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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

Trypanosoma cruzi parasites and its soluble antigens induce NET release.Neutrophils (2 × 105) were incubated with trypomastigote forms or their soluble antigens for 1–4 h and analyzed for NETs release by fluorescence microscopy and extracellular DNA quantification. (A) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne), soluble antigen (50 μg/mL), PMA (25 nM), or only HANKS for 4 h. NETs were observed by fluorescence staining using antibodies: anti-histone (green), anti-elastase (green), and fluorescein isothiocyanate-conjugated antibody and DAPI (blue). Stimulated neutrophils showed 5 ± 4 NETs by field (40× objective). (B) Neutrophils were incubated with T. cruzi in different ratios (0.1–5 parasites: 1 Ne), heat-killed T. cruzi (5 parasites: 1 Ne), and soluble antigen (50 μg/mL) for 4 h. HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants was quantified using a dsDNA High Sensibility Assay Kit. (C) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne) for different time periods (1–4 h). HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants were quantified as described in B. All experiments were conducted in triplicate with at least 3 independent assays. The results (B, 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, ***P < 0.001).
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pone.0139569.g001: Trypanosoma cruzi parasites and its soluble antigens induce NET release.Neutrophils (2 × 105) were incubated with trypomastigote forms or their soluble antigens for 1–4 h and analyzed for NETs release by fluorescence microscopy and extracellular DNA quantification. (A) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne), soluble antigen (50 μg/mL), PMA (25 nM), or only HANKS for 4 h. NETs were observed by fluorescence staining using antibodies: anti-histone (green), anti-elastase (green), and fluorescein isothiocyanate-conjugated antibody and DAPI (blue). Stimulated neutrophils showed 5 ± 4 NETs by field (40× objective). (B) Neutrophils were incubated with T. cruzi in different ratios (0.1–5 parasites: 1 Ne), heat-killed T. cruzi (5 parasites: 1 Ne), and soluble antigen (50 μg/mL) for 4 h. HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants was quantified using a dsDNA High Sensibility Assay Kit. (C) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne) for different time periods (1–4 h). HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants were quantified as described in B. All experiments were conducted in triplicate with at least 3 independent assays. The results (B, 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, ***P < 0.001).

Mentions: Initially, to determine whether T. cruzi triggers the release of NETs, neutrophils were incubated with trypomastigote forms (5 T. cruzi: 1 Ne ratio) for 4 h followed by DNA analysis using (DAPI; blue) and histone and elastase (specific antibodies; green) staining [18]. Based on fluorescence microscopy (Fig 1A), neutrophils incubated with only HANKS buffer showed multilobulated nuclei without extracellular projections and minimum staining for histone and elastase. In contrast, neutrophils incubated with T. cruzi had several extracellular DNA projections, which were stained with specific antibodies against histone and elastase. Similar results were obtained when human neutrophils were incubated with soluble antigens from trypomastigote forms of T. cruzi or PMA (Fig 1A). Separated images containing nucleus and protein staining are shown in S1 Fig. Weak intracellular staining, even in non-stimulated neutrophils, was also observed in previous studies [18–21]. In contrast, some authors showed that paraformaldehyde fixation is sufficient to allow for antibody detection of intracellular elastase [16, 22, 23]. In previous studies, different antibodies from several manufacturers were used; however, these studies did not include product codes, hindering comparison of the results.


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)

Trypanosoma cruzi parasites and its soluble antigens induce NET release.Neutrophils (2 × 105) were incubated with trypomastigote forms or their soluble antigens for 1–4 h and analyzed for NETs release by fluorescence microscopy and extracellular DNA quantification. (A) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne), soluble antigen (50 μg/mL), PMA (25 nM), or only HANKS for 4 h. NETs were observed by fluorescence staining using antibodies: anti-histone (green), anti-elastase (green), and fluorescein isothiocyanate-conjugated antibody and DAPI (blue). Stimulated neutrophils showed 5 ± 4 NETs by field (40× objective). (B) Neutrophils were incubated with T. cruzi in different ratios (0.1–5 parasites: 1 Ne), heat-killed T. cruzi (5 parasites: 1 Ne), and soluble antigen (50 μg/mL) for 4 h. HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants was quantified using a dsDNA High Sensibility Assay Kit. (C) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne) for different time periods (1–4 h). HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants were quantified as described in B. All experiments were conducted in triplicate with at least 3 independent assays. The results (B, 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, ***P < 0.001).
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Related In: Results  -  Collection

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pone.0139569.g001: Trypanosoma cruzi parasites and its soluble antigens induce NET release.Neutrophils (2 × 105) were incubated with trypomastigote forms or their soluble antigens for 1–4 h and analyzed for NETs release by fluorescence microscopy and extracellular DNA quantification. (A) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne), soluble antigen (50 μg/mL), PMA (25 nM), or only HANKS for 4 h. NETs were observed by fluorescence staining using antibodies: anti-histone (green), anti-elastase (green), and fluorescein isothiocyanate-conjugated antibody and DAPI (blue). Stimulated neutrophils showed 5 ± 4 NETs by field (40× objective). (B) Neutrophils were incubated with T. cruzi in different ratios (0.1–5 parasites: 1 Ne), heat-killed T. cruzi (5 parasites: 1 Ne), and soluble antigen (50 μg/mL) for 4 h. HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants was quantified using a dsDNA High Sensibility Assay Kit. (C) Neutrophils were incubated with T. cruzi (5 Tc: 1 Ne) for different time periods (1–4 h). HANKS and PMA (25 nM) were used as negative and positive controls, respectively. DNA in the supernatants were quantified as described in B. All experiments were conducted in triplicate with at least 3 independent assays. The results (B, 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, ***P < 0.001).
Mentions: Initially, to determine whether T. cruzi triggers the release of NETs, neutrophils were incubated with trypomastigote forms (5 T. cruzi: 1 Ne ratio) for 4 h followed by DNA analysis using (DAPI; blue) and histone and elastase (specific antibodies; green) staining [18]. Based on fluorescence microscopy (Fig 1A), neutrophils incubated with only HANKS buffer showed multilobulated nuclei without extracellular projections and minimum staining for histone and elastase. In contrast, neutrophils incubated with T. cruzi had several extracellular DNA projections, which were stained with specific antibodies against histone and elastase. Similar results were obtained when human neutrophils were incubated with soluble antigens from trypomastigote forms of T. cruzi or PMA (Fig 1A). Separated images containing nucleus and protein staining are shown in S1 Fig. Weak intracellular staining, even in non-stimulated neutrophils, was also observed in previous studies [18–21]. In contrast, some authors showed that paraformaldehyde fixation is sufficient to allow for antibody detection of intracellular elastase [16, 22, 23]. In previous studies, different antibodies from several manufacturers were used; however, these studies did not include product codes, hindering comparison of the results.

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