Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech

Abstract

In recent years, several studies have demonstrated that the RNASET2 gene is involved in the control of tumorigenicity in ovarian cancer cells. Furthermore, a role in establishing a functional cross-talk between cancer cells and the surrounding tumor microenvironment has been unveiled for this gene, based on its ability to act as an inducer of the innate immune response. Although several studies have reported on the molecular features of RNASET2, the details on the mechanisms by which this evolutionarily conserved ribonuclease regulates the immune system are still poorly defined. In the effort to clarify this aspect, we report here the effect of recombinant human RNASET2 injection and its role in regulating the innate immune response after bacterial challenge in an invertebrate model, the medicinal leech. We found that recombinant RNASET2 injection induces fibroplasias, connective tissue remodeling and the recruitment of numerous infiltrating cells expressing the specific macrophage markers CD68 and HmAIF1. The RNASET2-mediated chemotactic activity for macrophages has been further confirmed by using a consolidated experimental approach based on injection of the Matrigel biomatrice (MG) supplemented with recombinant RNASET2 in the leech body wall. One week after injection, a large number of CD68 and HmAIF-1 macrophages massively infiltrated MG sponges. Finally, in leeches challenged with lipopolysaccharides (LPS) or with the environmental bacteria pathogen Micrococcus nishinomiyaensis, numerous macrophages migrating to the site of inoculation expressed high levels of endogenous RNASET2. Taken together, these results suggest that RNASET2 is likely involved in the initial phase of the inflammatory response in leeches.

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

“…We have previously reported that AIF-1 and RNASET2 are significantly upregulated at 6 and 24 h following LPS injection, and that this change in their expression is involved in leech macrophage recruitment [2, 3]. However, the underlying mechanisms by which these two factors operate are still poorly characterized.…”

“…Cells in the chosen fields were counted by hand as macrophages if they were RNASET2 + FITC labelled [2] or as granulocytes if they were CD11b + /RNASET2 + (yellow labelled as a result of CY5/FITC double staining). Statistical analyses were performed using Statistica 7.0 software (StatSoft Inc., Tulsa, OK, USA), and differences were calculated by one-way ANOVA followed by Fisher’s post hoc test, and p < 0.05 was considered statistically significant.…”

“…Among these cytokines, two macrophage-derived interesting molecules have been recently demonstrated to be involved in inflammatory responses and tissue regeneration: allograft inflammatory factor-1 (AIF-1) and the RNASET2, a member of ribonuclease T2 family [2-4]. …”

“…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.…”

“…We have previously demonstrated that the injection in the leech body wall of LPS induces a massive migration of macrophages towards the stimulated area. These immune cells in turn express and produce AIF-1 and RNASET2, both involved in other macrophage recruitment [2, 4]. …”