Francesca Ruscitti

Azithromycin inhibits nuclear factor‐κB activation during lung inflammation: an in vivo imaging study

We studied in vivo the potential involvement of nuclear factor‐κB (NF‐κB) pathway in the molecular mechanism of the anti‐inflammatory and immunomodulatory activity of azithromycin in the lung. Mice transiently transfected with the luciferase gene under the control of a NF‐κB responsive element were used to assess in vivo NF‐κB activation by bioluminescence imaging. Bioluminescence as well as inflammatory cells and concentrations of proinflammatory cytokines in bronchoalveolar lavage fluids, were monitored in an acute model of pulmonary inflammation resulting from intratracheal instillation of lipopolysaccharide. Lipopolysaccharide (LPS) instillation induced a marked increase in lung bioluminescence in mice transiently transfected with the luciferase gene under the control of an NF‐κB responsive element, with significant luciferase expression in resident cells such as endothelial and epithelial cells, as assessed by duoplex immunofluorescence staining. Activation of NF‐κB and inflammatory cell lung infiltration linearly correlated when different doses of bortezomib were used to inhibit NF‐κB activation. Pretreatment with azithromycin significantly decreased lung bioluminescence and airways cell infiltration induced by LPS, also reducing proinflammatory cytokines concentrations in bronchoalveolar lavages and inhibiting NF‐κB nuclear translocation. The results obtained using a novel approach to monitor NF‐κB activation, provided, for the first time, in vivo evidence that azithromycin treatment results in pulmonary anti‐inflammatory activity associated with the inhibition of NF‐κB activation in the lung.

Assessment of asthmatic inflammation using hybrid fluorescence molecular tomography–x-ray computed tomography

Abstract. Nuclear imaging plays a critical role in asthma research but is limited in its readings of biology due to the short-lived signals of radio-isotopes. We employed hybrid fluorescence molecular tomography (FMT) and x-ray computed tomography (XCT) for the assessment of asthmatic inflammation based on resolving cathepsin activity and matrix metalloproteinase activity in dust mite, ragweed, and Aspergillus species-challenged mice. The reconstructed multimodal fluorescence distribution showed good correspondence with ex vivo cryosection images and histological images, confirming FMT-XCT as an interesting alternative for asthma research.

Enlightened Mannhemia haemolytica lung inflammation in bovinized mice

Polymorphonuclear cells diapedesis has an important contribution to the induced Mannhemia haemolytica (M. haemolytica) infection lung inflammation and IL-8 is the primary polymorphonuclear chemoattractant. Using a bovine IL-8/luciferase transiently transgenized mouse model, the orchestration among M. haemolytica, IL-8 promoter activation and neutrophilia was followed in real time by in vivo image analysis.

Heterologous Matrix Metalloproteinase Gene Promoter Activity Allows In Vivo Real-time Imaging of Bleomycin-Induced Lung Fibrosis in Transiently Transgenized Mice

Idiopathic pulmonary fibrosis is a very common interstitial lung disease derived from chronic inflammatory insults, characterized by massive scar tissue deposition that causes the progressive loss of lung function and subsequent death for respiratory failure. Bleomycin is used as the standard agent to induce experimental pulmonary fibrosis in animal models for the study of its pathogenesis. However, to visualize the establishment of lung fibrosis after treatment, the animal sacrifice is necessary. Thus, the aim of this study was to avoid this limitation by using an innovative approach based on a double bleomycin treatment protocol, along with the in vivo images analysis of bleomycin-treated mice. A reporter gene construct, containing the luciferase open reading frame under the matrix metalloproteinase-1 promoter control region, was tested on double bleomycin-treated mice to investigate, in real time, the correlation between bleomycin treatment, inflammation, tissue remodeling and fibrosis. Bioluminescence emitted by the lungs of bleomycin-treated mice, corroborated by fluorescent molecular tomography, successfully allowed real time monitoring of fibrosis establishment. The reporter gene technology experienced in this work could represent an advanced functional approach for real time non-invasive assessment of disease evolution during therapy, in a reliable and translational living animal model.

A multimodal imaging approach based on micro-CT and fluorescence molecular tomography for longitudinal assessment of bleomycin-induced lung fibrosis in mice

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the progressive and irreversible destruction of lung architecture, which causes significant deterioration in lung function and subsequent death from respiratory failure. The pathogenesis of IPF in experimental animal models has been induced by bleomycin administration. In this study, we investigate an IPF-like mouse model induced by a double intratracheal bleomycin instillation. Standard histological assessments used for studying lung fibrosis are invasive terminal procedures. The goal of this work is to monitor lung fibrosis through noninvasive imaging techniques such as Fluorescent Molecular Tomography (FMT) and Micro-CT. These two technologies validated with histology findings could represent a revolutionary functional approach for real time non-invasive monitoring of IPF disease severity and progression. The fusion of different approaches represents a step further for understanding the IPF disease, where the molecular events occurring in a pathological condition can be observed with FMT and the subsequent anatomical changes can be monitored by Micro-CT.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo 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 pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

CHF 6001 inhibits nuclear translocation of NF-κB in LPS-induced lung inflammation in mice

Introduction: CHF 6001 is a phosphodiesterase 4 (PDE4) inhibitor optimized for inhaled delivery and tolerability currently in phase IIb clinical trials for the treatment of chronic obstructive pulmonary disease (COPD). Aim of the study: Mice transiently transfected with the luciferase gene under the control of NF-κB responsive element were used to assess the in vivo activity of CHF 6001 against lipolysaccharide (LPS)-induced lung inflammation. Methods: CHF 6001 was administered as micronized dry powder by a nose-only inhalation system at 100-600 µg/kg. NF-κB activation was quantified by in vivo bioluminescence imaging (BLI) at 4 hours after LPS challenge. Bronchoalveolar lavage fluid (BALF) was collected 24 hours after challenge and cell numbers and cytokine levels were quantified. Results: CHF 6001 dose-dependently inhibited NF-κB nuclear translocation, achieving 59% ( p ) and 69% ( p ) inhibition at 100 and 600 µg/kg, respectively. CHF 6001 was able to dose-dependently and significantly inhibit BALF neutrophil influx and pro-inflammatory cytokine (TNF-a, MCP-1, IFN-γ, IL-17, G-CSF and Rantes) production induced by LPS. Conclusions: Topically administered CHF 6001 in mice significantly attenuates the NF-κB-activity after its LPS-driven induction.

WS10.3 Monitoring the pro-inflammatory effect of Pseudomonas aeruginosa culture supernatants and the inhibitory effect of azithromycin by in vivo imaging in IL-8 transiently transgenized mice

Objectives Airway inflammation is frequently associated with chronic bacterial infections. The IL-8 mediated lung inflammation induced by virulence factors, specifically metalloproteases (MP), secreted by Pseudomonas aeruginosa has been monitored in vivo in a mouse model transiently expressing the luciferase reporter gene under the control of an IL-8 bovine promoter. The model was also used to test the possible anti-inflammatory activity of AZMthromycin (AZM) mediated by its effect on Pseudomonas MP expression. Methods Culture supernatants (SNs) from two P. aeruginosa clinical strains, VR1 and VR2 respectively, were obtained after growth in the absence or presence of a sub-lethal dose of AZM. The MP activity was detectable in the SN from VR1 grown without AZM but was greatly reduced when cells grew with the antibiotic. Results The pro-inflammatory activity of VR1 SN was clearly visible in vivo in the transgenized mice while a significant decrease of the inflammation response was observed with the SN grown in the presence of AZM. Further, VR1 SN stimulated the recruitment of WBC and neutrophils and the expression of a number of cytokines. This later effect was clearly decreased when SN from VR1 grown with AZM was used. Contrarily, SN from VR2 showed neither protease activity or detectable inflammation in the mouse lung. Conclusion The present animal model has revealed useful to the in vivo, long term monitoring of the effect of bacterial virulence factors in the lung tissue and the possible beneficial, anti-inflammatory effect of molecules for therapeutic use. This study was supported by Lega Italiana Fibrosi Cistica and Italian CF Research Foundation (grant #18/2013).