(633) Extracellular vesicles as delivery vectors to enhance killing of bacteria and viruses by airway-recruited human neutrophils

Background: Polymorphonuclear neutrophils (PMNs) recruited to the airway lumen in cystic fibrosis (CF) undergo rapid transcriptional program resulting in exocytosis of granules and inhibition of bacterial killing. As a result, chronic infection, feed-forward inflammation and structural tissue damage occur. Since CF airway PMNs are also highly pinocytic, we hypothesized that we could deliver protein- and RNA-based therapies to modulate their function to benefit patients. We elected to use extracellular vesicles (EVs) as a delivery vector since they are highly customizable and airway PMNs have previously been shown by our group to efficiently process and utilize their cargo [1]. Furthermore, our prior work on CF airway PMNs [2] led to the identification of the long non-coding RNA MALAT1, the transcription factor EHF, and the histone deacetylase/long-chain fatty deacylase HDAC11, as potential targets to modulate CF airway PMN dysfunction.

Methods: H441 human club epithelial cells were chosen for EV production since they efficiently communicate with lung-recruited primary human PMNs [1]. Relevant constructs were cloned into an expression plasmid downstream of a constitutive CMV or U6 promoter with an additional puromycin selection cassette. EVs were generated in serum-depleted media and purified by differential centrifugation. Quality and concentration of EVs was determined by electron microscopy, nanoparticle tracking analysis and cargo content by western blot (protein) or qRT-PCR (RNA). EGFP protein and mRNA were used as controls. To test delivery to primary human PMNs, generated EVs were applied in the apical fluid of an airway transmigration model [2]. PMN activation was assessed by flow cytometry and bacterial (PA01 and S. aureus 8325-4) killing and viral (IAV H1N1/PR/8/34; SARS-CoV-2/Washington) clearance assays were conducted.

Results: To package protein, we used EV-loading motifs such as the tetraspanin CD63, Basp1 amino acids 1-9, and the palmitoylation signal of Lyn kinase. To load mRNA, a C’D box motif recognized by the RNA-binding protein L7Ae, was included in the 3’UTR of the expressed RNA and CD63-L7Ae was co-expressed. Airway-recruited PMNs treated with EVs containing siRNAs against MALAT1 or HDAC11 showed an increased ability to clear bacteria. Conversely, PMNs treated with constructs encasing MALAT1 or HDAC11 efficiently cleared IAV and SARS-CoV-2. Interestingly, PMNs expressing EHF showed increased clearance of both bacteria and viruses.

Conclusions: Our findings suggest mutually exclusive roles of MALAT-1 and HDAC11 in regulating bacterial vs. viral clearance by airway-recruited PMNs. Meanwhile, expression of EHF in airway PMNs may be a pathogen-agnostic approach to enhance clearance by airway-recruited PMNs. Overall, our study brings proof-of-concept data for therapeutic RNA/protein transfer to airway-recruited PMNs in CF and other lung diseases, and for the use of EVs as a promising method for cargo delivery to these cells. It is our expectation that by treating the immune-compartment of CF airway disease, that pathogen-therapies, such as antibiotics will be more effective, and epithelial-targeted therapies, such as CFTR modulators, will have greater penetrance to the cell-types of interest.

Acknowledgements: Support: CFF Award (TIROUV19A0) and Emory I3 Team Awards.

References: References
[1] Forrest et al. J Leuk Biol 2022; [2] Margaroli et al. Cell Rep Med 2021