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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

Ex vivo thymic epithelial cell sorting. Five replicates of thymic cell pools from control and infected mice were sorted using flow cytometry in order to isolate TEC. (A) Initially, CD45− cells were selected based on size, (B) from this population, MHC-II positive cells were isolated after EpCAM confirmation (C). (D) Then, according to UEA-1 and Ly51 surface markers, these cells were distinguished between cortical TEC (cTEC, Ly51+, and UEA−, orange) and medullary TEC (mTEC, Ly51−, and UEA+, blue) phenotypes. Post-sorting analysis revealed 98% purity in cTEC (E) while 95% purity in mTEC (F). (G) After miRNA isolation, the remaining mRNA from three experimental pools allowed us to analyze AIRE gene expression, confirming if the sorted cells matched the correct expected TEC profile. The bar plot represents the average expression in each condition. “a” indicates the magnitude of the expression ratio (log−2) due to TEC phenotype, whereas a positive value shows a higher expression in mTEC. “ab” indicates the expression ratio magnitude (in log−2) as consequence of the combination between infection and cell type, whereas a negative value shows that the AIRE expression in infected mTEC is lower than in control mTEC.
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Figure 2: Ex vivo thymic epithelial cell sorting. Five replicates of thymic cell pools from control and infected mice were sorted using flow cytometry in order to isolate TEC. (A) Initially, CD45− cells were selected based on size, (B) from this population, MHC-II positive cells were isolated after EpCAM confirmation (C). (D) Then, according to UEA-1 and Ly51 surface markers, these cells were distinguished between cortical TEC (cTEC, Ly51+, and UEA−, orange) and medullary TEC (mTEC, Ly51−, and UEA+, blue) phenotypes. Post-sorting analysis revealed 98% purity in cTEC (E) while 95% purity in mTEC (F). (G) After miRNA isolation, the remaining mRNA from three experimental pools allowed us to analyze AIRE gene expression, confirming if the sorted cells matched the correct expected TEC profile. The bar plot represents the average expression in each condition. “a” indicates the magnitude of the expression ratio (log−2) due to TEC phenotype, whereas a positive value shows a higher expression in mTEC. “ab” indicates the expression ratio magnitude (in log−2) as consequence of the combination between infection and cell type, whereas a negative value shows that the AIRE expression in infected mTEC is lower than in control mTEC.

Mentions: In order to prepare pure populations of primary (freshly harvested) cortical and medullary TEC (respectively cTEC and mTEC), thymuses from control and infected mice were harvested at 12 dpi and disaggregated by enzymatic digestion, where most thymocytes were eliminated and TEC were enriched, allowing cell sorting. Sorted population was then stained with Ulex europaeus Lectin 1 (UEA1) and antibodies against CD45, MHC-II, EpCAM, and Ly51 and sorted (Figures 2A–D). Post-sort analysis revealed more than 98% purity for cortical TEC (CD45−MHCII+EpCAM+Ly51+UEA1−) and 95% for medullary TEC (CD45−MHCII+EpCAM+Ly51−UEA1+) populations (Figures 2E–F, respectively).


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)

Ex vivo thymic epithelial cell sorting. Five replicates of thymic cell pools from control and infected mice were sorted using flow cytometry in order to isolate TEC. (A) Initially, CD45− cells were selected based on size, (B) from this population, MHC-II positive cells were isolated after EpCAM confirmation (C). (D) Then, according to UEA-1 and Ly51 surface markers, these cells were distinguished between cortical TEC (cTEC, Ly51+, and UEA−, orange) and medullary TEC (mTEC, Ly51−, and UEA+, blue) phenotypes. Post-sorting analysis revealed 98% purity in cTEC (E) while 95% purity in mTEC (F). (G) After miRNA isolation, the remaining mRNA from three experimental pools allowed us to analyze AIRE gene expression, confirming if the sorted cells matched the correct expected TEC profile. The bar plot represents the average expression in each condition. “a” indicates the magnitude of the expression ratio (log−2) due to TEC phenotype, whereas a positive value shows a higher expression in mTEC. “ab” indicates the expression ratio magnitude (in log−2) as consequence of the combination between infection and cell type, whereas a negative value shows that the AIRE expression in infected mTEC is lower than in control mTEC.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Ex vivo thymic epithelial cell sorting. Five replicates of thymic cell pools from control and infected mice were sorted using flow cytometry in order to isolate TEC. (A) Initially, CD45− cells were selected based on size, (B) from this population, MHC-II positive cells were isolated after EpCAM confirmation (C). (D) Then, according to UEA-1 and Ly51 surface markers, these cells were distinguished between cortical TEC (cTEC, Ly51+, and UEA−, orange) and medullary TEC (mTEC, Ly51−, and UEA+, blue) phenotypes. Post-sorting analysis revealed 98% purity in cTEC (E) while 95% purity in mTEC (F). (G) After miRNA isolation, the remaining mRNA from three experimental pools allowed us to analyze AIRE gene expression, confirming if the sorted cells matched the correct expected TEC profile. The bar plot represents the average expression in each condition. “a” indicates the magnitude of the expression ratio (log−2) due to TEC phenotype, whereas a positive value shows a higher expression in mTEC. “ab” indicates the expression ratio magnitude (in log−2) as consequence of the combination between infection and cell type, whereas a negative value shows that the AIRE expression in infected mTEC is lower than in control mTEC.
Mentions: In order to prepare pure populations of primary (freshly harvested) cortical and medullary TEC (respectively cTEC and mTEC), thymuses from control and infected mice were harvested at 12 dpi and disaggregated by enzymatic digestion, where most thymocytes were eliminated and TEC were enriched, allowing cell sorting. Sorted population was then stained with Ulex europaeus Lectin 1 (UEA1) and antibodies against CD45, MHC-II, EpCAM, and Ly51 and sorted (Figures 2A–D). Post-sort analysis revealed more than 98% purity for cortical TEC (CD45−MHCII+EpCAM+Ly51+UEA1−) and 95% for medullary TEC (CD45−MHCII+EpCAM+Ly51−UEA1+) populations (Figures 2E–F, respectively).

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