HMGB1 Antagonist, Box A, Reduces TLR4, RAGE, and Inflammatory Cytokines in the Cornea of P. aeruginosa-Infected Mice.

PURPOSE: High mobility group box 1 (HMGB1) contributes to adverse disease outcome in Pseudomonas aeruginosa keratitis. This study tests Box A, an HMGB1 antagonist, in a model of the disease. METHODS: C57BL/6 mice (B6) were injected subconjunctivally (1 day before infection) with Box A or phosphate-buffered saline (PBS), infected with P. aeruginosa strain ATCC 19660, and injected intraperitoneally with Box A or PBS at 1 and 3 days postinfection (p.i.). Clinical scores, photographs with a slit lamp camera, real-time polymerase chain reaction (RT-PCR), western blot, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), myeloperoxidase (MPO), and bacterial plate count were used to assess disease outcome. In separate experiments, the therapeutic potential of Box A was tested as described above, but with treatment begun at 6 h p.i. RESULTS: Box A versus PBS prophylactic treatment significantly reduced clinical scores, MPO activity, bacterial load, and expression of TLR4, RAGE, IL-1ß, CXCL2, and TNF-α in the infected cornea. Box A blocked co-localization of HMGB1/TLR4 in infiltrated cells in the stroma at 3 and 5 days p.i., but only at 5 days p.i. for HMGB1/RAGE. Box A versus PBS therapeutic treatment significantly reduced clinical scores, MPO activity, bacterial load, and protein levels of IL-1ß, CXCL2, and IL-6 in the infected cornea. CONCLUSION: Overall, Box A lessens the severity of Pseudomonas keratitis in mice by decreasing expression of TLR4, RAGE (their interaction with HMGB1), IL-1ß, CXCL2 (decreasing neutrophil infiltrate), and bacterial plate count when given prophylactically. Therapeutic treatment was not as effective at reducing opacity (disease), but shared similar features with pretreatment of the mice.

Topical Glycyrrhizin Is Therapeutic for Pseudomonas aeruginosa Keratitis.

PURPOSE: Glycyrrhizin (GLY), an inhibitor of high-mobility group box 1 (HMGB1) protects prophylactically against Pseudomonas aeruginosa keratitis. However, the therapeutic potential of GLY to enhance an antibiotic has not been tested and is our purpose. METHODS: C57BL/6 mice (B6) were infected with a clinical isolate (KEI 1025) of P. aeruginosa and treated topically at 6 h postinfection (p.i.) with GLY or phosphate-buffered saline (PBS). Clinical scores, photography with a slit lamp, enzyme-linked immunosorbent assay, myeloperoxidase assay, bacterial plate counts, histopathology, reactive oxygen/nitrogen species (ROS/RNS) assays, and in vitro macrophage (Mφ) stimulation assays were used to assess effects of GLY treatment. In separate similar experiments, the ability of GLY to bioenhance the antibiotic, tobramycin (TOB), was assessed. RESULTS: In vivo, GLY versus PBS topical treatment began at 6 h p.i., improved disease outcome by significantly reducing clinical scores, proinflammatory proteins (HMGB1, RAGE, TLR4, TNF-α, and CXCL2), neutrophil infiltrate, bacterial load, ROS/RNS, and nitric oxide. In vitro, GLY downregulated iNOS and COX-2 expression (mRNA) in both mouse and human (THP-1) Mφ. At 6 and 24 h p.i., treatment with GLY enhanced the effects of TOB compared with TOB alone by significantly reducing corneal bacterial load and/or protein levels of cytokines CXCL2 and IL-1ß. CONCLUSIONS: Data provide evidence that GLY is not only therapeutic for Pseudomonas keratitis through its ability to reduce HMGB1, bacterial load, and oxidative damage but also through its bioenhancement of an antibiotic, even when treatment is initiated at 24 h after infection.

Characterization of Three Ocular Clinical Isolates of P. aeruginosa: Viability, Biofilm Formation, Adherence, Infectivity, and Effects of Glycyrrhizin.

We selectively characterized three isolates from Pseudomonas aeruginosa keratitis patients and how glycyrrhizin (GLY) affected them. Type III toxins were determined using polymerase chain reaction (PCR). Minimum Inhibitory Concentration (MIC) of GLY and assays for its effects on: time kill, bacterial permeability, and biofilm/adhesion were done. In vivo, C57BL/6 (B6) mice were treated topically with GLY after G81007 infection. Clinical score, photography with a slit lamp and RT-PCR were used to assess treatment effects. Isolates expressed exoS and exoT, but not exoU. MIC for all isolates was 40 mg/mL GLY and bacteriostatic effects were seen for G81007 after treatment using time kill assays. From viability testing, GLY treatment significantly increased the number of permeabilized bacteria (live/dead assay). Isolates 070490 and G81007 formed more biofilms compared with R59733 and PAO1 (control). GLY-treated bacteria had diminished biofilm compared with controls for all isolates. GLY reduced adherence of the G81007 isolate to cultured cells and affected specific biofilm associated systems tested by reverse transcription PCR (RT-PCR). In vivo, after G81007 infection, GLY treatment reduced clinical score and messenger RNA (mRNA) expression of IL-1ß, TNF-α, CXCL2 and HMGB1. This study provides evidence that GLY is bacteriostatic for G81007. It also affects biofilm production, adherence to cultured cells, and an improved keratitis outcome.

Glycyrrhizin Reduces HMGB1 and Bacterial Load in Pseudomonas aeruginosa Keratitis.

PURPOSE: High mobility group box 1 (HMGB1) contributes to poor disease outcome in Pseudomonas aeruginosa keratitis. This study tests the prophylactic effect of treatment with HMGB1 inhibitors, glycyrrhizin (GLY) and its derivative, carbenoxolone (CBX), for Pseudomonas keratitis. METHODS: We treated C57BL/6 (B6) mice subconjunctivally with GLY or CBX, infected with a noncytotoxic clinical isolate (KEI 1025) or a cytotoxic strain (ATCC 19660) of P. aeruginosa, and injected intraperitoneally with either agent. Clinical score, photography with a slit lamp, real-time RT-PCR, ELISA, myeloperoxidase (MPO) assay, bacterial plate count, histopathology, and absorbance assays were used to assess treatment efficacy and bacteriostatic activity. RESULTS: After KEI 1025 infection, GLY treatment reduced HMGB1 (mRNA and protein levels) and improved disease outcome with significant reduction in mRNA levels of IL-1ß, TLR4, CXCL2, and IL-12; protein expression (IL-1ß, CXCL2); neutrophil infiltrate; and bacterial load. Treatment with GLY enhanced antimicrobial proteins, including CRAMP and mBD2, but not mBD3. Glycyrrhizin also reduced clinical scores and improved disease outcome in corneas infected with strain 19660. However, neither HMGB1 mRNA or protein levels were reduced, but rather, CXCL2 expression (mRNA and protein), neutrophil infiltrate, and bacterial load were reduced statistically. Treatment with GLY initiated 6 hours after infection reduced plate count; GLY also was bacteriostatic for KEI 1025 and ATCC 19660. CONCLUSIONS: Glycyrrhizin reduces HMGB1 and is protective against P. aeruginosa-induced keratitis with a clinical isolate that is noncytotoxic. It was similar, but less effective when used after infection with a cytotoxic strain, which did not reduce HMGB1.

Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis.

PURPOSE: The microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions. METHODS: Eight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays. RESULTS: MicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease. CONCLUSIONS: MicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

Thrombomodulin Protects Against Bacterial Keratitis, Is Anti-Inflammatory, but Not Angiogenic.

PURPOSE: Thrombomodulin (TM) is a multidomain, transmembrane protein with anti-inflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity. METHODS: Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only). RESULTS: Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1ß, CXCL2, NF-κB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1ß, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls. CONCLUSIONS: These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic.

Effects of VIP on corneal reconstitution and homeostasis following Pseudomonas aeruginosa induced keratitis.

PURPOSE: Studies from our laboratory have demonstrated that vasoactive intestinal peptide (VIP) directly converts the normally susceptible C57BL/6J (B6) mouse to resistant after ocular infection through modulation of the inflammatory response. This study examines mechanisms by which VIP influences the healing phase following infection--specifically reconstitution of the extracellular matrix (ECM). METHODS: B6 mice received daily intraperitoneal (IP) injections of VIP, while control mice were similarly injected with sterile phosphate buffered saline (PBS). Real-time RT-PCR, ELISA, and immunofluorescent staining were used to assess the effects of VIP treatment on ECM molecule expression after Pseudomonas aeruginosa-induced keratitis. We also compared the effect of VIP treatment on lipopolysaccharide (LPS)-stimulated B6- and BALB/c-derived fibroblasts. RESULTS: In vivo analyses revealed that VIP treatment of P. aeruginosa-infected B6 corneas led to a significant increase in ECM molecules associated with healing/homeostasis, while those associated with ECM degradation were significantly down-regulated when compared to wild-type (WT) controls. In vitro studies revealed that VIP treatment of lipopolysaccharide-stimulated fibroblasts derived from susceptible B6 and resistant BALB/c mice expressed distinct differences in ECM molecule expression, whereby the latter expressed higher levels of ECM molecules aimed at reconstitution. Furthermore, differential expression of VIP receptor-1/VIP receptor-2 (VIPR1/VIPR2) was observed between B6 and BALB/c after VIP treatment of LPS-stimulated fibroblasts. CONCLUSIONS: VIP treatment functions to enhance ECM reconstitution, which appears to be carried out in large part by fibroblasts via VIPR2. Overall, the data from this study suggest that VIP not only regulates disease pathogenesis, but also functions to restore integrity of the corneal stroma.

The role of VIP in cornea.

PURPOSE: Exogenous vasoactive intestinal peptide (VIP) down-regulates pro-inflammatory but up-regulates anti-inflammatory cytokines, growth factors (GFs) and Toll-like receptors promoting healing in experimental Pseudomonas aeruginosa (P. aeruginosa) keratitis. Whether VIP is required for GF or GF receptor (R) expression in normal and infected corneas is unknown and is the purpose of this study. METHODS: VIP knockout ((-/-)) and wild-type (WT) C57BL/6 (B6) mice were infected and tested using PCR array, real-time RT-PCR, ELISA, and immunostaining. VIP antagonist treatment studies also were done using B6 and BALB/c mice. RESULTS: Infected corneas of VIP(-/-) versus WT B6 mice perforated earlier (2 vs. 5 days postinfection [p.i.]), and array data showed that GFs were differentially changed between groups. RT-PCR revealed that the infected cornea of VIP(-/-) versus WT mice expressed higher mRNA levels of epidermal growth factor (EGF) and hepatocyte growth factor (HGF), reduced FGF, EGFR, and HGFR, with no difference in FGFR; differences between groups were not seen in normal cornea. Immunostaining for GF and GFR in the normal cornea of VIP(-/-) versus WT mice was similar. However, at 1 day p.i., VIP(-/-) versus WT mice had more intense EGF and HGF, similar FGFR, and reduced FGF, EGFR, and HGFR staining. VIP antagonist treatment decreased protein levels for GFR at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea. CONCLUSIONS: The data showed that endogenous VIP is not requisite for GF or GFR expression in the normal cornea but, after infection, its absence or reduction is critical for their regulation.

Substance P affects growth factors in Pseudomonas aeruginosa-infected mouse cornea.

PURPOSE: This study analyzed the influence of substance P (SP) on growth factors related to wound healing in mice in the presence of infectious keratitis. METHODS: Naturally resistant mice were injected intraperitoneally with SP or phosphate-buffered saline and infected with Pseudomonas aeruginosa, and corneal messenger RNA (mRNA) levels of growth factors and apoptosis genes were tested. Enzyme-linked immunosorbent assay determined the protein levels, whereas immunohistochemistry tested the distribution, macrophage phenotype, and cell quantitation. In vitro, macrophages were stimulated with lipopolysaccharide (LPS; with or without SP) and mRNA levels of proinflammatory and antiinflammatory cytokines and apoptosis genes were tested. RESULTS: After SP, epidermal growth factor mRNA and protein levels were disparately regulated early, with no differences later in the disease. Hepatocyte growth factor and fibroblast growth factor-7 mRNA and protein levels were increased after SP treatment. Enumerating dual-labeled stromal cells revealed no difference between SP-treated versus phosphate-buffered saline-treated groups in the percentage of epidermal growth factor-labeled fibroblasts or macrophages, but there were significant increases in both hepatocyte growth factor- and fibroblast growth factor-7-labeled cells. Type 2 (M2) macrophages and caspase-3 mRNA levels were decreased, whereas B-cell lymphoma-2 mRNA expression was increased after SP treatment. In vitro, mRNA levels of several proinflammatory cytokines and B-cell lymphoma-2 were elevated, whereas transforming growth factor ß was decreased after macrophage stimulation with SP (with LPS) over LPS alone. (Mice: n = 105 control; 105 experimental.) CONCLUSIONS: These data show that treatment with SP in infectious keratitis elevates growth factors but also adversely affects the disease by enhancing the inflammatory response and its sequelae.

Vasoactive intestinal peptide downregulates proinflammatory TLRs while upregulating anti-inflammatory TLRs in the infected cornea.

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.

VIP and growth factors in the infected cornea.

PURPOSE: Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide that downregulates proinflammatory cytokines and promotes healing in a susceptible model of P. aeruginosa keratitis. Growth factors also play a role in corneal healing and restoration of tissue homeostasis after wounding. However, whether VIP treatment modulates growth factors to promote healing in the infected cornea remains untested and is the purpose of this study. METHODS: C57BL/6 (B6) mice were injected with VIP and mRNA and protein levels, and immunostaining for EGF, FGF, HGF, and VEGF-A were done. Exogenous treatment with a mixture of the growth factors also was tested and levels of cytokines, defensins, and bacterial counts were determined. RESULTS: Real-time RT-PCR, immunostaining, and ELISA data demonstrated that treatment with VIP enhanced levels of EGF, FGF, and HGF during disease, and that VEGF-A, and associated angiogenic molecules also were increased by VIP. Moreover, immunohistochemical studies confirmed that both epithelial and stromal cells participated in growth factor production. Most notably, treatment with a mixture of EGF, FGF, and HGF after disease onset, prevented corneal perforation when compared with controls. This outcome was associated with downregulation of proinflammatory cytokines such as macrophage inflammatory protein-2 (MIP-2), upregulation of anti-inflammatory cytokines such as TGF-ß, and antimicrobials ß-defensins 2 and 3, as well as decreased plate counts at 1 day postinfection (p.i.) (P = 0.0001). CONCLUSIONS: Collectively, the data provide evidence that VIP treatment modulates growth factors, angiogenic molecules, and defensins in the infected cornea and that this in turn promotes healing and restoration of tissue homeostasis.

Testican-1 promotes resistance against Pseudomonas aeruginosa-induced keratitis through regulation of MMP-2 expression and activation.

PURPOSE: Testican-1 (or SPOCK) is a highly conserved chimeric proteoglycan encoded by the SPOCK1 gene. Protease regulatory activity has recently been demonstrated by this molecule and its family members testican-2 and -3. The present study tested the hypothesis that testican-1 regulates corneal matrix metalloproteinase (MMP)-2 expression, thus improving disease outcome after Pseudomonas aeruginosa-induced keratitis. METHODS: C57BL/6 (B6) and BALB/c mice were routinely infected with P. aeruginosa and were evaluated at various postinfection (pi) times for corneal expression of testican-1 and MMP-2, by PCR array, real-time RT-PCR, ELISA, activity assays, zymography, and immunohistochemistry. Next, B6 mice were treated with recombinant human (rh) testican-1, and expression was knocked down in BALB/c ice by siTestican-1 treatment, to determine the relationship between the two molecules. RESULTS: BALB/c versus B6 mice expressed significantly higher mRNA and protein levels of testican-1 after P. aeruginosa-induced ocular infection. MMP-2 expression and activation was also disparate between the two mouse strains. After rhTestican-1 treatment in B6 mice, overall disease response was significantly improved, whereas siRNA treatment of BALB/c mice converted the normally resistant response to susceptible. Testican-1 was shown to influence MMP-2 expression, activation, and regulation, as well. CONCLUSIONS: This study demonstrates corneal expression of testican-1 and its temporal regulation of MMP-2 expression and activation after induction of bacterial keratitis. Furthermore, the data collectively indicate that testican-1 is a novel target for disease treatment to promote better disease outcome regarding chronic inflammation and infection and diseases involving pathologic tissue destruction.

VIP promotes resistance in the Pseudomonas aeruginosa-infected cornea by modulating adhesion molecule expression.

PURPOSE: This study tested the hypothesis that the neuropeptide vasoactive intestinal peptide (VIP) regulates adhesion molecule expression, reduces inflammatory cell migration and infiltration into the Pseudomonas aeruginosa-infected cornea of susceptible B6 mice, and promotes corneal healing and resistance. METHODS: B6 mice received daily intraperitoneal (IP) injections of VIP from -1 through 5 days after infection. Control mice were similarly injected with sterile phosphate-buffered saline (PBS). Transcript levels of adhesion molecules were determined by PCR array, then select molecules were tested individually by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) or immunofluorescent staining with confocal laser scanning microscopy at various time points after infection to assess the effects of VIP treatment in the regulation of adhesion molecule expression. RESULTS: Injection of B6 mice with VIP compared with PBS resulted in significant downregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, platelet-endothelial cell adhesion molecule-1, and P-selectin and L-selectin mRNA expression. Protein levels for ICAM-1 and VCAM-1, detected by ELISA, supported the mRNA data at similar time points. Immunofluorescence staining further confirmed the effects of VIP treatment, showing reduced corneal expression of ICAM-1/leukocyte function-associated antigen (LFA-1) and VCAM-1/very late antigen-4 (VLA-4) at select time points compared with PBS-treated animals. CONCLUSIONS: VIP treatment downregulates the production of adhesion molecules integral to the transmigration process of host inflammatory cells (polymorphonuclear neutrophils, macrophages) into the infected cornea. This results directly in reduced cellular infiltration, less stromal destruction, and better disease outcome.

Role of the Fas pathway in Pseudomonas aeruginosa keratitis.

PURPOSE: The role of the Fas pathway was tested in Pseudomonas aeruginosa-infected mouse cornea by contrasting the responses of FasL(-/-) and wild-type (WT) mice. METHODS: TUNEL staining, real-time RT-PCR, immunostaining, and ELISA assay were used. RESULTS: Compared with WT (resistant) mice, BALB/c FasL(-/-) exhibited significantly elevated bacterial counts and polymorphonuclear leukocyte numbers at 1 and 3 days postinfection (p.i.) and worse outcomes from disease. Similar bacterial challenges in C57BL/6 FasL(-/-) compared with WT mice also led to worsened disease as evidenced by earlier corneal perforation in the susceptible mouse strain. Intense TUNEL staining of apoptotic cells was seen earlier (1 day vs. 3 days) p.i. in BALB/c WT than in knockout mice, This earlier apoptotic pattern correlated with increased expression of caspases 3, 8, and 9 and BAX and with decreased expression of the antiapoptotic molecule Bcl-2. Furthermore, expression levels of the proinflammatory molecule TNF-alpha and its receptor, MIP-2, inducible nitric oxide synthase (iNOS), and nitrite also were significantly elevated in the infected cornea of BALB/c FasL(-/-) compared with WT mice. In vitro, LPS-stimulated Mphi from BALB/c FasL(-/-) mice expressed significantly less caspase 3 and 9, BAX, and IL-10 and more TNF-alpha, MIP-2, and IL-1beta than did cells from WT mice. CONCLUSIONS: Fas-FasL interaction in the cornea balances the host innate immune response to improve disease outcome by promoting earlier apoptosis and regulating proinflammatory cytokines/chemokines and nitric oxide (nitrite) production. Dysregulation of this interaction contributes to bystander tissue damage, enhancing nutrients for bacterial growth and worsened disease outcome after P. aeruginosa infection.

IL-33 shifts macrophage polarization, promoting resistance against Pseudomonas aeruginosa keratitis.

PURPOSE: To determine the role of IL-33 in resistance to Pseudomonas aeruginosa keratitis. METHODS: Corneal IL-33 mRNA and protein levels were tested in susceptible C57BL/6 (B6) and resistant BALB/c mice. B6 mice were injected with recombinant mouse IL-33 (rmIL-33) and disease severity, bacterial load, polymorphonuclear neutrophils (PMN) infiltrate, gene expression of inflammatory, and T-helper (Th)1/Th2 cytokines were tested by RT-PCR. IL-33 signaling and macrophage (Mvarphi) polarization also were examined. RESULTS: IL-33 mRNA and protein were expressed constitutively in the normal corneas of both groups and were significantly elevated at 1 to 5 days after infection in BALB/c over B6 mice. rmIL-33-treated B6 mice showed less severe disease than did PBS controls and exhibited decreased bacterial load, PMN infiltrate, and corneal mRNA levels for IL-1beta, MIP-2, and TNF-alpha. Th2-type cytokines (IL-4, -5, -10) also were significantly upregulated, and protein levels for TNF-alpha and IL-10 confirmed the mRNA data. To further investigate IL-33 in corneal inflammation, it was overexpressed in Mvarphi (RAW264.7 cells). This significantly increased IL-5 and IL-10, while it decreased IFN-gamma and other pro-inflammatory cytokines. The role of the Mvarphi was further tested in infected rmIL-33 compared with PBS-injected mice. Immunostaining showed that rmIL-33 injection shifted Mvarphi polarization from NO synthase 2 to arginase production. Furthermore, peritoneally elicited cells (B6 mice) treated with lipopolysaccharide and rmIL-33 exhibited elevated ST2 levels and a shift from IL-12 to IL-10 mRNA production. CONCLUSIONS: These data provide evidence that IL-33 promotes a Th2-type immune response and reduces inflammation by polarizing the Mvarphi production of anti-inflammatory mediators in the cornea.

Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine beta-defensins (mBD) 3 and 4, the murine homologs of human beta-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-gamma, MIP-2, IL-1beta, TNF-alpha, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-kappaB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

Beta-defensin-2 promotes resistance against infection with P. aeruginosa.

Corneal infection with Pseudomonas aeruginosa results in corneal perforation in susceptible C57BL/6 (B6) mice, but not in resistant BALB/c mice. To explore the role of two important defensins, murine beta-defensin-1 (mBD1) and mBD2, in the ocular immune defense system, their mRNA and protein expression levels were tested by real-time RT-PCR and Western blot, respectively. mRNA, protein, and immunostaining data demonstrated that both mBD1 and mBD2 were constitutively expressed in normal BALB/c and B6 corneas, and they were disparately up-regulated in BALB/c (more) vs B6 (less) corneas after infection. To determine whether either defensin played a role in host resistance, BALB/c mice were treated with either mBD1 or mBD2 small interfering RNA by subconjunctival injection together with topical application. Increased corneal opacity and worsened disease were displayed after knockdown of mBD2 but not of mBD1. mBD2 silencing also increased bacterial counts and polymorphonuclear neutrophil infiltration in BALB/c corneas. Real-time RT-PCR data further demonstrated that mBD2, not mBD1, differentially modulated mRNA expression of proinflammatory cytokines/molecules such as IFN-gamma, MIP-2, IL-1beta, TNF-alpha, IL-6, and inducible NO synthase; TLR signaling molecules, including TLR2, TLR4, TLR9, and MyD88; and the transcription factor NF-kappaB. Additionally, in vivo studies indicated that mBD2 silencing enhanced corneal nitrite levels and NF-kappaB activation. Collectively, the data provide evidence that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection.

Rat silicone hydrogel contact lens model: effects of high- versus low-Dk lens wear.

OBJECTIVES: This study used a rat contact lens (CL) model to test if high- versus low-Dk lens wear caused changes in (1) conjunctival Langerhans cell (LC) number or location; (2) Bcl-2 expression; and (3) infection risk. METHODS: Female, Lewis rats wore a high- or low-Dk CL continuously for 2 weeks. Afterward, corneas were harvested and processed for ADPase activity to identify LCs, for immunostaining and for real time-polymerase chain reaction. Contact lens-wearing rats also were challenged with Pseudomonas aeruginosa by placing a bacterial-soaked CL on the eye followed by topical delivery of bacteria. After 48 hrs, slit lamp examination and real time-polymerase chain reaction were used to evaluate the corneal response. RESULTS: Conjunctival LC were significantly increased after low- versus high-Dk CL wear (P<0.0001). In contrast, conjunctival LC in non-lens wearing rats was not significantly different from the high-Dk lens wearing group. Bcl-2 mRNA levels were significantly decreased in low- versus high-Dk CL wearing rats, while Bax, FasL, caspase 3, and caspase 9 levels were unchanged. Immunostaining for Bcl-2 showed fewer positively stained epithelial cells in the low- versus high-Dk lens wearing group. After bacterial challenge, 30% of low- versus none of the high-Dk CL wearing corneas became infected and showed increased mRNA levels for several proinflammatory cytokines/chemokines, inducible nitric oxide synthase and matrix metalloproteinase-9. CONCLUSION: Low- versus high-Dk or non-CL wear led to an increased number of conjunctival LC, decreased Bcl-2 levels, and increased the risk of bacterial infection.

Substance P delays apoptosis, enhancing keratitis after Pseudomonas aeruginosa infection.

PURPOSE: Apoptosis was examined after Pseudomonas aeruginosa corneal infection in C57BL/6 (B6, susceptible) and BALB/c (resistant) mice. METHODS: TUNEL staining, real-time RT-PCR, polymorphonuclear neutrophils (PMNs) and macrophage (Mphi) depletion, and immunostaining were used. RESULTS: Intense TUNEL staining was seen in BALB/c versus B6 cornea at 1 versus 3 days after infection (PI) and correlated with mRNA levels for caspase-3. TUNEL staining (with or without PMN depletion) and PMN immunostaining revealed the PMN as the major apoptotic cell for both groups. Next, B6 mice with high corneal levels of the antiapoptosis neuropeptide, substance P (SP), were treated with the SP antagonist, Spantide I (with/without Mphi depletion), resulting in earlier apoptosis and diminished disease only when M(phi)s were present. SP interactions with M(phi)s were explored further by eliciting cells from both groups and stimulating them with lipopolysaccharide (LPS), with or without SP. LPS with SP treatment decreased the number of apoptotic M(phi)s in B6 but not BALB/c mice and correlated with reduced mRNA expression of NK-1R (major SP receptor) on BALB/c cells. In addition, mRNA expression for IL-12 was upregulated in LPS-stimulated B6 M(phi)s, although cells from BALB/c mice expressed more IL-10. CONCLUSIONS: These studies provide evidence that PMN apoptosis is delayed in the cornea of B6 versus BALB/c mice after bacterial infection; that in B6 mice, blocking SP interaction with the NK-1R promotes earlier apoptosis and improves disease outcome; that M(phi)s regulate PMN apoptosis; and that M(phi)s from B6 versus BALB/c mice differ in expression of the NK-1R and cytokines produced after LPS challenge.

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.