LL-37 and citrullinated-LL-37 enhances oxylipins: citrullination attenuates LL-37-mediated COX-2-dependent chemokine response in human bronchial epithelial cells

During airway inflammation, chemokines, oxylipins (bioactive lipids) and cationic host defence peptides (CHDP) are enhanced in the lungs. However, the interplay of these molecules in the process of airway inflammation is not fully resolved. The human cathelicidin CHDP, LL-37, can enhance the expression of chemokines which is turn facilitates influx of leukocytes into the lungs. Moreover, LL-37 can be citrullinated during inflammation and the effect of this post-translational modification on LL-37-mediated immunomodulatory functions remains unclear. Therefore, in this study we aimed to define the impact of LL-37 and citrullinated-LL-37 (citLL-37) on oxylipins and its association with downstream chemokine production in human bronchial epithelial cells (HBEC), and its functional impact on leukocyte influx. We used a lipidomics approach to identify oxylipins that are enhanced in response to LL-37 and citLL-37 in HBEC. We further examined the role of selected oxylipins in LL-37- and citLL-37-mediated chemokine production by ELISA, and related leukocyte migration using a transwell migration assay. We showed that LL-37, but not citLL-37, enhances oxylipins that are known to promote inflammation such as prostaglandins regulated by the cyclooxygenase (COX pathway). LL-37-mediated increase in COX-2 expression was significantly higher than that mediated by citLL-37. We showed that upregulation of COX-2 expression was dependent on the P2X7 purinergic receptor. Our mechanistic studies revealed that LL-37-mediated increase in chemokines, GROα, IL-8 and MIP-3α, was dependent on the COX-2 pathway, and that this was significantly attenuated by citrullination of the peptide. Our results also indicated that COX-2-induced PGE2 may act in an autocrine manner signaling through its EP receptors to facilitate LL-37-induced chemokine production. We functionally confirmed that factors secreted from HBEC in response to LL-37, but not citLL-37, promotes neutrophil migration which is COX-2 dependent. The results of this study indicate that pro-inflammatory responses mediated by LL-37 is attenuated by citrullination of the peptide. The findings of this study underscore the role of LL-37 in influencing the enhancement of bioactive lipids and metabolic pathways such as COX-2 and its link to the peptide-mediated immunomodulatory functions in the lungs.