Temporary Shutdown of ERK1/2 Phosphorylation Is Associated With Activation of Adaptive Immune Cell Responses and Disease Progression During Leishmania amazonensis Infection in BALB/c Mice

Abstract

Leishmania spp. infection outcomes are dependent on both host and parasite factors. Manipulation of host signaling pathways involved in the generation of immune responses is thought to be one of the most common mechanisms used by parasites for persistence within the host. Considering the diversity of pathologies caused by different Leishmania spp., it is plausible that significant differences may exist in the mechanisms of host cell manipulation by each parasite species, which may have implications when developing new vaccine or treatment strategies. Here we show that in L. braziliensis-infection in BALB/c mice, a model of resistance, activation of ERK1/2 coincides with the peak of inflammatory responses and resolution of tissue parasitism. In contrast, in the susceptibility model of L. amazonensis-infection, an early silent phase of infection is observed, detected solely by quantification of parasite loads. At this early stage, only basal levels of P-ERK1/2 are observed. Later, after a brief shutdown of ERK1/2 phosphorylation, disease progression is observed and is associated with increased inflammation, lesion size and tissue parasitism. Moreover, the short-term down-regulation of ERK1/2 activation affected significantly downstream inflammatory pathways and adaptive T cell responses. Administration of U0126, a MEK/ERK inhibitor, confirmed this phenomenon, since bigger lesions and higher parasite loads were seen in infected mice that received U0126. To investigate how kinetics of ERK1/2 activation could affect the disease progression, U0126 was administered to L. amazonensis-infected animals earlier than the P-ERK1/2 switch off time-point. This intervention resulted in anticipation of the same effects on inflammatory responses and susceptibility phenotype seen in the natural course of infection. Additionally, in vitro inhibition of ERK1/2 affected the phagocytosis of L. amazonensis by BMDMs. Collectively, our findings reveal distinct temporal patterns of activation of inflammatory responses in L. braziliensis and L. amazonensis in the same animal background and a pivotal role for a brief and specific shutdown of ERK1/2 activation at late stages of L. amazonensis infection. Since activation of inflammatory responses is a crucial aspect for the control of infectious processes, these findings may be important for the search of new and specific strategies of vaccines and treatment for tegumentary leishmaniasis.

Keywords: L. amazonensis; L. braziliensis; inflammation; kinetics of ERK1/2 activation; leishmaniasis infection; treatment; vaccine.

Figure 1

Kinetic evaluation of lesion size, tissue parasitism and histopathological changes during L. amazonensis and L. braziliensis infection in BALB/c mice. Females of BALB/c mice were inoculated into the left hind footpad with 1x10⁵ promastigotes of L. amazonensis or 1x10⁷ promastigotes of L. braziliensis. Data are represented as mean ± SD of at least four mice in each time point. (A) The development of the lesion size was monitored weekly, based on the difference in thickness between inoculated and the control paw with a digital caliper. (B) Parasite loads were evaluated at different times after infection. Statistically significant differences were assumed when p < 0.05 in relation to 2, 5 and 9 weeks after L. braziliensis infection and 2, 5 and 9 weeks after L. amazonensis infection. (C) Histopathological analyses (H&E) were performed 2-, 5- and 9-weeks post-infection for L. braziliensis (I e III) and L. amazonensis (II, IV, V and VI) respectively. E = eosinophils; M = macrophages; L = lymphocytes, bracket: dispersion of collagen fibers. Levels of IFN-g (D, E) and IL-10 (F, G) in culture supernatants of lymph node cells, at specific time points after L. braziliensis or L. amazonensis infection, respectively. Ag means Leishmania soluble antigen used to stimulate cultures. (a, c) indicate significant differences (p < 0.05) in cytokine levels in cultures with no stimulus (white bars), as compared to 24 hours (a) or 2 weeks (c); (b, d) indicate differences after stimulus (black bars), as compared to 24 hours or 2 weeks, respectively. ND, not detected.

Figure 2

Activation status of signaling proteins involved in inflammation and apoptotic pathways after L. braziliensis and L. amazonensis infection. Mice were infected in the left hind footpad with 1x10⁷ promastigotes of L. braziliensis or 1 x 10⁵ promastigotes of L. amazonensis. Skin samples from infected footpads were processed and evaluated by Western blotting analysis. (A) Western blotting of P-ERK1/2, P-p65/RelA (NF-kB), and cleaved caspase-3 at 24 hours, 2, and 12 weeks after L. braziliensis infection. (B) Western blotting of P-ERK1/2, P-p65/RelA (NF-kB), and cleaved caspase-3 at 24 hours, 2, 5 and 9 weeks after L. amazonensis infection. Densitometric analysis of the correspondent western blots for P-ERK1/2 (C, D), P-p65 (NF-kB) (E, F), and cleaved caspase-3 (G, H), for L. braziliensis or L. amazonensis infections, respectively. Data are representative of two experiments, with similar results. All data are presented as mean + SD of three mice per group in each time point. Statistically significant differences were assumed when p< 0.05 in relation to control (a), 24 hours post-infection (b), 2 weeks post-infection (c) or 5 weeks post-infection (d).

Figure 3

| In vitro evaluation of the impact of treatment of BMDMs obtained from BALB/c mice with U0126 on the L. amazonensis promastigotes intake and levels of cytokines. BMDMs were pre-treated with 15 mM OF U0126 for 2 hours and then infected with L. amazonensis promastigotes, at a 5:1 parasite/cell ratio. Profiles observed in western blotting analysis performed for ERK1/2 phosphorylation (A) and total ERK1/2 expression levels (B) of not-treated, not infected cells (1) or cells treated with U0126 prior to infection by L. amazonensis. Westerns were performed for infected cells either after 3 hours or 24 hours post-infection. Densitometric analyzes for ERK1/2 phosphorylation (C) and Total ERK1/2 expression (D), after normalization with β-actin. At 3, 6 and 24h post infection, cells were evaluated for the percentage of infected cells (E) and number of parasites per infected cells (F). At the same time points, culture supernatants were assessed for TNF-α (G) and IL-10 (H) concentrations. Data are representative of two experiments with similar results. In (C, D), statistically significant differences were assumed when p< 0.05 as compared to control cells (a), to 3 hours after infection of untreated cells (b). In (E, F), the asterisk indicates statistically significant differences by comparing infection rates in untreated and treated cells at each time-points after infection. In (G, H), (a) indicates statistically significant differences as compared to control non treated and infected cells for 3 hours and (b) as compared to treated and infected cells for 3 hours and (c) as compared to non-treated and infected cells for 6 hours (d) as compared to treated and infected cells for 6 hours. ND, not detected.

Figure 4

Evaluation of treatment with U0126 at 4 weeks of L. amazonensis infection. (A) Females of BALB/c mice (n = 6) were inoculated into the left hind footpad with 1x10⁵ promastigotes of L. amazonensis and after four weeks of infection animals received U0126 (3mg/kg) intraperitoneally for seven consecutive days and then they were euthanized (5th week). (B) The development of the lesion size was monitored weekly, before treatment beginning, based on the difference in thickness between inoculated and the control paw with a digital caliper, and daily, after treatment beginning for seven consecutive days. Black arrow indicates treatment beginning and dotted arrow indicates end of treatment. (C) Parasite loads between treated and not treated groups at five weeks after infection. Levels of IFN-g (D) and IL-10 (E) in culture supernatants of lymph node cells, at end of treatment (5 weeks). Ag means Leishmania soluble antigen used to stimulate cultures. Significant differences (p > 0.05) are indicated by (a) as compared to untread and not stimulated cultures, (b) to untread and stimulated cultures and (c) treated and stimulated cultures. (F) Histopathological analyses (H&E) were performed at 5 weeks post L. amazonensis infection in not treated and untreated groups. Quantification of inflammatory infiltrate (skin scores) in mice footpad at 5 weeks post-infection (G). The asterisks indicate statistically significant differences between treated and untread groups (p < 0.05).

Figure 5

Evaluation of treatment with U0126 at 2 weeks of L. amazonensis infection. (A) Females of BALB/c mice (n = 6) were inoculated into the left hind footpad with 1x10⁵ promastigotes of L. amazonensis and after two weeks of infection animals received U0126 (3mg/kg) intraperitoneally for seven consecutive days. (B) The development of the lesion size was monitored weekly, before and after treatment, for 6 weeks, based on the difference in thickness between inoculated and the control paw with a digital caliper. Black arrow indicates treatment beginning and dotted arrow indicates end of treatment. (C) Parasite loads between treated and not treated groups, three weeks after end of treatment (six weeks post-infection). Levels of IFN-g (D) and IL-10 (E) in culture supernatants of lymph node cells 6 weeks after infection. Ag means Leishmania soluble antigen used to stimulate cultures. Significant differences (p < 0.05) are indicated by (a) as compared to untread and not stimulated cultures, (b) to untread and stimulated cultures and (c) treated and stimulated cultures. (F) Histopathological analyses (H&E) were performed at 6 weeks post L. amazonensis infection in not treated and untreated groups. Quantification of inflammatory infiltrate (skin scores) in mice footpad at 6 weeks post-infection (G). The asterisks indicate statistically significant differences between treated and untread groups (p < 0.05).