Association of pro-inflammatory cytokines and iron regulatory protein 2 (IRP2) with Leishmania burden in canine visceral leishmaniasis

Abstract

Leishmania infantum infection in humans and dogs can evolve with a wide range of clinical presentations, varying from asymptomatic infections to visceral leishmaniasis. We hypothesized that the immune response elicited by L. infantum infection could modulate whether the host will remain asymptomatic or progress to disease. A total of 44 dogs naturally infected with L. infantum were studied. Leishmania burden was estimated in the blood and spleen by qPCR. The expression of IFN-γ, TNF-α, IL-10 and Iron Regulatory Protein 2 (IRP2) were determined in the spleen by quantitative PCR. Sera cytokines were evaluated by ELISA. Dogs were grouped in quartiles according parasite burden. Increased expression of IFN-γ and TNF-α was associated with reduced Leishmania burden, whereas increased IL-10 and IRP2 expressions were associated with higher Leishmania load. Increased plasma albumin and IFN-γ expression explained 22.8% of the decrease in parasite burden in the spleen. These data confirm that lower IFN-γ response and higher IL-10 correlated with increased parasite load and severity of the visceral leishmaniasis in dogs. The balance between the branches of immune response and the intracellular iron availability could determine, in part, the course of Leishmania infection.

Figure 1. Leishmania load in the blood and the spleen of dogs naturally infected with Leishmania infantum.

A. Dogs were grouped in quartiles according to spleen parasite burden and levels were compared to Leishmania level in the blood. Analysis was performed Mann-Whitney test. The Kolmogorov-Smirnov test was used to test the normality of the data. B. Dogs were grouped in accordance to Leishmania culture, positive or negative.

Figure 2. Anti-Leishmania antibodies in accordance to Leishmania load in the spleen.

A. Total anti-Leishmania IgG antibodies. B. Anti rK-39 antibodies. The cut-off values of 0.096 and 0.112 for SLA and rK-39, respectively, which corresponded to the mean of the results of five healthy dogs plus three standard deviations. The dashed line represents the cut off value for the test. C and D levels of IgG1 and IgG2 subclasses. Analysis was performed using One-way ANOVA or Kruskal-Wallis test. The Kolmogorov-Smirnov test was used to test the normality of the data.

Figure 3. Sera albumin concentration in accordance to Leishmania load in the spleen.

Analysis was performed by One-way ANOVA and the Kolmogorov-Smirnov test was used to test the normality of the data.

Figure 4. Spleen gene expression of cytokines and IRP2 in accordance to to Leishmania load.

A, IFN-γ.B, TNF-α.C, IL-10 and D, IRP2. ACTB was taken as housekeeping gene and the first quartile was used as the calibrator group, since it presented the lowest spleen parasitism estimated by kDNA. Analysis was performed by One-way ANOVA or Kruskal-Wallis test and the Kolmogorov-Smirnov test was used to test the normality of the data.

Figure 5. Levels of serum cytokines according to spleen Leishmania parasitism.

A, IFN-γ, B, TNF-α and C, IL-10. Serum samples from each animal were used to quantify the concentration of these cytokines by ELISA and a standard curve was built using recombinant canine cytokine. Analysis was performed by One-way ANOVA or Kruskal-Wallis test and the Kolmogorov-Smirnov test was used to test the normality of the data.