In vitro characterization of the cytokine Drosophila Helical factor

Drosophila Helical factor (Hf) is a protein discovered through the QT method, an algorithm specifically designed for finding helical cytokines. Since in vivo experiments suggested the involvement of Hf in Drosophila melanogaster immunity, we have proceeded with the characterization of Hf functions in the macrophage-like Drosophila embryonic hemocytes, SL2 cell line. qPCR results demonstrated that Hf gene is induced in the SL2 cell line, after either 6 or 24 h incubation with Escherichia coli-purified peptidoglycan. The silencing of Hf expression through RNAi resulted in the reduced capability of synthesizing antimicrobial peptides (AMP) after exposure to heat-inactivated E. coli. The effects of the recombinant peptide rHf have also been tested in the SL2 cell line. rHf promotes the expression and triggers the release of Hf from the hemocytes, and stimulates the synthesis of the antimicrobial peptides (AMP) Defensin and Drosomycin, without any further immune stimulation. Consistent with the output of the QT method, which predicts Hf as a secreted protein, chromatin immune-precipitation experiments confirmed that Hf does not bind DNA, excluding that it acts as an immune-regulated transcription factor. Finally, rHf neither exerts chemotactic action nor triggers bacterial phagocytosis in SL2 cells.Altogether, our data supports the prediction that Hf is an helical cytokine produced and secreted by the hemocytes and it is mainly involved in the regulation of the humoral component of the immune response of D. melanogaster.