Bone Marrow Of Old Mice Research Articles

3054 – MONOCYTE SUBSET PRODUCTION DURING AGING

A growing body of evidence suggests that monocytes are more heterogenous than previously appreciated and that monocyte subsets play distinct roles in both health and disease. We have previously demonstrated that two separate pathways of monocyte production by granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) yield functionally distinct monocyte subsets in mouse bone marrow. GMPs produce classical monocytes with neutrophil-like properties, and MDPs yield classical monocytes that give rise to monocyte-derived DCs (moDCs). We also showed that Toll-like receptor agonists differentially promote production of these monocyte subsets during emergency monopoiesis. Aging induces a myeloid bias leading to increased monocyte output by the bone marrow. However, it is unclear whether the GMP and MDP pathways both contribute to increased monocyte output during aging. We observed myeloid-biased hematopoiesis in the bone marrow of old mice and elevated classical monocytes in the circulation, recapitulating previous reports. Single cell RNA-sequencing revealed an increased proportion of classical monocytes expressing MHC II molecules (H2-A and H2-E genes) and Cd74 in the bone marrow of old mice. We also observed a higher frequency of MHC II-expressing monocytes in the bone marrow and blood of old mice by flow cytometry. Together the data suggest that production of moDCs by MDPs is enhanced during aging. We are also investigating whether infection and acetaminophen-induced liver injury alter the balance of monocyte subsets that promote inflammation and tissue damage versus tissue repair and regeneration.

Aging of the Hematopoietic Stem Cell Niche: An Unnerving Matter.

The drivers of aging in the hematopoietic system remain incompletely understood. In this issue of Cell Stem Cell, Ho etal. (2019) report that functional switching of β-adrenergic nerve signaling underlies remodeling of stem cell niches, driving age-associated alterations in hematopoiesis.

In senescence, age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5-10-fold increases in an ordinarily minor CD21/35(-) CD23(-) mature B-cell subset termed age-associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro-B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro-B-cell growth. ABC effects can be prevented by the anti-inflammatory cytokine IL-10. Notably, CD21/35(+) CD23(+) follicular (FO) splenic and FO-like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL-10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL-10 within this B-cell population ameliorates the TNFα-mediated effects on B-cell precursors. Loss of B-cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO-like B cells. Adoptive transfer of aged ABC into RAG-2 KO recipients resulted in significant losses of pro-B cells within the bone marrow. These results suggest that alterations in B-cell composition during old age, in particular, the increase in ABC within the B-cell compartments, contribute to a pro-inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B-cell 'feedback' that promotes down-regulation of B lymphopoiesis in old age.

Major age-related changes of mouse hematopoietic stem/progenitor cells.

To study age-related changes of mouse bone marrow (BM) cells and hematopoietic stem cells (HSCs), we isolated rhodamine-123(low) (Rh(low)) Thy1.1(low) Lin(-)Sca-1(+) (TLS) HSCs from the BM of old mice and compared their functional characteristics to cells of the same phenotype isolated from young mice. We observed impaired recovery of B lymphocytes and decreased self-renewal in recipients of old Rh(low) cells compared to young Rh(low) cells. Blockade of Rh efflux using verapamil improved lymphoid reconstitution by enriched HSCs, and isolation of aged HSCs based on efflux of a fluorescent multi-drug resistance (MDR) substrate (Bodipy-verapamil) resulted in enrichment of HSC activity equivalent to that obtained with Rh. These observations suggest a complex relationship between MDR activity and HSC function during aging. To address whether the difference between young and aged donors was intrinsic to the HSC compartment or was due to a shift in HSC phenotype, we co-transplanted normal BM derived from young or old donors and followed repopulation simultaneously in the same recipient animals. In a parallel experiment, we co-transplanted HSCs purified from old donors with BM derived from young donors. In both experiments, transplants were given to both young and old recipients. The results show a clear defect in B-cell engraftment from either BM or HSCs of old donors, irrespective of the age of the recipient. In contrast, myeloid engraftment was predominantly derived from BM or HSCs derived from aged donors, again irrespective of recipient age. These data suggest a stem cell basis for B-cell immuno-senescence and the increased incidence of myelocytic leukemia in elderly people.

Age-associated defects in B lymphocyte development.

The steady-state level of both RAG-1 and RAG-2 mRNA, the number of Pre-B cells, and the number of Pre-B cells expressing RAG-2 protein decrease in the bone marrow of old mice. These differences appear to be due, at least in part, to increased apoptosis of bone marrow Pre-B cells. To determine whether the age-associated increase in apoptosis reflects the impaired expression of the Pre-B cell receptor required for the survival of Pre-B cells, we examined the recombination of D to J and V to DJ in bone marrow from young and old mice. Both D to J recombination, which occurs early in the Pro-B cell stage of development, and V to DJ, which occurs just prior to the transition to the Pre-B cell stage, are diminished with age. These findings support the view that immunoglobulin recombination may impair the expression of the Pre-B cell receptor and may contribute to the increased rate of apoptosis of Pre-B cells in the bone marrow of old mice.

Age-related changes in the content of stromal clonogenic cells in hemopoietic and lymphoid organs

The content of stromal clonogenic cells in hemopoietic and lymphoid organs of mice and guinea pigs decreases with age. This drop is most pronounced in the thymus of mice and guinea pigs and in mouse spleen (more than 12-, 75-, and 8-fold, respectively). The contents of stromal clonogenic cells in the bone marrow of old mice and in the spleen of old guinea pigs are reduced by 50 and 40%, respectively, in comparison with young animals. These data indicate that the content of committed and inducible osteogenic precursors can decrease with age.

The aging of hematopoietic stem cells.

We have purified hematopoietic stem cells (HSCs) from the bone marrow of old mice and compared their properties to HSCs in young and middle-aged mice. Single, reconstituting HSCs (by limit dilution) from old and young mice exhibited indistinguishable progenitor activities in vivo. HSCs were five times as frequent in the bone marrow of old mice; however, HSCs from old mice were only one-quarter as efficient at homing to and engrafting the bone marrow of irradiated recipients. HSCs in young and middle-aged mice rarely were in the S/G2/M phases of the cell cycle, but HSCs in old mice were frequently in cycle. We speculate that the unexpected proliferation of HSCs in old mice might be related to the increased incidence of leukemia in old mice. HSCs change with age, but it is unknown whether these changes are determined intrinsically or caused by the aging of their environment.

Age-associated changes in the B-cell repertoire: effect of age on RAG-1 gene expression in murine bone marrow

The available repertoire of B cells that develop early or late in life reflects the restricted recombination of 5′ immunoglobulin heavy chain variable region genes. In addition, autoantibody reactivity is overrepresented in the preferentially recombined 3′ Vh gene families, resulting in over representation of autoreactive antibody specificities. The preferential utilization of 3′ Vh gene families is associated with, and perhaps caused by, the reduced expression of the RAG-1 gene. One can only speculate what effect the induction of RAG-1 activity in the bone marrow of old mice would have on their antibody repertoire.

Impaired Elkind recovery in hematopoietic colony-forming cells of aged mice.

SummarySpleen colony-forming cells (CFU) in the bone marrow of old mice (25–27 months) are deficient in their ability to recover from fractionated sublethal doses of X-rays in vivo. This lack of repair is not evident from single-dose radiation survival data. Fractionation survival curves suggest that old CFU which survive the first dose of fractionated radiation may not be as radioresistant as comparable young (3 months) survivors.