Pleiotropic Effect of Hormone Insulin-Like Growth Factor-I in Immune Response and Pathogenesis in Leishmaniases

Abstract

Leishmaniases are diseases caused by several Leishmania species, and many factors contribute to the development of the infection. Because the adaptive immune response does not fully explain the outcome of Leishmania infection and considering that the initial events are crucial in the establishment of the infection, we investigated one of the growth factors, the insulin-like growth factor-I (IGF-I), found in circulation and produced by different cells including macrophages and present in the skin where the parasite is inoculated. Here, we review the role of IGF-I in leishmaniasis experimental models and human patients. IGF-I induces the growth of different Leishmania species in vitro and alters the disease outcome increasing the parasite load and lesion size, especially in L. major- and L. amazonensis-infected mouse leishmaniasis. IGF-I affects the parasite interacting with the IGF-I receptor present on Leishmania. During Leishmania-macrophage interaction, IGF-I acts on the arginine metabolic pathway, resulting in polyamine production both in macrophages and Leishmania. IGF-I and cytokines interact with reciprocal influences on their expression. IL-4 is a hallmark of susceptibility to L. major in murine leishmaniasis, but we observed that IGF-I operates astoundingly as an effector element of the IL-4. Approaching human leishmaniasis, patients with mucosal, disseminated, and visceral diseases presented surprisingly low IGF-I serum levels, suggesting diverse effects than parasite growth. We observed that low IGF-I levels might contribute to the inflammatory response persistence and delayed lesion healing in human cutaneous leishmaniasis and the anemia development in visceral leishmaniasis. We must highlight the complexity of infection revealed depending on the Leishmania species and the parasite's developmental stages. Because IGF-I exerts pleiotropic effects on the biology of interaction and disease pathogenesis, IGF-I turns up as an attractive tool to explore biological and pathogenic processes underlying infection development. IGF-I pleiotropic effects open further the possibility of approaching IGF-I as a therapeutical target.

Figure 1

Growth curves of Leishmania spp promastigotes upon IGF-I stimulus. L. major, L. amazonensis, L. infantum, and L. braziliensis promastigotes (5 × 10⁵/mL) were cultured in 199 medium (Cultilab, Brazil) and Schneider's Insect medium (Sigma-Aldrich, USA), respectively, supplemented with 5% heat-inactivated fetal calf serum (FCS) (Cultilab, Brazil) at 26°C, with or without 50 ng/mL IGF-I (rIGF-I, R&D Systems, USA). The growth of parasites was monitored by daily counting, for 10 days, in a Neubauer chamber, and the results are presented as the number of parasites × 10⁷/mL (mean ± standard deviation) from three independent cultures with or without 50 ng/mL IGF-I. ^∗p < 0.05 (one-way ANOVA) compared with the culture without IGF-I (adapted from Reis et al. [65]).

Figure 2

Scheme of the effect of extrinsic IGF-I (rIGF-I) in the L-arginine metabolic pathway activation in macrophages infected by L. amazonensis and L. major. In RAW 264.7 cells or BALB/c mouse peritoneal macrophages infected with L. amazonensis or L. major promastigotes and stimulated with 50 ng/mL recombinant IGF-I (rIGF-I, R&D Systems, USA), the parasitism, arginase mRNA expression, and arginase activity, nitric oxide synthase 2 (NOS2) mRNA expression, and nitric oxide production (Griess reaction) were evaluated. Extrinsic IGF-I induced an increase in arginase expression and arginase activity in both parasites and macrophages, decreased the production of NO, and increased the parasitism in L. amazonensis- and L. major-infected cells, comparably.

Figure 3

Parasitism and nitric oxide production in L. infantum-infected cells upon extrinsic IGF-I stimulus. THP-1 cells (a–c) or BALB/c mouse bone marrow-derived macrophages (d–f) were infected with L. infantum promastigotes and stimulated with recombinant IGF-I (rIGF-I, 50 ng/mL; R&D Systems, USA) for 24 and 48 hours. One representative experiment from three independent assays is shown. THP-1 cells were differentiated into macrophages with 20 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich, USA) for 24 hours. Then, the cells were washed and allowed to rest in a fresh medium for 48 hours before infection with L. infantum promastigotes. The parasitism (median number of parasites per 100 cells), nitric oxide production (Griess Reagent), and arginase activity (urea production) were determined after 24 and 48 hours of incubation.

Figure 4

Detection of IGF-I within RAW 264.7 macrophages following infection with Leishmania major promastigotes. Colocalization of IGF-I and Leishmania was analyzed using immunofluorescence. Following a 24 h in vitro infection, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA), washed in 0.01 M phosphate-buffered saline, pH 7.2 (PBS), blocked for one hour with 2% bovine serum albumin (BSA; Sigma-Aldrich, USA) in PBS, and incubated overnight with monoclonal goat anti-mouse IGF-I antibody (1 : 75; R&D Systems, USA) and a polyclonal mouse anti-Leishmania antibody (1 : 400) [136]. Anti-goat IgG Alexa Fluor-546 (1 : 200, Invitrogen, USA—shown in red) and anti-mouse IgG Alexa Fluor-488 (1 : 400, Invitrogen, USA—shown in green) were used as secondary antibodies. 4,6-Diamidino-2-phenylindole (DAPI, Invitrogen, USA—shown in blue) was used to stain nuclei. Images were captured using a Leica LSM510 confocal microscope with a 63x objective and oil immersion (adapted from Reis et al. [82]).

Figure 5

Parasitism in L. major- or L. amazonensis-infected macrophages upon IGF-I siRNA transfection. RAW 264.7 cells infected with L. major or L. amazonensis promastigotes transfected with or without 150 μM IGF-I siRNA for 6 hours. The parasitism (median number of parasites per 100 cells) was evaluated after 24 and 48 hours. One representative experiment from three independent assays is shown. ^∗p < 0.05 (ANOVA and Tukey's tests).

Figure 6

The effects of IGF-I siRNA and IL-4 on components of the IGF-I signaling pathways: levels of phosphorylated p44 (ERK), p38 (MAPK), and AKT proteins. L. major promastigote-infected or noninfected RAW 264.7 cells transfected with or without IGF-I siRNA were stimulated for 30 minutes with IL-4 (2 ng/mL; R&D Systems, EUA) and recombinant IGF-I (50 ng/mL; R&D Systems, EUA). Cells were lysed, the proteins were separated in 10% SDS-PAGE, and subsequently, a Western blotting was performed using anti-phospho-p44 (137F5, Cell Signaling Technology, USA), anti-phospho-p38 (D13E1, Cell Signaling Technology, USA), and anti-phospho-AKT (Ser473, Cell Signaling Technology, USA) antibodies. Protein bands corresponding to protein expression levels were submitted to a densitometric analysis (AlphaEaseFC™ software 3.2 beta version; Alpha Innotech Corporation, USA), and data are expressed in arbitrary units (adapted from Reis et al. [65]).

Figure 7

Parasitism in response to cytokine treatments and IGF-I siRNA transfection. Parasitism (median number of parasites per 100 cells) in L. amazonensis-infected RAW 264.7 cells transfected with or without 150 μM IGF-I siRNA for 6 hours and then were stimulated with IFN-γ (200 U/mL; R&D Systems, EUA), IL-4 (2 ng/mL; R&D Systems, EUA), or IL-13 (5 ng/mL; R&D Systems, EUA) for 24 (a) and 48 (b) hours. One representative experiment from three independent assays is shown. ^∗p < 0.05 (ANOVA and Tukey's tests).