Legionella pneumophila-Induced NETs Do Not Bear LL-37 Peptides

Legionella pneumophila (L. pneumophila) infection is characterized by a wide spectrum of manifestations, from influenza-like illness to life-threatening atypical pneumonia with multiorgan failure. The aim of our study was the assessment of in vitro and ex vivo neutrophil activation in L. pneumophila infections, as well as the role of neutrophils' peptides such as LL-37 in infection. The ability of neutrophils to form ex vivo extracellular traps (NETs) in response to bacterial infection was examined by immunofluorescence. In parallel, patients' sera, as well as opsonized standard L. pneumophila strains, were used for in vitro activation of neutrophils from healthy individuals. The serum levels of interleukins were assessed using the LEGENDplexTM Multi-Analyte Flow Assay Kit. Furthermore, citrullinated cf-DNA as a marker of neutrophil extracellular traps (NETs) was detected in the serum of patients with acute infection. It was demonstrated that neutrophils released NETs in vitro and ex vivo upon L. pneumophila (interaction in an autophagy-independent manner. Notably, IL-1b was detected on NETs, but an antimicrobial peptide LL-37 was absent. The lack of antimicrobial activity failed to inhibit bacterial proliferation. In addition, in vitro and ex vivo NETs formation was observed during the Clarithromycin treatment. Those NETs were decorated with bioactive antimicrobial peptide LL-37, which inhibits bacterial proliferation. The findings provide evidence that neutrophils release NETs in vitro and ex vivo by expressing the IL1β protein in them. The lack of expression of the antimicrobial peptide LL-37 on the NETs demonstrates the inability of the cells to inhibit proliferation, and consequently the elimination of L. pneumophila. Clarithromycin plays a dual role in the elimination.