Mesenchymal stem cell-based adjunctive therapy for Pseudomonas aeruginosa-induced keratitis: A proof-of-concept in-vitro study.

PURPOSE: Pseudomonas aeruginosa-induced keratitis is one of the most severe and challenging forms of corneal infection, owing to its associated intense inflammatory reactions leading to corneal necrosis and dense corneal scar with loss of vision. Since mesenchymal stem cells (MSCs) are reported to possess antimicrobial and immunomodulatory properties, they can be tested as an adjuvant treatment along with the antibiotics which are the current standard of care. This study aims to investigate the anti-bacterial and immunomodulatory roles of human bone marrow MSC-derived conditioned medium (MSC-CM) in P. aeruginosa-infected human corneal epithelial cells (HCECs) in vitro. METHODS: The effect of MSC-CM on the growth of clinical isolates of P. aeruginosa was evaluated by colony-forming unit assay. The expression of inflammatory cytokines (IL-6 and TNF-α) and an antimicrobial peptide (Lipocalin 2) in lipopolysaccharide-treated MSCs and HCECs was analyzed through ELISA. Corneal epithelial repair following infection with P. aeruginosa was studied through scratch assay. RESULTS: Compared to control (P. aeruginosa (5*105) incubated in DMEM (1 ml) at 37 °C for 16 h), MSC-CM significantly: i) inhibits the growth of P. aeruginosa (159*109 vs. 104*109 CFU/ml), ii) accelerates corneal epithelial repair following infection with P. aeruginosa (9% vs. 24% closure of the wounded area after 12 h of infection), and iii) downregulates the lipopolysaccharide-induced expression of IL-6, TNF-α and Lipocalin 2 in HCECs. A combination of MSC-CM with an antibiotic, Ciprofloxacin moderately regulated the expression of IL-6, TNF-α, and Lipocalin 2. CONCLUSION: MSC-CM holds promise as an adjunctive therapeutic approach for P. aeruginosa-induced corneal epithelial damage.

Resolvin D1 protects against Aspergillus fumigatus keratitis in diabetes by blocking the MAPK-NF-κB pathway.

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

Thymic stromal lymphopoietin participates in the TLR2-and TLR4-dependent immune response triggered by Aspergillus fumigatus in human corneal cells.

Fungal keratitis constitutes a serious vision-threatening disease. Toll-like receptors (TLRs) comprise key mediators of innate immunity triggered by Aspergillus fumigatus (AF) in the cornea, but the messenger between innate and adaptive immunity remained unknown. Thymic stromal lymphopoietin (TSLP) represents a critical factor of adaptive immunity. Here we investigated the expression of TSLP in corneal epithelial and stromal cells challenged by AF and its relationship with TLRs. We stimulated corneal cells with TLR ligands zymosan or lipopolysaccharide (LPS), human recombinant TSLP, or AF hyphae for various periods, with or without prior TLR2, TLR4, or TSLP inhibition. TLR2, TLR4, TSLP, IL-8, and TNF-α release and expression were measured via enzyme-linked immunosorbent analysis, quantitative polymerase chain reaction, or western blot. Corneal cell stimulation with zymosan or LPS induced up-regulated TSLP expression. Enhanced TSLP expression was associated with AF treatment in human corneal cells; TLR2 or TLR4 inhibition impaired the AF-induced TSLP levels. Human recombinant TSLP augmented TLR2 and TLR4 expression; RNA interference of TSLP attenuated TLR, IL-8, and TNF-α expression stimulated by AF hyphae. These findings indicated that TSLP participates in the immune response of corneal cells triggered by AF, which is closely related to TLR function, and the innate immunity mediated by TLRs could be enhanced by TSLP. Innate immunity may therefore transmit inflammatory signals to adaptive immunity through activation of TSLP; in turn, adaptive immunity likely exerts certain regulatory effects on innate immunity via TSLP. That is, TSLP could interact with innate immunity mediated by TLR2 and TLR4 in human corneal cells challenged by AF and thus may serve as a messenger between the innate and adaptive immune responses in AF keratitis.

GSDMD, an executor of pyroptosis, is involved in IL-1ß secretion in Aspergillus fumigatus keratitis.

The protein GSDMD is an important performer of pyroptosis and a universal substrate for the inflammatory caspase. However, the role and regulatory mechanism of GSDMD in Aspergillus fumigatus keratitis is remains unknown. Here we detected GSDMD protein in the cornea of normal and fungal-infected C57BL/6 mice. Human corneal epithelial cell (HCECs) were preincubated with a hydrochloride solution (IFNR inhibitor), ruxolitinib (JAK/STAT inhibitor), belnacasan (caspase-1 inhibitor) before infection with A. fumigatus conidia. Mice corneas were infected with Aspergillus fumigatus after pretreatment of GSDMD siRNA via subconjunctival injection. After, samples were harvested at specific time points and the expression of GSDMD and IL-1ß was assessed by PCR, Western blot and immunofluorescence staining. Compared with the control group, we observed that the expression of GSDMD in fungal-infected mice cornea was significantly increased. After pretreatment with IFNR, JAK/STAT and caspase-1 inhibitors before fungal infection, the expression of GSDMD was significantly inhibited compared to the DMSO control in HCECs. Moreover, the GSDMD siRNA treatment have significantly weaken corneal inflammatory response, decreasing the proinflammatory factor IL-1ß secretion and reducing neutrophils and macrophages recruitment in mice infected corneas. In summary, the data here provided evidences that GSDMD, an executor of pyroptosis, is involved in the early immune response of A. fumigatus keratitis. Additionally, the inhibition of GSDMD expression can affect the secretion of IL-1ß and the recruitment of neutrophil and macrophages by blocking IFNR, JAK/STAT and caspase-1 signaling pathway. The protein GSDMD may emerge as a potential therapeutic target for A. fumigatus keratitis.

Dimethyl itaconate protects against fungal keratitis by activating the Nrf2/HO-1 signaling pathway.

Dimethyl itaconate (DI) is a membrane-permeable itaconate derivative with anti-inflammatory functions. However, the anti-inflammatory effect of DI has never been studied in fungal keratitis. In this study, we tested the protective effect of DI against fungal keratitis and assessed the role of NF-E2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling in this process. Eyes of C57BL/6 (B6) mice were treated with 2 mm DI after infection with Aspergillus fumigatus. Human corneal epithelial cells (HCECs) were pretreated with 0.25 mm DI and then incubated with A. fumigatus. Clinical scoring, slit-lamp photography, myeloperoxidase determination, flow cytometry and immunostaining were used to assess the disease response and treatment efficacy. PCR, Western blot and ELISA were used to assess the expression of interleukin-1ß (IL-1ß), chemokine (C-X-C motif) ligand 1, IL-6, IL-8, Nrf2 and HO-1. In addition, quantification of viable fungi, absorbance assays and fluorimetry were used to measure DI fungistatic activity. We observed that DI-treated eyes showed decreased clinical scores, fungal loads, polymorphonuclear neutrophil (PMN) infiltration and cytokine expression, compared with phosphate-buffered saline-treated infected eyes. DI treatment decreased the cytokine levels in infected corneas and in HCECs stimulated with A. fumigatus. Moreover, DI treatment increased Nrf2 and HO-1 expression in corneas and nuclear Nrf2 accumulation in HCECs. DI-induced cytokine downregulation was inhibited by pretreatment with an Nrf2 or HO-1 inhibitor. Finally, DI treatment reduced the A. fumigatus absorbance and fungal mass. These data indicate that DI protects against fungal keratitis by limiting inflammation via the Nrf2/HO-1 signaling pathway and that DI inhibits the growth of A. fumigatus.

Corneal epithelial injury-induced norepinephrine promotes Pseudomonas aeruginosa keratitis.

Tissue injury causes the secretion of stress hormone catecholamine and increases susceptibility to opportunistic infection. Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that is a leading cause of microbial keratitis usually associated with ocular injury or contact lens wear. However, the effect of catecholamine on P. aeruginosa induced corneal infection is unknown. Here, we test if norepinephrine (NE) would promote the progression of P. aeruginosa keratitis in mice. Adult C57BL/6 mouse corneas were scarified and then inoculated with P. aeruginosa. The content of NE was elevated in corneas after scarification and inoculation with P. aeruginosa. Then, exogenous NE was applied to the infected corneas at 24 h after inoculation; control eyes were treated with sterile saline. Topical application of NE aggravated the severity of P. aeruginosa keratitis, accompanied with the increase of clinical score, bacterial load, pathological changes, neutrophils infiltration, bacterial virulence factors and proinflammatory factors levels. In order to further verify the role of NE, N-(2-Chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), a neurotoxin selected to deplete NE, was injected subconjunctivally 12 h before scarification. Pre-depletion of local NE by DSP-4 significantly alleviated the severity of corneal infection. Moreover, NE was also confirmed to increase the bacterial growth and the expression of virulence factors gene in vitro. Together, these data showed that increased corneal NE content facilitated the progression of P. aeruginosa keratitis in mice by amplifying host excessive inflammatory response and bacterial virulence. Therefore, targeting NE may provide a potential strategy for the treatment of P. aeruginosa keratitis.

Involvement of Toll-like receptor 2 on human corneal epithelium during an infection of Pythium insidiosum.

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.

Mucosal fluid glycoprotein DMBT1 suppresses twitching motility and virulence of the opportunistic pathogen Pseudomonas aeruginosa.

It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition of twitching by tear fluid was dose-dependent with dilutions to 6.25% retaining activity. Purified lactoferrin, lysozyme, and contrived tears containing these, and many other, tear components lacked the activity. Systematic protein fractionation, mass spectrometry, and immunoprecipitation identified the glycoprotein DMBT1 (Deleted in Malignant Brain Tumors 1) in tear fluid as required. DMBT1 purified from human saliva also inhibited twitching, as well as P. aeruginosa traversal of human corneal epithelial cells in vitro, and reduced disease pathology in a murine model of corneal infection. DMBT1 did not affect PilA expression, nor bacterial intracellular cyclicAMP levels, and suppressed twitching motility of P. aeruginosa chemotaxis mutants (chpB, pilK), and an adenylate cyclase mutant (cyaB). However, dot-immunoblot assays showed purified DMBT1 binding of pili extracted from PAO1 suggesting that twitching inhibition may involve a direct interaction with pili. The latter could affect extension or retraction of pili, their interactions with biotic or abiotic surfaces, or cause their aggregation. Together, the data suggest that DMBT1 inhibition of twitching motility contributes to the mechanisms by which mucosal fluids protect against P. aeruginosa infection. This study also advances our understanding of how mucosal fluids protect against infection, and suggests directions for novel biocompatible strategies to protect our surface epithelia against a major opportunistic pathogen.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

In vivo confocal microscopy characteristics of equine epithelial and subepithelial nonulcerative keratomycosis.

OBJECTIVE: To describe the in vivo confocal microscopy features of horses with epithelial and subepithelial nonulcerative keratomycosis. ANIMALS STUDIED: Four horses with a clinical diagnosis of epithelial or subepithelial keratomycosis. PROCEDURES: Horses were examined on one or more occasions by in vivo laser scanning confocal microscopy of the cornea. Confocal microscopic examination characteristics were correlated with clinical, cytological, and histopathological findings for the horses. RESULTS: All horses had an irregular corneal epithelial surface during slit-lamp biomicroscopy examination. Epithelial or subepithelial corneal opacities were present in multifocal or diffuse patterns. Positive rose bengal corneal staining was present focally or diffusely in all cases. Fungal hyphae were detected in cytological or histopathological corneal samples from all horses. Aspergillus, Fusarium, and Penicillium spp. were cultured from corneal samples. Confocal microscopy detected hyphae diffusely distributed over the axial cornea in horses with epithelial clinical disease. Fungal hyphae were present in all layers of the corneal epithelium and associated with disorganized and sloughing epithelial cells with minimal leukocytes. Subepithelial keratomycosis was correlated with focal, dense accumulations of hyphae in the immediate subepithelial anterior stroma that were surrounded by moderate numbers of leukocytes. Two horses were examined by confocal microscopy on multiple occasions during the course of medical therapy, and fungal hyphae were observed to migrate from the epithelium into the subepithelial stroma as the clinical corneal disease progressed. CONCLUSIONS: With in vivo confocal microscopy, both epithelial and subepithelial keratomycosis appear as unique clinical entities. Equine epithelial keratomycosis is a potential precursor to subepithelial keratomycosis.

Autophagy induced by type III secretion system toxins enhances clearance of Pseudomonas aeruginosa from human corneal epithelial cells.

Corneal ulcers caused by Pseudomonas aeruginosa may lead to severe visual disability due to impaired bacterial clearance from corneal tissues. Our purpose was to study the role of autophagy in the intracellular clearance of P. aeruginosa from human corneal epithelial cells (HCET) and its regulation by the bacterial type III secretion system (T3SS) toxins. Nine different corneal ulcer isolates of P. aeruginosa, PAO1 and T3SS mutants of PAO1 were used to infect HCET cells. Induction of autophagy (Immunofluorescence and Western blot) and pro-inflammatory gene expression (real time PCR) by P. aeruginosa and the role of autophagy in intracellular bacterial clearance were studied in the context of T3SS genotypes. The clinical isolates and PAO1 induced autophagy irrespective of the T3SS genotype, whereas the T3SS mutants were relatively defective in inducing autophagy and becn1 gene expression. External induction of autophagy significantly reduced the intracellular load of P. aeruginosa strains that were associated with worst clinical outcomes. The T3SS negative isolate and PAO1ΔexoST were less sensitive to pharmacological modulation of autophagy and had relatively higher replication potential, suggesting a possible mechanism of bacterial survival in the absence of T3SS toxins. Overall, our results highlight a selective role for autophagy in bacterial clearance from corneal epithelial cells and emphasize the pro-autophagic role of bacterial toxins in the context of corneal ulcers.

Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.

The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more binding inhibition than by the same amount of intact human lactoferrin or by the plant-derived N-glycans released from the rice recombinant lactoferrin; 3) pre-incubation of the bacteria with N-linked glycans released from human tear proteins inhibiting the adhesion of the ocular P. aeruginosa strains to immobilised tear proteins; 4) inhibition by the N-glycans from lactoferrin of the ability of an ocular strain of P. aeruginosa to invade corneal epithelial cells; 5) removal of terminal sialic acid and fucose moieties from the tear glycoproteins with α2-3,6,8 neuraminidase (sialidase) and α1-2,3,4 fucosidase resulting in a reduction in binding of the UTI P. aeruginosa isolate, but not the adhesion of the ocular cytotoxic (6206) or invasive (6294) isolates. Glycosidase activity was validated by mass spectrometry. In all cases, the magnitude of inhibition of bacterial adhesion by the N-glycans was consistently greater for the cytotoxic ocular strain than for the invasive ocular strain. Ocular P. aeruginosa isolates seems to exhibit different adhesion mechanism than previously known PAI and PAII lectin adhesion. The work may contribute towards the development of glycan-focused therapies to prevent P. aeruginosa infection of the eye.

Protective efficacy of a peptide derived from a potential adhesin of Pseudomonas aeruginosa against corneal infection.

Dissecting the interactions between Pseudomonas aeruginosa and corneal cells is important to identify a novel target for prevention and treatment of Pseudomonas keratitis. The current study began with a peptide identified by phage display, and was to investigate the protective efficacy against P. aeruginosa infection in cornea. The original peptide Pc-E, with high homology to a hypothetical membrane protein (HmpA) in P. aeruginosa, and the derived peptide Pc-EP, with the same sequence as a region in HmpA, were synthesized. Peptide Pc-EP could directly bind to HCEC, stronger than Pc-E, and specifically activate toll-like receptor 5, and thereby significantly induce the production of pro-inflammatory factors, such as IL-1ß, IL-6, IFN-γ and IL-17. Moreover, Pc-EP could act as an antagonist to inhibit the adhesion of wild-type P. aeruginosa to HCEC and mouse corneas. No inhibitory effect was observed on the adhesion of the strain loss of HmpA. When compared to the wild-type strain, the adhesion of the hmpA mutant to corneal cells was significantly decreased. Treatment of infected mouse corneas with Pc-EP before infection significantly decreased the bacterial load in the cornea and attenuated the corneal pathology. These results indicate that Pc-EP can be a useful prophylactic agent for P. aeruginosa keratitis.

Aspergillus fumigatus promotes T helper type 2 responses through thymic stromal lymphopoietin production by human corneal epithelial cells.

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.

The importance of the Pseudomonas aeruginosa type III secretion system in epithelium traversal depends upon conditions of host susceptibility.

Pseudomonas aeruginosa is invasive or cytotoxic to host cells, depending on the type III secretion system (T3SS) effectors encoded. While the T3SS is known to be involved in disease in vivo, how it participates remains to be clarified. Here, mouse models of superficial epithelial injury (tissue paper blotting with EGTA treatment) and immunocompromise (MyD88 deficiency) were used to study the contribution of the T3SS transcriptional activator ExsA to epithelial traversal. Corneas of excised eyeballs were inoculated with green fluorescent protein (GFP)-expressing PAO1 or isogenic exsA mutants for 6 h ex vivo before bacterial traversal and epithelial thickness were quantified by using imaging. In the blotting-EGTA model, exsA mutants were defective in capacity for traversal. Accordingly, an ∼16-fold variability in exsA expression among PAO1 isolates from three sources correlated with epithelial loss. In contrast, MyD88-/- epithelia remained susceptible to P. aeruginosa traversal despite exsA mutation. Epithelial lysates from MyD88-/- mice had reduced antimicrobial activity compared to those from wild-type mice with and without prior antigen challenge, particularly 30- to 100-kDa fractions, for which mass spectrometry revealed multiple differences, including (i) lower baseline levels of histones, tubulin, and lumican and (ii) reduced glutathione S-transferase, annexin, and dermatopontin, after antigen challenge. Thus, the importance of ExsA in epithelial traversal by invasive P. aeruginosa depends on the compromise enabling susceptibility, suggesting that strategies for preventing infection will need to extend beyond targeting the T3SS. The data also highlight the importance of mimicking conditions allowing susceptibility in animal models and the need to monitor variability among bacterial isolates from different sources, even for the same strain.

Cell penetrating peptides as efficient nanocarriers for delivery of antifungal compound, natamycin for the treatment of fungal keratitis.

PURPOSE: Enhancing the penetration ability of the antifungal drug natamycin, known to possess poor penetration ability through the corneal epithelium, by complexing with cell penetrating peptides. METHODS: The drug, natamycin was conjugated to a cell penetrating peptide, Tat-dimer (Tat2). The uptake ability of the conjugate in human corneal epithelial cells and its antifungal activity against filamentous fungi, F.solani has been elucidated. RESULTS: The cellular penetration ability of natamycin increased upon conjugation with Tat2. The conjugation between natamycin and Tat2 also lead to enhanced solubility of the drug in aqueous medium. The antifungal activity of the conjugate increased two- folds in comparison to unconjugated natamycin against clinical isolates of F.solani. CONCLUSION: The formation of CPP-natamycin complex is clinically significant as it may enhance the bioavailability of natamycin in corneal tissues and aid in efficient management of fungal keratitis.

The role of Syk signaling in antifungal innate immunity of human corneal epithelial cells.

BACKGROUND: Fungal keratitis is a kind of intractable and sight-threatening diseases. Spleen-tyrosine kinase (Syk) is a non-receptor tyrosine kinase, which plays an important role in the signaling pathway of the receptors. In the current study, we investigate the expression and function of Syk in human corneal epithelial cells with Aspergillus fumigatus (A. fumigatus) infection. METHODS: Cultured telomerase-immortalized human corneal epithelial cells (THCEs) were treated with A. fumigatus hyphae with or without treatment of Syk inhibitors. Activation of Syk and the role of Syk in regulating inflammatory cytokines and chemokines expression were evaluated. The mRNA expression was determined by real time PCR, and protein activation was measured by western blotting. RESULTS: Syk protein was detected in THCEs, and its activation was enhanced after treatment of A. fumigatus hyphae. Expression of inflammatory cytokines (IL-1ß and IL-6) and chemokines (IL-8 and CXCL1) mRNA were significantly increased after stimulation of A. fumigatus hyphae in THCEs. Activation of Syk and expression of IL-1ß, IL-6, IL-8 and CXCL1 by A. fumigatus hyphae were blocked by Syk inhibitors. CONCLUSION: These findings demonstrate that normal human corneal epithelial cells produce Syk, and Syk activation plays an important role in regulating A. fumigatus hyphae-induced inflammatory responses in THCEs.

Homotrimeric macrophage migration inhibitory factor (MIF) drives inflammatory responses in the corneal epithelium by promoting caveolin-rich platform assembly in response to infection.

Acute inflammation that arises during Pseudomonas aeruginosa-induced ocular infection can trigger tissue damage resulting in long term impairment of visual function, suggesting that the appropriate treatment strategy should include the use of anti-inflammatory agents in addition to antibiotics. We recently identified a potential target for modulation during ocular infection, macrophage migration inhibitory factor (MIF). MIF deficiency protected mice from inflammatory-mediated corneal damage resulting from acute bacterial keratitis. To gain a better understanding of the molecular mechanisms of MIF activity, we analyzed the oligomeric states and functional properties of MIF during infection. We found that in human primary corneal cells infected with P. aeruginosa, MIF is primarily in a homotrimeric state. Homotrimeric MIF levels correlated with the severity of infection in the corneas of infected mice, suggesting that the MIF homotrimers were the functionally active form of MIF. During infection, human primary corneal cells released more IL-8 when treated with recombinant, locked MIF trimers than when treated with lower MIF oligomers. MIF promoted P. aeruginosa-induced IL-8 responses via the formation of caveolin-1-rich "signaling hubs" in the corneal cells that led to elevated MAPK p42/p44 activation and sustained inflammatory signaling. These findings suggest that inhibiting homotrimerization of MIF or the functional activities of MIF homotrimers could have therapeutic benefits during ocular inflammation.

Aspergillus fumigatus triggers innate immune response via NOD1 signaling in human corneal epithelial cells.

Fungal keratitis is a serious vision-threatening disease caused by fungi after corneal epithelium damage. We have previously shown a role of cell surface TLRs in Aspergillus fumigatus (A. fumigatus) keratitis. In the present study we showed that Human telomerase-immortalized corneal epithelial cells (HCECs) exposed to A. fumigatus elicited an inflammatory response consisting in increased interleukin-6 (IL-6), IL-8 and tumor necrosis factor (TNF)-α expression and innate defense molecules hBD2 and LL37 in a time-dependent manner. In this study we further investigated the role of intracellular nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors, NOD1 in innate immune and inflammatory response to A. fumigatus. We showed that NOD1 and its downstream signaling molecules RIP2 and NF-κB p65 are expressed in HCECs challenged with either NOD1 specific ligand iE-DAP or A. fumigatus. More importantly, NOD1 knockdown attenuated A. fumigatus-triggered the expression of NOD1, and downstream signaling effectors RIP2 and NF-κB p65, as well as the secretion of IL-6, IL-8 and TNF-α, and the production of hBD2 and LL37. In conclusion, our results demonstrated that NOD1 is a prominent factor of innate immune and inflammatory response in HCECs against A. fumigatus, suggesting that NOD1 might be a potential novel therapeutic target for the treatment of fungal keratitis.