(454) Relationships between Inflammation and Bacterial Ecology in BALF from the Cystic Fibrosis Airway

Background: Progressive, obstructive lung disease resulting from chronic airway infection and inflammation is the leading cause of death in cystic fibrosis (CF). Studies have shown that the CF airway is defined by heightened inflammatory response even in the absence of infection. Understanding the relationship between airway infection and inflammation early in the development of CF lung disease is crucial to our understanding of CF pathophysiology. Our aim was to integrate 16S rRNA bacterial sequencing data with inflammatory cell counts, cytokines, and airway proteases, from the lower airways of pediatric CF and disease control (DC) subjects to investigate the dynamic and complex interplay between infection and inflammation.

Methods: Bronchoalveolar lavage fluid (BALF) obtained during clinically indicated flexible bronchoscopies, as well as relevant clinical data, from pediatric CF and DC subjects across the age spectrum were obtained. Quantitative polymerase chain reaction to measure total bacterial load (TBL) and 16S sequencing to characterize bacterial communities were previously performed. GM-CSF, IL-10, IL-8, IL-6, IL-4, IL-2, IL-1β, TNF-α, Neutrophil elastase (NE), and Cathepsin B were quantified. Inflammatory measures were compared across disease groups using Wilcoxon signed rank test, and Fisher’s exact test was used to compare detection across groups. Shannon index was used to measure microbial diversity. Hierarchical clustering analysis was performed on the 16S data, including annotation with inflammatory, microbial, and clinical measures.

Results: One-hundred twenty-four BALF samples [N = 51 CF(41%)] were analyzed with a median (range) age of 6.6 (0.0-21.0) years at time of sample collection. There was sufficient sample to measure NE in 107 (86%), Cathepsin B in 71 (57%) and cytokines in 84 (67%) samples. Only 34 (32%) NE values were above the lower limit of detection (LLoD), 32 (94%) of which were from CF subjects (p < 0.01). Except for TNF-α, IL-2, IL-4, and IL-10, most cytokine values were above the LLoD. IL-8, IL-1β, and Cathepsin B were all elevated in CF BALF (p < 0.01). When inflammatory measures were compared to the clustering of samples based off the 16S data, CF samples mostly clustered together and were associated with increased inflammatory markers and decreased Shannon diversity, with one cluster associated with increased abundance of Staphylococcus (Figure 1).

Conclusions: CF subjects exhibit distinct airway inflammatory profiles compared to DC subjects, characterized by increased cytokines, WBC, percent neutrophils, and airway proteases. When integrated with detected bacterial communities, there are clear clusters including increased markers of inflammation associated with Staphylococcus, a pathogen previously shown to be a potential driver of changes in airway ecology, including increased TBL and decreased diversity observed around 2-5 years of age. While additional analyses are required, it is possible that Staphylococcus may elicit a particular immune response in CF critical in the development of chronic infection.

Acknowledgements: This abstract was supported by grants from the National Institutes of Health (NIH R01HL136499) and the Cystic Fibrosis
Foundation (CFF LAGUNA17A0).

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