an-il-8-transiently-transgenized-mouse-model-for-vivo-long-term

An IL-8 Transiently Transgenized Mouse Model for the <em>In Vivo</em> Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

University of Verona

Anti-inflammatory properties of azithromycin (AZM) have been proposed as possible mechanisms of clinical beneficial effects in patients with cystic fibrosis (CF). Altered glutathione (GSH) transport in cystic fibrosis transmembrane regulator protein (CFTR)-deficient cells leads to the occurrence of oxidative stress that finally induces glutathione S-transferase (GST) activity. The present investigation was aimed to verify the effects of AZM on GST activity and expression in CF airway cells in vitro and in vivo. AZM exposure significantly decreased GSTT1 and GSTM1 mRNA and protein expression in IB3-1, restoring the levels to those observed in non-CF C38 cells, which also express lower levels of gamma-glutamyltransferase (GGT) activity than IB3-1. In another CF cell line, 2CFSMEo-, AZM produced 45% reduction in GSTT1 and GSTM1 mRNA levels. AZM reduced GST activity by approximately 25% and 40% in IB3-1 and 2CFSMEo- cells, respectively. GSTP1 was similarly expressed in all CF and non-CF cells and was unaffected by AZM. The anti-inflammatory cytokine IL-10 down-modulated GST activity at similar levels, supporting a link between GST inhibition and anti-inflammatory properties of AZM. In bronchoalveolar lavage fluid of CF mice homozygous for the F508 del mutation, GSTM1 protein levels were undetectable after AZM treatment. The association between increased GST expression and activity, together with its reversal by AZM treatment in vitro and in vivo, suggest novel antioxidant properties for this drug. The issue whether decreased GST activity may directly concur to anti-inflammatory properties of AZM or is rather a marker of the oxidative status of CF cells will require additional studies.

Effects of azithromycin on glutathione S-transferases in cystic fibrosis airway cells.

Protein Tyrosine Phosphatase gamma (PTPgamma) is a receptor-like transmembrane protein belonging to the family of classical protein tyrosine phosphatases. PTPgamma is known to regulate haematopoietic differentiation in a murine embryonic stem cells model. We have recently demonstrated that PTPgamma mRNA is expressed in monocytes, tissue-localized myeloid dendritic cells and in both myeloid and plasmacytoid dendritic cells in peripheral blood. We now developed a PTPgamma specific antibody that recognizes the protein by flow cytometry. PTPgamma expression was detected in monocytes and both myeloid and plasmacytoid dendritic cells, while PMN showed a low but consistent staining in contrast with previous mRNA data. B cells were found to express the phosphatase while T cells were negative. In keeping with RNA data, PTPgamma was detected in monocyte-derived dendritic cells and its expression rose upon LPS stimulation. Finally, we discovered that CD34(+) haematopoietic precursors express high PTPgamma level that drops during in vitro expansion induced by IL-3 and SCF growth factors. We therefore propose PTPgamma as a new functionally regulated leukocyte marker whose role in normal and pathological context deserve further investigation.

Protein Tyrosine Phosphatase Gamma (PTPgamma) is a Novel Leukocyte Marker Highly Expressed by CD34 Precursors.

Pseudomonas aeruginosa (Pa) is the most common virulent pathogen contributing to the pathogenesis of cystic fibrosis (CF). During bacterial lung colonization, the products of its metabolism are released in the extracellular space contributing to the pathogenic events associated with its presence. To gain insights on the mechanisms involved in the Pa pathogenesis we focused our attention on proteins released by Pa using a MudPIT approach combined with cell biology assays. Conditioned medium (CM) collected under aerobic and microaerobic conditions from Pa clinical strains (in early and late colonization), unlike the laboratory strain, induced expression of IL-8 mRNA in CF airway epithelial cells. We have identified proteins released by clinically relevant Pa strains, focusing on the pro-inflammatory effects as metalloproteases (MMPs). In fact, their expression pattern was associated with the highest pro-inflammatory activity measured in the early clinically isolated strain. The relation was further supported by the result of the analysis of a larger and independent set of Pa isolates derived from sporadically and chronically infected CF patients: 76% of sporadic samples expressed protease activity (n = 44), while only 27% scored positive in the chronically infected individuals (n = 38, p < 0.0001, Fisher's exact test). Finally, looking for a possible mechanism of action of bacterial MMPs, we found that CM from early clinical isolates can cleave CXCR1 on the surface of human neutrophils, suggesting a potential role for the bacterially released MMPs in the protection of the pathogen from the host's response.

MudPIT analysis of released proteins in Pseudomonas aeruginosa laboratory and clinical strains in relation to pro-inflammatory effects.

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by a chronic neutrophilic airways inflammation, increasing levels of oxidative stress and reduced levels of antioxidants such as glutathione (GSH). Gamma-glutamyltransferase (GGT), an enzyme induced by oxidative stress and involved in the catabolism of GSH and its derivatives, is increased in the airways of CF patients with inflammation, but the possible implications of its increase have not yet been investigated in detail.

Contribution by polymorphonucleate granulocytes to elevated gamma-glutamyltransferase in cystic fibrosis sputum.

S-nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role for GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8; however, conflicting results have been reported. γ-Glutamyltransferase (GGT) enzyme activity can hydrolyze the γ-glutamyl bond present in the GSNO molecule thus greatly accelerating the release of bioactive nitric oxide. Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvesicles. This study was aimed at evaluating the effect of GSNO catabolism mediated by GGT on production of IL-8 in CF transmembrane regulation protein-mutated IB3-1 bronchial cells. The rapid, GGT-catalyzed catabolism of GSNO caused a decrease in both basal and lipopolysaccharide-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease in cell proliferation. In contrast, a slow decomposition of GSNO produced a significant increase in both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA. Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.

γ-Glutamyltransferase catabolism of S-nitrosoglutathione modulates IL-8 expression in cystic fibrosis bronchial epithelial cells.

Glutathione inhalation treatments in cystic fibrosis: the interference of airway γ-glutamyltransferase.

Chronic inflammation of the airways is a central component in lung diseases and is frequently associated with bacterial infections. Monitoring the pro-inflammatory capability of bacterial virulence factors in vivo is challenging and usually requires invasive methods.

In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin.

Deregulated immune response fails to control biofilm-forming bacteria, as Pseudomonas aeruginosa, in the lungs of cystic fibrosis (CF) patients. HLA-G is an immune-modulatory molecule involved in respiratory diseases and infections.

HLA-G expression and regulation during Pseudomonas aeruginosa infection in cystic fibrosis patients.

Pseudomonas aeruginosa is the predominant pathogen associated with the decline of pulmonary function in cystic fibrosis (CF) patients. Both environment-to-host acquisition and patient-to-patient transmission have been described for P. aeruginosa infection. Epidemic clones and bacterial phenotypic adaptation to the CF lung have been recognised as independent risk factors for disease progression. So far, there is no established link between genotypic prevalence and phenotypic traits. Here, we look at the major CF patient cohort in Italy to identify shared P. aeruginosa clones and associated common phenotypic traits.

Genotypic and phenotypic relatedness of Pseudomonas aeruginosa isolates among the major cystic fibrosis patient cohort in Italy.

When cystic fibrosis (CF) is suspected Nasal Potential Difference (NPD) measurements are proposed to support controversial diagnosis: we investigated appropriate outcomes at the CF Centre of Verona.

Nasal potential difference outcomes support diagnostic decisions in cystic fibrosis.

Experimentally, lung inflammation in laboratory animals is usually detected by the presence of inflammatory markers, such as immune cells and cytokines, in the bronchoalveolar lavage fluid (BALF) of sacrificed animals. This method, although extensively used, is time, money and animal life consuming, especially when applied to genetically modified animals. Thus a new and more convenient approach, based on in vivo imaging analysis, has been set up to evaluate the inflammatory response in the lung of CFTR-deficient (CF) mice, a murine model of cystic fibrosis.

In vivo monitoring of lung inflammation in CFTR-deficient mice.

Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Azithromycin improves the related clinical outcomes, but its mechanisms of action remain poorly understood. We tested the hypothesis that azithromycin downregulates P. aeruginosa-induced pro-inflammatory responses by modifying release of bacterial proteins.

Article	Year
Effects of azithromycin on glutathione S-transferases in cystic fibrosis airway cells. American journal of respiratory cell and molecular biology\| PubMed ID: 19097986	2009
Protein Tyrosine Phosphatase Gamma (PTPgamma) is a Novel Leukocyte Marker Highly Expressed by CD34 Precursors. Biomarker insights\| PubMed ID: 19662205	2007
MudPIT analysis of released proteins in Pseudomonas aeruginosa laboratory and clinical strains in relation to pro-inflammatory effects. Integrative biology : quantitative biosciences from nano to macro\| PubMed ID: 22298109	2012
Contribution by polymorphonucleate granulocytes to elevated gamma-glutamyltransferase in cystic fibrosis sputum. PloS one\| PubMed ID: 22496859	2012
γ-Glutamyltransferase catabolism of S-nitrosoglutathione modulates IL-8 expression in cystic fibrosis bronchial epithelial cells. Free radical biology & medicine\| PubMed ID: 23820266	2013
Glutathione inhalation treatments in cystic fibrosis: the interference of airway γ-glutamyltransferase. American journal of respiratory and critical care medicine\| PubMed ID: 24428657	2014
In vivo imaging of the lung inflammatory response to Pseudomonas aeruginosa and its modulation by azithromycin. Journal of translational medicine\| PubMed ID: 26239109	2015
HLA-G expression and regulation during Pseudomonas aeruginosa infection in cystic fibrosis patients. Future microbiology\| PubMed ID: 26934639	2016
Genotypic and phenotypic relatedness of Pseudomonas aeruginosa isolates among the major cystic fibrosis patient cohort in Italy. BMC microbiology\| PubMed ID: 27400750	2016
Nasal potential difference outcomes support diagnostic decisions in cystic fibrosis. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 27423539	2016
In vivo monitoring of lung inflammation in CFTR-deficient mice. Journal of translational medicine\| PubMed ID: 27468800	2016
Azithromycin Attenuates Pseudomonas-Induced Lung Inflammation by Targeting Bacterial Proteins Secreted in the Cultured Medium. Frontiers in immunology\| PubMed ID: 27895643	2016

Gabriella Bergamini

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