Project description:Adults with cystic fibrosis (CF) have chronic antibiotic-resistant polymicrobial lung infections, the leading cause of death in CF. We developed a polymicrobial culture model containing four genera that represents a ‘pulmotype’ detected in ~34% of lung infections in people with CF (pwCF), and accounts for 27% of the variability in lung function. This community, comprised of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, is grown in synthetic CF media (SCFM2) under anoxic conditions that mimic the environment in mucus plugs in CF. We have shown that Pseudomonas in monoculture communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. We report herein that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence and biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Trikafta, a highly effective drug, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited Trikafta-stimulated CFTR Cl- currents by CF pHBEC. These studies provide insight into why Trikafta does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF.
Project description:Adults with cystic fibrosis (CF) have chronic antibiotic-resistant polymicrobial lung infections, the leading cause of death in CF. We developed a polymicrobial culture model containing four genera that represents a ‘pulmotype’ detected in ~34% of lung infections in people with CF (pwCF), and accounts for 27% of the variability in lung function. This community, comprised of Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica, is grown in synthetic CF media (SCFM2) under anoxic conditions that mimic the environment in mucus plugs in CF. We have shown that Pseudomonas in monoculture communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. We report herein that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence and biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Trikafta, a highly effective drug, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited Trikafta-stimulated CFTR Cl- currents by CF pHBEC. These studies provide insight into why Trikafta does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF.
Project description:Chronic Pseudomas aeruginosa infection in the lung is a common in people with cystic fibrosis (CF). Current therapies for CF fail to eliminate persistent bacterial infections, chronic inflammation, or irreversible lung damage. Our group engineered mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) to carry the microRNA let-7b-5p as a dual anti-infective and anti-inflammatory treatment. In a preclinical CF mice model, we found that let-7b-5p-loaded MSC EVs reduced P. aeruginosa burden, immune cells and proinflammatory cytokines in the lungs. This research hypothesized two mechanisms of the observed effects in the mouse model: anti-inflammatory properties of the let-7b-5p-loaded MSC EVs and inhibition of antibiotic-resistant P. aeruginosa biofilm formation in CF airways. Primary human broncial epithelial cells (pHBECs) were exposed to P. aeruginosa and treated with differet MSC EV conditions. The results demonstrated that MSC EVs engineered to contain let-7b-5p effectively blocked the formation of P. aeruginosa biofilms on pHBECs while also reducing P. aeruginosa-induced inflammation by CF-pHBECs.