Relationship between air particulate matter and mycobacterial respiratory infection

VERÓNICA C. DELFOSSE; ANDREA K. GIOFFRÉ; MARTÍN ZUMARRAGA; DEBORAH R. TASAT

San Diego

Conferencia; Air Pollution and Health. Presented by the American Association for Aerosol Research; 2010

American Association for Aerosol Research

Epidemiological studies have shown that industrialization and vehicular transportation provokes adverse health effects. Increment of suspended air particulate matter (PM) and gases (CO, NOx, SOx and O$_3) cause broad ambient respiratory diseases. Therefore, ambient factors such as air pollution should be taken into account when incidence and evolution of microbial diseases are estimated. The environmental mycobacteria are opportunist pathogens in a variety of immunocompromised patients. They have significant impacts on the morbidity and mortality of humans and important economic impact on agriculture. Hydrophobic bacilli are more readily aerosolized, and aerosols are a major delivery mechanism for environmental mycobacteria to obtain pulmonary access to animal hosts. As the main target for air pollutants is the respiratory tract, we herein propose to study the effects of PM, particularly ROFA (Residual Oil Fly Ash), on the susceptibility of mycobacterial infection. The biologic response involved in these events was assessed in vitro on rat alveolar macrophages (AM) cultures. The environmental mycobacteria studied has been identified as Mycobacterium phlei, the typification was carried out using PCR restriction fragment length polymorphism analysis technique (PRA) and confirmed by partial sequencing of the gen hsp65. Subsequently, mycobacteria was electroporated with the replicative vector pMV27-GFP, allowing their visualization by fluorescence microscopy. AM from young Wistar rats were obtained by bronchoalveolar lavage, seeded at a density of 0.125 x 106 cells/ml in RPMI-160 supplemented with 10% FCS and antibiotics under 5% CO2 at 37°C. We co-culture AM with M. phlei (1:10) to determinate the best infection time. We also evaluated the effect of M. phlei alone and pre-exposition of different ROFA concentrations (0, 5, 10, 25, 50, 100ug/ml, for 24hs) analyzing the following endpoints; cellular metabolism (MTT assay), production of TNF-a (ELISA) and nitrites (Griess reaction). The optimal infection time as determined by optical microscopy was 24h. MTT assay indicated that cellular metabolism decreased in a dose-dependent manner when exposed to ROFA. A 20% reduction was found for 10ug/ml ROFA reaching values around 50% for higher doses. TNF-a production was not significantly modified by 0, 2.5, 5, or 50ug/ml of ROFA (34.3±1.2, 37.2±0.6, 36.7±13.23 and 45.6±9.8pg/ml respectively). However, as expected M. phlei infection elicited high levels of TNF-a. A significative reduction on TNF-a production was found when AM cultures were exposure to PM and then infected with M. phlei (0, 5, 50ug/ml ROFA; 369.6±148.7, 182.1±80.5, 16.6±1.3pg/ml respectively, p<0.05). Nitrites production rose for all ROFA concentrations reaching the highest value at 2.5ug/ml of ROFA (0, 2.5, 5, 50ug/ml ROFA; 2.3±1.23, 5.9±0.67, 3.3±0.99, 3.2±0.89uM, p<0.001). Although it was not statistically significant, PM exposure previous to M. phlei infection showed a dose dependant increase in nitrites production (0, 2.5, 5, 50ug/ml ROFA; 4.40±0.82, n.d, 5.6±1.3; 5.9±0.3uM, p<0.05). Our results indicate that pre -exposure to PM alters the innate pulmonary defense mechanisms after mycobacterial infection. This investigation underlined the potential impact of polluted environment on opportunistic mycobacterial infections and drives to novel questions toward other relevant mycobacterial infections such us tuberculosis.