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Trypanosoma cruzi and Its Soluble Antigens Induce NET Release by Stimulating Toll-Like Receptors.

Sousa-Rocha D, Thomaz-Tobias M, Diniz LF, Souza PS, Pinge-Filho P, Toledo KA - PLoS ONE (2015)

Bottom Line: NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4.In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested.Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Univ. Estadual Paulista-UNESP (FCL-Assis), Assis, São Paulo, Brazil.

ABSTRACT
Neutrophils release fibrous traps of DNA, histones, and granule proteins known as neutrophil extracellular traps (NETs), which contribute to microbicidal killing and have been implicated in autoimmunity. The role of NET formation in the host response to nonbacterial pathogens is not well-understood. In this study, we investigated the release of NETs by human neutrophils upon their interaction with Trypanosoma cruzi (Y strain) parasites. Our results showed that human neutrophils stimulated by T. cruzi generate NETs composed of DNA, histones, and elastase. The release occurred in a dose-, time-, and reactive oxygen species-dependent manner to decrease trypomastigote and increase amastigote numbers of the parasites without affecting their viability. NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4. In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested. Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi. We suggest that contact of T. cruzi with NETs during Chagas's disease can limit infection by affecting the infectivity/pathogenicity of the parasite.

No MeSH data available.


Related in: MedlinePlus

NET release induced by T. cruzi is an ROS-dependent process.(A) 2′,7′-Dichlorofluorescein-labeled neutrophils (2.5 × 105) were pretreated or not with DPI (20 μM) and stimulated by PMA (25 nM), T. cruzi (5 Tc:1 Ne), soluble antigens (50 μg/mL), or HANKS for 30 min. ROS production was measured by fluorescence. (B) Neutrophils (2.5 × 105) pretreated or not with DPI (20 μM) were stimulated by PMA (25 nM), T. cruzi (5 Tc: 1 Ne), soluble antigens (50 μg/mL), or HANKS for 4 h. Extracellular DNA was measured in the supernatant using the dsDNA High Sensibility Assay Kit. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05, **P < 0.01).
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pone.0139569.g005: NET release induced by T. cruzi is an ROS-dependent process.(A) 2′,7′-Dichlorofluorescein-labeled neutrophils (2.5 × 105) were pretreated or not with DPI (20 μM) and stimulated by PMA (25 nM), T. cruzi (5 Tc:1 Ne), soluble antigens (50 μg/mL), or HANKS for 30 min. ROS production was measured by fluorescence. (B) Neutrophils (2.5 × 105) pretreated or not with DPI (20 μM) were stimulated by PMA (25 nM), T. cruzi (5 Tc: 1 Ne), soluble antigens (50 μg/mL), or HANKS for 4 h. Extracellular DNA was measured in the supernatant using the dsDNA High Sensibility Assay Kit. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05, **P < 0.01).

Mentions: We evaluated ROS production and NETs release stimulated by T. cruzi or its soluble antigens in neutrophils pretreated with DPI, an NADPH inhibitor. After 30 min, T. cruzi induced ROS production, which was inhibited in the presence of DPI (Fig 5A). Similar results were obtained when neutrophils were stimulated with PMA or soluble antigens from T. cruzi. Additionally, the blocking of NADPH decreased NET release induced by T. cruzi, its soluble antigens, and PMA (Fig 5B). These data indicate that the T. cruzi parasite stimulates NET release in an ROS-dependent process.


Trypanosoma cruzi and Its Soluble Antigens Induce NET Release by Stimulating Toll-Like Receptors.

Sousa-Rocha D, Thomaz-Tobias M, Diniz LF, Souza PS, Pinge-Filho P, Toledo KA - PLoS ONE (2015)

NET release induced by T. cruzi is an ROS-dependent process.(A) 2′,7′-Dichlorofluorescein-labeled neutrophils (2.5 × 105) were pretreated or not with DPI (20 μM) and stimulated by PMA (25 nM), T. cruzi (5 Tc:1 Ne), soluble antigens (50 μg/mL), or HANKS for 30 min. ROS production was measured by fluorescence. (B) Neutrophils (2.5 × 105) pretreated or not with DPI (20 μM) were stimulated by PMA (25 nM), T. cruzi (5 Tc: 1 Ne), soluble antigens (50 μg/mL), or HANKS for 4 h. Extracellular DNA was measured in the supernatant using the dsDNA High Sensibility Assay Kit. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05, **P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591979&req=5

pone.0139569.g005: NET release induced by T. cruzi is an ROS-dependent process.(A) 2′,7′-Dichlorofluorescein-labeled neutrophils (2.5 × 105) were pretreated or not with DPI (20 μM) and stimulated by PMA (25 nM), T. cruzi (5 Tc:1 Ne), soluble antigens (50 μg/mL), or HANKS for 30 min. ROS production was measured by fluorescence. (B) Neutrophils (2.5 × 105) pretreated or not with DPI (20 μM) were stimulated by PMA (25 nM), T. cruzi (5 Tc: 1 Ne), soluble antigens (50 μg/mL), or HANKS for 4 h. Extracellular DNA was measured in the supernatant using the dsDNA High Sensibility Assay Kit. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05, **P < 0.01).
Mentions: We evaluated ROS production and NETs release stimulated by T. cruzi or its soluble antigens in neutrophils pretreated with DPI, an NADPH inhibitor. After 30 min, T. cruzi induced ROS production, which was inhibited in the presence of DPI (Fig 5A). Similar results were obtained when neutrophils were stimulated with PMA or soluble antigens from T. cruzi. Additionally, the blocking of NADPH decreased NET release induced by T. cruzi, its soluble antigens, and PMA (Fig 5B). These data indicate that the T. cruzi parasite stimulates NET release in an ROS-dependent process.

Bottom Line: NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4.In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested.Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Univ. Estadual Paulista-UNESP (FCL-Assis), Assis, São Paulo, Brazil.

ABSTRACT
Neutrophils release fibrous traps of DNA, histones, and granule proteins known as neutrophil extracellular traps (NETs), which contribute to microbicidal killing and have been implicated in autoimmunity. The role of NET formation in the host response to nonbacterial pathogens is not well-understood. In this study, we investigated the release of NETs by human neutrophils upon their interaction with Trypanosoma cruzi (Y strain) parasites. Our results showed that human neutrophils stimulated by T. cruzi generate NETs composed of DNA, histones, and elastase. The release occurred in a dose-, time-, and reactive oxygen species-dependent manner to decrease trypomastigote and increase amastigote numbers of the parasites without affecting their viability. NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4. In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested. Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi. We suggest that contact of T. cruzi with NETs during Chagas's disease can limit infection by affecting the infectivity/pathogenicity of the parasite.

No MeSH data available.


Related in: MedlinePlus