Arginase I induction during Leishmania major infection mediates the development of disease

Abstract

In a previous work, we demonstrated that the induction of arginase I favored the replication of Leishmania inside macrophages. Now we have analyzed the differential expression of this enzyme in the mouse model of L. major infection. Ours results show that arginase I is induced in both susceptible and resistant mice during the development of the disease. However, in BALB/c-infected tissues, the induction of this protein parallels the time of infection, while in C57BL/6 mice, the enzyme is upregulated only during footpad swelling. The induction of the host arginase in both strains is mediated by the balance between interleukin-4 (IL-4) and IL-12 and opposite to nitric oxide synthase II expression. Moreover, inhibition of arginase reduces the number of parasites and delays disease outcome in BALB/c mice, while treatment with l-ornithine increases the susceptibility of C57BL/6 mice. Therefore, arginase I induction could be considered a marker of disease in leishmaniasis.

FIG. 1.

Arginase I induction correlates with footpad swelling and is opposite to NOS II induction. Mice (n = 10) were infected with 10⁶ stationary-phase L. major promastigotes, and lesion size (n = 10), parasites/mg of tissue (n = 3) (A), or arginase activity/mg (n = 5) (B) were measured as described in Materials and Methods. (C) Tissue homogenates (20 μg protein/lane) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with anti-arginase I, anti-NOS II, or anti-actin as internal control. C, positive controls; L, L. major lysate. One of three independent repeats is represented. The data presented are representative of three independent experiments. Values are the means ± standard deviations of n (mice per time point). **, P < 0.01; ***, P < 0.001, versus those for day 0 by Student's t test.

FIG. 2.

Immunohistochemical staining of arginase I in BALB/c and C57BL/6 footpads. Tissues (n = 2) were fixed in formalin, embedded in paraffin, and treated with anti-arginase I monoclonal antibody as described in Materials and Methods. (A) BALB/c day 14. Positive staining in mononuclear infiltrate and the intracellular connective tissue of the dermis. Magnification, ×10. (B) BALB/c at day 28. Massive mononuclear infiltrate with intense arginase reaction, mainly in phagocytes. The magnification (×100) shows a macrophage with intracellular arginase staining. (C) BALB/c at day 42. Massive arginase expression in subepidermal and dermal areas. Magnification, ×20. The inset represents a negative isotype control for arginase I showing the necrotic areas. (D) C57BL/6 at day 14. Presence of immunostaining mainly in connective tissue. Magnification, ×10. (E) C57BL/6 at day 28. Moderate arginase staining in fibroblasts and some phagocytes within the dermis. Magnification, ×100. (F) C57BL/6 at day 42. Low levels of arginase expression in the interstitial connective tissue of the dermis. Magnification, ×20. Micrographs are the best representatives of two independent repeats with n = 2.

FIG. 3.

Kinetics of circulating IL-4 (A) and IL-12 (B) levels in sera of L. major-infected BALB/c and C57BL/6 mice. Blood samples were taken at different times of infection, and cytokine concentrations were measured by enzyme-linked immunosorbent assay as described in Materials and Methods. Data are representative of three independent experiments. Values are the means ± standard errors of the means of five mice per point. Significance values are comparing the increase in cytokine concentrations from day 2 postinfection for IL-4 or day 0 for IL-12. **, P < 0.01; ***, P < 0.001.

FIG. 4.

Nor-NOHA delays disease in BALB/c mice, while l-ornithine increases the susceptibility to the infection in C57BL/6 mice. Animals (five per group) were injected daily with a dose of 10 μg/50 μl nor-NOHA (Fig. 4A) or 500 μg/50 μl l-ornithine (Fig. 4B) next to the footpad. Control mice (BALB/c and C57BL/6) were treated with the same amount of PBS. Parasites/lesion at indicated times are represented in numbers. One in five comparable experiments for nor-NOHA and one in two for ornithine treatments are shown. Significant values are comparing treated animals versus nontreated animals. **, P < 0.01; ***, P < 0.001.