(560) Keratan sulfate decoration on airway epithelial surfaces and its role in host defense and infections

Background: Understanding the ability of bacteria to colonize/transform mucus and the periciliary layer (PCL) in CF lung disease is important in stopping disease progression. Previous work has shown that Pseudomonas aeruginosa (Ps.a) can attach to mucins to form a biofilm, but doesn’t adhere to heavily sialylated or sulfated mucins[1]. Our prior work has shown that the membrane tethered mucins of the PCL are heavily decorated with highly charged keratan sulfate (KS) chains[2], and thus, we hypothesize that KS decoration may serve a protective function in the PCL. Here we show that Ps.a is able to remove keratan sulfate from the membrane mucins. This removal may change the properties of the PCL, which may in turn promote further infection and stagnant mucus.

Methods: Primary human bronchial epithelial cell cultures were grown at an air-liquid interface until they both produced mucus apically and were well ciliated. Prior to the start of experimental challenges, accumulated mucus was washed off of the culture with PBS. Cultures were challenged either by administering IL-1B basolaterally, or the filtrate of Pseudomonas aeruginosa apically (PsaF). Apical washings were collected daily over the course of a 5-day challenge period. Washings were analyzed via slot and western blots and with proteomics. Cells were fixed and prepared for immunohistochemical imaging with confocal microscopy.

Results: While performing the cell washings, we observed that cells challenged with either IL-1B or PsaF both produced more mucus as compared to their respective controls. Additionally, while collecting the apical washings off of these cultures we observed a high degree of filance, i.e. elastic strands, which suggests that some of the material was adhered to the surface. Our analysis of the washings confirmed prior studies[3] that indicated that MUC5B increased in both the PsaF challenge and the IL-1B challenge. MUC4 antibody signal increased after 5 days of PsaF challenge, while KS signal decreased. Both of these could be attributed to the PsaF’s ability to remove KS side chains, which could increase epitope accessibility for the MUC4 antibody. We did not see an increase in MUC16, however, which could indicate that either the KS disproportionately decorates MUC4, or that there is an increase in MUC4 expression.

Conclusions: Our data suggests that Pseudomonas aeruginosa is capable of enzymatically removing keratan sulfate from membrane bound mucins. Stripping heavily sulfated KS could enable bacterial adherence to MBMs. Furthermore, we show that there is an associated increase in MUC5B production. The additional MUC5B serves as a further substrate for bacterial biofilms. Additionally, the combination of additional MUC5B and MBM’s stripped of KS sidechains could promote the collapse of the PCL and adherence of the gel forming mucins to the epithelial surface.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation [CARPEN19I0]

References: [1]. Ramphal R, Houdret N, Koo L, Lamblin G, Roussel P. Differences in adhesion of Pseudomonas aeruginosa to mucin glycopeptides from sputa of patients with cystic fibrosis and chronic bronchitis. Infect Immun. 1989;57(10):3066-71.

[2]. Carpenter J, Kesimer M. Membrane-bound mucins of the airway mucosal surfaces are densely decorated with keratan sulfate: revisiting their role in the Lung's innate defense. Glycobiology. 2021;31(4):436-43.

[3]. Batson BD. AIRWAY MUCIN DYNAMICS IN INFECTION AND INFLAMMATION: Chapel Hill, NC: University of North Carolina at Chapel Hill, 2019.; 2019.