Vitamins A and D and Zinc Affect the Leshmanicidal Activity of Canine Spleen Leukocytes

Abstract

Canine leishmaniasis (CanL) is a chronic disease caused by Leishmania infantum, and the limitations of the current treatments have encouraged new alternatives, such as the use of immunomodulatory nutrients. The objective of this study was to determine the serum levels of vitamin A (retinol), vitamin D (25(OH)VD₃), and zinc (Zn) in dogs with CanL and the effect of in vitro supplementation with the respective active forms ATRA, 1,25(OH)₂VD₃, and SZn on spleen leukocyte cultures. Serum retinol, 25(OH)VD₃, and Zn were determined by HPLC, ELISA, and ICP-MS, respectively. Spleen leukocyte cultures were used for the detection of NO and ROS by flow cytometry; the IFN-γ, TNF-α, and IL-10 levels were determined by ELISA; and the parasite load was determined by microscopy. We detected low serum levels of retinol and Zn and high levels of 25(OH)VD₃ in the CanL group. The in vitro supplementation of CanL spleen leukocytes with ATRA, 1,25(OH)₂VD₃, and SZn, in addition to a soluble leishmania antigen (SLA) treatment, increased the NO and ROS levels, while the treatments with only ATRA and SZn increased the TNF-a levels. Increased IL-10 and IFN-g levels were observed with the addition of SLA to the medium, although the addition of the three nutrients led to a reduction of the IL-10 levels, and the addition of 1,25(OH)₂VD₃ and SZn led to a reduction of IFN-g. A supplementation with 1,25(OH)₂VD₃ and SZn reduced the parasite load but only in the absence of SLA. We suggest that the nutrients we tested are involved in the leishmanicidal mechanism, showing a potential for investigation in future studies.

Keywords: Leishmania spp.; all-trans retinoic acid; retinol; vitamin D3; zinc.

Figure 1

Serum levels of nutrients in the CanL group and in healthy dogs. Low levels of retinol (A) and Zn (C) and higher levels of 25(OH)VD₃ (B) were observed in the CanL group (n = 15) compared to healthy dogs (n = 5). Data represent the median and interquartile range (IQR). Asterisks indicate a significant difference between the CanL group and healthy dogs (Mann–Whitney test, *** p < 0.001).

Figure 2

NO production by spleen leukocytes of dogs with CanL and healthy dogs supplemented with nutrients. Spleen leukocytes from CanL (n = 15) and healthy dogs (n = 5) were cultured in the medium alone or with the addition of ATRA, 1,25(OH)₂VD₃ and SZn, with and without SLA. NO production in the cell culture was determined by flow cytometry after 20 h. Bars represent the median and IQR with minimum and maximum error bars. Asterisks indicate a significant difference for the comparisons within the treatment (Wilcoxon test, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 3

ROS production by spleen leukocyte cultures of the dogs with CanL and healthy dogs supplemented with nutrients. Spleen leukocytes from CanL (n = 15) and healthy dogs (n = 5) were cultured in the medium alone or with the addition of with ATRA, 1,25(OH)₂VD₃ and SZn, both with and without SLA. ROS production in the cell cultures was determined by flow cytometry after 20 h. Bars represent the median and IQR with minimum and maximum error bars. Asterisks indicate a significant difference for the comparisons within the treatment (Wilcoxon test, * p < 0.05 and ** p < 0.01).

Figure 4

Cytokine production in the supernatant of spleen leukocyte cultures of the CanL group and the control group of healthy dogs supplemented with nutrients. Spleen leukocytes from the CanL (n = 10, 7 and 15 for INF-γ, TNF-α and IL-10, respectively) and healthy dogs (n = 5) were cultured in medium alone or with the addition of ATRA, 1,25(OH)₂VD₃ and SZn, with and without SLA. After 72 h, IFN-γ (A), TNF-α (B) and IL-10 (C) production in the culture supernatant was determined by ELISA. Bars represent the median and IQR with minimum and maximum error bars. Asterisks indicate a significant difference for the comparisons within the treatment (Wilcoxon test, * p < 0.05 and ** p < 0.01).

Figure 4

Cytokine production in the supernatant of spleen leukocyte cultures of the CanL group and the control group of healthy dogs supplemented with nutrients. Spleen leukocytes from the CanL (n = 10, 7 and 15 for INF-γ, TNF-α and IL-10, respectively) and healthy dogs (n = 5) were cultured in medium alone or with the addition of ATRA, 1,25(OH)₂VD₃ and SZn, with and without SLA. After 72 h, IFN-γ (A), TNF-α (B) and IL-10 (C) production in the culture supernatant was determined by ELISA. Bars represent the median and IQR with minimum and maximum error bars. Asterisks indicate a significant difference for the comparisons within the treatment (Wilcoxon test, * p < 0.05 and ** p < 0.01).

Figure 5

Parasite load in the spleen leukocytes of the CanL group supplemented with nutrients. Spleen leukocytes from dogs with CanL (n = 15) were cultured in the medium alone or in medium supplemented with ATRA, 1,25(OH)₂VD₃ and SZn, either with or without SLA. After 72 h, the parasite load was quantitated by counting the amastigotes present inside the infected macrophages on slides obtained by cytocentrifugation and was determined by dividing the number of amastigotes by the number of infected macrophages counted. The results are expressed as a percentage (%), considering the medium as 100% in relation to the other treatments. Bars represent the median and IQR with minimum and maximum error bars. Asterisks indicate a significant difference for the comparisons within the treatment (Wilcoxon test, * p < 0.05).