ABSTRACT
PURPOSE OF REVIEW: The ocular surface is prone to inflammation due to exposure to environmental irritants and pathogens. Inflammasomes are intracellular, multiprotein complexes that communicate potentially dangerous signals to the immune system. The identification of inflammasomes in various inflammatory ocular surface conditions can aid in the development of therapeutics to treat these chronic inflammatory conditions. RECENT FINDINGS: Several inflammasomes have been associated with ocular surface disorders including dry eye disease, keratitis, and allergies. Mechanisms for activation of these inflammasomes with regards to specific disorders have been explored in models to aid in the development of targeted treatments. SUMMARY: Research efforts continue to characterize the types of inflammasomes and activators of these in inflammatory ocular surface conditions. Various therapies targeting specific inflammasome types or pyroptosis are being tested preclinically to assess effects on decreasing the associated chronic inflammation.
Subject(s)
Dry Eye Syndromes , Inflammasomes , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Animals , Dry Eye Syndromes/immunology , Keratitis/immunology , Inflammation/immunology , Hypersensitivity/immunologyABSTRACT
Immune-mediated ocular inflammation is a common clinical diagnosis reached for horses with keratitis and uveitis. This diagnosis is made as a diagnosis of exclusion following a thorough effort to rule out an underlying cause for the inflammation, most importantly infectious and neoplastic disease. Practically, response to ophthalmic and systemic anti-inflammatory or immunomodulatory medications is used to support a diagnosis of immune-mediated ocular inflammation; however, such medications are often contraindicated in the face of infection or neoplasia. This article will summarize our current understanding and approach to the diagnosis and management of immune-mediated keratitis and recurrent or insidious uveitis in horses.
Subject(s)
Eye Diseases , Horse Diseases , Animals , Horse Diseases/immunology , Horse Diseases/diagnosis , Horse Diseases/therapy , Horses , Eye Diseases/veterinary , Eye Diseases/diagnosis , Eye Diseases/immunology , Eye Diseases/therapy , Uveitis/veterinary , Uveitis/diagnosis , Uveitis/immunology , Uveitis/drug therapy , Keratitis/veterinary , Keratitis/diagnosis , Keratitis/immunologyABSTRACT
Background: Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Methods: Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1ß, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. Results: A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. Conclusions: During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.
Subject(s)
Conjunctiva , Cornea , Cytokines , Keratitis , Tears , Transcriptome , Humans , Tears/metabolism , Cytokines/metabolism , Cytokines/genetics , Cornea/metabolism , Cornea/immunology , Female , Male , Middle Aged , Adult , Conjunctiva/metabolism , Conjunctiva/immunology , Keratitis/genetics , Keratitis/immunology , Keratitis/metabolism , Aged , Gene Expression ProfilingABSTRACT
PURPOSE: To investigate the potential treatment of formononetin (FMN) on Aspergillus fumigatus (A. fumigatus) keratitis with anti-inflammatory and antifungal activity. METHODS: The effects of FMN on mice with A. fumigatus keratitis were evaluated through keratitis clinical scores, hematoxylin-eosin (HE) staining, and plate counts. The expression of pro-inflammatory factors was measured using RT-PCR, ELISA, or Western blot. The distribution of macrophages and neutrophils was explored by immunofluorescence staining. The antifungal properties of FMN were assessed through minimum inhibitory concentration (MIC), propidium iodide (PI) staining, fungal spore adhesion, and biofilm formation assay. RESULTS: In A. fumigatus keratitis mice, FMN decreased the keratitis clinical scores, macrophages and neutrophils migration, and the expression of TNF-α, IL-6, and IL-1ß. In A. fumigatus-stimulated human corneal epithelial cells (HCECs), FMN reduced the expression of IL-6, TNF-α, IL-1ß, and NLRP3. FMN also decreased the expression of thymic stromal lymphopoietin (TSLP) and thymic stromal lymphopoietin receptor (TSLPR). Moreover, FMN reduced the levels of reactive oxygen species (ROS) induced by A. fumigatus in HCECs. Furthermore, FMN inhibited A. fumigatus growth, prevented spore adhesion and disrupted fungal biofilm formation in vitro. In vivo, FMN treatment reduced the fungal load in mice cornea at 3 days post infection (p.i.). CONCLUSION: FMN demonstrated anti-inflammatory and antifungal properties, and exhibited a protective effect on mouse A. fumigatus keratitis.
Subject(s)
Anti-Inflammatory Agents , Aspergillosis , Aspergillus fumigatus , Isoflavones , Keratitis , Animals , Aspergillus fumigatus/drug effects , Keratitis/drug therapy , Keratitis/microbiology , Keratitis/immunology , Aspergillosis/drug therapy , Aspergillosis/immunology , Isoflavones/pharmacology , Isoflavones/therapeutic use , Humans , Mice , Anti-Inflammatory Agents/therapeutic use , Anti-Inflammatory Agents/pharmacology , Cytokines/metabolism , Antifungal Agents/therapeutic use , Antifungal Agents/pharmacology , Neutrophils/immunology , Neutrophils/drug effects , Disease Models, Animal , Reactive Oxygen Species/metabolism , Female , Macrophages/drug effects , Macrophages/immunology , Biofilms/drug effects , Mice, Inbred C57BL , Cornea/pathology , Cornea/drug effects , Cornea/microbiologyABSTRACT
Keratitis induced by bacterial toxins, including lipopolysaccharide (LPS), is a major cause of corneal opacity and vision loss. Our previous study demonstrates hepatocyte growth factor (HGF) promotes epithelial wound healing following mechanical corneal injury. Here, we investigated whether HGF has the capacity to suppress infectious inflammatory corneal opacity using a new model of LPS-induced keratitis. Keratitis, induced by two intrastromal injections of LPS on day 1 and 4 in C57BL/6 mice, resulted in significant corneal opacity for up to day 10. Following keratitis induction, corneas were topically treated with 0.1% HGF or PBS thrice daily for 5 days. HGF-treated mice showed a significantly smaller area of corneal opacity compared to PBS-treated mice, thus improving corneal transparency. Moreover, HGF treatment resulted in suppression of α-SMA expression, compared to PBS treatment. HGF-treated corneas showed normalized corneal structure and reduced expression of pro-inflammatory cytokine, demonstrating that HGF restores corneal architecture and immune quiescence in corneas with LPS-induced keratitis. These findings offer novel insight into the potential application of HGF-based therapies for the prevention and treatment of infection-induced corneal opacity.
Subject(s)
Corneal Opacity/drug therapy , Corneal Opacity/etiology , Hepatocyte Growth Factor/administration & dosage , Keratitis/drug therapy , Lipopolysaccharides/adverse effects , Actins/genetics , Actins/immunology , Animals , Cornea/drug effects , Cornea/immunology , Corneal Opacity/genetics , Corneal Opacity/immunology , Cytokines/genetics , Cytokines/immunology , Disease Models, Animal , Humans , Keratitis/etiology , Keratitis/genetics , Keratitis/immunology , Mice , Mice, Inbred C57BLABSTRACT
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.
Subject(s)
Calcitonin Gene-Related Peptide/metabolism , Keratitis/metabolism , Macrophages/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Sensory Receptor Cells/metabolism , Signal Transduction , Animals , Keratitis/immunology , Keratitis/microbiology , Keratitis/pathology , Macrophage Activation , Male , Mice , Mice, Inbred C57BL , Pseudomonas Infections/immunology , Pseudomonas Infections/metabolism , Pseudomonas Infections/microbiology , Sensory Receptor Cells/physiologyABSTRACT
PURPOSE: Disulfiram, an inhibitor of gasdermin D-induced pore formation, is known to suppress interleukin (IL)-1ß secretion and pyroptosis. However, its effects on fungal keratitis remain unknown. Therefore, we investigated the role of disulfiram in Aspergillus fumigatus keratitis. METHODS: In vitro, Cell Count Kit-8 (CCK8) assay and cell scratch test were performed to determine optimal concentration. In vivo and in vitro experiments were conducted in a mouse model, human neutrophils, and mouse peritoneal macrophages. We pre-treated the mice or cells with disulfiram and infected them with A. fumigatus at specific times. We subsequently evaluated the development of fungal keratitis lesions, the recruitment of inflammatory cells, and the production of inflammatory cytokines using slit lamp microscopy, clinical evaluation, quantitative reverse transcription polymerase chain reaction, immunofluorescence staining, enzyme-linked immunosorbent assay, and western blotting. We also used slit lamp microscopy and clinical evaluation to assess the effect of natamycin with or without disulfiram. RESULTS: Disulfiram at 20 µM has no significant cytotoxic effect and does not affect cell migration. In the mouse model, disulfiram significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and down-regulated myeloperoxidase and nitric oxide synthase levels at earlier stages of infection. Disulfiram had no effect on IL-1ß production and maturation, but it inhibited IL-1ß secretion in macrophages. Disulfiram combined with natamycin significantly increased corneal transparency in the mice model. CONCLUSION: Overall, disulfiram reduced the host immune response in fungal keratitis by attenuating neutrophil and macrophage recruitment and inhibiting IL-1ß secretion in macrophages. Disulfiram in combination with antifungal agents may serve as a novel therapeutic method for reducing corneal opacity in fungal keratitis.
Subject(s)
Antifungal Agents/therapeutic use , Aspergillosis/drug therapy , Aspergillus fumigatus/drug effects , Disulfiram/therapeutic use , Inflammation/immunology , Interleukin-1beta/metabolism , Keratitis/drug therapy , Animals , Aspergillosis/immunology , Aspergillosis/microbiology , Aspergillus fumigatus/immunology , Female , Fluorescent Antibody Technique , Inflammation/microbiology , Keratitis/immunology , Keratitis/microbiology , Mice , Mice, Inbred C57BLABSTRACT
Our previous work has shown that topical thymosin beta 4 (Tß4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tß4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tß4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tß4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tß4 alone and adjunctive Tß4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tß4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study.
Subject(s)
Eye Infections, Bacterial/complications , Keratitis/drug therapy , Macrophages/immunology , Pseudomonas Infections/complications , Thymosin/therapeutic use , Animals , Ciprofloxacin/therapeutic use , Drug Therapy, Combination , Eye Infections, Bacterial/immunology , Female , Inflammation , Keratitis/etiology , Keratitis/immunology , Mice , Mice, Inbred C57BL , Pseudomonas Infections/immunology , Pseudomonas aeruginosa , RAW 264.7 CellsABSTRACT
Purpose: We aimed to evaluate activation of conjunctiva-associated lymphoid tissue (CALT) in patients with keratitis using in vivo confocal microscopy (IVCM) and conjunctival impression cytology (CIC). Methods: In addition to anterior segment photography and corneal fluorescein staining, IVCM revealed the palpebral conjunctiva in all subjects, and CIC and immunofluorescence staining were performed. Results: Diffuse lymphoid tissue cell density in the eyes of patients with keratitis was significantly greater compared with healthy volunteers (P < 0.001). Similar trends were found in perifollicular lymphocyte density (P < 0.001), follicular density (P = 0.029), follicular center reflection intensity (P = 0.011), and follicular area (P < 0.001). Immunofluorescence staining showed that the proportions of CD4+ (61.7% ± 8.0% vs. 17.3% ± 10.2%, respectively, P < 0.001) and CD8+ (46.9% ± 10.0% vs. 19.6% ± 11.5%, respectively, P < 0.001) cells in patients with keratitis was greater compared with healthy volunteers. Interestingly, we also observed changes in the contralateral eye in subjects with keratitis. Conclusions: Our research suggests that CALT, as an ocular immune structure, is activated and plays an important role in the pathogenesis of keratitis. This has been overlooked previously. CALT is also active in the contralateral eye of subjects with keratitis.
Subject(s)
Conjunctiva/pathology , Cornea/pathology , Eye Infections, Bacterial/pathology , Immunity, Cellular , Keratitis/pathology , Lymphoid Tissue/pathology , Adult , Conjunctiva/immunology , Cornea/metabolism , Eye Infections, Bacterial/immunology , Eye Infections, Bacterial/metabolism , Female , Humans , Keratitis/immunology , Keratitis/metabolism , Lymphoid Tissue/immunology , Lymphoid Tissue/metabolism , Male , Microscopy, Confocal , Middle Aged , Retrospective StudiesABSTRACT
Purpose: To determine the role of scavenger receptor expressed by endothelial cell-1 (SREC-â ) in vitro and in a mouse model of Aspergillus fumigatus keratitis. Methods: SREC-â mRNA and protein expression were tested in both normal and A fumigatus stimulated human corneal epithelial cells (HCECs). Immunofluorescence was used to detect SREC-â expression in human corneas with or without A fumigatus infection. HCECs were incubated with SREC-â small interfering RNA, then the mRNA levels of LOX-1, IL-1ß, and TNF-α were detected after A fumigatus stimulation. A mouse fungal keratitis (FK) model was established and SREC-â 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-1ß, and TNF-α mRNA expression levels were tested before and after anti-SREC-â treatment. Results: SREC-â expressed in normal and A fumigatus treated HCECs and human corneal epithelium. In vitro experiment showed that SREC-â mRNA and protein levels were significantly increased after A fumigatus stimulation. SREC-â small interfering RNA treatment inhibited the expressions of LOX-1, IL-1ß, and TNF-α in HCECs. The expressions of CLCX1, LOX-1, IL-1ß, and TNF-α were elevated in mice with A fumigatus keratitis, which could be decreased by SREC-â -neutralizing antibody treatment. Conclusions: SREC-â is a key mediator in inflammatory response induced by A fumigatus keratitis. SREC-â blockade could be a potential therapeutic approach for FK.
Subject(s)
Aspergillosis/genetics , Aspergillus fumigatus/isolation & purification , Eye Infections, Fungal/genetics , Gene Expression Regulation/genetics , Immunity, Innate/genetics , Keratitis/genetics , RNA, Messenger/genetics , Scavenger Receptors, Class F/genetics , Adult , Aspergillosis/immunology , Aspergillosis/microbiology , Blotting, Western , Cells, Cultured , Eye Infections, Fungal/immunology , Eye Infections, Fungal/microbiology , Female , Humans , Keratitis/immunology , Keratitis/microbiology , Male , Middle Aged , Scavenger Receptors, Class F/biosynthesisABSTRACT
PURPOSE: To explore the anti-inflammatory effect of lipoxin A4 (LXA4) in Aspergillus fumigatus (A. fumigatus) keratitis and the underlying mechanisms. METHODS: In A. fumigatus keratitis mouse models, enzyme-linked immunosorbent assay (ELISA) was used to detect the level of LXA4. Clinical scores were utilized to evaluate fungal keratitis (FK) severity. Fungal load was assessed by plate count. Immunofluorescence staining, HE staining and myeloperoxidase (MPO) assays were carried out to evaluate the neutrophil infiltration and activity. In A. fumigatus infected mouse corneas and inactivated A. fumigatus-stimulated RAW264.7 cells, quantitative real time polymerase chain reaction (qRT-PCR) and ELISA were applied to assess the expression of pro-inflammatory mediators and anti-inflammatory factors.Reactive oxygen species (ROS) was determined by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining in RAW264.7 cells. RESULTS: LXA4 level was significantly increased in mice with A. fumigatus keratitis. In an A. fumigatus keratitis mouse model, LXA4 treatment alleviated FK severity, reduced fungal load and repressed neutrophil infiltration and activity. Additionally, LXA4 inhibited the expression of pro-inflammatory mediators including IL-1ß, TNF-α, IL-6, cyclooxygenase-2 (COX-2), TLR-2, TLR-4, Dectin-1 and iNOS, and promoted the expression of anti-inflammatory factors IL-10 and Arg-1. In RAW264.7 cells, LXA4 receptor/formyl peptide receptor 2 (ALX/FPR2) blockade reversed the anti-inflammatory effect of LXA4. LXA4 suppressed inactivated A. fumigatus induced elevated ROS production in RAW264.7 cells, which was abrogated by ALX/FPR2 antagonist Boc-2. CONCLUSION: LXA4 ameliorated inflammatory response by suppressing neutrophil infiltration, downregulating the expression of pro-inflammatory mediators and ROS production through ALX/FPR2 receptor in A. fumigatus keratitis.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Aspergillosis/immunology , Aspergillus fumigatus , Eye Infections, Fungal/immunology , Keratitis/immunology , Lipoxins/immunology , Receptors, Formyl Peptide/immunology , Animals , Cornea/immunology , Cytokines/genetics , Cytokines/immunology , Female , Inflammation/immunology , Mice , Mice, Inbred C57BL , Neutrophil Infiltration , RAW 264.7 CellsABSTRACT
Purpose: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection. Methods: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans-infected corneas. B6 CD11c-DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively. Results: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1ß levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1ß, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas. Conclusions: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection-induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.
Subject(s)
Corneal Ulcer/prevention & control , Eye Infections, Fungal/prevention & control , Immunity, Innate/physiology , Interleukin-1/physiology , Keratitis/prevention & control , Receptors, Interleukin-1/physiology , Signal Transduction/physiology , Animals , Blotting, Western , Candida albicans , Corneal Ulcer/immunology , Corneal Ulcer/microbiology , Dendritic Cells/immunology , Enzyme-Linked Immunosorbent Assay , Eye Infections, Fungal/immunology , Eye Infections, Fungal/microbiology , Gene Expression Regulation/physiology , Keratitis/immunology , Keratitis/microbiology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Real-Time Polymerase Chain ReactionABSTRACT
PURPOSE: To determine the role of galectin-3 (Gal-3) in cornea infected by Aspergillus fumigatus (A. fumigatus). METHODS: Gal-3 was tested in normal and infected corneas of C57BL/6 mice. Mice corneas were pretreated with or without rmGal-3 or Gal-3 siRNA and infected with A. fumigatus. Recombinant mouse (rm) Gal-3 stimulated polymorphonuclear neutrophilic leukocytes (PMNs). PMNs were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of Gal-3 siRNA. Disease severity was documented by clinical score and photographs with a slit lamp. PCR, Western blot, and ELISA tested expression of Gal-3, interleukin (IL)-1ß, IL-6, macrophage inflammatory protein 2 (MIP-2) and p-p38. PMNs infiltration was assessed by flow cytometry and myeloperoxidase (MPO) assay. RESULTS: Gal-3 expression was significantly elevated by A. fumigatus in mice corneas. rmGal-3 treatment increased clinical scores, PMNs infiltration, and cytokines expression, which were decreased by Gal-3 siRNA treatment. In PMNs, Gal-3 expression was also significantly increased by A. fumigatus. The rmGal-3 treatment upregulated proinflammatory cytokines secretion and p-p38 expression, which was significantly inhibited by Gal-3 siRNA. CONCLUSION: These data proved that A. fumigatus increased Gal-3 expression and elevated disease clinical scores, PMNs infiltration and cytokines expression through Gal-3. In PMNs, A. fumigatus upregulated IL-1ß and IL-6 secretion through the Gal-3 / p38 pathway.
Subject(s)
Aspergillosis/immunology , Aspergillus fumigatus/immunology , Galectin 3/metabolism , Keratitis/immunology , Neutrophils/immunology , Animals , Aspergillosis/microbiology , Aspergillosis/pathology , Disease Models, Animal , Female , Galectin 3/administration & dosage , Humans , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Keratitis/microbiology , Keratitis/pathology , Mice , Mice, Inbred C57BL , Neutrophils/metabolism , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism , Up-Regulation/immunology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Purpose: C-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro. Methods: Quantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages. Results: The expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1ß production. Conclusions: These findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1ß production in response to A. fumigatus keratitis.
Subject(s)
Aspergillosis/metabolism , Eye Infections, Fungal/metabolism , Gene Expression Regulation/physiology , Keratitis/metabolism , Lectins, C-Type/genetics , Lectins, C-Type/physiology , Membrane Proteins/physiology , Animals , Aspergillosis/immunology , Aspergillosis/microbiology , Aspergillus fumigatus , Blotting, Western , Cytokines/metabolism , Dependovirus/genetics , Disease Models, Animal , Eye Infections, Fungal/immunology , Eye Infections, Fungal/microbiology , Female , Genetic Vectors , Humans , Keratitis/immunology , Keratitis/microbiology , Macrophages/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Neutrophil Infiltration , Peroxidase/metabolism , Real-Time Polymerase Chain Reaction , Receptors, Mitogen/physiology , Slit Lamp MicroscopyABSTRACT
Human corneal epithelial (HCE) cells play a significant role in the innate immune response by secreting cytokines and antimicrobial peptides when they encounter fungal pathogens. But the detailed mechanism of attachment and engulfment of the fungal conidia by HCE cells is not well understood. Here, we show the phagocytosis of Aspergillus flavus conidia by RCB2280 cells and primary HCE cultures using confocal microscopy and proteomic analysis of conidium-containing phagosomes. Phalloidin staining showed actin polymerization, leading to an actin ring around engulfed conidia. Cytochalasin D inhibited the actin-mediated endocytosis of the conidia. Immunolabeling of the early endosomal markers CD71 and early endosomal antigen (EEA1) and the late endosomal markers lysosome-associated membrane protein 1 (LAMP1), Rab7, and cathepsin G showed that endosomal proteins were recruited to the site of conidia and showed maturation of the conidium-containing phagosomes. Lysotracker red DND 99 labeling showed the acidification of the phagosomes containing conidia. Phagosome-specific proteome analysis confirmed the recruitment of various phagosomal and endosomal proteins to the conidium-containing phagosomes. These results show that the ocular surface epithelium contributes actively to antifungal defense by the phagocytosis of invading fungal conidia.
Subject(s)
Aspergillus flavus/immunology , Cornea/cytology , Endocytosis , Epithelial Cells/microbiology , Spores, Fungal/immunology , Disease Susceptibility , Endosomes/metabolism , Epithelial Cells/immunology , Epithelial Cells/metabolism , Humans , Keratitis/immunology , Keratitis/metabolism , Keratitis/microbiology , Phagosomes/metabolism , Proteome , Proteomics/methodsABSTRACT
Pseudomonas aeruginosa (P. aeruginosa) keratitis is a sight-threatening and rapidly progressive corneal disease. Neutrophils and neutrophil extracellular traps (NETs) are widely thought to play a vital role in hosts' immune defenses against bacteria, such as P. aeruginosa. The present study aimed to investigate the dynamics of the formation and the role of NETs in P. aeruginosa keratitis. First, scratched corneas of mice models were treated with 1 × 108 colony-forming units (CFU)/ml of P. aeruginosa suspension or normal saline (NS). Second, after 48 h postinfection, the infected corneas were treated with TobraDex, Tobrex, 0.1% dexamethasone, or NS four times a day, respectively. Clinical examination, hematoxylin and eosin (H&E) staining, immunofluorescence staining, scanning electron microscopy, and bacterial burden testing were performed on the corneas. Tobrex reduced neutrophil infiltration and corneal P. aeruginosa burden. Dexamethasone reduced NETs, bacterial burden, and severe neutrophil infiltration. TobraDex produced a greater reduction in the amount of neutrophils, NETs, and bacterial burden and the results of Tobrex-treated group were between them. These findings corresponded with the clinical findings that TobraDex- and Tobrex-treated mice exhibited slight corneal damage, while dexamethasone-treated mice exhibited very severe corneal damage. Cumulatively, our data suggest that NETs may play a dual role of infection control and corneal damage in P. aeruginosa keratitis. Furthermore, combination treatment targeting NET formation and bacteria may serve as a way of improving the clinical outcomes of bacterial keratitis.
Subject(s)
Extracellular Traps , Keratitis/drug therapy , Neutrophils , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/immunology , Animals , Dexamethasone/administration & dosage , Dexamethasone/therapeutic use , Disease Models, Animal , Drug Therapy, Combination , Keratitis/immunology , Male , Mice , Mice, Inbred C57BL , Pseudomonas Infections/immunology , Tobramycin/administration & dosage , Tobramycin/therapeutic useABSTRACT
Fungal keratitis (FK) is a keratopathy caused by pathogenic fungal infection. The aim of this work is to explore the role of thymic stromal lymphopoietin (TSLP) in FK. Human corneal epithelial cells (HCECs) were treated with Aspergillus fumigatus hyphae, and we found that TSLP was highly expressed and secreted in the hyphae-treated HCECs. Hyphae-treated HCECs or TSLP treatment enhanced the expression of caspase-1 P20, GSDMD-N (p30), IL-1ß, and IL-18 in the human THP-1 macrophages. The influence conferred by hyphae-treated HCECs or TSLP treatment was rescued by TSLP neutralizing antibody or VX-765 (caspase-1 inhibitor) treatment. Moreover, TSLP treatment promoted the expression of NLRP3, ASC, caspase-1 P20, GSDMD-N (p30), IL-1ß, and IL-18 in the THP-1 macrophages, which was abolished by NLRP3 knockdown. Furthermore, TSLPR silencing suppressed the expression of NLRP3, ASC, caspase-1 P20, GSDMD-N (p30), IL-1ß, and IL-18 in the TSLP-treated THP-1 macrophages. In conclusion, our article confirms that Aspergillus fumigatus-stimulated HCECs induce pyroptosis of THP-1 macrophages by secreting TSLP. TSLP/TSLPR induces caspase-1-dependent pyroptosis through activation of NLRP3 inflammasome. Thus, our work suggests that TSLP may be a potential target for FK treatment.
Subject(s)
Aspergillosis/immunology , Aspergillus fumigatus , Cytokines/immunology , Epithelial Cells/immunology , Keratitis/immunology , Macrophages/immunology , Pyroptosis/immunology , Aspergillosis/metabolism , Biomarkers/metabolism , Blotting, Western , Cells, Cultured , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Epithelium, Corneal/immunology , Epithelium, Corneal/metabolism , Epithelium, Corneal/microbiology , Humans , Keratitis/metabolism , Keratitis/microbiology , Macrophages/metabolism , Real-Time Polymerase Chain Reaction , THP-1 CellsABSTRACT
The regulatory role of toll-like receptor 4 (TLR4) in the inactivate staphylococcus epidermidis (ISE)-induced cornea inflammation is not well investigated. Here, TLR4 silence could decrease inflammatory cytokines in corneal epithelial cells treated with ISE. The mouse corneal epithelial cells were exposed to ISE for 24 h, either alone or with the NF-κB inhibitor, TLR4 lentivirus to bilaterally (knock-down or and overexpression). The expression of TLR4 in mouse corneal epithelial cells was investigated using western blot and qRT-PCR assay. The inflammatory cytokine levels were evaluated by qRT-PCR and ELISA, respectively. The relative impact factors of TLR4-mediated NF-κB signaling detected using western blot assay. Results show the expression levels of TLR4 and some inflammatory cytokines were significantly increased in corneal epithelial cells treated with ISE. TLR4 Silence markedly decreased ISE-induced production of IL12, TNF-α, CCL5, and CCL9 in corneal epithelial cells. Furthermore, the nuclear translocation of NF-κB p65 and myeloid differentiation protein 88 (MyD88) in the cells treated with ISE were further reduced by silencing TLR4. Inhibition of TLR4-mediated NF-κB signaling by using BAY11-7082 also alleviated ISE-induced inflammation. In the rescue experiment, transfected the stable TLR4 silenced corneal epithelial cells with TLR4 overexpression lentivirus, we found that TLR4 overexpression can restore the down-regulation of TLR4 and inflammatory cytokines (IL12, TNF-α, CCL9) caused by TLR4 knocked down. Therefore, ISE-induced cornea inflammation was due to the activation of the TLR4/MyD88/NF-κB signaling pathway, and dramatically stimulated IL12, TNF-α, CCL9 secretion. TLR4 silence presented mitigates damage in corneal epithelial cells treated with ISE.