The RNASET2 ribonuclease, belonging to the highly conserved RH/T2/s RNase gene family, has been recently shown to modulate inflammatory processes in both vertebrates and invertebrates. Indeed, the RNASET2 protein acts as a chemoattractor for macrophages in both in vitro and in vivo experimental settings and its expression significantly increases following bacterial infections. Moreover, we recently observed that injection of human recombinant RNASET2 protein in the body wall of the medicinal leech (a consolidated invertebrate model for both immune response and tissue regeneration) not only induced immune cell recruitment but also apparently triggered massive connective tissue remodelling as well. Based on these data, we evaluate here a possible role of leech recombinant RNASET2 protein (rHvRNASET2) in connective tissue remodelling by characterizing the cell types involved in this process through histochemical, morphological and immunofluorescent assays. Moreover, a time-course expression analysis of newly synthesized pro-collagen1α1 (COL1α1) and basic FGF receptor (bFGFR, a known fibroblast marker) following rHvRNASET2 injection in the leech body wall further supported the occurrence of rHvRNASET2-mediated matrix remodelling. Human MRC-5 fibroblast cells were also investigated in order to evaluate their pattern of collagen neosynthesis driven by rHvRNASET2 injection.
Taken together, the data reported in this work provide compelling evidence in support of a pleiotropic role for RNASET2 in orchestrating an evolutionarily conserved crosstalk between inflammatory response and regenerative process, based on macrophage recruitment and fibroblast activation, coupled to a massive extracellular reorganization.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Acquati F, Bertilaccio S, Grimaldi A et al (2011) Microenvironmental control of malignancy exerted by RNASET2, a widely conserved extracellular RNase. Proc Natl Acad Sci U S A 108:1104–1109. https://doi.org/10.1073/pnas.1013746108
Aurora AB, Olson EN (2014) Immune modulation of stem cells and regeneration. Cell Stem Cell 15:14–25. https://doi.org/10.1016/j.stem.2014.06.009
Baranzini N, Pedrini E, Girardello R et al (2017) Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech. Cell Tissue Res 368:337–351. https://doi.org/10.1007/s00441-016-2557-9
Baranzini N, Monti L, Vanotti M et al (2018) AIF-1 and RNASET2 play complementary roles in the innate immune response of medicinal leech. J Innate Immun:1–18. https://doi.org/10.1159/000493804
Baranzini N, De Vito A, Orlandi VT et al (2020). Antimicrobial role of RNASET2 protein during innate immune response in the medicinal leech Hirudo verbana. Submitted in Frontiers Immunology
Berse B, Brown LF, Van de Water L et al (1992) Vascular permeability factor (vascular endothelial growth factor) gene is expressed differentially in normal tissues, macrophages, and tumors. Mol Biol Cell 3:211–220. https://doi.org/10.1091/mbc.3.2.211
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Campomenosi P, Cinquetti R, Tallarita E et al (2011) Comparison of the baculovirus-insect cell and Pichia pastoris heterologous systems for the expression of the human tumor suppressor protein RNASET2. Biotechnol Appl Biochem 58:39–49. https://doi.org/10.1002/bab.7
Chujo S, Shirasaki F, Kondo-Miyazaki M et al (2009) Role of connective tissue growth factor and its interaction with basic fibroblast growth factor and macrophage chemoattractant protein-1 in skin fibrosis. J Cell Physiol 220:189–195. https://doi.org/10.1002/jcp.21750
Cooke JP, Sayed N, Lee J et al (2014) Innate immunity and epigenetic plasticity in cellular reprogramming. Curr Opin Genet Dev ;28:89–91. https://doi.org/10.1016/j.gde.2014.11.002. Review.
Daley WP, Peters SB, Larsen M (2008) Extracellular matrix dynamics in development and regenerative medicine. J Cell Sci 121:255–264. https://doi.org/10.1242/jcs.006064
de Eguileor M, Grimaldi A, Boselli A et al (1999a) Possible roles of extracellular matrix and cytoskeleton in leech body wall muscles. J Microsc 196:6–18
de Eguileor M, Tettamanti G, Grimaldi A et al (1999b) Histopathological changes after induced injury in Leeches1. J Invertebr Pathol 74:14–28. https://doi.org/10.1006/jipa.1999.4850
de Eguileor M, Grimaldi A, Tettamanti G et al (2000a) Different types of response to foreign antigens by leech leukocytes. Tissue Cell 32:40–48
de Eguileor M, Grimaldi A, Tettamanti G et al (2000b) Lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers. https://doi.org/10.1054/tice.2000.0132
de Eguileor M, Grimaldi A, Tettamanti G et al (2001) Hirudo medicinalis: a new model for testing activators and inhibitors of angiogenesis. Angiogenesis 4:299–312
de Eguileor M, Tettamanti G, Grimaldi A et al (2003) Leeches: immune response, angiogenesis and biomedical applications. Curr Pharm Des 9:133–147
de Eguileor M, Tettamanti G, Grimaldi A et al (2004) Hirudo medicinalis: avascular tissues for clear-cut angiogenesis studies? Curr Pharm Des 10:1979–1988
Eming SA, Hammerschmidt M, Krieg T, Roers A (2009) Interrelation of immunity and tissue repair or regeneration. Semin Cell Dev Biol 20(5):517–527. https://doi.org/10.1016/j.semcdb.2009.04.009.
Farup J, Madaro L, Puri PL, Mikkelsen UR (2015) Interactions between muscle stem cells, mesenchymal-derived cells and immune cells in muscle homeostasis, regeneration and disease. Cell Death Dis 6:1–13. https://doi.org/10.1038/cddis.2015.198
Frantz S, Vincent KA, Feron O, Kelly RA (2005) Innate immunity and angiogenesis. Circ Res 96:15–26. https://doi.org/10.1161/01.RES.0000153188.68898.ac
Girardello R, Baranzini N, Molteni M et al (2019) The medicinal leech as a valuable model for better understanding the role of a TLR4-like receptor in the inflammatory process. Cell Tissue Res:1–13. https://doi.org/10.1007/s00441-019-03010-0
Glaros T, Larsen M, Li L (2009) Macrophages and fibroblasts during inflammation, tissue damage and organ injury. Front Biosci 14:3988–3993
Godwin JW, Brockes JP (2006) Regeneration, tissue injury and the immune response. J Anat 209:423–432. https://doi.org/10.1111/j.1469-7580.2006.00626.x
Godwin JW, Rosenthal N (2014) Scar-free wound healing and regeneration in amphibians: immunological influences on regenerative success. Differentiation 87:66–75. https://doi.org/10.1016/J.DIFF.2014.02.002
Godwin JW, Debuque R, Salimova E, Rosenthal NA (2017) Heart regeneration in the salamander relies on macrophage-mediated control of fibroblast activation and the extracellular landscape. NPJ Regen Med 2:22. https://doi.org/10.1038/s41536-017-0027-y
Grimaldi A, Tettamanti G, Rinaldi L et al (2004) Role of cathepsin B in leech wound healing. Invertebr Surviv J:38–46
Grimaldi A, Tettamanti G, Perletti G et al (2006) Hematopoietic cell formation in leech wound healing. Curr Pharm Des 12:3033–3041
Grimaldi A, Tettamanti G, de Eguileor M (2018) Annelida: Hirudinea (leeches): heterogeneity in leech immune responses. In: Advances in comparative immunology. Springer International Publishing, Cham, pp 173–191
Gurtner GC, Werner S, Barrandon Y, Longaker MT (2008) Wound repair and regeneration. Nature 453:314–321. https://doi.org/10.1038/nature07039
Jenkins SJ, Ruckerl D, Cook PC et al (2011) Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 332(6035):1284–1288
Jenkins SJ, Ruckerl D, Thomas GD et al (2013) IL-4 directly signals tissue-resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1. J Exp Med 210:2477–2491. https://doi.org/10.1084/jem.20121999
Julier Z, Park AJ, Briquez PS, Martino MM (2017) Promoting tissue regeneration by modulating the immune system. Acta Biomater 53:13–28. https://doi.org/10.1016/j.actbio.2017.01.056
Lai SL, Marín-Juez R, Moura PL et al (2017) Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration. Elife 6:1–20. https://doi.org/10.7554/eLife.25605
Lee J, Sayed N, Hunter A et al (2012) Activation of innate immunity is required for efficient nuclear reprogramming. Cell 151(3):547–558. https://doi.org/10.1016/j.cell.2012.09.034
Liu S, Tobias R, McClure S et al (1997) Removal of endotoxin from recombinant protein preparations. Clin Biochem 30:455–463. https://doi.org/10.1016/S0009-9120(97)00049-0
Luhtala N, Parker R (2010) T2 family ribonucleases: ancient enzymes with diverse roles. Trends Biochem Sci 35:253–259. https://doi.org/10.1016/j.tibs.2010.02.002
Macagno ER, Gaasterland T, Edsall L et al (2010) Construction of a medicinal leech transcriptome database and its application to the identification of leech homologs of neural and innate immune genes. BMC Genomics 11:407. https://doi.org/10.1186/1471-2164-11-407
Malagoli D (2018) Going beyond a static picture: the apple snail Pomacea canaliculata can tell us the life history of molluscan hemocytes. Invertebr Surviv J 15:61–65
Mastellos DC, DeAngelis RA, Lambris JD (2013) Complement-triggered pathways orchestrate regenerative responses throughout phylogenesis. Semin Immunol 25:29–38. https://doi.org/10.1016/j.smim.2013.04.002
Mescher AL, Neff AW (2005) Regenerative capacity and the developing immune system. Adv Biochem Eng Biotechnol 93:39–66. https://doi.org/10.1007/b99966
Mescher AL, Neff AW, King MW (2017) Inflammation and immunity in organ regeneration. Dev Comp Immunol 66:98–110. https://doi.org/10.1016/j.dci.2016.02.015
Moore RD, Mumaw V, Schoenberg MD (1960) Optical microscopy of ultrathin tissue sections. J Ultrastruct Res 4:113–116. https://doi.org/10.1016/S0022-5320(60)90047-2
Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11:723–737. https://doi.org/10.1038/nri3073
Peiris TH, Hoyer KK, Oviedo NJ (2014) Innate immune system and tissue regeneration in planarians: an area ripe for exploration. Semin Immunol 26:295–302. https://doi.org/10.1016/j.smim.2014.06.005
Portou MJ, Baker D, Abraham D, et al (2015) The innate immune system, toll-like receptors and dermal wound healing: A review. Vascul Pharmacol 71:31–36. https://doi.org/10.1016/j.vph.2015.02.007.
Rappolee DA, Mark D, Banda MJ, Werb Z (1988) Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science 241:708–712. https://doi.org/10.1126/science.3041594
Roggiani F, Riva C, Raspagliesi F et al (2019) A cell-autonomous oncosuppressive role of human RNASET2 affecting ECM-mediated oncogenic signaling. Cancers (Basel) 11:255. https://doi.org/10.3390/cancers11020255
Sawyer RT (1986) Leech biology and behaviour. Clarendon Press
Schorn T, Drago F, de Eguileor M et al (2015a) The allograft inflammatory factor-1 ( AIF-1 ) homologous in Hirudo medicinalis ( medicinal leech ) is involved in immune response during wound healing and graft rejection processes. Invertebr Surviv J 1:129–141
Schorn T, Drago F, Tettamanti G et al (2015b) Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech. Cell Tissue Res 359:853–864. https://doi.org/10.1007/s00441-014-2054-y
Sciorati C, Rigamonti E, Manfredi AA, Rovere-Querini P (2016) Cell death, clearance and immunity in the skeletal muscle. Cell Death Differ 23:927–937. https://doi.org/10.1038/cdd.2015.171
Shimokado K, Raines EW, Madtes DK et al (1985) A significant part of macrophage-derived growth factor consists of at least two forms of PDGF. Cell 43:277–286. https://doi.org/10.1016/0092-8674(85)90033-9
Tettamanti G, Grimaldi A, Ferrarese R et al (2003a) Leech responses to tissue transplantation. Tissue Cell 35:199–212. https://doi.org/10.1016/S0040-8166(03)00027-2
Tettamanti G, Grimaldi A, Valvassori R et al (2003b) Vascular endothelial growth factor is involved in neoangiogenesis in Hirudo medicinalis (Annelida, Hirudinea). Cytokine 22:168–179
Tettamanti G, Grimaldi A, Rinaldi L et al (2004) The multifunctional role of fibroblasts during wound healing in Hirudo medicinalis (Annelida, Hirudinea). Biol Cell 96:443–455. https://doi.org/10.1016/j.biolcel.2004.04.008
Tettamanti G, Grimaldi A, Congiu T et al (2005) Collagen reorganization in leech wound healing. Biol Cell 97:557–568. https://doi.org/10.1042/BC20040085
Willenborg S, Lucas T, van Loo G et al (2012) CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair. Blood 120:613–625. https://doi.org/10.1182/blood-2012-01-403386
Würden S, Homberg U (1993) A simple method for immunofluorescent double staining with primary antisera from the same species. J Histochem Cytochem 41:627–630. https://doi.org/10.1177/41.4.8450202
Wynn TA, Vannella KM (2016) Macrophages in tissue repair, regeneration, and fibrosis. Immunity 44:450–462. https://doi.org/10.1016/j.immuni.2016.02.015
Zhao A, Qin H, Fu X (2016) What determines the regenerative capacity in animals? Bioscience 66:735–746. https://doi.org/10.1093/biosci/biw079
Nicolò Baranzini is a PhD student of the Biotechnology, Biosciences and Surgical Technology course at the University of Insubria.
This work was supported by the CARIPLO foundation (URL http://wwwfondazionecariplo.it), FRAMYEVO to MdE, grant code 2016–0835 and by FAR 2017–2018 (Fondi dell’Ateneo per la Ricerca, University of Insubria) to AG, FA and GT.
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies performed by any of the authors with animals requiring ethical approval.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Baranzini, N., Weiss-Gayet, M., Chazaud, B. et al. Recombinant HvRNASET2 protein induces marked connective tissue remodelling in the invertebrate model Hirudo verbana. Cell Tissue Res 380, 565–579 (2020). https://doi.org/10.1007/s00441-020-03174-0
- Connective tissue remodelling
- Medicinal leech