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. 2020 Aug 31:11:1986.
doi: 10.3389/fmicb.2020.01986. eCollection 2020.

Modulation of Cytokines and Extracellular Matrix Proteins Expression by Leishmania amazonensis in Susceptible and Resistant Mice

Flávia de Oliveira Cardoso  1 , Tânia Zaverucha-do-Valle  1 , Fernando Almeida-Souza  1   2 , Ana Lúcia Abreu-Silva  2 , Kátia da Silva Calabrese  1
Affiliations

Modulation of Cytokines and Extracellular Matrix Proteins Expression by Leishmania amazonensis in Susceptible and Resistant Mice

Flávia de Oliveira Cardoso et al. Front Microbiol. .

Abstract

Leishmaniases are a complex of diseases with a broad spectrum of clinical forms, which depend on the parasite species, immunological status, and genetic background of the host. In the Leishmania major model, susceptibility is associated with the Th2 pattern of cytokines production, while resistance is associated with Th1 response. However, the same dichotomy does not occur in L. amazonensis-infected mice. Cytokines are key players in these diseases progression, while the extracellular matrix (ECM) components participate in the process of parasite invasion as well as lesion healing. In this article, we analyzed the influence of host genetics on the expression of cytokines, inducible nitric oxide synthase (iNOS), and ECM proteins, as well as the parasite load in mice with different genetic backgrounds infected by L. amazonensis. C57BL/10 and C3H/He mice were subcutaneously infected with 106 L. amazonensis promastigotes. Lesion kinetics, parasite load, cytokines, iNOS, and ECM proteins expression were measured by quantitative PCR (qPCR) in the footpad, draining lymph nodes, liver, and spleen at early (24 h and 30 days) and late phase (120 and 180 days) of infection. Analysis of lesion kinetics showed that C57BL/10 mice developed ulcerative lesions at the inoculation site after L. amazonensis infection, while C3H/He showed slight swelling in the footpad 180 days after infection. C57BL/10 showed progressive enhancement of parasite load in all analyzed organs, while C3H/He mice showed extremely low parasite loads. Susceptible C57BL/10 mice showed high levels of TGF-β mRNA in the footpad early in infection and high levels of proinflammatory cytokines mRNA (IL-12, TNF-α, and IFN-γ) and iNOS in the late phase of the infection. There is an association between increased expression of fibronectin, laminin, collagen III and IV, and TGF-β. On the other hand, resistant C3H/He mice presented a lower repertory of cytokines mRNA expression when compared with susceptible C57BL/10 mice, basically producing TNF-α, collagen IV, and laminin early in infection. The findings of our study indicate that L. amazonensis infection induces different cytokine expression in resistant and susceptible mice but not like the L. major model. An organ-compartmentalized cytokine response was observed in our model. Host genetics determine this response, which modulates ECM proteins expression.

Keywords: cytokines; extracellular matrix; immunopathology; leishmaniasis; mice; quantitative PCR; real-time quantitative PCR.

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Figures

Figure 1
Figure 1
Lesion kinetics. Progression of lesion size in the footpad of C57BL/10 and C3H/He mice subcutaneously infected with 106 Leishmania amazonensis promastigote forms in the right hind footpad (RHF). Data represent mean ± SD of two independent experiments with four animals. ****p < 0.0001 when compared between groups by two-way ANOVA and Bonferroni’s multiple comparison test.
Figure 2
Figure 2
Parasite Load. Quantification of parasites by quantitative PCR (qPCR) in the footpad (lesion site; A), draining lymph node (B), liver (C), and spleen (D) of C57BL/10 and C3H/He mice subcutaneously infected with 106 L. amazonensis promastigote forms in the RHF. Data represent mean ± SD of two independent experiments with four animals assayed in triplicate. ****p < 0.0001 when compared with the control group or between groups in brackets by two-way ANOVA and Bonferroni’s multiple comparison test.
Figure 3
Figure 3
Cytokine expression profile. Cytokine expression by real-time quantitative PCR (RT-qPCR) in the footpad (lesion site), liver, and spleen of C57BL/10 and C3H/He mice subcutaneously infected with 106 L. amazonensis promastigote forms in the RHF and normal mice from the same genetic background. Expression of TNF-α (A,G,M), IFN-γ (B,H,N), IL-12 (C,I,O), IL-4 (D,J,P), IL-10 (E,K,Q), and TGF-β (F,L,R) was estimated by ΔΔCT method, using Rplp0 as a reference gene. Data represent mean ± SD of two independent experiments with four animals assayed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 when compared with the control group or between groups in brackets by two-way ANOVA and Bonferroni’s multiple comparison test. RQ, relative quantification; Ifng – interferon gamma; Tnf – tumor necrosis factor, transcript variant 1; Il12a – interleukin 12a, transcript variant 1; Il4 – interleukin 4, transcript variant 1; Il10 – interleukin 10; Tgfb1 – transforming growth factor, beta 1; Rplp0 – ribosomal protein, large, P0.
Figure 4
Figure 4
Inducible nitric oxide synthase (iNOS) expression. iNOS expression by RT-qPCR in the footpad (lesion site; A), liver (B), and spleen (C) of C57BL/10 and C3H/He mice subcutaneously infected with 106 L. amazonensis promastigote forms in the RHF and normal mice from the same genetic background. Expression of iNOS was estimated by the ΔΔCT method, using Rplp0 as a reference gene. Data represent mean ± SD of two independent experiments with four animals assayed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 when compared with the control group or between groups in brackets by two-way ANOVA and Bonferroni’s multiple comparison test. RQ, relative quantification; Nos2 – nitric oxide synthase 2; inducible, transcript variant 1; Rplp0 – ribosomal protein, large, P0.
Figure 5
Figure 5
Extracellular matrix (ECM) protein expression. Expression profile of ECM proteins by RT-qPCR from the lesion site, liver, and spleen of C57BL/10 and C3H/He mice subcutaneously infected with 106 L. amazonensis promastigote forms in the RHF and normal mice from the same genetic background. Expression of collagens I (A,F,K), III (B,G,L), and IV (C,H,M), fibronectin (D,I,N), and laminin (E,J,O) were estimated by the ΔΔCT method, using Rplp0 as a reference gene. Data represent mean ± SD of two independent experiments with four animals assayed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 when compared with the control group or between groups in brackets by two-way ANOVA and Bonferroni’s multiple comparison test. RQ, relative quantification; Col1a1 – collagen, type I, alpha 1; Col3a1 – collagen, type III, alpha 1; Col4a2 – collagen, type IV, alpha 2; Fn1 – fibronectin 1; Lama5 – laminin, alpha 5, transcript variant 1; Rplp0 – ribosomal protein, large, P0.
Figure 6
Figure 6
Immunopathological mechanisms occurred in the skin of C3H/He and C57BL/10 mice infected with L. amazonensis. In C3H/He mice, innate immune response acts quickly to eliminate Leishmania. Infected dendritic cells (DC1) produce TNF-α, which activates infected macrophages to kill intracellular amastigotes; and IL-12 which stimulates the differentiation of naïve CD4+ T cells into Th1 effectors. Th1 cells help macrophage activation with more TNF-α production. In response, macrophages produce more of these cytokines and kill intracellular amastigotes through a NO-independent mechanism. TGF-β produced by regulatory T cells acts on fibroblasts present on the infection site, stimulating the production of collagens III and IV and laminin and helping the healing process. Due to the efficient response, in the late phase of infection, no more parasites are found on the infection site and tissue structure is restored. In C57BL/10, innate immune response is also activated. IFN-γ is produced by NK cells and dendritic cells (DC1), which also produce TNF-α to active macrophages (M1) to kill intracellular amastigotes. At the same time, IL-12 produced by DC1 stimulates Th1 cells to produce more IFN-γ and TNF-α, reinforcing the parasite killing. Meanwhile, TGF-β produced by DC2 and T reg cells, stimulates macrophages alternatively activating them into the M2 profile, which are incapable of eliminating intracellular amastigotes. Upon stimulation by TGF-β, fibroblasts secrete collagen III and IV, fibronectin, and laminin in an attempt to recover the damaged tissue. In the late phase, as parasites continue to grow in the tissue, Th2 lymphocytes and eosinophils produce IL-4 stimulating M2 macrophages that are incapable of fighting infection. Amastigotes proliferate inside M2 macrophages, while M1 cells continue to fight the parasite, producing more TNF-α and IL-12. Excessive TNF-α provokes more tissue damage. Continue IFN-γ stimulus block TGF-β transcription, suppressing the fibronectin and collagen IV production by fibroblasts. The high proinflammatory stimulus attracts more macrophages to the site, which amplifies infection and destroys the tissue. DC1, dendritic cell type 1; DC2, dendritic cell type 2; ECM, extracellular matrix; MØ, macrophage; M1, classically-activated macrophage; M2, alternatively activated macrophage; NK, natural killer; T reg cells, regulatory T cells.
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