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Sensing of the microbiota by NOD1 in mesenchymal stromal cells regulates murine hematopoiesis

Chiaki Iwamura, Nicolas Bouladoux, Yasmine Belkaid, Alan Sher and Dragana Jankovic

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    NOD1L administration restores numbers of HSPCs in GF mice to the levels observed in SPF mice. (A) Levels of HSC, MPP, and CLP in SPF vs GF mice. Each symbol indicates the absolute number of LSK or CLP in BM from individual SPF (n = 6) and GF mice (n = 11). The horizontal bars represent the mean values for each group. (B) Serum levels of NOD1 or NOD2 ligand in naïve SPF and GF mice. Bars represent the mean ± standard error of the mean (SEM) of the values obtained in the reporter gene assay used (n = 8 per group). (C) Levels of Nod1 and Nod2 mRNA expression in indicated in BM HSPC populations isolated from WT, NOD1−/−, or NOD2−/− mice (n = 10). BMMϕ from WT animals were included as positive controls. Bars represent the mean ± standard deviation (SD) of triplicate values of Nod1 or Nod2 expression levels relative to the Rplp2 housekeeping gene. (D) NOD1 ligand levels in GF mice (n = 5-9) given either synthetic NOD1 ligand (C12-iE-DAP, 100 μg) or phosphate-buffered saline by gavage every 2 or 3 days for 2 weeks. (E-F) HSPC populations in GF mice after gavage with NOD1 ligand (C12-iE-DAP, 100 μg) as noted before. (E) The representative dot plots shown were gated on lineageneg and IL-7Rαneg (upper panels) or IL-7Rα+ (lower panels) cells, respectively. (F) Absolute numbers of LT-HSC, ST-HSC, MPP, CLP, and CMP in SPF mice, GF mice, and GF mice treated with NOD1 ligand. The symbols represent the cell numbers for the individual animals in each group (n = 7-12). The bars represent the means ± SEM of these values. *P < .05; **P < .01; ***P < .001 denote the statistical significance of the differences in group means. The data shown in (A), (B), (D), and (F) are pooled from 2 to 3 independently performed experiments, whereas the values shown in (C) are from an experiment representative of 2 performed.

  • 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.