![Figure 2. NOD1 ligand stimulation of MSCs induces secretion of cytokines that promote proliferation of HSPCs. (A) FACS-purified HSC, MPP, and CLP were cultured for 48 hours in the presence of NOD1 (10 μg/mL) or NOD2 (10 μg/mL) ligand alone or in combination with SCF (10 ng/mL), Flt3L (10 ng/mL), or ThPO (10 ng/mL) in the case of HSC and MPP, and with IL-7 (10 ng/mL) and Flt3L in the case of CLP. [3H]-thymidine was added during the last 16 hours of culture. Bars represent mean ± SD of thymidine incorporation for triplicate cultures. N.D., not determined. (B) Expression of Nod1 and Nod2 mRNA in cultured CD45neg MSC and CD45+ Mϕ from WT, NOD1−/−, or NOD2−/− mice. Bars represent mean ± SD of triplicate values of Nod1 and Nod2 expression relative to RPLP2 expression. (C) MSCs generated from WT or NOD1−/− mice were stimulated with NOD1 ligand (C12-iE-DAP, 10 μg/mL) or LPS (10 μg/mL) and 72 hours later harvested for RNA extraction. Bars represent mean ± SD of triplicate measurements of Nod1 relative to Rplp2 expression for each stimulus. (D) Expression of mRNA for IL-3, IL-6, IL-7, SCF, Thpo, and Flt3L in WT MSCs treated with NOD1 ligand (10 μg/mL) or LPS (10 μg/mL). Bars represent the means ± SD of triplicate values of mRNA expression relative to Hprt for each cytokine. (E-F) Sorted LSK (2 × 103/well), HSC (2 × 103/well), CLP (2 × 103/well), or CMP (4 × 103/well) from WT mice were stimulated for 48 hours with the culture supernatants harvested from WT (WT CS) or NOD1−/− (KO CS) MSC cultures treated with NOD1 ligand (10 μg/mL) for 72 hours (E). [3H]-thymidine (0.5 μCi/well, New England Nuclear Corp., sp act: 2 Ci/mmol) was added at 48 hours and the incorporated radioactivity measured 16 hours later. Bars represent the mean ± SD of the values from triplicate cultures. The data shown in (A-E) are from 1 representative out of 3 experiments performed. (F) Serum levels of IL-7, SCF, ThPO, and Flt3L in GF mice after gavage with NOD1 ligand. The concentrations of IL-7, Flt3L, ThPO, and SCF in culture supernatants or serum were measured by Quantikine ELISA kit (R&D systems). Bars represent the mean ± SEM of the ELISA values obtained from the individual SPF, GF, and NOD1 ligand–treated GF mice shown in Figure 1F and pooled from 2 independently performed replicate experiments. *P < .05; **P < .01; ***P < .001.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/129/2/10.1182_blood-2016-06-723742/4/blood723742f2.jpeg?Expires=1724943832&Signature=o7-YPsTj25xpUc4-AHlW~-FZTx2MyvMPzw9U6AUI5qIkeLIgVJ66EC2apvLXK7igqAdkcWS-AOZdNcO~J8B~27A1TQ8SvmGEvbYxF~oU~N0B7l7CzrlR8PpGgSVwYdGyzlL29B7OiAUBEprmdDyvJhC39VixH7RK7W3fv1bnKfepyFGYzdPKhtcdsUuJozvqlVBzTLU~jWHsj500muG8PUwGGmAID7unO88HngC631~ki~R426dvsO4Du64Jab02GLgVe6BCD84of8m795a6JQ4Hnw4ydY6l2ZB0nOMGdxDGuBO6gdGtRh6deGoaCuPGrmvkMi3fiI5LhvYxw66-bA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
NOD1 ligand stimulation of MSCs induces secretion of cytokines that promote proliferation of HSPCs. (A) FACS-purified HSC, MPP, and CLP were cultured for 48 hours in the presence of NOD1 (10 μg/mL) or NOD2 (10 μg/mL) ligand alone or in combination with SCF (10 ng/mL), Flt3L (10 ng/mL), or ThPO (10 ng/mL) in the case of HSC and MPP, and with IL-7 (10 ng/mL) and Flt3L in the case of CLP. [3H]-thymidine was added during the last 16 hours of culture. Bars represent mean ± SD of thymidine incorporation for triplicate cultures. N.D., not determined. (B) Expression of Nod1 and Nod2 mRNA in cultured CD45neg MSC and CD45+ Mϕ from WT, NOD1−/−, or NOD2−/− mice. Bars represent mean ± SD of triplicate values of Nod1 and Nod2 expression relative to RPLP2 expression. (C) MSCs generated from WT or NOD1−/− mice were stimulated with NOD1 ligand (C12-iE-DAP, 10 μg/mL) or LPS (10 μg/mL) and 72 hours later harvested for RNA extraction. Bars represent mean ± SD of triplicate measurements of Nod1 relative to Rplp2 expression for each stimulus. (D) Expression of mRNA for IL-3, IL-6, IL-7, SCF, Thpo, and Flt3L in WT MSCs treated with NOD1 ligand (10 μg/mL) or LPS (10 μg/mL). Bars represent the means ± SD of triplicate values of mRNA expression relative to Hprt for each cytokine. (E-F) Sorted LSK (2 × 103/well), HSC (2 × 103/well), CLP (2 × 103/well), or CMP (4 × 103/well) from WT mice were stimulated for 48 hours with the culture supernatants harvested from WT (WT CS) or NOD1−/− (KO CS) MSC cultures treated with NOD1 ligand (10 μg/mL) for 72 hours (E). [3H]-thymidine (0.5 μCi/well, New England Nuclear Corp., sp act: 2 Ci/mmol) was added at 48 hours and the incorporated radioactivity measured 16 hours later. Bars represent the mean ± SD of the values from triplicate cultures. The data shown in (A-E) are from 1 representative out of 3 experiments performed. (F) Serum levels of IL-7, SCF, ThPO, and Flt3L in GF mice after gavage with NOD1 ligand. The concentrations of IL-7, Flt3L, ThPO, and SCF in culture supernatants or serum were measured by Quantikine ELISA kit (R&D systems). Bars represent the mean ± SEM of the ELISA values obtained from the individual SPF, GF, and NOD1 ligand–treated GF mice shown in Figure 1F and pooled from 2 independently performed replicate experiments. *P < .05; **P < .01; ***P < .001.
