Linda Garthwaite

Evaluation of synergistic activity of bovine lactoferricin with antibiotics in corneal infection

OBJECTIVES The objectives of this study were to determine whether a synergistic effect could be obtained in vitro between bovine lactoferricin (B-LFcin) and antibiotics against Pseudomonas aeruginosa and Staphylococcus aureus isolates from ocular infections, and to evaluate the use of B-LFcin as an adjunct to the antibiotic treatment of corneal infection in vivo. METHODS Chequerboard and time-kill assays were performed to investigate the combined effects of B-LFcin and conventional antibiotics, including ciprofloxacin, ceftazidime and gentamicin, against 17 strains of P. aeruginosa (8) and S. aureus (9) isolated from ocular infection and inflammation, and 1 reference strain of S. aureus. Corneas of C57BL/6 mice were topically challenged with a multidrug-resistant strain of P. aeruginosa. Nine hours post-challenge, mice were treated topically and hourly with either vehicle, B-LFcin, ciprofloxacin or ciprofloxacin containing B-LFcin for 8 h. Corneas were then clinically examined, and bacterial numbers and levels of myeloperoxidase (MPO) evaluated. RESULTS Synergy between B-LFcin and ciprofloxacin or ceftazidime was identified in most P. aeruginosa isolates, including multidrug-resistant strains, whereas no synergistic effect was seen between B-LFcin and gentamicin. Synergy was only observed with B-LFcin and ciprofloxacin against 2/10 S. aureus strains, and there was no synergy between B-LFcin and any of the other antibiotics tested. Combined B-LFcin and ciprofloxacin treatment significantly improved the clinical outcome, and reduced bacterial numbers and MPO in infected mouse corneas. B-LFcin alone was also able to reduce levels of MPO in infected corneas. CONCLUSIONS These findings indicate that B-LFcin may have advantages as an adjunct therapy with both antimicrobial and anti-inflammatory properties in the treatment of corneal infection.

Differential injurious effects of ambient and traffic-derived particulate matter on airway epithelial cells

Exposure to airborne particulate matter (PM) may promote development of childhood asthma and trigger acute exacerbations of existing asthma via injury to airway epithelial cells (AEC).

The role of CXC chemokine receptor 2 in Pseudomonas aeruginosa corneal infection

Pseudomonas is one of the leading causes of contact lens‐related microbial keratitis. Despite the use of antibiotics, the host inflammatory response continues to cause damage to the cornea, which may lead to blindness. CXCR2‐binding chemokines have been implicated in the pathogenesis of Pseudomonas keratitis, and the exact role of this receptor remains to be elucidated. Corneas of CXCR2 knockout and wild‐type mice (Cmkar 2−/− and Cmkar 2+/+) were scratched, and 2 × 106 CFU/mL Pseudomonas 6294 or 6206 was added to corneas. Twenty‐four hours postinfection, mice were killed, and eyes were harvested for enumeration of bacteria, myeloperoxidase (MPO) levels, and inflammatory mediators. Cmkar 2−/− had 20‐ to 100‐fold more bacteria than Cmkar 2+/+ mice. There were no differences in MPO levels between gene knockout and Cmkar 2+/+ mice. Histology revealed PMN were restricted to the limbal area. Levels of CXCR2 chemokines (keratinocyte‐derived chemokine and MIP‐2) were elevated significantly in gene knockout mice. A lack of CXCR2 leads to an inability to control bacterial numbers as a result of the inability of PMN to reach the site of infection in the avascular cornea. These results imply that CXCR2 is critical to the extravasation of neutrophils into the avascular cornea.

Interleukin-33 Drives Activation of Alveolar Macrophages and Airway Inflammation in a Mouse Model of Acute Exacerbation of Chronic Asthma

We investigated the role of interleukin-33 (IL-33) in airway inflammation in an experimental model of an acute exacerbation of chronic asthma, which reproduces many of the features of the human disease. Systemically sensitized female BALB/c mice were challenged with a low mass concentration of aerosolized ovalbumin for 4 weeks to induce chronic asthmatic inflammation and then received a single moderate-level challenge to trigger acute airway inflammation simulating an asthmatic exacerbation. The inflammatory response and expression of cytokines and activation markers by alveolar macrophages (AM) were assessed, as was the effect of pretreatment with a neutralizing antibody to IL-33. Compared to chronically challenged mice, AM from an acute exacerbation exhibited significantly enhanced expression of markers of alternative activation, together with enhanced expression of proinflammatory cytokines and of cell surface proteins associated with antigen presentation. In parallel, there was markedly increased expression of both mRNA and immunoreactivity for IL-33 in the airways. Neutralization of IL-33 significantly decreased both airway inflammation and the expression of proinflammatory cytokines by AM. Collectively, these data indicate that in this model of an acute exacerbation of chronic asthma, IL-33 drives activation of AM and has an important role in the pathogenesis of airway inflammation.

Development of asthmatic inflammation in mice following early-life exposure to ambient environmental particulates and chronic allergen challenge

SUMMARY Childhood exposure to environmental particulates increases the risk of development of asthma. The underlying mechanisms might include oxidant injury to airway epithelial cells (AEC). We investigated the ability of ambient environmental particulates to contribute to sensitization via the airways, and thus to the pathogenesis of childhood asthma. To do so, we devised a novel model in which weanling BALB/c mice were exposed to both ambient particulate pollutants and ovalbumin for sensitization via the respiratory tract, followed by chronic inhalational challenge with a low mass concentration of the antigen. We also examined whether these particulates caused oxidant injury and activation of AEC in vitro. Furthermore, we assessed the potential benefit of minimizing oxidative stress to AEC through the period of sensitization and challenge by dietary intervention. We found that characteristic features of asthmatic inflammation developed only in animals that received particulates at the same time as respiratory sensitization, and were then chronically challenged with allergen. However, these animals did not develop airway hyper-responsiveness. Ambient particulates induced epithelial injury in vitro, with evidence of oxidative stress and production of both pro-inflammatory cytokines and Th2-promoting cytokines such as IL-33. Treatment of AEC with an antioxidant in vitro inhibited the pro-inflammatory cytokine response to these particulates. Ambient particulates also induced pro-inflammatory cytokine expression following administration to weanling mice. However, early-life dietary supplementation with antioxidants did not prevent the development of an asthmatic inflammatory response in animals that were exposed to particulates, sensitized and challenged. We conclude that injury to airway epithelium by ambient environmental particulates in early life is capable of promoting the development of an asthmatic inflammatory response in sensitized and antigen-challenged mice. These findings are likely to be relevant to the induction of childhood asthma.

Contribution of the cornea to cytokine levels in the whole eye induced during the early phase of Pseudomonas aeruginosa challenge

Pseudomonas aeruginosa keratitis is one of the most destructive diseases of the cornea. The host response to this infection is critical to the outcome, and is regulated by cytokines produced in the ocular tissue. In this study, we assessed the relative contribution of the cytokines produced in the cornea to the inflammatory response of the whole eye to gain a better understanding of the inflammatory and regulatory processes in the ocular environment during localized corneal infection. C57BL/6 mice were challenged by topical application of P. aeruginosa to wounded corneas. Corneas and whole eyes were harvested 24 h post‐challenge and bacterial numbers, myeloperoxidase levels and the levels of cytokines known to be important in keratitis were determined. The site of production of IL‐6 and KC in the retina was determined by in situ hybridization. Before infection, 90% of macrophage inflammatory protein (MIP)‐2 and approximately 80% of all IFN‐γ and IL‐10 produced constitutively in the eye was found outside the cornea. Twenty‐four hours after infection, bacterial numbers, levels of myeloperoxidase, and levels of MIP‐2 and IL‐1 were not different, whether measured in cornea or whole eye. However, expression of IL‐6, KC, IFN‐γ and IL‐10 was significantly greater in whole eyes than in the corneas of infected eyes. The cells expressing IL‐6 and KC in the retina were identified by in situ hybridization. This study indicates that during corneal inflammation, the response of the whole eye as well as the cornea needs to be considered.

Response of airway epithelial cells to double-stranded RNA in an allergic environment

BackgroundRespiratory viral infections are the most common trigger of acute exacerbations in patients with allergic asthma. The anti-viral response of airway epithelial cells (AEC) may be impaired in asthmatics, while cytokines produced by AEC may drive the inflammatory response. We investigated whether AEC cultured in the presence of Th2 cytokines associated with an allergic environment exhibited altered responses to double-stranded RNA, a virus-like stimulus.MethodsWe undertook preliminary studies using the MLE-12 cell line derived from mouse distal respiratory epithelial cells, then confirmed and extended our findings using low-passage human AEC. Cells were cultured in the absence or presence of the Th2 cytokines IL-4 and IL-13 for 48 hours, then stimulated with poly I:C for 4 hours. Expression of relevant anti-viral response and cytokine genes was assessed by quantitative real-time PCR. Secretion of cytokine proteins was assessed by immunoassay.ResultsFollowing stimulation with poly I:C, MLE-12 cells pre-treated with Th2 cytokines exhibited significantly higher levels of expression of mRNA for the cytokine genes Cxcl10 and Cxcl11, as well as a trend towards increased expression of Cxcl9 and Il6. Expression of anti-viral response genes was mostly unchanged, although Stat1, Ifit1 and Ifitm3 were significantly increased in Th2 cytokine pre-treated cells. Human AEC pre-treated with IL-4 and IL-13, then stimulated with poly I:C, similarly exhibited significantly higher expression of IL8, CXCL9, CXCL10, CXCL11 and CCL5 genes. In parallel, there was significantly increased secretion of CXCL8 and CCL5, as well as a trend towards increased secretion of CXCL10 and IL-6. Again, expression of anti-viral response genes was not decreased. Rather, there was significantly enhanced expression of mRNA for type III interferons, RNA helicases and other interferon-stimulated genes.ConclusionThe Th2 cytokine environment appears to promote increased production of pro-inflammatory chemokines by AEC in response to double-stranded RNA, which could help explain the exaggerated inflammatory response to respiratory viral infection in allergic asthmatics. However, any impairment of anti-viral host defences in asthmatics appears unlikely to be a consequence of Th2 cytokine-induced downregulation of the expression of viral response genes by AEC.

The corneal response to infection with Staphylococcus aureus in the absence of interleukin-4.

Interleukin‐4 (IL‐4) has previously been implicated in a protective response to Staphylococcus aureus corneal infection. Consequently, the specific role of IL‐4 during S. aureus corneal infection was investigated using IL‐4 gene knockout mice. The eyes of IL‐4−/− mice and wild‐type mice were challenged topically with S. aureus and examined at 24 h post‐infection. Keratitis was examined clinically and histologically. Bacterial and polymorphonuclear leucocytes (PMN) numbers were enumerated and cytokine and chemokine levels determined by enzyme‐linked immunosorbent assay. Exogenous IL‐4 was administered to both IL‐4−/− and wild‐type mice and clinical parameters were determined. A lack of IL‐4 resulted in a significant increase in clinical scores, pathology, bacterial load and neutrophil numbers. The absence of IL‐4 also resulted in an upregulation of interferon (IFN)‐γ and a downregulation of IL‐6, IL‐10 and the chemokines KC and macrophage inflammatory protein‐2. Administration of exogenous IL‐4 to IL‐4−/− mice was protective but time‐dependent. This study highlights the protective role of IL‐4 during S. aureus infection and emphasizes the balance between IL‐4 and IFN‐γ in achieving bacterial control and maintaining the integrity of the cornea. This information may lead to the development of novel therapeutic strategies potentially improving the prognosis for infection of this unique avascular site.

Effect of multipurpose solutions on cell morphology and cytokine production by corneal epithelial cells.

Purpose. We sought to determine whether human corneal limbal epithelial cells (HCLE) differ in their physiological response and production of inflammatory mediators during exposure to two multipurpose disinfecting solutions that differ in only four excipients. Methods. HCLE were exposed to dilutions (1%–20%) of OPTI-FREE Express and OPTI-FREE RepleniSH for 2, 6, or 18 h. Cell numbers were measured using CyQuant and metabolic activity assays. Morphology, viability, and apoptotic changes were examined by confocal microscopy after staining with Calcein AM/propidium iodide or Hoechst 33,342/propidiun iodide/YO-PRO-1. Cytokine responses and arachidonic acid metabolites were examined by enzyme-linked immunosorbent assay. Results. OPTI-FREE Express showed greater reductions in corneal cell metabolic activity (up to 3-fold) than OPTI-FREE RepleniSH over 18 h. Cells exposed to OPTI-FREE Express were highly vacuolated, whereas those exposed to OPTI-FREE RepleniSH had morphology consistent with a presumed apoptotic response. OPTI-FREE Express elicited higher levels of interleukin (IL)-6 (maximum 4225 ± 300 pg/mL) and IL-8 (maximum 1094 ± 250 pg/mL) from cells than OPTI-FREE RepleniSH (maximum of 1717 ± 225 pg/mL and 930 ± 300 pg/mL, respectively) at all concentrations tested after 18 h. HCLE did not produce leukotriene B4 or prostaglandin E2 on stimulation, and IL-1&bgr; was produced in low levels, but its production was not different between multipurpose disinfecting solution types. Conclusions. The findings reported here are the first to describe the pattern of cytokine production produced in corneal epithelial cells in response to contact lens solutions. Alteration of only four excipients in the formulations had such effects on corneal epithelia; however, these differences do not explain differences in the incidences of corneal infiltrative events between these solutions. This might indicate that changes in the rates of infiltrative events result from interactions of the solutions with the contact lens surface, or directly with the corneal immune system, or as the result of gram-negative contamination of lens cases.

Anti-Inflammatory and Anti-Remodelling Effects of ISU201, a Modified Form of the Extracellular Domain of Human BST2, in Experimental Models of Asthma: Association with Inhibition of Histone Acetylation

There are few alternatives to glucocorticosteroids for treatment of asthma. We assessed the activity of a novel protein drug designated ISU201, the extracellular domain of the human cell surface protein BST2, stabilised by fusion with the Fc region of IgG, in mouse models of mild chronic asthma and an acute exacerbation of asthma. The ability of ISU201 to suppress airway inflammation and remodelling was compared with that of dexamethasone. Female BALB/c mice were systemically sensitised with ovalbumin, then received controlled low-level challenge with aerosolised ovalbumin for 6 weeks, which induced lesions of mild chronic asthma, and were treated with drugs during the final 2 weeks. Alternatively, sensitised mice received 4 weeks of chronic low-level challenge and were treated 24 and 2 hours before a final single moderate-level challenge, which triggered acute airway inflammation simulating an asthmatic exacerbation. Inflammation and remodelling were quantified, as was the expression of pro-inflammatory cytokines in bronchoalveolar lavage fluid and tissues. To identify cellular targets of ISU201, we assessed the effects of the drug on activated lymphocytes, macrophages and airway epithelial cells. In the model of mild chronic asthma, ISU201 was as effective as dexamethasone in suppressing airway inflammation and most changes of remodelling. In the model of an allergen-induced acute exacerbation of chronic asthma, ISU201 was also an effective anti-inflammatory agent, although it was less active than dexamethasone. The drug acted on multiple cellular targets, suppressing production of pro-inflammatory cytokines by lymphocytes and macrophages. ISU201 significantly reduced acetylation of histone H4 in airway epithelial cells, suggesting at least one potential mechanism of action. We conclude that in these models of asthma, ISU201 is a broad-spectrum inhibitor of both airway inflammation and remodelling. Thus, unlike drugs which target specific mediators, it could potentially be an alternative or an adjunct to glucocorticoids for the treatment of asthma.