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

Abstract

Recent studies demonstrated that allograft inflammatory factor-1 (AIF-1) and RNASET2 act as chemoattractants for macrophages and modulate the inflammatory processes in both vertebrates and invertebrates. The expression of these proteins significantly increases after bacterial infection; however, the mechanisms by which they regulate the innate immune response are still poorly defined. Here, we evaluate the effect of bacterial lipopolysaccharide injection on the expression pattern of these genes and the interrelation between them during innate immune response in the medicinal leech, an invertebrate model with a simple anatomy and a marked similarity with vertebrates in inflammatory processes. Collectively, prokaryotic-eukaryotic co-cultures and in vivo infection assays suggest that RNASET2 and AIF-1 play a crucial role in orchestrating a functional cross-talk between granulocytes and macrophages in leeches, resulting in the activation of an effective response against pathogen infection. RNASET2, firstly released by granulocytes, likely plays an early antibacterial role. Subsequently, AIF-1⁺ RNASET2-recruited macrophages further recruit other macrophages to potentiate the antibacterial inflammatory response. These experimental data are in keeping with the notion of RNA­SET2 acting as an alarmin-like molecule whose role is to locally transmit a “danger” signal (such as a bacterial infection) to the innate immune system in order to trigger an appropriate host response.

Antimicrobial Role of RNASET2 Protein During Innate Immune Response in the Medicinal Leech Hirudo verbana

“…Noteworthy, we were able to confirm the pro-inflammatory role of T2 RNases in this invertebrate model, carried out by means of recruitment and activation of cells from the monocyte/macrophage lineage, as previously defined in mammalian experimental models (10). This finding clearly suggests that stimulation of innate immunitymediated host defense represents a key evolutionarily conserved role for T2 RNases (16,17).…”

“…By contrast, starting from 30 min following LTA injection, newly formed vessels and many infiltrating immune cells were clearly observable underneath the epithelium and in the ECM surrounding the muscle fibers ( Figures 1B-H). In particular, several granulocytes were clearly recognizable both by light ( Figure 1D) and TEM microscope analysis ( Figure 1G) (17,30). At 6 h post-injection, a high number of macrophages infiltrating the injected area (characterized by a ruffled surface due to the presence of pseudopodia, a typical feature of migrating cells) were readily detected as well ( Figures 1E,F,H).…”

“…We next carried out double immunofluorescence assays with anti-RNASET2 and anti-CD11b antibodies, coupled to immunogold experiments at TEM, in order to show the presence of HvRNASET2 in the granules of leech granulocytes ( Figures 5A-B ′′ ), as previously reported in LPS-stimulated leeches (17). In addition, HvRNASET2 immunolocalization on cryosections from control and LTA-injected leech body wall showed that, in control, PBS-injected animals, HvRNASET2 was expressed at a basal level (Figure 5C), whereas 30 min after LTA treatment, its expression gradually increased, reaching a peak after 6 and 24 h from stimulation ( Figures 5D-H).…”

“…Furthermore, we found experimental evidence in support of a putative role for T2 RNases in host defense against bacterial infections as well, since lipopolysaccharide (LPS) injection into the leech body wall triggered a marked increase in the expression levels of endogenous leech T2 RNase (named HvRNASET2) protein in both host macrophages and granulocytes. Moreover, recombinant HvRNASET2 was apparently able to affect bacterial cell integrity in vivo (16,17).…”

Diminished Adherence of Biomphalaria Glabrata Embryonic Cell Line to Sporocysts of Schistosoma Mansoni Following Programmed Knockout of the Allograft Inflammatory Factor

“…The Bge cell line has been studied extensively to interrogate the host-parasite relationship because the Bge cell exhibits a hemocyte-like behavior that includes encapsulation of the larval parasite [20][21][22][23][24][25][26][27][28]. The genome sequence of B. glabrata has been reported [29], along with ongoing transcriptome and proteome catalogues that include factors participating in immunological surveillance, phagocytosis, cytokine responses, and pathogen recognition receptor elements including Toll-like receptors and brinogen-related proteins [30][31][32][33][34][35][36]. An orthologue of the evolutionary conserved allograft in ammatory factor (AIF), which is a conserved protein typically expressed in phagocytes and granular leukocytes in both vertebrate and invertebrate.…”