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Differential Expression of microRNAs in Thymic Epithelial Cells from Trypanosoma cruzi Acutely Infected Mice: Putative Role in Thymic Atrophy.

Linhares-Lacerda L, Palu CC, Ribeiro-Alves M, Paredes BD, Morrot A, Garcia-Silva MR, Cayota A, Savino W - Front Immunol (2015)

Bottom Line: Thymic epithelial cells (TEC) play a major role in the intrathymic T cell differentiation.In silico analysis revealed that these miRNAs may control target mRNAs known to be responsible for chemotaxis, cell adhesion, and cell death.Considering that we sorted TEC in the initial phase of thymocyte loss, it is conceivable that changes in TEC miRNA expression profile are functionally related to thymic atrophy, providing new clues to better understanding the mechanisms of the thymic involution seen in experimental Chagas disease.

View Article: PubMed Central - PubMed

Affiliation: Laboratory on Thymus Research, Institute Oswaldo Cruz, Oswaldo Cruz Foundation , Rio de Janeiro , Brazil.

ABSTRACT
A common feature seen in acute infections is a severe atrophy of the thymus. This occurs in the murine model of acute Chagas disease. Moreover, in thymuses from Trypanosoma cruzi acutely infected mice, thymocytes exhibit an increase in the density of fibronectin and laminin integrin-type receptors, with an increase in migratory response ex vivo. Thymic epithelial cells (TEC) play a major role in the intrathymic T cell differentiation. To date, the consequences of molecular changes promoted by parasite infection upon thymus have not been elucidated. Considering the importance of microRNA for gene expression regulation, 85 microRNAs (mRNAs) were analyzed in TEC from T. cruzi acutely infected mice. The infection significantly modulated 29 miRNAs and modulation of 9 was also dependent whether TEC sorted out from the thymus exhibited cortical or medullary phenotype. In silico analysis revealed that these miRNAs may control target mRNAs known to be responsible for chemotaxis, cell adhesion, and cell death. Considering that we sorted TEC in the initial phase of thymocyte loss, it is conceivable that changes in TEC miRNA expression profile are functionally related to thymic atrophy, providing new clues to better understanding the mechanisms of the thymic involution seen in experimental Chagas disease.

No MeSH data available.


Related in: MedlinePlus

Trypanosoma cruzi acute infection induces progressive thymic atrophy and CD4+CD8+ thymocyte loss. Nine C57BL/6 mice were infected with 1,000 metacyclic trypomastigotes and in the following 6–18 days the parasitemia was accessed to verify the infection progression. (A) The box plot shows the median amount of parasites per blood milliliter, upper and lower quartiles, maximum and minimum values (whiskers) excluding outliers (circles). Once the infection peak was identified (8 dpi), the thymocyte subpopulation variation in the following days was studied in order to identify when the CD4+CD8+ thymocyte loss starts. (B) The cytofluorometric profiles of thymocytes were obtained staining with anti-CD4 and anti-CD8. Thymocytes from three or four mice were submitted to FACS analysis on days 9–12 post-infection (dpi). The plots represent the mean number of cells ±SD in each day. The described decrease on CD4+CD8+ thymocyte subpopulation due to infection progression was confirmed (ANOVA p-value = 0.0002) and the Tukey’s honestly significant difference (HSD) post-test revealed significant differences in each dpi. (adjusted p-value ≤*0.05, **0.01, and ***0.001) (C) Thymocyte subpopulation percentages data derived from the cytofluorometric profiles show that by 14 dpi the thymic atrophy is reached. Each bar represents thymocytes from a single mouse.
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Figure 1: Trypanosoma cruzi acute infection induces progressive thymic atrophy and CD4+CD8+ thymocyte loss. Nine C57BL/6 mice were infected with 1,000 metacyclic trypomastigotes and in the following 6–18 days the parasitemia was accessed to verify the infection progression. (A) The box plot shows the median amount of parasites per blood milliliter, upper and lower quartiles, maximum and minimum values (whiskers) excluding outliers (circles). Once the infection peak was identified (8 dpi), the thymocyte subpopulation variation in the following days was studied in order to identify when the CD4+CD8+ thymocyte loss starts. (B) The cytofluorometric profiles of thymocytes were obtained staining with anti-CD4 and anti-CD8. Thymocytes from three or four mice were submitted to FACS analysis on days 9–12 post-infection (dpi). The plots represent the mean number of cells ±SD in each day. The described decrease on CD4+CD8+ thymocyte subpopulation due to infection progression was confirmed (ANOVA p-value = 0.0002) and the Tukey’s honestly significant difference (HSD) post-test revealed significant differences in each dpi. (adjusted p-value ≤*0.05, **0.01, and ***0.001) (C) Thymocyte subpopulation percentages data derived from the cytofluorometric profiles show that by 14 dpi the thymic atrophy is reached. Each bar represents thymocytes from a single mouse.

Mentions: Since the interaction between thymocytes and TEC play a major role in T-cell development, variations in TEC gene expression may alter the thymic environment with consequences on thymocyte fate (4). Accordingly, we analyzed miRNA profiles variation due to infection in the initial point of thymic atrophy to avoid secondary effects caused by thymocyte loss or consequent microenvironmental modifications. We used the decay of CD4+CD8+ thymocytes number to define when the thymus should be harvested. Intraperitoneal acute infection led to a parasite load picking at 8 dpi, and characterized by the high number of metacyclic trypomastigotes found circulating in the peripheral blood (Figure 1A). During the following days a decrease of CD4+CD8+ thymocytes was observed (Figure 1B) and later, a severe thymic atrophy with an average loss of 80% of CD4+CD8+ thymocytes was seen on the 14th dpi (Figure 1C). On the 12th dpi, this cell subpopulation was significantly reduced when compared with cell counting from control and 9–10 dpi mice (Figure 1B), preceding the thymic atrophy, thus we perform the following experiments using samples at this time point of infection.


Differential Expression of microRNAs in Thymic Epithelial Cells from Trypanosoma cruzi Acutely Infected Mice: Putative Role in Thymic Atrophy.

Linhares-Lacerda L, Palu CC, Ribeiro-Alves M, Paredes BD, Morrot A, Garcia-Silva MR, Cayota A, Savino W - Front Immunol (2015)

Trypanosoma cruzi acute infection induces progressive thymic atrophy and CD4+CD8+ thymocyte loss. Nine C57BL/6 mice were infected with 1,000 metacyclic trypomastigotes and in the following 6–18 days the parasitemia was accessed to verify the infection progression. (A) The box plot shows the median amount of parasites per blood milliliter, upper and lower quartiles, maximum and minimum values (whiskers) excluding outliers (circles). Once the infection peak was identified (8 dpi), the thymocyte subpopulation variation in the following days was studied in order to identify when the CD4+CD8+ thymocyte loss starts. (B) The cytofluorometric profiles of thymocytes were obtained staining with anti-CD4 and anti-CD8. Thymocytes from three or four mice were submitted to FACS analysis on days 9–12 post-infection (dpi). The plots represent the mean number of cells ±SD in each day. The described decrease on CD4+CD8+ thymocyte subpopulation due to infection progression was confirmed (ANOVA p-value = 0.0002) and the Tukey’s honestly significant difference (HSD) post-test revealed significant differences in each dpi. (adjusted p-value ≤*0.05, **0.01, and ***0.001) (C) Thymocyte subpopulation percentages data derived from the cytofluorometric profiles show that by 14 dpi the thymic atrophy is reached. Each bar represents thymocytes from a single mouse.
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Figure 1: Trypanosoma cruzi acute infection induces progressive thymic atrophy and CD4+CD8+ thymocyte loss. Nine C57BL/6 mice were infected with 1,000 metacyclic trypomastigotes and in the following 6–18 days the parasitemia was accessed to verify the infection progression. (A) The box plot shows the median amount of parasites per blood milliliter, upper and lower quartiles, maximum and minimum values (whiskers) excluding outliers (circles). Once the infection peak was identified (8 dpi), the thymocyte subpopulation variation in the following days was studied in order to identify when the CD4+CD8+ thymocyte loss starts. (B) The cytofluorometric profiles of thymocytes were obtained staining with anti-CD4 and anti-CD8. Thymocytes from three or four mice were submitted to FACS analysis on days 9–12 post-infection (dpi). The plots represent the mean number of cells ±SD in each day. The described decrease on CD4+CD8+ thymocyte subpopulation due to infection progression was confirmed (ANOVA p-value = 0.0002) and the Tukey’s honestly significant difference (HSD) post-test revealed significant differences in each dpi. (adjusted p-value ≤*0.05, **0.01, and ***0.001) (C) Thymocyte subpopulation percentages data derived from the cytofluorometric profiles show that by 14 dpi the thymic atrophy is reached. Each bar represents thymocytes from a single mouse.
Mentions: Since the interaction between thymocytes and TEC play a major role in T-cell development, variations in TEC gene expression may alter the thymic environment with consequences on thymocyte fate (4). Accordingly, we analyzed miRNA profiles variation due to infection in the initial point of thymic atrophy to avoid secondary effects caused by thymocyte loss or consequent microenvironmental modifications. We used the decay of CD4+CD8+ thymocytes number to define when the thymus should be harvested. Intraperitoneal acute infection led to a parasite load picking at 8 dpi, and characterized by the high number of metacyclic trypomastigotes found circulating in the peripheral blood (Figure 1A). During the following days a decrease of CD4+CD8+ thymocytes was observed (Figure 1B) and later, a severe thymic atrophy with an average loss of 80% of CD4+CD8+ thymocytes was seen on the 14th dpi (Figure 1C). On the 12th dpi, this cell subpopulation was significantly reduced when compared with cell counting from control and 9–10 dpi mice (Figure 1B), preceding the thymic atrophy, thus we perform the following experiments using samples at this time point of infection.

Bottom Line: Thymic epithelial cells (TEC) play a major role in the intrathymic T cell differentiation.In silico analysis revealed that these miRNAs may control target mRNAs known to be responsible for chemotaxis, cell adhesion, and cell death.Considering that we sorted TEC in the initial phase of thymocyte loss, it is conceivable that changes in TEC miRNA expression profile are functionally related to thymic atrophy, providing new clues to better understanding the mechanisms of the thymic involution seen in experimental Chagas disease.

View Article: PubMed Central - PubMed

Affiliation: Laboratory on Thymus Research, Institute Oswaldo Cruz, Oswaldo Cruz Foundation , Rio de Janeiro , Brazil.

ABSTRACT
A common feature seen in acute infections is a severe atrophy of the thymus. This occurs in the murine model of acute Chagas disease. Moreover, in thymuses from Trypanosoma cruzi acutely infected mice, thymocytes exhibit an increase in the density of fibronectin and laminin integrin-type receptors, with an increase in migratory response ex vivo. Thymic epithelial cells (TEC) play a major role in the intrathymic T cell differentiation. To date, the consequences of molecular changes promoted by parasite infection upon thymus have not been elucidated. Considering the importance of microRNA for gene expression regulation, 85 microRNAs (mRNAs) were analyzed in TEC from T. cruzi acutely infected mice. The infection significantly modulated 29 miRNAs and modulation of 9 was also dependent whether TEC sorted out from the thymus exhibited cortical or medullary phenotype. In silico analysis revealed that these miRNAs may control target mRNAs known to be responsible for chemotaxis, cell adhesion, and cell death. Considering that we sorted TEC in the initial phase of thymocyte loss, it is conceivable that changes in TEC miRNA expression profile are functionally related to thymic atrophy, providing new clues to better understanding the mechanisms of the thymic involution seen in experimental Chagas disease.

No MeSH data available.


Related in: MedlinePlus