Age-related alteration of arginase activity impacts on severity of leishmaniasis - PubMed (original) (raw)

Ingrid Müller et al. PLoS Negl Trop Dis. 2008.

Abstract

Background: The leishmaniases are a group of vector-borne parasitic diseases that represent a major international public health problem; they belong to the most neglected tropical diseases and have one of the highest rates of morbidity and mortality. The clinical outcome of infection with Leishmania parasites depends on a variety of factors such as parasite species, vector-derived products, genetics, behaviour, and nutrition. The age of the infected individuals also appears to be critical, as a significant proportion of clinical cases occur in children; this age-related higher prevalence of disease is most remarkable in visceral leishmaniasis. The mechanisms resulting in this higher incidence of clinical disease in children are poorly understood. We have recently revealed that sustained arginase activity promotes uncontrolled parasite growth and pathology in vivo. Here, we tested the hypothesis that arginase-mediated L-arginine metabolism differs with age.

Methodology: The age distribution of patients with visceral or cutaneous leishmaniasis was determined in cohorts of patients in our clinics in endemic areas in Ethiopia. To exclude factors that are difficult to control in patients, we assessed the impact of ageing on the manifestations of experimental leishmaniasis. We determined parasite burden, T cell responses, and macrophage effector functions in young and aged mice during the course of infection.

Results: Our results show that younger mice develop exacerbated lesion pathology and higher parasite burdens than aged mice. This aggravated disease development in younger individuals does not correlate with a change in T helper cytokine profile. To address the underlying mechanisms responsible for the more severe infections in younger mice, we investigated macrophage effector functions. Our results show that macrophages from younger mice do not have an impaired capacity to kill parasites; however, they express significantly higher levels of arginase 1 than aged mice and promote parasite growth more efficiently. Thus, our results demonstrate that ageing differentially impacts on L-arginine metabolism and subsequent effector functions of physiologically distinct macrophage subsets.

Conclusions: Here, we show that arginase-mediated L-arginine metabolism is modulated with age and affects the capacity of macrophages to express arginase; the increased capacity to upregulate this enzyme in younger individuals results in a more permissive environment for parasite growth, increased disease severity and pathology. These results suggest that the difference in arginase-mediated L-arginine catabolism is likely to be an important factor contributing to the increased incidence of clinical cases in children. Thus, targeting L-arginine metabolism might be a promising therapeutic strategy against leishmaniasis, especially in children and young adults.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Severity of experimental leishmaniasis is increased in young BALB/c mice.

Groups of young (6–8 weeks) and older (12 months) BALB/c mice were infected with 2×106 L. major promastigotes in one hind footpad. (a) The lesion development was monitored by measuring the increase in footpad thickness at regular intervals; the error bars represent standard error of the mean; (b) the parasite load was determined by limiting dilution assay 4 weeks post infection; the horizontal bar represents the average value. Data show the results of one representative experiment out of six independent experiments.

Figure 2

Figure 2. Age distribution of visceral and cutaneous leishmaniasis.

The age distribution of patients suffering from visceral (n = 37, circle) or cutaneous (n = 91, triangle) leishmaniasis was determined in endemic areas of Ethiopia.

Figure 3

Figure 3. Exacerbation of disease in young BALB/c mice is not associated to a switch in Th phenotype.

Groups of young (6–8 weeks) and older (12 months) BALB/c mice were infected with 2×106 L. major promastigotes in one hind footpad. Four weeks post infection, the popiteal lymph nodes were harvested and restimulated with L. major parasites. The supernatant were harvested and tested for their content of cytokines by ELISA. The error bars represent standard error of the mean and data show the results of one representative experiment out of three independent experiments.

Figure 4

Figure 4. Higher arginase activity at the site of pathology correlates with exacerbation of disease.

Groups of young (6–8 weeks) and older (12 months) BALB/c mice were infected with 2×106 L. major promastigotes in one hind footpad. Four weeks post infection, the infected footpads were harvested and the level of arginase activity was determined by enzymatic assay (a) or western blot (b). (c) Groups of pre-pubertal mice (3–4 weeks), adult (10 weeks) or older (40 weeks) BALB/c mice were infected with 2×106 L. major promastigotes in one hind footpad and the lesion development was monitored by measuring the increase in footpad thickness at regular intervals; the error bars represent standard error of the mean (left panel); 4 weeks post infection, the infected footpads were harvested and the parasite load (upper right panel) and arginase activity (lower right panel) were measured. (d) BALB/c mice were infected with 2×106 L. major promastigotes in one hind footpad. Four weeks post infection, the infected footpads were harvested and arginase activity was determined both in the footpads and in the amastigotes purified from the footpads. The horizontal bar represents the average value and data show the results of one representative experiment out of three independent experiments.

Figure 5

Figure 5. Classically activated macrophages are not affected by ageing.

BMMΦ were derived from young (6–8 weeks) and older (12 months) BALB/c mice and stimulated into CAMΦ with IFN-γ and TNF-α or into AAMΦ with IL-4 or IL-13. (a) Activation markers were determined by flow cytometry; (b) NO production was assessed by using the Griess reagents. The error bars represent standard deviation and data show the results of one representative experiment out of four independent experiments.

Figure 6

Figure 6. Arginase expression is increased in alternatively activated macrophages from young mice.

BMMΦ were derived from young (6–8 weeks) and older (12 months) BALB/c mice and differentiated into AAMΦ with IL-4 or IL-13. (a) Activation markers were determined in L. major infected and uninfected cells by flow cytometry; (b) arginase activity was measured by enzymatic assay. The error bars represent standard deviation and data show the results of one representative experiment out of four independent experiments.

Figure 7

Figure 7. Alternatively activated macrophages from young mice have an increased capacity to promote parasite growth.

BMMΦ were derived from young (6–8 weeks) and older (12 months) BALB/c mice, differentiated into CAMΦ with IFN-γ and TNF-α or into AAMΦ with IL-4 and infected with L. major. Four days later, the number of viable parasite per 1×106 MΦ was determined by LDA.

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