Mouse Cornea Research Articles

Induction of Innate Inflammatory Pathways in the Corneal Epithelium in the Desiccating Stress Dry Eye Model.

To determine whether 24-hour exposure to the desiccating stress (DS) dry eye model induces NF-kB and NLRP3 inflammasome pathways in the mouse cornea epithelium. Six- to 8-week-old C57BL/6J mice were housed under normal humidity (nonstressed) or subjected to DS from a drafty, low-humidity environment combined with subcutaneous scopolamine four times/day for one day to suppress tear production (DS1). Cornea whole mounts were prepared for immunofluorescent staining, or the corneal epithelium was scraped for NF-kB p-p65 ELISA, Western blot, or real-time PCR to detect NF-kB and inflammasome pathway proteins and gene transcripts, respectively. NF-kB phospho-p65 protein, nuclear NF-kB p-p65, and expression of the NF-kB inducible cytokines (IL-12a, IL-12b, and lymphotoxin b [Ltb]) and chemokine (CCL-2) genes were significantly increased in DS1 compared to nonstressed control. NLRP3 protein and RNA transcripts significantly increased in DS1. NLRP3 and Caspase-1 immunostaining increased in the cornea epithelium at DS1. At DS1 there was no change in IL-18 and a decrease in IL-1β mRNA transcripts; however, levels of bound and total IL-18 protein increased at DS1, and the level of mature IL-1β increased from DS1 to DS5. These findings indicate innate NF-kB and NLRP3 inflammasome inflammatory pathways are induced in the corneal epithelium within one day in the DS dry eye model. NF-kB activation was associated with increased expression of inflammatory mediators involved in dry eye. Induction of these pathways is accompanied by increased bound/total IL-18 and mature IL-1β.

Transglutaminase-2 is critical for corneal epithelial barrier function via positive regulation of Claudin-1

PurposeTransglutaminase (TG)-2 is a ubiquitous multi-functional protein expressed in all living cells. The purpose of the current study was to investigate the role of TG-2 in corneal barrier function and its potential regulation of epithelial junctional proteins and transcription factors. MethodsCorneal barrier function to ions in TG-2−/− and TG-2+/+ mice was assessed by Ussing chamber assay. Hypo-osmolar water or FITC-dextran was applied on top of mouse eyes to evaluate the corneal barrier function to water and macromolecules. Western blots, qPCR and immunofluorescent staining were used to investigate the expression of tight junction proteins in TG-2−/− and TG-2+/+ mouse corneas, and also in TG-2 knockdown human corneal epithelial cells. ResultsCorneal explants from TG-2−/− mice had a lower trans-epithelial electrical resistance compared to TG-2+/+ mice. When challenged by hypo-osmolar water, the central corneal thickness of TG-2−/− mice increased faster, and these mice had a faster rise of fluorescence in the anterior chamber after ocular exposure to FITC-dextran, compared to TG-2+/+. Claudin-1 protein and transcript levels were reduced in the cornea of TG-2−/− mice and in TG-2 knockdown human corneal epithelial cells. Slug which previously reported suppressing Claudin-1 transcription, was increased at both protein and transcript level in TG-2 knockdown cells. TG-2 and Claudin-1 protein levels were unchanged in shRNA and shTG cells after MG132 treatment, while Slug accumulated in treated cells. ConclusionTG-2 may positively regulate Claudin-1 through repressing Slug at transcript level, and thus it is critical for normal corneal barrier function.

IFN-γ Facilitates Corneal Epithelial Cell Pyroptosis Through the JAK2/STAT1 Pathway in Dry Eye.

To investigate the effect of gamma interferon (IFN-γ) on corneal epithelial pyroptosis in an experimental dry eye (DE) model and explore the underlying molecular mechanisms. Experimental DE was established in adult wild-type (WT) C57BL/6 mice and Ifng-knockout mice on a C57BL/6 background by subcutaneous injection of scopolamine (1.5 mg/0.3 mL, three times per day) and exposure to desiccating stress. An immortalized human corneal epithelial cell line (HCE-T) was treated with IFN-γ under hyperosmolar conditions. Corneal epithelial defects, tear production, and conjunctival goblet cells were detected by fluorescein sodium staining, the phenol red cotton test, and periodic acid-Schiff staining. The mRNA expression was measured by quantitative real-time PCR. Changes in protein expression were analyzed by Western blotting and immunofluorescence staining. Cell Counting Kit-8 and lactate dehydrogenase assays and in situ TUNEL staining were used to assess cell death. The expression of IFNG and its related genes was increased in the corneas of DE mice, whereas genetic deletion of Ifng ameliorated desiccating stress-induced dry eye symptoms. We further found that IFN-γ activated the JAK2/STAT1 signaling pathway inducing corneal epithelial pyroptosis. Topical application of a STAT1 inhibitor in vivo or siRNA targeting STAT1 in vitro suppressed pyroptosis of corneal epithelial cells. In addition, the production of reactive oxygen species (ROS) was elevated in DE, and a reduction in excessive ROS release prevented pyroptosis. The increase in IFN-γ participates in the pathogenesis of dry eye and promotes corneal epithelial pyroptosis by activating the JAK2/STAT1 signaling pathway. Oxidative stress might be in downstream of JAK2/STAT1, thereby contributing to pyroptosis.

Peroxisome proliferator-activated receptor-α (PPARα) regulates wound healing and mitochondrial metabolism in the cornea

Diabetes can result in impaired corneal wound healing. Mitochondrial dysfunction plays an important role in diabetic complications. However, the regulation of mitochondria function in the diabetic cornea and its impacts on wound healing remain elusive. The present study aimed to explore the molecular basis for the disturbed mitochondrial metabolism and subsequent wound healing impairment in the diabetic cornea. Seahorse analysis showed that mitochondrial oxidative phosphorylation is a major source of ATP production in human corneal epithelial cells. Live corneal biopsy punches from type 1 and type 2 diabetic mouse models showed impaired mitochondrial functions, correlating with impaired corneal wound healing, compared to nondiabetic controls. To approach the molecular basis for the impaired mitochondrial function, we found that Peroxisome Proliferator-Activated Receptor-α (PPARα) expression was downregulated in diabetic human corneas. Even without diabetes, global PPARα knockout mice and corneal epithelium-specific PPARα conditional knockout mice showed disturbed mitochondrial function and delayed wound healing in the cornea, similar to that in diabetic corneas. In contrast, fenofibrate, a PPARα agonist, ameliorated mitochondrial dysfunction and enhanced wound healing in the corneas of diabetic mice. Similarly, corneal epithelium-specific PPARα transgenic overexpression improved mitochondrial function and enhanced wound healing in the cornea. Furthermore, PPARα agonist ameliorated the mitochondrial dysfunction in primary human corneal epithelial cells exposed to diabetic stressors, which was impeded by siRNA knockdown of PPARα, suggesting a PPARα-dependent mechanism. These findings suggest that downregulation of PPARα plays an important role in the impaired mitochondrial function in the corneal epithelium and delayed corneal wound healing in diabetes.

Interference of sympathetic overactivation restores limbal stem/progenitor cells function and accelerates corneal epithelial wound healing in diabetic mice

Diabetic keratopathy (DK), the diabetic complication in the cornea, is characterized by the delayed epithelial regeneration and sensory nerve degeneration. The involvement of limbal stem/progenitor cells (LSPCs) dysfunction has been reported, however the pathogenic mechanisms remain unclear. Here, we confirmed the dysfunction of LSPCs in diabetic mouse and human corneas. The sympathetic nerve in the cornea was adjacent to LSPCs, and the sympathetic overactivation was found in diabetic mice. Surgical and pharmacological ablation of sympathetic nerves rescued the LSPCs function and promoted corneal epithelial regeneration in diabetic mice. In contrast, both topical norepinephrine (NE) application and chemogenetic sympathetic overactivation directly impaired the stemness and proliferation characteristics of LSPCs, as well as the normal epithelial regeneration. Moreover, we identified that β2-adrenoceptor (Adrb2) was the predominant adrenergic receptor expressed in LSPCs by corneal limbal single-cell sequencing and real time PCR (RT-PCR) analysis of sorted LSPCs. The Adrb2 knockout mice exhibited the enhancement of epithelial regeneration and LSPCs function, compared with the wild-type mice. Similarly, topical application of the Adrb2 specific antagonist ICI 118, 551 effectively accelerated diabetic corneal epithelial regeneration with the restored LSPCs function. Mechanistically, sonic hedgehog (Shh) activity mediated the downstream effects of NE-Adrb2 signaling pathway in regulating LSPCs and epithelial regeneration. Taken together, our data revealed the involvement of sympathetic overactivation in the impairment of diabetic LSPCs function and corneal epithelial regeneration through the NE-Adrb2-Shh signaling pathway. The interference of sympathetic overactivation may provide novel treatment strategies for diabetic keratopathy.

Overactivation of Norepinephrine-β2-Adrenergic Receptor Axis Promotes Corneal Neovascularization.

To investigate the role of the sympathetic nervous system in corneal neovascularization (CNV) and to identify the downstream pathway involved in this regulation. Three types of CNV models were constructed with C57BL/6J mice, including the alkali burn model, suture model, and basic fibroblast growth factor (bFGF) corneal micropocket model. Subconjunctival injection of the sympathetic neurotransmitter norepinephrine (NE) was administered in these three models. Control mice received injections of water of the same volume. The corneal CNV was detected using slit-lamp microscopy and immunostaining with CD31, and the results were quantified by ImageJ. The expression of β2-adrenergic receptor (β2-AR) was stained with mouse corneas and human umbilical vein endothelial cells (HUVECs). Furthermore, the anti-CNV effects of β2-AR antagonist ICI-118,551 (ICI) were examined with HUVEC tube formation assay and with a bFGF micropocket model. Additionally, partial β2-AR knockdown mice (Adrb2+/-) were used to establish the bFGF micropocket model, and the corneal CNV size was quantified based on the slit-lamp images and vessel staining. Sympathetic nerves invaded the cornea in the suture CNV model. The NE receptor β2-AR was highly expressed in corneal epithelium and blood vessels. The addition of NE significantly promoted corneal angiogenesis, whereas ICI effectively inhibited CNV invasion and HUVEC tube formation. Adrb2 knockdown significantly reduced the cornea area occupied by CNV. Our study found that sympathetic nerves grow into the cornea in conjunction with newly formed vessels. The addition of the sympathetic neurotransmitter NE and activation of its downstream receptor β2-AR promoted CNV. Targeting β2-AR could potentially be used as an anti-CNV strategy.

Functional Peptide-Loaded Gelatin Nanoparticles as Eyedrops for Cornea Neovascularization Treatment.

Corneal neovascularization (NV) is a process of abnormal vessel growth into the transparent cornea from the limbus and can disturb the light passing through the cornea, resulting in vision loss or even blindness. The use of nanomedicine as an effective therapeutic formulation in ophthalmology has led to higher drug bioavailability and a slow drug release rate. In this research, we designed and explored the feasibility of a new nanomedicine, gp91 ds-tat (gp91) peptide-encapsulated gelatin nanoparticles (GNP-gp91), for inhibiting corneal angiogenesis. GNP-gp91 were prepared by a two-step desolvation method. The characterization and cytocompatibility of GNP-gp91 were analyzed. The inhibition effect of GNP-gp91 on HUVEC cell migration and tube formation was observed by an inverted microscope. The drug retention test in mouse cornea was observed by in vivo imaging system, fluorescence microscope, and DAPI/TAMRA staining. Finally, the therapeutic efficacy and evaluation of neovascularization-related factors were conducted through the in vivo corneal NV mice model via topical delivery. The prepared GNP-gp91 had a nano-scale diameter (550.6 nm) with positive charge (21.7 mV) slow-release behavior (25%, 240hr). In vitro test revealed that GNP-gp91 enhanced the inhibition of cell migration and tube formation capacity via higher internalization of HUVEC. Topical administration (eyedrops) of the GNP-gp91 significantly prolongs the retention time (46%, 20 min) in the mouse cornea. In chemically burned corneal neovascularization models, corneal vessel area with a significant reduction in GNP-gp91 group (7.89%) was revealed when compared with PBS (33.99%) and gp91 (19.67%) treated groups via every two days dosing. Moreover, GNP-gp91 significantly reduced the concentration of Nox2, VEGF and MMP9 in NV's cornea. The nanomedicine, GNP-gp91, was successfully synthesized for ophthalmological application. These data suggest that GNP-gp91 contained eyedrops that not only have a longer retention time on the cornea but also can treat mice corneal NV effectively delivered in a low dosing frequency, GNP-gp91 eyedrops provides an alternative strategy for clinical ocular disease treatment in the culture.

Airborne Exposure of the Cornea to PM10 Induces Oxidative Stress and Disrupts Nrf2 Mediated Anti-Oxidant Defenses.

The purpose of this study is to test the effects of whole-body animal exposure to airborne particulate matter (PM) with an aerodynamic diameter of <10 μm (PM10) in the mouse cornea and in vitro. C57BL/6 mice were exposed to control or 500 µg/m3 PM10 for 2 weeks. In vivo, reduced glutathione (GSH) and malondialdehyde (MDA) were analyzed. RT-PCR and ELISA evaluated levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and inflammatory markers. SKQ1, a novel mitochondrial antioxidant, was applied topically and GSH, MDA and Nrf2 levels were tested. In vitro, cells were treated with PM10 ± SKQ1 and cell viability, MDA, mitochondrial ROS, ATP and Nrf2 protein were tested. In vivo, PM10 vs. control exposure significantly reduced GSH, corneal thickness and increased MDA levels. PM10-exposed corneas showed significantly higher mRNA levels for downstream targets, pro-inflammatory molecules and reduced Nrf2 protein. In PM10-exposed corneas, SKQ1 restored GSH and Nrf2 levels and lowered MDA. In vitro, PM10 reduced cell viability, Nrf2 protein, and ATP, and increased MDA, and mitochondrial ROS; while SKQ1 reversed these effects. Whole-body PM10 exposure triggers oxidative stress, disrupting the Nrf2 pathway. SKQ1 reverses these deleterious effects in vivo and in vitro, suggesting applicability to humans.

The NLRP3 Inflammasome Is Required for Protection Against Pseudomonas Keratitis.

The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of Pseudomonas aeruginosa (PA) infection in mouse corneas. Corneas of wild type and NLRP3-/- mice were infected with PA. The severity of bacterial keratitis was graded on days 1 and 3 post-infection by slit lamp, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) immunoblotting analysis of cleaved caspase-1 and IL-1β, and (iv) IL-1β quantification by ELISA. In parallel experiments, severity of keratitis was examined in the wild-type mice receiving a subconjunctival injection of a highly selective NLRP3 inhibitor immediately prior to infection. Compared to wild type mice, NLRP3-/- mice exhibited more severe infection, as indicated by an increase in opacity score and an increase in bacterial load. The hallmark of inflammasome assembly is the activation of proinflammatory caspase-1 and IL-1β by cleavage of their precursors, pro-caspase-1 and pro-IL-1β, respectively. Accordingly, increased severity of infection in the NLRP3-/- mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1β and reduced IL-1β+ neutrophil infiltration in infected corneas. Likewise, corneas of mice receiving subconjunctival injections of NLRP3 inhibitor exhibited increased bacterial load, and reduced IL-1β expression. Activation of NLRP3 pathway is required for the clearance of PA infection in mouse corneas.

Therapeutic effect of Atractylenolide I on Aspergillus fumigatus keratitis by affecting MyD88/ NF-κB pathway and IL-1β, IL-10 expression.

Atractylenolide I (AT-I) is a natural sesquiterpene with anti-inflammatory effects. The purpose of this study was to research the anti-inflammatory effect of AT-I on Aspergillus fumigatus(A. fumigatus) keratitis in mice. Cytotoxicity test and cell scratch test were used to determine the therapeutic concentrations of corneal infections. In vivo and in vitro studies, mouse cornea and human corneal epithelial cells (HCECs) infected with A. fumigatus were treated with AT-I or dimethyl sulfoxide (DMSO). Then, to analyze the effect of AT-I on inflammatory response, namely neutrophil or macrophage recruitment and the expression of cytokines involving MyD88, NF-κB, interleukin 1β (IL-1β) and interleukin 10 (IL-10). To study the effects of the drug, the techniques used include slit-lamp photography, immunofluorescence, myeloperoxidase (MPO) detection, quantitative real-time polymerase chain reaction (QRT-PCR), and western blot. At the same time, in order to explore the combined effect of the drug and natamycin, slit-lamp photographs and clinical scores were used to visually display the disease process. No cytotoxicity was observed under the action of AT-I at a concentration of 800μM. In mouse models, AT-I significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and decreased myeloperoxidase levels early in infection. Studies have shown that AT-I may reduce the levels of IL-1β and IL-10 by inhibiting the MyD88/ NF-κB pathway. The drug combined with natamycin can increase corneal transparency in infected mice. AT-I may inhibit MyD88 / NF-κB pathway and the secretion of inflammatory factors IL-1 β and IL-10 to achieve the therapeutic effect of fungal keratitis.

Accelerating corneal wound healing using exosome-mediated targeting of NF-κB c-Rel

The integrity of the corneal epithelium is essential for the maintenance of the physiological function of the cornea. Studies have found that inflammation greatly delays corneal wound healing. NF-κB c-Rel is preferentially expressed by immune cells and promotes the expression of inflammatory cytokines. In the current study, we sought to investigate whether c-Rel could be used as a potential therapeutic target for treating a corneal injury. Our studies reveal that expressions of c-Rel and its inflammatory targets are significantly increased in the cornea of mice with corneal injury. In addition, we find that c-Rel-deficient mice exhibit accelerated corneal wound healing and reduced expression of inflammatory cytokines. Further studies show that topical treatment on the corneal surface using nano-polymers or exosomes loaded with c-Rel-specific siRNA (siRel) can effectively accelerate regular and diabetic corneal wound healing. More importantly, we find that exosomes, as carriers of siRel, showed better efficacy than nano-polymers in treating corneal injury. We further demonstrate that exosomes secreted by mesenchymal stem cells can efficiently transfer siRNA into macrophages and dendritic cells but not T cells. Taken together, these results indicate that blocking c-Rel may represent an attracting strategy for the treatment of both regular and diabetic corneal injury.

Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea

Corneal injury–associated inflammation could induce inward-growing neovascularization from the periphery of the tissue. Such neovascularization could cause stromal opacification and curvature disturbance, and both potentially impair visual function. In this study, we determined the effects of the loss of transient receptor potential vanilloid 4 (TRPV4) expression on the development of neovascularization in the corneal stroma in mice by producing a cauterization injury in the central area of the cornea. New vessels were immunohistochemically labeled with anti-TRPV4 antibodies. TRPV4 gene knockout suppressed the growth of such CD31-labeled neovascularization in association with the suppression of infiltration of macrophages and tissue messenger RNA expression of the vascular endothelial cell growth factor A level. Treatment of cultured vascular endothelial cells with supplementation of HC-067047 (0.1 μM, 1 μM, or 10 μM), a TRPV4 antagonist, attenuated the formation of a tube-like structure with sulforaphane (15 μM, for positive control) that modeled the new vessel formation. Therefore, the TRPV4 signal is involved in injury-induced macrophagic inflammation and neovascularization activity by vascular endothelial cells in a mouse corneal stroma. TRPV4 could be a therapeutic target to prevent unfavorable postinjury neovascularization in the cornea.

Osteopontin contributes to virus resistance associated with type I IFN expression, activation of downstream ifn-inducible effector genes, and CCR2+CD115+CD206+ macrophage infiltration following ocular HSV-1 infection of mice.

Ocular pathology is often associated with acute herpes simplex virus (HSV)-1 infection of the cornea in mice. The present study was undertaken to determine the role of early T lymphocyte activation 1 protein or osteopontin (OPN) in corneal inflammation and host resistance to ocular HSV-1 infection. C57BL/6 wild type (WT) and osteopontin deficient (OPN KO) mice infected in the cornea with HSV-1 were evaluated for susceptibility to infection and cornea pathology. OPN KO mice were found to possess significantly more infectious virus in the cornea at day 3 and day 7 post infection compared to infected WT mice. Coupled with these findings, HSV-1-infected OPN KO mouse corneas were found to express less interferon (IFN)-α1, double-stranded RNA-dependent protein kinase, and RNase L compared to infected WT animals early post infection that likely contributed to decreased resistance. Notably, OPN KO mice displayed significantly less corneal opacity and neovascularization compared to WT mice that paralleled a decrease in expression of vascular endothelial growth factor (VEGF) A within 12 hr post infection. The change in corneal pathology of the OPN KO mice aligned with a decrease in total leukocyte infiltration into the cornea and specifically, in neutrophils at day 3 post infection and in macrophage subpopulations including CCR2+CD115+CD206+ and CD115+CD183+CD206+ -expressing cells. The infiltration of CD4+ and CD8+ T cells into the cornea was unaltered comparing infected WT to OPN KO mice. Likewise, there was no difference in the total number of HSV-1-specific CD4+ or CD8+ T cells found in the draining lymph node with both sets functionally competent in response to virus antigen comparing WT to OPN KO mice. Collectively, these results demonstrate OPN deficiency directly influences the host innate immune response to ocular HSV-1 infection reducing some aspects of inflammation but at a cost with an increase in local HSV-1 replication.

Synthetic Strategies for Improving Solubility: Optimization of Novel Pyrazolo[1,5-a]pyrimidine CFTR Activator That Ameliorates Dry Eye Disease.

Dry eye disease (DED) is one of the most prevalent ocular diseases but has limited treatment options. Cystic fibrosis transmembrane conductance regulator (CFTR), a major chloride channel that stimulates fluid secretion in the ocular surface, may pave the way for new therapeutic strategies for DED. Herein, we report the optimization of Cact-3, a potent CFTR activator with poor solubility, to 16d, a potent CFTR activator with suitable solubility for eye drop formulation. Notably, 16d was well distributed in target tissues including cornea and conjunctiva with minimal systemic exposure in rabbit. Topical ocular instillation of 16d significantly enhanced tear secretion and improved corneal erosion in a mouse model of DED. In addition, 16d significantly reduced mRNA expression of pro-inflammatory cytokines including IL-1β, IL-17, and TNF-α and MMP2 in cornea and conjunctiva of DED mice.

Characterization of C-X-C chemokine receptor type 5 in the cornea and role in the inflammatory response after corneal injury

C-X-C chemokine receptor type 5 (CXCR5) regulates inflammatory responses in ocular and non-ocular tissues. However, its expression and role in the cornea are still unknown. Here, we report the expression of CXCR5 in human cornea in vitro and mouse corneas in vivo, and its functional role in corneal inflammation using C57BL/6J wild-type (CXCR5+/+) and CXCR5-deficient (CXCR5−/−) mice, topical alkali injury, clinical eye imaging, histology, immunofluorescence, PCR, qRT-PCR, and western blotting. Human corneal epithelial cells, stromal fibroblasts, and endothelial cells demonstrated CXCR5 mRNA and protein expression in PCR, and Western blot analyses, respectively. To study the functional role of CXCR5 in vivo, mice were divided into four groups: Group-1 (CXCR5+/+ alkali injured cornea; n = 30), Group-2 (CXCR5−/− alkali injured cornea; n = 30), Group-3 (CXCR5+/+ naïve cornea; n = 30), and Group-4 (CXCR5−/− naïve cornea; n = 30). Only one eye was wounded with alkali. Clinical corneal evaluation and imaging were performed before and after injury. Mice were euthanized 4 h, 3 days, or 7 days after injury, eyes were excised and used for histology, immunofluorescence, and qRT-PCR. In clinical eye examinations, CXCR5−/− mouse corneas showed ocular health akin to the naïve corneas. Alkali injured CXCR5+/+ mouse corneas showed significantly increased mRNA (p < 0.001) and protein (p < 0.01 or p < 0.0001) levels of the CXCR5 compared to the naïve corneas. Likewise, alkali injured CXCR5−/− mouse corneas showed remarkably amplified inflammation in clinical eye exams in live animals. The histological and molecular analyses of these corneas post euthanasia exhibited markedly augmented inflammatory cells in H&E staining and significant CD11b + cells in immunofluorescence (p < 0.01 or < 0.05); and tumor necrosis factor-alpha (TNFα; p < 0.05), cyclooxygenase 2 (COX-2; p < 0.0001), interleukin (IL)-1β (p < 0.0001), and IL-6 (p < 0.0001 or < 0.01) mRNA expression compared to the CXCR5+/+ mouse corneas. Interestingly, CXCR5−/− alkali injured corneas also showed altered mRNA expression of fibrotic alpha smooth muscle actin (α-SMA; p > 0.05) and angiogenic vascular endothelial growth factor (VEGF; p < 0.01) compared to the CXCR5+/+ alkali injured corneas. In summary, the CXCR5 gene is expressed in all three major layers of the cornea and appears to influence corneal inflammatory and repair events post-injury in vivo. More studies are warranted to tease the mechanistic role of CXCR5 in corneal inflammation and wound healing.

Synthetic Heparan Sulfate Mimetic Polymer Enhances Corneal Nerve Regeneration and Wound Healing after Experimental Laser Ablation Injury in Mice.

(1) Background: Abnormal corneal wound healing compromises visual acuity and can lead to neuropathic pain. Conventional treatments usually fail to restore the injured corneal tissue. In this study, we evaluated the effectiveness of a synthetic heparan sulfate mimetic polymer (HSmP) in a mouse model of corneal wound healing. (2) Methods: A surgical laser ablation affecting the central cornea and subbasal nerve plexus of mice was used as a model of the wound-healing assay. Topical treatment with HSmP was contrasted to its vehicle and a negative control (BSS). Corneal repair was studied using immunofluorescence to cell proliferation (Ki67), apoptosis (TUNEL assay), myofibroblast transformation (αSMA), assembly of epithelial cells (E-cadherin) and nerve regeneration (β-tubulin III). (3) Results: At the end of the treatment, normal epithelial cytoarchitecture and corneal thickness were achieved in HSmP-treated animals. HSmP treatment reduced myofibroblast occurrence compared to eyes irrigated with vehicle (p &lt; 0.01) or BSS (p &lt; 0.001). The HSmP group showed 50% more intraepithelial nerves than the BSS or vehicle groups. Only HSmP-treated corneas improved the visual quality to near transparent. (4) Conclusions: These results suggest that HSmP facilitates the regeneration of the corneal epithelium and innervation, as well as restoring transparency and reducing myofibroblast scarring after laser experimental injury.

Pseudomonas aeruginosa Can Diversify after Host Cell Invasion to Establish Multiple Intracellular Niches.

Within epithelial cells, Pseudomonas aeruginosa depends on its type III secretion system (T3SS) to escape vacuoles and replicate rapidly in the cytosol. Previously, it was assumed that intracellular subpopulations remaining T3SS-negative (and therefore in vacuoles) were destined for degradation in lysosomes, supported by data showing vacuole acidification. Here, we report in both corneal and bronchial human epithelial cells that vacuole-associated bacteria can persist, sometimes in the same cells as cytosolic bacteria. Using a combination of phase-contrast, confocal, and correlative light-electron microscopy (CLEM), we also found they can demonstrate biofilm-associated markers: cdrA and cyclic-di-GMP (c-di-GMP). Vacuolar-associated bacteria, but not their cytosolic counterparts, tolerated the cell-permeable antibiotic ofloxacin. Surprisingly, use of mutants showed that both persistence in vacuoles and ofloxacin tolerance were independent of the biofilm-associated protein CdrA or exopolysaccharides (Psl, Pel, alginate). A T3SS mutant (ΔexsA) unable to escape vacuoles phenocopied vacuole-associated subpopulations in wild-type PAO1-infected cells, with results revealing that epithelial cell death depended upon bacterial viability. Intravital confocal imaging of infected mouse corneas confirmed that P. aeruginosa formed similar intracellular subpopulations within epithelial cells in vivo. Together, these results show that P. aeruginosa differs from other pathogens by diversifying intracellularly into vacuolar and cytosolic subpopulations that both contribute to pathogenesis. Their different gene expression and behavior (e.g., rapid replication versus slow replication/persistence) suggest cooperation favoring both short- and long-term interests and another potential pathway to treatment failure. How this intracellular diversification relates to previously described "acute versus chronic" virulence gene-expression phenotypes of P. aeruginosa remains to be determined. IMPORTANCE Pseudomonas aeruginosa can cause sight- and life-threatening opportunistic infections, and its evolving antibiotic resistance is a growing concern. Most P. aeruginosa strains can invade host cells, presenting a challenge to therapies that do not penetrate host cell membranes. Previously, we showed that the P. aeruginosa type III secretion system (T3SS) plays a pivotal role in survival within epithelial cells, allowing escape from vacuoles, rapid replication in the cytoplasm, and suppression of host cell death. Here, we report the discovery of a novel T3SS-negative subpopulation of intracellular P. aeruginosa within epithelial cells that persist in vacuoles rather than the cytoplasm and that tolerate a cell-permeable antibiotic (ofloxacin) that is able to kill cytosolic bacteria. Classical biofilm-associated markers, although demonstrated by this subpopulation, are not required for vacuolar persistence or antibiotic tolerance. These findings advance our understanding of how P. aeruginosa hijacks host cells, showing that it diversifies into multiple populations with T3SS-negative members enabling persistence while rapid replication is accomplished by more vulnerable T3SS-positive siblings. Intracellular P. aeruginosa persisting and tolerating antibiotics independently of the T3SS or biofilm-associated factors could present additional challenges to development of more effective therapeutics.

Blocking Autophagy in M1 Macrophages Enhances Virus Replication and Eye Disease in Ocularly Infected Transgenic Mice.

Macrophages are one of the first innate immune infiltrates in the cornea of mice following ocular infection with herpes simplex virus 1 (HSV-1). Using gamma interferon (IFN-γ) and interleukin-4 (IL-4) injections to polarize macrophages into M1 and M2, respectively, and in M1 and M2 conditional knockout mice, we have shown that M1 macrophages play an important role in suppressing both virus replication in the eye and eye disease in HSV-1-infected mice. Autophagy is also important in controlling HSV infection and integrity of infected cells. To determine if blocking autophagy in M1 and M2 macrophages affects HSV-1 infectivity and eye disease, we generated two transgenic mouse strains expressing the HSV-1 γ34.5 autophagy gene under the M1 promoter (M1-γ34.5) or the M2 promoter (M2-γ34.5). We found that blocking autophagy in M1 macrophages increased both virus replication in the eyes and eye disease in comparison to blocking autophagy in M2 macrophages or wild-type (WT) control mice, but blocked autophagy did not affect latency-reactivation. However, blocking autophagy affected fertility in both M1 and M2 transgenic mice. Analysis of 62 autophagy genes and 32 cytokines/chemokines from infected bone marrow-derived macrophages from M1-γ34.5, M2-γ34.5, and WT mice suggested that upregulation of autophagy-blocking genes (i.e., Hif1a, Mtmr14, mTOR, Mtmr3, Stk11, and ULK2) and the inflammatory tumor necrosis factor alpha (TNF-α) gene in M1-γ34.5 transgenic mice correlated with increased pathogenicity, while upregulation of proautophagy genes (Nrbf2 and Rb1cc1) in M2-γ34.5 macrophages correlated with reduced pathogenicity. The in vivo and in vitro responses of M1-γ34.5 and M2-γ34.5 transgenic mice to HSV-1 infection were independent of the presence of the γ34.5 gene in wild-type HSV-1. Our results suggest that M1 macrophages, but not M2 macrophages, play an important role in autophagy relative to primary virus replication in the eye and eye disease in infected mice. IMPORTANCE Autophagy plays a critical role in clearing, disassembling, and recycling damaged cells, thus limiting inflammation. The HSV-1 γ34.5 gene is involved in neurovirulence and immune evasion by blocking autophagy in infected cells. We found that blocking autophagy in M1 macrophages enhances HSV-1 virus replication in the eye and eye disease in ocularly infected transgenic mice. Our results also show the suppressive effects of γ34.5 on immune responses to infection, suggesting the importance of intact autophagy in M1 but not M2 macrophages in controlling primary infection and eye disease.

Caspase-8 is involved in pyroptosis, necroptosis and the maturation and release of IL-1β in Aspergillus fumigatus keratitis

PurposeTo explore the role of caspase-8 in mediating the transition between different death modes in fungal keratitis. MethodsThe expression of caspase-8 in Aspergillus fumigatus (A. fumigatus) keratitis was detected using western blotting and immunofluorescence. After subconjunctival injection of Z-IETD-FMK (caspase-8 inhibitor) or VX765 (caspase-1 inhibitor), the mice corneas of A. fumigatus keratitis were observed and scored under a slit lamp. Colony plate count, immunofluorescence staining, western blotting and qRT-PCR experiments were used to detect fungal load, inflammatory cells, and the production of related mRNAs and proteins. In vitro experiments, the LDH release test, Cell Count Kit-8(CCK-8) assay, ELISA, qRT-PCR and western blotting were used to detect cell viability, related mRNAs and proteins. ResultsThe caspase-8 protein was upregulated following fungal infection. Compared with the A. fumigatus keratitis group, the mice treated with Z-IETD-FMK had heavier corneal turbidity, higher clinical scores, more fungal load and fewer inflammatory cells. The expression of NLRP3, cleaved-caspase-1, N-GSDMD, and IL-1β in the fungal infection group after Z-IETD-FMK pretreatment were downregulated, while RIPK3 and p-MLKL were upregulated. In the fungal infection group after VX765 pretreatment, the expression of cleaved-caspase-8 was up-regulated, while N-GSDMD was downregulated. ConclusionsCaspase-8 is involved in the early immune defense response of A. fumigatus keratitis. It is essential for the recruitment of inflammatory cells and the clearance of the fungus. In A. fumigatus keratitis, activated caspase-8 promoted the caspase-1/GSDMD signaling pathway to participate in pyroptosis, inhibited RIPK3/MLKL signaling pathway-mediated necroptosis, and promoted IL-1β maturation and release by activating the NLRP3 inflammasomes.

MCPIP1 alleviates inflammatory response through inducing autophagy in Aspergillus fumigatus keratitis

Fungal keratitis (FK) is a serious corneal infection caused by pathogenic fungi. Monocyte chemoattractant protein-induced protein 1 (MCPIP1) plays an important role in restricting the inflammatory response in various immune disorders. However, the function of MCPIP1 in Aspergillus fumigatus (A. fumigatus) keratitis is unclear. In the present study, we found that A. fumigatus infection promotes the expression of MCPIP1 in human corneal epithelial cells (HCECs) and in mouse corneas. Overexpression of MCPIP1 decreased the production of inflammatory cytokines, including TNF-α, IL-6, and IL-1β, while knockdown of MCPIP1 increased the expression of these cytokines. MCPIP1 enhanced autophagy flux by inhibiting the mTOR signaling in HCECs with A. fumigatus infection. Further study showed that inhibition of autophagy using chloroquine reverses the anti-inflammatory effect of MCPIP1 in HCECs infected with A. fumigatus. Moreover, MCPIP1 alleviated the severity of keratitis and inhibited the expression of inflammatory cytokines by activating autophagy in an FK mouse model. In conclusion, our study demonstrated that MCPIP1 alleviates the severity of A. fumigatus keratitis by inducing mTOR-mediated autophagy in HCECs and in a mouse model. Exogenous use of MCPIP1 protein may have potential applications in FK clinical therapy.