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.

Wedelolactone ameliorates Pseudomonas aeruginosa-induced inflammation and corneal injury by suppressing caspase-4/5/11/GSDMD-mediated non-canonical pyroptosis.

Pseudomonas aeruginosa (P. aeruginosa) keratitis, a worldwide leading cause of corneal perforation and blindness, which is associated with contact lens usage. Increasing evidence has indicated that pyroptosis, a novel proinflammatory programmed cell death, is linked with ocular diseases, little is known about the role of noncanonical pyroptosis in microbial keratitis. Here, we first indicated the involvement of noncanonical pyroptosis in P. aeruginosa keratitis and investigated whether wedelolactone (WDL), a major active component of Eclipta prostrate known to target caspase-11, could alleviate P. aeruginosa keratitis development. We found the expression of caspase-4/5/11 and cleaved GSDMD in corneas of P. aeruginosa keratitis patients, animal models and lipopolysaccharide (LPS)-induced primary cultured human corneal keratocytes (piHCKs) were increased. Combining ciprofloxacin with WDL significantly ameliorated the severity of P. aeruginosa keratitis, as manifested by decreased inflammatory responses and reduced corneal epithelial defects. Consistent with these findings, WDL also dose-dependently alleviated LPS-induced noncanonical pyroptosis by reversing the increased expression of caspase-4/5 and GSDMD in piHCKs. In summary, our results demonstrated that by targeting the activation of caspase-4/5/11, wedelolactone inhibited the development of P. aeruginosa keratitis and suppressed the release of proinflammatory cytokines. Wedelolactone may be a promising anti-inflammatory candidate to combat P. aeruginosa keratitis.

Prophylactic and therapeutic vaccine against Pseudomonas aeruginosa keratitis using bacterial membrane vesicles.

PURPOSE: Pseudomonas aeruginosa (P. aeruginosa) infection is one of the major causes of keratitis. However, effective prophylactic and therapeutic vaccines against P. aeruginosa keratitis have yet to be developed. In this study, we explored the use of P. aeruginosa membrane vesicles (MVs) as a prophylactic vaccine as well as the use of immune sera derived from P. aeruginosa MV-immunized animals as a treatment for P. aeruginosa corneal infections in C57BL/6 mice. METHODS: C57BL/6 mice were intramuscularly immunized with P. aeruginosa MVs; the mouse corneas were then scarified and topically infected with several P. aeruginosa strains, followed by determination of corneal clinical score and corneal bacterial load. Next, immune sera derived from P. aeruginosa MV-immunized ICR mice were administered intraperitoneally to naïve C57BL/6 mice, followed by topical P. aeruginosa challenge. Finally, the immune sera were also used as a topical treatment in the mice with established P. aeruginosa corneal infections. RESULTS: P. aeruginosa-specific IgG and IgA antibodies induced by intramuscular immunization were detected not only in the sera but also in the eye-wash solution. Both active and passive immunization significantly inhibited P. aeruginosa corneal infection. Finally, topical treatment with immune sera in the mice with established P. aeruginosa corneal infections notably decreased the corneal clinical score and corneal bacterial load. CONCLUSIONS: P. aeruginosa keratitis can be attenuated by vaccination of P. aeruginosa MVs and topical application of P. aeruginosa MV-specific immune sera.

A novel murine model for contact lens wear reveals clandestine IL-1R dependent corneal parainflammation and susceptibility to microbial keratitis upon inoculation with Pseudomonas aeruginosa.

PURPOSE: Contact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear. METHODS: C57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification. RESULTS: Without deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days. CONCLUSIONS: This murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.

The involvement of proline-rich protein Mus musculus predicted gene 4736 in ocular surface functions.

AIM: To research the two homologous predicted proline-rich protein genes, Mus musculus predicted gene 4736 (MP4) and proline-rich protein BstNI subfamily 1 (Prb1) which were significantly upregulated in cultured corneal organs when encountering fungal pathogen preparations. This study was to confirm the expression and potential functions of these two genes in ocular surface. METHODS: A Pseudomonas aeruginosa keratitis model was established in Balb/c mice. One day post infection, mRNA level of MP4 was measured using real-time polymerase chain reaction (PCR), and MP4 protein detected by immunohistochemistry (IHC) or Western blot using a customized polyclonal anti-MP4 antibody preparation. Lacrimal glands from normal mice were also subjected to IHC staining for MP4. An online bioinformatics program, BioGPS, was utilized to screen public data to determine other potential locations of MP4. RESULTS: One day after keratitis induction, MP4 was upregulated in the corneas at both mRNA level as measured using real-time PCR and protein levels as measured using Western blot and IHC. BioGPS analysis of public data suggested that the MP4 gene was most abundantly expressed in the lacrimal glands, and IHC revealed that normal murine lacrimal glands were positive for MP4 staining. CONCLUSION: MP4 and Prb1 are closely related with the physiology and pathological processes of the ocular surface. Considering the significance of ocular surface abnormalities like dry eye, we propose that MP4 and Prb1 contribute to homeostasis of ocular surface, and deserve more extensive functional and disease correlation studies.

Serrated Margins in Pseudomonas aeruginosa Keratitis.

PURPOSE: To describe 3 cases of culture-positive P. aeruginosa keratitis, who presented with a focus having serrated margins. METHODS: Observational case report. RESULTS: Three cases wearing contact lenses complained of symptoms such as pain, redness, and a decrease in vision. Slit-lamp biomicroscopic examination revealed a focus with serrated margins in all cases. The patients underwent corneal scraping for cultures, which confirmed the presence of P. aeruginosa. All 3 patients were treated with a combination of fluoroquinolone and tobramycin, 6-8 times per day. Corneal defects and infiltration disappeared within 2 weeks after initiating the therapy. CONCLUSION: Serrated margins may also be a characteristic initial presentation of P. aeruginosa keratitis.

Expression of IRFs and iNOS in the Mouse Cornea with Pseudomonas Aeruginosa Keratitis

PURPOSE: To investigate the expression of IRF-1, IRF-7 and iNOS in the mice model of Pseudomonas aeruginosa keratitis. The enzymatic activity of iNOS and its expression were also investigated. METHODS: With western blot analysis, the protein expression of IRF-1, IRF-7 (at 24 hours), and iNOS (at 12 hours and 24 hours) were evaluated in the mouse model of P. aeruginosa keratitis. iNOS enzymatic activity was determined with a scintillation counter. IRF-1 and IRF-7 expression were localized with immunofluorescent labeling. The wounded control group was given the same corneal wound without bacterial inoculation, and the fellow eyes served as normal controls. RESULTS: Expression of IRF-1, IRF-7 and iNOS was highly upregulated in corneas with P. aeruginosa keratitis compared to normal or wounded corneas. iNOS enzymatic activity also was higher in infected than normal corneas. In wounded corneas, NOS2 expression and activity slightly increased at 12 hours after the infection. Intense IRF-1 immunopositivity was seen in the epithelial layer of infected corneas. Some corneal stromal cells and endothelial cells showed moderate positive labeling in infected corneas. IRF-7 showed intense labeling in the epithelial layer and endothelial cells of normal as well as infected corneas. Increased IRF-7 labeling was observed in epithelial cells in the ulcerated region of infected corneas. CONCLUSIONS: These results suggest that IRF-1, IRF-7 and iNOS may play a regulatory role in the immune responses and wound healing process in P. aeruginosa keratitis.