TLR4 is required for host resistance in Pseudomonas aeruginosa keratitis

PURPOSE: To determine the role of Toll-like receptor 4 (TLR4) in Pseudomonas aeruginosa (P. aeruginosa) keratitis in resistant (cornea-healing) BALB/c mice. METHODS: Corneal TLR4 mRNA levels were tested by real-time PCR in BALB/c mice before and after infection. Clinical score, slit lamp, histopathology, bacterial counts, and polymorphonuclear neutrophil (PMN) quantitation were performed in the infected cornea of TLR4-deficient (TLR4(lps-d)) and wild-type BALB/c mice. mRNA for IL-1beta, MIP-2, IFN-gamma, IL-18, inducible nitric oxide synthase (iNOS), and beta-defensin-2 levels were measured by real-time PCR. Protein levels for IL-1beta, MIP-2, and IFN-gamma were tested by ELISA. RESULTS: In resistant BALB/c mice, TLR4 mRNA expression was significantly upregulated in the cornea after P. aeruginosa infection. In contrast, TLR4-deficient mice were susceptible to infection with P. aeruginosa and showed increased corneal opacity, PMN infiltration, bacterial counts, and perforated infected corneas. After infection, TLR4-deficient mice also showed increased mRNA expression of proinflammatory cytokines (IL-1beta and MIP-2) and type-1-associated cytokines (IFN-gamma and IL-18) when compared with wild-type BALB/c mice. ELISA analyses showed that IL-1beta, MIP-2, and IFN-gamma protein levels also were significantly upregulated in the cornea of TLR4-deficient versus wild-type mice. In contrast, levels of iNOs and beta-defensin-2 were significantly decreased in TLR4-deficient compared with wild-type mice. CONCLUSIONS: TLR4 is critical in host resistance to P. aeruginosa, as its deficiency results in increased PMN infiltration and proinflammatory cytokine production, decreased iNOs and beta-defensin-2 production, impaired bacterial killing, and a susceptible phenotype.     

ST2 is essential for Th2 responsiveness and resistance to pseudomonas aeruginosa keratitis

PURPOSE: To elucidate the role of ST2, a member of the TLR/IL-1R (TIR) superfamily, in protecting against Pseudomonas aeruginosa keratitis in BALB/c mice. METHODS: ST2 mRNA and protein expression levels were tested by real-time PCR and Western-blot in C57BL/6 (B6; susceptible) versus BALB/c (resistant) mice before and after P. aeruginosa (strain 19660; American Type Culture Collection, Philadelphia, PA) challenge. Infected BALB/c mice also were tested after subconjunctival injection with recombinant murine (rm)ST2 or PBS. Disease was monitored by clinical score, slit lamp, bacterial plate count, a myeloperoxidase (MPO) assay to measure polymorphonuclear neutrophil (PMN) infiltrate, real-time RT-PCR, and ELISA. RESULTS: ST2 mRNA and protein were constitutively expressed in the uninfected normal corneas of both mouse groups. ST2 levels in the cornea of BALB/c compared with B6 mice were elevated significantly at 1 to 3 days post infection (PI), peaked at 3 and decreased at 5 days PI. BALB/c mice treated with rmST2 showed increased corneal opacity and perforation (at 5 days PI) when compared with PBS controls. rmST2- versus PBS-injected mice exhibited increased bacterial load, PMN infiltrate, and higher corneal mRNA levels for IL-1beta, MIP-2, IL-6, IL-1R1, and Th1-type cytokine such as IFN-gamma. Protein levels for IL-1beta, MIP-2, and IL-6 also were significantly upregulated, whereas the Th2 cytokines IL-4 (mRNA), IL-5 (mRNA), and IL-10 (mRNA and protein) were significantly reduced. CONCLUSIONS: ST2 is critical in resistance to P. aeruginosa keratitis, functioning to reduce corneal infection (bacterial load) and inflammation by negatively regulating proinflammatory cytokines and inhibiting type-1 immunity, but upregulating type-2 cytokine production, particularly IL-10.     

Amnionmembrantransplantation bessert experimentelle herpetische Keratitis

PURPOSE: Transplantation of human amniotic membrane (AMT) accelerates the healing of experimental ulcerative herpetic keratitis. Here the expression and activity of matrix metalloproteinase (MMP)-9 was studied. METHODS: BALB/c mice were corneally infected with HSV-1. Whereas the infected corneas of mice in group 1 were covered with AM, tarsorrhaphies were performed in others (group 2). After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically, and the corneal PMN infiltration was studied histologically. The expression of MMP-9 in the corneas was localized by immunohistochemistry and analyzed by Western-blot technique. The MMP-9 activity in the corneas was determined by zymography. RESULTS: On day 14, the ulcerating corneas had a dense PMN infiltration, the ulcers and the majority of PMNs were highly positive for MMP-9, and the active forms of MMP-9 were detected. Gelatinolytic activity was found in these corneas by zymography. Compared with the mice of group 2, ulceration, stromal inflammation and neovascularization markedly improved clinically and histologically within 2 days in mice of group 1. This was associated with a reduced expression of MMP-9 in corneal tissue and in PMNs. The gelatinolytic activity of MMP-9 was reduced after AMT. CONCLUSIONS: These observations suggest that improvement of herpetic corneal ulcers and reduced corneal neovascularization after AMT may result from a reduced expression and activity of MMP-9.     

Regulation of MMPs and TIMPs by IL-1beta during corneal ulceration and infection

PURPOSE: This study was conducted to investigate the role of IL-1beta in the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in a mouse model of experimental keratitis and corneal injury. METHODS: Mice were injected subconjunctivally with 10 micro g of anti-mouse IL-1beta antibody 2 hours before challenge with Pseudomonas aeruginosa (strain 6294). Control animals received an equal volume and concentration of isotype control antibody at the same time. Eyes were enucleated at 0, 8, 24, and 72 hours, after bacterial challenge and processed for histologic examination. Some eyes were homogenized and used to evaluate production of MMP-2, MMP-9, TIMP-1, and TIMP-2 protein, by zymography and reverse zymography. RESULTS: Injury without bacterial infection resulted in increases in both MMP-2 and -9 and a slight but significant downregulation of TIMP-1. Administration of anti-IL-1beta just before injury and without bacterial infection resulted in a significant reduction in expression of MMP-2 (at 8 hours), MMP-9 (at 8 hours), TIMP-1 (at 8 and 72 hours), and TIMP-2 (at 8 hours). Mice treated with anti-IL-1beta antibody, before bacterial challenge, demonstrated markedly reduced corneal damage compared with the severe corneal injury and massive neutrophil infiltration observed in infected mice treated with control antibody. Administration of the neutralizing anti-IL-1beta antibody resulted in a significant reduction of MMP-9 and a change in the time course of TIMP-1 and -2 expression. The reduction in MMP-9 by anti-IL-1beta during infection was much greater than the reduction without infection. CONCLUSIONS: The results imply that IL-1beta has a central role in corneal destruction during bacterial keratitis and suggests that targeting IL-1beta may be a novel therapeutic strategy for microbial keratitis.     

Silencing Toll-like receptor-9 in Pseudomonas aeruginosa keratitis

PURPOSE: To determine the effects of silencing Toll-like receptor (TLR) 9 signaling in Pseudomonas aeruginosa keratitis. METHODS: Corneal TLR9 mRNA levels were tested by RT-PCR in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice and compared. The response of B6 mice to CpG DNA, which binds TLR9, was tested after subconjunctival injection of mice with control or CpG DNA; TLR9, IL-1beta, macrophage inflammatory protein (MIP)-2, IL-4, IL-10, IL-12, IL-18, and IFN-gamma levels were measured by RT-PCR. Langerhans cells (LCs) were stimulated with CpG DNA and treated with TLR9 or control siRNA, and mRNA levels of TLR9, IL-1beta, and MIP-2 were detected by RT-PCR. In addition, IL-1beta levels were tested by ELISA. Then B6 mice were injected subconjunctivally with control or TLR9 siRNA before infection and treated topically afterward. Slit lamp, clinical score, RT-PCR, ELISA, myeloperoxidase assay, and plate counts were performed. RESULTS: TLR9 mRNA levels were sixfold higher in B6 than in BALB/c corneas the day after injection. B6 mice injected with CpG DNA exhibited an increase in corneal mRNA for TLR9, IL-1beta, MIP-2, IL-12, and IFN-gamma over controls. LCs stimulated with CpG DNA and treated with TLR9 siRNA exhibited reduced TLR9, IL-1beta, and MIP-2 levels compared with controls. Finally, B6 mice treated with TLR9 siRNA showed decreases in corneal opacity, polymorphonuclear leukocyte number, IL-12 and IFN-gamma mRNA, IL-1beta, and MIP-2 protein compared with those treated with control siRNA. Fewer corneas perforated in these mice, but bacterial loads were higher than in controls. CONCLUSIONS: Signaling through TLR9 appears important in P. aeruginosa keratitis, and silencing TLR9 signaling reduces inflammation but likely contributes to decreased bacterial killing in the cornea.     

Corneal response to Pseudomonas aeruginosa infection

Pseudomonas aeruginosa (P. aeruginosa) is a common organism associated with bacterial keratitis, especially in those who use extended wear contact lenses. Recent advances in our understanding of host innate and adaptive immune responses to experimental infection have been made using a variety of animal models, including inbred murine models that are classed as resistant (cornea heals) vs. susceptible (cornea perforates). Evidence has been provided that sustained IL-12-driven IFN-gamma production in dominant Th1 responder strains such as C57BL/6 (B6) contributes to corneal destruction and perforation, while IL-18-driven production of IFN-gamma in the absence of IL-12 is associated with bacterial killing and less corneal destruction in dominant Th2 responder strains such as BALB/c. The critical role of IL-1 and chemotactic cytokines such as MIP-2 in PMN recruitment and the critical role of this cell in the innate immune response to bacterial infection is reviewed. Regulation of PMN persistence is also discussed and evidence provided that persistence of PMN in B6 cornea is regulated by CD4+ T cells, while macrophages regulate PMN number in the cornea of BALB/c mice. The studies provide a better understanding of the inflammatory mechanisms that are operative in the cornea after P. aeruginosa challenge and are consistent with long-term goals of providing targets for alternative or adjunctive treatment for this disease. Future studies will be aimed at better defining the role of Toll receptors, neuropeptides (as unconventional modulators of the immune response) and exploitation of disease control by new techniques, such as RNA silencing.     

Role of innate and adaptive immunity in the pathogenesis of keratitis

Pseudomonas aeruginosa is a common organism associated with bacterial keratitis primarily resulting from contact lens usage. Advances in our understanding of host innate and adaptive immune responses to experimental infection have been achieved using animal models, including inbred mouse models that are classed as resistant (cornea heals) vs. susceptible (cornea perforates). Evidence has shown that sustained IL-12-driven IFN-gamma production in dominant Th1 responder strains such as C57BL/6 (B6) contributes to corneal destruction and perforation. In contrast, in Th2-responder BALB/c mice, IL-18-driven IFN-gamma production regulates bacterial killing with less corneal destruction. IL-1 and chemotactic cytokines (e.g., MIP-2) recruit PMN to the cornea. The critical role of these cells in the innate immune response and their regulation after bacterial infection has been established. The studies provide a better understanding of the regulatory mechanisms that operate in the cornea after P. aeruginosa challenge, determining susceptibility vs. resistance to disease, and are consistent with long-term goals of providing targets for better treatment of disease.     

Role of Innate and Adaptive Immunity in the Pathogenesis of Keratitis

Pseudomonas aeruginosais a common organism associated with bacterial keratitis primarily resulting from contact lens usage. Advances in our understanding of host innate and adaptive immune responses to experimental infection have been achieved using animal models, including inbred mouse models that are classed as resistant (cornea heals) vs. susceptible (cornea perforates). Evidence has shown that sustained IL-12-driven IFN-γ production in dominant Th1 responder strains such as C57BL/6 (B6) contributes to corneal destruction and perforation. In contrast, in Th2-responder BALB/c mice, IL-18-driven IFN-γ production regulates bacterial killing with less corneal destruction. IL-1 and chemotactic cytokines (e.g., MIP-2) recruit PMN to the cornea. The critical role of these cells in the innate immune response and their regulation after bacterial infection has been established. The studies provide a better understanding of the regulatory mechanisms that operate in the cornea after P. aeruginosa challenge, determining susceptibility vs. resistance to disease, and are consistent with long-term goals of providing targets for better treatment of disease.     

Transplantation of amniotic membrane in murine herpes stromal keratitis modulates matrix metalloproteinases in the cornea

PURPOSE: To study matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the corneas from mice with ulcerative herpes stromal keratitis (HSK) treated with amniotic membrane transplantation (AMT). METHODS: The corneas from BALB/c mice were infected with HSV-1. Mice with ulcerative HSK on postinfection (PI) day 14 were used for the experiments. In one group of mice, the corneas were treated with transplantation of amniotic membrane (AMT) that was secured with a tarsorrhaphy, and a control group underwent tarsorrhaphy alone. After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically. Corneal sections were studied by immunohistochemistry for the expression of MMP-2, -8, and -9 and TIMP-1 and -2. MMP activity in the corneas was investigated by zymography, and the expression of the enzymes was measured by the Western blot technique. RESULTS: At day 14 PI, the ulcers stained intensely positive for MMP-2, -8, and -9 and TIMP-1 and -2. Ulceration (P < 0.001), stromal inflammation (P < 0.01) and inflammatory cell infiltration (P < 0.001) markedly improved by day 2 after AMT. This was associated with reduced expression (P < 0.01) and activity of MMP-8, and -9 and increased localization of TIMP-1 (P < 0.01), whereas TIMP-2 was not affected. In contrast, high levels of expression of MMP-8 and -9 remained in the cornea after tarsorrhaphy, and the TIMP-1 expression was only slightly upregulated. CONCLUSIONS: Rapid improvement of HSV-1-induced ulcerative keratitis is noted after amniotic membrane transplantation. This may be caused by reduced expression and activity of MMP-8 and -9, increased expression of TIMP-1, and sustained expression of TIMP-2.     

The role of nitric oxide in resistance to P. aeruginosa ocular infection

PURPOSE: This study determined the role of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in the resistance response of BALB/c mice to P. aeruginosa-induced keratitis. METHODS: RT-PCR, nitrite detection, iNOS inhibition, ELISA, and immunohistochemistry were used. RESULTS: Early after infection, iNOS mRNA expression and nitrite levels in cornea were elevated compared to levels in the uninfected cornea. Treatment with aminoguanidine sulfate (AG), an inhibitor of iNOS, resulted in extensive corneal destruction, reduced nitrite levels, and reduced nitrotyrosine staining. Infected mice also had increased bacterial burden and elevated levels of MIP-1alpha, IL-1beta, and MIP-2 in the cornea. Dual-labeling immunohistochemistry established the macrophage as the major source of iNOS in the infected cornea. CONCLUSIONS: These data provide evidence that iNOS is constitutively expressed in the BALB/c cornea; that iNOS-derived NO is required for bacterial killing/stasis; and that the macrophage is the major cell source of NO.     

Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis

To determine the distribution and activities of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus (HSV) type-1 keratitis, BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain) and then observed for the clinical signs of keratitis. Corneas were harvested at days 0, 2, 7 and 14 post-infection (p.i.). MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and the Western blot technique. The enzymatic activities were analyzed by zymography. Epithelial HSV keratitis was present at day 2 after corneal infection and healed by day 5 p.i. While the expression and activity of MMP-2, MMP-8 and MMP-9 increased in the corneas at day 2 p.i., it was reduced at day 7 p.i. TIMP-1 and -2 were expressed in the corneas before and seven days after infection. Necrotizing stromal keratitis with corneal ulceration and dense polymorphonuclear leukocyte (PMN) infiltration was present at day 14 p.i. This correlated with increased expression of MMP-2, MMP-8 and MMP-9 in the corneas. MMP-8, MMP-9 and MMP-2 staining was particularly intense in the proximity of the ulcers and in areas of PMN infiltration. At day 14 p.i., MMP-2, -8 and -9 activities were upregulated, and TIMP-2 was expressed. These data suggest that MMPs produced by resident corneal cells and PMNs may possibly play a role in early epithelial keratitis and in the ulcerative process in the late phase after corneal HSV-1 infection. The ratio of MMPs to TIMPs may be important for the course of necrotizing HSV keratitis. TIMPs might participate in the repair process.     

Aging and PMN response to P. aeruginosa infection

PURPOSE: Alterations in immune system function associated with aging may contribute to increased morbidity in this population of individuals. The current studies were performed to determine aging-related changes in polymorphonuclear neutrophil (PMN) function after corneal infection with Pseudomonas aeruginosa. METHODS: Total PMN number, macrophage inflammatory protein (MIP)-2 mRNA and protein expression, and ocular bacterial load were determined in 8-week- and 12-month-old inbred BALB/c mice at various times after infection with P. aeruginosa. In addition, 12-month-old mice were treated systemically with the MIP-2 polyclonal antibody (pAb) to determine the effects of MIP-2 neutralization on ocular disease and PMN recruitment. RESULTS: Histologically, PMN infiltration into the cornea of 12-month-old mice was delayed initially and was associated with an inability to reduce bacterial load at later postinfection (PI) times. In addition, a significantly greater number of PMNs were found in the cornea of 12-month-old mice at later PI times. The increase in PMN number in 12-month-old mice correlated with a persistence of MIP-2 expression in cornea at these later times. Systemic treatment of 12-month-old mice with neutralizing MIP-2 pAb versus normal rabbit serum (NRS) resulted in reduced corneal PMN number and ocular disease. CONCLUSIONS: These data provide evidence that persistence of PMN in the cornea of 12-month-old mice contributes to corneal tissue destruction after P. aeruginosa challenge. Further evidence also is provided that the chemoattractant MIP-2 contributes to the altered PMN response in these animals.     

Role and regulation of CXC-chemokines in acute experimental keratitis

The aim of this study was to elucidate the expression of chemokines, their role and regulation in bacterial corneal infection using three bacterial strains (Pseudomonas. aeruginosa- invasive, cytotoxic and contact lens induced acute red eye strains) which have been shown to produce three distinct patterns of corneal disease in the mouse. The predominant chemokine expressed in response to all three strains was MIP-2. Prolonged expression of high levels of MIP-2 was associated with increased severity of corneal inflammation. Significantly reduced disease severity upon administration of anti-MIP-2 antibodies suggested that MIP-2 may play an important role in the pathogenesis of Pseudomonas keratitis at least in part by being a major chemoattractant for polymorphonuclear leukocytes (PMN) recruitment. Interestingly, the numbers of bacteria in eyes with neutralized MIP-2 activity did not decrease even though the severity of the disease was decreased. This implies PMNs as the major destructive factor in microbial keratitis. Further, neutralization of IL-1beta activity alone using monoclonal antibodies resulted in significant reduction of both MIP-2 and KC activity indicating that chemokine levels were regulated by IL-1beta. These studies demonstrate that the regulation of MIP-2 activity may be beneficial in reducing corneal damage during microbial keratitis in rodents and perhaps that regulation of the human homologue of MIP-2, IL-8, may be useful for controlling keratitis in humans.     

Spantide I decreases type I cytokines, enhances IL-10, and reduces corneal perforation in susceptible mice after Pseudomonas aeruginosa infection

PURPOSE: To determine the effects of blocking substance P (SP) interactions with its major receptor (NK1-R) using the antagonist spantide I in susceptible mice infected with Pseudomonas aeruginosa. METHODS: Immunohistochemistry and enzyme immunosorbent assay (EIA) tested levels of SP in the cornea of B6 and BALB/c mice. B6 mice were treated with spantide, and after infection, slit lamp examination; clinical score; bacterial counts; and myeloperoxidase (MPO), RT-PCR, ELISA, and polymorphonuclear (PMN) cell chemotaxis assays were performed. RESULTS: SP corneal levels were significantly elevated constitutively and after infection in the B6 more than in BALB/c mice. Spantide treatment of B6 mice significantly decreased the number of perforated corneas, bacterial counts, and PMNs. mRNA levels for type I cytokines (e.g., IFN-gamma) as well as MIP-2, IL-6, TNF-alpha, and IL-1beta (mRNA and protein) also were significantly reduced after spantide treatment. The type II cytokine IL-10 (mRNA and protein) was elevated, whereas TGF-beta mRNA levels were unchanged after spantide treatment. PMN chemotaxis was induced by SP and other neuropeptides in vitro, but was not affected by spantide I. mRNA for neurokinin-1-receptor-1 (NK-1R) was detected in the normal and infected corneas and on macrophages (Mphis), but not on PMNs (unstimulated or stimulated with endotoxin [LPS]). Spantide treatment of Mphis reduced IL-1beta after LPS+SP treatment but not after either alone. CONCLUSIONS: The SP antagonist Spantide provides a novel approach to reduce type 1 and enhance the type 2 cytokine IL-10 in the infected cornea of B6 mice, leading to a significant reduction in corneal perforation and improved disease outcome.     

Substance P promotes susceptibility to Pseudomonas aeruginosa keratitis in resistant mice: anti-inflammatory mediators downregulated

PURPOSE: Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa-induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice. METHODS: The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed. RESULTS: Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFkappaB and type 1 cytokines (e.g., IFN-gamma), as well as for TNF-alpha, MIP-2, IL-18, IL-6, and IL-1beta, were significantly elevated, whereas VIP and cytokines TGF-beta and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFkappaB, IL-1beta, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators. CONCLUSIONS: These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.     

兔棘阿米巴角膜炎IL-1β和MIP-1的表达

目的建立一种模拟临床人角膜棘阿米巴感染的动物模型,探讨角膜棘阿米巴原虫感染后角膜炎症细胞因子巨噬细胞炎性蛋白-1(macrophage inflammatory protein-1,MIP-1)、白细胞介素-1β(interleukin-1β,IL-1β)的表达。方法 20只新西兰白兔应用角膜表层镜片法,即刮除角膜上皮,覆盖角膜植片,右眼在层间注入棘阿米巴滋养体混悬液,左眼注入生理盐水,缝合眼睑24h,建立棘阿米巴角膜炎模型,观察角膜溃疡形态,并行角膜HE染色或PAS染色组织病理切片检查,应用逆转录聚合酶链反应(RT-PCR)技术检测不同病程角膜组织中的IL-1β、MIP-1的表达。结果 20只兔右眼均感染棘阿米巴性角膜炎,病变角膜组织中IL-1β含量与MIP-1含量于术后第1天、3天、5天、7天、9天均明显升高(均为P<0.01),分别于术后第5天(53.360±1.083)与术后第3天(34.445±1.072)达最高值,差异均有显著统计学意义(均为P<0.01),以后逐渐下降。结论 IL-1β是反映兔棘阿米巴感染角膜局部炎症反应程度的敏感指标;而MIP-1的表达则是兔棘阿米巴角膜炎中机体重要的防御和保护性因素。     

Expression of matrix metalloproteinases 2 and 9 in experimental corneal injury and fungal keratitis

PURPOSE: Levels of matrix metalloproteinases (MMPs) can be modulated during corneal infection, but little is known about MMP profiles during fungal keratitis. The purpose of this study was to determine the effect of corneal trauma and immunosuppressive treatment on the expression kinetics of MMP-2 and MMP-9 during experimental keratomycosis. METHODS: Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with 1 x 10 culturable units of Fusarium solani or mock-inoculated with or without superficial corneal scarification. Eyes were scored daily for disease severity and processed for zymography after 1.5 hours, 6 hours, 1 day, 4 days, or 8 days. Gelatinase activity was densitometrically quantitated and normalized to MMP-2 and MMP-9 controls. RESULTS: MMP-9 levels in nontraumatized eyes transiently increased at 6 hours after fungal exposure, but this increase was inhibited by cyclophosphamide treatment. Corneal injury significantly induced early MMP-9 expression that returned to baseline levels within 4 days. Cyclophosphamide pretreatment reduced and delayed MMP-9 after scarification. Fusarium exposure dampened the MMP-9 response to corneal trauma in immunocompetent and cyclophosphamide-treated animals. Ocular levels of MMP-2 were not affected by scarification, fungal exposure, or immunosuppressive treatment. CONCLUSIONS: Ocular MMP-9 levels, but not MMP-2 levels, increased soon after corneal injury. A similar, although muted, MMP-9 response occurs during early filamentous fungal keratitis, with a kinetic profile similar to corneal disease progression. The early stage of ulcerative keratitis may involve selective regulation of corneal matrix metalloproteinases, suggesting an initial opportunity for therapeutic intervention.     

The Role of Nitric Oxide in Resistance to P. aeruginosa Ocular Infection

Purpose: This study determined the role of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in the resistance response of BALB/c mice to P. aeruginosa-induced keratitis. Methods: RT-PCR, nitrite detection, iNOS inhibition, ELISA, and immunohistochemistry were used. Results: Early after infection, iNOS mRNA expression and nitrite levels in cornea were elevated compared to levels in the uninfected cornea. Treatment with aminoguanidine sulfate (AG), an inhibitor of iNOS, resulted in extensive corneal destruction, reduced nitrite levels, and reduced nitrotyrosine staining. Infected mice also had increased bacterial burden and elevated levels of MIP-1α, IL-1β, and MIP-2 in the cornea. Dual-labeling immunohistochemistry established the macrophage as the major source of iNOS in the infected cornea. Conclusions: These data provide evidence that iNOS is constitutively expressed in the BALB/c cornea; that iNOS-derived NO is required for bacterial killing/stasis; and that the macrophage is the major cell source of NO.     

Experimental dry eye stimulates production of inflammatory cytokines and MMP-9 and activates MAPK signaling pathways on the ocular surface

PURPOSE: To evaluate whether experimentally induced dry eye in mice activates mitogen-activated protein kinase (MAPK) signaling pathways, c-Jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK), and p38 and stimulates ocular surface inflammation. METHODS: 129SvEv/CD-1 mixed mice aged 6 to 8 weeks were treated with systemic scopolamine and exposure to an air draft for different lengths of time, from 4 hours to 10 days. Untreated mice were used as the control. The concentrations of IL-1beta and TNF-alpha in tear fluid washings and in corneal and conjunctival epithelia were measured by ELISA. MMP-9 in tear washings was evaluated by zymography, and gelatinase activity in the cornea and conjunctiva was determined by in situ zymography. Corneal and conjunctival epithelia were lysed in RIPA buffer for Western blot with MAPK antibodies, or they were lysed in 4 M guanidium thiocyanate solution for extraction of total RNA, which was used to determine gene expression by semiquantitative RT-PCR, real-time PCR, and gene array. RESULTS: Compared with those in age-matched control subjects, the concentrations of IL-1beta and MMP-9 in tear fluid washings and the concentrations of IL-1beta and TNF-alpha and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice receiving treatments to induce dry eye after 5 or 10 days. The expression of IL-1beta, TNF-alpha, and MMP-9 mRNA by the corneal and conjunctival epithelia was also stimulated in mice treated for 5 or 10 days. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were markedly increased as early as 4 hours after treatment, and they remained elevated up to 5 days. CONCLUSIONS: Experimental dry eye stimulates expression and production of IL-1beta, TNF-alpha, and MMP-9 and activates MAPK signaling pathways on the ocular surface. MAPKs are known to stimulate the production of inflammatory cytokines and MMPs, and they could play an important role in the induction of these factors that have been implicated in the pathogenesis of dry eye disease.