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.

The Role of Elastase in Corneal Epithelial Barrier Dysfunction Caused by Pseudomonas aeruginosa Exoproteins.

Purpose: To investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa. Methods: Exoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1ß levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro. Results: Elastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ± 2.7 to 104 ± 6.8 Ω/cm2 (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm2) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor. Conclusions: Elastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.

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.

IL-36α Exerts Proinflammatory Effects in Aspergillus fumigatus Keratitis of Mice Through the Pathway of IL-36α/IL-36R/NF-κB.

Purpose: To explore the role of IL-36α in corneas infected by Aspergillus fumigatus. Methods: The experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1ß, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry. Results: IL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1ß, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1ß, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra. Conclusions: IL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1ß, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.

Aspergillus fumigatus-Stimulated Human Corneal Epithelial Cells Induce Pyroptosis of THP-1 Macrophages by Secreting TSLP.

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.

Silencing TLR4/MyD88/NF-κB Signaling Pathway Alleviated Inflammation of Corneal Epithelial Cells Infected by ISE.

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.

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.

Perillaldehyde Ameliorates Aspergillus fumigatus Keratitis by Activating the Nrf2/HO-1 Signaling Pathway and Inhibiting Dectin-1-Mediated Inflammation.

Purpose: The purpose of this study was to investigate the therapeutic effect of perillaldehyde (PAE) on Aspergillus fumigatus (A. fumigatus) keratitis. Methods: Human corneal epithelial cells (HCECs) were pretreated with PAE and stimulated with A. fumigatus mycelium. C57BL/6 mice were infected with A. fumigatus and treated with or without PAE 1 day after infection. Clinical scores, PCR, ELISA, and Western blotting were used to detect the expression of pro-inflammatory mediators, dendritic cell-associated c-type lectin-1 (Dectin-1), nuclear factor (erythroid-derived 2) like 2 (Nrf2), and heme oxygenase (HO-1). Nrf2 expression in HCECs pretreated with PAE was observed by immunofluorescence. NIMP-R14 protein expression and localization in mouse corneas were observed by immunofluorescence staining after treatment with PAE. Corneal colony counting, time-kill tests, and mycelial transformation inhibition tests were used to evaluate the antifungal effect of PAE. Results: C57BL/6 mice treated with PAE at 1 day after infection had a lower clinical score and decreased IL-1ß, TNF-α, IL-6, Dectin-1, and MPO levels. PAE treatment significantly reduced neutrophil recruitments to the corneal stroma. Compared with the DMSO-treated group, PAE treatment significantly decreased mRNA and protein levels of pro-inflammatory cytokines and Dectin-1 in HCECs. PAE pretreatment before A. fumigatus stimulation obviously elevated the mRNA and protein levels of components of the Nrf2/HO-1 axis. HCECs pretreated with PAE before infection showed a weakened ability to inhibit inflammation in the presence of brusatol (BT; an Nrf2 inhibitor) or ZnPP (an HO-1 inhibitor). PAE treatment significantly reduced the fungal load of C57BL/6 mouse corneas and inhibited fungal growth in vitro. Conclusions: These data proved that PAE may ameliorate A. fumigatus keratitis by activating the Nrf2/HO-1 signaling pathway and inhibiting the Dectin-1 mediated inflammatory response and neutrophil recruitment. Furthermore, PAE exerts direct fungicidal activity on 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.

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.

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.

Knockdown of IQGAP-1 Enhances Tight Junctions and Prevents P. aeruginosa Invasion of Human Corneal Epithelial Cells.

PURPOSE: To determine the role of IQ-domain GTPase-activating protein1 (IQGAP-1) in tight junctions of human corneal epithelial cells (HCECs) and its effect against P. aeruginosa (PAK) invasion. MATERIAL AND METHODS: Primary human corneal epithelial cells (HCECs), immortalized HCECs, and IQGAP-1 RNA knockdown HCECs (siHCECs) were used. Confocal microscopy, transepithelial electrical resistance (TER), trypan blue exclusion assay and gentamicin invasion assay were done. RESULTS: In primary and immortalized HCECs, IQGAP-1 co-localized with zonular occludin-1 (ZO-1) and actin. Enhanced actin and ZO-1 aggregation were seen in siHCECs. IQGAP-1 knockdown significantly increased TER of immortalized HCECs (P < .0001). Cell viability after PAK infection increased for siHCECs for up to 4 h after infection. PAK intracellular invasion was significantly lowered by 50% in siHCECs at 1 h post-infection. CONCLUSION: IQGAP-1 knockdown increased the strength and integrity of tight junctions and may provide an early protective effect against P. aeruginosa invasion.

Antimicrobial Nitric Oxide Releasing Contact Lens Gels for the Treatment of Microbial Keratitis.

Microbial keratitis is a serious sight threatening infection affecting approximately two million individuals worldwide annually. While antibiotic eye drops remain the gold standard treatment for these infections, the significant problems associated with eye drop drug delivery and the alarming rise in antimicrobial resistance has meant that there is an urgent need to develop alternative treatments. In this work, a nitric oxide releasing contact lens gel displaying broad spectrum antimicrobial activity against two of the most common causative pathogens of microbial keratitis is described. The contact lens gel is composed of poly-ε-lysine (pεK) functionalized with nitric oxide (NO) releasing diazeniumdiolate moieties which enables the controlled and sustained release of bactericidal concentrations of NO at physiological pH over a period of 15 h. Diazeniumdiolate functionalization was confirmed by Fourier transform infrared (FTIR), and the concentration of NO released from the gels was determined by chemiluminescence. The bactericidal efficacy of the gels against Pseudomonas aeruginosa and Staphylococcus aureus was ascertained, and between 1 and 4 log reductions in bacterial populations were observed over 24 h. Additional cell cytotoxicity studies with human corneal epithelial cells (hCE-T) also demonstrated that the contact lens gels were not cytotoxic, suggesting that the developed technology could be a viable alternative treatment for microbial  keratitis.

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.

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.

Contact lens wear and the diabetic corneal epithelium: A happy or disastrous marriage?

Diabetes mellitus is an epidemic in the US and abroad. With the advent of new contact lens technology, the use of contact lenses as glucose sensors in lieu of the traditional finger stick is quickly becoming realized. This has the potential to rapidly expand the contact lens market into this growing patient population. The independent cellular and physiological effects of contact lens wear and diabetes on the corneal epithelium have been described. However, little evidence exists to date to support whether there is increased risk associated with contact lens wear in diabetes. The focus of this review is to discuss what is known about the cellular effects of contact lenses on the corneal epithelium, the pathophysiological changes in the corneal epithelium that occur in diabetes, and whether an increased risk for corneal epithelial damage and/or infection may negatively impact safety in diabetic contact lens wearers. Available data indicates that there are inherent risks associated with contact lens wear in diabetics. Importantly, eye care practitioners fitting contact lenses in the diabetic patient need to carefully consider the duration of disease, the level of glycemic control, the presence of retinopathy, and the patient's overall health.

Role of pvdE Pyoverdine Synthesis in Pseudomonas aeruginosa Keratitis.

PURPOSE: Pseudomonas aeruginosa produces pyoverdine, encoded by the pvdE gene, for high-affinity iron uptake from transferrin and lactoferrin. This study investigated the contribution of pyoverdine to P. aeruginosa keratitis pathogenesis using in vitro and in vivo models. METHODS: The P. aeruginosa strains examined were parental strain PAO1 and isogenic mutant strain pvdE (ΔpvdE) defective in pyoverdine. Bacterial growth in vitro was determined by PAO1 and ΔpvdE optical densities in Luria-Bertani (LB) broth. PAO1 or ΔpvdE (10 colony-forming units/mL) was inoculated onto cultured human corneal epithelial cells (HCECs) for 1 hour. The monolayers were examined for bacterial adhesion and invasion. In addition, the corneas of C57BL/6 mice were infected with PAO1 or ΔpvdE. Corneal virulence was evaluated by determining clinical scores and bacterial counts during infection. RESULTS: The growth of PAO1 and ΔpvdE in LB broth was similar. Although adhesion of ΔpvdE onto HCECs was significantly increased compared with PAO1, the invasive capacity of ΔpvdE was significantly decreased. Clinical scores and bacterial numbers were significantly lower in ΔpvdE-infected eyes compared with PAO1-infected eyes at 6, 24, and 48 hours (P < 0.001). ΔpvdE was not detected in mouse corneas and did not induce corneal opacity at 6, 24, or 48 hours. CONCLUSIONS: ΔpvdE lost invasive ability toward HCECs. Moreover, ΔpvdE did not cause keratitis in vivo. Thus, pvdE pyoverdine synthesis has critical roles in proliferation and invasion on ocular surfaces and could be a target for prevention of P. aeruginosa keratitis.

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.