Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Review
. 2019;11(2):150-167.
doi: 10.1159/000493804. Epub 2018 Oct 26.

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

Nicolò Baranzini  1 , Laura Monti  1 , Marta Vanotti  1 , Viviana T Orlandi  1 , Fabrizio Bolognese  1 , Debora Scaldaferri  1 , Rossana Girardello  1 , Gianluca Tettamanti  1 , Magda de Eguileor  1 , Jacopo Vizioli  2 , Roberto Taramelli  1 , Francesco Acquati  1 , Annalisa Grimaldi  3
Affiliations
Review

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

Nicolò Baranzini et al. J Innate Immun. 2019.

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 RNA-SET2 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.

Keywords: Allograft inflammatory factor-1; Granulocytes; Leech innate immunity; Macrophages; RNASET2.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Drawing representing a general view of leech body in cross section. Under the cuticle and epithelium, the muscle fibers and gut are visible, and between them a loose connective tissue containing the botryoidal and the vasofibrous tissue can be seen. Modified from Grimaldi et al. [55].
Fig. 2
Fig. 2
Morphological analysis of leech body wall at optical and TEM microscopes 30 min after PBS and LPS injection. Few resident macrophages (arrow) and vasofibrous tissue cells (arrowheads) are visible underneath the epithelium (e) or among the muscle fibres (m) in unlesioned (a) and in PBS-injected animals (b). Detailed TEM of macrophages (arrows in c) and of the vasofibrous tissue (d) surrounded by extracellular matrix (ECM) formed by vasocentral cell (vc), with cytoplasm containing a few granules, surrounded by vasofibrous cells (arrowheads), with a cytoplasm containing numerous small highly electron-dense granules. 30 min after LPS injection (e, f), numerous vasofibrous tissue cells are recognizable by their dark color (arrowheads in e) among muscle fibers and underneath the epithelium (e). f Detailed view of type I granulocytes (arrowheads) detached from vasocentral cells (vc) and next to resident macrophages (arrow) localized in the subepithelial region (e). Bars in a, b, e: 100 μm; bar in c, d: 2 μm; bar in f: 10 nm.
Fig. 3
Fig. 3
Morphological analysis of leech body wall at optical and TEM microscopes 6 h after LPS injection. a, b Optical images show numerous macrophages (arrows) and type I granulocytes (arrowheads) underneath the epithelium (e) and localized in the extracellular matrix surrounding the muscle fibers (m). Ultrastructure TEM images show that macrophages (arrows in c) are characterized by pseudopodia (arrow in b1) and are in close contact with type I granulocytes (arrowheads in b1, c, and d), some of which are undergoing the degranulation process (e). n, nuclei. Bars in a, b: 20 μm; bar in b1: 2 μm; bar in c: 4 μm; bar in d, e: 2 μm.
Fig. 4
Fig. 4
AIF-1 and RNASET2 Western blot analysis. Proteins extracted from 3 PBS- and LPS-injected leeches and probed with anti- HmAIF-1 (a) and anti-RNASET2 (b) antibodies. The housekeeping protein D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control, and band intensity appeared to be similar in each loaded sample. The anti- HmAIF-1 antibody detected a specific immunoreactive band of about 18 kDa, while two bands of approximately 36 kDa (the extracellular form) and 29 kDa (the intracellular form) were detected by the anti-RNASET2 antibody. c, d The levels of expression were quantified by densitometry using the Image J software package, and the obtained graphs show the level of expression of the two factors. d The graphic is based on the RNASET2 extracellular form. The individual signals from each lane have been cropped from larger digital images, which are available as supplementary information (see www.karger.com/doi/10.1159/000493804 for all supplementary material). Statistical differences were calculated by one-way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (between PBS and LPS treatments). Means with different letters indicate significant difference between PBS and LPS treatments at different times. Experiments were performed in triplicate, and data represent mean values ± SEM. 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.
Fig. 5
Fig. 5
Immunofluorescence analyses. Immunofluorescence assays (a–h) of leech body wall sections. In unlesioned (a) and PBS-injected animals (b), a few cells located close to the epithelium (e) and among muscle fibers (m) are visible and express HmAIF-1 (in red) and RNASET2 (in green), whereas after LPS injection several migrating immune-responsive cells are visible in the epithelial region and among muscles (m) (d–h). No signal is detected in negative control experiments in which the primary antibodies were omitted (c, i) or substituted by preimmune serum (c1, i1). Cell nuclei stained blue by treatment with DAPI. Bars: 100 μm.
Fig. 6
Fig. 6
Double immunolocalization of RNASET2 and CD11b or RNASET2 and HmAIF-1 in animals 30 min and 6 h following LPS injection. Numerous RNASET2+/CD11b+ type I granulocytes (yellow in a, b) and RNASET2+/ HmAIF-1+ macrophages (yellow in d, e) migrating towards the injected area are visible under the epithelium (e) and among the muscle fibers (m). Double immunostaining was performed with anti-RNASET2 (green) anti-CD11b or anti HmAIF-1 (red). c, f No signal was detected in negative control experiments in which the primary antibodies were omitted. Cell nuclei stained blue by treatment with DAPI. Bars: 50 μm. g The percentages of granulocytes (RNASET2+/CD11b+) and of macrophages (RNASET2+/CD11b–) were assessed by analyzing 5 different slides (10 random fields of 45,000 μm2 for each slide) using the Image J software package. Statistical differences were calculated by factorial ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant (between PBS- and LPS-challenged leeches). Means with different letters indicate significant difference between the number of granulocytes and macrophages in untreated animals.
Fig. 7
Fig. 7
Immunogold staining of RNASET2. Type I granulocytes (arrowhead in a) present in the extracellular matrix (ECM). b Detailed TEM showing the localization of gold particles in the granules (arrows). c, d Negative controls. Bar in a: 2.5 μm; bars in b, c, d: 300 nm.
Fig. 8
Fig. 8
In vitro and in vivo analysis. P. aeruginosa PAO1 cells expressing GFP and co-cultured with RNASET2 silenced THP-1 cells (b, d, and inset) maintained their typical rod morphology, and GFP signal was localized inside the bacteria. On the other hand, in the presence of THP-1 expressing RNASET2 cells (a, c, and inset), GFP signal is widespread, indicating the PAO1 are in a stressful condition. Cryosection of leech body wall injected with GFP/PAO1 cells and an antibody anti-RNASET2 (e–g) or injected with GFP/PAO1 alone (h–j). e Violet and fuchsin staining shows the presence of bacteria agglomerates. GFP (f) and DAPI (g) signals highlight the characteristic rod shape of these bacteria. In absence of the functional blocking antibody anti-RNASET2, no bacteria are visible (h), and GFP (i) and DAPI (j) show a diffuse staining. The squares in e, h indicate the magnified areas of f, g, i, j. a, b. The merge of fluorescent channel with transmission image shows the position of bacteria with respect to eukaryotic cells. Bars in a–d: 15 μm; bars in e–j: 4 μm.
Fig. 9
Fig. 9
Representation to explain the different but complementary roles of RNASET2 and AIF-1 in early inflammatory response. 30 min after LPS stimulation, activated granulocytes secrete RNASET2, whose first effect is to carry out a direct antibacterial activity. In parallel, LPS-activated macrophages release AIF-1 in order to recruit other macrophages. These cells, releasing RNASET2, maintain the inflammatory state by recruiting other macrophages involved in cleaning the infected area from bacterial debris.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Frantz S, Vincent KA, Feron O, Kelly RA. Innate immunity and angiogenesis. Circ Res. 2005 Jan;96((1)):15–26. - PubMed
    1. Baranzini N, Pedrini E, Girardello R, Tettamanti G, de Eguileor M, Taramelli R, et al. Human recombinant RNASET2-induced inflammatory response and connective tissue remodeling in the medicinal leech. Cell Tissue Res. 2017 May;368((2)):337–51. - PubMed
    1. Schorn T, Drago F, Tettamanti G, Valvassori R, de Eguileor M, Vizioli J, et al. Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech. Cell Tissue Res. 2015 Mar;359((3)):853–64. - PubMed
    1. Schorn T, Drago F, de Eguileor M, Valvassori R, Vizioli J, Tettamanti G, et al. 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. Invertebrate Surviv J. 2015;1:129–41.
    1. Utans U, Arceci RJ, Yamashita Y, Russell ME. Cloning and characterization of allograft inflammatory factor-1: a novel macrophage factor identified in rat cardiac allografts with chronic rejection. J Clin Invest. 1995 Jun;95((6)):2954–62. - PMC - PubMed

Publication types

MeSH terms