Local increase of arginase activity in lesions of patients with cutaneous leishmaniasis in Ethiopia

Abstract

Background: Cutaneous leishmaniasis is a vector-borne disease that is in Ethiopia mainly caused by the parasite Leishmania aethiopica. This neglected tropical disease is common in rural areas and causes serious morbidity. Persistent nonhealing cutaneous leishmaniasis has been associated with poor T cell mediated responses; however, the underlying mechanisms are not well understood.

Methodology/principal findings: We have recently shown in an experimental model of cutaneous leishmaniasis that arginase-induced L-arginine metabolism suppresses antigen-specific T cell responses at the site of pathology, but not in the periphery. To test whether these results translate to human disease, we recruited patients presenting with localized lesions of cutaneous leishmaniasis and assessed the levels of arginase activity in cells isolated from peripheral blood and from skin biopsies. Arginase activity was similar in peripheral blood mononuclear cells (PBMCs) from patients and healthy controls. In sharp contrast, arginase activity was significantly increased in lesion biopsies of patients with localized cutaneous leishmaniasis as compared with controls. Furthermore, we found that the expression levels of CD3ζ, CD4 and CD8 molecules were considerably lower at the site of pathology as compared to those observed in paired PBMCs.

Conclusion: Our results suggest that increased arginase in lesions of patients with cutaneous leishmaniasis might play a role in the pathogenesis of the disease by impairing T cell effector functions.

Figure 1. Arginase activity in PBMCs from controls and LCL patients.

PBMCs from controls (n = 10) and LCL patients (n = 11) were isolated by Ficoll gradient and the activity of arginase was measured by enzymatic assay. Values represent median with interquartile range. Statistical significance was determined by a two-tailed Mann-Whitney test, ns = not significant.

Figure 2. Arginase-expressing cells in PBMCs are neutrophils.

PBMCs were isolated by density gradient from the blood of LCL patients. The phenotype of arginase-expressing cells was determined by flow cytometry. (A) Dot plot profile of CD15⁺ arginase⁺ cells; (B) dot plot profile of CD14⁺ arginase⁺ cells; (C) dot plot profile of forward (FSC) and side scatter (SSC). Data show the results of one representative experiment out of 15 independent experiments. Isotype control for arginase: 0.98%.

Figure 3. Frequency of neutrophils and monocytes in PBMCs.

PBMCs were isolated by density gradient from the blood of controls (n = 10) and LCL patients (n = 15) and the frequencies of CD15⁺ (A), CD14⁺ (B) cells and the ratio of CD15/CD14 (C) were determined by flow cytometry. Values represent median with interquartile range. Statistical significance was determined by a two-tailed Mann-Whitney test, ns = not significant.

Figure 4. Arginase activity in skin lesions from controls and LCL patients.

Skin biopsies from controls (n = 6) and LCL patients (n = 10) were homogenized and the activity of arginase was measured by enzymatic assay. Values represent median with interquartile range. Statistical significance was determined by a two-tailed Mann-Whitney test.

Figure 5. Arginase-expressing cells in biopsy are neutrophils.

The phenotype of arginase-expressing cells in homogenates of skin biopsies was determined by flow cytometry by using a combination of antibodies against CD14, CD15 and arginase. Data show the results of one representative experiment out of three independent experiments.

Figure 6. Arginase activity in cells isolated from peripheral blood and skin biopsies from LCL patients.

Arginase activity was measured by enzymatic assay in skin homogenates and PBMCs from LCL patients (n = 10). Statistical significance was determined by a Wilcoxon paired test.

Figure 7. Frequency of CD4⁺ and CD8⁺ T cells in PBMCs and biopsies of LCL patients.

The frequencies of CD4⁺ and CD8⁺ T cells was determined in cells isolated from the blood and from the biopsies of LCL patients (n = 15) by flow cytometry. (A) % of CD4⁺ T cells; (B) % of CD8⁺ T cells; (C) ratio of CD4/CD8. Values represent median with interquartile range. Statistical significance was determined by a two-tailed Mann-Whitney test, ns = not significant.

Figure 8. CD3ζ and CD4 MFI in PBMCs and biopsies of LCL patients.

The MFI of CD3ζ in CD4⁺ T cells (A) and MFI of CD4 molecule (D) were compared between cells isolated from the blood and from the biopsies of LCL patients (n = 12) by flow cytometry; (B) representative histogram of MFI of CD3ζ in CD4⁺ T cells (open histogram  =  biopsies, closed histogram = PBMCs); (E) representative histogram of MFI of CD4 (open histogram = biopsies, closed histogram = PBMCs); (C) % decrease in CD3ζ MFI in CD4 (black bar = median); (F) % decrease in CD4. Statistical significance was determined by a Wilcoxon paired test.

Figure 9. CD3ζ and CD8 MFI in PBMCs and biopsies of LCL patients.

The MFI of CD3ζ in CD8⁺ T cells (A) and MFI of CD8 molecule (D) were compared between cells isolated from the blood and from the biopsies of LCL patients (n = 12) by flow cytometry; (B) representative histogram of MFI of CD3ζ in CD8⁺ T cells (open histogram = biopsies, closed histogram = PBMCs); (E) representative histogram of MFI of CD8 (open histogram = biopsies, closed histogram = PBMCs); (C) % decrease in CD3ζ MFI in CD8 (black bar = median); (F) % decrease in CD8. Statistical significance was determined by a Wilcoxon paired test.