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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 were not able to kill T. cruzi.(A) Neutrophils were incubated with T. cruzi (10 Tc: 1 Ne) for 4 h. NETs were observed by fluorescence staining using DAPI (blue). White arrows indicate trapped T. cruzi in the NETs. (B) NET solution (1 μg) was evaluated by 1.5% agarose gel electrophoresis. NET solution and ladder DNA (1 kb) were stained using GelRed (1:10,000). (C)Trypanosoma cruzi (104 parasites) were incubated with NET solution or HANKS for 1 h at 37°C. Motile parasites were counted in a Neubauer chamber as live parasites. The results are shown as T. cruzi survival (%), where HANKS results were considered to be 100%. The results were analyzed by Student’s t-test and did not reveal significant differences. All experiments were conducted in triplicate in independent assays.
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pone.0139569.g003: NETs were not able to kill T. cruzi.(A) Neutrophils were incubated with T. cruzi (10 Tc: 1 Ne) for 4 h. NETs were observed by fluorescence staining using DAPI (blue). White arrows indicate trapped T. cruzi in the NETs. (B) NET solution (1 μg) was evaluated by 1.5% agarose gel electrophoresis. NET solution and ladder DNA (1 kb) were stained using GelRed (1:10,000). (C)Trypanosoma cruzi (104 parasites) were incubated with NET solution or HANKS for 1 h at 37°C. Motile parasites were counted in a Neubauer chamber as live parasites. The results are shown as T. cruzi survival (%), where HANKS results were considered to be 100%. The results were analyzed by Student’s t-test and did not reveal significant differences. All experiments were conducted in triplicate in independent assays.

Mentions: NETs are structures that immobilize a broad range of pathogens, but it is unknown whether microorganisms immobilized by NETs are dead [29]. When neutrophils were stimulated by T. cruzi (10 Tc: 1 Ne), some parasites were captured by the NETs (Fig 3A; white arrows). This result was similar that observed by Guimarães-Costa et al. [3]. We examined whether NETs generated from neutrophils stimulated by PMA could kill the parasite T. cruzi. Thus, parasites were incubated for 1 h in the supernatant containing NETs. The NETs profile was evaluated by agarose electrophoresis as shown in Fig 3B. The NETs solution contained large heterogeneous fragments greater than 1,000 base pairs (bp). Parasite viability was assessed by observing their mobility using a light microscope. Under these conditions, NETs could not kill the parasite T. cruzi, as their mobility was similar to that found in parasites incubated with HANKS solution only (Fig 3C).


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 were not able to kill T. cruzi.(A) Neutrophils were incubated with T. cruzi (10 Tc: 1 Ne) for 4 h. NETs were observed by fluorescence staining using DAPI (blue). White arrows indicate trapped T. cruzi in the NETs. (B) NET solution (1 μg) was evaluated by 1.5% agarose gel electrophoresis. NET solution and ladder DNA (1 kb) were stained using GelRed (1:10,000). (C)Trypanosoma cruzi (104 parasites) were incubated with NET solution or HANKS for 1 h at 37°C. Motile parasites were counted in a Neubauer chamber as live parasites. The results are shown as T. cruzi survival (%), where HANKS results were considered to be 100%. The results were analyzed by Student’s t-test and did not reveal significant differences. All experiments were conducted in triplicate in independent assays.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591979&req=5

pone.0139569.g003: NETs were not able to kill T. cruzi.(A) Neutrophils were incubated with T. cruzi (10 Tc: 1 Ne) for 4 h. NETs were observed by fluorescence staining using DAPI (blue). White arrows indicate trapped T. cruzi in the NETs. (B) NET solution (1 μg) was evaluated by 1.5% agarose gel electrophoresis. NET solution and ladder DNA (1 kb) were stained using GelRed (1:10,000). (C)Trypanosoma cruzi (104 parasites) were incubated with NET solution or HANKS for 1 h at 37°C. Motile parasites were counted in a Neubauer chamber as live parasites. The results are shown as T. cruzi survival (%), where HANKS results were considered to be 100%. The results were analyzed by Student’s t-test and did not reveal significant differences. All experiments were conducted in triplicate in independent assays.
Mentions: NETs are structures that immobilize a broad range of pathogens, but it is unknown whether microorganisms immobilized by NETs are dead [29]. When neutrophils were stimulated by T. cruzi (10 Tc: 1 Ne), some parasites were captured by the NETs (Fig 3A; white arrows). This result was similar that observed by Guimarães-Costa et al. [3]. We examined whether NETs generated from neutrophils stimulated by PMA could kill the parasite T. cruzi. Thus, parasites were incubated for 1 h in the supernatant containing NETs. The NETs profile was evaluated by agarose electrophoresis as shown in Fig 3B. The NETs solution contained large heterogeneous fragments greater than 1,000 base pairs (bp). Parasite viability was assessed by observing their mobility using a light microscope. Under these conditions, NETs could not kill the parasite T. cruzi, as their mobility was similar to that found in parasites incubated with HANKS solution only (Fig 3C).

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