Drosophila Helical factor is an inducible protein acting as an immune-regulated cytokine in S2 cells Davide Malagoli a , Alice Accorsi a , Sandro Sacchi b , Valentina Basile a , Mauro Mandrioli a , Marcello Pinti c , Darrell Conklin d,e , Enzo Ottaviani a,⇑ a Department of Biology, University of Modena and Reggio Emilia, Modena, Italy b Department of Medicine, Endocrinology, Metabolism and Geriatrics, University of Modena and Reggio Emilia, Modena, Italy c Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy d Department of Computer Science and Artificial Intelligence, University of the Basque Country UPV/EHU, San Sebastián, Spain e IKERBASQUE, Basque Foundation for Science, Bilbao, Spain article info Article history: Received 23 May 2011 Received in revised form 3 February 2012 Accepted 4 February 2012 Available online 2 March 2012 Keywords: Cytokine Immunity Insect Immune signaling abstract The innate immunity of Drosophila melanogaster is based on cellular and humoral components. Drosophila Helical factor (Hf), is a molecule previously discovered using an in silico approach and whose expression is controlled by the immune deficiency (Imd) pathway. Here we present evidence demonstrating that Hf is an inducible protein constitutively produced by the S2 hemocyte-derived cell line. Hf expression is stim- ulated by bacterial extracts that specifically trigger the Imd pathway. In absence of any bacterial chal- lenge, the recombinant form of Hf can influence the expression of the antimicrobial peptides (AMPs) defensin but not drosomycin. These data suggest that in vitro Hf is an inducible and immune-regulated fac- tor, with functions comparable to those of secreted vertebrate cytokines. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction As in mammals, insect innate immunity relies on cellular and humoral components [1,2]. These two elements are intercon- nected, and the latter, in particular, has been well characterized in insects starting from studies on the larval fat body of the fruit fly Drosophila melanogaster [3]. Among humoral factors, the antimi- crobial peptides (AMPs) are the molecules whose structures and functions have been elucidated [4], whereas Drosophila cytokines are less characterized. In mammals, cytokines are chemical media- tors involved in immune, neuroendocrine and developmental func- tions. In invertebrates other than D. melanogaster, cytokines have also been retrieved [5–12]. Drosophila and the majority of other invertebrate cytokines display no relevant similarity of primary se- quences with mammalian counterparts, and the term cytokine it- self has been attributed on a functional criterion, based on an involvement in immunity and development. The Drosophila cytokine Spätzle is the activator of the Toll path- way, a cascade activated mainly by Gram positive bacteria and fun- gi [13]. The protein Spätzle acts through the binding of the receptor Toll and one of its typical effects is the induction of the AMP gene drosomycin [14]. Recently it has also been demonstrated that the secretion of Spätzle by the hemocytes is fundamental to set up a proper systemic response against septic injury or oral infection [15] and also molecular details underpinning Spätzle processing before its interactions with Toll have been provided [16]. A second cytokine observed in D. melanogaster, Unpaired (Upd)- 3, is secreted by hemocytes after septic injury and activates the JAK-STAT pathway in the fat body. However, the control of Upd-3 secretion has so far not been fully clarified [17], whereas its expression seems not to depend from the Imd-related kinase dTAK1 [18]. Finally, we have isolated the gene of a putative helical cytokine, Drosophila Helical factor [19], now indexed as Helical fac- tor (Dmel/Hf) in Flybase. Hf expression in the S2 hemocytes is dependent on the Imd pathway [18], the signaling pathway con- trolling the response to Gram negative bacteria [20]. In the present study, our aim has been to obtain more direct evi- dence of the role of Hf as a cytokine, providing further information on the action it may play on the immune function of cultured hemocytes. Our results indicate that Hf expression is significantly increased after exposure to Escherichia coli-purified peptidoglycan 1043-4666/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2012.02.002 Abbreviations: AMP, antimicrobial peptide; Hf, Drosophila Helical factor; Imd, immune deficiency; S2, Schneider’s Line 2. ⇑ Corresponding author. Address: Department of Biology, University of Modena and Reggio Emilia, Via Campi 213/D, 41125 Modena, Italy. Tel.: +39 059 2055536; fax: +39 059 2055548. E-mail address: enzo.ottaviani@unimore.it (E. Ottaviani). Cytokine 58 (2012) 280–286 Contents lists available at SciVerse ScienceDirect Cytokine journal homepage: www.elsevier.com/locate/issn/10434666