AIF-1 and RNASET2 play different roles in the modulation of leech innate immune response

In recent years, several studies have demonstrated that the AIF-1 (Allograft Inflammatory Factor-1) and RNASET2 (a member of the ribonuclease T2 family) factors are involved in the activation and modulation of inflammation processes, both in vertebrates and in invertebrates. In particular, it has been demonstrated that both AIF-1 and RNASET2 have a chemotactic activity for macrophages and that their expression significantly increases after bacterial infections. However, the details on the mechanisms by which these evolutionarily conserved proteins regulate the innate immune response are still poorly defined. In order to better understand the specific role of these two factors, we report here the effect of Gram- bacterial lipopolysaccharides (LPS) injection on AIF-1 and RNASET2 expression in an invertebrate model, the medicinal leech. This animal has been chosen for its very simple anatomy and for its notable similarities during inflammatory processes with those of vertebrates. Western blot assays demonstrate that AIF-1 and RNASET2 have a different temporal expression profile. In fact, while RNASET2 has two expression peaks after 30 min and 6 h from LPS injection, AIF-1 shows a peak after 30 min and its expression remains high after 24 h from stimulation. Furthermore, double immunostaining coupling anti-AIF-1 or anti-RNASET2 to an antibody directed against the common granulocyte marker CD11b demonstrates that these two factors are expressed by two different types of cells, whose amount varies over time after LPS stimulation. RNASET2 is mainly localized in the granulocytes, the first cells migrating towards the stimulated area, AIF-1 is instead expressed by macrophages. Taken together, our results clearly suggest that AIF-1 and RNASET2 play a different role in the initial phase of the inflammatory response. We suggest that RNASET2 is at first released by granulocytes in order to induce a massive recruitment of macrophages. Once chemoattracted to the stimulated area, activated macrophages highly express AIF-1 to sustain the recruitment of further macrophages whose role is to rid the area of bacteria.