In vitro model to study the correlation between neutrophils and miR21 expression: a role in Bronchiolitis obliterans syndrome

Bronchiolitis obliterans syndrome (BOS) is associated with poor long term survival. In its pathogenesis neutrophils (NEU) are recognized as relevant players. Moreover, our group has recently showed that some microRNAs (miRs) may act as pro-fibrotic effectors in the context of BOS and highlighted the role of miR21, whose expression was higher in BOS lungs respective to healthy lungs. Our aim was to assess whether NEU driven inflammation might induce expression of miR21 in bronchial epithelial cells. As bronchial epithelial model we chose to use 16HBE cell line, due to their poor basal expression of miR21. We co-coltured for 96hr 16HBE cells with different ratio (1:1 and 1:10) of healthy donor-derived NEU previously incubated for 1 hr +/- phorbol myristate acetate (PMA) or N-Formylmethionyl-leucyl-phenylalanine (fMLP). 16HBE were also exposed to NEU conditioned medium. At 48, 72 and 96h total RNAs from treated 16HBE cells was extracted and miR21 expression was assessed by RT-PCR (using U6 as reference gene). We observed that NEU induced significant miR21 over-expression in 16HBE in contrast to untreated cells (4.3 ± 0.55 fold-increase for activated NEU 1:1; and 1.94 ± 0.10 fold-increase for activated NEU 1:10 vs. untreated p=0.01). Interestingly, miR21 expression was even more activated by conditioned medium derived from activated NEU (7.17 ± 0.55 fold-increase vs. untreated cells). These results light new interest on a molecular mechanism underlying BOS development, and, if further confirmed suggest a possible therapeutic strategy aimed to modulate miR21 expression on bronchiolar epithelium.