(387) Pre-infection Knockdown of Muc5b Reduces Severity of Chronic Pseudomonas aeruginosa Infection in the CFTR-KO Rat

Background: One of the major gel-forming mucins in mucus is Muc5b. Muc5b is secreted from submucosal glands in strands and bundles to clear bacteria and other debris from the airway. In CF, these mucins are overexpressed by hypersecreting cells and glands, contributing to mucus plugging and preventing normal clearance of pathogens such as Pseudomonas aeruginosa (PA). Our Cftr^-/- (CFTR KO) rat model exhibits submucosal glands that, when mature, lead to the development of a mucus defect that recapitulates the human CF lung environment. Data from our lab shows this mucus defect, apparent at 6 months of age in the KO rat model, is correlated with chronic infection with PA. Using our novel rat model, we want to assess the effect of altering mucin secretion into the airways on acute and chronic PA infection outcomes.

Methods: CFTR KO rats aged to at least 6 months received two doses of either 20 µg/300 µL Muc5b siRNA or scramble siRNA via intratracheal inoculation with 48 hours (h) between treatments. Rats were then intratracheally inoculated with 10⁶ colony forming units (CFUs) of the PA mucoid clinical isolate PAM57-15 48h after last treatment and euthanized 3- or 14-days post infection (dpi). Muc5b concentration in the bronchoalveolar lavage fluid (BALF) was determined by dot blot. Inflammatory cells in the BALF were quantified by Diff-Quik staining. ELISAs were utilized to assess levels of important cytokines in BALF. Bacterial burden was assessed by homogenizing and plating lung tissue. Lung tissue was prepared for routine histopathology.

Results: There was no difference in infection outcomes between the treatment groups at 3dpi. At the 14dpi chronic infection timepoint, Muc5b concentrations were significantly decreased following Muc5b siRNA treatment as compared to the control. While there was no difference in macrophage percentages present in the BALF between the Muc5b siRNA and control at the 14dpi timepoint, there was a significant decrease in the percentage of neutrophils, indicating less inflammatory cell influx in the Muc5b siRNA treatment group. However, by the 14dpi chronic infection timepoint, CFUs in the lung were significantly decreased in the Muc5b siRNA treated group. Histopathological studies of lung and tracheal tissue by AB-PAS staining at the 14dpi timepoint display reduced plugging of submucosal glands in the tracheae as well as less mucus accumulation in the small airways of Muc5b siRNA treated rats.

Conclusions: siRNA knockdown of Muc5b in 6mo+ CFTR KO rats alters the development of chronic PA infection resulting in reduced bacterial burden, fewer mucus plugs, and reduced inflammation. Additionally, IL-1β levels are reduced in the BALF of chronically infected rats treated with Muc5b siRNA, indicating that knockdown of Muc5b prior to bacterial exposure appears to specifically target the vicious muco-inflammatory cycle between IL-1β and mucin secretion. These results indicate that Muc5b is a crucial mucin for the development of chronic PA infection in the CFTR KO rat.

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