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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 enhances the development of DCs in FLT3-L derived BM culturesBM derived DC cultivated in the presence of Flt3-L were harvested on day 11 of culture and labelled with antibodies against CD11c, Siglec-H and CD11b.Total numbers of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ DCs in FLT3-L derived BM-DC cultures with and without CsA and FK506. Representative of of five experiment (n = 6; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.IFN-β production in FLT3-L derived DC grown in presence or absence of CsA. Protein levels IFN-β in supernatants were analysed 18 h after Poly I:C or CpG stimulation. Differences in IFN-β were analysed by Student t-test, *p ≤ 0.05 (mean ± SEM).Percentage of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ cells in FLT3-L derived BM-DC cultures with and without CsA and FK506. Example of flow analysis of experiment presented in (A).Representative example of changes in apoptosis (gate I), proliferation (gate II for S phase) and M + G2 phase (gate III), measured by BrdU incorporation in CsA-treated cells (right) compared to not-treated controls (left).Analysis of cell cycle and proliferation. Non-treated and CsA DC cultures were pulsed with BrdU for the last 72 h of culture. The total number of cells in S phases (left) and undergoing apoptosis (right) is presented. Data are representative of three experiments (n = 3–5; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.
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fig05: Calcineurin/NFAT inhibition enhances the development of DCs in FLT3-L derived BM culturesBM derived DC cultivated in the presence of Flt3-L were harvested on day 11 of culture and labelled with antibodies against CD11c, Siglec-H and CD11b.Total numbers of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ DCs in FLT3-L derived BM-DC cultures with and without CsA and FK506. Representative of of five experiment (n = 6; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.IFN-β production in FLT3-L derived DC grown in presence or absence of CsA. Protein levels IFN-β in supernatants were analysed 18 h after Poly I:C or CpG stimulation. Differences in IFN-β were analysed by Student t-test, *p ≤ 0.05 (mean ± SEM).Percentage of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ cells in FLT3-L derived BM-DC cultures with and without CsA and FK506. Example of flow analysis of experiment presented in (A).Representative example of changes in apoptosis (gate I), proliferation (gate II for S phase) and M + G2 phase (gate III), measured by BrdU incorporation in CsA-treated cells (right) compared to not-treated controls (left).Analysis of cell cycle and proliferation. Non-treated and CsA DC cultures were pulsed with BrdU for the last 72 h of culture. The total number of cells in S phases (left) and undergoing apoptosis (right) is presented. Data are representative of three experiments (n = 3–5; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.

Mentions: To clarify the processes by which myeloid cell enrichment occurs following impairment of calcineurin/NFAT signalling, we studied BM-DC differentiation in vitro in medium supplemented with Flt3-L (200 ng/ml) in the presence or absence of CsA (2 µg/ml) or FK506 (0.2 ug/ml). To achieve efficient inhibition of calcineurin/NFAT signalling, inhibitors were added to the cells 30 min prior to Flt3-L administration and were maintained throughout the 9–11 days culture period, at which time cell subset frequency and total number were analysed by flow cytometry. Part of the generated DC exhibited a pDC phenotype and expressed Siglec-H, B220 and PDCA-1 on surface of CD11c+ cells, also cDC were present in the culture as CD11c+CD11b+ cells. The addition of CsA or FK506 to the cultures induced significantly higher numbers of CD11c+ DC with higher SSC and FSC, while other DC subsets were not affected by treatment (Fig 5A and C). Both controls and CsA-inhibited cultures gave rise to DC that are able to produce interferon (IFN)-β, detected 18 h after stimulation with Poly I:C or CpG (Fig 5B). The basis of this DC population increase was investigated using BrdU incorporation to determine proliferation and apoptosis levels in the cultures. DC were pulsed with BrdU for the last 72 h of culture before analysis by flow cytometry (Fig 5D). DC treated with CsA included higher numbers of proliferating cells in the S phase, but decreased apoptosis (Fig 5E). Data from the in vitro Flt3-L/CsA-treated BM-DC cultures confirm our in vivo findings for DC expansion. Since we observed a strong impact of calcineurin/NFAT impairment on Flt3-L-derived DC development, we further investigated the possible mechanism of action by global gene transcription analysis.


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 enhances the development of DCs in FLT3-L derived BM culturesBM derived DC cultivated in the presence of Flt3-L were harvested on day 11 of culture and labelled with antibodies against CD11c, Siglec-H and CD11b.Total numbers of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ DCs in FLT3-L derived BM-DC cultures with and without CsA and FK506. Representative of of five experiment (n = 6; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.IFN-β production in FLT3-L derived DC grown in presence or absence of CsA. Protein levels IFN-β in supernatants were analysed 18 h after Poly I:C or CpG stimulation. Differences in IFN-β were analysed by Student t-test, *p ≤ 0.05 (mean ± SEM).Percentage of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ cells in FLT3-L derived BM-DC cultures with and without CsA and FK506. Example of flow analysis of experiment presented in (A).Representative example of changes in apoptosis (gate I), proliferation (gate II for S phase) and M + G2 phase (gate III), measured by BrdU incorporation in CsA-treated cells (right) compared to not-treated controls (left).Analysis of cell cycle and proliferation. Non-treated and CsA DC cultures were pulsed with BrdU for the last 72 h of culture. The total number of cells in S phases (left) and undergoing apoptosis (right) is presented. Data are representative of three experiments (n = 3–5; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.
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Related In: Results  -  Collection

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fig05: Calcineurin/NFAT inhibition enhances the development of DCs in FLT3-L derived BM culturesBM derived DC cultivated in the presence of Flt3-L were harvested on day 11 of culture and labelled with antibodies against CD11c, Siglec-H and CD11b.Total numbers of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ DCs in FLT3-L derived BM-DC cultures with and without CsA and FK506. Representative of of five experiment (n = 6; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.IFN-β production in FLT3-L derived DC grown in presence or absence of CsA. Protein levels IFN-β in supernatants were analysed 18 h after Poly I:C or CpG stimulation. Differences in IFN-β were analysed by Student t-test, *p ≤ 0.05 (mean ± SEM).Percentage of cDC (CD11c+CD11b+Siglec-H−), pDCs (CD11c+CD11b−Siglec-H+) and SSC, FCShi CD11c+ cells in FLT3-L derived BM-DC cultures with and without CsA and FK506. Example of flow analysis of experiment presented in (A).Representative example of changes in apoptosis (gate I), proliferation (gate II for S phase) and M + G2 phase (gate III), measured by BrdU incorporation in CsA-treated cells (right) compared to not-treated controls (left).Analysis of cell cycle and proliferation. Non-treated and CsA DC cultures were pulsed with BrdU for the last 72 h of culture. The total number of cells in S phases (left) and undergoing apoptosis (right) is presented. Data are representative of three experiments (n = 3–5; mean ± SEM). Changes in the cell number were analysed by Student t-test, ***p ≤ 0.001.
Mentions: To clarify the processes by which myeloid cell enrichment occurs following impairment of calcineurin/NFAT signalling, we studied BM-DC differentiation in vitro in medium supplemented with Flt3-L (200 ng/ml) in the presence or absence of CsA (2 µg/ml) or FK506 (0.2 ug/ml). To achieve efficient inhibition of calcineurin/NFAT signalling, inhibitors were added to the cells 30 min prior to Flt3-L administration and were maintained throughout the 9–11 days culture period, at which time cell subset frequency and total number were analysed by flow cytometry. Part of the generated DC exhibited a pDC phenotype and expressed Siglec-H, B220 and PDCA-1 on surface of CD11c+ cells, also cDC were present in the culture as CD11c+CD11b+ cells. The addition of CsA or FK506 to the cultures induced significantly higher numbers of CD11c+ DC with higher SSC and FSC, while other DC subsets were not affected by treatment (Fig 5A and C). Both controls and CsA-inhibited cultures gave rise to DC that are able to produce interferon (IFN)-β, detected 18 h after stimulation with Poly I:C or CpG (Fig 5B). The basis of this DC population increase was investigated using BrdU incorporation to determine proliferation and apoptosis levels in the cultures. DC were pulsed with BrdU for the last 72 h of culture before analysis by flow cytometry (Fig 5D). DC treated with CsA included higher numbers of proliferating cells in the S phase, but decreased apoptosis (Fig 5E). Data from the in vitro Flt3-L/CsA-treated BM-DC cultures confirm our in vivo findings for DC expansion. Since we observed a strong impact of calcineurin/NFAT impairment on Flt3-L-derived DC development, we further investigated the possible mechanism of action by global gene transcription analysis.

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