Figure 7.
Retinoic acid impairs the ability of BMSCs to support emergency myelopoiesis against L monocytogenes during BM transplantation in a RIG-I–dependent manner. (A) Schematic of experimental design for co-transplantation of BMNCs with BMSCs derived from Rig-I+/+ and Rig-I−/− mice pretreated with ATRA or control vehicle (B-C). (B) The absolute numbers of donor-derived macrophage (CD11b+F4/80+) and donor-derived monocytes (CD11b+Ly6Chi) after the 8-week recovery in recipients received BMNC and BMSC co-transplantation as indicated; n = 3–5 mice per group. (C) The recovery of neutrophils in the peripheral blood of transplanted recipients as indicated; n = 4–7 mice per group. (D) Schematic of experimental design for evaluating stromal niche function in Rig-I+/+ or Rig-I−/− mice after ATRA treatment. Rig-I+/+ and Rig-I−/− mice pretreated with vehicle or ATRA consecutively 6 times were lethally irradiated and transplanted with 1 × 106 CD45.1+ BMNCs for recovery. After 8 weeks of recovery, the chimeric recipients were infected with L monocytogenes (L.M.) at 1 × 104 CFUs (D-H). (E) Absolute numbers of donor-derived HSPCs in the BM in chimeric recipients at 3 days after L.M. infection; n = 5 mice per group. (F-G) Absolute numbers in the BM (F) and frequency in the peripheral blood (G) of donor-derived macrophages (CD11b+F4/80+) and neutrophils (CD11b+Gr-1+) in chimeric recipients at 3 days after L.M. infection; n = 5 mice per group. (H) Representative images (i) and quantification (ii) of CFUs in liver and spleen at 3 days after L.M. infection in chimeric recipients; n = 5 mice per group. (I) Western blots for RIG-I, NRF2, Keap1, and Trim25 in BMSCs from Rig-I+/+ or Rig-I−/− mice treated with IFNγ or L.M. as indicated, with β-actin used as a loading control. (J) qPCR of RIG-I in BMSCs with IFNγ, L.M., and STAT1 inhibitor (fludarabine) as indicated; n = 3 biologically independent replicates. (K) Schematic of experimental design for the role of RIG-I in BMSCs on L.M. infection challenge; 1 × 106Rig-I+/+ BMNCs were transplanted into lethal irradiated Rig-I+/+ or Rig-I−/− mice. Eight weeks after transplantation, chimeric Rig-I+/+ and Rig-I−/− mice were infected with L.M. at 1 × 104 CFUs, and BMSCs were analyzed at 7 days after infection (L-O). (L-O) The ROS levels (L), cell death (M), absolute numbers (N), and CFU-F activity (O) of BMSCs from chimeric Rig-I+/+ and Rig-I−/− recipient mice after indicated treatments; n = 4–5 mice per group. (P) Schematic of experimental design for evaluating the role of RIG-I in stromal niche on L.M. infection challenge; 1 × 106 CD45.1+ BMNCs were transplanted into lethally irradiated Rig-I+/+ and Rig-I−/− mice, which were preinfected with L.M. at 1 × 104 CFU for 7 days. Engraftment was analyzed at 4 weeks after transplantation (Q-R). (Q-R) Absolute numbers of CD45.1+ donor-derived HSPCs (Q) and cell cycle analysis of CD45.1+ donor-derived HSCs (R) in recipient mice as indicated; n = 5-6 mice per group. Error bars indicate mean ± SD. Repeated-measures 1-way (B and J) or 2-way (C, E-H, L-O, Q, and R) ANOVA followed by Dunnett’s test multiple comparisons. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

Retinoic acid impairs the ability of BMSCs to support emergency myelopoiesis against L monocytogenes during BM transplantation in a RIG-I–dependent manner. (A) Schematic of experimental design for co-transplantation of BMNCs with BMSCs derived from Rig-I+/+ and Rig-I−/− mice pretreated with ATRA or control vehicle (B-C). (B) The absolute numbers of donor-derived macrophage (CD11b+F4/80+) and donor-derived monocytes (CD11b+Ly6Chi) after the 8-week recovery in recipients received BMNC and BMSC co-transplantation as indicated; n = 3–5 mice per group. (C) The recovery of neutrophils in the peripheral blood of transplanted recipients as indicated; n = 4–7 mice per group. (D) Schematic of experimental design for evaluating stromal niche function in Rig-I+/+ or Rig-I−/− mice after ATRA treatment. Rig-I+/+ and Rig-I−/− mice pretreated with vehicle or ATRA consecutively 6 times were lethally irradiated and transplanted with 1 × 106 CD45.1+ BMNCs for recovery. After 8 weeks of recovery, the chimeric recipients were infected with L monocytogenes (L.M.) at 1 × 104 CFUs (D-H). (E) Absolute numbers of donor-derived HSPCs in the BM in chimeric recipients at 3 days after L.M. infection; n = 5 mice per group. (F-G) Absolute numbers in the BM (F) and frequency in the peripheral blood (G) of donor-derived macrophages (CD11b+F4/80+) and neutrophils (CD11b+Gr-1+) in chimeric recipients at 3 days after L.M. infection; n = 5 mice per group. (H) Representative images (i) and quantification (ii) of CFUs in liver and spleen at 3 days after L.M. infection in chimeric recipients; n = 5 mice per group. (I) Western blots for RIG-I, NRF2, Keap1, and Trim25 in BMSCs from Rig-I+/+ or Rig-I−/− mice treated with IFNγ or L.M. as indicated, with β-actin used as a loading control. (J) qPCR of RIG-I in BMSCs with IFNγ, L.M., and STAT1 inhibitor (fludarabine) as indicated; n = 3 biologically independent replicates. (K) Schematic of experimental design for the role of RIG-I in BMSCs on L.M. infection challenge; 1 × 106Rig-I+/+ BMNCs were transplanted into lethal irradiated Rig-I+/+ or Rig-I−/− mice. Eight weeks after transplantation, chimeric Rig-I+/+ and Rig-I−/− mice were infected with L.M. at 1 × 104 CFUs, and BMSCs were analyzed at 7 days after infection (L-O). (L-O) The ROS levels (L), cell death (M), absolute numbers (N), and CFU-F activity (O) of BMSCs from chimeric Rig-I+/+ and Rig-I−/− recipient mice after indicated treatments; n = 4–5 mice per group. (P) Schematic of experimental design for evaluating the role of RIG-I in stromal niche on L.M. infection challenge; 1 × 106 CD45.1+ BMNCs were transplanted into lethally irradiated Rig-I+/+ and Rig-I−/− mice, which were preinfected with L.M. at 1 × 104 CFU for 7 days. Engraftment was analyzed at 4 weeks after transplantation (Q-R). (Q-R) Absolute numbers of CD45.1+ donor-derived HSPCs (Q) and cell cycle analysis of CD45.1+ donor-derived HSCs (R) in recipient mice as indicated; n = 5-6 mice per group. Error bars indicate mean ± SD. Repeated-measures 1-way (B and J) or 2-way (C, E-H, L-O, Q, and R) ANOVA followed by Dunnett’s test multiple comparisons. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal