title: The inflammatory cytokine effect of Pam3CSK4 TLR2 agonist alone or in combination with Leishmania infantum antigen on ex-vivo whole blood from sick and resistant dogs.
abstract: BACKGROUND
  A wide spectrum of clinical manifestations and immune responses exist in canine L. infantum infection. Ibizan hounds are more "resistant" to disease than other dog breeds. Recognition of pathogen-associated molecule patterns by toll like receptors (TLRs) rapidly triggers a variety of anti-microbial immune responses through the induction of pro-inflammatory cytokines such as TNF-α and IL-6 which may play an important role in controlling Leishmania infection. The main objective of this study was to investigate and compare the effect of a TLR2 agonist (TLR2a) alone or in combination with L. infantum antigen (LSA) on ex vivo whole blood cytokine production from healthy seronegative IFN-γ non-producer dogs from an area of low in canine leishmaniosis endemicity (n = 11); sick seropositive dogs with low production of IFN-γ (n = 17) and healthy seronegative or low positive Ibizan hounds with a predominant IFN-γ production (n = 21) from a highly endemic area. Whole blood was stimulated with medium alone (Ø), LSA, concanavalin A, TLR2 (Pam3CSK4) receptor agonist (Ø + TLR2a) and TLR2a and LSA (LSA + TLR2a) for 48 h. Supernatants were harvested for measurement of canine TNF-α and IL-6 cytokines by ELISA.
  
  
  RESULTS
  A significant increase of TNF-α was found in the supernatants of stimulated blood from all groups (Ø + TLR2a and LSA + TLR2a) when compared with medium alone. A similar pattern was observed for IL-6. Interestingly, a significant increase of TNF-α production was only observed when stimulation with LSA + TLR2a was compared with TLR2a alone in Ibizan hounds. A significant increase of TNF-α production was observed with stimulation of LSA + TLR2a when compared with LSA in all groups. Significantly higher concentrations of TNF-α and IL-6 were detected in Ibizan hounds, especially for the Ø + TLR2a and LSA + TLR2a treatments compared with other groups.
  
  
  CONCLUSIONS
  This study demonstrated that TLR2a alone enhances the production of the inflammatory cytokines TNF-α and IL-6 in sick, "resistant" and healthy non-infected dogs. In addition, a combination of LSA+TLR2a promoted a synergistic pro-inflammatory effect with TNF-α in Ibizan hounds but not in seropositive sick dogs and seronegative healthy dogs. These findings might suggest the importance of Pam3CSK4 as a possible immunomodulator for CanL.
authors.affiliation: Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
authors.forename: Pamela
authors.initials: P
authors.lastname: Martínez-Orellana
authors.affiliation: Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
authors.forename: Paulina
authors.initials: P
authors.lastname: Quirola-Amores
authors.affiliation: Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Montserrat-Sangrà
authors.affiliation: Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Ordeix
authors.affiliation: Hospital Mon Veterinari, Manacor, Mallorca, Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Llull
authors.affiliation: Clinica Veterinaria Noreña, Noreña, Asturias, Spain.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Álvarez-Fernández
authors.affiliation: Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain. laia.solano@uab.cat.
authors.forename: Laia
authors.identifier: http://orcid.org/0000-0001-8479-4896
authors.initials: L
authors.lastname: Solano-Gallego
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D000953:Antigens, Protozoan / Q000276:immunology* / Q000494:pharmacology; D016207:Cytokines / Q000097:blood* / Q000276:immunology; D004283:Dog Diseases / Q000276:immunology*; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D015850:Interleukin-6 / Q000096:biosynthesis / Q000097:blood / Q000276:immunology; D018314:Leishmania infantum / Q000276:immunology*; D007898:Leishmaniasis, Visceral / Q000276:immunology / Q000662:veterinary*; D055666:Lipopeptides / Q000276:immunology / Q000494:pharmacology*; D051195:Toll-Like Receptor 2 / Q000819:agonists*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000097:blood / Q000276:immunology
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D016207:Cytokines; D015850:Interleukin-6; D055666:Lipopeptides; C508075:Pam(3)CSK(4) peptide; D051195:Toll-Like Receptor 2; D014409:Tumor Necrosis Factor-alpha
keywords: Ibizan hound; Inflammatory cytokines; Leishmania infantum; Sick dog; TLR-2 agonist (Pam3CSK4)