Functional polarization of primary bronchial epithelial cells during the stimulation with P.aeruginosa

Toll-like receptors (TLR) are part of the innate immune system of the lung and recognize pathogens to activate host defense. They are mostly located on immune cells but can also be found in airway epithelial cells. The aim of this work was to investigate the location and of TLR4 after the stimulation with Pseudomonas aeruginosa (P.a.) and the resulting functional polarization of primary bronchial epithelial cells.

Primary human bronchial epithelial cells (pHBE) were cultured and differentiated in the Air Liquid Interface (ALI) system to form an epithelial-like structure. To investigate the cellular location of TLR4 the cells were stimulated with P.a. apically and basolateral, which would mimic respiratory infection apically and infection via the bloodstream, respectively. The effect of directional stimulation was investigated by the quantification of inflammatory biomarkers using enzyme-linked immunosorbent assay (ELISA) and barrier function using trans epithelial resistance (TEER) measurement. In addition, the localization of TLRs in the cell was visualized by immunofluorescence staining and high resolution structured-illumination microscopy (SIM) over time.

The experiments showed that pHBEs can be successfully stimulated by P.a. The release of IL-6 and IL-8 was dependent on the direction of stimulation. The stimulation from the apical side led to a higher inflammatory response directed to the apical surface compared to the stimulation from the basolateral side. The cells also showed a lower TEER after apical stimulation. TLR4 was detected in the ALI culture system in pHBEs. High resolution SIM showed that TLR4 localized on the surface of the cells. After stimulation with P.a. TLR4 translocated into the cytoplasm in a time dependent manner.

In summary, the experiments show that TLRs localize on the cell surface in differentiated airway epithelial cells, creating a functional polarization that is also reflected in the release of chemokines and cytokines.

Publication History

Article published online:
09 March 2023