Cystic fibrosis (CF) is an autosomal recessive disorder that effects about 1 in 4000 births in the U.S. with more than 150,000 people affected worldwide (1–3). CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) which leads to muco-obstructive airway disease making this an ideal environment for opportunistic pathogens such as Pseudomonas aeruginosa (PA) (4–6). PA is the most common colonizing pathogen of people with CF (pwCF) greater than 35 years old (7). Colonization with PA is associated with an increase in pulmonary exacerbations, accelerated lung function decline and increased risk of mortality in pwCF (8–10). PA colonization and infection in the CF lung increases expression of the chemokine interleukin [IL] -8, which recruits neutrophils and cytokines (IL-1β, IL-6, and tumor necrosis factor (TNF) α), leading to further inflammation and immune cell responses (11, 12).

IL-8 is a cytokine well known to initiate neutrophil recruitment in the lungs especially in CF. CF neutrophils exhibit the GRIM phenotype named for their primary granule release, immunoregulatory function, and metabolic activities upon recruitment and transmigration to the CF lung tissue (13). These neutrophils express CD66b, which is a neutrophil specific surface protein that aids in adhesion to endothelial cells for transmigration, degranulation, and production of reactive oxygen species. Additionally, GRIM neutrophils downregulate surface expression of CD16 and increase primary granule release, measured by surface CD63 expression (13–15). Despite chronic neutrophilia in the CF lung, GRIM neutrophils cannot efficiently phagocytose bacteria, such as PA (15, 16). Therefore, chronic PA colonization and recurrent infections lead to a perpetuating cycle of chronic inflammation, chronic infection, and lung damage ultimately increasing mortality due to respiratory failure.

Improved nutrition and the use of inhaled antibiotics led to increased life expectancy in pwCF. The introduction of highly effective modulator therapy (HEMT), especially the restoration of CFTR activity using a triple combination of CFTR activators and potentiators (elexacaftor/tezacaftor/ivacaftor or ETI) led to further improvements in morbidity and mortality for patients, who are eligible by their CF genotype. Longitudinal studies though have shown that the bacterial burden with PA, while initially decreased on HEMT, increases again by two years post-HEMT initiation (6, 17). Additionally, some pwCF continue to be colonized even 3.5 years post initiation of ETI therapy (18). Therefore, more investigations are needed to identify the underlying mechanisms of PA-mediated lung injury in pwCF independent of their eligibility for HEMT. Recent reports show controversial results addressing consistent pan-anti-inflammatory effects of ETI and whether those are systemic or tissue specific (19–24). Further investigations are needed to determine the impact of ETI therapy on the inflammatory profile in the CF bronchial epithelium.

Extracellular vesicles (EVs), 30–1000 nm particles containing proteins, DNA, and several types of RNA, are produced through exocytosis from most cell types, including epithelial cells. They have been found in high abundance in bodily fluids (25, 26) and can mediate intercellular communication in many different inflammatory lung diseases including chronic obstructive pulmonary disease (COPD), asthma, interstitial lung disease and CF (25–29).

EVs have also been implicated in the disease pathology of CF. EVs have been found to be increasing in number in the bronchial alveolar lavage fluid (BALF) of people with CF compared to healthy controls and concurrently also increase in number with age (30, 31). EVs from CF bronchial epithelial cells demonstrate increased myeloperoxidase activity [MPO] and promote neutrophil migration (30). EVs isolated from corneal epithelial cells, infected with a PA lab strain (PAO1) induced cytokine production and neutrophil migration though using a different migration model (32). So far, there are not studies assessing prolonged PA infection on bronchial epithelial EV mediated autocrine and paracrine signaling and whether these effects can be attenuated by CFTR modulator therapy.

Our study is one of the first studies to establish crosstalk between the bronchial epithelium and neutrophils in the PA-infected CF lung via EVs. We demonstrate that EVpPAs derived from CFBEs induce neutrophil activation and mediate an increase in pro-inflammatory markers via autocrine signaling, which persists despite CFTR correction with ETI (Figure 4).

We and others have shown previously that the CF bronchial epithelium expresses increased levels of pro-inflammatory markers such as IL-8, when compared to non-CF bronchial epithelial cells (36, 37). Furthermore, PA infection itself leads to further upregulation of IL-1β, IL-6 and IL-8 (12). Multiple reports have described the vicious cycle of infection and inflammation in the CF lung as one of the main contributors to lung injury (38). Several of the underlying PA virulence factors that are mechanistically responsible for the proinflammatory phenotype observed in CF include alginate and flagellin proteins (9, 39) and activation of host signaling pathways such as NF-κB (40). We have shown previously that PA infection can lead to an increase in the phosphorylation and resultant activation of PLCγ (12). It remains yet to be determined whether EV-induced inflammation is associated with the activation of canonical epithelial signaling mechanisms such as PLCγ.

One limitation of our study is that EVs were derived from a bronchial epithelial cell line, grown in submerged culture to establish our model. Future studies will need to be conducted in differentiated cultures and ultimately primary differentiated cultures, which could change outcomes (expression of pro-inflammatory markers and neutrophil activation) due to the role of differentiation and polarization on EV cargo and internalization.

In CF, neutrophilia is well described; however, neutrophils that are recruited to the airway display impaired phagocytosis of bacteria such as PA (8, 16, 41, 42). Furthermore, CF neutrophils possess an intrinsic granule-releasing, immunoregulatory and metabolically active (GRIM) phenotype with impaired bactericidal activity and adaptation to their environment resulting in a decreased bacterial clearance (15, 43, 44). In this study, primary granule release was significantly increased upon addition of EVs from CFBEs treated with PA compared to 16HBE controls, suggesting that response to PA may change the quality of EV cargo and in turn influence neutrophil activation. Further, the appearance of a CD16^low/CD63^low neutrophil population in our in vitro model could be due to the use of different epithelial cells (i.e. large vs. small airways), which should be further investigated in future studies.

It has been shown that PA can increase primary granule release in airway neutrophils (15). PA itself can release outer membrane vesicles (OMVs), which have been shown previously to affect CFTR activity and can alleviate lung injury in a sepsis model (45, 46). It will remain to be studied in future investigations whether OMVs have a modulating effect in EV-mediated paracrine and endocrine crosstalk in our model. In the CF lung, EVs originating from neutrophils have been described as promoting the pro-inflammatory vicious cycle in CF airways via an autocrine loop leading activation of neutrophils via sustained inflammasome signaling (13), but to date, not many research studies have investigated EVs as potential vehicles for paracrine crosstalk, specifically between the CF bronchial epithelium and neutrophils. Koeppen et al. has shown that EVs from primary bronchial epithelial cells have a direct effect on PA via miRNA delivery reducing biofilm formation and increasing antibiotic sensitivity (47). The same group has also shown recently that HBEC EVs reduced PA burden and bronchoalveolar inflammation in a CF mouse model (48). Taken together, those findings suggest that EVs isolated from non-CF bronchial epithelial cells have a beneficial role in the PA-infected CF lung with direct effects towards the pathogen and the host.

Comparing our data, when we used HBE-derived EVs, they did not induce the pro-inflammatory and neutrophil activating response we observed with CFBE-derived EVs, which is consistent with the current literature. It was also surprising to see that CD9, which is a well-established marker for EVs, was expressed at much higher levels in EVs isolated from CFBEs, compared to 16HBEs, independent of PA infection. One possible explanation could be that CD9 is not only an exosome marker but also involved in growth, adhesion, signaling and inflammation (49) It will be of future interest to see further characterize differences in EV content between CF and control cells and whether CFBE-derived EVs exert a similar protective role in PA infection for both host and pathogen.

Lastly, the introduction of HEMT specifically ETI, has improved both quality of life and reduction of symptom burden for pwCF, especially respiratory symptoms, and some studies have shown that ETI may decrease pro-inflammatory markers such as IL-6, MMP9, and CXCL1 (50) while others show no systemic decrease with ETI therapy (51). Therefore, our finding that ETI pre-treatment of CFBEs does not attenuate EV-induced expression of pro-inflammatory markers fits within these previous reports. Veltman et al, suggested that persistent inflammation could be due to epigenetic changes, which are not reversed by CFTR correction (52). We did not observe significant differences in levels of IL-8 and TNFα between groups, which is consistent with previous reports from Allegretta et al. showing that stimulation with exoproducts of PA did not lead to significant differences in levels of IL-8 and TNFα between the control and ETI treated cells (53). The trending increase of IL-1β with ETI therapy could be an enhanced response to the inflammatory stimuli that the EVpPAs produce as suggested by Gentzsch et al, 2021 (54). Therefore, further studies are needed to fully elucidate the role of ETI therapy on pro-inflammatory signaling in the CF bronchial epithelium.

Overall, our work demonstrates that EVs isolated from the PA-infected bronchial epithelium potentially play a role in autocrine and paracrine crosstalk mediating a pro-inflammatory response and neutrophil activation. This pro-inflammatory response is not affected by ETI. Further studies are needed to decipher the underlying signaling pathways activated by EVpPAs and whether this mode of intercellular crosstalk can be therapeutically targeted.