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

BackgroundA 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.ResultsA 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2062-3) contains supplementary material, which is available to authorized users.

Exploring the relationship between susceptibility to canine leishmaniosis and anti-Phlebotomus perniciosus saliva antibodies in Ibizan hounds and dogs of other breeds in Mallorca, Spain

“…Indeed, in the present study, Ibizan hounds contained significantly more IFN-γ producers and produced significantly higher levels of IFN-γ than other breeds. This result aligns well with a previous work, which reported marked Leishmania-specific IFN-γ production in healthy Ibizan hounds when compared to control and sick dogs [28]. In general, production of this pro-inflammatory cytokine is correlated with disease resistance or mild disease [12,19,20,52], while lack of expression is described in experimentally infected symptomatic dogs [12] and in dogs with a strong humoral response and severe disease [17,18].…”

“…It has been proposed that the breed's relative immunocompetence is derived from a predominantly cellular, parasite-specific immune response, which is associated with a clinically healthy status and favorable prognosis [26,27]. Compared to other breeds living in the same area, Ibizan hounds display higher frequency of positive LST reactions, greater IFN-γ production, and weaker or lack of humoral immune response [26,28]. However, a precise etiology for this apparent resistance has not yet been identified.…”

Leishmania species‐dependent functional duality of toll‐like receptor 2

“…Ibizan hounds are more resistant to L. infantum ‐related VL than other dog breeds. The stimulation of whole blood using L. infantum antigen with Pam3CSK4 (a TLR2 agonist) results in higher TNF‐α production in healthy Ibizan hounds when compared to seropositive sick dogs and seronegative healthy dogs, suggesting that Pam3CSK4 may act as an immunomodulator in canine leishmaniasis …”

Toll-Like Receptors 2, 4, and 7, Interferon-Gamma, Interleukin 10, and Programmed Death Ligand 1 Transcripts in Leishmanin Skin Test-Positive Reactions of Ibizan Hound Dogs

“…Ibizan hounds are considered resistant dogs to Leishmania infection as they rarely manifest clinical disease and mount a significant cellular immune response to the infection demonstrated by a high prevalence of positive LST reactions as well as a potent Leishmania-specific IFN-γ production and low or null humoral response when compared with other breeds from the same geographical area [12,18]. The immunohistological features of LST-positive reactions in Ibizan hound dogs have been recently described [19].…”