Modelo experimental de alvéolo pulmonar in vitro utilizado para demonstrar a habilidade de interaçãocelular e citotoxicidade de amostras de Corynebacterium diphtheriae e Corynebacterium ulcerans

Abstract

The presente study aimed to standardize an experimental model of human lung auveolus performed in vitro to investigate the ability and interaction of C. dipphtheriae and C. ulcerans samples from different origins (n=6), correlating this ability with bacterial adhesive properties to abiotic surfaces. In the tests carried out with the in vitro pulmonar alveolus model, type II pneumocytes of the A-549 lineage and pulmonary macrophagesof the U-937 lineage were cultured in Dulbecco’s modified Eagle’s médium (DMEM) containing 50 µg/ml of fugizone and gentamicin and 10% fetal bovine sérum. The formation of monolayers of U-937 cells was induced by the addition of 0.25 ng/ml de PMA (Phorbol 12-myristate 13-acetate). In the monolayers in co-cultures, pneumocytes (1 x 104 cel/ml) and macrophages (5 x 105 cel/ml) were deposited in holes of polystyrene plates (24 wells) containing glass coverslips. The readingwas performed at different times of infection. The results demonstrated that the experiments using the in vitro alveolar model can be used as an effective methodologyfor the study of pathogenicity mecanisms of C. diphtheriae and C. ulcerans since all the studied samples were capable of interacting with the cell lineage monolayers in co-culture of type II pneumocytes A-549 with pulmonary macrophages U-937. The pathogenes showed differences in terms of bacterial adherence to lung cells, and the numberof injured pneumocytes and macrophages in co-cultures when compared with cells cultured individually. C. diphtheriae and C. ulcerans samples showed different patterns of virulence, with the propertyof interaction with the pulmonary alveoçus being characterized as sample-dependet. Sampleswere classified according to the intensity of virulence to lung tissues: V-I: C. diphtheriae TR-241 > V-II: C. ulcerans 809 > V-III: C. diphtheriae INCA-402 > V-IV: C. ulcerans 2590 > V-V: C. ulcerans BR-AD22 > C. diphtheriae CDC-E8392. The heterogeneity of the ability to interact with pulmonary macrophages of the U-937 lineage and type II pneumocytes of the A-549 lineage was independent of the ability to produce biofilm on the hydrophilic surface of glass and the hydrophobic surface of polystyrene, in addition to the absence of slime production in medium Congo red for C. diphtheriae and C. ulcerans samples. However, bacterial surface properties related to sucrose metabolism and Shiga-like toxin production seem to contribute to increased virulence of samples in lung tissues. Thus, additional future research will be carried out using the in vitro alveolar model, in the investigation of virulence factors involved in these infectious processes.