Advertisement

The medicinal leech as a valuable model for better understanding the role of a TLR4-like receptor in the inflammatory process

  • Rossana Girardello
  • Nicolò Baranzini
  • Monica Molteni
  • Carlo Rossetti
  • Gianluca Tettamanti
  • Magda de Eguileor
  • Annalisa GrimaldiEmail author
Regular Article
  • 80 Downloads

Abstract

Despite extensive investigation focused on both the molecular characteristics and the expression level of Toll-like receptors (TLRs) during the inflammatory response in vertebrates, few data are available in the literature on the role of these proteins in invertebrate’s immune response. Here, we propose the medicinal leech as a valuable model to better elucidate the role of TLR4 and its related products, such as tumor necrosis factor (TNF-α), after activation of the leech peripheral immune system with the endogenous medicinal leech recombinant allograft inflammatory factor-1 (rHmAIF-1) or with an exogenous stimulus, such as lipopolysaccharide (LPS). Our results indicate that activated macrophages (HmAIF-1+) and granulocytes (CD11b+) express both TLR4 and its coreceptor CD14. Moreover, functional studies performed by injecting a cyanobacterium selective TLR4 antagonist CyP demonstrated that only the TLR4 pathway was blocked, while the immune response caused by lipoteichoic acid (LTA) treatment is not affected. These results are consistent with literature on vertebrates, indicating that TLR4 functions as a LPS receptor while the recognition of LTA may involve other pathways.

Keywords

TLR4 LPS Cyanobacterial product Macrophage Granulocyte 

Notes

Acknowledgements

N.B. (PhD students of the “Biotechnology, Biosciences and Surgical Technology” course) is supported by a PhD fellowship of the DBSV, University of Insubria. The authors wish to thank Prof. Jacopo Vizioli (University of Lille, France), for providing us the anti HmAIF1 antibody.

Author contributions

AG, MM and CR conceived and designed the experiments. RG and NB performed the experiments. AG and RG analyzed the data and wrote the manuscript. GT and MdeE provided expertise for TEM and imaging and contributed reagents/materials/analysis tools. All authors critically reviewed the manuscript.

Funding information

This study was technically supported by the Centro Grandi Attrezzature (CGA) core facilities of the University of Insubria. The authors’ research was funded by CARIPLO (Fondazione Cassa di Risparmio delle Province Lombarde) -2016-0835 Ricerca malattie invecchiamento 2016: FRAMYEVO and Fondi per l’Ateneo (FAR 2017) to Grimaldi, de Eguileor and Tettamanti.

Compliance with ethical standards

Ethical approval

All applicable international, national and/or institutional guidelines for the care and use of animals were followed.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

441_2019_3010_Fig8_ESM.png (2.2 mb)
Figure S1

H. verbanaTRL4 sequence and conserved domains. H. verbana TLR4 protein sequence from the transcriptome database obtained by Northcutt and colleagues (Northcutt et al. 2018) (a). SMART analysis of the conserved domains about the relative TLR4 protein sequence using SMART software (http://smart.embl-heidelberg.de) (b). Alignment of TLR1 from H. medicinalis and TLR4 from H. verbana protein sequences (c). Alignment of H. sapiens and H. verbana TLR4 protein sequences in the specific region recognized by the anti-TLR4 antibody (in red) (d). (PNG 2212 kb)

441_2019_3010_MOESM1_ESM.tif (15.2 mb)
Additional file: high-resolution image was received; however, no citation was provided in the manuscript. Please provide the location of where to insert the citation in the main body of the text. Otherwise, kindly advise us on how to proceed.Please note that additional files should be cited in ascending numerical order in the main body of the text. High-resolution image (TIF 15612 kb)
441_2019_3010_Fig9_ESM.png (1.3 mb)
Figure S2

Representative full-length of one of three independent Western blot analyses of TLR4 and TNF-α and from which different lanes were cropped (squares) to make Figs. 1g, 2g, 6g respectively. (PNG 1337 kb)

441_2019_3010_MOESM2_ESM.tif (1.8 mb)
Additional file: high-resolution image was received; however, no citation was provided in the manuscript. Please provide the location of where to insert the citation in the main body of the text. Otherwise, kindly advise us on how to proceed.Please note that additional files should be cited in ascending numerical order in the main body of the text. High-resolution image (TIF 1838 kb)

References

  1. Adams SL (1988) The medicinal leech. A page from the annelids of internal medicine. Ann Intern Med 109:399–405CrossRefGoogle Scholar
  2. 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 CrossRefGoogle Scholar
  3. 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
  4. Chen G, Zhuchenko O, Kuspa A (2007) Immune-like phagocyte activity in the social amoeba. Science 317:678–681.  https://doi.org/10.1126/science.1143991 CrossRefPubMedCentralGoogle Scholar
  5. Coscia M, Giacomelli S, Oreste U (2011) Toll-like receptors: an overview from invertebrates to vertebrates. ISJ 8:210–226Google Scholar
  6. da Silva Correia J, Ulevitch RJ (2001) MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor*.  https://doi.org/10.1074/jbc.M109910200
  7. Davidson CR, Best NM, Francis JW et al (2008) Toll-like receptor genes (TLRs) from Capitella capitata and Helobdella robusta (Annelida). Dev Comp Immunol 32:608–612.  https://doi.org/10.1016/j.dci.2007.11.004 CrossRefGoogle Scholar
  8. de Eguileor M, Grimaldi A, Tettamanti G et al (2000a) Different types of response to foreign antigens by leech leukocytes. Tissue Cell 32:40–48CrossRefGoogle Scholar
  9. de Eguileor M, Grimaldi A, Tettamanti G, et al (2000b) Lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers. doi:  https://doi.org/10.1054/tice.2000.0132
  10. Drago F, Sautière PE, Le Marrec-Croq F et al (2014) Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium-binding adapter molecule 1 (Iba1/alias aif-1). Dev Neurobiol 74:987–1001.  https://doi.org/10.1002/dneu.22179 CrossRefGoogle Scholar
  11. Gemma S, Molteni M, Rossetti C (2016) Lipopolysaccharides in cyanobacteria: a brief overview. Adv Microbiol 06:391–397.  https://doi.org/10.4236/aim.2016.65038 CrossRefGoogle Scholar
  12. Girardello R, Drago F, De Eguileor M et al (2015a) Cytokine impregnated biomatrix: a new tool to study multi-wall carbon nanotubes effects on invertebrate immune cells. J Nanomed Nanotechnol 6.  https://doi.org/10.4172/2157-7439.1000323
  13. Girardello R, Tasselli S, Baranzini N et al (2015b) Effects of carbon nanotube environmental dispersion on an aquatic invertebrate, Hirudo medicinalis. PLoS One 10:e0144361.  https://doi.org/10.1371/journal.pone.0144361 CrossRefPubMedCentralGoogle Scholar
  14. Girardello R, Baranzini N, Tettamanti G et al (2017) Cellular responses induced by multi-walled carbon nanotubes: in vivo and in vitro studies on the medicinal leech macrophages. Sci Rep 7:8871.  https://doi.org/10.1038/s41598-017-09011-9 CrossRefPubMedCentralGoogle Scholar
  15. Grimaldi A (2016) Origin and fate of hematopoietic stem precursor cells in the leech Hirudo medicinalis. Invertebr Surviv J 13:257–268Google Scholar
  16. Grimaldi A, Tettamanti G, Perletti G et al (2006) Hematopoietic cell formation in leech wound healing. Curr Pharm Des 12:3033–3041CrossRefGoogle Scholar
  17. Grimaldi A, Banfi S, Vizioli J et al (2011) Cytokine loaded biopolymers as a novel strategy to study stem cells during wound-healing processes. Macromol Biosci 11:1008–1019.  https://doi.org/10.1002/mabi.201000452 CrossRefGoogle Scholar
  18. 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 173–191CrossRefGoogle Scholar
  19. Heckmann JG, Dutsch M, Neundorfer B et al (2005) Leech therapy in the treatment of median nerve compression due to forearm haematoma. J Neurol Neurosurg Psychiatry 76:1465CrossRefPubMedCentralGoogle Scholar
  20. Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on toll-like receptors. Nat Immunol 11:373–384.  https://doi.org/10.1038/ni.1863 CrossRefGoogle Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685CrossRefGoogle Scholar
  22. Ling GS, Bennett J, Woollard KJ et al (2014) Integrin CD11b positively regulates TLR4-induced signalling pathways in dendritic cells but not in macrophages. Nat Commun 5:1–12.  https://doi.org/10.1038/ncomms4039 CrossRefGoogle Scholar
  23. Macagno A, Molteni M, Rinaldi A et al (2006) A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression. J Exp Med 203:1481–1492.  https://doi.org/10.1084/jem.20060136 CrossRefPubMedCentralGoogle Scholar
  24. 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 CrossRefPubMedCentralGoogle Scholar
  25. Mahla RS, Reddy MC, Prasad DVR, Kumar H (2013) Sweeten PAMPs: role of sugar complexed PAMPs in innate immunity and vaccine biology. Front Immunol 4:248.  https://doi.org/10.3389/fimmu.2013.00248 CrossRefPubMedCentralGoogle Scholar
  26. Mann KH, Kerkut GA (1962) Leeches (Hirudinea) : their structure, physiology, ecology and embryology, with an appendix on the systematics of marine leeches. Elsevier ScienceGoogle Scholar
  27. Molteni M, Gemma S, Rossetti C (2016) The role of toll-like receptor 4 in infectious and noninfectious inflammation. Mediat Inflamm 2016:1–9.  https://doi.org/10.1155/2016/6978936 CrossRefGoogle Scholar
  28. 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 CrossRefGoogle Scholar
  29. Northcutt AJ, Fischer EK, Puhl JG et al (2018) An annotated CNS transcriptome of the medicinal leech, Hirudo verbana: De novo sequencing to characterize genes associated with nervous system activity. PLoS One 13:e0201206.  https://doi.org/10.1371/journal.pone.0201206 CrossRefPubMedCentralGoogle Scholar
  30. Ohnishi T, Muroi M, Tanamoto K-I (2003) MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation essential for its translocation to the cell surface. Clin Diagn Lab Immunol 10:405–410.  https://doi.org/10.1128/CDLI.10.3.405-410.2003 PubMedCentralGoogle Scholar
  31. Porshinsky BS, Saha S, Grossman MD et al (2011) Clinical uses of the medicinal leech: a practical review. J Postgrad Med 57:65–71.  https://doi.org/10.4103/0022-3859.74297 CrossRefGoogle Scholar
  32. Rast JP, Smith LC, Loza-Coll M et al (2006) Genomic insights into the immune system of the sea urchin. Science 314:952–956.  https://doi.org/10.1126/science.1134301 CrossRefPubMedCentralGoogle Scholar
  33. Rodet F, Tasiemski A, Boidin-Wichlacz C et al (2015) Hm-MyD88 and Hm-SARM: two key regulators of the neuroimmune system and neural repair in the medicinal leech. Sci Rep 5:1–13.  https://doi.org/10.1038/srep09624 CrossRefGoogle Scholar
  34. Sawyer RT (1986) Leech biology and behaviour. Clarendon PressGoogle Scholar
  35. Schikorski D, Cuvillier-hot V, Slomianny C et al (2009) Deciphering the immune function and regulation by a TLR of the cytokine EMAPII in the lesioned central nervous system using a leech model. J Immunol 183:7119–7128.  https://doi.org/10.4049/jimmunol.0900538 CrossRefGoogle Scholar
  36. 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 1:129–141Google Scholar
  37. 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 CrossRefGoogle Scholar
  38. Tasiemski A, Salzet M (2017) Neuro-immune lessons from an annelid: the medicinal leech. Dev Comp Immunol 66:33–42.  https://doi.org/10.1016/j.dci.2016.06.026 CrossRefGoogle Scholar
  39. Thorgersen EB, Macagno A, Rossetti C, Mollnes TE (2008) Cyanobacterial LPS antagonist (CyP)—a novel and efficient inhibitor of Escherichia coli LPS-induced cytokine response in the pig. Mol Immunol 45:3553–3557.  https://doi.org/10.1016/j.molimm.2008.05.005 CrossRefGoogle Scholar
  40. Trianiafilou M, Manukyan M, Mackie A et al (2004) Lipoteichoic acid and Toll-like receptor 2 internalization and targeting to the Golgi are lipid raft-dependent. J Biol Chem 279:40882–40889.  https://doi.org/10.1074/jbc.M400466200 CrossRefGoogle Scholar
  41. Wakabayashi Y, Kobayashi M, Akashi-Takamura S et al (2006) A protein associated with toll-like receptor 4 (PRAT4A) regulates cell surface expression of TLR4. J Immunol 177:1772–1779.  https://doi.org/10.4049/jimmunol.177.3.1772 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biotechnology and Life SciencesUniversity of InsubriaVareseItaly
  2. 2.Department of Medicine and SurgeryUniversity of InsubriaVareseItaly

Personalised recommendations