Demodex Blepharitis: A Comprehensive Review of the Disease, Current Management, and Emerging Therapies.

ABSTRACT: Demodex blepharitis is a common disease of the eyelid, affecting approximately 25 million Americans. This article reviews what is known about the mechanisms and impact of Demodex blepharitis, risk factors, signs and symptoms, diagnostic techniques, current management options, and emerging treatments. Demodex mites contribute to blepharitis in several ways: direct mechanical damage, as a vector for bacteria, and by inducing hypersensitivity and inflammation. Risk factors for Demodex blepharitis include increasing age, rosacea, and diabetes. The costs, symptom burden, and psychosocial effects of Demodex blepharitis are considerable. The presence of collarettes is pathognomonic for Demodex blepharitis. Redness, dryness, discomfort, foreign body sensation, lash anomalies, and itching are also hallmarks of the disease. Although a number of oral, topical, eyelid hygiene and device-based options have been used clinically and evaluated in studies for the management of Demodex blepharitis, none have been FDA approved to treat the disease. Recent randomized controlled clinical trials suggest that lotilaner ophthalmic solution, 0.25%, is a topical treatment with the potential to eradicate Demodex mites and eliminate collarettes and eyelid redness for an extended period.

A novel murine model for contact lens wear reveals clandestine IL-1R dependent corneal parainflammation and susceptibility to microbial keratitis upon inoculation with Pseudomonas aeruginosa.

PURPOSE: Contact lens wear carries a risk of complications, including corneal infection. Solving these complications has been hindered by limitations of existing animal models. Here, we report development of a new murine model of contact lens wear. METHODS: C57BL/6 mice were fitted with custom-made silicone-hydrogel contact lenses with or without prior inoculation with Pseudomonas aeruginosa (PAO1-GFP). Contralateral eyes served as controls. Corneas were monitored for pathology, and examined ex vivo using high-magnification, time-lapse imaging. Fluorescent reporter mice allowed visualization of host cell membranes and immune cells. Lens-colonizing bacteria were detected by viable counts and FISH. Direct-colony PCR was used for bacterial identification. RESULTS: Without deliberate inoculation, lens-wearing corneas remained free of visible pathology, and retained a clarity similar to non-lens wearing controls. CD11c-YFP reporter mice revealed altered numbers, and distribution, of CD11c-positive cells in lens-wearing corneas after 24 h. Worn lenses showed bacterial colonization, primarily by known conjunctival or skin commensals. Corneal epithelial cells showed vacuolization during lens wear, and after 5 days, cells with phagocyte morphology appeared in the stroma that actively migrated over resident keratocytes that showed altered morphology. Immunofluorescence confirmed stromal Ly6G-positive cells after 5 days of lens wear, but not in MyD88 or IL-1R gene-knockout mice. P. aeruginosa-contaminated lenses caused infectious pathology in most mice from 1 to 13 days. CONCLUSIONS: This murine model of contact lens wear appears to faithfully mimic events occurring during human lens wear, and could be valuable for experiments, not possible in humans, that help solve the pathogenesis of lens-related complications.

Surfactant protein D contributes to ocular defense against Pseudomonas aeruginosa in a murine model of dry eye disease.

Dry eye disease can cause ocular surface inflammation that disrupts the corneal epithelial barrier. While dry eye patients are known to have an increased risk of corneal infection, it is not known whether there is a direct causal relationship between these two conditions. Here, we tested the hypothesis that experimentally-induced dry eye (EDE) increases susceptibility to corneal infection using a mouse model. In doing so, we also examined the role of surfactant protein D (SP-D), which we have previously shown is involved in corneal defense against infection. Scopolamine injections and fan-driven air were used to cause EDE in C57BL/6 or Black Swiss mice (wild-type and SP-D gene-knockout). Controls received PBS injections and were housed normally. After 5 or 10 days, otherwise uninjured corneas were inoculated with 10(9) cfu of Pseudomonas aeruginosa strain PAO1. Anesthesia was maintained for 3 h post-inoculation. Viable bacteria were quantified in ocular surface washes and corneal homogenates 6 h post-inoculation. SP-D was measured by Western immunoblot, and corneal pathology assessed from 6 h to 4 days. EDE mice showed reduced tear volumes after 5 and 10 days (each by ∼75%, p<0.001) and showed fluorescein staining (i.e. epithelial disruption). Surprisingly, there was no significant difference in corneal pathology between EDE mice and controls (∼10-14% incidence). Before bacterial inoculation, EDE mice showed elevated SP-D in ocular washes. After inoculation, fewer bacteria were recovered from ocular washes of EDE mice (<2% of controls, p = 0.0004). Furthermore, SP-D knockout mice showed a significant increase in P. aeruginosa corneal colonization under EDE conditions. Taken together, these data suggest that SP-D contributes to corneal defense against P. aeruginosa colonization and infection in EDE despite the loss of barrier function to fluorescein.

MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2.

Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization.

Cytokeratins mediate epithelial innate defense through their antimicrobial properties.

Epithelial cells express antimicrobial proteins in response to invading pathogens, although little is known regarding epithelial defense mechanisms during healthy conditions. Here we report that epithelial cytokeratins have innate defense properties because they constitutively produce cytoprotective antimicrobial peptides. Glycine-rich C-terminal fragments derived from human cytokeratin 6A were identified in bactericidal lysate fractions of human corneal epithelial cells. Structural analysis revealed that these keratin-derived antimicrobial peptides (KDAMPs) exhibited coil structures with low α-helical content. Synthetic analogs of these KDAMPS showed rapid bactericidal activity against multiple pathogens and protected epithelial cells against bacterial virulence mechanisms, while a scrambled peptide showed no bactericidal activity. However, the bactericidal activity of a specific KDAMP was somewhat reduced by glycine-alanine substitutions. KDAMP activity involved bacterial binding and permeabilization, but the activity was unaffected by peptide charge or physiological salt concentration. Knockdown of cytokeratin 6A markedly reduced the bactericidal activity of epithelial cell lysates in vitro and increased the susceptibility of murine corneas to bacterial adherence in vivo. These data suggest that epithelial cytokeratins function as endogenous antimicrobial peptides in the host defense against infection and that keratin-derived antimicrobials may serve as effective therapeutic agents.

3D quantitative imaging of unprocessed live tissue reveals epithelial defense against bacterial adhesion and subsequent traversal requires MyD88.

While a plethora of in vivo models exist for studying infectious disease and its resolution, few enable factors involved in the maintenance of health to be studied in situ. This is due in part to a paucity of tools for studying subtleties of bacterial-host interactions at a cellular level within live organs or tissues, requiring investigators to rely on overt outcomes (e.g. pathology) in their research. Here, a suite of imaging technologies were combined to enable 3D and temporal subcellular localization and quantification of bacterial distribution within the murine cornea without the need for tissue processing or dissection. These methods were then used to demonstrate the importance of MyD88, a central adaptor protein for Toll-Like Receptor (TLR) mediated signaling, in protecting a multilayered epithelium against both adhesion and traversal by the opportunistic bacterial pathogen Pseudomonas aeruginosa ex vivo and in vivo.

Adenylate cyclase activity of Pseudomonas aeruginosa ExoY can mediate bleb-niche formation in epithelial cells and contributes to virulence.

We previously showed that ADP-ribosylation (ADP-r) activity of ExoS, a type III secreted toxin of Pseudomonas aeruginosa, enables bacterial replication in corneal and respiratory epithelial cells and correlates with bacterial trafficking to plasma membrane blebs (bleb-niche formation). Here, we explored another type III secreted toxin, ExoY, for its impact on intracellular trafficking and survival, and for virulence in vivo using a murine corneal infection model. Chromosomal or plasmid-mediated expression of exoY in invasive P. aeruginosa (strain PAO1) enabled bacteria to form and traffic to epithelial membrane blebs in the absence of other known effectors. In contrast, plasmid expression of any of four adenylate cyclase mutant forms of exoY did not enable bleb-niche formation, and bacteria localized to perinuclear vacuoles as for effector-null mutant controls. None of the plasmid-complemented bacteria used in this study showed ADP-r activity in the absence of ExoS and ExoT. In contrast to ADP-r activity of ExoS, bleb-niche formation induced by ExoY's adenylate cyclase activity was not accompanied by enhanced intracellular replication. In vivo results showed that ExoY-adenylate cyclase activity promoted P. aeruginosa corneal virulence in susceptible mice. Together the data show that adenylate cyclase activity of P. aeruginosa ExoY, similarly to the ADP-r activity of ExoS, can mediate bleb-niche formation in epithelial cells. While this activity did not promote intracellular replication in vitro, ExoY conferred increased virulence in vivo in susceptible mice. Mechanisms for bleb-niche formation and relationships to intracellular replication and virulence in vivo require further investigation for both ExoS and ExoY.

Modulation of epithelial immunity by mucosal fluid.

Mucosal epithelial cells, including those at the ocular surface, resist infection by most microbes in vivo but can be susceptible to microbial virulence in vitro. While fluids bathing mucosal surfaces (e.g. tears) contain antimicrobials, potentially pathogenic microbes often thrive in these fluids, suggesting that additional mechanisms mediate epithelial resistance in vivo. Here, tear fluid acted directly upon epithelial cells to enhance their resistance to bacterial invasion and cytotoxicity. Resistance correlated with tear fluid-magnified activation of NFκB and AP-1 transcription factors in epithelial cells in response to bacterial antigens, suggesting priming of innate defense pathways. Further analysis revealed differential regulation of potential epithelial cell defense genes by tears. siRNA knockdown confirmed involvement of at least two factors, RNase7 and ST-2, for which tears increased mRNA levels, in protection against bacterial invasion. Thus, the role of mucosal fluids in defense can include modulation of epithelial immunity, in addition to direct effects on microbes.

Factors impacting corneal epithelial barrier function against Pseudomonas aeruginosa traversal.

PURPOSE: Mechanisms determining epithelial resistance versus susceptibility to microbial traversal in vivo remain poorly understood. Here, a novel murine model was used to explore factors influencing the corneal epithelial barrier to Pseudomonas aeruginosa penetration. METHODS: Murine corneas were blotted with tissue paper before inoculation with green fluorescent protein-expressing P. aeruginosa. The impact of blotting on epithelial integrity was evaluated by susceptibility to fluorescein staining and histology. Using fluorescence imaging, blotted corneas were compared to nonblotted corneas for susceptibility to bacterial binding and epithelial penetration after 5 hours or were monitored for disease development. In some experiments, inoculation was performed ex vivo to exclude tear fluid or corneas were pretreated with EGTA to disrupt Ca(2+)-dependent factors. The role of surfactant protein D (SP-D), which inhibits P. aeruginosa cell invasion in vitro, was examined using knockout mice. RESULTS: Blotting enabled fluorescein penetration through the epithelium into the underlying stroma without obvious disruption to corneal morphology. Although blotting enabled bacterial binding to the otherwise adhesion-resistant epithelial surface, adherent bacteria did not penetrate the surface or initiate pathology. In contrast, bacteria penetrated blotted corneas after EGTA treatment and in SP-D knockouts. Visible disease occurred and progressed only in aged, blotted, and EGTA-treated, SP-D knockout mice. CONCLUSIONS: Neither fluorescein staining nor bacterial adhesion necessarily predict or enable corneal susceptibility to bacterial penetration or disease. Corneal epithelial defenses limiting traversal by adherent bacteria include EGTA-sensitive factors and SP-D. Understanding mechanisms modulating epithelial traversal by microbes could improve our understanding of susceptibility to infection and may indicate new strategies for preventing disease.

The impact of inoculation parameters on the pathogenesis of contact lens-related infectious keratitis.

PURPOSE: Contact lens wear predisposes to Pseudomonas aeruginosa keratitis, but the mechanisms involved remain unclear. An in vivo model was used to study lens inoculation conditions enabling disease. METHODS: Custom-made hydrogel contact lenses were fitted to rats after incubation in P. aeruginosa approximately 10(11) cfu/mL (3 hours) or approximately 10(3) cfu/mL (24 hours). Another group was inadvertently inoculated with a suction pen previously used with high inocula, but rinsed in ethanol and stored dry (6 months). Some corneas were tissue paper-blotted to cause fluorescein staining before lens fitting. Contralateral eyes were untreated. Twenty-four hours after disease detection, lenses were transferred to naive rats or examined by confocal microscopy before homogenization to quantify viable bacteria. After lens removal, corneas were washed to collect nonadherent bacteria and were analyzed by immunohistochemistry. RESULTS: All eyes challenged with unworn contaminated lenses developed keratitis after approximately 7 to 10 days. Disease delay and severity were unaffected by inoculum parameters or tissue blotting but occurred sooner with lenses transferred from infected eyes ( approximately 2 days). Worn lenses and corneal washes contained infecting bacteria. Posterior, not anterior, lens surfaces harbored P. aeruginosa biofilms that penetrated the lens matrix. Diseased corneas showed an infiltration of phagocytes and T-lymphocytes. CONCLUSIONS: P. aeruginosa induces keratitis in this lens-wearing model after a single inoculation. Delayed disease onset was interesting considering the greater keratitis risk during extended wear. Infection did not require the disruption of corneal barrier function before lens wear and occurred without exposure to lens care solutions. The data suggest that keratitis involves biofilm formation or other bacterial adaptations in vivo.

Clearance of Pseudomonas aeruginosa from a healthy ocular surface involves surfactant protein D and is compromised by bacterial elastase in a murine null-infection model.

Our previous studies showed that surfactant protein D (SP-D) is present in human tear fluid and that it can protect corneal epithelial cells against bacterial invasion. Here we developed a novel null-infection model to test the hypothesis that SP-D contributes to the clearance of viable Pseudomonas aeruginosa from the healthy ocular surface in vivo. Healthy corneas of Black Swiss mice were inoculated with 10(7) or 10(9) CFU of invasive (PAO1) or cytotoxic (6206) P. aeruginosa. Viable counts were performed on tear fluid collected at time points ranging from 3 to 14 h postinoculation. Healthy ocular surfaces cleared both P. aeruginosa strains efficiently, even when 10(9) CFU was used: e.g., <0.01% of the original inoculum was recoverable after 3 h. Preexposure of eyes to bacteria did not enhance clearance. Clearance of strain 6206 (low protease producer), but not strain PAO1 (high protease producer), was delayed in SP-D gene-targeted (SP-D(-/-)) knockout mice. A protease mutant of PAO1 (PAO1 lasA lasB aprA) was cleared more efficiently than wild-type PAO1, but this difference was negligible in SP-D(-/-) mice, which were less able to clear the protease mutant. Experiments to study mechanisms for these differences revealed that purified elastase could degrade tear fluid SP-D in vivo. Together, these data show that SP-D can contribute to the clearance of P. aeruginosa from the healthy ocular surface and that proteases can compromise that clearance. The data also suggest that SP-D degradation in vivo is a mechanism by which P. aeruginosa proteases could contribute to virulence.

Pathogenic phenotype and genotype of Pseudomonas aeruginosa isolates from spontaneous canine ocular infections.

PURPOSE: This study was designed to determine whether the ability to adversely affect corneal epithelial cell health is a factor common to Pseudomonas aeruginosa keratitis strains and to assess the prevalence of each pathogenic phenotype and genotype in a canine model of naturally-acquired P. aeruginosa ocular infection. METHODS: P. aeruginosa ocular isolates were collected by sampling 100 dogs without disease (six isolates collected) and by sampling dogs with conjunctivitis (two isolates), endophthalmitis (one isolate), active keratitis (12 isolates), and resolved P. aeruginosa keratitis (four isolates). Phenotype was determined in vitro by quantifying corneal epithelial cell invasion by gentamicin survival assays, and cytotoxic activity by Trypan blue exclusion assays. Genotyping was performed for genes encoding the type III secreted effectors. RESULTS: The ratio of invasive to cytotoxic strains with 95% confidence intervals (CI) was 0.83 (CI, 0.42-0.99) for conjunctival microflora isolates, 0.80 (CI, 0.54-0.94) for ocular infection isolates, and 1.0 (CI, 0.45-1.0) for strains isolated post-resolution of keratitis. Among ocular infection isolates, invasive and cytotoxic strains were significantly (P

Genetic analysis of crawling and swimming locomotory patterns in C. elegans.

Alternative patterns of neural activity drive different rhythmic locomotory patterns in both invertebrates and mammals. The neuro-molecular mechanisms responsible for the expression of rhythmic behavioral patterns are poorly understood. Here we show that Caenorhabditis elegans switches between distinct forms of locomotion, or crawling versus swimming, when transitioning between solid and liquid environments. These forms of locomotion are distinguished by distinct kinematics and different underlying patterns of neuromuscular activity, as determined by in vivo calcium imaging. The expression of swimming versus crawling rhythms is regulated by sensory input. In a screen for mutants that are defective in transitioning between crawl and swim behavior, we identified unc-79 and unc-80, two mutants known to be defective in NCA ion channel stabilization. Genetic and behavioral analyses suggest that the NCA channels enable the transition to rapid rhythmic behaviors in C. elegans. unc-79, unc-80, and the NCA channels represent a conserved set of genes critical for behavioral pattern generation.