[en] Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum, of worldwide distribution and highly endemic in the Mediterranean basin. This parasite is transmitted by the bite of sandfly females. The dog is the main host and reservoir. The clinical manifestations of L. infantum infection in the dog are very variable and range from a chronic subclinical infection to a very severe disease, which can be fatal. Due to its wide pathogenesis, the innate and adaptive immune responses play a role in this canine infection. However, there is much more information on the adaptive immune response than on the innate one. Toll-like receptors (TLR) are essential in the machinery of the immune system that facilitates the early arrest of several infections as well as the activation of the inflammatory cascade. However, the role of these receptors in L. infantum infection in dogs is not well known and the information is very limited. The hypothesis of this doctoral thesis is that TLRs have an important role in L. infantum infection in dogs because they stimulate the inflammatory cascade. The general objective of this doctoral thesis was to investigate the expression of TLRs in the CanL and compare with parasitological, clinical, biochemical and immunological parameters as well as to evaluate the possible use of TLR agonists in the treatment of this disease. The specific objectives have been developed in five studies and are described below. The first specific objective is described in chapter 3.1. and consisted in evaluating the expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) in unstimulated blood of dogs with moderate clinical leishmaniosis at the time of diagnosis and during one year of treatment as well as correlating clinical, biochemical and parasitological parameters. In chapter 3.2., we described the second experiment where the relative quantification of TLR2 and TLR4 was evaluated in dogs infected by L. infantum in different stages of the disease according to the LeishVet classification at the time of diagnosis. This study also investigated the antibodies and the production of interferon gamma (IFN-γ) specific to the parasite as well as the parasitemia in clinical stage I (dogs with papular dermatitis) and in clinical stages II-III (dogs with exfoliative dermatitis or ulcerative) The specific objective of the third study (Chapter 3.3.) was to determine the transcription of the TLR2, TLR4 and programmed death ligand 1 (PD-L1) genes in blood stimulated with L. infantum soluble antigen (LSA) and the mitogen Concanavalin A (Con A) in sick dogs IFN-γ producers and non-IFN-γ producers and healthy dogs. The purpose of the study described in chapter 3.4. was to determine the production of the cytokines TNF-α and IL-6 in blood stimulated with LSA or TLRs agonists (TLR3, TLR4 and TLR7) alone or combined of dogs naturally infected by Leishmania. Finally, an in vitro experiment (Chapter 3.5) was carried out where the parasites' susceptibility to different conventional anti-Leishmania drugs (allopurinol, miltefosine or meglumine antimonate) was evaluated in promastigote and amastigote assays in a canine macrophage cell line as well as in the treatment of TLRs agonists 2, 3, 4 and 7 (TLRsa) alone or in combination with conventional drugs. Descriptive studies of this thesis have confirmed that TLRs seem to have a role in the disease. Sick dogs infected with Leishmania present different expression of TLRs compared to healthy individuals, also directly depending on the immunological profile of the animal as well as the clinical stage. Thus, in this doctoral thesis, overexpression of TLR2 was obtained in unstimulated blood at the time of diagnosis in dogs with moderate disease (stage II-III) when compared with healthy dogs. However, expression of TLR2 in unstimulated blood was the same for dogs with mild disease (stage I) and healthy dogs. In addition, there was a decrease in the expression of TLR2 during one year of treatment and clinical improvement in dogs with moderate disease. However, no changes were observed in TLR4 expression at diagnosis or during treatment in any of the dogs studied. It is also important to highlight that the expression of TLR2 was correlated with clinical, parasitological and immunological parameters associated with moderate to severe disease. In addition, dogs with mild stage I and papular dermatitis had a predominantly Th1 immunological profile whereas animals classified in more severe stages had a Th2 profile predominant. The results obtained in blood stimulated with LSA were very interesting and correlated with the findings obtained in unstimulated blood. A reduction in gene expression of the TLR2 and TLR4 genes in blood stimulated with LSA was observed in sick dogs that were IFN-γ producers compared to healthy dogs and a high expression of PD-L1 in all the groups studied for both LSA as for Con A. The blood stimulated with TLRs agonists (TLRsa) and LSA of sick dogs turned out to have a high production of the cytokines IFN-γ, TNF-α and IL-6, compared with the medium alone. The combinations that gave the most production of cytokines of the Th1 profile are the agonists TLR4 and TLR7 each combined with LSA. In vitro studies demonstrated the susceptibility of the parasite to conventional drugs, being miltefosine the most effective drug. In addition, it was shown that TLRsa agonists alone reduced infection, and a synergistic effect was also observed in the reduction of infection with allopurinol and agonists for TLR4. However, the production of TNF-α or NO was not detected in the supernatants collected after 72 hours. However, the transcription of TLR2, 4 and 7 was detected in all the conditions studied. In general, a decrease in the transcription of TLR2s was demonstrated or no changes in the expression of TLR4 and TLR7 with infection. However, the expression of TLRs after treatment with conventional anti-Leishmania drugs alone or TLR agonists alone or combination of both was more variable. In conclusion, this doctoral thesis has shown that the expression of TLR2 in blood is not stimulated in a marker of moderate to severe disease. However, TLR4 does not appear to be a good marker for CanL in unstimulated blood. In addition, the reduced expression of TLR2 and 4 in blood stimulated with LSA was associated with sick dogs responding to IFN-γ which have a more protective profile than dogs not responding to IFN-γ. The in vitro study revealed that drugs combined with TLRsa or even TLRsa alone can reduce infection. For this reason, the findings found in this thesis are that TLRs can be used as immunotherapy or as adjuvants in future vaccines.

[es] La leishmaniosis canina (CanL) es una enfermedad causada por el protozoa Leishmania infantum. Las manifestaciones clínicas en el perro son muy variables, abarcan desde una infección subclínica a un cuadro severo, que puede ser fatal. Al ser una enfermedad compleja, es difícil de diagnosticar. La respuesta inmunitaria de cada individuo juega un papel muy importante, tanto para el diagnóstico como para su tratamiento. Los receptores tipo Toll (TLRs) son los principales iniciadores de la respuesta inmunitaria innata cuya función es frenar cualquier intrusión de agentes extraños al cuerpo. Sin embargo, poca información existe sobre el papel de estos receptores en la CanL. El objetivo general de esta tesis doctoral es investigar el papel que los TLRs tiene en la CanL, tanto en experimentos ex vivo como in vitro. En la presente tesis doctoral, se ha estudiado la expresión genética en sangre no estimulada de los TLR2 y TLR4, en el momento del diagnóstico y durante el tratamiento, demostrando un notable aumento de la transcripción del TLR2 en perros con leishmaniosis clinica. Cuando se compararon perros enfermos en diferentes estadíos clínicos, se observó una expresión más notoria del TLR2 en aquellos perros que padecían la enfermedad de forma más severa comparado con los perros con estadíos más leves o con dermatitis papular. En, estos últimos, se notó de forma característica que tenían una alta producción de la citoquina interferon-gamma (IFN-γ). En sangre estimulada con antígeno soluble de Leishmania (LSA), la expresión del TLR2 y TLR4 disminuye en los perros enfermos que son productores de IFN-γ. Estimulando la sangre con TLRs agonistas, se halló un aumento en la producción de las citoquinas IFN-γ, TNF-α y IL-6 particularmente después de la estimulación con los agonistas TLR4 y TLR7 en sangre de perros naturalmente infectados. En el estudio in vitro incluido en esta tesis doctoral, en el cual macrófagos caninos fueron infectados experimentalmente con L. infantum y tratados con diferentes fármacos convencionales y TLRs agonistas, solos o combinados entre ellos, se demostró que la tasa de infección y la intensidad de infección se redujeron después de la estimulación los agonistas de TLRs. Además, el agonista TLR4 parece tener un efecto sinérgico combinado con el alopurinol. Así pues, con estos resultados, se ha ampliado el conocimiento de los TLRs en la infección por L. infantum en el perro. Y se han obtenido resultados esperanzadores para seguir investigando en esta línea sobre cómo los TLRs agonistas podrían actuar como inmunomoduladores en el tratamiento de esta enfermedad o incluso como adyuvantes en vacunas.