Medical College of Georgia :: Vascular Biology Center :: Biography

Introduction

Streptococcus pneumoniae, a commensal of the human nasopharynx, is a major cause of community-acquired pneumonia, leading in a multitude of cases to pneumococcal sepsis. Infections with S. pneumoniae have accounted historically for more morbidity and mortality than any other bacterium. Moreover, the increasing prevalence of antibiotic resistance necessitates the development of novel therapeutic strategies to combat pneumonia. Acute lung injury (ALI) and acute respiratory distress syndrome ARDS) are sentinel features of the septic lung failure in patients with pneumogenic sepsis. Bacterial virulence factors, such as pneumolysin (PLY) secreted by S. pneumoniae, contribute to the transition from pneumonia to ALI to sepsis and finally to death. Apart from infections with S. pneumoniae, also Listeria monocytogenes infections, which mostly start as an oral infection caused by contaminated food, can cause severe lung complications in individuals having pre-disposing conditions, such as age, pregnancy, cancer, chronic diseases or organ transplantation. In this case, Listeriolysin O (LLO), a cholesterol-dependent cytolysin with homology to PLY and Streptolysin O, is the main virulence factor and can cause permeability edema. LLO directly increases epithelial and endothelial permeability and, like PLY, induces a strong inflammatory response, which can cause a dysfunction in ion channels implicated in alveolar fluid clearance. In view of their crucial role in bacterial virulence and their profound effects on the immune system of the host, LLO and PLY can thus be considered as model toxins for G+ infection-associated acute lung injury and permeability edema, which still result in high mortality.

1. Inhibitory activity of the TIP peptide in exotoxin-induced endothelial hyperpermeability.

In this project, we propose to further unravel and evaluate the protective mechanisms of the lectin-like domain of TNF in LLO- and PLY-induced models of permeability edema. Since the lectin-like domain of TNF is able to prevent endothelial hyperpermeability induced by LLO and PLY in monolayers of primary human lung microvascular endothelial cells, as measured in the electrical cell-substrate impedance device (Applied Biophysics), one part of this project aims at unraveling its protective mechanism., using state-of-the art molecular biology and immunocytochemistry approaches, thereby concentrating on actions of the TIP peptide on cytoskeletal changes induced by exotoxins in endothelial cells.

2. Role of the lectin-like domain of TNF in the regulation of sodium transporters in type II alveolar epithelial cells.

During the course of pulmonary diseases, such as during L. monocytogenes and S. pneumoniae infection-associated ALI, the alveolar space, as well as the interstitium are sites of intense inflammation. During this inflammatory process, proinflammatory substances such as TNF, IL-1beta and TGF-beta are produced locally. Interestingly, the occurrence of an inflammation can have profound effects on the functioning of ion transporters. Indeed, pro-inflammatory cytokines, such as TNF and IL-1 beta have been recently shown to influence under certain inflammatory conditions epithelial sodium uptake. Moreover, TNF was shown to directly promote edema formation, by means of by means of decreasing transendothelial resistance, inducing the production of reactive oxygen species in lung epithelial cells, or inducing ventilator-induced lung injury. In view of the previously demonstrated protective effect of the TNF-derived TIP peptide in LLO- and PLY-induced acute lung injury, as assessed by measuring the lung wet-to-dry ratio, we will also investigate whether this substance can restore the dysregulated lung liquid clearance under these conditions, thereby concentrating on the regulation of ENaC, in view of our observation that the specific ENaC inhibitor amiloride blocks the activity of the TIP peptide. To this purpose we will investigate gene and protein expression of the ENaC subunits in LLO/PLY-treated primary airway epithelial cells and in the H441 cell line, as well as the expression and activation status of ENaC regulators.

3. Relevance of the lectin-like domain of TNF in exotoxin-induced acute lung injury in vivo.

We have generated Triple mTNF knock-in C57BL6 mice, expressing a T104AE106AE109A mutant of mouse TNF that has lost its lectin-like activity, but has retained the TNF receptor-mediated effects, in order to investigate the physiological role of this TNF domain in TNF-mediated effects during G+-induced acute lung injury.

The results from these studies can give important information about alternative activation mechanisms of edema reabsorption during G+ infection and can thus contribute to the development of alternative therapeutic strategies for the treatment of permeability edema.