Expression of key cytokines in dog macrophages infected by Leishmania tarentolae opening new avenues for the protection against Leishmania infantum

Abstract

The detection of Leishmania tarentolae in sympatric areas where Leishmania infantum is endemic raised questions regarding the protective effect exerted in dogs by L. tarentolae when in coinfection. This study aimed monitoring the in vitro gene expression of pro- (IFN- γ; TNF-α; IL-12) and anti-inflammatory (IL-4; IL-6; IL-10) cytokines in primary canine macrophages infected by L. tarentolae and L. infantum in single and in co-infections. Macrophages differentiated from dog blood mononuclear cells were infected with the L. tarentolae field-isolated (RI-325) and laboratory (LEM-124) strains, with L. infantum laboratory strain (IPT1), or both. Infection and the number of amastigotes per infected cell were evaluated microscopically by counting a total of 200 cells between 4 and 96 h. Cytokine gene expression was analyzed by real-time PCR from infected macrophages mRNA. Single infections presented higher expression of the cytokines IL-4 and IL-6, and lower of IL-12. Co-infections induced a lower gene expression of IL-4 and IL-6, and a higher gene expression of IL-12, correlating with the low amastigote burden despite the slight increase of infected cells. Data highlight the potential protective effect of L. tarentolae against L. infantum in co-infection by the reduced anti-inflammatory and increased pro-inflammatory cytokines gene expression, opening new perspectives for a canine vaccine development exploiting the non-pathogenic L. tarentolae.

Fig. 1

Leishmania infection efficiency (A) and intracellular parasite burden at 96 h post-infection (B) were assessed microscopically in primary dog macrophages performed in triplicate. RI-325 and LEM-124: Leishmania tarentolae; IPT1: Leishmania infantum. a/infc: average number of amastigotes in each infected cell. Asterisks indicate the statistical difference between groups (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Fig. 2

Relative mRNA gene expression (relative units) of anti-inflammatory cytokines IL-4 (A, B), IL-6 (C, D) and IL-10 (E, F) for each strain and its coinfections from 4 to 96 h in triplicate (standard deviation shown). RI-325 and LEM-124: Leishmania tarentolae; IPT1: Leishmania infantum. Asterisks indicate the statistical difference between species/strains (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Fig. 3

Relative mRNA gene expression (relative units) of pro-inflammatory cytokines IL-12 (A, B) for each strain and its coinfections from 4 to 96 h in triplicate (standard deviation shown). RI-325 and LEM-124: Leishmania tarentolae; IPT1: Leishmania infantum. Asterisks indicate the statistical difference between groups (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).