Paradise revealed: first-class science rocked by the sound of the waves

Univ São Paulo, Inst Ciencias Biomed, Dept Imunol, BR-05508900 São Paulo, BrazilInst Nacl Ciencia & Tecnol, Inst Invest Imunol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, Diaderna, SP, BrazilFundacao Oswaldo Cruz, Inst Oswaldo Cruz, Lab Imunofarmacol, Rio de Janeiro, BrazilUniv São Paulo, Fac Med, BR-05508900 São Paulo, BrazilInst Nacl Canc, Div Biol Celular, Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, Diaderna, SP, BrazilWeb of Scienc

Role of interplay between IL-4 and IFN-gamma in the in regulating M1 macrophage polarization induced by Nattectin

Recently our group described that Nattectin, a C-type lectin of the venom of Thalassophryne nattereri shows a potent pro-inflammatory capacity. Here, we demonstrated that Nattectin is able to induce M1 macrophage marker iNOS, and up-regulate the expression of MHC class II, CD80, CD86 and CD40 molecules. the increase in MHC class II and CD49a integrin expression with MMP-9 production and endocytic capacity depend on lectin function of Nattectin. Moreover, the polarization of peritoneal and bone marrow-derived macrophages induced by Nattectin to M1 profile is dependent on Th1 cytokines (IL-12 and IFN-gamma), and negatively regulated by Th2 cytokines (IL-4, IL-10 and IL-13). Also we reveal that IL-4 play a dual role in this polarization: a regular action of IL-4 was seen in the negative regulation of the CD40 expression, but an unexpected positive regulation was seen in the expression of CCR7 and MHC class II. Finally, our in vivo studies showed that the influx of neutrophils and small peritoneal macrophage - F4/80(low)MHCII(hi) induced by Nattectin is totally dependent on IL-4 and IFN-gamma cytokines. Furthermore, the induction of IL-6 release is negatively regulated by IL-4 and positively regulated by IL-12 and IFN-gamma. Together, the results allowed us to expand the knowledge about the regulation of macrophage activation, as well as confirmed the ability of Nattectin, a fish C-type lectin, as an important immunomodulatory agent. (c) 2012 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Inst Butantan, Unidade Imunorregulacao, Lab Especial Toxinol Aplicada, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Mol Immunol Innate Recognit Unit, Dept Biol Sci, São Paulo, BrazilUniversidade Federal de São Paulo, Mol Immunol Innate Recognit Unit, Dept Biol Sci, São Paulo, BrazilWeb of Scienc

NAIP/NLRC4 inflammasome participates in macrophage responses to Trypanosoma cruzi by a mechanism that relies on cathepsin-dependent caspase-1 cleavage

Inflammasomes are large protein complexes that, once activated, initiate inflammatory responses by activating the caspase-1 protease. They play pivotal roles in host defense against pathogens. The well-established role of NAIP/NLRC4 inflammasome in bacterial infections involves NAIP proteins functioning as sensors for their ligands. However, recent reports have indicated the involvement of NLRC4 in non-bacterial infections and sterile inflammation, even though the role of NAIP proteins and the exact molecular mechanisms underlying inflammasome activation in these contexts remain to be elucidated. In this study, we investigated the activation of the NAIP/NLRC4 inflammasome in response to Trypanosoma cruzi, the protozoan parasite responsible for causing Chagas disease. This parasite has been previously demonstrated to activate NLRP3 inflammasomes. Here we found that NAIP and NLRC4 proteins are also required for IL-1β and Nitric Oxide (NO) release in response to T. cruzi infection, with their absence rendering macrophages permissive to parasite replication. Moreover, Nlrc4-/- and Nlrp3-/- macrophages presented similar impaired responses to T. cruzi, underscoring the non-redundant roles played by these inflammasomes during infection. Notably, it was the live trypomastigotes rather than soluble antigens or extracellular vesicles (EVs) secreted by them, that activated inflammasomes in a cathepsins-dependent manner. The inhibition of cathepsins effectively abrogated caspase-1 cleavage, IL-1β and NO release, mirroring the phenotype observed in Nlrc4-/-/Nlrp3-/- double knockout macrophages. Collectively, our findings shed light on the pivotal role of the NAIP/NLRC4 inflammasome in macrophage responses to T. cruzi infection, providing new insights into its broader functions that extend beyond bacterial infections

Specialty section:

This article was submitted to Antige

Correction: A Human Trypanosome Suppresses CD8+ T Cell Priming by Dendritic Cells through the Induction of Immune Regulatory CD4+ Foxp3+ T Cells.

[This corrects the article DOI: 10.1371/journal.ppat.1005698.]

BMDC exposed to <i>T</i>. <i>cruzi</i> are fully matured and actively suppress CD8⁺ T cell priming <i>in trans</i>.

<p>a and b- To assess viability, BMDC were left untreated or exposed to <i>T</i>. <i>cruzi</i> or actinomycin D prior to staining with Annexin V and 7AAD or probes to detect the active forms of caspase 3 and 7 (gated on CD11c⁺ cells). c and d- The upregulation of MHC and co-stimulatory molecules on the surface of LPS and/or <i>T</i>. <i>cruzi-</i>exposed BMDC was assessed by flow cytometry (histograms were gated in CD11c⁺ cells) and the cytokine response was assessed by RT-PCR. e- BMDC were incubated with <i>T</i>. <i>cruzi</i>, stimulated with LPS, loaded with SIINFEKL peptide and the complex H-2K^b-SIINFEKL on the surface of CD11c⁺ cells was stained with 25-D1.16 antibody. f- The ability of naïve OTI CD8⁺ T cells to proliferate <i>in vitro</i> was assessed by CFSE dilution after 3 days of co-culture with the indicated BMDC loaded with SIINFEKL peptide and exposed or not to <i>T</i>. <i>cruzi</i>. g- 1 x 10⁴ OTI cells were adoptively transferred into C57BL/6 mice before the transfer of 5 x 10⁵ BMDC (Gr.1), 5 x 10⁵ BMDC-SIINFEKL (Gr.2) or 5 x 10⁵ BMDC-SIINFEKL and 5 x 10⁵ <i>T</i>. <i>cruzi</i>-exposed BMDC (Gr.3). The SIINFEKL-specific immune response was assessed after 5 days. h- Numbers of SIINFEKL-specific CD8⁺ T cells were determined by TCR Vα2 Vβ5 staining. i- The ability of naïve OTI cells to differentiate into effector cells was evaluated by CD44 and CD62L staining of TCR Vα2⁺ Vβ5⁺ CD8⁺ T cells. j- Spleen cells were restimulated <i>ex vivo</i> with SIINFEKL peptide and numbers of TNF and/or IFN-γ-producing CD8⁺ T cells were determined by ICS. Results are one of three separate experiments expressed as individual values and the mean ± SEM of each group. Asterisks represent significant difference between the indicated groups (***P<0.001, ****P<0.0001 One-way ANOVA followed by Tukey post-hoc test).</p

CTLA-4 and TGF-β contribute to the suppression of OTI CD8⁺ T cell priming induced by <i>T</i>. <i>cruzi</i>-exposed BMDC.

<p>a- 1 x 10⁴ OTI cells were adoptively transferred into C57BL/6 mice treated with IgG or blocking antibodies against CTLA-4 or TGF-β. These mice were transferred with 5 x 10⁵ BMDC (Gr.1), 5 x 10⁵ BMDC-SIINFEKL (Gr.2) or 5 x 10⁵ <i>T</i>. <i>cruzi</i>-exposed BMDC-SIINFEKL (Gr.3). The SIINFEKL-specific immune response was assessed after 5 days. b- The numbers of SIINFEKL-specific CD8⁺ T cells were determined by H-2K^b-SIINFEKL tetramer staining. c- Spleen cells were also restimulated <i>ex vivo</i> with SIINFEKL peptide and the numbers of TNF and/or IFN-γ-producing CD8⁺ T cells were assessed by ICS. d- The proportion of polyfunctional cells simultaneously stained for CD107a, IL-2, TNF and IFN-γ was determined by Boolean analysis. Results are one of two separate experiments expressed as individual values and the mean ± SEM of each group. Asterisks indicate significant differences between the indicated groups (*P<0.05, **P<0.01 One-way ANOVA followed by Tukey post-hoc test).</p

CD4⁺Foxp3⁺ cells induced by <i>T</i>. <i>cruzi</i>-exposed BMDC mediate the suppression of OTI CD8⁺ T cell priming.

<p>a and b- 5 x 10⁵ untreated BMDC or 5 x 10⁵ <i>T</i>. <i>cruzi</i>-exposed BMDC were adoptively transferred into Foxp3-GFP CD45.1⁺ mice and the splenic CD4⁺GFP⁺ cells were sorted after 5 days. These cells (1 x 10⁶) were transfer into C57BL/6 mice on the same day of OTI cell transfer (1 x 10⁴ cells) and 24 h before 5 x 10⁵ BMDC-SIINFEKL transfer. The SIINFEKL-specific immune response was assessed after 5 days. c- The numbers of SIINFEKL-specific CD8⁺ T cells were determined by H-2K^b-SIINFEKL tetramer staining. d- The ability of naïve OTI cells to differentiate into effector cells was evaluated by CD44 and CD62L staining of TCR Vα2⁺ Vβ5⁺ CD8⁺ T cells. e- Spleen cells were restimulated <i>ex vivo</i> with SIINFEKL peptide and the numbers of IL-2 and/or TNF and/or IFN-γ-producing CD8⁺ T cells were determined by ICS. f- Proportion of polyfunctional CD8⁺ T cells stained for CD107a and/or one, two or three cytokines (IL-2, IFN-γ and TNF) combined after restimulation with SIINFEKL peptide. Results are one of two separate experiments expressed as individual values and the mean ± SEM of each group. Asterisks indicate significant differences between the indicated groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 One-way ANOVA followed by Tukey post-hoc test).</p