IL-36α Exerts Proinflammatory Effects in Aspergillus fumigatus Keratitis of Mice Through the Pathway of IL-36α/IL-36R/NF-κB

PurposeTo explore the role of IL-36α in corneas infected by Aspergillus fumigatus.MethodsThe experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1β, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry.ResultsIL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1β, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra.ConclusionsIL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1β, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.     

The relationship between corneal subbasal nerve density and corneal sensitivity in patients with Fuchs endothelial corneal dystrophy

Purpose:The aim of this study was to investigate the association between alterations in corneal subbasal nerve plexus and tactile corneal sensitivity in patients with Fuchs’ endothelial corneal dystrophy (FECD).Methods:This retrospective, cross-sectional study included 24 (10 M/14 F) patients with FECD and 25 age- and sex-matched (10 M/15 F) healthy subjects as controls. Subjects with FECD were classified as having early (grades 1 and 2) and late (grades 3 and 4) disease. All subjects underwent central corneal tactile sensitivity measurements with the Cochet–Bonnet esthesiometer (Luneau Ophthalmologie, Chartres, France) and subbasal nerve density evaluation using in vivo confocal microscopy (IVCM). Association between corneal nerve plexus density and corneal sensitivity alterations were evaluated using the Mann–Whitney U test and the Spearman correlation test.Results:Compared to healthy subjects (mean age = 60.4 ± 7.5 years), patients with FECD (mean age = 60.6 ± 8.0 years) had worse central corneal sensitivity scores (5.9 ± 0.1 cm vs. 4.2 ± 0.8 cm; P < 0.001), reduced corneal nerve fibers (3.4 ± 1.3 nerves/frame vs. 5.0 ± 0.9 nerves/frame; P < 0.001) and lower corneal subbasal nerve plexus densities (2229.4 ± 364.3 μm/mm² vs. 1901.6 ± 486.8 μm/mm²; P = 0.050). Patients with late stage FECD demonstrated lower subbasal nerve densities as compared to those with early disease (2204.3 ± 313.1 μm/mm² (range = 1523–2552 μm/mm²); 1397.1 ± 227.4 μm/mm² (range = 1120-1834 μm/mm²); P < 0.001). In the FECD group, subbasal nerve density was found to be directly correlated with corneal sensitivity scores (r = 0.457, P = 0.025).Conclusion:Progressive loss of the corneal subbasal nerve plexus appears to be a consistent feature of FECD. Reduction of the corneal nerve plexus parallels the decrease in corneal sensitivity in this patient population.     

ID1 and ID3 are Negative Regulators of TGFβ2-Induced Ocular Hypertension and Compromised Aqueous Humor Outflow Facility in Mice

PurposeIn POAG, elevated IOP remains the major risk factor in irreversible vision loss. Increased TGFβ2 expression in POAG aqueous humor and in the trabecular meshwork (TM) amplifies extracellular matrix (ECM) deposition and reduces ECM turnover in the TM, leading to a decreased aqueous humor (AH) outflow facility and increased IOP. Inhibitor of DNA binding proteins (ID1 and ID3) inhibit TGFβ2-induced fibronectin and PAI-1 production in TM cells. We examined the effects of ID1 and ID3 gene expression on TGFβ2-induced ocular hypertension and decreased AH outflow facility in living mouse eyes.MethodsIOP and AH outflow facility changes were determined using a mouse model of Ad5-hTGFβ2^C226S/C288S-induced ocular hypertension. The physiological function of ID1 and ID3 genes were evaluated using Ad5 viral vectors to enhance or knockdown ID1/ID3 gene expression in the TM of BALB/cJ mice. IOP was measured in conscious mice using a Tonolab impact tonometer. AH outflow facilities were determined by constant flow infusion in live mice.ResultsOver-expressing ID1 and ID3 significantly blocked TGFβ2-induced ocular hypertension (P < 0.0001). Although AH outflow facility was significantly decreased in TGFβ2-transduced eyes (P < 0.04), normal outflow facility was preserved in eyes injected concurrently with ID1 or ID3 along with TGFβ2. Knockdown of ID1 or ID3 expression exacerbated TGFβ2-induced ocular hypertension.ConclusionsIncreased expression of ID1 and ID3 suppressed both TGFβ2-elevated IOP and decreased AH outflow facility. ID1 and/or ID3 proteins thus may show promise as future candidates as IOP-lowering targets in POAG.     

Investigations on Retinal Pigment Epithelial Damage at Laser Irradiation in the Lower Microsecond Time Regime

PurposeNew lasers with a continuous wave power exceeding 15 W are currently investigated for retinal therapies, promising highly localized effects at and close to the Retinal Pigment Epithelium (RPE). The goal of this work is to evaluate mechanisms and thresholds for RPE cell damage by means of pulse durations up to 50 µs.MethodsA diode laser with a wavelength of 514 nm, a power of 15 W, and adjustable pulse durations between 2 µs and 50 µs was used. Porcine RPE-choroidal explants (ex vivo) and chinchilla bastard rabbits (in vivo) were irradiated to determine threshold radiant exposures for RPE damage H¯Cell by calcein vitality staining and fluorescence angiography, respectively. Thresholds for microbubble formation (MBF) H¯MBF were evaluated by time-resolved optoacoustics. Exemplary histologies support the findings.Results H¯MBF is significantly higher than H¯Cell at pulse durations ≥ 5 µs (P < 0.05) ex vivo, while at 2 µs, no statistically significant difference was found. The ratios between H¯MBF and H¯Cell increase with pulse duration from 1.07 to 1.48 ex vivo and 1.1 to 1.6 in vivo, for 5.2 and 50 µs.ConclusionsCellular damage with and without MBF related disintegration are both present and very likely to play a role for pulse durations ≥ 5 µs. With the lower µs pulses, selective RPE disruption might be possible, while higher values allow achieving spatially limited thermal effects without MBF. However, both modi require a very accurate real-time dosing control in order to avoid extended retinal disintegration in this power range.     

Variations in the central corneal thickness during the menstrual cycle in Indian women

Purpose:To determine the changes in central corneal thickness (CCT) during the menstrual cycle in Indian women.Methods:A prospective observational clinical study at a tertiary care center between December 2015 and December 2018. One hundred and twenty sixty women between 18 and 45 years were included. The CCT was measured using an ultrasound pachymeter at three specific timelines of the menstrual cycle: at the beginning (1^st to 3^rd day), during ovulation time (14^th to 16^th day), and at the end of the cycle (28^th to 33^rd day). Phases of the cycle were confirmed by the urine luteinizing hormone level.Results:The mean CCT of both eyes was 541.76 ± 4.21 μm, 559.21 ± 4.50 μm, and 544.52 ± 8.06 μm at the beginning, mid, and end of cycle, respectively. The mean CCT of the right eye was 541.68 ± 4.15 μm, 559.08 ± 4.50 μm, and 544.44 ± 8.06 μm and of the left eye was 541.84 ± 4.27 μm, 559.35 ± 4.50 μm, and 544.61 ± 8.06 μm at the beginning, mid, and end of cycle, respectively.Conclusion:The CCT value was significantly (P < 0.001) higher during ovulation compared to the beginning and end of the menstrual cycle. Our study recommends adding menstrual history in the workup of women undergoing refractive surgery as physiological variations in the CCT may result in unexpected surgical outcomes.     

The Role of SREC-Ⅰ in Innate Immunity to Aspergillus fumigatus Keratitis

PurposeTo determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-Ⅰ) in vitro and in a mouse model of Aspergillus fumigatus keratitis.MethodsSREC-Ⅰ mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-Ⅰ expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-Ⅰ small interfering RNA, then the mRNA levels of LOX-1, IL-1β, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-Ⅰ mRNA and protein expression were detected by RT-PCR, Western blot and immunofluorescence. The severity of FK was evaluated by clinical score. CLCX1, LOX-1, IL-1β, and TNF-α mRNA expression levels were tested before and after anti–SREC-Ⅰ treatment.ResultsSREC-Ⅰ expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-Ⅰ mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-Ⅰ small interfering RNA treatment inhibited the expressions of LOX-1, IL-1β, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1β, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-Ⅰ–neutralizing antibody treatment.ConclusionsSREC-Ⅰ is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-Ⅰ blockade could be a potential therapeutic approach for FK.     

Investigating the effects of carvacrol in rats using oxygen-induced retinopathy model

Purpose:Investigating the effects of intraperitoneal carvacrol administration in rats using the oxygen-induced retinopathy (OIR) model.Methods:A total of 28 newborn Sprague Dawley rats were used and the OIR model was created using the 50/10% oxygen model. The study composed of four groups in total. While the OIR model was not used in Group I (control group), it was created for Groups II, III, and IV. About 0.01 mL carvacrol, bevacizumab, or 0.9% NaCl was administered intraperitoneal (IP) to the rats in all groups on postnatal day (PND) 14 as follows: Group I and Group II were administered 0.9% NaCl, Group III was administered bevacizumab, and Group IV was administered carvacrol. On PND 18, rats were sacrificed and their right eyes were enucleated.Results:Histopathological and immunohistochemical studies showed that the number of vascular endothelial cells (VECs), vascular endothelial growth factor (VEGF), and tumor necrosis factor-α (TNF-α) decreased similarly in Group III and Group IV compared with Group II. VECs values for Group I, Group II, Group III, and Group IV were measured as 0 ± 0, 26.45 ± 4.57, 7.75 ± 1.98, and 5.78 ± 1.72, respectively, and it differed significantly between groups (P < 0.001). Likewise, VEGF levels were observed as 0.06 ± 0.01, 3.31 ± 0.53, 2.47 ± 0.44, and 2.49 ± 0.52, respectively, and it differed significantly between groups (P < 0.001). TNF-α levels were recorded as 0.06 ± 0.01, 3.58 ± 0.38, 2.46 ± 0.49, and 2.29 ± 0.25, respectively, and it differed significantly between groups (P < 0.001). VECs, VEGF, and TNF-α were similar between Group III and IV (range of P values were 0.486–0.998).Conclusion:The study demonstrated that carvacrol significantly reduced retinal pathological angiogenesis, NV, VEC nuclei count, VEGF, and TNF-α levels. Moreover, the observed effects were comparable to those of bevacizumab.     

TGF-β2 Promotes Oxidative Stress in Human Trabecular Meshwork Cells by Selectively Enhancing NADPH Oxidase 4 Expression

PurposeThe multifunctional profibrotic cytokine TGF-β2 is implicated in the pathophysiology of primary open angle glaucoma (POAG). While the underlying cause of POAG remains unclear, TGF-β2 dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Mitochondrial-targeted antioxidants have been recently shown by our group to markedly attenuate TGF-β2 profibrotic responses, strongly implicating oxidative stress as a key facilitator of TGF-β2 signaling in human TM cells. In this study, we determined the mechanism by which oxidative stress facilitates TGF-β2 profibrotic responses in cultured primary human TM cells.MethodsSemiconfluent cultures of primary or transformed human TM cells were conditioned overnight in serum-free media and subsequently challenged without or with TGF-β2 (5 ng/mL). Relative changes in the mRNA content of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) isoforms, connective tissue growth factor (CTGF), collagen 1α1 and 4α1 isoforms or relative changes in the protein content of Nox4, phospho- and total-Smad2 and -Smad3, collagens I and IV were determined in the absence or presence of GKT137831, a Nox1-Nox4 dual enzyme inhibitor, and quantified by real-time qPCR or by immunoblot, respectively. Relative in situ changes in collagens I and IV and in alpha smooth muscle actin (αSMA) were semiquantified by immunocytochemistry, whereas relative changes in filamentous actin stress fiber formation was semiquantified by phalloidin staining.ResultsQuiescent primary human TM cells cultured in the presence of TGF-β2 exhibited a marked selective increase in endogenous Nox4 mRNA and Nox4 protein expression. Actinomycin D prevented TGF-β2 mediated increases in Nox4 mRNA expression. TM cells reverse transfected with siRNA against Smad3 prevented TGF-β2 mediated increases in Nox4 mRNA expression. Pre-incubating TM cells with GKT137831 attenuated TGF-β2 mediated increases in intracellular reactive oxygen species (ROS), in COL1A1, COL4A1, and CTGF mRNA expression, in Smad3 protein phosphorylation, in collagens I, collagens IV, and αSMA protein expression, and in filamentous actin stress fiber formation.ConclusionsTGF-β2 promotes oxidative stress in primary human TM cells by selectively increasing expression of NADPH oxidase 4. Dysregulation of redox equilibrium by induction of NADPH oxidase 4 expression appears to be a key early event involved in the pathologic profibrotic responses elicited by TGF-β2 canonical signaling, including ECM remodeling, filamentous actin stress fiber formation, and αSMA expression. Selective inhibition of Nox4 expression/activation, in combination with mitochondrial-targeted antioxidants, represents a novel strategy by which to slow the progression of TGF-β2 elicited profibrotic responses within the TM.     

CD40 Expressed in Endothelial Cells Promotes Upregulation of ICAM-1 But Not Pro-Inflammatory Cytokines,NOS2 and P2X7 in the Diabetic Retina

PurposeCD40 is an upstream inducer of inflammation in the diabetic retina. CD40 is upregulated in retinal endothelial cells in diabetes. The purpose of this study was to determine whether expression of CD40 in endothelial cells is sufficient to promote inflammatory responses in the retina of diabetic mice.MethodsTransgenic mice with CD40 expression restricted to endothelial cells (Trg-CD40 EC), transgenic control mice (Trg-Ctr), B6, and CD40^−/− mice were made diabetic using streptozotocin. Leukostasis was assessed using FITC-conjugated ConA. Pro-inflammatory molecule expression was examined by real-time PCR, immunohistochemistry, ELISA, or flow cytometry. Release of ATP was assessed by ATP bioluminescence.ResultsDiabetic B6 and Trg-CD40 EC mice exhibited increased retinal mRNA levels of ICAM-1, higher ICAM-1 expression in endothelial cells, and increased leukostasis. These responses were not detected in diabetic mice that lacked CD40 (CD40^−/− and Trg-Ctr). Diabetic B6 but not Trg-CD40 EC mice upregulated TNF-α, IL-1β, and NOS2 mRNA levels. CD40 stimulation in retinal endothelial cells upregulated ICAM-1 but not TNF-α, IL-1β, or NOS2. CD40 ligation did not trigger ATP release by retinal endothelial cells or pro-inflammatory cytokine production in bystander myeloid cells. In contrast to diabetic B6 mice, diabetic Trg-CD40 EC mice did not upregulate P2X₇ mRNA levels in the retina.ConclusionsEndothelial cell CD40 promotes ICAM-1 upregulation and leukostasis. In contrast, endothelial cell CD40 does not lead to pro-inflammatory cytokine and NOS2 upregulation likely because it does not activate purinergic-mediated pro-inflammatory molecule expression by myeloid cells or induce expression of these pro-inflammatory molecules in endothelial cells.     

Stress Signal Regulation by Na/K-ATPase As a New Approach to Promote Physiological Revascularization in a Mouse Model of Ischemic Retinopathy

PurposeThe identification of target pathways to block excessive angiogenesis while simultaneously restoring physiological vasculature is an unmet goal in the therapeutic management of ischemic retinopathies. pNaKtide, a cell-permeable peptide that we have designed by mapping the site of α1 Na/K-ATPase (NKA)/Src binding, blocks the formation of α1 NKA/Src/reactive oxygen species (ROS) amplification loops and restores physiological ROS signaling in a number of oxidative disease models. The aim of this study was to evaluate the importance of the NKA/Src/ROS amplification loop and the effect of pNaKtide in experimental ischemic retinopathy.MethodsHuman retinal microvascular endothelial cells (HRMECs) and retinal pigment epithelium (ARPE-19) cells were used to evaluate the effect of pNaKtide on viability, proliferation, and angiogenesis. Retinal toxicity and distribution were assessed in those cells and in the mouse. Subsequently, the role and molecular mechanism of NKA/Src in ROS stress signaling were evaluated biochemically in the retinas of mice exposed to the well-established protocol of oxygen-induced retinopathy (OIR). Finally, pNaKtide efficacy was assessed in this model.ResultsThe results suggest a key role of α1 NKA in the regulation of ROS stress and the Nrf2 pathway in mouse OIR retinas. Inhibition of α1 NKA/Src by pNaKtide reduced pathologic ROS signaling and restored normal expression of hypoxia-inducible factor 1-α/vascular endothelial growth factor (VEGF). Unlike anti-VEGF agents, pNaKtide did promote retinal revascularization while inhibiting neovascularization and inflammation.ConclusionsTargeting α1 NKA represents a novel strategy to develop therapeutics that not only inhibit neovascularization but also promote physiological revascularization in ischemic eye diseases.     

Treatment of ocular symptoms of Behçet's disease with interferon α2a: a pilot study

AIM—To study long term effects of interferon α_2a (IFNα_2a) on panuveitis in seven patients with Behçet's disease in a prospective, open clinical trial.
METHODS—Seven patients were treated with IFNα_2a for a mean of 23.6 months (14-37 months). They received an initial dose of IFNα_2a of 6×10⁶ IU/day, followed by 3×10⁶ IU/day after 1 month and 3×10⁶ IU every other day after 3 months. Two patients received low dose prednisolone (between 0.2 and 0.4 mg/kg/body weight) additionally at the beginning of the therapy. Complete cessation of IFNα_2a was possible in three patients (observation period 22, 6, and 4 months).
RESULTS—Marked improvement occurred in six patients who had ocular manifestations of Behçet's disease for the first time or with minor damage during their course of chronic relapsing panuveitis. In one patient with advanced ocular Behçet's disease, new relapses were prevented. Retinal infiltrates resolved within 2 weeks; vasculitis, macular oedema, infiltration of the anterior chamber and vitreous resolved within 4 weeks. Mean posterior uveitis score before treatment (nine affected eyes) was 6.6, 4 weeks after IFN it was reduced to 0.4. The mean observation period is 27.6 months, ranging from 14 to 42 months.
CONCLUSION—Treatment of ocular symptoms of Behçet's disease with IFNα_2a alone or in combination with low dose steroids led to complete remission of ocular vasculitis in all patients treated in this open, uncontrolled trial. Treatment with IFNα_2a may prevent permanent retinal or optic nerve damage due to vascular occlusion. No severe side effects occurred. Controlled randomised studies are warranted in order to prove the efficacy of IFNα_2a in ocular Behçet's disease and to compare it with other, established treatments such as azathioprine or cyclosporin A.

Keywords: Behçet's disease; uveitis; interferon α_2a     

Effects of Trabecular Meshwork Width and Schlemm’s Canal Area on Intraocular Pressure Reduction in Glaucoma Patients

PurposeTo evaluate the effects of baseline trabecular meshwork (TM) and Schlemm’s canal (SC) microstructures on intraocular pressure (IOP) reduction amount in treatment-naïve patients with primary open-angle glaucoma (POAG).MethodsA total 69 eyes of POAG patients who had not been treated with IOP-lowering agent were enrolled in this retrospective study. The patients had been prescribed topical IOP-lowering agent and used it for 1 year. The morphologic features of the TM and SC were collected using anterior segment module of spectral-domain optical coherence tomography with enhanced depth imaging at baseline. Images of the nasal and temporal corneoscleral limbus were obtained with serial horizontal enhanced depth imaging B-scans and TM width and SC area were measured in each scan. We investigated the effects of baseline TM and SC microstructures on IOP reduction amount.ResultsThe baseline IOP of 69 glaucomatous eyes was 17.9 ± 3.8 mmHg, and the mean amount of IOP reduction was 3.5 ± 2.1 mmHg after 1 year. Mean TM widths of nasal and temporal sector were 470.33 ± 80.05 and 479.74 ± 79.59 μm, respectively. SC area was measured as 4,818.50 ± 1,464.28, 4,604.23 ± 1,567.73 μm² at nasal sector and temporal sector, respectively. The correlation analysis revealed a positive correlation between SC area and average amount of IOP reduction, indicating that the larger baseline SC area, the greater the IOP drop with topical IOP-lowering agents. However, no correlation was found between TM width and IOP lowering amount in patients with POAG.ConclusionsThe baseline SC area showed positive correlation with the IOP reduction amount in patients with POAG. This finding suggests that the SC area can be a clinical parameter to predict the IOP reduction amount before using IOP-lowering agents in POAG patient.     

Orbital fibroblast chemokine modulation: effects of dexamethasone and cyclosporin A

AIM—Orbital inflammation is common, but the mechanisms underlying leucocytic infiltration of orbital tissue are poorly understood. Human orbital fibroblasts (OF) express chemokines, interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1), when exposed to proinflammatory cytokines. The effects of dexamethasone (DEX) and cyclosporin A (CSA) on OF IL-8 and MCP-1 were examined.
METHODS—Cultured human OF were incubated with recombinant interleukin 1β (rIL-1β; 0.2, 2.0, 20 ng/ml) alone or incubated with rIL-1β and DEX (10^-8, 10^-7, 10^-6 M) or CSA (3, 30, 300 ng/ml) for 24 hours. ELISA and northern blot analyses were performed to determine OF IL-8 and MCP-1 protein secretion and mRNA expression, respectively.
RESULTS—OF lacked constitutive IL-8 or MCP-1 expression, but secreted significant amounts of these chemokines and expressed substantial steady state mRNA for both chemokines upon rIL-1β stimulation. DEX caused dose dependent inhibition of IL-1 induced IL-8 (p<0.001) and MCP-1 (p<0.05) secretion and mRNA expression at all concentrations of rIL-1β. CSA enhanced IL-1 induced OF IL-8 (p<0.001) and suppressed rIL-1β induced OF MCP-1 (p<0.05) secretion when lower doses of rIL-1β were used. These effects on secreted chemokines at different concentrations of rIL-1β and immunomodulating agents were corroborated by steady state OF IL-8 and MCP-1 mRNA expression.
CONCLUSIONS—DEX is a potent inhibitor of OF IL-8 and MCP-1. In contrast, CSA enhances IL-1 induced OF IL-8 and suppresses OF MCP-1. These observations may explain the relative lack of CSA effectiveness in human orbital diseases that respond to corticosteroids.

Keywords: orbital fibroblasts; chemokines; dexamethasone; cyclosporin A     

Involvement of Müller Glial Autoinduction of TGF-β in Diabetic Fibrovascular Proliferation Via Glial–Mesenchymal Transition

PurposeMüller glial–mesenchymal transition (GMT) is reported as the fibrogenic mechanism promoted by TGF-β–SNAIL axis in Müller cells transdifferentiated into myofibroblasts. Here we show the multifaceted involvement of TGF-β in diabetic fibrovascular proliferation via Müller GMT and VEGF-A production.MethodsSurgically excised fibrovascular tissues from the eyes of patients with proliferative diabetic retinopathy were processed for immunofluorescence analyses of TGF-β downstream molecules. Human Müller glial cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and ELISA, respectively. Immunoblot analyses were performed to detect TGF-β signal activation.ResultsMüller glial cells in patient fibrovascular tissues were immunopositive for GMT-related molecular markers, including SNAIL and smooth muscle protein 22, together with colocalization of VEGF-A and TGF-β receptors. In vitro administration of TGF-β1/2 upregulated TGFB1 and TGFB2, both of which were suppressed by inhibitors for nuclear factor-κB, glycogen synthase kinase-3, and p38 mitogen-activated protein kinase. Of the various profibrotic cytokines, TGF-β1/2 application exclusively induced Müller glial VEGFA mRNA expression, which was decreased by pretreatment with small interfering RNA for SMAD2 and inhibitors for p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Supporting these findings, TGF-β1/2 stimulation to Müller cells increased the phosphorylation of these intracellular signaling molecules, all of which were also activated in Müller glial cells in patient fibrovascular tissues.ConclusionsThis study underscored the significance of Müller glial autoinduction of TGF-β as a pathogenic cue to facilitate diabetic fibrovascular proliferation via TGF-β–driven GMT and VEGF-A–driven angiogenesis.     

Baicalein Protects Against Aspergillus fumigatus Keratitis by Reducing Fungal Load and Inhibiting TSLP-Induced Inflammatory Response

PurposeTo investigate the antifungal and anti-inflammatory effects of baicalein on Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms.MethodsThe noncytotoxic antifungal concentration of baicalein was determined using CCK8, cell scratch assay, minimum inhibitory concentration, biofilm formation, scanning electron microscopy, propidium iodide uptake test and adherence assay in vitro and Draize test in vivo. In fungal keratitis (FK) mouse models, clinical score and plate count were used to evaluate FK severity, and myeloperoxidase assay and immunofluorescence staining were performed to examine neutrophil infiltration and activity. Real-time PCR, ELISA, and Western blot were performed to explore the anti-inflammatory activity of baicalein and the underlying mechanisms in vivo and in vitro.ResultsBaicalein at 0.25 mM (noncytotoxic) significantly inhibited A. fumigatus growth, biofilm formation, and adhesion in vitro. In A. fumigatus keratitis mice, baicalein mitigated FK severity, reduced fungal load, and inhibited neutrophil infiltration and activity. Baicalein not only suppressed mRNA and protein levels of proinflammatory factors IL-1β, IL-6, and TNF-α, but also inhibited the expression of thymic stromal lymphopoietin (TSLP) and TSLP receptor (TSLPR) in vivo and in vitro. In HCECs, mRNA and protein levels of IL-1β, IL-6, and TNF-α were significantly lower in the TSLP siRNA–treated group, while higher in the rTSLP-treated group than in the corresponding control. Baicalein treatment significantly inhibited rTSLP induced the expression of IL-1β, IL-6, and TNF-α.ConclusionsBaicalein plays a protective role in mouse A. fumigatus keratitis by inhibiting fungal growth, biofilm formation, and adhesion, and suppressing inflammatory response via downregulation of the TSLP/TSLPR pathway.     

Immunosubunit β5i Knockout Suppresses Neovascularization and Restores Autophagy in Retinal Neovascularization by Targeting ATG5 for Degradation

PurposeTo investigate the functional role of immunoproteasome subunit β5i in pathologic retinal neovascularization (RNV) and its ability to link the immunoproteasome and autophagy.MethodsOxygen-induced retinopathy (OIR) was induced in wild-type (WT) and β5i knockout (KO) mouse pups on a C57BL/6J background. Proteasome catalytic subunit expression and proteasome activity were evaluated by quantitative real-time PCR (qPCR) and proteasome activity. Retinal vascular anatomy and neovascularization were characterized and quantified by retinal vascular flat-mount staining, fluorescence angiography, platelet endothelial cell adhesion molecule (PECAM) immunostaining, and hematoxylin and eosin staining. Correlation factors, including VEGF and ICAM-1, were detected by qPCR. Autophagy was examined by transmission electron microscopy (TEM). Autophagy biomarkers, including LC3, P62, ATG5, and ATG7, were measured by immunostaining and immunoblotting. The protein interaction between β5i and ATG5 was detected by immunoprecipitation.ResultsWe observed that β5i had the greatest effect in WT OIR mice. Fundus fluorescence angiography, retinal flat-mount staining, and PECAM staining revealed that pathologic RNV decreased in β5i KO OIR mice compared with WT OIR mice. Concurrently, TEM, immunostaining, and immunoblotting showed that autophagy was induced in β5i KO OIR mice compared to WT OIR mice through increases in autophagosome and LC3 expression and a decrease in P62. Mechanistically, β5i interacted with ATG5 and promoted its degradation, leading to autophagy inhibition and pathogenic RNV.ConclusionsThis study identifies a functional role for β5i in RNV regulation. β5i deletion ameliorates RNV and restores autophagy by stabilizing ATG5. These results demonstrate the potential of β5i to serve as a bridge linking the immunoproteasome and autophagy.     

Murine Model of Primary Acquired Ocular Toxoplasmosis: Fluorescein Angiography and Multiplex Immune Mediator Profiles in the Aqueous Humor

PurposeTo establish a murine model of primary acquired ocular toxoplasmosis (OT) and to investigate the immune mediator profiles in the aqueous humor (AH).MethodsC57BL/6 mice were perorally infected with Toxoplasma gondii. The ocular fundus was observed, and fluorescein angiography (FA) was performed. The AH, cerebrospinal fluid (CSF), and serum were collected before infection and at 28 days post-infection (dpi); the immune mediator levels in these samples were analyzed using multiplex bead assay.ResultsFundus imaging revealed soft retinochoroidal lesions at 14 dpi; many of these lesions became harder by 28 dpi. FA abnormalities, such as leakage from retinal vessels and dilation and tortuosity of the retinal veins, were observed at 14 dpi. Nearly all these abnormalities resolved spontaneously at 28 dpi. In the AH, interferon-γ, interleukin (IL)-1α, IL-1β, IL-6, IL-10, IL-12(p40), IL-12(p70), CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, and CXCL1/KC levels increased after infection. All these molecules except IL-1α, IL-4, and IL-13 showed almost the same postinfection patterns in the CSF as they did in the AH. The tumor necrosis factor α, IL-4, and IL-5 levels in the AH and CSF of the T. gondii–infected mice were lower than those in the serum. The postinfection IL-1α, IL-6, CCL2/MCP-1, CCL4/MIP-1β, and granulocyte colony-stimulating factor levels in the AH were significantly higher than those in the CSF and serum.ConclusionsA murine model of primary acquired OT induced via the natural infection route was established. This OT model allows detailed ophthalmologic, histopathologic, and immunologic evaluations of human OT. Investigation of AH immune modulators provides new insight into OT immunopathogenesis.     

Location of Parapapillary Gamma Zone and Vertical Fovea Location. The Beijing Eye Study 2011

PurposeTo assess the spatial relationship between the locations of the parapapillary gamma zone and the fovea.MethodsIn a non-glaucomatous subgroup of the population-based Beijing Eye Study population, we measured the mean angle between the optic disc–fovea line and the horizontal (disc–fovea angle), the vertical distance of the fovea from the horizontal through the optic disc center (fovea vertical distance), and the location and width of the widest part of parapapillary gamma zone.ResultsThe study included 203 individuals (203 eyes; mean axial length, 24.4 ± 1.5 mm; range, 22.03–28.87 mm). The widest gamma zone part was located most often temporal horizontally (51.7%), then inferiorly (43.8%), superiorly (2.5%), and nasally (2.0%). The disc–fovea angle (mean, 7.50° ± 4.00°; range, –6.30° to –23.25°) was significantly higher (P = 0.003; i.e., fovea located more inferiorly) in eyes with the widest gamma zone inferiorly (8.46° ± 4.37°) than in eyes with the widest gamma zone temporally (6.71° ± 3.46°) and in eyes with the widest gamma zone temporally, superiorly, or nasally combined (6.75° ± 3.53°; P = 0.003). The fovea vertical distance (mean, 0.65 ± 0.33 mm; range, –0.20 to 1.67 mm) was longer (P = 0.001; i.e., fovea located more inferiorly) in eyes with the widest gamma zone inferiorly (0.73 ± 0.33 mm) than in eyes with the widest gamma zone temporally (0.58 ± 0.30 mm) and in eyes with a temporal, superior, or nasal gamma zone combined (0.58 ± 0.31 mm; P = 0.001). The fovea vertical distance increased (multivariate analysis) with the widest gamma zone location inferiorly (β = 0.25; P = 0.001) and wider width of the gamma zone (β = 0.19; P = 0.01).ConclusionsAn inferior fovea location is associated with a wider inferior gamma zone and vice versa, supporting the notion of an inferior shifting of Bruch''s membrane as the cause for an inferior gamma zone.