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Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

Fric J, Lim CX, Koh EG, Hofmann B, Chen J, Tay HS, Mohammad Isa SA, Mortellaro A, Ruedl C, Ricciardi-Castagnoli P - EMBO Mol Med (2012)

Bottom Line: Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment.Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis.In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

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Impaired calcineurin/NFAT signalling increases the number and proliferation of myeloid progenitorsCFU in methyl cellulose from BM of mice reconstituted with a mixture of VIVIT-eGFP expressing and control-tdTomato expressing HSC, injected in the proportions of 30% VIVIT-eGFP to 70% tdTomato control, the methylcellulose cultures were set 4 weeks (upper panel) and 8 weeks (lower panel) after reconstitution and counted after 4–7 days of culture. Images of colonies from eight individual representative mice are shown as example (original magnification, ×10, scale bar 200 µm).Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from the BM of mice 4 and 8 weeks after reconstitution with a mixture of HSC consisting of 30% VIVIT-eGFP and 70% tdTomato control expressing cells.Analysis of average cell numbers per colony. After the ratio between VIVIT-eGFP and control tdTomato colonies was determined, cultures were harvested and labelled with antibodies against CD11b+. Differentiated cells (over 90% of total) were counted by flow cytometry with four replicates per mouse. Average number of cells per colony is plotted. Data are summarized from three (B and C) independent experiments with at least three mice per group in each experiment.Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from HSC lines used for reconstitution of mice.Total numbers of LSK (lin−, Sca-1+ and c-Kit+) in BM of mice reconstituted with control tdTomato or VIVIT-eGFP HSC.Total numbers of VIVIT-eGFP and Tomato control LSK (lin−, Sca-1+ and c-Kit+) in mice reconstituted with mixture of control tdTomato or VIVITeGFP HSC in initial ratio 1:1. (E and F) Analysis was performed 6 weeks after reconstitution with five mice per group. Changes in total numbers of DCs, in IFN-β production and changes in percentage of proliferating cells have been analysed by Student t-test *p ≤ 0.05 and ***p ≤ 0.001.
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fig04: Impaired calcineurin/NFAT signalling increases the number and proliferation of myeloid progenitorsCFU in methyl cellulose from BM of mice reconstituted with a mixture of VIVIT-eGFP expressing and control-tdTomato expressing HSC, injected in the proportions of 30% VIVIT-eGFP to 70% tdTomato control, the methylcellulose cultures were set 4 weeks (upper panel) and 8 weeks (lower panel) after reconstitution and counted after 4–7 days of culture. Images of colonies from eight individual representative mice are shown as example (original magnification, ×10, scale bar 200 µm).Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from the BM of mice 4 and 8 weeks after reconstitution with a mixture of HSC consisting of 30% VIVIT-eGFP and 70% tdTomato control expressing cells.Analysis of average cell numbers per colony. After the ratio between VIVIT-eGFP and control tdTomato colonies was determined, cultures were harvested and labelled with antibodies against CD11b+. Differentiated cells (over 90% of total) were counted by flow cytometry with four replicates per mouse. Average number of cells per colony is plotted. Data are summarized from three (B and C) independent experiments with at least three mice per group in each experiment.Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from HSC lines used for reconstitution of mice.Total numbers of LSK (lin−, Sca-1+ and c-Kit+) in BM of mice reconstituted with control tdTomato or VIVIT-eGFP HSC.Total numbers of VIVIT-eGFP and Tomato control LSK (lin−, Sca-1+ and c-Kit+) in mice reconstituted with mixture of control tdTomato or VIVITeGFP HSC in initial ratio 1:1. (E and F) Analysis was performed 6 weeks after reconstitution with five mice per group. Changes in total numbers of DCs, in IFN-β production and changes in percentage of proliferating cells have been analysed by Student t-test *p ≤ 0.05 and ***p ≤ 0.001.

Mentions: To better understand the preference for in vivo expansion of myeloid cells, we analysed myeloid CFU obtained from reconstituted mice to determine the differentiation stage at which preferential expansion of VIVIT-expressing cells occurred. BM cells from reconstituted mice were cultured in methylcellulose media containing SCF, IL-3 and IL-6. Numbers of granulocytic and monocytic colony-forming units – granulocyte, macrophage (CFU-GM), corresponding to numbers of myeloid progenitors in BM, were determined after 4–7 days of culture (Fig 4A). Figure 4B shows numbers of CFU-GM from mice reconstituted with 30% VIVIT-eGFP competitors mixed with 70% control-tdTomato HSC and analysed 4 and 8 weeks later. Numbers of VIVIT-eGFP myeloid progenitors were significantly enriched over time compared with controls, consistent with the pattern of reconstitution achieved with NFAT-impaired myeloid cells in previous experiments (Fig 2B and C). After CFU-GM enumeration the colonies were harvested and the total number of CD11b+ cells was determined by flow cytometry. Figure 4C depicts average cell number per colony, which indicates significantly faster growth among VIVIT-eGFP expressing colonies. In mice singly engrafted with either VIVIT-eGFP or tdTomato control HSC alone, the respective progenitor populations remained detectable in BM for 12 weeks (unpublished observation).


Calcineurin/NFAT signalling inhibits myeloid haematopoiesis.

Fric J, Lim CX, Koh EG, Hofmann B, Chen J, Tay HS, Mohammad Isa SA, Mortellaro A, Ruedl C, Ricciardi-Castagnoli P - EMBO Mol Med (2012)

Impaired calcineurin/NFAT signalling increases the number and proliferation of myeloid progenitorsCFU in methyl cellulose from BM of mice reconstituted with a mixture of VIVIT-eGFP expressing and control-tdTomato expressing HSC, injected in the proportions of 30% VIVIT-eGFP to 70% tdTomato control, the methylcellulose cultures were set 4 weeks (upper panel) and 8 weeks (lower panel) after reconstitution and counted after 4–7 days of culture. Images of colonies from eight individual representative mice are shown as example (original magnification, ×10, scale bar 200 µm).Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from the BM of mice 4 and 8 weeks after reconstitution with a mixture of HSC consisting of 30% VIVIT-eGFP and 70% tdTomato control expressing cells.Analysis of average cell numbers per colony. After the ratio between VIVIT-eGFP and control tdTomato colonies was determined, cultures were harvested and labelled with antibodies against CD11b+. Differentiated cells (over 90% of total) were counted by flow cytometry with four replicates per mouse. Average number of cells per colony is plotted. Data are summarized from three (B and C) independent experiments with at least three mice per group in each experiment.Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from HSC lines used for reconstitution of mice.Total numbers of LSK (lin−, Sca-1+ and c-Kit+) in BM of mice reconstituted with control tdTomato or VIVIT-eGFP HSC.Total numbers of VIVIT-eGFP and Tomato control LSK (lin−, Sca-1+ and c-Kit+) in mice reconstituted with mixture of control tdTomato or VIVITeGFP HSC in initial ratio 1:1. (E and F) Analysis was performed 6 weeks after reconstitution with five mice per group. Changes in total numbers of DCs, in IFN-β production and changes in percentage of proliferating cells have been analysed by Student t-test *p ≤ 0.05 and ***p ≤ 0.001.
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Related In: Results  -  Collection

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fig04: Impaired calcineurin/NFAT signalling increases the number and proliferation of myeloid progenitorsCFU in methyl cellulose from BM of mice reconstituted with a mixture of VIVIT-eGFP expressing and control-tdTomato expressing HSC, injected in the proportions of 30% VIVIT-eGFP to 70% tdTomato control, the methylcellulose cultures were set 4 weeks (upper panel) and 8 weeks (lower panel) after reconstitution and counted after 4–7 days of culture. Images of colonies from eight individual representative mice are shown as example (original magnification, ×10, scale bar 200 µm).Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from the BM of mice 4 and 8 weeks after reconstitution with a mixture of HSC consisting of 30% VIVIT-eGFP and 70% tdTomato control expressing cells.Analysis of average cell numbers per colony. After the ratio between VIVIT-eGFP and control tdTomato colonies was determined, cultures were harvested and labelled with antibodies against CD11b+. Differentiated cells (over 90% of total) were counted by flow cytometry with four replicates per mouse. Average number of cells per colony is plotted. Data are summarized from three (B and C) independent experiments with at least three mice per group in each experiment.Numbers of VIVIT-eGFP and control tdTomato myeloid colonies (CFU) generated from HSC lines used for reconstitution of mice.Total numbers of LSK (lin−, Sca-1+ and c-Kit+) in BM of mice reconstituted with control tdTomato or VIVIT-eGFP HSC.Total numbers of VIVIT-eGFP and Tomato control LSK (lin−, Sca-1+ and c-Kit+) in mice reconstituted with mixture of control tdTomato or VIVITeGFP HSC in initial ratio 1:1. (E and F) Analysis was performed 6 weeks after reconstitution with five mice per group. Changes in total numbers of DCs, in IFN-β production and changes in percentage of proliferating cells have been analysed by Student t-test *p ≤ 0.05 and ***p ≤ 0.001.
Mentions: To better understand the preference for in vivo expansion of myeloid cells, we analysed myeloid CFU obtained from reconstituted mice to determine the differentiation stage at which preferential expansion of VIVIT-expressing cells occurred. BM cells from reconstituted mice were cultured in methylcellulose media containing SCF, IL-3 and IL-6. Numbers of granulocytic and monocytic colony-forming units – granulocyte, macrophage (CFU-GM), corresponding to numbers of myeloid progenitors in BM, were determined after 4–7 days of culture (Fig 4A). Figure 4B shows numbers of CFU-GM from mice reconstituted with 30% VIVIT-eGFP competitors mixed with 70% control-tdTomato HSC and analysed 4 and 8 weeks later. Numbers of VIVIT-eGFP myeloid progenitors were significantly enriched over time compared with controls, consistent with the pattern of reconstitution achieved with NFAT-impaired myeloid cells in previous experiments (Fig 2B and C). After CFU-GM enumeration the colonies were harvested and the total number of CD11b+ cells was determined by flow cytometry. Figure 4C depicts average cell number per colony, which indicates significantly faster growth among VIVIT-eGFP expressing colonies. In mice singly engrafted with either VIVIT-eGFP or tdTomato control HSC alone, the respective progenitor populations remained detectable in BM for 12 weeks (unpublished observation).

Bottom Line: Reconstituting lethally irradiated mice with haematopoietic stem cells expressing an NFAT-inhibitory peptide resulted in enhanced development of the myeloid compartment.Global gene expression analysis of untreated DC and NFAT-inhibited DC revealed differential expression of transcripts that regulate cell cycle and apoptosis.In conclusion, these results provide evidence that calcineurin/NFAT signalling negatively regulates myeloid lineage development.

View Article: PubMed Central - PubMed

Affiliation: Singapore Immunology Network (SIgN), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.

Show MeSH
Related in: MedlinePlus