A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Findings: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent on the PI3-kinase signaling pathway. Conclusions: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.Fundacao de Amparo a Pesquisa do Estado de Minas Gerais [APQ-00621-11]Fundacao de Amparo a Pesquisa do Estado de Minas GeraisFundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao PauloCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior [23038005295/2011-40]Coordenacao de Aperfeicoamento de Pessoal de Nivel SuperiorConselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Cientifico e Tecnologic

P21-His₆ binds to CXCR4 chemokine receptor.

<p>CCR4 knockout did not affect P21-His₆ pro-phagocytic activity (A). CXCR4 inhibitor abolished the pro-phagocytic P21-His₆ activity (B). Peritoneal macrophages treated with P21-His₆ showed an increased expression of CXCR4 revealed by Western blotting (C). *P<0.5, **P<0.01, ***P<0.001.</p

P21-His₆ augments phagocytosis of different intracellular parasites.

<p>Peritoneal macrophages were incubated with <i>Trypanosoma cruzi</i>, <i>Leishmania amazonensis</i> or <i>Toxoplasma gondii</i> and treated or not with P21-His₆. The number of internalized parasites in treated cells was higher than in control cells, indicating that P21-His₆ increases parasite internalization. *P<0.5, **P<0.01, ***P<0.001.</p

Peritoneal macrophages treated with P21-His₆ show increased actin polymerization.

<p>Peritoneal macrophages were treated or not with P21-His₆ for 0.5, 1.0 and 3.0 hours in the presence or not of anti-P21-His₆ polyclonal antibody and FcR blocking. The actin cytoskeleton was stained with TRITC-phalloidin. Flow cytometry showed that the cells treated with the recombinant protein had a higher fluorescence intensity than the control cells following 0.5 (A) and 1.0 hour (B) of incubation. After 3.0 hours of incubation, the protein lost its activity (C). Representative histograms are also shown. Macrophages not treated with P21-His₆ (D); macrophages treated with P21-His₆ (E); macrophages with FcR-blocking and treated with P21-His₆ previously incubated with anti-P21-His₆ polyclonal antibody (F).*P<0.5, **P<0.01, ***P<0.001.</p

P21-His₆ activity depends on PI3-kinase pathway.

<p>Peritoneal macrophages were incubated with zymosan, treated or not with P21-His₆, and also treated or not with inhibitors to PI3-kinase (A), AKT (B), n-Ras (C), ERK 2 (D), MEK 1 (E), MEK1/2 (F), m-Tor (G). P21-His₆ activity was not detected in the presence of PI3-K inhibitor. *P<0.5, **P<0.01, ***P<0.001.</p

P21-His₆ enhances phagocytosis of zymosan particles.

<p>Phagocytosis assay using zymosan particles treated or not with P21-His₆. (A) peritoneal macrophages, treated in different periods,1, 3, 6, 12, 24 and 48 hours. P21-His₆ enhanced zymosan internalization, in all treatment periods. (B) Peritoneal macrophages (grey bar), treated with BSA (white bars) or P21-His6 (black bars) at different concentrations: 1, 20, 40 and 80 µg/ml. P21-His6, but not BSA, enhanced zymosan internalization at all concentrations tested. (C) Peritoneal macrophages obtained from wild-type (WT) or <i>toll4^−/−</i> animals, treated or not with P21-His₆, and those also treated with polymyxin B. P21-His₆ treatment increased zymosan phagocytosis in both, the WT and <i>toll4^−/−</i> macrophages. Also, treatment with polymyxin B did not inhibit P21-His₆ activity. (D) Phagocytosis assay using folded and denatured P21-His₆ (dP21-His₆) at 100°C. Note that only folded protein was able to upregulate phagocytosis. (E) Peritoneal macrophages with (white bars) or without (black bars) FcR blocked were incubated with P21-His₆ (40 µg/ml) previously opsonized in different concentrations of serum. Peritoneal macrophages with FcR did not show an enhanced zymosan internalization as those without FcR blocked. *P<0.05 **P<0.01 ***P<0.001.</p