(432) Molecular Signatures of Human Neutrophils from Healthy Subjects and CF Patients

Background: Persistent neutrophilic inflammation, mucopurulent airway obstruction and chronic bacterial infection dominate cystic fibrosis (CF) lung disease. CFTR dysfunction has been linked to alterations in neutrophil function. Here we investigated the transcriptional landscapes of heathy and CF neutrophils in their native states and in response to LPS, and have elucidated in part their molecular signatures and gene networking.

Methods: Human peripheral blood neutrophils from 3 subjects with CF under Trikafta regimen and 3 healthy donors were isolated by Percoll-gradient centrifugation. The obtained cells were divided for single-cell RNA sequencing (scRNAseq) and culture and stimulation with LPS (40 µg/mL) for 18 hours followed by scRNAseq. Primary data analyses were performed by Cell Ranger 6.1.2. The downstream analyses were achieved using R 4.1.2 (R packages: Seurat 4.1.2, Monocle 2.2.2, ClusterProfiler 4.2.2, and AUCell 1.16.0), and Python 3.8.5 (Python packages: Velocyto 0.17.16, and scVelo 0.2.5).

Results: We profiled ~15,000 high quality neutrophils from each group revealing distinct molecular signatures. Compared to WT neutrophils, CF neutrophils without external stimulation appeared to be pre-activated, showing significantly higher expression of S100A9 and CD16b, and stronger innate immune responses, including the pathways related to leukocyte chemotaxis, immune response-activating signal transduction, phagocytosis, and degranulation. LPS stimulation provoked both healthy and CF neutrophils. There was a clear and shared molecular pathway for both neutrophils to transform to the terminal stage. However, CF neutrophils displayed exhausted innate immune responses as compared to healthy neutrophils, which had normal and robust responses.

Conclusions: Overall, our study delineated the transcriptional landscapes of primary peripheral blood WT and CF neutrophils. Despite the highly effective modulator therapy, loss of CFTR rendered neutrophils intrinsically hypersensitive to stimulation but less responsive to LPS challenge.

Acknowledgements: This work was supported by the National Institutes of Health [grant number: HL150370]; and the Cystic Fibrosis Foundation [grant number: WANG18I0].

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