Peptidoglycan-Mediated Bone Marrow Autonomic Neuropathy Impairs Hematopoietic Stem/Progenitor Cells via a NOD1-Dependent Pathway in db/db Mice

Figure 2

Peptidoglycan worsened diabetes-induced HSPC depletion and impairment in the bone marrow. (a) Representative flow cytometry gating scheme for the HSPC subpopulations in each group. (b–d) Flow cytometry analysis shows the percentage of (c) LT-HSC subpopulation was decreased in PGN-treated db/db mice group, while the percentages of (b) LSK and (c) ST-HSC were not affected by PGN injections ( per group). (e) Colony-forming capacity of bone marrow cells and blood cells from db/m and db/db mice treated with either PGN or vehicle control were tested in CFU assay. Colonies of BFU-E, CFU-G/M/GM, and CFU-GEMM using the bone marrow cells and total colony numbers using the peripheral blood were calculated after 10 days culture ( per group). (f) LSK cells from each group were grown in LTC-IC cultures for 4 weeks, followed by methylcellulose plating and culture for 12 days. PGN administration worsened diabetes-induced reduction in the total number of colonies per BM LSK cells. (g) The bar graph shows the percentage of BrdU⁺ CD34^-LSK was the lowest in db/db+PGN mice by FACS in the in vivo BrdU incorporation study ( per group). (h, i) In the competitive transplantation study, lethally irradiated recipient mice (F1 progeny, CD45.1⁺ CD45.2⁺) were transplanted with LSK cells (CD45.2) from db/m+v, db/db+v, db/m+PGN, and db/db+PGN mice mixed with bone marrow cells (CD45.1, B6.SJL competitor). The bar graph in (i) shows the mean percentage of donor-derived cells in the peripheral blood 16 weeks after transplantation ( per group). Data represent . PGN: peptidoglycan; V: vehicle; LT-HSC: long-term repopulating hematopoietic stem cell; ST-HSC: short-term repopulating hematopoietic stem cell. , ; ^&compared to db/m+V; ^#compared to db/m+PGN; ^§compared to db/db+V.

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