Xinyi Wu

Advanced glycation end products induce human corneal epithelial cells apoptosis through generation of reactive oxygen species and activation of JNK and p38 MAPK pathways.

Advanced Glycation End Products (AGEs) has been implicated in the progression of diabetic keratopathy. However, details regarding their function are not well understood. In the present study, we investigated the effects of intracellular reactive oxygen species (ROS) and JNK, p38 MAPK on AGE-modified bovine serum albumin (BSA) induced Human telomerase-immortalized corneal epithelial cells (HUCLs) apoptosis. We found that AGE-BSA induced HUCLs apoptosis and increased Bax protein expression, decreased Bcl-2 protein expression. AGE-BSA also induced the expression of receptor for advanced glycation end product (RAGE). AGE-BSA-RAGE interaction induced intracellular ROS generation through activated NADPH oxidase and increased the phosphorylation of p47phox. AGE-BSA induced HUCLs apoptosis was inhibited by pretreatment with NADPH oxidase inhibitors, ROS quencher N-acetylcysteine (NAC) or neutralizing anti-RAGE antibodies. We also found that AGE-BSA induced JNK and p38 MAPK phosphorylation. JNK and p38 MAPK inhibitor effectively blocked AGE-BSA-induced HUCLs apoptosis. In addition, NAC completely blocked phosphorylation of JNK and p38 MAPK induced by AGE-BSA. Our results indicate that AGE-BSA induced HUCLs apoptosis through generation of intracellular ROS and activation of JNK and p38 MAPK pathways.

Inflammatory Responses of Corneal Epithelial Cells to Pseudomonas aeruginosa Infection

Purpose: We hypothesized that corneal epithelium plays a role in the innate immune response by sensing the presence of pathogens and providing signals that activate the corneal defense system. We sought to determine the mechanisms involved in the activation of the signaling pathways and subsequent production of proinflammatory cytokines in human corneal epithelial cells (HCECs) in response to Pseudomonas aeruginosa infection. Methods: Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary cultures of HCECs were exposed to P. aeruginosa (PA01 strain) with or without the presence of the NF-κ B inhibitor kamebakaurin, the p38 inhibitor SB203580, or the JNK inhibitor SP600125. Iκ B-α phosphorylation and degradation and p38 and JNK phosphorylation were assessed at different time points by Western blot analysis. Interleukin (IL)-6, IL-8, and TNF-α levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Results: Exposure of HUCL cells and primary HCECs to P. aeruginosa resulted in rapid activation of NF-κ B as indicated by an increase in IκB-α phosphorylation observed within 15 min and by IκB-α degradation, which peaked in 1 hr. Two stress-activated mitogen-activated protein kinases, p38 and JNK, were also activated as their phosphorylation was induced by P. aeruginosa infection. Concomitant with the activation of these Toll-like receptor–mediated signaling pathways, transcriptional expression and subsequent secretion of IL-6 and IL-8 in HUCL cells were also induced by P. aeruginosa. Presence of the NF-κ B inhibitor kamebakaurin in culture medium blocked P. aeruginosa–induced NF-κ B activation and inhibited IL-6, IL-8, and TNF-α expression and secretion. Inhibition of p38 or JNK also resulted in a decrease in bacteria-induced expression and secretion of these cytokines. Conclusions: P. aeruginosa triggers an innate immune response in HCECs, and NF-κ B and, to a lesser extent, the p38/JNK signal pathways are responsible for P. aeruginosa–induced proinflammatory cytokine production in these cells.

Activation of Toll-like receptors 2 and 4 in Aspergillus fumigatus keratitis

Toll-like receptor (TLR) 2 and TLR4 are major receptors of Aspergillus fumigatus. Aspergillus fumigatus signaling in cornea induces the production of many pro-inflammatory molecules. In this study, we have shown that exposure of telomerase-immortalized human corneal epithelial cells (HCECs) to A. fumigatus antigens resulted in up-regulation of TLR2 and TLR4, and release of IL-1β and IL-10 in HCECs, effects that could be inhibited by treatment with TLR2, and TLR4 antibodies. In addition, the A. fumigatus antigens-induced production of IL-1β and IL-10 in supernatants of corneal epithelial cells was also attenuated by NF-κB inhibitor. Aspergillus fumigatus keratitis developed in Wistar rats, as evidenced by high SLE scores, influx of polymorphonuclear leukocytes (PMNs), activation of TLR2 and TLR4, and production of IL-1β and IL-10 over controls. These findings indicate that the cornea has functional TLR2 and TLR4, and activation of TLR2 and TLR4 through NF-κB may contribute to pathogenesis of keratomycosis.

Activation of JNK signaling mediates connective tissue growth factor expression and scar formation in corneal wound healing.

Connective Tissue Growth Factor (CTGF) and Transforming growth factor-β1 (TGF-β1) are key growth factors in regulating corneal scarring. Although CTGF was induced by TGF-β1 and mediated many of fibroproliferative effects of TGF-β1, the signaling pathway for CTGF production in corneal scarring remains to be clarified. In the present study, we firstly investigated the effects of c-Jun N-terminal kinase (JNK) on CTGF expression induce by TGF-β1 in Telomerase-immortalized human cornea stroma fibroblasts (THSF). Then, we created penetrating corneal wound model and determined the effect of JNK in the pathogenesis of corneal scarring. TGF-β1 activated MAPK pathways in THSF cells. JNK inhibitor significantly inhibited CTGF, fibronectin and collagen I expression induced by TGF-β1 in THSF. In corneal wound healing, the JNK inhibitor significantly inhibited CTGF expression, markedly improved the architecture of corneal stroma and reduced corneal scar formation, but did not have a measurable impact on corneal wound healing in vivo. Our results indicate that JNK mediates the expression of CTGF and corneal scarring in corneal wound healing, and might be considered as specific targets of drug therapy for corneal scarring.

TLR4: the receptor bridging Acanthamoeba challenge and intracellular inflammatory responses in human corneal cell lines.

Acanthamoeba keratitis (AK) is a painful, vision‐threatening infection caused by pathogenic strains of the protozoan, Acanthamoeba. Toll‐like receptors (TLRs), which are important components of innate immunity, have an important role in the detection of foreign pathogens and the signaling cascades in host cells. However, no report on the interaction between Acanthamoeba and TLR has been found. In this study we analyzed the role of the TLR and its signaling pathway in human telomerase‐immortalized corneal epithelial cells (HUCLs) and stromal fibroblasts (THSFs) challenged by Acanthamoeba. We show that the expressions of TLR4, myeloid differentiation protein 88 (MyD88), nuclear factor (NF)‐κB, phospho‐IκB, phospho‐extracellular signal‐regulated kinases 1/2 (p‐Erk1/2) and the inflammatory cytokines interleukin (IL)‐8, tumor necrosis factor (TNF)‐α and interferon (IFN)‐β were significantly increased in Acanthamoeba‐treated cells. Pretreatment with anti‐TLR antibodies or the specific inhibitors pyrrolidine dithiocarbamate (PDTC) (for the NF‐κB pathway) and U0126 (for the ERK pathway) was conducted. It was found that anti‐TLR4 antibody attenuated the production of cytokines induced by Acanthamoeba infection. PDTC inhibited the production of IL‐8 and TNF‐α whereas U0126 inhibited the synthesis of IFN‐β. Thus, TLR4 is a receptor for Acanthamoeba and exerts an effect through TLR4–MyD88–NF‐κB and TLR4–ERK1/2 pathways to induce the secretion of cytokines in human corneal cell lines challenged by Acanthamoeba.

Derivation of Corneal Endothelial Cell-Like Cells from Rat Neural Crest Cells In Vitro

The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC) to differentiate to functional corneal endothelial cell (CEC)-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na+/K+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet’s membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.

Aspergillus fumigatus antigens activate immortalized human corneal epithelial cells via toll-like receptors 2 and 4.

Purpose: To evaluate the role of toll-like receptors (TLR) 2 and 4 in host responses to Aspergillus fumigatus by use of cultured telomerase-immortalized human corneal epithelial cells (HCECs). Methods: HCECs were stimulated with inactive antigens from A. fumigatus. The expression of TLR2 and TLR4, phosphorylation of Iκ B-α (pIκ B-α), and release of interleukin (IL)-1β and IL-6 was measured with and without inhibitors to TLR2 and TLR4. Results: Exposure of HCECs to A. fumigatus antigens resulted in up-regulation of TLR2 and TLR4, activation of pIκ B, and release of IL-1β and IL-6 in HCECs, effects that could be inhibited by treatment with TLR2 and TLR4 antibodies. Conclusions: TLR2 and TLR4-nuclear factor-κ B signaling pathways in corneal epithelium play important roles in inflammatory responses against A. fumigatus antigens.

Toll-like receptor 2 siRNA suppresses corneal inflammation and attenuates Aspergillus fumigatus keratitis in rats

Toll‐like receptors (TLRs) are key components of innate immunity that detect microbial infection and trigger host defense responses. However, they are capable of initiating both protective and damaging immune responses, as exaggerated expression of inflammatory components can have devastating effects on the host. We previously reported that TLR2 in corneal epithelium has an important role in the pathogenesis of fungal keratitis, however, how the corneal inflammation is modulated remains to be elucidated. This study aims to investigate the effect of targeting TLR2 on Aspergillus fumigatus keratitis in rats. The control or TLR2 small interfering RNA (siRNA) was applied sub‐conjunctively and topically to the cornea. TLR2 immunostaining was performed to determine the feasibility of TLR2 siRNA delivery. Production of inflammatory cytokines and chemokines were determined by real‐time quantitative PCR. Polymorphonuclear leukocyte (PMN) infiltration was assessed by myeloperoxidase activity. It was found that rat corneas treated with TLR2 siRNA showed a significant reduction of TLR2 expression in corneal epithelium. TLR2 siRNA treatment improved the outcome of keratitis, which was characterized by decreased corneal opacity, less corneal perforation, suppressed PMN infiltration, reduced production of inflammatory cytokines and chemokines, and less fungal burden. In conclusion, TLR2 siRNA treatment attenuated A. fumigatus keratitis by suppressing corneal inflammation and preventing fungal invasion, suggesting a novel avenue to control fungal infection and avert damage caused by excessive inflammation.

Topical flagellin-mediated innate defense against Candida albicans keratitis

PURPOSE This study was conducted to investigate whether flagellin, the sole ligand of Toll-like receptor-5 (TLR5), induces an innate defense that is sufficient to protect injured corneas from Candida albicans. METHODS Scarified corneas of adult B6, TLR5(-/-), Camp(-/-) (cathelicidin-related antimicrobial peptide), or PMN-depleted mice were pretreated with Pseudomonas aeruginosa flagellin or a mutant and then were inoculated with C. albicans. The corneas were compared for disease progression, cytokine and Camp expression, and PMN infiltration before and after C. albicans infection. Disease progress was recorded by digital photography and clinical scoring, cytokine levels were determined by ELISA, the levels of Camp gene product were assessed by Western blot, and PMN infiltration was measured by MPO determination and immunohistochemistry. RESULTS Topical application of flagellin induced profound protection against Candida keratitis in a TLR5-dependent manner. The improved disease outcome including reduced tissue inflammation and rapid functional recovery can be attributed to a marked decrease in fungal burden at the early stage of C. albicans infection in flagellin-exposed B6 mouse corneas. Although both PMN infiltration and Camp upregulation contributed to corneal innate defense against fungal infection, Camp ablation totally, and PMN depletion partially, abrogated flagellin-induced fungal clearance in B6 mouse corneas. CONCLUSIONS Flagellin induces a strong innate defense and promotes robust resistance to C. albicans infection in the cornea. Topical flagellin or its mimetic may become a new prophylactic agent for preventing contact lens or trauma/injury-associated microbial keratitis.

Toll-like receptor 2 mediates the induction of IL-10 in corneal fibroblasts in response to Fusarium solu

Toll‐like receptors (TLRs) are key components of the innate immune system that detect microbial infection and trigger antimicrobial host responses. To determine the role of TLR2 in the expression of pro‐ and anti‐inflammatory cytokines in corneal fibroblasts challenged by fungi, we used siRNA specific for TLR2 to knockdown TLR2 expression in telomerase‐immortalized human stroma fibroblasts (THSF). TLR2 expression was assessed by immunocytochemistry, reverse transcription (RT)‐PCR and western blotting analyses, and we found that THSF transfected with TLR2‐siRNA plasmid exhibited a reduced level of TLR2 when compared with the control cells transfected with empty plasmid. Then the transfected and control cells were stimulated by hyphae or conidia of Fusarium solu and mRNA levels of IL‐1β and IL‐10 were measured by real‐time RT‐PCR. We observed that stimulation of control cells with F. solu resulted in elevation of IL‐1β and IL‐10 mRNA with hyphae being more stimulatory than conidia. In contrast, F. solu‐stimulated IL‐10 production by transfected THSF was severely impaired (with a decrease of approximately 82% in hyphae and 70% in conidia stimulation), while IL‐1β production was partially inhibited (with a reduction of 60 and 54% in hyphae and conidia stimulations). Our results suggested that TLR2 is a major pattern recognition receptor able to detect F. solu in vitro and may play the anti‐inflammatory role through the induction of IL‐10. This may lead to a better understanding of the pathogenesis of cornea fungal infections and immune evading.