Evolution of invasive pulmonary aspergillosis produced in mice treated with monoclonal antibodies anti GR-1/Ly-6G and infected with Aspergillus fumigatus strains which presented distincts patterns of production of elastase.

Author(s)

Raphael Luiz de Holanda e Silva

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2012-04-02

Examining board members

Claudia Maria Leite Maffei; Roberto Martinez; Mario Leon Silva Vergara

• Advisor: Claudia Maria Leite Maffei

Abstract

Invasive Pulmonary Aspergillosis (IPA) is an opportunistic fungal disease, caused mainly by Aspergillus fumigatus, that affects immunocompromised patients. To better understand this mycoses, we originally established in C57BL/6 mice an experimental model of neutrophils depletion by intraperitoneal inoculation of antibodies anti GR-1/Ly-6G, confirmed by total and differential leukocyte counts from blood. Next, we evaluated the evolution of experimental pulmonary infection using two strains of A. fumigatus, previously characterized as weak (strain 699) and strong (strain 1753) elastase producers. None of immunocompetent infected mice died with 7 days of observation, while neutropenic mice, infected with both strains, showed 100% mortality after 5 days, with survival curves nearly overlap, suggesting that the major contribution to the virulence was the immune status instead of elastase activity of each fungal strain. For analysis of lung parenchyma, mice were sacrificed 24, 48 and 72 hours post-infection. During the course of experimental infection it was observed a reduction of fungal burden in the lungs, for both strains of A. fumigatus, but this reduction was not statistically significant between the infected groups (neutropenic and immunocompetent). The cellular pattern of the inflammatory infiltrate observed in lungs from neutropenic mice, infected with both strains of A. fumigatus, revealed a predominance of mononuclear cells, a diffuse pattern and clear evidences of angioinvasion, bronchial disruption with break of elastic fibers in both structures, besides exuberance of conidia filamentation for both fungal strains, since the early period of experimental infection. The inflammatory process observed in lungs from immunocompetent mice, infected with both fungal strains, was composed on early times by neutrophils and became exuberant after 72 hours, with predominance of macrophages. It was observed integrity of blood vessels and moderate bronchial wall disruption in lung parenchyma. A relevant observation was the lack of transformation of conidia in hyphae for 699 A. fumigatus strain, in all periods of observation. Total leukocytes count in bronchoalveolar lavage (BAL) was significantly higher at 72 hours post-infection for both groups infected with both strains. The differential count revealed the presence of macrophages and neutrophils, with the former always in greater percentage in BAL from neutropenic mice and the latter always more elevated in immunocompetent group. Analysis by transmission electron microscopy demonstrated that the interaction of fungal structures (conidia or hyphae) with the defense cells (neutrophlis or macrophages) of BAL involved an intimate adhesion and fusion between the surface components from both cells. The presence of hemoglobin in BAL was a result of alveolar injury caused by the fungal development and invasion, but also by injuries determined by the inflammatory process itself. We concluded that neutrophils have a critical role against A. fumigatus since the pathogen quickly invades and damages the lung parenchyma in its absence. However, we must consider that the mere presence of A. fumigatus in immunocompetent mice induces the neutrophils migration to the infection site, which can also cause a tissue injury. Strains of A. fumigatus with distinct patterns of elastase production did not reflect in significant differences in mortality or origin of pulmonary lesions observed in neutropenic mice, suggesting that although elastase contributes to elastic disruptions observed in pulmonary tissue, another virulence factors, such as morphogenesis, can assume a more relevant role for pathogenesis of experimental IPA. (AU)

• FAPESP's process: 09/04667-4 - Role of the neutrophils in the progression of the murine invasive pulmonary aspergillosis established by different Aspergillus fumigatus strains.
• Grantee: Raphael Luiz de Holanda e Silva
• Support Opportunities: Scholarships in Brazil - Master