Michele Goodhardt

Age-related changes in human hematopoietic stem/progenitor cells

Adult stem cells are critical for maintaining cellular homeostasis throughout life, yet the effects of age on their regenerative capacity are poorly understood. All lymphoid and myeloid blood cell lineages are continuously generated from hematopoietic stem cells present in human bone marrow. With age, significant changes in the function and composition of mature blood cells are observed. In this study, we report that age‐related changes also occur in the human hematopoietic stem cell compartment. We find that the proportion of multipotent CD34+CD38− cells increases in the bone marrow of elderly (>70 years) individuals. CD34+CD38+CD90−CD45RA+/−CD10− and CD34+CD33+ myeloid progenitors persist at the same level in the bone marrow, while the frequency of early CD34+CD38+CD90−CD45RA+CD10+ and committed CD34+CD19+ B‐lymphoid progenitors decreases with age. In contrast to mice models of aging, transplantation experiments with immunodeficient NOD/SCID/IL‐2Rγ null (NSG) mice showed that the frequency of NSG repopulating cells does not change significantly with age, and there is a decrease in myeloid lineage reconstitution. An age‐related decrease in the capacity of CD34+ cells to generate myeloid cells was also seen in colony‐forming assays in vitro. Thus, with increasing age, human hematopoietic stem/progenitor cells undergo quantitative changes as well as functional modifications.

Do age-associated changes in ‘physiologic’ autoantibodies contribute to infection, atherosclerosis, and Alzheimer's disease?

More than 25 years ago, Pierre Grabar proposed that the age-associated increase in serum autoantibodies reflected a homeostatic function of the immune system that defended the internal milieu by targeting senescent molecules and cells for elimination. This mini-review examines recent evidence that autoantibodies may influence the risk of the elderly developing infectious, atherosclerotic, or Alzheimers disease. Auto-anti-idiotypic antibodies suppress the antibody response to the nominal antigen and, thus, may contribute to the increased risk of infection and poor response to vaccines in the elderly. In contrast, low levels of autoantibodies to oxidised low-density lipoproteins or to the amyloid beta peptide may contribute to the increased risk of developing atherosclerosis of Alzheimers disease, respectively.

Reduced EBF expression underlies loss of B-cell potential of hematopoietic progenitors with age.

Aging is accompanied by a reduction in the generation of B lymphocytes leading to impaired immune responses. In this study, we have investigated whether the decline in B lymphopoiesis is due to age‐related defects in the hematopoietic stem cell compartment. The ability of hematopoietic stem cells from old mice to generate B cells, as measured in vitro, is decreased 2–5‐fold, while myeloid potential remains unchanged. This age‐related decrease in B‐cell potential is more marked in common lymphoid progenitors (CLP) and was associated with reduced expression of the B‐lineage specifying factors, EBF and Pax5. Notably, retrovirus‐mediated expression of EBF complemented the age‐related loss of B‐cell potential in CLP isolated from old mice. Furthermore, transduction of CLP from old mice with a constitutively active form of STAT5 restored both EBF and Pax5 expression and increased B‐cell potential. These results are consistent with a mechanism, whereby reduced expression of EBF with age decreases the frequency with which multipotent hematopoietic progenitors commit to a B‐cell fate, without altering their potential to generate myeloid cells.

Hind limb unloading, a model of spaceflight conditions, leads to decreased B lymphopoiesis similar to aging

Within the bone marrow, the endosteal niche plays a crucial role in B‐cell differentiation. Because spaceflight is associated with osteoporosis, we investigated whether changes in bone microstructure induced by a ground‐based model of spaceflight, hind limb unloading (HU), could affect B lymphopoiesis. To this end, we analyzed both bone parameters and the frequency of early hematopoietic precursors and cells of the B lineage after 3, 6, 13, and 21 d of HU. We found that limb disuse leads to a decrease in both bone microstructure and the frequency of B‐cell progenitors in the bone marrow. Although multi‐potent hematopoietic progenitors were not affected by HU, a decrease in B lymphopoiesis was observed as of the common lymphoid progenitor (CLP) stage with a major block at the progenitor B (pro‐B) to precursor B (pre‐B) cell transition (5‐ to 10‐fold decrease). The modifications in B lymphopoiesis were similar to those observed in aged mice and, as with aging, decreased B‐cell generation in HU mice was associated with reduced expression of B‐cell transcription factors, early B‐cell factor (EBF) and Pax5, and an alteration in STAT5‐mediated IL‐7 signaling. These findings demonstrate that mechanical unloading of hind limbs results in a decrease in early B‐cell differentiation resembling age‐related modifications in B lymphopoiesis.—Lescale, C., Schenten, V., Djeghloul, D., Bennabi, M., Gaignier, F., Vandamme, K., Strazielle, C., Kuzniak, I., Petite, H., Dosquet, C., Frippiat, J.‐P., Goodhardt, M. Hind limb unloading, a model of spaceflight conditions, leads to decreased B lymphopoiesis similar to aging. FASEB J. 29, 455‐463 (2015). www.fasebj.org

Re-expression of DNA methylation-silenced CD44 gene in a resistant NB4 cell line: rescue of CD44-dependent cell death by cAMP

In the acute promyelocytic leukemia cell line, NB4, activation of the CD44 receptor triggers apoptosis. This pathway does not operate in the retinoid-maturation-resistant NB4-LR1 subclone. In this work, we show that the CD44 gene is silenced in these cells. The molecular defect involves DNA methylation of cytosine phosphate guanine (CpG) island and underacetylation of histone H3 at CD44 promoter. The methylating inhibitor 5-aza-CdR and cyclic AMP (cAMP) reverse the CD44 gene silencing. Contrary to 5-aza-CdR, cAMP does not induce DNA demethylation or histone modification at the CD44 promoter, whereas an H3pS10/AcK14 dual modification is observed on a global level. cAMP also induces the expression of c-Jun transcription factor and its recruitment at the CD44 promoter. Chromatin immunoprecipitation assays further show the association of brahma (Brm), a subunit of SWI/SNF chromatin-remodelling complex involved in the crosstalk between transcription and RNA polymerase II (RNA Pol II) processing, as well as the binding of phosphorylated RNA Pol II to the proximal promoter region of CD44. Finally, our study reveals that cAMP re-establishes the CD44-mediated cell death signalling. We propose that one of the actions of cAMP in restoring normal cell phenotype of leukaemia cells may consist in a broad trans-reactivation of silenced genes, despite marked hypermethylation of their promoters, as illustrated here with CD44 re-expression.

Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line.

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD− murine B cell line, 38C-13, which has previously been found to undergo κ light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vκ-Jκ and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD− B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.

Absence of Bone Sialoprotein (BSP) Alters Profoundly Hematopoiesis and Upregulates Osteopontin

Matrix proteins of the SIBLING family interact with bone cells, extracellular matrix and mineral and are thus in a key position to regulate the microenvironment of the bone tissue, including its hematopoietic component. In this respect, osteopontin (OPN) has been implicated in the hematopoietic stem cell (HSC) niche as negative regulator of the HSC function. We investigated the impact on hematopoietic regulation of the absence of the cognate bone sialoprotein (BSP). BSP knockout (−/−) mice display increased bone marrow cellularity, and an altered commitment of hematopoietic precursors to myeloid lineages, leading in particular to an increased frequency of monocyte/macrophage cells. The B cell pool is increased in −/− bone marrow, and its composition is shifted toward more mature lymphocyte stages. BSP‐null mice display a decreased HSC fraction among LSK cells and a higher percentage of more committed progenitors as compared to +/+. The fraction of proliferating LSK progenitors is higher in −/− mice, and after PTH treatment the mutant HSC pool is lower than in +/+. Strikingly, circulating levels of OPN as well as its expression in the bone tissue are much higher in the −/−. Thus, a BSP‐null bone microenvironment affects the hematopoietic system both quantitatively and qualitatively, in a manner in part opposite to the OPN knockout, suggesting that the effects might in part reflect the higher OPN expression in the absence of BSP. J. Cell. Physiol. 230: 1342–1351, 2015.