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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

NETs released by T. cruzi and its soluble antigens containing granular and nuclear proteins.Neutrophils (2 × 105) were incubated in a ratio of 5 T. cruzi: 1 neutrophil (A) or its soluble antigens (B) for 1–4 h. The supernatant from these cells containing isolated NETs was analyzed for elastase activity using an enzymatic colorimetric method with the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline (1 mM). HANKS and PMA (25 nM) incubated for 4 h were used as negative and positive controls, respectively. (C) Densitometry analyses from three independent immunoblottings for citrullinated H3 (3 donors for control and PMA; 4 donors for Tcruzi and soluble antigens). (D) NET solution from 2 different donors (D1 and D2) stimulated with T. cruzi (5 Tc: 1 Ne) or its soluble antigens (50 μg/mL) for 4 h was subjected to identification of citrullinated H3 by immunoblotting. All experiments were conducted in triplicate with at least 3 independent assays. The results (A, B and C) were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences when compared with the control group (HANKS) (*P < 0.05, **P < 0.01).
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pone.0139569.g002: NETs released by T. cruzi and its soluble antigens containing granular and nuclear proteins.Neutrophils (2 × 105) were incubated in a ratio of 5 T. cruzi: 1 neutrophil (A) or its soluble antigens (B) for 1–4 h. The supernatant from these cells containing isolated NETs was analyzed for elastase activity using an enzymatic colorimetric method with the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline (1 mM). HANKS and PMA (25 nM) incubated for 4 h were used as negative and positive controls, respectively. (C) Densitometry analyses from three independent immunoblottings for citrullinated H3 (3 donors for control and PMA; 4 donors for Tcruzi and soluble antigens). (D) NET solution from 2 different donors (D1 and D2) stimulated with T. cruzi (5 Tc: 1 Ne) or its soluble antigens (50 μg/mL) for 4 h was subjected to identification of citrullinated H3 by immunoblotting. All experiments were conducted in triplicate with at least 3 independent assays. The results (A, B and C) were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences when compared with the control group (HANKS) (*P < 0.05, **P < 0.01).

Mentions: The protein composition of NETs generated by neutrophils incubated with T. cruzi and its soluble molecules was determined by measuring released elastase (Fig 2A and 2B) and specific immunostaining with citrullinated histone–3 (Fig 2C and 2D). Neutrophils incubated with T. cruzi (5 Tc: 1 Ne) for 4 h increased the release of elastase by 1.73-fold into the supernatant compared to in the negative control, HANKS (Fig 2A). This amount was lower than the release observed for PMA, the positive control. Soluble antigens from T. cruzi also induced elastase release by a lower amount (1.48-fold) (Fig 2B). The presence of citrullinated H3 [24] from T. cruzi and soluble antigen stimulated-neutrophils were confirmed in our analysis from different donors (D1 and D2) (Fig 2C and 2D). The induction of citrullination of histone by PMA is controversial [18, 25–27]. On our hands, neutrophils incubated with PMA but not HANKS showed immunostaining for citrullinated H3. The presence of citrullinated H3 may be associated with NETose processes [28]. Together, these results (Figs 1 and 2) support that neutrophils incubated with T. cruzi or its soluble antigens receive sufficient stimuli to generate “classic NETs”, with the main structure consisting of DNA which is "decorated" by nuclear and granular proteins such as histone and elastase. In the pathogenesis of Chagas disease, the extracellular presence of granular proteins, mainly myeloperoxidase, can contribute to myoblast injury [27]. Thus, NETs release induced by T. cruzi from infiltrated neutrophils in the cardiac lesions may increase tissue damage.


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)

NETs released by T. cruzi and its soluble antigens containing granular and nuclear proteins.Neutrophils (2 × 105) were incubated in a ratio of 5 T. cruzi: 1 neutrophil (A) or its soluble antigens (B) for 1–4 h. The supernatant from these cells containing isolated NETs was analyzed for elastase activity using an enzymatic colorimetric method with the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline (1 mM). HANKS and PMA (25 nM) incubated for 4 h were used as negative and positive controls, respectively. (C) Densitometry analyses from three independent immunoblottings for citrullinated H3 (3 donors for control and PMA; 4 donors for Tcruzi and soluble antigens). (D) NET solution from 2 different donors (D1 and D2) stimulated with T. cruzi (5 Tc: 1 Ne) or its soluble antigens (50 μg/mL) for 4 h was subjected to identification of citrullinated H3 by immunoblotting. All experiments were conducted in triplicate with at least 3 independent assays. The results (A, B and C) were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences when compared with the control group (HANKS) (*P < 0.05, **P < 0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591979&req=5

pone.0139569.g002: NETs released by T. cruzi and its soluble antigens containing granular and nuclear proteins.Neutrophils (2 × 105) were incubated in a ratio of 5 T. cruzi: 1 neutrophil (A) or its soluble antigens (B) for 1–4 h. The supernatant from these cells containing isolated NETs was analyzed for elastase activity using an enzymatic colorimetric method with the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroaniline (1 mM). HANKS and PMA (25 nM) incubated for 4 h were used as negative and positive controls, respectively. (C) Densitometry analyses from three independent immunoblottings for citrullinated H3 (3 donors for control and PMA; 4 donors for Tcruzi and soluble antigens). (D) NET solution from 2 different donors (D1 and D2) stimulated with T. cruzi (5 Tc: 1 Ne) or its soluble antigens (50 μg/mL) for 4 h was subjected to identification of citrullinated H3 by immunoblotting. All experiments were conducted in triplicate with at least 3 independent assays. The results (A, B and C) were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences when compared with the control group (HANKS) (*P < 0.05, **P < 0.01).
Mentions: The protein composition of NETs generated by neutrophils incubated with T. cruzi and its soluble molecules was determined by measuring released elastase (Fig 2A and 2B) and specific immunostaining with citrullinated histone–3 (Fig 2C and 2D). Neutrophils incubated with T. cruzi (5 Tc: 1 Ne) for 4 h increased the release of elastase by 1.73-fold into the supernatant compared to in the negative control, HANKS (Fig 2A). This amount was lower than the release observed for PMA, the positive control. Soluble antigens from T. cruzi also induced elastase release by a lower amount (1.48-fold) (Fig 2B). The presence of citrullinated H3 [24] from T. cruzi and soluble antigen stimulated-neutrophils were confirmed in our analysis from different donors (D1 and D2) (Fig 2C and 2D). The induction of citrullination of histone by PMA is controversial [18, 25–27]. On our hands, neutrophils incubated with PMA but not HANKS showed immunostaining for citrullinated H3. The presence of citrullinated H3 may be associated with NETose processes [28]. Together, these results (Figs 1 and 2) support that neutrophils incubated with T. cruzi or its soluble antigens receive sufficient stimuli to generate “classic NETs”, with the main structure consisting of DNA which is "decorated" by nuclear and granular proteins such as histone and elastase. In the pathogenesis of Chagas disease, the extracellular presence of granular proteins, mainly myeloperoxidase, can contribute to myoblast injury [27]. Thus, NETs release induced by T. cruzi from infiltrated neutrophils in the cardiac lesions may increase tissue damage.

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