Setting the proportion of CD4+ and CD8+ T-cells co-cultured with canine macrophages infected with Leishmania chagasi

Abstract

New methods for evaluating the canine immune system are necessary, not only to monitor immunological disorders, but also to provide insights for vaccine evaluations and therapeutic interventions, reducing the costs of assays using dog models, and provide a more rational way for analyzing the canine immune response. The present study intended to establish an in vitro toll to assess the parasitological/immunological status of dogs, applicable in pre-clinical trials of vaccinology, prognosis follow-up and therapeutics analysis of canine visceral leishmaniasis. We have evaluated the performance of co-culture systems of canine Leishmania chagasi-infected macrophages with different cell ratios of total lymphocytes or purified CD4(+) and CD8(+) T-cells. Peripheral blood mononuclear cells from uninfected dogs were used for the system set up. Employing the co-culture systems of L. chagasi-infected macrophages and purified CD4(+) or CD8(+) T-cell subsets we observed a microenvironment compatible with the expected status of the analyzed dogs. In this context, it was clearly demonstrated that, at this selected T-cell:target ratio, the adaptive immune response of uninfected dogs, composed by L. chagasi-unprimed T-cells was not able to perform the in vitro killing of L. chagasi-infected macrophages. Our data demonstrated that the co-culture system with T-cells from uninfected dogs at 1:5 and 1:2 ratio did not control the infection, yielding to patent in vitro parasitism (≥ 80%), low NO production (≤ 5 μM) and IL-10 modulated (IFN-γ/IL-10 ≤ 2) immunological profile in vitro. CD4(+) or CD8(+) T-cells at 1:5 or 1:2 ratio to L. chagasi-infected macrophages seems to be ideal for in vitro assays. This co-culture system may have great potential as a canine immunological analysis method, as well as in vaccine evaluations, prognosis follow-up and therapeutic interventions.

In vitro Infectivity of Strains Isolated From Dogs Naturally Infected With Leishmania infantum Present a Distinct Pathogenic Profile in Hamsters

“…Parasites were grown in LIT and NNN (McNeal, Novy & Nicolle) medium up to eight in vitro passages to preserve the most important parasite characteristics regarding infectivity and pathogenicity profiles, in addition to providing appropriate adaptation in the media culture. Stationary growth phase promastigotes, comprising predominantly metacyclic promastigotes ( 21 – 23 ), were obtained from an initial inoculum of 10 mL of LIT medium containing between 10 7 and 10 8 promastigotes/mL in logarithmic growth. The promastigotes were added to 40 mL of NNN/LIT culture medium and stored at 23 ± 1°C.…”

“…One-month-old male Syrian golden hamsters ( Mesocricetus auratus ), weighing ~50–80 g, were obtained from the Animal Science Center at the Universidade Federal de Ouro Preto (UFOP). Experimental infection was done at the end of seven days of promastigote cultivation [stationary phase presenting predominantly metacyclic forms as described by ( 23 )] that were transferred to sterile polypropylene tubes (FalconH, Becton Dickinson, USA) and centrifuged at 900x g for 15 min. The parasites were adjusted to 10 7 promastigotes per inoculum.…”

“…Immunohistochemistry was performed as previously described to evaluate parasite load ( 23 ) in sections of the spleen and liver ( 26 ). The numbers of immunolabeled Leishmania amastigotes in the spleen and liver sections were classified according to the following scores: 0 (absent), 1 (mild, 1–50 amastigotes per microscopic field under 400x magnification), 2 (moderate, 51–100 per microscopic field under 400x magnification), and 3 (severe, more than 101 amastigotes per microscopic field under 400x magnification).…”

Within-Host Bayesian Joint Modeling of Longitudinal and Time-to-Event Data ofLeishmaniaInfection

“…Canine CD8+ T cells are capable of influencing Leishmania -infected macrophage microbicidal activity [39], therefore we also included CD8+ T cell parameters in this model to see if any associations were revealed. We observed a strong negative effect of tick preventative treatment on future CD8+ T cell proliferation (posterior probability 0.928).…”