The Role of TRPM4 Channel in Pseudomonas aeruginosa Infection and the Regulation of the Inflammatory Response in Airway Epithelial Cells

Abstract

Bacterial pneumonia is among the leading causes of death worldwide. The emerging resistant strains require the development of new treatment strategies. Ion channels play a role in many signaling pathways of the cell. TRPM4 belongs to the family of TRP-channels (transient receptor potential ion channels), which are membrane bound ion channels expressed in a wide variety of cells. The activity of TRPM4 is induced by rising Ca2+ concentrations in the cytosol. The channel is permissive for Na+ and K+ ions, which lead to a depolarization of the cellular membrane. However, activated by Ca2+, TRPM4 is not permissive for Ca2+. The goal of this work is to investigate the influence of the activity of TRPM4 on the pulmonary innate immune response in the course of bacterial infection. After detecting the expression of TRPM4 in the human bronchial epithelial cell line NCI-H292, the cytotoxicity of the pharmacologic TRPM4 inhibitor 9-Phenanthrol (9-Ph.) and bacterial stimulation with heat inactivated Pseudomonas aeruginosa (Ps.a.) was investigated. The stimulation with heat inactivated Ps.a. or 9-Ph. in the IC50-range had no cytotoxic effects on the cells for at least 18 hr. Most interestingly, the release of IL-6, TNF-α, CXCL2, and S100A8 was inhibited after the pre-treatment with 9-Ph. and stimulation with Ps.a.. This effect was dependent on the concentration of 9-Ph. and was not observed at concentrations of 1/4 - 1/8 of the IC50 value of 9 Ph. Furthermore, the inhibition was not detected on transcriptional level, except for S100A8, whose transcription was also inhibited by the pre-incubation with 9-Ph. These findings were confirmed by TRPM4-specific siRNA, which also resulted in significantly less concentrations of IL-6 after the stimulation with Ps.a.. This indicates that the function of TRPM4 was necessary for the export of inflammatory mediators. This was proved by FACS-Analysis and ELISA. The bronchial-epithelial cell line NCI-H292 and human primary airway epithelial cells were also stimulated with TLR1/2, TLR4, and TLR5-ligands. These experiments showed that the treatment with distinct TLR-ligands can also be influenced by the inhibition of TRPM4 and that the effects observed so far are not due to the inhibition of a certain TLR-signaling pathway. The antimicrobial activity of airway-epithelial cells is mostly mediated by the expression of antimicrobial peptides. The expression of the antimicrobial peptide hBD-2 (human beta-defensin-2) was highly induced in differentiated human primary airway epithelial cells after the stimulation with live Ps.a.. This correlated with increased concentrations of hBD-2 in the cell culture supernatant and increased antimicrobial activity of the cells. In contrast, the inhibition of TRPM4 significantly downregulated the expression of hBD2, the concentration in cell culture supernatant, and the antimicrobial activity of the cells. This indicates a direct connection of the activity of TRPM4 to the innate immune response of the lung. To investigate the influence of TRPM4 on the release of inflammatory cytokines in different cell types and species, monocyte derived macrophages from the bone marrow of mice and the human monocyte cell line U937 were used. Again, the inhibition of TRPM4 resulted in a significantly decreased release of IL-6, TNF-α, CXCL1, and CXCL2. These results were also confirmed with macrophages isolated from TRPM4-deficient mice. To further explore these findings, the lungs of TRPM4-deficient mice were stimulated with heat inactivated Ps.a.. TRPM4-deficient mice showed less inflammation than the corresponding wildtype control animals. These findings are based upon less influx of neutrophilic granulocytes into the lung and lower concentrations of IL-6, TNF-a, CXCL1, and CXCL2 in the broncho-alveolar lavage (BALF) of the TRPM4-deficient animals. Furthermore, the expression of mBD-4, the murine homologue of the human beta-defensin-2, was significantly reduced in the TRPM4-deficient mice. The stimulation of differentiated primary murine tracheal epithelial cells with heat inactivated Ps.a. induced a higher release of KC and MIP-2 if TRPM4 was inhibited. Furthermore, the stimulation of TRPM4-deficient mice with LPS resulted in an increased inflammatory response from the TRPM4-knockout mice compared to the wildtype controls. These differences may result from the different stimulations or the reactivity of the different cell types and possible variations in the immune response of human and mouse cells. These results show that the function of TRPM4 is important for the regulation of inflammation and the innate immune response of the lung.