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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 decrease T. cruzi infectivity.(A–D) LLC-MK2 cell (2 × 104) monolayers were infected with trypomastigotes (105) pretreated for 1 h with NET solution released from neutrophils stimulated with 25 nM PMA (NETs) or only HANKS. After 5 days, we evaluated: (A) the total number of released trypomastigotes in the supernatant, (B) the presence of amastigote forms (black arrows), (C) the number of infected cells by light microscopy traversing the diameter of each well, and (D) the viability of cells using the MTT assay where cells cultured with HANKS correspond to 100% viability. (E) Mice (N = 5) were infected with trypomastigotes (1000 forms) pretreated with NET or HANKS. Parasitemia was monitored in the blood for 16 days. All experiments were conducted 3 times in independent assays. The results show the media ± SD. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05).
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pone.0139569.g004: NETs decrease T. cruzi infectivity.(A–D) LLC-MK2 cell (2 × 104) monolayers were infected with trypomastigotes (105) pretreated for 1 h with NET solution released from neutrophils stimulated with 25 nM PMA (NETs) or only HANKS. After 5 days, we evaluated: (A) the total number of released trypomastigotes in the supernatant, (B) the presence of amastigote forms (black arrows), (C) the number of infected cells by light microscopy traversing the diameter of each well, and (D) the viability of cells using the MTT assay where cells cultured with HANKS correspond to 100% viability. (E) Mice (N = 5) were infected with trypomastigotes (1000 forms) pretreated with NET or HANKS. Parasitemia was monitored in the blood for 16 days. All experiments were conducted 3 times in independent assays. The results show the media ± SD. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05).

Mentions: We evaluated whether contact with NETs could result in a decrease in parasite pathogenicity, such as whether other microorganisms are killed and/or lose their virulence when captured by NETs [17]. For this purpose, LLC-MK2 cells were infected with T. cruzi that had been pretreated with NETs or HANKS solution. After 5 days, the number of trypomastigotes (infective form; Fig 4A) and the presence of amastigotes (non-infective form; Fig 4B) were counted in the supernatant of the cells. In the supernatant of HANKS T. cruzi infected-cells, we counted a large number of trypomastigote forms. Additionally, we observed a low frequency of amastigote forms, identified as rounded parasites without motility (see the black arrows in Fig 4B and S1 Movie). In contrast, when the parasites were pretreated with NETs, their cultures showed low numbers of trypomastigote forms (Fig 4A and S2 Movie) and large numbers of amastigote forms (see the black arrows in Fig 4B and S2 Movie).


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 decrease T. cruzi infectivity.(A–D) LLC-MK2 cell (2 × 104) monolayers were infected with trypomastigotes (105) pretreated for 1 h with NET solution released from neutrophils stimulated with 25 nM PMA (NETs) or only HANKS. After 5 days, we evaluated: (A) the total number of released trypomastigotes in the supernatant, (B) the presence of amastigote forms (black arrows), (C) the number of infected cells by light microscopy traversing the diameter of each well, and (D) the viability of cells using the MTT assay where cells cultured with HANKS correspond to 100% viability. (E) Mice (N = 5) were infected with trypomastigotes (1000 forms) pretreated with NET or HANKS. Parasitemia was monitored in the blood for 16 days. All experiments were conducted 3 times in independent assays. The results show the media ± SD. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139569.g004: NETs decrease T. cruzi infectivity.(A–D) LLC-MK2 cell (2 × 104) monolayers were infected with trypomastigotes (105) pretreated for 1 h with NET solution released from neutrophils stimulated with 25 nM PMA (NETs) or only HANKS. After 5 days, we evaluated: (A) the total number of released trypomastigotes in the supernatant, (B) the presence of amastigote forms (black arrows), (C) the number of infected cells by light microscopy traversing the diameter of each well, and (D) the viability of cells using the MTT assay where cells cultured with HANKS correspond to 100% viability. (E) Mice (N = 5) were infected with trypomastigotes (1000 forms) pretreated with NET or HANKS. Parasitemia was monitored in the blood for 16 days. All experiments were conducted 3 times in independent assays. The results show the media ± SD. Asterisks indicate significant differences compared with the control group (HANKS) (*P < 0.05).
Mentions: We evaluated whether contact with NETs could result in a decrease in parasite pathogenicity, such as whether other microorganisms are killed and/or lose their virulence when captured by NETs [17]. For this purpose, LLC-MK2 cells were infected with T. cruzi that had been pretreated with NETs or HANKS solution. After 5 days, the number of trypomastigotes (infective form; Fig 4A) and the presence of amastigotes (non-infective form; Fig 4B) were counted in the supernatant of the cells. In the supernatant of HANKS T. cruzi infected-cells, we counted a large number of trypomastigote forms. Additionally, we observed a low frequency of amastigote forms, identified as rounded parasites without motility (see the black arrows in Fig 4B and S1 Movie). In contrast, when the parasites were pretreated with NETs, their cultures showed low numbers of trypomastigote forms (Fig 4A and S2 Movie) and large numbers of amastigote forms (see the black arrows in Fig 4B and S2 Movie).

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