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Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruzi.

Magalhães LM, Viana A, Chiari E, Galvão LM, Gollob KJ, Dutra WO - PLoS Negl Trop Dis (2015)

Bottom Line: It is known that these DTUs have different geographical distribution, as well as biological features.TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively.These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia das Interações Celulares, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT

Background: Trypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease.

Methodology/principal findings: We evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain.

Conclusion/significance: Our study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression.

No MeSH data available.


Related in: MedlinePlus

Evaluation of the frequency and intensity of T. cruzi infection in human monocytes.Results are expressed as average ± standard deviation. The symbol * indicates p < 0.05 between groups. Trypomastigotes from Y strain and Col cl1.7 were previously stained with CFSE and used for infections for 15 and 72 hours. (A) Representative figure of CFSE staining on trypomastigotes of Y strain and Col cl1.7. The plot shows the population of trypomastigotes; the white peaks in the histograms represent isotype control and the gray and black peaks represent Y and Col cl1.7 strains, respectively. (B) Representative figure of gating strategy for defining the CFSE+ and CFSE- in CD14+ cells, showing the first gate considering size (FSC) versus CD14 expression, followed by the gates considering CD14+ and CFSE+ and CFSE-. (C) Frequency of CFSE+ CD14+ cells. (D) Representative confocal microscopy analysis, showing DAPI-stained parasite’s nuclei inside monocytes. Cells were infected with Y strain or Col cl1.7 for 15 or 72 hours with 10 parasites/cell, preparation were stained with DAPI, and read on a confocal microscope, as described in material and methods; magnification x62. (E) Mean intensity of expression of CFSE fluorescence.
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pntd.0003816.g001: Evaluation of the frequency and intensity of T. cruzi infection in human monocytes.Results are expressed as average ± standard deviation. The symbol * indicates p < 0.05 between groups. Trypomastigotes from Y strain and Col cl1.7 were previously stained with CFSE and used for infections for 15 and 72 hours. (A) Representative figure of CFSE staining on trypomastigotes of Y strain and Col cl1.7. The plot shows the population of trypomastigotes; the white peaks in the histograms represent isotype control and the gray and black peaks represent Y and Col cl1.7 strains, respectively. (B) Representative figure of gating strategy for defining the CFSE+ and CFSE- in CD14+ cells, showing the first gate considering size (FSC) versus CD14 expression, followed by the gates considering CD14+ and CFSE+ and CFSE-. (C) Frequency of CFSE+ CD14+ cells. (D) Representative confocal microscopy analysis, showing DAPI-stained parasite’s nuclei inside monocytes. Cells were infected with Y strain or Col cl1.7 for 15 or 72 hours with 10 parasites/cell, preparation were stained with DAPI, and read on a confocal microscope, as described in material and methods; magnification x62. (E) Mean intensity of expression of CFSE fluorescence.

Mentions: After 15 and 72 hours of incubation, the erythrocytes were lysed using RBC “Lysing buffer” (Bio Legend) at 20mL/1mL of peripheral blood. The tubes were incubated for 15 min at 20°C in the dark. After the incubation, cells were washed three times with PBS by centrifugation at 600g for 10 min at 4°C and resuspended in PBS to 107cells/ml. Cells were then immunostained and analyzed using multiparametric flow cytometry. 200.000 cells were incubated for 15 min at 4°C with different antibody combinations. Samples were washed three times in PBS-1% bovine serum albumin (BSA) and fixed by 20-min incubation with 2% formaldehyde solution. After removal of the fixation solution by centrifugation and washing once with PBS, we permeabilized the cells by incubation for 10 min with 0.5% saponin solution, centrifuged and incubated with antibodies to intracellular molecules for 30 min at 20°C. The antibodies to surface molecules used were: anti-CD4, anti-TLR-2 or anti-CD69 – labeled with PE; anti-CD14 – labeled with APC; anti-CD8, anti-HLA-DR, anti-CD80 – labeled with PE-Cy7; anti-CD86 – labeled with Pacific Blue and anti-CD4 labeled with APC-Cy7. For intracellular staining we used the following antibodies: anti-TNF-alpha, anti-IL-12/IL-23p40, anti-IL-10 and anti-Granzyme A. All antibodies were purchased from BioLegend, San Diego, CA, USA. After intracellular staining, cells were washed and resuspended in PBS and acquired using a FACSCanto II (Becton & Dickinson, San Jose, CA, USA). A total of 30,000 lymphocyte events were acquired and the parameters were analyzed in the monocyte or lymphocyte population. Lymphocyte analysis was done by gating the region occupied classically by lymphocytes in a size versus granularity plot, followed by gating in CD4+ or CD8+ cells. For monocytes, we first gated on CD14high cells in plot of size versus CD14 and further gated on CD14+CFSE+, CD14+CFSE- (Fig 1B). The analyses were performed using FlowJo 7.6.5 software (Tree Star Inc., Ashland, OR, USA).


Differential Activation of Human Monocytes and Lymphocytes by Distinct Strains of Trypanosoma cruzi.

Magalhães LM, Viana A, Chiari E, Galvão LM, Gollob KJ, Dutra WO - PLoS Negl Trop Dis (2015)

Evaluation of the frequency and intensity of T. cruzi infection in human monocytes.Results are expressed as average ± standard deviation. The symbol * indicates p < 0.05 between groups. Trypomastigotes from Y strain and Col cl1.7 were previously stained with CFSE and used for infections for 15 and 72 hours. (A) Representative figure of CFSE staining on trypomastigotes of Y strain and Col cl1.7. The plot shows the population of trypomastigotes; the white peaks in the histograms represent isotype control and the gray and black peaks represent Y and Col cl1.7 strains, respectively. (B) Representative figure of gating strategy for defining the CFSE+ and CFSE- in CD14+ cells, showing the first gate considering size (FSC) versus CD14 expression, followed by the gates considering CD14+ and CFSE+ and CFSE-. (C) Frequency of CFSE+ CD14+ cells. (D) Representative confocal microscopy analysis, showing DAPI-stained parasite’s nuclei inside monocytes. Cells were infected with Y strain or Col cl1.7 for 15 or 72 hours with 10 parasites/cell, preparation were stained with DAPI, and read on a confocal microscope, as described in material and methods; magnification x62. (E) Mean intensity of expression of CFSE fluorescence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492932&req=5

pntd.0003816.g001: Evaluation of the frequency and intensity of T. cruzi infection in human monocytes.Results are expressed as average ± standard deviation. The symbol * indicates p < 0.05 between groups. Trypomastigotes from Y strain and Col cl1.7 were previously stained with CFSE and used for infections for 15 and 72 hours. (A) Representative figure of CFSE staining on trypomastigotes of Y strain and Col cl1.7. The plot shows the population of trypomastigotes; the white peaks in the histograms represent isotype control and the gray and black peaks represent Y and Col cl1.7 strains, respectively. (B) Representative figure of gating strategy for defining the CFSE+ and CFSE- in CD14+ cells, showing the first gate considering size (FSC) versus CD14 expression, followed by the gates considering CD14+ and CFSE+ and CFSE-. (C) Frequency of CFSE+ CD14+ cells. (D) Representative confocal microscopy analysis, showing DAPI-stained parasite’s nuclei inside monocytes. Cells were infected with Y strain or Col cl1.7 for 15 or 72 hours with 10 parasites/cell, preparation were stained with DAPI, and read on a confocal microscope, as described in material and methods; magnification x62. (E) Mean intensity of expression of CFSE fluorescence.
Mentions: After 15 and 72 hours of incubation, the erythrocytes were lysed using RBC “Lysing buffer” (Bio Legend) at 20mL/1mL of peripheral blood. The tubes were incubated for 15 min at 20°C in the dark. After the incubation, cells were washed three times with PBS by centrifugation at 600g for 10 min at 4°C and resuspended in PBS to 107cells/ml. Cells were then immunostained and analyzed using multiparametric flow cytometry. 200.000 cells were incubated for 15 min at 4°C with different antibody combinations. Samples were washed three times in PBS-1% bovine serum albumin (BSA) and fixed by 20-min incubation with 2% formaldehyde solution. After removal of the fixation solution by centrifugation and washing once with PBS, we permeabilized the cells by incubation for 10 min with 0.5% saponin solution, centrifuged and incubated with antibodies to intracellular molecules for 30 min at 20°C. The antibodies to surface molecules used were: anti-CD4, anti-TLR-2 or anti-CD69 – labeled with PE; anti-CD14 – labeled with APC; anti-CD8, anti-HLA-DR, anti-CD80 – labeled with PE-Cy7; anti-CD86 – labeled with Pacific Blue and anti-CD4 labeled with APC-Cy7. For intracellular staining we used the following antibodies: anti-TNF-alpha, anti-IL-12/IL-23p40, anti-IL-10 and anti-Granzyme A. All antibodies were purchased from BioLegend, San Diego, CA, USA. After intracellular staining, cells were washed and resuspended in PBS and acquired using a FACSCanto II (Becton & Dickinson, San Jose, CA, USA). A total of 30,000 lymphocyte events were acquired and the parameters were analyzed in the monocyte or lymphocyte population. Lymphocyte analysis was done by gating the region occupied classically by lymphocytes in a size versus granularity plot, followed by gating in CD4+ or CD8+ cells. For monocytes, we first gated on CD14high cells in plot of size versus CD14 and further gated on CD14+CFSE+, CD14+CFSE- (Fig 1B). The analyses were performed using FlowJo 7.6.5 software (Tree Star Inc., Ashland, OR, USA).

Bottom Line: It is known that these DTUs have different geographical distribution, as well as biological features.TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively.These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Biologia das Interações Celulares, Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

ABSTRACT

Background: Trypanosoma cruzi strains are currently classified into six discrete typing units (DTUs) named TcI to VI. It is known that these DTUs have different geographical distribution, as well as biological features. TcI and TcII are major DTUs found in patients from northern and southern Latin America, respectively. Our hypothesis is that upon infection of human peripheral blood cells, Y strain (Tc II) and Col cl1.7 (Tc I), cause distinct immunological changes, which might influence the clinical course of Chagas disease.

Methodology/principal findings: We evaluated the infectivity of CFSE-stained trypomastigotes of Col cl1.7 and Y strain in human monocytes for 15 and 72 hours, and determined the immunological profile of lymphocytes and monocytes exposed to the different isolates using multiparameter flow cytometry. Our results showed a similar percentage and intensity of monocyte infection by Y and Col cl1.7. We also observed an increased expression of CD80 and CD86 by monocytes infected with Col cl1.7, but not Y strain. IL-10 was significantly higher in monocytes infected with Col cl1.7, as compared to Y strain. Moreover, infection with Col cl1.7, but not Y strain, led to an increased expression of IL-17 by CD8+ T cells. On the other hand, we observed a positive correlation between the expression of TNF-alpha and granzyme A only after infection with Y strain.

Conclusion/significance: Our study shows that while Col cl1.7 induces higher monocyte activation and, at the same time, production of IL-10, infection with Y strain leads to a lower monocyte activation but higher inflammatory profile. These results show that TcI and TcII have a distinct immunological impact on human cells during early infection, which might influence disease progression.

No MeSH data available.


Related in: MedlinePlus