RESUMEN
PURPOSE: To assess the efficacy and safety of human leukocyte antigen-matched allogeneic cultivated limbal epithelial stem cell grafts in the treatment of aniridia-associated keratopathy (AAK). METHODS: Six eyes of 6 patients with severe AAK received an allogeneic stem cell graft between January 2010 and March 2017. Anatomical and functional results were assessed at 6 months, 1 year, 2 years, and the final follow-up visit available. Safety analysis was performed by considering all perioperative and postoperative adverse events and additional surgeries required during the follow-up period. RESULTS: The mean follow-up was 53.6 months (range 24-104 months). In most patients (80%), there was an early improvement of the keratopathy postoperatively, which slowly regressed during longer follow-up. At the final follow-up, 4 of the eyes were graded as failure and 1 eye was graded as partial success. Grading the sixth eye was not possible because of an adverse event. None of the patients maintained a total anatomical success in the long-term. Only 1 patient maintained a modest improvement in best-corrected visual acuity from hand motion to counting fingers. Four serious adverse events were recorded in 2 patients. CONCLUSIONS: Severe AAK remains a challenging condition to manage. Transplantation of allogenic ex vivo cultivated limbal stem cells may provide a temporary improvement in ocular surface stability, but anatomical and functional results are poor in the long-term. The eyes are prone to adverse events, and any surgical treatment should take this into consideration.
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Aniridia/complicaciones , Enfermedades de la Córnea/cirugía , Epitelio Corneal/citología , Antígenos HLA/inmunología , Limbo de la Córnea/citología , Trasplante de Células Madre/métodos , Células Madre/inmunología , Adulto , Anciano , Células Cultivadas , Enfermedades de la Córnea/diagnóstico , Enfermedades de la Córnea/etiología , Epitelio Corneal/inmunología , Femenino , Estudios de Seguimiento , Supervivencia de Injerto , Humanos , Limbo de la Córnea/inmunología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Células Madre/citología , Factores de Tiempo , Trasplante Autólogo , Agudeza Visual , Adulto JovenRESUMEN
Photodamage-induced and viral keratitis could benefit from treatment with novel nonsteroid anti-inflammatory agents. Therefore, we determined whether human corneal epithelial cells (HCECs) express members of the endocannabinoid system (ECS), and examined how the endocannabinoid anandamide (AEA, N-arachidonoyl ethanolamine) influences the Toll-like receptor 3 (TLR3) agonism- or UVB irradiation-induced inflammatory response of these cells. Other than confirming the presence of cannabinoid receptors, we show that endocannabinoid synthesizing and catabolizing enzymes are also expressed in HCECs in vitro, as well as in the epithelial layer of the human cornea in situ, proving that they are one possible source of endocannabinoids. p(I:C) and UVB irradiation was effective in promoting the transcription and secretion of inflammatory cytokines. Surprisingly, when applied alone in 100 nM and 10 µM, AEA also resulted in increased pro-inflammatory cytokine production. Importantly, AEA further increased levels of these cytokines in the UVB model, whereas its lower concentration partially prevented the transcriptional effect of p(I:C), while not decreasing the p(I:C)-induced cytokine release. HCECs express the enzymatic machinery required to produce endocannabinoids both in vitro and in situ. Moreover, our data show that, despite earlier reports about the anti-inflammatory potential of AEA in murine cornea, its effects on the immune phenotype of human corneal epithelium may be more complex and context dependent.
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Antiinflamatorios/farmacología , Ácidos Araquidónicos/farmacología , Endocannabinoides/farmacología , Epitelio Corneal/inmunología , Inflamación/inmunología , Alcamidas Poliinsaturadas/farmacología , Receptor Toll-Like 3/agonistas , Rayos Ultravioleta , Bloqueadores de los Canales de Calcio/farmacología , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Epitelio Corneal/efectos de la radiación , Regulación de la Expresión Génica , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/radioterapiaRESUMEN
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.
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Células Epiteliales/inmunología , Epitelio Corneal/inmunología , Queratitis/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , FN-kappa B/inmunología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Biomarcadores/metabolismo , Western Blotting , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Epitelio Corneal/efectos de los fármacos , Epitelio Corneal/metabolismo , Epitelio Corneal/microbiología , Silenciador del Gen , Queratitis/tratamiento farmacológico , Queratitis/metabolismo , Queratitis/microbiología , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Nitrilos/farmacología , Nitrilos/uso terapéutico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Staphylococcus epidermidis , Sulfonas/farmacología , Sulfonas/uso terapéutico , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Regulación hacia ArribaRESUMEN
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.
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Aspergilosis/inmunología , Aspergillus fumigatus , Citocinas/inmunología , Células Epiteliales/inmunología , Queratitis/inmunología , Macrófagos/inmunología , Piroptosis/inmunología , Aspergilosis/metabolismo , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Epitelio Corneal/inmunología , Epitelio Corneal/metabolismo , Epitelio Corneal/microbiología , Humanos , Queratitis/metabolismo , Queratitis/microbiología , Macrófagos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Células THP-1RESUMEN
BACKGROUND: Pythium insidiosum, a pathogenic oomycete, is a common causative organism of infectious corneal ulcer. Studying the innate immune response at the ocular surface is important for better understanding of the underlying pathogenesis and host defense against P. insidiosum infection. OBJECTIVE: The present study aims to investigate the role of Toll-like receptor (TLR)2 on human corneal epithelial cells (HCECs) in P. insidiosum infection. METHODS: Human embryonic kidney (HEK) cells were stimulated with either P. insidiosum zoospores or hyphae. NF-κB activation was determined by spectrophotometric measurement of secreted embryonic alkaline phosphatase (SEAP) levels. The role of TLR2 in P. insidiosum infection was studied in HCECs and monocyte derived macrophages (MDMs) using anti-TLR2 neutralizing antibody. The expression levels of pro-inflammatory cytokines were determined. RESULTS: Both P. insidiosum hypha and zoospore stimulated TLR2-dependent NF-κB activation in HEK-Blue™-hTLR2 cells in dose-dependent manner. IL-6 and IL-8, but not IL-1ß, were upregulated in HCECs after stimulation with P. insidiosum. Blockade of TLR2 on HCECs altered neither IL-6 nor IL-8 expressions. In contrast, the 3 cytokines were upregulated in the stimulated MDMs and the expression levels of IL-1ß and IL-8 but not IL-6 were attenuated in TLR2 blockade MDMs. CONCLUSIONS: P. insidiosum was recognized by human TLR2 on HEK cells. The mRNA expression levels of certain cytokines were dependent of TLR2 in P. insidiosum infected MDMs but not HCECs at early stage of infection.
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Epitelio Corneal/inmunología , Oftalmopatías/inmunología , Pitiosis/inmunología , Pythium/fisiología , Receptor Toll-Like 2/metabolismo , Citocinas/metabolismo , Epitelio Corneal/microbiología , Células HEK293 , Humanos , Hifa/inmunología , Mediadores de Inflamación/metabolismo , FN-kappa B/metabolismo , Esporas Fúngicas/inmunologíaRESUMEN
PURPOSE OF REVIEW: To review the updated literature regarding eye rubbing complications and its association with ocular allergy disorders. RECENT FINDINGS: Atopy and ocular allergy disorders, mainly vernal keratoconjunctivitis (VKC), are strongly associated with rubbing-related complications, most probably via itching and watery eye sensations that trigger the habit of chronic eye rubbing. Vigorous and prolonged rubbing may lead to establishment of corneal remodeling and ectatic disorders, such as keratoconus. Keratoconus development in rubbed eyes can be caused by mechanical mechanisms of corneal thinning and its loss of rigidity, by elevated temperature of the epithelium during rubbing, by increased intraocular pressure (IOP) because of distending forces, and by inflammatory molecules that may serve as a causal mediator between eye rubbing and keratoconus. Other eye rubbing complications include acute hydrops and perforation, IOP spikes, iris prolapse and iridoschisis rupture of lens capsule and IOL dislocation, and even posterior segment disorders, such as glaucomatous optic neuropathy, retinal detachment and extrusion of implanted silicone oil in the eye. SUMMARY: Chronic eye rubbing in allergic eye diseases can lead to progression of keratoconus, and to other rare anterior and posterior segment complications. Strategies eliminating eye rubbing and its consequences are vital, mainly among at-risk populations, such as young children and individuals with allergic ocular disorders or corneal transplants.
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Conjuntivitis Alérgica , Epitelio Corneal , Queratocono , Enfermedad Crónica , Conjuntivitis Alérgica/complicaciones , Conjuntivitis Alérgica/inmunología , Conjuntivitis Alérgica/patología , Conjuntivitis Alérgica/fisiopatología , Epitelio Corneal/inmunología , Epitelio Corneal/patología , Epitelio Corneal/fisiopatología , Humanos , Presión Intraocular , Queratocono/etiología , Queratocono/inmunología , Queratocono/patología , Queratocono/fisiopatologíaRESUMEN
The in vitro induction of corneal epithelial cells (CECs) from human induced pluripotent stem cells (iPSCs) represents a new strategy for obtaining CE stem/progenitor cells for the surgical reconstruction of a diseased or injured ocular surface. The clinical promise of this strategy is considerable, but if the approaches' potential is to be realised, robust methods for the purification of iPSC-derived CE lineage cells need to be developed to avoid contamination with other cells that may carry the risk of unwanted side effects, such as tumorigenesis. Experiments conducted here revealed that during CEC isolation, CD200-negative selection using a cell sorter considerably reduced the contamination of the cell population with various non-CECs compared with what could be achieved using TRA-1-60, a conventional negative marker for CECs. Furthermore, CD200-negative sorting did not affect the yield of CECs nor that of their stem/progenitor cells. Single-cell gene expression analysis for CEC sheets obtained using CD200-negative sorting showed that all analysed cells were CE-lineage cells, expressing PAX6, delta-N p63, and E-cadherin. Non-CECs, on the other hand, expressed non-CEC genes such as FGFR1 and RPE65. CD200, thus, represents a robust negative marker for purification of induced CE lineage cells, which is expressed by undifferentiated iPSCs and non-CECs, including iPSC-derived neural and retinal cells.
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Antígenos CD/metabolismo , Epitelio Corneal/citología , Células Madre Pluripotentes Inducidas/citología , Antígenos CD/genética , Antígenos de Superficie/genética , Cadherinas/genética , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Epitelio Corneal/inmunología , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Factor de Transcripción PAX6/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/inmunología , Proteoglicanos/genética , Análisis de la Célula IndividualRESUMEN
PURPOSE: In this study, we examine the expression of corneal epithelium-derived thrombospondin-1 (TSP-1) and its immunomodulatory functions in a validated murine model of dry eye disease (DED). METHODS: DED was induced in female C57BL/6 using a controlled environment chamber (CEC) for 14 days. mRNA and protein expression of TSP-1 by corneal epithelial cells was quantified using real-time PCR and flow cytometry. Corneal epithelial cells from either naïve or DED mice were cultured with bone marrow derived dendritic cells (BMDCs) in the presence of IFNγ for 48â¯h, and BMDC expression of MHC-II and CD86 was determined using flow cytometry. Next, either recombinant TSP-1 or anti-TSP-1 antibody was added to the co-culture, and BMDC expression of above activation markers was evaluated. Finally, either DED mice were topically treated with either recombinant TSP-1 or human serum albumin (HSA), and maturation of corneal DCs, expression of inflammatory cytokines, and DED severity were investigated. RESULTS: mRNA expression of TSP-1 by the corneal epithelium was upregulated in DED. Corneal epithelial cells derived from mice with DED demonstrated an enhanced capacity in suppressing BMDC expression of MHC-II and CD86 relative to wild type mice, and this effect was abrogated by TSP-1 blockade and potentiated by recombinant TSP-1. Finally, topical application of recombinant TSP-1 significantly suppressed corneal DC maturation and mRNA expression of pro-inflammatory cytokines, and ameliorated disease severity in mice with DED. CONCLUSIONS: Our study elucidates the function of epithelium-derived TSP-1 in inhibiting DC maturation and shows its translational potential to limit corneal epitheliopathy in DED.
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Síndromes de Ojo Seco/inmunología , Epitelio Corneal/inmunología , Trombospondina 1/fisiología , Animales , Córnea/metabolismo , Células Dendríticas/inmunología , Síndromes de Ojo Seco/metabolismo , Células Epiteliales/metabolismo , Epitelio Corneal/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Trombospondina 1/metabolismoRESUMEN
Immune privilege helps protect the cornea from damaging inflammation but can also impair pathogen clearance from this mucosal surface. Programmed death-ligand 1 (PD-L1 or B7-H1) contributes to corneal immune privilege by inhibiting the function of a variety of immune cells. We asked whether programmed death-1 (PD-1)/PD-L1 interaction regulates HSV-1 clearance from infected corneas. We show that PD-L1 is constitutively expressed in the corneal epithelium and is upregulated upon HSV-1 corneal infection, with peak expression on CD45+ cells NK cells, dendritic cells, neutrophils, and macrophages and CD45- corneal epithelial cells at 4 d postinfection (dpi). As early as 1 dpi, HSV-1-infected corneas of B7-H1-/- mice as compared with wild-type mice showed increased chemokine expression and this correlated with increased migration of inflammatory cells into the viral lesions and decreased HSV-1 corneal titers. Local PD-L1 blockade caused a similar increase in viral clearance, suggesting a local effect of PD-1/PD-L1 in the cornea. The enhanced HSV-1 clearance at 2 dpi resulting from PD-1/PD-L1 blockade is mediated primarily by a monocyte/macrophage population. Studies in bone marrow chimeras demonstrated enhanced viral clearance when PD-L1 was absent only from nonhematopoietic cells. We conclude that PD-L1 expression on corneal cells negatively impacts the ability of the innate immune system to clear HSV-1 from infected corneas.
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Antígeno B7-H1/metabolismo , Córnea/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Macrófagos/inmunología , Animales , Antígeno B7-H1/inmunología , Córnea/metabolismo , Córnea/virología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/virología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Epitelio Corneal/inmunología , Epitelio Corneal/metabolismo , Epitelio Corneal/virología , Femenino , Herpes Simple/metabolismo , Herpes Simple/virología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/virología , Antígenos Comunes de Leucocito/inmunología , Antígenos Comunes de Leucocito/metabolismo , Macrófagos/metabolismo , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/virología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/virologíaRESUMEN
Purpose: To explore the impact of ocular surface insults on the immunomodulatory capacity and phenotype of corneal epithelial cells (CECs) with a focus on epithelial-mesenchymal transition (EMT). Methods: Corneas were harvested from mice 6 days following scratch injury, ragweed pollen-induced allergy, or herpes simplex virus type 1 (HSV-1) infection and compared to healthy tissue controls. Corneas were enzymatically digested and CECs phenotypically characterized using flow cytometry. CECs were defined as epithelial cell adhesion molecule (EpCAM)-positive CD45-negative cells. CECs were assessed by PCR to evaluate EMT-associated transcripts. Recombinant HSV-1 and transgenic mice were utilized to investigate the role of vascular endothelial growth factor A (VEGFA) on the phenotype observed. The immunomodulatory potential of CECs was assessed in coculture assays with ovalbumin-specific CD4 T cells. Results: Ectopic expression of classic "myeloid" antigens Ly6G, CCR2, and CX3CR1 was identified in CEC subsets from all groups with evidence supporting an underlying partial EMT event resulting from loss of cell-cell contacts. Corneal HSV-1 infection induced Ly6C expression and major histocompatibility complex (MHC)-II upregulation in CECs through a VEGFA-linked mechanism. These Ly6C+ MHC-II+ CECs were found to function as amateur antigen-presenting cells and induced CD4 T cell proliferation in vitro. Conclusions: This study characterizes a novel immunomodulatory CEC phenotype with possible implications for immune privilege, chronic inflammation, and tissue fibrosis. Moreover, the identification of CECs masquerading with multiple "myeloid" antigens warrants careful evaluation of flow cytometry data involving corneal digests.
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Enfermedades de la Córnea/inmunología , Células Epiteliales/inmunología , Transición Epitelial-Mesenquimal/inmunología , Epitelio Corneal/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Células Mieloides/inmunología , Animales , Modelos Animales de Enfermedad , RatonesRESUMEN
Pentraxin3 (PTX3), a member of long pentraxin family, plays a non-redundant role in human humoral innate immunity. However, whether PTX3 is expressed by corneal epithelial cells and its role during corneal fungi infection has not yet been investigated. To identify the presence of PTX3 in cornea, the possible mechanisms involved in its expression, and also the effects on corneal anti-fungi innate immune response, clinic human corneal tissues and cultured human corneal epithelial cells (HCECs) were resorted. PTX3 mRNA and protein were detected in corneal samples and cultured HCECs, which was significantly up-regulated after exposing to Aspergillus fumigatus (A. fumigatus). Pretreated with specific inhibitors, only Syk contributed to the regulation of PTX3 expression in Dectin-1/Syk signal axis. Furthermore, among the MAPK members (p38 MAPK, ERK1/2 and JNK), only ERK1/2 and JNK were responsible for A. fumigatus induced PTX3 production. Blocking of endogenous PTX3 by siRNA down-regulated the production of IL-1ß at both mRNA and protein levels. Meanwhile, blocking of PTX3 also inhibited the phosphorylation of ERK1/2 and JNK, but not p38 MAPK. These findings demonstrate that PTX3 is expressed in human corneal epithelial cells and Syk, ERK1/2, JNK signaling pathways play an important role in the regulation of PTX3 induction. PTX3 plays a proinflammatory role in corneal epithelial anti-fungi immune response by affecting the production of IL-1ß and activation of some proinflammatory signaling pathways (ERK1/2 and JNK).
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Aspergilosis/inmunología , Proteína C-Reactiva/fisiología , Úlcera de la Córnea/inmunología , Epitelio Corneal/inmunología , Infecciones Fúngicas del Ojo/inmunología , Inmunidad Innata/fisiología , Componente Amiloide P Sérico/fisiología , Aspergilosis/microbiología , Aspergillus fumigatus/patogenicidad , Western Blotting , Línea Celular , Úlcera de la Córnea/microbiología , Ensayo de Inmunoadsorción Enzimática , Infecciones Fúngicas del Ojo/microbiología , Humanos , Interleucina-1beta/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
Previously we reported that corneal epithelial barrier function against Pseudomonas aeruginosa was MyD88-dependent. Here, we explored contributions of MyD88-dependent receptors using vital mouse eyes and confocal imaging. Uninjured IL-1R (-/-) or TLR4 (-/-) corneas, but not TLR2 (-/-), TLR5 (-/-), TLR7 (-/-), or TLR9 (-/-), were more susceptible to P. aeruginosa adhesion than wild-type (3.8-fold, 3.6-fold respectively). Bacteria adherent to the corneas of IL-1R (-/-) or TLR5 (-/-) mice penetrated beyond the epithelial surface only if the cornea was superficially-injured. Bone marrow chimeras showed that bone marrow-derived cells contributed to IL-1R-dependent barrier function. In vivo, but not ex vivo, stromal CD11c+ cells responded to bacterial challenge even when corneas were uninjured. These cells extended processes toward the epithelial surface, and co-localized with adherent bacteria in superficially-injured corneas. While CD11c+ cell depletion reduced IL-6, IL-1ß, CXCL1, CXCL2 and CXCL10 transcriptional responses to bacteria, and increased susceptibility to bacterial adhesion (>3-fold), the epithelium remained resistant to bacterial penetration. IL-1R (-/-) corneas also showed down-regulation of IL-6 and CXCL1 genes with and without bacterial challenge. These data show complex roles for TLR4, TLR5, IL-1R and CD11c+ cells in constitutive epithelial barrier function against P. aeruginosa, with details dependent upon in vivo conditions.
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Antígeno CD11c/inmunología , Permeabilidad de la Membrana Celular , Epitelio Corneal/inmunología , Regulación de la Expresión Génica , Factor 88 de Diferenciación Mieloide/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Animales , Adhesión Bacteriana , Médula Ósea/inmunología , Médula Ósea/metabolismo , Médula Ósea/microbiología , Antígeno CD11c/metabolismo , Células Cultivadas , Epitelio Corneal/metabolismo , Epitelio Corneal/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Receptores de Interleucina-1/fisiología , Transducción de Señal , Receptor Toll-Like 4/fisiología , Receptor Toll-Like 5/fisiologíaRESUMEN
In a transgenic mouse line hK14mIL33tg, with the expression of interleukin-33 (IL-33) driven by a keratin 14 promoter, keratoconjunctivitis developed spontaneously between 18 and 22 weeks of age under specific-pathogen-free conditions. These mice showed blepharitis and corneal impairments, and the histology revealed epithelial thickening in the conjunctiva and the cornea with infiltration of eosinophils, mast cells and basophils. IL-5, IL-13 and CCL11 were abundant in lacrimal fluid in the mice, and the gene expressions of IL-4, IL-5, IL-13, IL-33, Prg2 and Mmcp8 were significantly increased in the cornea. Furthermore, group 2 innate lymphoid cells (ILC2) producing IL-5 and IL-13 were markedly increased in the cornea. These phenotypes closely resemble human atopic keratoconjunctivitis (AKC). The characteristic ocular phenotype in these mice strongly suggests that IL-33 is crucial for the development of AKC. The mouse line may be useful as a novel model for research and development of therapeutic strategies for AKC.
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Modelos Animales de Enfermedad , Epitelio Corneal/inmunología , Efecto Fundador , Interleucina-33/inmunología , Queratoconjuntivitis/genética , Linfocitos/inmunología , Animales , Basófilos/inmunología , Basófilos/patología , Quimiocina CCL11/genética , Quimiocina CCL11/inmunología , Conjuntiva/inmunología , Conjuntiva/patología , Eosinófilos/inmunología , Eosinófilos/patología , Epitelio Corneal/patología , Regulación de la Expresión Génica , Inmunidad Innata , Interleucina-13/genética , Interleucina-13/inmunología , Interleucina-33/genética , Interleucina-4/genética , Interleucina-4/inmunología , Interleucina-5/genética , Interleucina-5/inmunología , Queratina-14/genética , Queratina-14/inmunología , Queratoconjuntivitis/inmunología , Queratoconjuntivitis/patología , Linfocitos/patología , Mastocitos/inmunología , Mastocitos/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Transducción de Señal , Triptasas/genética , Triptasas/inmunologíaRESUMEN
Tear film hyperosmolarity and anterior ocular inflammation are two clinical signs that may be indicative of dry eye disease (DED). This condition can cause pathological and functional changes to the anterior ocular surface tissues. A contributing factor may be dysfunctional aquaporin 5 (AQP5) water channels as they are the AQP subtype that expressed in the corneal epithelium and contribute to fluid efflux needed for corneal function. We determined if described hyperosmolarity-induced increases in proinflammatory cytokine expression and cell death are mediated through AQP5 upregulation and JNK1/2 MAPK signaling activation in both primary human corneal epithelial cells (HCECs), and in a HCEC line. Real time RT-PCR identified rises in IL-1ß, IL-6, IL-8, TNF-α, caspase-1, and AQP5 mRNA levels upon step increases in osmolarity up to 550 mOsm. Western blot analysis and the TUNEL assay identified corresponding rises in AQP5 and p-JNK1/2 protein expression and cell death respectively. JNK1/2 inhibition with SP600125, or siRNA AQP5 gene silencing reduced hypertonic-induced rises in proinflammatory cytokine expression and cell death. Taken together, hypertonicity-induced AQP5 upregulation leads to increases in proinflammatory cytokine expression and cell death through JNK1/2 MAPK activation. These results suggest that drug targeting AQP5 upregulation may be a therapeutic option in DED management.
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Acuaporina 5/genética , Epitelio Corneal/citología , Inflamación/metabolismo , Regulación hacia Arriba , Acuaporina 5/metabolismo , Muerte Celular , Células Cultivadas , Citocinas/genética , Epitelio Corneal/inmunología , Epitelio Corneal/metabolismo , Humanos , Inflamación/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Presión Osmótica , Transducción de SeñalRESUMEN
Pseudomonas aeruginosa is the principal cause of bacterial keratitis worldwide and overstimulation of the innate immune system by this organism is believed to contribute significantly to sight loss. In the current study, we have used primary human corneal fibroblast (hCF) cells as an ex vivo model of corneal infection to examine the role of P. aeruginosa flagellum and type three secretion system (TTSS) in inducing inflammasome-associated molecules that trigger IL-1ß and IL-18 production during the early stages of the infection. Our results show that P. aeruginosa infection stimulated the non-canonical pathway for IL-1ß and IL-18 expression and pathway stimulation was influenced predominantly by the flagellum. Both IL-1ß and IL-18 cytokines were expressed intracellularly during bacterial infection, but only the former was released and detected in the extracellular environment. We also investigated the signaling pathways in hCFs mediated by Toll-Like Receptor (TLR)4 and TLR5 sensing of P. aeruginosa, and our data show that the signal triggered by TLR5-flagellin sensing significantly contributed to IL-1ß and IL-18 cytokine production in our model. Our study suggests that IL-18 expression is wholly dependent on extracellular flagellin sensing by TLR5, whereas IL-1ß expression is also influenced by P. aeruginosa lipopolysacharide. Additionally, we demonstrate that IL-1ß and IL-18 production by hCFs can be triggered by both MyD88-dependent and -independent pathways. Overall, our study provides a rationale for the development of targeted therapies, by proposing an inhibition of flagellin-PRR-signaling interactions, in order to ameliorate the inflammatory response characteristic of P. aeruginosa keratitis.
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Epitelio Corneal/efectos de los fármacos , Fibroblastos/metabolismo , Flagelina/farmacología , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Pseudomonas aeruginosa/química , Transducción de Señal/inmunología , Receptor Toll-Like 5/metabolismo , Adhesinas Bacterianas , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Células Cultivadas , Citocinas/metabolismo , Epitelio Corneal/inmunología , Flagelina/genética , Flagelina/inmunología , Flagelina/aislamiento & purificación , Humanos , Inflamasomas/metabolismo , Lipopolisacáridos/inmunología , Pseudomonas aeruginosa/patogenicidad , Proteínas Recombinantes , Receptor Toll-Like 4/metabolismo , Sistemas de Secreción Tipo III/inmunologíaRESUMEN
The concept of innate immunity has been expanded to recognize environmental pathogens other than microbial components. However, whether and how the innate immunity is initiated by epithelium in response to environmental physical challenges such as low humidity and high osmolarity in an autoimmune disease, dry eye, is still largely unknown. Using two experimental dry eye models, primary human corneal epithelial cultures exposed to hyperosmolarity and mouse ocular surface facing desiccating stress, we uncovered novel innate immunity pathway by ocular surface epithelium, where oxidized mitochondrial DNA induces imbalanced activation of NLRP3/NLRP6 inflammasomes via stimulation of caspase-8 and BRCC36 in response to environmental stress. Activated NLRP3 with suppressed NLRP6 stimulates caspase-1 activation that leads to IL-1ß and IL-18 maturation and secretion. NLRP3-independent caspase-8 noncanonically activates caspase-1 via reciprocal regulation of NLRP3/NLRP6-mediated inflammasomes. Reactive oxygen species-induced mitochondrial DNA oxidative damage and BRCC36 deubiquitinating activity provide a missing link and mechanism by which innate immunity responds to environmental stress via caspase-8-involved NLRP3/NLRP6 inflammasomes.
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Caspasa 8/metabolismo , ADN Mitocondrial/metabolismo , Síndromes de Ojo Seco/inmunología , Epitelio Corneal/inmunología , Inflamasomas/metabolismo , Proteínas de la Membrana/metabolismo , Adolescente , Adulto , Anciano , Animales , Autoinmunidad , Células Cultivadas , Daño del ADN , ADN Mitocondrial/genética , Enzimas Desubicuitinizantes , Exposición a Riesgos Ambientales/efectos adversos , Epitelio Corneal/patología , Femenino , Humanos , Inmunidad Innata , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Modelos Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Adulto JovenRESUMEN
PURPOSE: To evaluate the treatment of autologous serum eye drops (ASED) on dry eyes in patients with graft-versus-host disease (GVHD). METHODS: A retrospective chart review of 35 patients with a history of ocular GVHD following hematopoietic stem cell transplantation that used ASED to alleviate dry eye symptoms was performed. Patients were categorized into three different groups. If patients had available ophthalmic data before and after starting treatment was group 1 (n = 14), had available ophthalmic data after starting treatment in group 2 (n = 10) and had available ophthalmic data before treatment or did not have any data after starting treatment in group 3 (n = 11). Data were collected on patient's age, gender, primary diagnosis, visual acuity and fluorescein corneal staining were collected on individual eyes in order to evaluate the efficacy of the ASED on alleviating dry eye-related signs and symptoms. RESULTS: No adverse ocular effect from the ASED was found in our series (except one fungal keratitis). All patients reported either improvement (55%) or stability (45%) in their ocular symptoms upon the use of ASED. In patients with available data before and after starting treatment, the corneal staining score improved by a median of 1 (p = 0.003) and the LogMAR visual acuity had a non-significant improvement. CONCLUSION: In our study, ASED used by patients with ocular GVHD were both safe and effective. ASED should be considered in patients with GVHD who suffer from dry eyes.
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Síndromes de Ojo Seco/terapia , Enfermedad Injerto contra Huésped/terapia , Inmunoterapia/métodos , Soluciones Oftálmicas/efectos adversos , Suero/inmunología , Adulto , Anciano , Enfermedad Crónica , Síndromes de Ojo Seco/inmunología , Epitelio Corneal/inmunología , Epitelio Corneal/fisiopatología , Femenino , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Soluciones Oftálmicas/uso terapéutico , Estudios Retrospectivos , Suero/química , Trasplante Autólogo/métodos , Trasplante Homólogo/efectos adversos , Agudeza Visual/inmunología , Adulto JovenRESUMEN
Our previous studies reported that pattern recognition receptors (PRRs), including the cell surface Toll-like receptors (TLRs) and cytoplasmic NOD-like receptors (NLRs), recognize pathogen-associated molecular patterns (PAMPS) to initiate downstream signal cascades to active immunity responses. Thymic stromal lymphopoietin (TSLP) has recently emerged as a key cytokine in the development of type 2 adaptive immune responses. However, the crosstalk between PRRs and TSLP has not been well elucidated in Aspergillus fumigates keratitis. Our studies demonstrated that HCECs not only respond to TSLP, but also initiate immunological regulation through TSLP/TSLPR/STAT5 signaling pathway. In addition, we revealed that zymosan TLR2 agonist enhanced the expression of TSLP and TSLPR and phosphorylation of STAT5. Furthermore, neutralization of TLR2 with monoclonal Ab prevented the production of TSLP and TSLPR and phosphorylation of STAT5 from increasing which induced by A. fumigatus hyphae. Interestingly, we also found that human recombinant TSLP induced the increase of TLR2 downstream signal molecules, and TSLP knockdown could reduce the increase of TLR2 downstream signaling molecules(MyD88 and NF-κB-p65) induced by A. fumigatus hyphae. These studies indicated that HCECs represent a novel target of TSLP, TSLP/TSLPR/STAT5 signaling plays an important role in response to A. fumigatus infection in HCECs, and TLR2 downstream signaling molecules up regulate TSLP/TSLPR/STAT5 signaling as well as TSLP downstream signaling molecules up regulate TLR2/MyD88/NFκB-p65 signaling in this phenomenon.
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Aspergilosis/inmunología , Epitelio Corneal/inmunología , Tolerancia Inmunológica/inmunología , Queratitis/inmunología , Transducción de Señal/inmunología , Aspergillus fumigatus , Western Blotting , Células Cultivadas , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/microbiología , Humanos , Factor 88 de Diferenciación Mieloide/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Citocinas/inmunología , Factor de Transcripción STAT5/inmunología , Receptor Toll-Like 2/inmunología , Factor de Transcripción ReIA/inmunología , Linfopoyetina del Estroma TímicoRESUMEN
BACKGROUND: Fungal keratitis is a major cause of blindness. To understand the mechanism of both innate and adaptive immunity in corneal infection is of great significance in the treatment and prevention of fungal keratitis. Our previous study concerned innate immunity. Here, we explored the potential role of thymic stromal lymphopoietin (TSLP) in adaptive immunity of fungal keratitis. METHODS: Human corneal epithelial cells (HCECs) were stimulated with Aspergillus fumigatus hyphae (10(6) pieces per millilitre) with or without TSLP siRNA, and peripheral blood mononuclear cells (PBMCs) were cultured with or without TSLP. HCECs and PBMCs were co-cultured in a transwell system for various periods. Then we collected PBMCs and detected the proliferation and activation as well as T helper type 2 (Th2) differentiation by flow cytometry and quantitative real-time reverse transcription polymerase chain reaction. IgG and IgA levels in supernatants of PBMCs were measured by means of ELISA. RESULTS: Thymic stromal lymphopoietin could induce a Th2 response in vitro, and the expression of TSLP was highly increased in HCECs stimulated with A. fumigatus hyphae. A. fumigatus-infected HCECs were capable of promoting human lymphocyte proliferation and activating human CD4(+) T cells, CD8(+) T cells and B cells by up-regulating the expression of activation marker CD69. Importantly, Th2 differentiation of CD4(+) T cells was induced during co-culture with A. fumigatus-infected HCECs in a transwell system. Interestingly, blockade of TSLP using siRNA prevented the proliferation and activation of lymphocytes as well as Th2 differentiation. We also detected an increased IgG level that was associated with TSLP. CONCLUSION: These findings suggested that HCEC-derived TSLP has a key role in adaptive immune responses of fungal keratitis via skewing Th2 differentiation and promoting humoral immunity.
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Inmunidad Adaptativa/fisiología , Aspergillus fumigatus/fisiología , Citocinas/genética , Epitelio Corneal/microbiología , Regulación de la Expresión Génica/fisiología , Células Th2/inmunología , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Técnicas de Cocultivo , Ensayo de Inmunoadsorción Enzimática , Epitelio Corneal/inmunología , Epitelio Corneal/metabolismo , Femenino , Citometría de Flujo , Humanos , Inmunidad Humoral/fisiología , Inmunoglobulina G/sangre , Lectinas Tipo C/metabolismo , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/fisiología , Masculino , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Linfopoyetina del Estroma TímicoRESUMEN
Dry eye is an allegedly autoimmune disorder for which the initiating mechanisms and the targeted antigens in the ocular surface are not known, yet there is extensive evidence that a localized T helper type 1 (Th1)/Th17 effector T cell response is responsible for its pathogenesis. In this work, we explore the reconciling hypothesis that desiccating stress, which is usually considered an exacerbating factor, could actually be sufficient to skew the ocular surface's mucosal response to any antigen and therefore drive the disease. Using a mouse model of dry eye, we found that desiccating stress causes a nuclear factor kappa B (NF-κB)- and time-dependent disruption of the ocular surface's immune tolerance to exogenous ovalbumin. This pathogenic event is mediated by increased Th1 and Th17 T cells and reduced regulatory T cells in the draining lymph nodes. Conversely, topical NF-κB inhibitors reduced corneal epithelial damage and interleukin (IL)-1ß and IL-6 levels in the ocular surface of mice under desiccating stress. The observed effect was mediated by an augmented regulatory T cell response, a finding that highlights the role of mucosal tolerance disruption in dry eye pathogenesis. Remarkably, the NF-κB pathway is also involved in mucosal tolerance disruption in other ocular surface disorders. Together, these results suggest that targeting of mucosal NF-κB activation could have therapeutic potential in dry eye.