The di-leucine motif in the host defense peptide LL-37 is essential for initiation of autophagy in human macrophages.

The human cathelicidin peptide LL-37 induces autophagy in human macrophages. Different post-translational modifications (PTMs) such as citrullination, acetylation, and formylation impact LL-37, yet their effect on autophagy remains unknown. Thus, we set out to study how the cellular source could impact PTM of LL-37 and subsequent effects on autophagy initiation. Neutrophil-released LL-37 failed to induce autophagy, unlike macrophage-released LL-37. Mass spectrometry analysis revealed modifications on neutrophil-derived LL-37, especially at the N terminus, while macrophage-derived LL-37 remained mostly native. Native LL-37 initiated autophagy, while formylated and acetylated versions did not. Truncated peptides lacking the N-terminal di-leucine motif or substituted with di-alanine did not initiate autophagy. Native LL-37 failed to initiate autophagy in macrophages with genetic inactivation of dipeptidyl peptidase-1. An intact N-terminal di-leucine motif in LL-37 was crucial for autophagy initiation, and modifications abrogated the effects. This pathway presents a novel way to regulate the effects of LL-37 in infection or inflammation.