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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 their soluble antigens induce NET release by activating TLR–2 and TLR–4.(A) Neutrophils (2 × 105) preincubated with monoclonal antibodies (anti-TLR2 and/or anti-TLR4) were co-cultivated with T. cruzi or its soluble antigens for 4 h and analyzed for NET release by extracellular DNA quantification. Neutrophils incubated with non-specific anti-human IgG were used as the negative control. DNA in the supernatant was quantified using the dsDNA High Sensibility Assay Kit. All experiments were conducted in triplicate in independent assays. (B) MIP–1β was measured in the supernatant from Pam2CSK4- and LPS-stimulated neutrophils previously blocked with monoclonal antibodies (anti-TLR2 and/or anti-TLR4). Neutrophils incubated with non-specific anti-human IgG were used as the negative control. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences compared with the control group (IgG control for each treatment) (**P < 0.01).
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pone.0139569.g006: Trypanosoma cruzi parasites and their soluble antigens induce NET release by activating TLR–2 and TLR–4.(A) Neutrophils (2 × 105) preincubated with monoclonal antibodies (anti-TLR2 and/or anti-TLR4) were co-cultivated with T. cruzi or its soluble antigens for 4 h and analyzed for NET release by extracellular DNA quantification. Neutrophils incubated with non-specific anti-human IgG were used as the negative control. DNA in the supernatant was quantified using the dsDNA High Sensibility Assay Kit. All experiments were conducted in triplicate in independent assays. (B) MIP–1β was measured in the supernatant from Pam2CSK4- and LPS-stimulated neutrophils previously blocked with monoclonal antibodies (anti-TLR2 and/or anti-TLR4). Neutrophils incubated with non-specific anti-human IgG were used as the negative control. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences compared with the control group (IgG control for each treatment) (**P < 0.01).

Mentions: Studies have demonstrated that the absence of TLR4 and TLR4-MyD88 signaling are associated with decreased release of DNA from neutrophils stimulated by Haemophilus influenzae [39, 40]. Other pathways involving TLRs are also important in the generation of NETs, such as TLR2 [40, 41]. In order to investigate whether TLRs are involved in NET release induced by T. cruzi, human neutrophils were blocked with specific antibodies against TLR2 and TLR4 molecules before incubation with T. cruzi parasites and their soluble extracts (Fig 6). The results showed that blocking of TLR–2 or TLR–4 significantly influenced the release of DNA promoted by both the parasite and its soluble extract. Blocking of TLR2 and TLR4 accentuated the decrease in NET release, but did not inhibit it completely. This result indicates that other receptors are involved in neutrophil stimulation by the T. cruzi parasite during NET release. The control IgG antibody did not alter NET release induced by T. cruzi or its soluble extract. Blocking by monoclonal antibodies was confirmed, as the presence of chemokine MIP-β was decreased in the supernatant of neutrophils stimulated with TLR-2- and TLR-4-specific agonists (Pam2CSK4 and LPS, respectively). Parasite molecules such as glycosylphosphatidylinositol anchors and their DNA may be responsible for stimulating the generation of NETs induced by T. cruzi, heat-killed T. cruzi, or its soluble antigens. The appointment of these pathogen-associated molecular patterns does not exclude the possibility that many other molecules present in the soluble extract of the parasite can participate individually or synergistically in the induction of NETs.


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 their soluble antigens induce NET release by activating TLR–2 and TLR–4.(A) Neutrophils (2 × 105) preincubated with monoclonal antibodies (anti-TLR2 and/or anti-TLR4) were co-cultivated with T. cruzi or its soluble antigens for 4 h and analyzed for NET release by extracellular DNA quantification. Neutrophils incubated with non-specific anti-human IgG were used as the negative control. DNA in the supernatant was quantified using the dsDNA High Sensibility Assay Kit. All experiments were conducted in triplicate in independent assays. (B) MIP–1β was measured in the supernatant from Pam2CSK4- and LPS-stimulated neutrophils previously blocked with monoclonal antibodies (anti-TLR2 and/or anti-TLR4). Neutrophils incubated with non-specific anti-human IgG were used as the negative control. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences compared with the control group (IgG control for each treatment) (**P < 0.01).
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getmorefigures.php?uid=PMC4591979&req=5

pone.0139569.g006: Trypanosoma cruzi parasites and their soluble antigens induce NET release by activating TLR–2 and TLR–4.(A) Neutrophils (2 × 105) preincubated with monoclonal antibodies (anti-TLR2 and/or anti-TLR4) were co-cultivated with T. cruzi or its soluble antigens for 4 h and analyzed for NET release by extracellular DNA quantification. Neutrophils incubated with non-specific anti-human IgG were used as the negative control. DNA in the supernatant was quantified using the dsDNA High Sensibility Assay Kit. All experiments were conducted in triplicate in independent assays. (B) MIP–1β was measured in the supernatant from Pam2CSK4- and LPS-stimulated neutrophils previously blocked with monoclonal antibodies (anti-TLR2 and/or anti-TLR4). Neutrophils incubated with non-specific anti-human IgG were used as the negative control. The results were analyzed by ANOVA followed by Bonferroni multiple comparisons test. Asterisks indicates significant differences compared with the control group (IgG control for each treatment) (**P < 0.01).
Mentions: Studies have demonstrated that the absence of TLR4 and TLR4-MyD88 signaling are associated with decreased release of DNA from neutrophils stimulated by Haemophilus influenzae [39, 40]. Other pathways involving TLRs are also important in the generation of NETs, such as TLR2 [40, 41]. In order to investigate whether TLRs are involved in NET release induced by T. cruzi, human neutrophils were blocked with specific antibodies against TLR2 and TLR4 molecules before incubation with T. cruzi parasites and their soluble extracts (Fig 6). The results showed that blocking of TLR–2 or TLR–4 significantly influenced the release of DNA promoted by both the parasite and its soluble extract. Blocking of TLR2 and TLR4 accentuated the decrease in NET release, but did not inhibit it completely. This result indicates that other receptors are involved in neutrophil stimulation by the T. cruzi parasite during NET release. The control IgG antibody did not alter NET release induced by T. cruzi or its soluble extract. Blocking by monoclonal antibodies was confirmed, as the presence of chemokine MIP-β was decreased in the supernatant of neutrophils stimulated with TLR-2- and TLR-4-specific agonists (Pam2CSK4 and LPS, respectively). Parasite molecules such as glycosylphosphatidylinositol anchors and their DNA may be responsible for stimulating the generation of NETs induced by T. cruzi, heat-killed T. cruzi, or its soluble antigens. The appointment of these pathogen-associated molecular patterns does not exclude the possibility that many other molecules present in the soluble extract of the parasite can participate individually or synergistically in the induction of NETs.

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