Sensory nerves promote corneal inflammation resolution via CGRP mediated transformation of macrophages to the M2 phenotype through the PI3K/AKT signaling pathway.

OBJECTIVES: To explore the role of the corneal sensory nerves in Pseudomonas aeruginosa (P. aeruginosa) keratitis, the synergistic effect between the sensory neurons and macrophages in calcitonin gene-related peptide (CGRP) release, and the functional mechanisms of CGRP-mediated transformation of macrophages to the M2 phenotype. METHODS: Corneal nerve loss, macrophage recruitment, and CGRP expression were evaluated. To explore the synergistic effect between the sensory neurons and macrophages, RAW 264.7 cells were challenged with lipopolysaccharide (LPS), then trigeminal ganglion (TG) sensory neurons were isolated and co-incubated with macrophages, and CGRP expression was tested. To investigate the biological function of cornea neuron-initiated immune responses mediated by CGRP, BIBN 4096BS was used to inhibit CGRP in vivo and α-CGRP was used to simulate CGRP in vitro. The expressions of inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IL-10), M1 (CD80/CD86), M2 (CD163/CD206) macrophage markers, and signal transducers (PI3K/AKT) were detected. RESULTS: P. aeruginosa infection induced corneal nerve loss, macrophage recruitment, and CGRP up-expression. CGRP was co-localized with macrophages. Co-culture showed that sensory neurons and macrophages can mediate CGRP release. More CGRP was released when the two types of cells were combined to respond to LPS. BIBN 4096BS promoted pro-inflammatory cytokines and inhibited the anti-inflammatory cytokines and signal transducers, while, α-CGRP inhibited the pro-inflammatory cytokines and M1 markers and promoted the anti-inflammatory cytokine, M2 markers, and signal transducers. CONCLUSIONS: P. aeruginosa infection induces corneal sensory neuron activation, macrophage recruitment, and CGRP up-expression. The synergistic effect between the sensory neurons and macrophages promotes CGRP release. CGRP inhibits corneal inflammation and promotes the transformation of macrophages to the M2 phenotype through the PI3K/AKT signaling pathway.

Effects on the Ocular Surface from Reading on Different Smartphone Screens: A Prospective Randomized Controlled Study.

The purpose of this study was to investigate the influence of smartphone reading on the ocular surface and to compare the various effects of different screens and light conditions on the ocular surface. One hundred nineteen volunteers were randomly divided into: light + organic light-emitting diode (OLED), light + electronic ink (eINK), dark + OLED, and dark + eINK. Ocular surface examinations, including noninvasive break-up time (NIBUT), noninvasive keratograph tear meniscus height (NIKTMH), ocular redness, fluorescein break-up time (FBUT), corneal fluorescein staining, meibomian gland assessment, Schirmer I Test, and blinking frequency, were performed before and after a reading task. Symptoms were evaluated using the Ocular Surface Disease Index (OSDI) and Computer Vision Syndrome Questionnaire (CVS-Q). NIBUT and FBUT were decreased statistically significantly after participants read on an OLED screen for 2 hours compared with the baseline in light and dark environments, whereas no statistically significant decrease was observed on an eINK screen. NIKTMH was statistically significantly decreased after reading on an OLED screen in light and dark settings, and the eINK screen had a lesser effect on NIKTMH. An obvious increase in the ocular redness, OSDI and CVS-Q scores was observed after reading on an OLED screen, whereas the eINK screen had a lesser effect on these indicators. Blink rate increased gradually in OLED subgroups during the reading task, whereas no statistically significant difference was observed in the eINK subgroups. Our research suggested that reading on an OLED screen can cause ocular surface disorder and obvious subjective discomfort, whereas reading on an eINK screen can minimize ocular surface disorder in both dark and light environments.

Influence of Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone on human corneal epithelial cells.

The quorum-sensing (QS) signaling-dependent extracellular virulence factors of Pseudomonas aeruginosa can cause infections such as P. aeruginosa keratitis. P. aeruginosa communicates by secreting and sensing small chemical molecules called autoinducers in QS system. The key QS signal molecule, N-3-oxododecanoyl-homoserine lactone (3OC12HSL), can affect the behavior of host cells and initiate immune response. In this report we investigated the influence of 3OC12HSL on human corneal epithelial cells (HCECs) and the mechanisms of 3OC12HSL on activated toll-like receptor 2 (TLR2)-dependent interleukin-8 (IL-8) secretion in HCECs. Cells were cultured under different concentrations of 3OC12HSL. Cell viability was assessed using Crystal violet staining and the cell counting kit-8 assay. We demonstrated the administration of 3OC12HSL decreased HCEC viability and survival in a concentration- and time-dependent manner. At high concentrations, 3OC12HSL rapidly promoted a time-dependent increase in the expressions of TLR2 and TLR4. It was found that the nuclear translocation and expression of nuclear factor-κB (NF-κB) were also increased in response to 3OC12HSL treatment. The significantly elevated expressions of TLR2, TLR4, and NF-κB, encouraged us to further test their mechanisms that cause inflammatory response. Among the inflammatory factors examined (IL-6, IL-8, IL-10, and TNF-α), we found that IL-8 was significantly increased after treatment with 3OC12HSL and its expression was inhibited when TLR2 was specifically blocked or silenced. These results indicated that the QS signaling molecule 3OC12HSL could be recognized by the host innate immune system in HCECs. This recognition then triggered an immune inflammatory response involving the activation of TLR2 and an increase in expression of IL-8. This crosstalk between 3OC12HSL and host immunity in HCECs contributes to the development and progression of P. aeruginosa keratitis.

Acetylsalicylic Acid Promotes Corneal Epithelium Migration by Regulating Neutrophil Extracellular Traps in Alkali Burn.

Neutrophils are the first cells to migrate into the cornea in response to alkali burns, and excessive neutrophil infiltration is associated with inflammatory injury and a poorer prognosis. In an effort to understand the mechanisms underlying the inflammation mediated by neutrophils after alkali burns, we examined the role of alkali-activated neutrophils on human corneal epithelial cells (HCEs) proliferation and migration, as well as the effects of acetylsalicylic acid (ASA) and dexamethasone (DXM) on NETosis. We stimulated human neutrophils with sodium hydroxide (NaOH) and observed dose- and time-dependent neutrophil extracellular traps (NETs) formation. We also observed that ASA, but not DXM, significantly inhibited NaOH-induced NETosis. Furthermore, the activation of nuclear factor (NF)-κB, but not the production of reactive oxygen species, was involved in ASA-regulated NETosis. Moreover, NETs were found to be involved in alkali-activated neutrophils (ANs) induced neutrophil-HCE adhesion. ANs enhanced HCEs proliferation via phagocytosis. Meanwhile, ANs inhibited HCEs migration through the release of NETs, which was partially rescued by 5 mM ASA. In conclusion, ANs may interfere with HCEs proliferation and migration by phagocytosis and NETs formation, respectively. ASA may enhance HCEs migration by decreasing NETs formation through inhibition of NF-κB activation and could be a promising strategy for improving the prognosis of corneal alkali burns.

Fenretinide Inhibits Neutrophil Recruitment and IL-1ß Production in Aspergillus fumigatus Keratitis.

PURPOSE: Fungal keratitis is a major cause of corneal ulcers, resulting in significant visual impairment and blindness. Fenretinide, a derivative of vitamin A, has been shown to suppress inflammation in a multitude of diseases. In this study, we aimed to characterize the effect of fenretinide in Aspergillus fumigatus keratitis of the eye in a mouse model. METHODS: In vivo and in vitro experiments were performed in mouse models and THP-1 macrophage cell cultures infected with A. fumigatus, respectively. Experimental subjects were first pretreated with fenretinide, and then the effect of the compound was assessed with clinical evaluation, neutrophil staining, myeloperoxidase assay, quantitative polymerase chain reaction (qRT-PCR), and western blot. RESULTS: We confirmed that fenretinide contributed to protection of corneal transparency during early mouse A. fumigatus keratitis by reducing neutrophil recruitment, decreasing myeloperoxidase (MPO) levels and increasing apoptosis. Compared with controls, fenretinide impaired proinflammatory cytokine interleukin 1 beta (IL-1ß) production in response to A. fumigatus exposure with contributions by lectin-type oxidized LDL receptor 1 (LOX-1) and c-Jun N-terminal kinase (JNK). CONCLUSIONS: Together, these findings demonstrate that fenretinide may suppress inflammation through reduced neutrophil recruitment and inflammatory cytokine production in A. fumigatus keratitis.

Wnt5a contributes to dectin-1 and LOX-1 induced host inflammatory response signature in Aspergillus fumigatus keratitis.

Fungal keratitis causes devastating corneal ulcers which can result in significant visual impairment and even blindness. As a ligand that activates the non-canonical Wnt signaling pathways, Wnt5a triggers the production of important inflammatory chemokines and the chemotactic migration of neutrophils. In this study we aimed to characterize the role of Wnt5a production, in situ, in vivo and in vitro in response to fungal keratitis. Wnt5a expression in corneas of Aspergillus fumigatus (A. fumigatus) keratitis patients was determined by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. In vivo and in vitro experiments were then performed in mouse models and THP-1 macrophages cell cultures infected with A. fumigatus, respectively. C57BL/6 mice were pretreated with siRNAs or neutralizing antibodies for dectin-1, LOX-1 and Wnt5a, or inhibitors of erk1/2 and JNK. Changes in Wnt5a expression were assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blot and bioluminescence imaging system image acquisition. We confirmed that corneal Wnt5a expression increased with A. fumigatus keratitis in patients and a murine model. Wnt5a production was dependent on dectin-1 and LOX-1 expression with contributions by Erk1/2 and JNK pathways. Additionally, Wnt5a knockdown revealed decreased levels of MPO, lower neutrophil recruitment, and a higher fungal load in mouse models. Compared with controls, Wnt5a knockdown impaired pro-inflammatory cytokine IL-1ß production in response to A. fumigatus exposure. Wnt5a also produces dectin-1 and LOX-1 induced inflammatory signature via effective neutrophil recruitment and inflammatory cytokine production in response to A. fumigatus keratitis. These findings demonstrate that Wnt5a is a critical component of the antifungal immune response.

Osteopontin contributes to effective neutrophil recruitment, IL-1ß production and apoptosis in Aspergillus fumigatus keratitis.

Fungal keratitis is a major cause of corneal ulcers, resulting in significant visual impairment and blindness. A phosphorylated glycoprotein secreted by immunocompetent cells, osteopontin (OPN) mediates cluster formation of the host fungal receptors and enhances the phagocytosis and clearance of pathogenic fungi. However, whether OPN production and function occurs in fungal keratitis is unknown. OPN expression in Aspergillus fumigatus keratitis patient corneas was assessed by quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence. Human neutrophils, THP-1 macrophages and corneal epithelial cells (HCECs) stimulated with A. fumigatus were utilized for in vitro experiments. Mouse models of A. fumigatus keratitis were developed by intrastromal injection for in vivo experiments. Using siRNAs, neutralizing antibodies, recombinant proteins and inhibitors, the production and role of OPN in A. fumigatus infection was assessed by clinical evaluation, qRT-PCR, immunofluorescence, western blotting and bioluminescence image acquisition. We observed increased corneal OPN expression in A. fumigatus keratitis patients and mouse models compared to controls. OPN production in response to A. fumigatus infection was dependent on LOX-1 and Erk1/2. Compared to controls, OPN knockdown impaired proinflammatory cytokine IL-1ß production, which was dependent on 4E-BP1. OPN knockdown decreased myeloperoxidase levels, and resulted in decreased neutrophil recruitment, higher fungal load and increased apoptosis in mouse A. fumigatus keratitis. Our results indicate that OPN is a critical component of the antifungal immune response and is essential for effective neutrophil recruitment, inflammatory cytokine production and apoptosis in A. fumigatus keratitis.

Dectin-1 is essential for IL-1ß production through JNK activation and apoptosis in Aspergillus fumigatus keratitis.

PURPOSE: To investigate the role of phosphorylated JNK in Dectin-1-induced IL-1ß production and the role of Dectin-1 in apoptosis in mouse Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Mice corneas were pretreated with Dectin-1 siRNA or SP600125 (the inhibitor of JNK) before A. fumigatus infection. THP-1 macrophages were preincubated with SP600125 before the stimulation of A. fumigatus conidia. Dectin-1, IL-1ß, JNK, Bax, Bcl-2, cytochrome-c (cyt-c), caspase-9, caspase-8 and caspase-3 expressions were tested by PCR, Western blot, or Immunofluorescence staining. RESULTS: Pretreatment with Dectin-1 siRNA significantly decreased A. fumigatus-induced IL-1ß production and JNK phosphorylation compared with scrambled control in C57BL/6 mice corneas. SP600125 treatment before infection significantly inhibited IL-1ß production compared with DMSO control both in mice corneas and THP-1 macrophages. Furthermore, Dectin-1 deficiency resulted in increased ratio of Bax/Bcl-2, release of cyt-c, activation of caspase-9 and caspase-3 in mouse A. fumigatus keratitis. However, Dectin-1 deficiency didn't affect the activation of caspase-8. CONCLUSIONS: Being an important inflammatory PRR to mediate host inflammatory response, Dectin-1 induced IL-1ß production is JNK dependent in mouse A. fumigatus keratitis, and suppressed apoptosis mediated anti-inflammatory response.