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Polymicrobial extracellular vesicles reduce the innate immune response of human cystic fibrosis bronchial epithelial

Lily A Charpentier¹, Roxanna L Barnaby¹, Carolyn T Roche¹

¹Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA.

Biorxiv : the Preprint Server for Biology

|April 17, 2026

Abstract:

Chronic antibiotic-resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which 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. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a 'pulmotype' detected in ~34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report 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, biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Elexacaftor/Tezacaftor/Ivacaftor (ETI), a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited ETI CFTR Cl^- currents by CF pHBEC. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF.

Insights

Polymicrobial bacterial extracellular vesicles (bEVs) promote chronic cystic fibrosis (CF) lung infections and inflammation. Current modulator therapies do not stop this process or its impact on CF lung cells.

Area of Science:

Microbiology
Cell Biology
Pulmonology

Background:

Chronic lung infections are a major cause of mortality in cystic fibrosis (CF) patients.
Despite modulator therapies, microbial communities persist, exacerbating CF pathogenesis.
Bacterial extracellular vesicles (bEVs) deliver virulence factors to lung cells, but polymicrobial contributions are unknown.

Purpose of the Study:

To investigate the role of polymicrobial bEVs in CF lung infections.
To determine the impact of bEVs on primary human bronchial epithelial cells (pHBEC), including CF cells.
To assess the effect of Elexacaftor/Tezacaftor/Ivacaftor (ETI) on bEV-induced responses.

Main Methods:

A polymicrobial culture model simulating a common CF 'pulmotype' (P. aeruginosa, S. aureus, S. sanguinis, P. melaninogenica) was used.
bEVs were isolated and analyzed for protein and RNA content.
CF and wild-type (WT) pHBEC were exposed to bEVs, and cellular responses were measured.
ETI's effect on bEV-induced responses and CFTR currents was evaluated.

Main Results:

Polymicrobial bEVs contain proteins and RNAs that enhance bacterial virulence, biofilm formation, and pro-inflammatory pathways in pHBEC.
CF pHBEC exhibited a more pronounced response to bEVs compared to WT cells.
ETI therapy did not reduce the bEV-induced cellular response or restore it to WT levels.
bEVs inhibited ETI-mediated CFTR currents in CF pHBEC.

Conclusions:

Polymicrobial bEVs contribute to chronic CF lung infections and hyperinflammation.
Current ETI therapy does not overcome the detrimental effects of bEVs on CF lung cells.
These findings highlight the need for novel therapeutic strategies targeting bEVs to combat chronic CF lung infections.

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