Evaluation of immune response in dogs under different leishmaniosis protocol treatment and according to their clinical stage

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Cytokine gene expression and cellular immune response in dogs with leishmaniosis before and under the two first-line treatment protocols : new insights into the animal disease

Publication . Santos, Marcos André Ferreira; Sampaio, Isabel Maria Soares Pereira da Fonseca de; Gomes, Gabriela Maria Santos

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic visceral disease of worldwide concern. The drugs used for its treatment improve the animal’s clinical condition, although, in most cases, the parasites are not completely destroyed. The current study aimed to evaluate the immune response of the dog with leishmaniosis before and during treatment with first-line drugs, by analyzing the profile of cytokines and subsets of CD4+ and CD8+ T-cells in peripheral blood, lymph node and bone marrow. Two groups of six dogs diagnosed with CanL were treated with either miltefosine or meglumine antimoniate in combination with allopurinol. Simultaneously, another group of ten clinically healthy dogs was used as a control group. Upon diagnosis and during the following three months of treatment, clinical signs, hematological and biochemical parameters, urinalysis results and anti-Leishmania antibody titers using IFAT were recorded. Furthermore, peripheral blood, popliteal lymph node and bone marrow mononuclear cells were collected to evaluate the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β and IFN-γ by qPCR. In parallel, these cells were also immunophenotypically analyzed be flow cytometry, using surface monoclonal antibodies anti-CD45, CD3, CD4, CD8, CD25 and intracellular monoclonal antibody anti-nuclear factor FoxP3. Both treatment protocols promoted the remission of clinical signs, normalization of hematological and biochemical parameters and urinalysis values. Sick dogs showed a generalized increase in IFN-γ gene expression and a decrease of IL-2, IL-4, and TGF-β. The expression of IL-12, TNF-α, IL-5, and IL-10 showed variations between groups of dogs and the tissue analyzed. CanL also resulted in an overall increase in the percentage of CD8+ T-cells in all tissues. In the peripheral blood there was also a decrease in CD4+ T-cells and an increase of CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ T-cells, with the latter also increasing on the bone marrow. CD4+CD25-FoxP3- T-cells showed a marked decrease in blood and bone marrow. During treatment, a trend towards normalization of cytokine gene expression and T-cell subsets was observed. However, high levels of IFN-γ gene expression were still observed in all tissues. In turn, the treatments caused an increase in the percentage of CD4+CD25+FoxP3+ and a decrease in CD8+CD25-FoxP3- T-cells, leading to normalization of CD4+ and CD8+ T cells in all tissues. Furthermore, the effect of treatment on gene expression of cytokines that were not significantly altered by infection indicates that these combined treatment protocols directly affect cytokine production. Both combined treatments are effective in remitting clinical sings and appear to influence the dog’s immune response, sustaining a pro-inflammatory immune environment while promoting the normalization of T-cell subsets. These findings indicate that L. infantum may be able to manipulate elements of the dog's immune system to avoid differentiating an efficient protective response, preventing the rapid development of severe pathology while ensuring the parasite’s survival and securing the possibility of several transmission cycles. Allied to these results, other studies carried out in collaboration with the working group on the role of neutrophils, hepatocytes and Kupffer cells in CanL, as well as the evaluation of treatment in feline leishmaniosis, have allowed to enhance the knowledge in the area of animal leishmaniosis.

2020-10-01Doctoral thesis

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