemic protective immunity that prevented metastatic spread of tumor cells to the liver. Unexpectedly, tumors transfected with wild-type FasL (wtFasL; cleavable), which could produce both sFasL and mFasL, elicited considerably more inflammation and grew more slowly within the eye. However, the cleavable wtFasL eye tumors failed to trigger protective immunity and gave rise to liver metastases. Interestingly, exposure to the ocular environment was required for the wtFasL tumors to gain metastatic potential. We conclude that the fate of FasL-expressing tumors is determined by a combination of the following: (a) the relative proportion of membrane and sFasL, and (b) the local environment that determines the extent of FasL cleavage.

Survival and cell mediated immunity after burn injury in aged mice.

The elderly are less able to survive burn injury than young healthy individuals. Regardless of age, burn victims often succumb to secondary infections rather than the primary injury. Since immune responses diminish with age, it is likely that aged individuals are predisposed to a poor outcome by virtue of their weak immune system. Elevated production of macrophage-derived mediators, including interleukin-6 (IL-6), may lead to post-injury immunosuppression in young adults. Healthy aged individuals produce high circulating levels of these mediators; therefore, the combination of the age and burn trauma could further suppress immune responses and contribute to the rapid demise of aged burn patients. Herein, the effects of age and burn trauma using a murine scald injury model were examined. After injury, aged mice are less likely to survive, are unable to mount immune responses, and produce more IL-6 when compared to young adult mice given the same size injuries. Enhancing our understanding of the mechanisms responsible for regulating cell-mediated immune responses after injury could lead to the development of therapies designed to treat aged burn patients.

Membrane Fas ligand activates innate immunity and terminates ocular immune privilege.

It has been proposed that the constitutive expression of Fas ligand (FasL) in the eye maintains immune privilege, in part through inducing apoptosis of infiltrating Fas(+) T cells. However, the role of FasL in immune privilege remains controversial due to studies that indicate FasL is both pro- and anti-inflammatory. To elucidate the mechanism(s) by which FasL regulates immune privilege, we used an ocular tumor model and examined the individual roles of the membrane-bound and soluble form of FasL in regulating ocular inflammation. Following injection into the privileged eye, tumors expressing only soluble FasL failed to trigger inflammation and grew progressively. By contrast, tumors expressing only membrane FasL 1) initiated vigorous neutrophil-mediated inflammation, 2) terminated immune privilege, and 3) were completely rejected. Moreover, the rejection coincided with activation of both innate and adaptive immunity. Interestingly, a higher threshold level of membrane FasL on tumors is required to initiate inflammation within the immune privileged eye, as compared with nonprivileged sites. The higher threshold is due to the suppressive microenvironment found within aqueous humor that blocks membrane FasL activation of neutrophils. However, aqueous humor is unable to completely block the proinflammatory effects of tumor cells that express high levels of membrane FasL. In conclusion, our data indicate that the function of FasL on intraocular tumors is determined by the microenvironment in conjunction with the form and level of FasL expressed.

Elevated monocyte chemoattractant protein-1 levels following thermal injury precede monocyte recruitment to the wound site and are controlled, in part, by tumor necrosis factor-alpha.

In previous studies, mice given a full-thickness scald injury had an influx of neutrophils into the skin that followed a local increase in a neutrophil chemoattractant. Because macrophages are known to infiltrate the wound area after neutrophils and are essential for normal wound repair, studies were designed to characterize the time course of macrophage accumulation in the wound and to identify the factor(s) responsible for this influx. A macrophage infiltrate into the wound was observed at 4 days post-injury and persisted through at least 10 days. This influx was preceded by an initial fourfold increase in dermal monocyte chemoattractant protein-1 levels at 24 hours post-injury (p < 0.05). This elevation in monocyte chemoattractant protein-1 was enhanced at 4 and 10 days postburn resulting in a sixfold increase over baseline (p < 0.01). Levels of tumor necrosis factor-alpha, a proinflammatory cytokine known to induce chemokine production, were elevated at 90 minutes after injury in burn- versus sham-injured groups (p < 0.05). Furthermore, administration of tumor necrosis factor-alpha neutralizing antibody in vivo reduced the dermal levels of monocyte chemoattractant protein-1 seen at 10 days postburn by 57% (p < 0.01); however, macrophage accumulation was not altered. Thus, elevated systemic TNF-alpha levels may influence the local chemokine milieu following burn injury.

Effect of acute ethanol exposure on the dermal inflammatory response after burn injury.

BACKGROUND: More than 100,000 people each year are admitted to U.S. hospitals for severe burn injury. Strikingly, ethanol use prior to injury is apparent in nearly 50% of burn patients, rendering them six times more likely to die from infection than patients not exposed to ethanol. We previously reported that the kinetics and magnitude of neutrophil chemokine production and subsequent accumulation of neutrophils in the lung was dramatically altered when ethanol exposure preceded injury. Here, we tested whether burn injury and ethanol exposure combined, altered susceptibility to infection, neutrophil chemoattractant production, and neutrophil accumulation at the site of the burn wound. METHODS: Male B(6)D(2)F1 mice were administered a dose of ethanol designed to achieve 90-100 mg/dl circulating levels and 30 min later subjected to a 15% total body surface area dorsal scald injury. Susceptibility to topically applied Pseudomonas aeruginosa was examined. At various times after injury, burn wound and normal tissues were collected for assessments of neutrophil counts, myeloperoxidase quantitation, and neutrophil chemoattractant (KC and MIP-2) production. RESULTS: Ethanol exposure prior to burn injury enhanced susceptibility to infection after burn and was associated with significantly elevated production of KC, but not MIP-2, at the wound site. Despite the enhanced elevation of KC, neutrophil accumulation in the wounds of ethanol exposed, burn injured mice did not differ from those that received burn injury alone. TNFalpha (a potent activator of neutrophils), however, was found to be significantly elevated in the wounds of mice that received only burn injury, but not in those that received injury in combination with prior ethanol exposure. CONCLUSION: In the presence of ethanol, neutrophils are adequately recruited to the site of burn injury, but their host defense functions are impaired, perhaps due to the lack of proinflammatory cytokines such as TNFalpha.