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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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Calcineurin/NFAT inhibition in HSC confers a growth advantage to myeloid cellsHost mice were lethally irradiated and rescued by intravenous injection of HSC. HSC populations comprised a mixture of cells expressing either VIVIT or a control plasmid with a fluorescent tag in the indicated ratios.Mice were reconstituted with a mixture of VIVIT-eGFP and control tdTomato HSC in a 1:1 ratio and analysed 8 weeks later. Subsets of DC (CD11c+MHCII+), granulocytes (CD11b+Gr-1+) and monocytes (CD11b+Gr1−) were gated. The ratio between VIVIT-eGFP and control tdTomato cells in each lineage is plotted as the logarithm of the percentage of competitor VIVIT-eGFP cells, divided by percentage of control tdTomato cells (value 0 reflects 1:1 ratio), for BM, spleen, lymph nodes and blood. Initial ratio of HSC used for reconstitution is shown.Example of flow analysis of the ratio between VIVIT-eGFP and control tdTomato granulocytes, monocytes, cDC and pDC 4 weeks after reconstitution with mixture of HSC of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines.Analysis of kinetic of reconstitution in mice injected with a mixture of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines. Mice were analysed 4 and 8 weeks after reconstitution (value −0.36 reflects the injected 30:70 ratio). Granulocytes, monocytes and DC were analysed in BM and spleen, CD4+ and CD8+ T cells were analysed in spleen alone. Data (A) are summarized from two of three experiments with similar results (n = 8; mean ± SEM), (C) data are from one of 3 experiments (n = 4 for 4 weeks and n = 8 for 8 weeks time point). Changes in the ratios between two-time point have been analysed by Student t-test ***p ≤ 0.001.
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fig02: Calcineurin/NFAT inhibition in HSC confers a growth advantage to myeloid cellsHost mice were lethally irradiated and rescued by intravenous injection of HSC. HSC populations comprised a mixture of cells expressing either VIVIT or a control plasmid with a fluorescent tag in the indicated ratios.Mice were reconstituted with a mixture of VIVIT-eGFP and control tdTomato HSC in a 1:1 ratio and analysed 8 weeks later. Subsets of DC (CD11c+MHCII+), granulocytes (CD11b+Gr-1+) and monocytes (CD11b+Gr1−) were gated. The ratio between VIVIT-eGFP and control tdTomato cells in each lineage is plotted as the logarithm of the percentage of competitor VIVIT-eGFP cells, divided by percentage of control tdTomato cells (value 0 reflects 1:1 ratio), for BM, spleen, lymph nodes and blood. Initial ratio of HSC used for reconstitution is shown.Example of flow analysis of the ratio between VIVIT-eGFP and control tdTomato granulocytes, monocytes, cDC and pDC 4 weeks after reconstitution with mixture of HSC of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines.Analysis of kinetic of reconstitution in mice injected with a mixture of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines. Mice were analysed 4 and 8 weeks after reconstitution (value −0.36 reflects the injected 30:70 ratio). Granulocytes, monocytes and DC were analysed in BM and spleen, CD4+ and CD8+ T cells were analysed in spleen alone. Data (A) are summarized from two of three experiments with similar results (n = 8; mean ± SEM), (C) data are from one of 3 experiments (n = 4 for 4 weeks and n = 8 for 8 weeks time point). Changes in the ratios between two-time point have been analysed by Student t-test ***p ≤ 0.001.

Mentions: To extend these observations, we performed competitive reconstitution of irradiated hosts using mixed HSC lines in a 1:1 ratio; VIVIT-eGFP expressing HSCs as competitors to control tdTomato HSC lines. The ratio of VIVIT-eGFP to control-tdTomato derived granulocytes (CD11b+Gr1+), monocytes (CD11b+Gr1−), cDC (CD11c+MHCII+) and pDC (CD11c+B220+) was analysed in BM, spleen, lymph nodes and peripheral blood 8 weeks after engraftment. We observed a selective advantage for calcineurin/NFAT impaired cells in the repopulation of pDC, cDC, granulocytic and monocytic compartments throughout the BM, spleen, lymph nodes and peripheral blood (shown as log ratio of engrafted NFAT-impaired competitors to controls, Fig 2A). Critically, the relative proportions of lymphoid T and B cells remained unchanged and simply reflected the ratio of injected HSC (Fig 2A), indicating comparable engraftment of both HSC lines and confirming that lymphopoiesis is not impaired by this level of calcineurin/NFAT inhibition. The unaffected ratio of lymphocytes indicates that self-renewal and maintenance of stem cell progenitors is not affected by impaired NFAT signalling.


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)

Calcineurin/NFAT inhibition in HSC confers a growth advantage to myeloid cellsHost mice were lethally irradiated and rescued by intravenous injection of HSC. HSC populations comprised a mixture of cells expressing either VIVIT or a control plasmid with a fluorescent tag in the indicated ratios.Mice were reconstituted with a mixture of VIVIT-eGFP and control tdTomato HSC in a 1:1 ratio and analysed 8 weeks later. Subsets of DC (CD11c+MHCII+), granulocytes (CD11b+Gr-1+) and monocytes (CD11b+Gr1−) were gated. The ratio between VIVIT-eGFP and control tdTomato cells in each lineage is plotted as the logarithm of the percentage of competitor VIVIT-eGFP cells, divided by percentage of control tdTomato cells (value 0 reflects 1:1 ratio), for BM, spleen, lymph nodes and blood. Initial ratio of HSC used for reconstitution is shown.Example of flow analysis of the ratio between VIVIT-eGFP and control tdTomato granulocytes, monocytes, cDC and pDC 4 weeks after reconstitution with mixture of HSC of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines.Analysis of kinetic of reconstitution in mice injected with a mixture of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines. Mice were analysed 4 and 8 weeks after reconstitution (value −0.36 reflects the injected 30:70 ratio). Granulocytes, monocytes and DC were analysed in BM and spleen, CD4+ and CD8+ T cells were analysed in spleen alone. Data (A) are summarized from two of three experiments with similar results (n = 8; mean ± SEM), (C) data are from one of 3 experiments (n = 4 for 4 weeks and n = 8 for 8 weeks time point). Changes in the ratios between two-time point have been analysed by Student t-test ***p ≤ 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376854&req=5

fig02: Calcineurin/NFAT inhibition in HSC confers a growth advantage to myeloid cellsHost mice were lethally irradiated and rescued by intravenous injection of HSC. HSC populations comprised a mixture of cells expressing either VIVIT or a control plasmid with a fluorescent tag in the indicated ratios.Mice were reconstituted with a mixture of VIVIT-eGFP and control tdTomato HSC in a 1:1 ratio and analysed 8 weeks later. Subsets of DC (CD11c+MHCII+), granulocytes (CD11b+Gr-1+) and monocytes (CD11b+Gr1−) were gated. The ratio between VIVIT-eGFP and control tdTomato cells in each lineage is plotted as the logarithm of the percentage of competitor VIVIT-eGFP cells, divided by percentage of control tdTomato cells (value 0 reflects 1:1 ratio), for BM, spleen, lymph nodes and blood. Initial ratio of HSC used for reconstitution is shown.Example of flow analysis of the ratio between VIVIT-eGFP and control tdTomato granulocytes, monocytes, cDC and pDC 4 weeks after reconstitution with mixture of HSC of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines.Analysis of kinetic of reconstitution in mice injected with a mixture of 30% VIVIT-eGFP and 70% tdTomato control expressing HSC lines. Mice were analysed 4 and 8 weeks after reconstitution (value −0.36 reflects the injected 30:70 ratio). Granulocytes, monocytes and DC were analysed in BM and spleen, CD4+ and CD8+ T cells were analysed in spleen alone. Data (A) are summarized from two of three experiments with similar results (n = 8; mean ± SEM), (C) data are from one of 3 experiments (n = 4 for 4 weeks and n = 8 for 8 weeks time point). Changes in the ratios between two-time point have been analysed by Student t-test ***p ≤ 0.001.
Mentions: To extend these observations, we performed competitive reconstitution of irradiated hosts using mixed HSC lines in a 1:1 ratio; VIVIT-eGFP expressing HSCs as competitors to control tdTomato HSC lines. The ratio of VIVIT-eGFP to control-tdTomato derived granulocytes (CD11b+Gr1+), monocytes (CD11b+Gr1−), cDC (CD11c+MHCII+) and pDC (CD11c+B220+) was analysed in BM, spleen, lymph nodes and peripheral blood 8 weeks after engraftment. We observed a selective advantage for calcineurin/NFAT impaired cells in the repopulation of pDC, cDC, granulocytic and monocytic compartments throughout the BM, spleen, lymph nodes and peripheral blood (shown as log ratio of engrafted NFAT-impaired competitors to controls, Fig 2A). Critically, the relative proportions of lymphoid T and B cells remained unchanged and simply reflected the ratio of injected HSC (Fig 2A), indicating comparable engraftment of both HSC lines and confirming that lymphopoiesis is not impaired by this level of calcineurin/NFAT inhibition. The unaffected ratio of lymphocytes indicates that self-renewal and maintenance of stem cell progenitors is not affected by impaired NFAT signalling.

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