Modelos de infección "in vitro" para el análisis de la respuesta inmunológica frente a micobacterias

Abstract

M. tuberculosis is the species of the genus Mycobacterium sp that causes most infections worldwide. However, in recent decades, the prevalence of nontuberculous mycobacterial (NTM) infections has increased considerably, mainly the pulmonary form associated with chronic pathologies, such as bronchiectasis. Many immunological mechanisms involved in these infections are still unknown, partly due to the diversity of available in vitro assays, which fail to adequately reproduce the physiological response. In the present work, we optimized several blood cell derived models to analyze the anti-mycobacterial activity against different species of the genus, selected based on their level of pathogenicity, in order to reproduce the phenomena occurring in vivo in susceptible populations, such as smokers or patients with bronchiectasis. Cell purification was, generally, performed by double gradient sedimentation and bacterial individualization by sonication. In vitro infection used a low number of bacteria per cell, which were incubated in serum-free medium. In order to ensure that clinical isolates corresponded to different strains, we designed a simple genotyping method based on the Amplified Fragment Length Polymorohism (AFLP) technique, which allowed us to obtain easily interpretable results with a good level of discrimination. No mycobactericidal activity was observed in the monocyte-derived macrophage model. However, with the total leukocyte and whole blood models, with all the immune cell fractions and humoral components, we obtained different resistance patterns depending on the pathogenicity of the infecting strains. In these models, growth restriction was observed in all species using EDTA as anticoagulant, related to their bacteriostatic activity. These models were also used to evaluate the immune response of the smoking population against M. tuberculosis, without observing differences with the control group (non-smokers) in the different cellular models analyzed (neutrophils, peripheral blood mononuclear cells or monocytes). To reproduce aspects of the immune response in bronchiectasis, we used a neutrophil model with different clinical isolates of M. abscessus and M. mageritense. We did not observe mycobactericidal activity against any species, but did observe a limitation of M. abscessus growth and aggregation when high cellular concentrations were used, which was enhanced by the addition of fresh neutrophils over time, before bacterial multiplication occurred. When mycobacteria aggregated, the release of proteolytic enzymes, related to tissue damage in this pathology, was increased. While supernatants from cells infected with M. mageritense increased neutrophil chemotaxis, this effect was not observed using M. abscessus.