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Dogs are the most common domestic reservoir of Leishmania infantum, making canine visceral leishmaniasis (CVL) a serious public health issue. Identifying new methodologies that can mimic lymphoid and myeloid competence in naturally infected dogs could lower costs and save time in preliminary screenings of potential immunotherapeutic agents and vaccines against CVL. For that, we established a cell-to-cell communication approach between lymphocytes and myeloid cells from healthy, asymptomatic (infected, without apparent clinical signs) and symptomatic (infected with apparent clinical signs) dogs. Peripheral blood mononuclear cells (PBMC) from these dogs were used as source of CD4 + , CD8 + T lymphocytes and macrophages, that were posteriorly infected with L. infantum GFP + promastigotes (green fluorescent protein). Macrophages co-cultured with purified lymphocytes were tested for the ability to control cellular parasitism, and their microbicidal function by producing nitric oxide (NO) and reactive oxygen species (ROS). The kind of T cell response within the co-culture was also evaluated, by assessing their ability to produce interferon-gamma (IFN-γ) and interleukin 4 (IL-4). The data suggests that T lymphocytes from symptomatic dogs are more prone to produce IL-4 than the ones from asymptomatic dogs. Macrophages from asymptomatic dogs also demonstrated a higher microbicidal potential, with increased levels of NO and ROS production, compared to symptomatic dogs, mainly in highly parasitized cells. Together, our results identify the ratio of IL-4/IFN-γ produced by CD4 + and CD8 + T cells, as well as, the ratio between parasite GFP signal/NO and ROS signal in macrophages as potential immunological biomarkers of failure and success of the screened agents. Our findings also propose a reliable methodology that can be used to follow the immune response in trials of potential drugs or vaccines targeting CVL., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

An important strategy to reduce the risk of visceral leishmaniasis (VL) in humans is to control the infection and disease progression in dogs, the domestic reservoir of Leishmania infantum parasites. Certain therapeutic strategies that modulate the host immune response show great potential for the treatment of experimental VL, restoring the impaired effector functions or decreasing host excessive responses. It is known that the overproduction of interleukin-10 (IL-10) promotes parasite replication and disease progression in human VL as well as in canine visceral leishmaniasis (CVL). Thus, in the present study we investigated the potential of the anti-canine IL-10 receptor-blocking monoclonal antibody (Bloq IL-10R) to control and reduce in vitro infectivity of L. infantum and improve the ability of PBMC isolated from VL dogs to alter the lymphoproliferative response and intracytoplasmic cytokines. Overall, GFP + Leishmania showed lower capacity of in vitro infectivity in the presence of Bloq IL-10R. Moreover, addition of Bloq IL-10R in cultured PBMC enhanced T-CD4 and CD8 proliferative response and altered the intracytoplasmic cytokine synthesis, reducing CD4 + IL-4 + cells and increasing CD8 + IFN-γ + cells after specific antigen stimulation in PBMC of dogs. Furthermore, we observed an increase of TNF-α levels in supernatant of cultured PBMC under IL-10R neutralizing conditions. Together, our findings are encouraging and reaffirm an important factor that could influence the effectiveness of immune modulation in dogs with VL and suggest that blocking IL-10R activity has the potential to be a useful approach to CVL treatment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The complex interplay between cytokines and chemokines regulates innate and adaptive immune responses against pathogens; specifically, cytokine and chemokine expression drives activation of immune effector cells and their recruitment to tissue infection sites. Herein, we inoculated dogs with Leishmania braziliensis antigens plus saponin (the LBSap vaccine), as well as with the vaccine components, and then used real-time PCR to evaluate the kinetics of dermal expression of mRNAs of cytokines (IL-12, IFN-γ, TNF-α, IL-4, IL-13, TGF-β and IL-10) and chemokines (CCL2, CCL4, CCL5, CCL21 and CXCL8) 1, 12, 24 and 48 h after inoculation. We also evaluated the correlation between cytokine and chemokine expression and dermal cellularity. The LBSap vaccine induced high levels of IL-12 and IL-10 expression at 12 and 24 h, respectively. Furthermore, we observed positive correlations between IL-12 and IL-13 expression, IFN-γ and IL-13 expression, and IL-13 and TGF-β expression, suggesting that a mixed cytokine microenvironment developed after immunization with the vaccine. Inoculation with the saponin adjuvant alone induced a chemokine and cytokine expression profile similar to that observed in the LBSap group. CCL4 and CXCL8 chemokine expression was up regulated by the LBSap vaccine. CCL5 expression was initially highest in the LBSap group, but at 48 h, expression was highest in the LB group. Information about the kinetics of the immune response to this vaccine gained using this dog model will help to elucidate the mechanisms of and factors involved in a protective response against Leishmania infection and will aid in establishing rational approaches for the development of vaccines against canine visceral leishmaniasis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)