Resumen de In vitro effects of pollutants and novel therapeutic approaches for tuberculosis
Tuberculosis, a disease caused by Mycobacterium tuberculosis, kills about 1.6 million people every year. The outcome of the infection is conditioned by host factors, diversity of mycobacteria and by external factors like pollution. Smoking is an established risk factor for tuberculosis infection, disease, relapse, fail in the treatment and drug-resistant tuberculosis and it is estimated that 15% of global tuberculosis deaths are attributed to smoking. Electronic cigarettes are usually promoted as smoking cessation devices, although their harmless have not been proven. On the other hand, the emergence of drug resistance and limited pipeline make necessary to look for new therapeutic approaches. Encapsulation of current drugs and host-directed therapies are promising strategies for tuberculosis treatment.
There are two main aims in this work. One is to evaluate the impact of diesel, electronic cigarette vapor (e-vapor) and cigarette smoke (CS) on macrophages infected with M. tuberculosis, as an in vitro model of a tuberculosis patient exposed to these pollutants. Chapter I summarizes the relationship between poor air quality and susceptibility to respiratory infections. In Chapter II, diesel, a main contributor to urban pollution, was evaluated for a possible association with tuberculosis, and results suggested that contention of bacilli could be impaired to some extent by diesel pollutants. In Chapter III, it was observed that exposure to CS/e-vapor reduced the intracellular M. tuberculosis burden, due to an impairment of the phagocytosis. The production of cytokines was also modified by exposure to CS/e-vapor. In Chapter IV, it is discussed the metabolomics in extracellular and intracellular fractions of infected cultures exposed to CS/e-vapor. Main differences in metabolites were due to maturation of the cultures, but e-vapor induced changes in the metabolome of THP-1 cells comparable to CS exposure. It was investigated in Chapter V the effects of CS and anti-tuberculosis drugs in macrophages infected with M. tuberculosis. Macrophages treated with drugs showed a reduction in the intracellular burden while rifampicin concentration detected was lower in smoked samples, maybe due to an interaction drugs-CS.
Secondly, it was evaluated the activity of nanoparticles (NPs) and one virulence inhibitor compared to free drugs. In Chapter VI, it was tested the activity of silica NPs loaded with rifampicin or isoniazid in human macrophages infected with M. tuberculosis exposed to CS. Poor drug load efficiency and high toxicity of the NPs made that administration of NPs had no advantage comparing to free drugs. No interaction NPs-CS was found. In Chapter VII it was tested the activity of PLGA NPs loaded with rifampicin. NPs were not cytotoxic to macrophages, NPs were resistant to simulated gastric conditions, unloaded NPs had some bactericidal action and loaded NPs had a sustained release of the drug. Loaded NPs showed a more effective reduction of M. tuberculosis intracellular burden than free drugs. Finally, in Chapter VIII we explored a new approach targeting a protein that prevents phagosome maturation, allowing M. tuberculosis to survive inside macrophages. We studied the combined effect of the inhibitor and CS in THP-1 macrophages infected. The inhibitor is a promising candidate as a drug for tuberculosis treatment, especially in combination with rifampicin.
In conclusion, this work highlights the importance of making global environmental policies and to implement tobacco cessation strategies as part of the tuberculosis treatment. Results show e-vapor provokes a pro-inflammatory response that could favour tuberculosis disease. Due to drug resistance is inevitable, it is necessary to focus on new therapeutic approaches, as nanoparticles carriers or host-directed therapies. Our results support that the use of loaded PLGA NPs is an improvement compared to free drugs, and targeting virulence factors in addition to drugs administration potentiate the bactericidal effect against M. tuberculosis.
