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Age-related accumulation of B-1 cell progenitors in mice reflects changes in miR15a/16-1 expression and radioresistance capacity
Experimental Hematology & Oncology volume 12, Article number: 24 (2023)
Hyperproliferative diseases such as Chronic Lymphocytic Leukemia (CLL) and Systemic Lupus Erythematosus (SLE) are potentially related to some disturbance in the apoptosis pathway, specifically in B-1a cells (CD5+). Accumulation of B-1a cells in lymphoid organs, bone marrow or periphery is observed in some leukemia experimental murine models along aging. It is known that aging also increases the healthy B-1 cell population. However, it is not yet clear if it happens due to self-renewal of mature cells or proliferation of progenitor cells. Herein we demonstrated that the B-1 cell precursor population (B-1p) from bone marrow of middle-aged mice is higher than from young mice. Also, these aged cells are more resistant to irradiation and have downregulation of microRNA15a/16. Alterations in these microRNAs expression and in Bcl-2 regulation were already described in human hematological malignancies and new therapeutically approaches focus on that axis. This finding could explain the early events related to cell transformation during aging and correlate with beginning of symptoms in hyperproliferative diseases. Moreover, studies have already reported these pro-B-1 as a contributor to the origin of other leukemia (Acute Myeloid Leukemia - AML). Our results point to a possible relation between B-1 cell precursors and hyperproliferation during aging. We hypothesized that this population could be maintained until the mature status of the cell or reveal changes that result in re-activation of precursor in adult bone marrow, culminating in accumulation of B-1 cells later. Based on this, B-1 cell progenitor could represent an origin for B cell malignancies and a new candidate target to diagnose and treatments in the future.
To the editor,
B cells are subdivided in B-1 and B-2 cells, that play different roles in the immune system. Mice B-1 progenitor population (pro-B-1-stage) is described and is distinct from B-2 progenitor . The human B-1p population is yet to be fully characterized but studies showed the development from the same hematopoietic stem cells (HSCs) that give rise to B-2 cells .
There is a proposal that some mature human B-cell malignancies and autoimmune diseases could arise from B-1 cells [3, 4]. Accumulation of B-1 cells was seen in New Zealand Black/White mice (NZB/NZW) , in patients with SLE  and it was described an aged-dependent increase of B-1 cells in NZB/NZW, BALB/c and CBA mice . Other characteristics could be seen in CLL patients and B-1 from NZB/NZW strain, such as presence of the asymptomatic precursor, downregulation of microRNA15a/16-1 and radioresistant capacity to gamma ionization (8Gy) [8, 9]. Despite the association between B-1 cells, CLL and aging, the origin of B-1 cell accumulation is not elucidated. We investigated the role of B-1p cells as a source of B-1 cell malignancies.
We isolated B-1p cells from young (8 weeks old) and middle-aged mice (30 weeks old). First, there is an increase of B-1p cells in middle-aged mice in relation to the young group (p=0.0391), as shown in Fig. 1B. Fig. 1A and C also show increased B-1p in middle-aged as represented by percentage of parent. The absolute number of events also reveals that difference (p=0.0453). Other study showed the accumulation of B-1p cells in older mice (~2 years) , however, our aim is to identify changes in B-1p population before that time point.
In samples from older animals, miR15a/16-1 is downregulated (p=0.033) whereas Bcl-2 has tendency to increase (Fig. 1E). The analysis of Bcl-2 phosphorylation in the progenitor revealed that the protein is less phosphorylated in older precursors (Fig. 1F). Since phosphorylation indicate activity, we did not show that Bcl-2 is the responsible for cell accumulation with aging. Bcl-2 gene expression also did not present statistical significance between groups. Considering this, other targets for miR15a/16-1 remains to be investigated. The finding that B-1p cells from middle-aged mice accumulates and already present miR15a/16 downregulation is in accordance with the hypothesis that this could be the time of appearing changes that could culminate in B-1 accumulation in future, in aging and disease. Corroborating this, a study demonstrated that miR-15a deficient B-1p cells repopulated irradiated recipients and produced elevated numbers of B-1 cells . Yet, other investigations about this microRNA, BCL-2 and activity of other apoptosis regulators are necessary to elucidate progenitor accumulation.
When we observed the viability of B-1p in vitro, we found more viable cells from middle-aged mice in cell culture after 72h in comparison with young cells (Fig. 2A). Further, these older B-1p cells also presented minor levels of median fluorescence intensity, indicating higher proliferation (Additional file 1: Fig. S1 A-C) and more proliferating cells after 72h in culture (Fig. 2B). So, we conclude that the middle-aged B-1p is more able to survive and proliferate in vitro than B-1 p cells from young mice. Accumulated changes in aging impact survival and proliferation index of cells.
Previous data from our group reported that a small percentage of B-1 cells are able to survive in vitro after 3.5Gy dose of irradiation and acquired characteristics of neoplastic cells (B-CLL like cells) . We found a decrease in live cell numbers in cell culture with young irradiated B-1p cells, in comparison with non-irradiated control group. However, when B-1p cells from middle-aged mice were irradiated, these cells survived and an increase in the number of cells is observed in comparison to control group (Fig. 2C). The irradiation also influences proliferation, as demonstrated in Fig. 2D. Young cells do not proliferate after irradiation but old cells augment proliferation after this process (data are presented at Fig. 2D and in Additional file 1: Fig. S3). Based on the fold change analysis between irradiated and non-irradiated group, it is possible to observe that middle-aged irradiated cells have higher expression of anti-apoptotic gene Bcl-2 (Fig. 2E). The Bax gene did not change between these groups. Moreover, older irradiated cells demonstrated a downregulation of miR15a/16-1 (consistent with levels of Bcl-2). However, a threshold may need to be attained to reveal the role of Bcl-2. These data could explain that B-1 precursors obtained from middle-aged animals survive to irradiation in vitro and also have higher proliferation index.
These findings demonstrated that B-1p have molecular changes induced by aging that are augmented with radiation and could lead progenitors to acquire malignant potential.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Chronic Lymphocytic Leukemia
Systemic Lupus Erythematosus
B-1 cell progenitor
B cell lymphoma 2
Hematopoietic Stem Cells
New Zealand Black/White mice
Systemic Lupus Erythematosus
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We thank Daniela Teixeira for technical assistance in the cell sorting and flow cytometry analysis and Geová Pereira Santos for animal care.
This work was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) 2017/11725-7, 2017/24451-2, 2019/27009-4 and 2021/05377-1.
Ethics approval and consent to participate
The study was conducted under the approval of the Ethical Committee from UNIFESP (2017/3576280617, 2018/7292050618 and 2019/1374110219).
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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Additional file 1: Figure S1.
B-1p cells from middle-aged mice proliferate in vitro more than young B-1p cells. A) Celltrace fluorescence histogram of young (blue) or middle-aged (green) B-1p after 24h in cell culture. Purple bar indicates cells stained with celltrace in t0. B) Celltrace fluorescence histogram of young (blue) or middle-aged (green) B-1p after 72h in cell culture. Purple bar indicates cells stained with celltrace in t0. C) Median fluorescence intensity of celltrace reagent in proliferating progenitors. Two-way ANOVA F(3,8)=6.32 p=0.017. Differences are observed between aged 24h (6766 ± 548) and all other groups: young 24h (2302 ± 490; p<0.001), young 72h (3757 ± 147; p<0.001) and aged 72h (2734 ± 195; p<0.001) and young 24h with young 72h (p=0.023). This figure represents two different experiments performed in triplicate. Figure S2. B-1 progenitor cells from young and middle-aged mice survives in vitro after irradiation. A) Gates strategies revealing live B-1p cells (Annexin-7AAD-), from young (above) and middle-aged (below) mice. B) Positive control of cell death. B-1p cells from this same experiment were collected after cell sorting and placed on dry-bath at 95º for 5 minutes. Cells were stained with Annexin V and 7AAD as previously described. C) Percentage of live cells (Annexin-7AAD-) found after irradiation and cell culture. Figure S3. B-1 progenitor cells from young and middle-aged mice survives in vitro after irradiation but differs in proliferation rates. A) Gates strategies revealing B-1p cells, from young (above) and middle-aged (below) mice, that incorporated BrdU marker. B) Percentage of cells that incorporated BrdU marker after irradiation and cell culture period. Two-way ANOVA F(3,5)=6.03; p=0.041. Difference between young ctrl non irr (1.63 ± 0.375) and young irr (4.1 ± 0.955; p=0.036) is shown.
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Souza, O.F., de Oliveira, V.C., Rodrigues, G.J.F. et al. Age-related accumulation of B-1 cell progenitors in mice reflects changes in miR15a/16-1 expression and radioresistance capacity. Exp Hematol Oncol 12, 24 (2023). https://doi.org/10.1186/s40164-023-00390-6
- B-1 cell progenitor
- Chronic Lymphocytic Leukemia