Bacterial keratitis: Photodynamic inactivation reduced experimental inflammation.

The successful treatment of bacterial keratitis remains an unsolved clinical problem. The current study aimed to establish a murine keratitis model and to investigate the effect of chlorin e6 (Ce6) and photodynamic inactivation (PDI) on corneal inflammation. The cornea of anesthetized mice was scratched and covered with a bacterial suspension of Pseudomonas aeruginosa. A paste containing Ce6 was applied to the cornea with subsequent exposure to specified light. Two days later the animals were sacrificed, and the globes were processed for light microscopy. Evaluation parameters were the maximal corneal thickness and the severity of the hypopyon. The maximal corneal thickness of 290±16 µm in the infected and untreated group was significantly reduced to 220±8 µm in the infected and treated group (P<0.05). In addition, the hypopyon was less severe in the infected and treated group. In conclusion, the present study indicates that PDI using Ce6 may be a potential approach to treat patients suffering with severe bacterial keratitis.

Aspherical, Nanostructured Microparticles for Targeted Gene Delivery to Alveolar Macrophages.

Introducing novel shapes to particulate carrier systems adds unique features to modern drug and gene delivery. Depending on the route of administration, particle geometry can influence deposition and fate within biological environments. In this work, a template-assisted engineering technique is applied, providing full control of size and shape in the preparation of aspherical, nanostructured microparticles. Based on the interconnection of nanoparticles, stabilized by a functional layer-by-layer (LbL) coating, the resulting cylindrical micrometer architecture is especially qualified for pulmonary delivery. Designed as gene delivery system, plasmid-DNA (pCMV-luciferase) and branched polyethylenimine are used to reach both structural integrity of the carrier system and delivery of genes into the cells of interest. Due to their size, particles are exclusively taken up by phagocytes, which also adds a targeting effect to the introduced system. The luciferase expression is demonstrated in macrophages showing increasing levels over a time period of at least 7 d. Furthermore, it is shown for the first time that the expression is depending on the LbL design. From in vivo experiments, corresponding luciferase expression is observed in mice alveolar macrophages. Combining site specific transport with the possibility of genetically engineering immunocompetent phagocytes, the presented system offers promising potential to improve applications for cell-based immunotherapy.

Probenecid-treatment reduces demyelination induced by cuprizone feeding.

Recent experiments showed that a pannexin-1 inhibitor, probenecid, reduced clinical symptoms in the murine experimental autoimmune encephalomyelitis when applied during the initial phase of neuronal inflammation. An inflammatory component is also present in a toxically induced inflammation and demyelination using cuprizone diet. Probenecid is a pannexin-1 antagonist and a probenecid therapy was investigated. Mice were fed for 10days with a cuprizone diet. In the following, the diet was continued but combined with a daily injection of a low dose of probenecid or solvent for 10days. Electron microscopy revealed demyelination in the optic nerve. The demyelination as measured by the axonal diameter was significantly reduced in the animals treated with 100mg per kg body weight probenecid. In comparison to controls, the number of leukocytes and lymphocytes in the peripheral blood was reduced in all cuprizone groups including the treatment group. In conclusion, early demyelination in the optic nerve was moderately reduced by 10days treatment with a low dose probenecid. This is a hint for the involvement of pannexin-1 modulated inflammation in cuprizone feeding induced toxic demyelination. Thus, probenecid is a candidate for the treatment of neuro-inflammation and multiple sclerosis.

CcpA Affects Infectivity of Staphylococcus aureus in a Hyperglycemic Environment.

Many bacteria regulate the expression of virulence factors via carbon catabolite responsive elements. In Gram-positive bacteria, the predominant mediator of carbon catabolite repression is the catabolite control protein A (CcpA). Hyperglycemia is a widespread disorder that predisposes individuals to an array of symptoms and an increased risk of infections. In hyperglycemic individuals, the bacterium Staphylococcus aureus causes serious, life-threatening infections. The importance of CcpA in regulating carbon catabolite repression in S. aureus suggests it may be important for infections in hyperglycemic individuals. To test this suggestion, hyperglycemic non-obese diabetic (NOD; blood glucose level ≥20 mM) mice were challenged with the mouse pathogenic S. aureus strain Newman and the isogenic ccpA deletion mutant (MST14), and the effects on infectivity were determined. Diabetic NOD mice challenged with the ccpA deletion mutant enhanced the symptoms of infection in an acute murine pneumonia model relative to the parental strain. Interestingly, when diabetic NOD mice were used in footpad or catheter infection models, infectivity of the ccpA mutant decreased relative to the parental strain. These differences greatly diminished when normoglycemic NOD mice (blood glucose level ≤ 10 mM) were used. These data suggest that CcpA is important for infectivity of S. aureus in hyperglycemic individuals.

IL-17A-mediated expression of epithelial IL-17C promotes inflammation during acute Pseudomonas aeruginosa pneumonia.

Lung epithelial cells are suggested to promote pathogen-induced pulmonary inflammation by the release of chemokines, resulting in enhanced recruitment of circulating leukocytes. Recent studies have shown that the interleukin-17C (IL-17C) regulates innate immune functions of epithelial cells in an autocrine manner. The aim of this study was to investigate the contribution of IL-17C to pulmonary inflammation in a mouse model of acute Pseudomonas aeruginosa pneumonia. Infection with P. aeruginosa resulted in an increased expression of IL-17C in lung tissue of wild-type mice. Numbers of neutrophils and the expression of the neutrophil-recruiting chemokines keratinocyte-derived chemokine and macrophage inflammatory protein 2 were significantly decreased in lungs of IL-17C-deficient (IL-17C-/-) mice infected with P. aeruginosa at 24 h. Systemic concentrations of interleukin-6 (IL-6) were significantly decreased in infected IL-17C-/- mice at 24 h and the survival of IL-17C-/- mice was significantly increased at 48 h. The expression of IL-17C was reduced in infected mice deficient for interleukin-17A (IL-17A), whereas pulmonary concentrations of IL-17A were not affected by the deficiency for IL-17C. Stimulation of primary alveolar epithelial cells with IL-17A resulted in a significantly increased expression of IL-17C in vitro. Our data suggest that IL-17A-mediated expression of epithelial IL-17C amplifies the release of chemokines by epithelial cells and thereby contributes to the recruitment of neutrophils and systemic inflammation during acute P. aeruginosa pneumonia.

IL-17A attracts inflammatory cells in murine lung infection with P. aeruginosa.

IL-17A-dependent immunity is of importance in the protection against extracellular bacterial pathogens. However, IL-17A is also suggested to mediate the pathogenesis of lung diseases, such as acute respiratory distress syndrome. Here, we studied the role of IL-17A in a mouse model of acute pneumonia. IL-17A mediated the expression of keratinocyte-derived chemokine (KC) and the recruitment of inflammatory cells in mice infected with a sub-lethal dose of Pseudomonas aeruginosa. IL-17A deficiency protected mice from lethal P. aeruginosa lung infection. A sub-lethal infection with Streptococcus pneumoniae resulted in increased bacterial burden associated with increased pulmonary inflammation. Thus, the type of infectious bacteria seemed to influence the way in which IL-17A functions during pulmonary infection. Reducing pulmonary inflammation by targeting IL-17A may be a therapeutic option in acute P. aeruginosa pneumonia.

A calcium-redox feedback loop controls human monocyte immune responses: The role of ORAI Ca2+ channels.

In phagocytes, pathogen recognition is followed by Ca(2+) mobilization and NADPH oxidase 2 (NOX2)-mediated "oxidative burst," which involves the rapid production of large amounts of reactive oxygen species (ROS). We showed that ORAI Ca(2+) channels control store-operated Ca(2+) entry, ROS production, and bacterial killing in primary human monocytes. ROS inactivate ORAI channels that lack an ORAI3 subunit. Staphylococcal infection of mice reduced the expression of the gene encoding the redox-sensitive Orai1 and increased the expression of the gene encoding the redox-insensitive Orai3 in the lungs or in bronchoalveolar lavages. A similar switch from ORAI1 to ORAI3 occurred in primary human monocytes exposed to bacterial peptides in culture. These alterations in ORAI1 and ORAI3 abundance shifted the channel assembly toward a more redox-insensitive configuration. Accordingly, silencing ORAI3 increased the redox sensitivity of the channel and enhanced oxidation-induced inhibition of NOX2. We generated a mathematical model that predicted additional features of the Ca(2+)-redox interplay. Our results identified the ORAI-NOX2 feedback loop as a determinant of monocyte immune responses.

Il-17A contributes to maintenance of pulmonary homeostasis in a murine model of cigarette smoke-induced emphysema.

Smoking is the main risk factor for the development of the chronic obstructive pulmonary disease (COPD) in Western countries. Recent studies suggest that IL-17A and Th17 cells play a role in the pathogenesis of COPD. We used a murine model of chronic cigarette smoke (CS) exposure to explore the contribution of IL-17A to CS-induced lung damage and loss of pulmonary function. Histology and morphometry showed that IL-17A deficiency spontaneously resulted in a loss of lung structure under basal conditions. Even though inflammatory markers [IL-1ß and granulocyte colony-stimulating factor (G-CSF)] were decreased in IL-17A-deficient mice (IL-17A(-/-)) exposed to CS compared with wild-type (WT) mice, IL-17A(-/-) mice were per se not protected from CS-induced emphysematous disease. Assessment of pulmonary function showed that IL-17A(-/-) mice were partially protected from CS-induced changes in total lung capacity. However, the respiratory elastance decreased and respiratory compliance increased in IL-17A(-/-) mice after exposure to CS. Morphometry revealed destruction of lung tissue in CS-exposed IL-17A(-/-) mice similar to WT mice. The expression of elastin was decreased in air-exposed IL-17A(-/-) mice and in CS-exposed WT and IL-17A(-/-) mice. Thus, in the present model of sterile CS-exposure, IL-17A contributes to normal lung homeostasis and does not mediate CS-induced loss of lung structure and pulmonary function.

Cigarette smoke-promoted acquisition of bacterial pathogens in the upper respiratory tract leads to enhanced inflammation in mice.

BACKGROUND: Bacterial colonization and recurrent infections of the respiratory tract contribute to the progression of chronic obstructive pulmonary disease (COPD). There is evidence that exacerbations of COPD are provoked by new bacterial strains acquired from the environment. Using a murine model of colonization, we examined whether chronic exposure to cigarette smoke (CS) promotes nasopharyngeal colonization with typical lung pathogens and whether colonization is linked to inflammation in the respiratory tract. METHODS: C57BL/6 N mice were chronically exposed to CS. The upper airways of mice were colonized with nontypeable Haemophilus influenzae (NTHi) or Streptococcus pneumoniae. Bacterial colonization was determined in the upper respiratory tract and lung tissue. Inflammatory cells and cytokines were determined in lavage fluids. RT-PCR was performed for inflammatory mediators. RESULTS: Chronic CS exposure resulted in significantly increased numbers of viable NTHi in the upper airways, whereas NTHi only marginally colonized air-exposed mice. Colonization with S. pneumoniae was enhanced in the upper respiratory tract of CS-exposed mice and was accompanied by increased translocation of S. pneumoniae into the lung. Bacterial colonization levels were associated with increased concentrations of inflammatory mediators and the number of immune cells in lavage fluids of the upper respiratory tract and the lung. Phagocytosis activity was reduced in whole blood granulocytes and monocytes of CS-exposed mice. CONCLUSIONS: These findings demonstrate that exposure to CS impacts the ability of the host to control bacterial colonization of the upper airways, resulting in enhanced inflammation and susceptibility of the host to pathogens migrating into the lung.

Probenecid reduces infection and inflammation in acute Pseudomonas aeruginosa pneumonia.

The activation of inflammasome signaling mediates pathology of acute Pseudomonas aeruginosa pneumonia. This suggests that the inflammasome might represent a target to limit the pathological consequences of acute P. aeruginosa lung infection. Pannexin-1 (Px1) channels mediate the activation of caspase-1 and release of IL-1ß induced by P2X7 receptor activation. The approved drug probenecid is an inhibitor of Px1 and ATP release. In this study, we demonstrate that probenecid reduces infection and inflammation in acute P. aeruginosa pneumonia. Treatment of mice prior to infection with P. aeruginosa resulted in an enhanced clearance of P. aeruginosa and reduced levels of inflammatory mediators, such as IL-1ß. In addition, probenecid inhibited the release of inflammatory mediators in murine alveolar macrophages and human U937 cell-derived macrophages upon bacterial infection but not in human bronchial epithelial cells. Thus, Px1 blockade via probenecid treatment may be a therapeutic option in P. aeruginosa pneumonia by improving bacterial clearance and reducing negative consequences of inflammation.

IL-17C is a mediator of respiratory epithelial innate immune response.

The IL-17 family of cytokines consists of at least six members (IL-17A to -F). IL-17 directly activates epithelial cells leading to the expression of inflammatory mediators and antimicrobial factors. Recent studies showed that IL-17C is expressed by epithelial cells. It was the purpose of this study to examine the expression of IL-17 family members in respiratory epithelial cells during bacterial infection. We show that common bacterial pathogens, such as Pseudomonas aeruginosa and Haemophilus influenzae, and ligands of Toll-like receptors 3 and 5 (flagellin, polyI:C) induced the expression and release of IL-17C in cultured human bronchial epithelial cells (HBECs). The expression of IL-17A, -B, -D, or -E was not induced by bacterial stimuli in HBECs. IL-17C enhanced inflammatory responses of respiratory epithelial cells infected with P. aeruginosa. Furthermore, we demonstrate that cigarette smoke suppressed the expression of IL-17C in HBECs in response to bacterial infection and in vivo in the upper airways of mice colonized with H. influenzae. IL-17C could also be detected in bronchial tissue of subjects with infection-related lung diseases. These data show that IL-17C is involved in the innate immune response of respiratory epithelial cells and is suppressed by cigarette smoke.