IL-36α Enhances Host Defense against Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas.

The IL-36 cytokines are known to play various roles in mediating the immune response to infection in a tissue- and pathogen-dependent manner. The present study seeks to investigate the role of IL-36R signaling in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. IL-36α-/-, IL-36γ-/-, and IL-36R-/- mice had significantly more severe keratitis than wild-type mice. At six hours postinfection, IL-36α pretreatment augmented P. aeruginosa-induced expression of IL-1Ra, IL-36γ, LCN2, and S100A8/A9. At one day postinfection, exogenous IL-36α suppressed, whereas IL-36α deficiency promoted, the expression of IL-1ß. At three days postinfection, exogenous IL-36α suppressed Th1 but promoted Th2 immune response. IL-36α stimulated the infiltration of IL-22-expressing immune cells, and IL-22 neutralization resulted in more severe keratitis. IL-36α alone stimulated dendritic cell infiltration in B6 mouse corneas. Taken together, our study suggests that IL-36R signaling plays a protective role in the pathogenesis of P. aeruginosa keratitis by promoting the innate immune defense, Th2, and/or Th22/IL-22 immune responses. Exogenous IL-36α might be a potential therapy for improving the outcome of P. aeruginosa keratitis.

IL-17 Promotes Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas.

The aim of this study was to elucidate the expression and functions of IL-17 in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. We found that P. aeruginosa infection induced and increased signaling of IL-23/23R/17/17R in mouse corneas. Targeting IL-17A or the IL-17A-specific receptor IL-17RA/IL-17RC with neutralizing Abs resulted in a significant decrease in the severity of P. aeruginosa keratitis, including a decrease in bacterial burden and polymorphonuclear leukocyte infiltration. IL-17A-signaling blockade also significantly reduced the expression of the proinflammatory cytokines L-1ß, IL-24, and MMP-13 and increased the expression of the anti-inflammatory cytokine IL-1RA in mouse corneal epithelium. The presence of mouse IL-17A exacerbated P. aeruginosa-mediated tissue destruction. A cytokine protein array revealed that the expression of osteoprotegerin (OPG) was regulated by IL-17A, and OPG neutralization also resulted in a decrease in the severity of P. aeruginosa keratitis. Although both IL-17 and OPG affected the balanced expression of IL-1ß and IL-1RA, only IL-17 inhibited the expression of TH2 cytokines. Taken together, our results revealed that IL-17A, along with its downstream factor OPG, plays a detrimental role in the pathogenesis of P. aeruginosa keratitis. Targeting IL-17A and/or the OPG/RANKL/RANK/TRAIL system is a potential therapeutic strategy in controlling the outcome of P. aeruginosa keratitis, which was demonstrated by concurrent topical application of IL-17A-neutralizing Ab and ciprofloxacin in B6 mice.

Opposing Effects of IL-1Ra and IL-36Ra on Innate Immune Response to Pseudomonas aeruginosa Infection in C57BL/6 Mouse Corneas.

Pseudomonas aeruginosa keratitis is characterized by severe corneal ulceration and may lead to blindness if not treated properly in a timely manner. Although the roles of the IL-1 subfamily of cytokines are well established, as a newly discovered subfamily, IL-36 cytokine regulation, immunological relevance, and relation with IL-1 cytokines in host defense remain largely unknown. In this study, we showed that P. aeruginosa infection induces the expression of IL-36α and IL-36γ, as well as IL-1ß and secreted IL-1Ra (sIL-1Ra), but not IL-36Ra. Downregulation of IL-1Ra increases, whereas downregulation of IL-36Ra decreases the severity of P. aeruginosa keratitis. IL-1R and IL-36Ra downregulation have opposing effects on the expression of IL-1ß, sIL-1Ra, IL-36γ, S100A8, and CXCL10 and on the infiltration of innate immune cells. Administration of recombinant IL-1Ra improved, whereas IL-36Ra worsened the outcome of P. aeruginosa keratitis. Local application of IL-36γ stimulated the expression of innate defense molecules S100A9, mouse ß-defensin 3, but suppressed IL-1ß expression in B6 mouse corneas. IL-36γ diminished the severity of P. aeruginosa keratitis, and its protective effects were abolished in the presence of S100A9 neutralizing Ab and partially affected by CXCL10 and CXCR3 neutralizations. Thus, our data reveal that IL-1Ra and IL-36Ra have opposing effects on the outcome of P. aeruginosa keratitis and suggest that IL-36 agonists may be used as an alternative therapeutic to IL-1ß-neutralizing reagents in controlling microbial keratitis and other mucosal infections.

IL-24 Promotes Pseudomonas aeruginosa Keratitis in C57BL/6 Mouse Corneas.

The aim of this study was to elucidate the expression and functions of IL-24 in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. Among IL-20R cytokines, only IL-24 was induced at both mRNA and protein levels by infection at early time points. The upregulation of IL-24 was dampened by flagellin pretreatment, which protects the corneas from microbial infection. Time course studies revealed bimodal early and later peaks of IL-24 expression, a pattern shared with suppressor of cytokine signaling (SOCS)3 but not IL-1ß or IL-6. Silencing of IL-24 enhanced S100A8/A9 expression and suppressed SOCS3, IL-1ß, IL-1RN, and matrix metalloproteinase 13 expression at 6 h postinfection. Downregulation of the IL-24 signaling pathway significantly reduced the severity of keratitis, whereas rIL-24 exacerbated P. aeruginosa-mediated tissue destruction. In vitro, rIL-1ß induced the expression of SOCS3, IL-24, IL-1ß, and IL-6 in primary cultured human corneal epithelial cells. rIL-24, alternatively, stimulated the expression of SOCS3, but not the others. In conclusion, IL-24 promotes P. aeruginosa keratitis through the suppression of early protective mucosal immunity, culminating in increased severity of P. aeruginosa keratitis.

CXCL10 suppression of hem- and lymph-angiogenesis in inflamed corneas through MMP13.

Though not present in the normal adult cornea, both hem- and lymph-angiogenesis can be induced in this tissue after an inflammatory, infectious, or traumatic insult. We previously showed that the chemokine CXCL10 plays a key role in eradicating invading Candida (C.) albicans in C57BL6 mouse corneas. However, even after the clearance of pathogens, infection-induced inflammation and angiogenesis continue to progress in the cornea. The aim of this study is define the role of CXCL10 as a major angiostatic factor in modulating cornea angiogenesis in B6 mouse corneas under pathogenic conditions. We showed that epithelial expression of CXCL10, driven by AAV9 vector, suppressed both infection- and inflammation-induced hem and lymph angiogenesis, whereas the neutralization of CXCL10 as well as its receptor CXCR3 greatly promoted these processes. The inhibitory effect of CXCL10 was unrelated to its antimicrobial activity, but through the suppression of the expression of many angiogenic factors, including VEGFa and c, and MMP-13 in vivo. Inhibition of MMP13 but not TIMPs, attenuated suture-induced neovascularization but had no effects on CXCL10 expression. Strikingly, topical application of CXCL10 post-C. albicans infection effectively blocked both hem- and lymph-angiogenesis and preserved the integrity of sensory nerves in the cornea. Taken together, CXCL10 has strong inhibitory effects on neovascularization, whereas MMP13 is required for neovascularization in C. albicans-infected corneas and the local application of CXCL10 or MMP13 inhibitors, alone or as adjuvant therapy, may target hem- and lymph-angiogenesis in the inflamed corneas.

Matrix Metalloproteinase-13 as a Target for Suppressing Corneal Ulceration Caused by Pseudomonas aeruginosa Infection.

PURPOSE: Pseudomonas aeruginosa keratitis is characterized by severe corneal ulceration. This study investigated whether matrix metalloproteinase-13 (MMP13) is involved in P. aeruginosa-induced corneal ulceration and whether it therefore can be targeted for preventing P. aeruginosa keratitis. METHODS: MMP13 expression in P. aeruginosa-infected C57BL/6 mouse corneas was assessed by quantitative polymerase chain reaction and immunohistochemistry analyses. An MMP13-inhibitor (MMP13i) was either injected subconjunctivally prior to or coapplied topically with gatifloxacin 16 hours after infection. Disease severity was assessed by corneal imaging, clinical scoring, bacterial burden, neutrophil infiltration, and CXCL2 expression. Corneal damage and infiltration were also determined by immunohistochemistry analysis and whole-mount confocal microscopy. RESULTS: P. aeruginosa infection induced an increased expression of MMP13 in mouse corneas from 6 to 24 hours after infection in a Toll-liked receptor 5-dependent manner. Subconjunctival injection of MMP13i prior to P. aeruginosa inoculation significantly decreased keratitis severity, as evidenced by preserved epithelium integrity and intact basement membrane, leading to reduced bacterial dissemination to the stroma. Furthermore, topical coapplication of MMP13i with gatifloxacin greatly improved disease outcomes, including accelerated opacity dissolution; decreased inflammation, cellular infiltration, and collagen disorganization; and basement membrane preservation. CONCLUSIONS: Elevated MMP13 activity may contribute to P. aeruginosa keratitis through basement membrane degradation, and its inhibition could potentially be used as an adjunctive therapy to treat microbial keratitis and other mucosal infections.

Chitinase 3-Like 1 Promotes Candida albicans Killing and Preserves Corneal Structure and Function by Controlling Host Antifungal Responses.

Chitinase 3-like 1 (CHI3L1) has been shown to play a role in promoting antibacterial responses, decreasing tissue injury, and enhancing pulmonary repair. This study sought to elucidate the role of CHI3L1 in augmenting the corneal innate immune response to Candida albicans infection in an animal model of fungal keratitis. Flagellin applied topically 24 h prior to C. albicans inoculation significantly protected the corneal from C. albicans and induced CHI3L1 expression in C57BL/6 mouse corneas. CHI3L1, however, played a detectable but minor role in flagellin-induced protection. While C. albicans keratitis was more severe in the corneas treated with Chi3l1 small interfering RNA (siRNA), corneas treated with recombinant CHI3L1 before C. albicans inoculation had markedly ameliorated keratitis, reduced fungal load, and decreased polymorphonucleocyte (PMN) infiltration in an interleukin 13 receptor α2 (IL-13Rα2)-dependent manner. CHI3L1 treatment resulted in the induction of the antimicrobial peptides ß-defensin 3, CRAMP, and chemokine CXCL10 and its receptor CXCR3 in corneal epithelial cells. Importantly, CHI3L1 administered after C. albicans inoculation also had strong protection against fungal keratitis, suggesting a therapeutic window. This is the first report demonstrating that CHI3L1 is induced during fungal infection, where it acts as an immunomodulator to promote fungal clearance and to regulate antifungal innate immune responses in the cornea.

Flagellin-induced expression of CXCL10 mediates direct fungal killing and recruitment of NK cells to the cornea in response to Candida albicans infection.

We previously showed that topical flagellin induces profound mucosal innate protection in the cornea against microbial infection, a response involving multiple genes and cell types. In this study, we used a Candida albicans (CA)-C57BL/6 mouse keratitis model to delineate the contribution of CXCL10- and CXCR3-expressing cells in flagellin-induced protection. Flagellin pretreatment markedly enhanced CXCL10 expression at 6 h post CA infection (hpi), but significantly dampened CXCL10 expression at 24 hpi. At the cellular level, CXCL10 was expressed in the epithelia at 6 hpi in flagellin-pretreated corneas, and concentrated at lesion sites 24 hpi. CXCR3-expressing cells were detected in great numbers at 24 hpi, organized within clusters at the lesion sites in CA-infected corneas. CXCL10 or CXCR3 neutralization increased keratitis severity and dampened flagellin-induced protection. CXCR3-positive cells were identified as NK cells, the depletion of which resulted in severe CA keratitis. Contributions from NK T-cells were excluded by finding no change in flagellin-induced protection in Rag1 KO mice. Recombinant CXCL10 inhibited CA growth in vitro and accelerated fungal clearance and inflammation resolution in vivo. Taken together, our data indicate that epithelium-expressed CXCL10 plays a critical role in fungal clearance and that CXCR3-expressing NK cells contribute to CA eradication in mouse corneas.

Reduced innervation and delayed re-innervation after epithelial wounding in type 2 diabetic Goto-Kakizaki rats.

Patients with diabetes are at an increased risk for developing corneal complications including delayed wound healing and potential vision loss. To understand the cause of diabetic keratopathy, we investigated innervation and its correlation with delayed corneal epithelial wound healing in type 2 diabetic Goto-Kakizaki (GK) rats. GK rats are smaller than the age-matched control Wistar rats from which the GK rats were derived. The blood sugar levels of GK rats are significantly higher than those of Wistar rats. GK rats had increased rose bengal staining and cornea fragility. Fewer nerve fibers were detected compared with Wistar rats. Although nerve fiber densities detected by whole-mount immunohistochemistry were similar near the limbal region, in the central cornea the subbasal nerve plexuses were thinner, less abundant, and showed less branching in GK rats. Corneal epithelial wound closure was delayed and re-innervation was slow and incomplete in GK rats. These abnormalities were more apparent in older GK rats (12 months). Our data suggest that diabetic neuropathy occurs in the cornea of type 2 diabetic GK rats, and defects in the sensory nerve and/or tear film may contribute to diabetic keratopathy and delayed epithelial wound healing in diabetic corneas.

Diabetes Exacerbates Pseudomonas aeruginosa Keratitis in Streptozotocin-Induced and db/db Mice via Altering Programmed Cell Death Pathways.

Purpose: Patients with diabetes have a higher incidence of infections, which are often more severe. This study aimed to investigate the impact of hyperglycemia on bacterial keratitis caused by Pseudomonas aeruginosa (Pa) in two mouse models of diabetes, streptozotocin-induced type 1 diabetes mellitus (T1DM) and db/db type 2 diabetes mellitus. Methods: The susceptibility of corneas to Pa was assessed by determining the inocula required to cause infectious keratitis. Dead or dying cells were identified using TUNEL staining or immunohistochemistry. Specific inhibitors were used to evaluate the role of cell death modulators in Pa keratitis. Cytokines and Treml4 expressions were analyzed using quantitative PCR, and the role of Treml4 in keratitis was determined using small interfering RNA technology. Results: DM corneas required significantly fewer inocula to develop Pa keratitis, with T1DM corneas requiring 750 inocula and type 2 diabetes mellitus corneas requiring 2000 inocula, compared with 10,000 inocula required for normal (NL) mice. T1DM corneas had more TUNEL-positive and fewer F4/80-positive cells than NL corneas. Phospho-caspase 8 (apoptosis) and -RIPK3 (necroptosis) staining was more intense in the epithelial and stromal layers of NL and T1DM corneas, respectively. Pa keratitis was augmented by targeting caspase-8 and prevented by RIPK3 inhibition in both NL and T1DM mice. Hyperglycemia suppressed IL-17A/F and augmented IL-17C, IL-1ß, IL-1Ra, and TREML4, the downregulation of which protected T1DM corneas from Pa infection by suppressing necroptosis. RIPK3 inhibition blocked Pa infection in db/+ mice and significantly decreased the severity of keratitis in db/db mice. Conclusions: Hyperglycemia exacerbates bacterial keratitis in B6 mice by skewing apoptosis toward necroptosis. Preventing or reversing this transition may serve as an adjunct therapy for treating microbial keratitis in patients with diabetes.

Protective effects of resolvin D1 in Pseudomonas aeruginosa keratitis.

PURPOSE: Here, we explored the protective effects of resolvin D1 (RvD1) in Pseudomonas aeruginosa (PA) keratitis. METHODS: C57BL/6 (B6) mice were used as an animal model of PA keratitis. Plate counting and clinical scores were used to assess the severity of the infection and the therapeutic effects of RvD1 in the model. Myeloperoxidase assay was used to detect neutrophil infiltration and activity. Quantitative PCR (qPCR) was used to examine the expression of proflammatory and anti-inflammatory mediators. Immunofluorescence staining and qPCR were performed to identify macrophage polarization. RESULTS: RvD1 treatment alleviated PA keratitis severity by decreasing corneal bacterial load and inhibiting neutrophil infiltration in the mouse model. Furthermore, RvD1 treatment decreased mRNA levels of TNF-α, IFN-γ, IL-1ß, CXCL1, and S100A8/9 while increasing those of IL-1RA, IL-10, and TGF-ß1. RvD1 treatment also reduced the aggregation of M1 macrophages and increased that of M2 macrophages. RvD1 provided an auxiliary effect in gatifloxacin-treated mice with PA keratitis. CONCLUSION: Based on these findings, RvD1 may improve the prognosis of PA keratitis by inhibiting neutrophil recruitment and activity, dampening the inflammatory response, and promoting M2 macrophage polarization. Thus, RvD1 may be a potential complementary therapy for PA keratitis.

Dendritic cell-epithelium interplay is a determinant factor for corneal epithelial wound repair.

The functions of intraepithelial dendritic cells (DCs) are critical for mucosal innate and adaptive immunity, but little is known about the role of tissue-specific DCs in epithelial homeostasis and tissue repair. By using the epithelial debridement wound model and CD11c-diphtheria toxin receptor mice that express a CD11c promoter-driven diphtheria toxin receptor, we showed that DCs migrate along with the epithelial sheet to cover the wound and that local depletion of DCs resulted in a significant delay in epithelial wound closure. In response to wounding, migratory epithelia produce CXCL10, thymic stromal lymphopoietin, and IL-1ß and its antagonist soluble IL-1 receptor antagonist (sIL-1Ra); depletion of corneal DCs reversed their elevated expressions to a different extent, suggesting a DC-mediated positive feedback loop in epithelial gene expression. Furthermore, both CXCL10 and thymic stromal lymphopoietin were localized in migratory epithelia, suggesting that epithelial cells play a key role in DC infiltration and activation in injured corneas. On the other hand, DC depletion resulted in suppressed epithelial AKT activation, increased cell apoptosis, and decreased polymorphonuclear leukocyte infiltration in the healing cornea. These results indicate that DCs and epithelium form a functional entity at mucosal surfaces for maintaining corneal homeostasis and for tissue repair.

The impact of sensory neuropathy and inflammation on epithelial wound healing in diabetic corneas.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes, with several underlying pathophysiological mechanisms, some of which are still uncertain. The cornea is an avascular tissue and sensitive to hyperglycemia, resulting in several diabetic corneal complications including delayed epithelial wound healing, recurrent erosions, neuropathy, loss of sensitivity, and tear film changes. The manifestation of DPN in the cornea is referred to as diabetic neurotrophic keratopathy (DNK). Recent studies have revealed that disturbed epithelial-neural-immune cell interactions are a major cause of DNK. The epithelium is supplied by a dense network of sensory nerve endings and dendritic cell processes, and it secretes growth/neurotrophic factors and cytokines to nourish these neighboring cells. In turn, sensory nerve endings release neuropeptides to suppress inflammation and promote epithelial wound healing, while resident immune cells provide neurotrophic and growth factors to support neuronal and epithelial cells, respectively. Diabetes greatly perturbs these interdependencies, resulting in suppressed epithelial proliferation, sensory neuropathy, and a decreased density of dendritic cells. Clinically, this results in a markedly delayed wound healing and impaired sensory nerve regeneration in response to insult and injury. Current treatments for DPN and DNK largely focus on managing the severe complications of the disease. Cell-based therapies hold promise for providing more effective treatment for diabetic keratopathy and corneal ulcers.

Toll-like receptor 2 ligand-induced protection against bacterial endophthalmitis.

BACKGROUND: Activation of innate immunity plays a key role in determining the outcome of an infection. Here, we investigated whether Toll-like receptors (TLRs) are involved in retinal innate response and explored the prophylactic use of TLR2 ligand in preventing bacterial endophthalmitis. METHODS: C57BL/6 mice were given intravitreal injections of Pam3Cys, a synthetic ligand of TLR2, or vehicle (phosphate-buffered saline) 24 h prior to Staphylococcus aureus inoculation. The severity of endophthalmitis was graded by slit lamp, electroretinography, histological examinations, and determination of bacterial load in the retina. The expression of cytokines/chemokines and cathelicidin-related antimicrobial peptide was assessed by enzyme-linked immunosorbent assay and Western blot, respectively. RESULTS: Intravitreal injections of Pam3Cys up-regulated TLR2 expression in the retina of C57BL/6 mice, and Pam3Cys pretreatment significantly improved the outcome of S. aureus endophthalmitis, preserved retinal structural integrity, and maintained visual function as assessed by electroretinography in C57BL/6 mice. Furthermore, Pam3Cys pretreatment activated retinal microglia cells, induced the expression of cathelicidin-related antimicrobial peptide, and remarkably reduced the bacterial load. CONCLUSIONS: This is the first report that highlights the existence and role of TLR2 in retinal innate immune response to S. aureus infection and suggests that modulation of TLR activation provides a novel prophylactic approach to prevent bacterial endophthalmitis.

Role of IL-36γ/IL-36R Signaling in Corneal Innate Defense Against Candida albicans Keratitis.

Purpose: Interleukin (IL)-36 cytokines have been shown to play either beneficial or detrimental roles in the infection of mucosal tissues in a pathogen-dependent manner, but their involvement in fungal keratitis remains elusive. We herein investigated their expression and function in mediating corneal innate immunity against Candida albicans infection. Methods: Gene expression in mouse corneas with or without C. albicans infection was determined by regular RT- and real-time (q)-PCR, Western blot analysis, ELISA or proteome profile assay. The severity of C. albicans keratitis was assessed using clinical scoring, bacterial counting, and myeloperoxidase (MPO) activity as an indicator of neutrophil infiltration. IL36R knockout mice and IL-33-specific siRNA were used to assess the involvement IL-33 signaling in C. albicans-infected corneas. B6 CD11c-DTR mice and clodronate liposomes were used to define the involvement of dendritic cells (DCs) and macrophages in IL-36R signaling and C. albicans keratitis, respectively. Results: IL-36γ were up-regulated in C57BL6 mouse corneas in response to C. albicans infection. IL-36 receptor-deficient mice display increased severity of keratitis, with a higher fungal load, MPO, and IL-1ß levels, and lower soluble sIL-1Ra and calprotectin levels. Exogenous IL-36γ prevented fungal keratitis pathogenesis with lower fungal load and MPO activity, higher expression of sIL-1Ra and calprotectin, and lower expression of IL-1ß, at mRNA or protein levels. Protein array analysis revealed that the expression of IL-33 and REG3G were related to IL-36/IL36R signaling, and siRNA downregulation of IL-33 increased the severity of C. albicans keratitis. Depletion of dendritic cells or macrophages resulted in severe C. albicans keratitis and yet exhibited minimal effects on exogenous IL-36γ-induced protection against C. albicans infection in B6 mouse corneas. Conclusions: IL-36/IL36R signaling plays a protective role in fungal keratitis by promoting AMP expression and by suppressing fungal infection-induced expression of proinflammatory cytokines in a dendritic cell- and macrophage-independent manner.

Lipo-PEG nano-ocular formulation successfully encapsulates hydrophilic fluconazole and traverses corneal and non-corneal path to reach posterior eye segment.

The present study describes a special lipid-polyethylene glycol matrix solid lipid nanoparticles (SLNs; 138 nm; -2.07 mV) for ocular delivery. Success of this matrix to encapsulate (entrapment efficiency - 62.09%) a hydrophilic drug, fluconazole (FCZ-SLNs), with no burst release (67% release in 24 h) usually observed with most water-soluble drugs, is described presently. The system showed 164.64% higher flux than the marketed drops (Zocon®) through porcine cornea. Encapsulation within SLNs and slow release did not compromise efficacy of FCZ-SLNs. Latter showed in vitro and in vivo antifungal effects, including antibiofilm effects comparable to free FCZ solution. Developed system was safe and stable (even to sterilisation by autoclaving); and showed optimal viscosity, refractive index and osmotic pressure. These SLNs could reach up to retina following application as drops. The mechanism of transport via corneal and non-corneal transcellular pathways is described by fluorescent and TEM images of mice eye cross sections. Particles streamed through the vitreous, crossed inner limiting membrane and reached the outer retinal layers.

ISG15 Acts as a Mediator of Innate Immune Response to Pseudomonas aeruginosa Infection in C57BL/6J Mouse Corneas.

Purpose: IFN-stimulated gene (ISG) 15 is a type 1 IFN-induced protein and known to modify target proteins in a manner similar to ubiquitylation (protein conjugation by ISG15 is termed ISGylation). We sought to determine the role of ISG15 and its underlying mechanisms in corneal innate immune defense against Pseudomonas aeruginosa keratitis. Methods: ISG15 expression in cultured human corneal epithelial cells (HCECs) and mouse corneas was determined by PCR and Western blot analysis. Gene knockout mice were used to define the role of ISG15 signaling in controlling the severity of P. aeruginosa keratitis, which was assessed with photographing, clinical scoring, bacterial counting, myeloperoxidase assay, and quantitative PCR determination of cytokine expression. Integrin LFA-1 inhibitor was used to assess its involvement of ISG15 signaling in P. aeruginosa-infected corneas. Results: Heat-killed P. aeruginosa induced ISG15 expression in cultured HCECs and accumulation in the conditioned media. Isg15 deficiency accelerated keratitis progress, suppressed IFNγ and CXCL10, and promoted IL-1ß while exhibiting no effects on IFNα expression. Moreover, exogenous ISG15 protected the corneas of wild-type mice from P. aeruginosa infection while markedly reducing the severity of P. aeruginosa keratitis in type 1 IFN-receptor knockout mice. Exogenous ISG15 increased bacteriostatic activity of B6 mouse corneal homogenates, and inhibition of LFA-1 exacerbated the severity of and abolished protective effects of ISG15 on P. aeruginosa keratitis. Conclusions: Type 1 INF-induced ISG15 regulates the innate immune response and greatly reduces the susceptibility of B6 mouse corneas to P. aeruginosa infection in an LFA-1-dependent manner.

Role of VIP and Sonic Hedgehog Signaling Pathways in Mediating Epithelial Wound Healing, Sensory Nerve Regeneration, and Their Defects in Diabetic Corneas.

Diabetic keratopathy, a sight-threatening corneal disease, comprises several symptomatic conditions including delayed epithelial wound healing, recurrent erosions, and sensory nerve (SN) neuropathy. We investigated the role of neuropeptides in mediating corneal wound healing, including epithelial wound closure and SN regeneration. Denervation by resiniferatoxin severely impaired corneal wound healing and markedly upregulated proinflammatory gene expression. Exogenous neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP), and vasoactive intestinal peptide (VIP) partially reversed resiniferatoxin's effects, with VIP specifically inducing interleukin-10 expression. Hence, we focused on VIP and observed that wounding induced VIP and VIP type 1 receptor (VIPR1) expression in normal (NL) corneas, but not corneas from mice with diabetes mellitus (DM). Targeting VIPR1 in NL corneas attenuated corneal wound healing, dampened wound-induced expression of neurotrophic factors, and exacerbated inflammatory responses, while exogenous VIP had the opposite effects in DM corneas. Remarkably, wounding and diabetes also affected the expression of Sonic Hedgehog (Shh) in a VIP-dependent manner. Downregulating Shh expression in NL corneas decreased while exogenous Shh in DM corneas increased the rates of corneal wound healing. Furthermore, inhibition of Shh signaling dampened VIP-promoted corneal wound healing. We conclude that VIP regulates epithelial wound healing, inflammatory response, and nerve regeneration in the corneas in an Shh-dependent manner, suggesting a therapeutic potential for these molecules in treating diabetic keratopathy.

Opposing Effects of Neuropilin-1 and -2 on Sensory Nerve Regeneration in Wounded Corneas: Role of Sema3C in Ameliorating Diabetic Neurotrophic Keratopathy.

The diabetic cornea exhibits pathological alterations, such as delayed epithelial wound healing and nerve regeneration. We investigated the role of semaphorin (SEMA) 3C in corneal wound healing and reinnervation in normal and diabetic B6 mice. Wounding induced the expression of SEMA3A, SEMA3C, and their receptor neuropilin-2 (NRP2), but not NRP1, in normal corneal epithelial cells; this upregulation was suppressed for SEMA3C and NRP2 in diabetic corneas. Injections of Sema3C-specific small interfering RNA and NRP2-neutralizing antibodies in wounded mice resulted in a decrease in the rate of wound healing and regenerating nerve fibers, whereas exogenous SEMA3C had opposing effects in diabetic corneas. NRP1 neutralization, on the other hand, decreased epithelial wound closure but increased sensory nerve regeneration in diabetic corneas, suggesting a detrimental role in nerve regeneration. Taken together, epithelium-expressed SEMA3C plays a role in corneal epithelial wound closure and sensory nerve regeneration. The hyperglycemia-suppressed SEMA3C/NRP2 signaling may contribute to the pathogenesis of diabetic neurotrophic keratopathy, and SEMA3C might be used as an adjunctive therapeutic for treating the disease.

Flagellin suppresses the inflammatory response and enhances bacterial clearance in a murine model of Pseudomonas aeruginosa keratitis.

Pseudomonas aeruginosa is a common organism associated with bacterial keratitis, especially in extended-wear contact lens users. In the present study, we determined that pretreatment of cultured human corneal epithelial cells with flagellin isolated from the P. aeruginosa PAO1 strain attenuated cytokine production when the cells were challenged with a cytotoxic strain (ATCC 19660), suggesting a potential use of bacterial flagellin to downregulate infection-associated inflammation in vivo. Administration of flagellin via the subconjunctival and intraperitoneal routes 24 h prior to Pseudomonas inoculation significantly improved the disease outcome, preserved structural integrity and transparency, and thus maintained vision in otherwise perforated corneas of C57BL/6 (B6) mice. The flagellin pretreatment resulted in suppression of polymorphonuclear leukocyte infiltration at a late stage of infection but not at an early stage of infection, decreased the expression of proinflammatory cytokine genes (genes encoding interleukin-1beta [IL-1beta], macrophage inflammatory protein 2, IL-12, and gamma interferon), and greatly enhanced bacterial clearance in the corneas of B6 mice probably through induced expression of the cathelicidin-related antimicrobial peptide and inducible nitric oxide synthase. This is the first report that describes the protective mechanisms induced by a Toll-like receptor agonist that not only curbs the host inflammatory response but also eliminates invading bacteria in the B6 mouse cornea.