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


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.


TLR4 LPS Cyanobacterial product Macrophage Granulocyte 



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 ( (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)


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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

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