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

Antimicrobial Role of RNASET2 Protein During Innate Immune Response in the Medicinal Leech Hirudo verbana

“…By contrast, starting from 30 min following LTA injection, newly formed vessels and many infiltrating immune cells were clearly observable underneath the epithelium and in the ECM surrounding the muscle fibers ( Figures 1B-H). In particular, several granulocytes were clearly recognizable both by light ( Figure 1D) and TEM microscope analysis ( Figure 1G) (17,30). At 6 h post-injection, a high number of macrophages infiltrating the injected area (characterized by a ruffled surface due to the presence of pseudopodia, a typical feature of migrating cells) were readily detected as well ( Figures 1E,F,H).…”

“…Group 3: injection with 100 µl of PBS containing 1 µg/ml of CyP, an LPS-like molecule extracted from the cyanobacterium Oscillatoria Planktothrix FP1 [cyanobacterial product (CyP)] that acts as a potent and selective antagonist of bacterial LPS (30, 37) followed by evaluation at 30-min, 1-h, 3, 6, and 24-h time points. This treatment was performed to exclude any possible interaction between LTA and the LPS receptor TLR4 (30). Group 4: injection with 100 µl of PBS containing 1 µg/ml of CyP plus 100 ng/ml of LTA obtained from B. subtilis (Sigma-Aldrich) followed by evaluation at 30-min, 1-h, 3, 6, and 24-h time points.…”

“…The samples were analyzed 6 h after LTA treatment. Group 6: injection with 100 µl of PBS containing methicillin-susceptible Staphylococcus aureus ATCC 6538P (10 7 CFU/ml) followed by evaluation at 3 h. Group 7: injection with 100 µl of a PBS solution containing S. aureus ATCC 6538P (10 7 CFU/ml) and 10 µM recombinant H. verbana RNASET2 protein (HvRNASET2, accession number BankIt2095553 Hirudo MH325331) followed by evaluation at 3 h. Group 8: injection with 100 µl of a PBS solution containing S. aureus ATCC 6538P (10 7 CFU/ml) and 10 µM rHvRNASET2 pre-treated with 1 µg of an anti-RNASET2 antibody followed by evaluation at 3 h, as a specificity control to functionally block HvRNASET2 activity (30). Group 9: animals injected with 300 µl of liquid MG supplemented with 50 µl of S. aureus pre-treated with 50 ng of rHvRNASET2, used to selectively isolate macrophages recruited by HvRNASET2 (16) and to evaluate their enhanced phagocytic activity, were analyzed at T7 days.…”

“…For immunofluorescence assays, slices were incubated for 30 min in blocking solution and then for 1 h at 37 • C with the following polyclonal primary antibodies diluted in the same blocking solution: rabbit anti-RNASET2 (40), expressed by macrophages and granulocytes of leech (17) diluted 1:200; goat anti-CD11b (Santa Cruz Biotechnology, CA, USA, sc-28664) that specifically stains leech granulocytes (41) diluted 1:100; rabbit anti-HmAIF-1 (kindly donated by Prof. Jacopo Vizioli, University of Lille 1, France), reacting with leech macrophages (36,42), diluted 1:1000; rabbit anti-TNF-α (Abcam, Cambridge, UK, ab6671) diluted 1:200 reacting with leech homologous protein (30); and rabbit anti-TLR2 (Abcam, Cambridge, UK, ab213676, recognizing an epitope corresponding to amino acids 730-780 mapping to an internal region of TLR2 of human origin) diluted 1:200. After washing in PBS, samples were incubated for 45 min at room temperature, respectively with an antigoat or anti-rabbit Cy5-conjugated (Jackson ImmunoResearch Laboratories, West Grove, USA) secondary antibodies (excitation filter 650 nm, emission filter 672 nm) diluted 1:250 in blocking solution.…”

“…As such, alarmins promote inflammatory responses, usually mediated by Toll-like receptor family members (TLRs) (26,27). Among TLRs, TLR2, and TLR4 represent the most significant group of PRRs (pattern recognition receptors), which are evolutionary conserved both in vertebrate and in invertebrate species (28)(29)(30) and are expressed by immune cell membranes. These receptors mediate the recognition of conserved biomolecules known as pathogen-associated molecular patterns (PAMPs), such as lipoteichoic acid (LTA) and LPS, which are normally displayed in the external membrane of Gram-positive and Gram-negative bacteria, respectively.…”