Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium‐binding adapter molecule 1 (Iba1/alias aif‐1)

Abstract

The Ionized calcium-Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF-1), is a 17 kDa cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF-1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events.

AIF-1 and RNASET2 Play Complementary Roles in the Innate Immune Response of Medicinal Leech

“…Indeed, a gene showing high similarity with vertebrates’ AIF-1, named Hm Iba1/alias Hm AIF-1, has been recently characterized in the central nervous system of the medicinal leech [11]. Of note, the expression of AIF-1 in leech has been reported to increase significantly after transplantation, wound healing, or bacterial infections, suggesting its involvement in the inflammatory response and in immune system regulation.…”

“…Proteins separated by SDS-PAGE were transferred onto Bio-Rad nitrocellulose filters. Membranes were then saturated with 5% nonfat dried milk in Tris-buffered saline (TBS; 20 mM TRIS-HCl buffer, 500 mM NaCl, pH 7.5) at room temperature for 2 h and incubated for 90 min with a rabbit polyclonal anti-RNASET2 [30] or an anti- Hm AIF-1 [11] antibody (1: 5,000 dilution in 5% TBS-milk). After three washes with TBS-Tween 0.1%, antigens were revealed with a secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories), diluted at 1: 5,000.…”

“…Immunocomplexes were revealed with luminol LiteAblot PLUS Enhanced Chemiluminescent Substrate (EuroClone, Pero, Italy). In control experiments, anti-RNASET2 [13, 14] and anti- Hm AIF-1 (specific for medicinal leech macrophage [4, 11]) antibodies were substituted with rabbit preimmune serum (1: 20,000). Bands were normalized by using the ImageJ software package (http://rsbweb.nih.gov/ij/download.html), and the housekeeping protein D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), used as an internal reference, was detected with a rabbit polyclonal anti-human GAPDH IgG (Proteintech, Chicago, IL, USA), diluted at 1: 2,000.…”

“…In fact, our previous investigations showed that injection in leeches of human cytokines and growth factors promotes hematopoiesis, vascular growth, immune cell migration, and myofibroblast differentiation [17]. Among these factors, AIF-1 and RNASET2 have recently been shown to play a pivotal role in the leech immune response and tissue repair [2, 3, 11]. Indeed, expression of both factors is significantly increased following lipopolysaccharides (LPS) or bacterial infection in leeches.…”

“…Subsequently, several AIF-1-like factors showing high amino acid sequence conservation have been identified in other metazoans (both vertebrates [6, 7] and invertebrates [8-11]). AIF-1 expression was shown to increase significantly after transplantation, wound healing, or bacterial infections, strongly suggesting its involvement in the inflammatory response and in immune system regulation by attracting macrophages to the challenged area.…”