Peripheral B cells repress B-cell regeneration in aging through a TNF-α/IGFBP-1/IGF-1 immune-endocrine axis

Abstract

Loss of B lymphocyte regeneration in the bone marrow (BM) is an immunologic hallmark of advanced age, which impairs the replenishment of peripheral B-cell subsets and results in impaired humoral responses, thereby contributing to immune system dysfunction associated with aging. A better understanding of the mechanism behind this loss may suggest ways to restore immune competence and promote healthy aging. In this study, we uncover an immune-endocrine regulatory circuit that mediates cross-talk between peripheral B cells and progenitors in the BM, to balance B-cell lymphopoiesis in both human and mouse aging. We found that tumor necrosis factor α (TNF-α), which is increasingly produced by peripheral B cells during aging, stimulates the production of insulin-like growth factor-binding protein 1 (IGFBP-1), which binds and sequesters insulin-like growth factor 1 (IGF-1) in the circulation, thereby restraining its activity in promoting B-cell lymphopoiesis in the BM. Upon B-cell depletion in aging humans and mice, circulatory TNF-α decreases, resulting in increased IGF-1 and reactivation of B-cell lymphopoiesis. Perturbation of this circuit by administration of IGF-1 to old mice or anti-TNF-α antibodies to human patients restored B-cell lymphopoiesis in the BM. Thus, we suggest that in both human and mouse aging, peripheral B cells use the TNF-α/IGFBP-1/IGF-1 axis to repress B-cell lymphopoiesis. This trial was registered at www.clinicaltrials.govas#NCT00863187.

© 2021 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Peripheral B cells in old mice have extended survival. (A-B) Splenic B cells from young, old, or old hCD20Tg mice treated for B-cell depletion that have reconstituted their peripheral B-cell compartment from de novo B-cell lymphopoiesis in the BM (old depleted) were cultured for the indicated time intervals. Cells were collected and fixed, and spontaneous death was determined by propidium iodide (PI) staining. Shown are representative results after 24 hours (A) and kinetic measurements of accumulated spontaneous apoptosis (B). Significance of differences between groups of young and old mice or between old-depleted mice and old mice are marked with stars (n = 6 mice from each group in a total of 4 independent experiments). (C-D) Young and old Mx-cre/RAG-2fl/fl or control mice were injected intraperitoneally with polyinosinic:polycytidylic acid [poly(I:C)] to ablate floxed alleles, and 17 weeks later, spleens were analyzed to quantify B cells. Shown are representative plots for individual mice with the indicated genotypes (C) and absolute cell numbers (D). Graph depicts means from 5 mice in each group ± standard error (SE). **P < .01; ***P < .001.

Figure 2.

Peripheral B cells from old mice suppress B-cell lymphopoiesis. Splenic B cells from young and old mice were adoptively transferred to young hCD20Tg mice that were treated (and confirmed by blood stain) for B-cell depletion. Bone marrow and spleen from recipient mice were quantified for B-cell lymphopoiesis 28 days after depletion. (A) Schematic diagram showing details of the kinetics of the experiment. (B-C) Analysis of BM cells for the indicated mice. For analysis, viable lymphocytes were defined by forward scatter and light side scatter, and gates were set to analyze pro-B (pro) and pre-B (pre) cells (B220+CD93+IgM–) and immature B cells (B220+CD93+IgM+). Shown are representative plots for a single mouse from each group; arrows indicate populations (B). Absolute cell numbers (C). Graph depicts mean from 4 mice in each group ± SE. (D-E) Analysis of spleen cells for the indicated mice. Gates were set to quantify newly generated host B cells as B220+hCD20+. Shown are representative plots for individual mice from each group (D) and absolute cell numbers (E). Graph depicts means from 4 mice in each group ± SE. (F-I) IL-7–driven BM cultures to grow B cells in vitro were prepared from young (F-G) or old (H-I) mice. In these experiments, the fetal calf serum (FCS) in culture media was replaced with 1% fresh mouse serum from the indicated mice. After 5 days, cells were harvested, counted, and stained for surface markers and analyzed for pro-B and pre-B (B220+/IgM–) and immature (B220+/IgM+) B cells. Shown are representative results from a single experiment (F,H) and absolute B-cell counts (G,I) in the cultures. Graphs depict means from 5 experiments ± SE. dep, depleted.

Figure 3.

B-cell lymphopoiesis in aging is regulated by IGF-1. (A) Sera collected from the indicated mice were analyzed for IGF-1 by enzyme-linked immunosorbent assay (ELISA). Shown are results for individual mice and group means (n = 5 for each group). (B) IL-7–driven BM cultures were prepared, replacing the FCS in media with 1% serum from young mice in the presence or absence of goat–anti-mouse IGF-1 (50 ng/mL). After 5 days, cells were harvested, counted, and stained to quantify B-cell numbers. Graph depicts mean from 4 experiments ± SE. (C-F) Old mice were subcutaneously injected with human GH (hGH) (C-D) or with human IGF-1 (hIGF-1) (E-F) for 10 days. Control mice were injected with phosphate-buffered saline (PBS). One day after the last injection, we analyzed the BM of the mice for B-cell lymphopoiesis (described in the Figure 2 legend) with gates marked for pro-B , pre-B, and immature B cells. Shown are representative plots for a single mouse from each group (C,E) and absolute cell numbers (D,F) for pro-B , pre-B, and immature B-cell populations. Graphs depict means from 5 mice in each group ± SE (in 2 different experiments). Reference values for pro-B , pre-B, and immature B cell numbers in young mice are shown in Figure 2. (G-H) IL-7–driven BM cultures containing 1% fresh serum from young or old mice in the absence or presence of hIGF-1 were prepared. After 5 days, cells were harvested, counted, and stained to quantify B-cell numbers. Shown are representative results from a single experiment (G) and absolute B-cell counts (H) in the cultures. Graph depicts mean from 4 experiments ± SE.

Figure 4.

Regulation of IGF-1 by peripheral B cells in aging is mediated by TNF-α through IGFBP-1. (A) Sera collected from the indicated mice were analyzed for TNF-α by ELISA. Shown are results for individual mice and group means (n = 6-7 for each group). (B) Sera were collected from old hCD20Tg mice before and 14 days after B-cell depletion and analyzed for levels of TNF-α by ELISA. Shown are longitudinal results for individual mice (n = 3). (C) Purified splenic B cells from the indicated mice were analyzed for relative expression of TNF-α messenger RNA (mRNA) by quantitative polymerase chain reaction (qPCR) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Graph depicts results for individual mice (n = 9-11) and group mean ± SE. (D) Purified splenic B cells from the indicated mice were cultured in vitro for 24 hours. Supernatants were collected and analyzed for TNF-a by ELISA. Shown are results for individual mice and group means (n = 8-13 for each group). (E) Sera collected from the indicated mice were analyzed for IGFBP-1 by ELISA. Shown are results for individual mice and group means (n = 8-9 for each group). (F) Measurements of IGF-1, TNF-α, and IGFBP-1 for individual mice from the indicated groups were plotted in a 3-dimensional chart. Each group is clustered with a color-matched covariance ellipsoid centered around the mean of each group.

Figure 5.

The TNF-α/IGFBP-1/IGF-1 axis in regulating B-cell lymphopoiesis in older humans. (A-B) Plasma samples were collected from healthy young or older humans or older patients with lymphoma treated for B-cell depletion (old depleted) (see the Methodology section of NCT00863187). Plasma samples were analyzed for IGF-1 (A) and IGFBP-1 (B) by ELISA. Shown are results for individual patients and group means (indicated by thick solid horizontal line) (n = 9-18 for each group). (C) Peripheral blood B cells were collected from patients from the indicated human cohorts and cultured in vitro for 24 hours. Supernatants were collected and analyzed for TNF-a by ELISA. Shown are results for individual patients and group means (n = 5-6 for each group).

Figure 6.

Transitional B cells in patients with IJD treated or not treated with anti–TNF-α. (A-B) Cross-sectional analysis of transitional B cells in healthy controls (n = 4) and patients with IJD treated with TNF-α (IJD-TNF-α, n = 19) or naïve to anti-TNF-α (IJD-NB, n = 14). Blood cells were analyzed by flow cytometry and mature (CD19+/CD24lo/med/CD38lo/med) and transitional (CD19+/CD24hi/CD38hi) B-cell frequency was determined. (A) Representative analysis for a single patient from each group gated on CD19+ cells. Shown are gates for mature and transitional B cells indicated by arrows. (B) Frequency of transitional B cells. Quantity of transitional B cells was calculated as the fraction of transitional B cells divided by the sum of transitional and mature B cells. Shown are results for individual patients and group means. (C-D) Longitudinal analysis of transitional B-cell ratio of patients with IJD undergoing treatment with anti–TNF-α. (C) Transitional B-cell ratio averages in patient subset (n = 6) analyzed upon initiation of anti–TNF-α therapy and again after 3 months of anti–TNF-α therapy. Results are expressed as mean ± SE. (D) Longitudinal analysis for individual patients with IJD transitional B-cell ratios before and after anti–TNF-α therapy. (E-F) Longitudinal analysis of plasma IGF-1 in patients with IJD undergoing anti–TNF-α therapy. (E) Plasma IGF-1 averages in the patient subset (n = 6) analyzed upon initiation of anti–TNF-α therapy and again after 3 months of anti–TNF-α therapy. Results are expressed as mean ± SE. (F) Longitudinal analysis for individual patients with IJD transitional B-cell ratios before and after anti–TNF-α therapy.

Figure 7.

A proposed model for the function of the TNF-α/IGFBP-1/IGF-1 axis in regulating B-cell lymphopoiesis in aging. B-cell lymphocyte production in the BM is regulated in aging by the level of IGF-1 in the circulation. The long-lived B cells, which accumulate in the periphery with aging, produce large amounts of the pro-inflammatory cytokine TNF-α, which stimulates the liver to increase production of IGFBP-1. The increased IGFBP-1 binds IGF-1 and sequesters its activity in the BM, resulting in a suppressed B-cell lymphopoiesis. Upon B-cell depletion, the peripheral compartment is replenished with newly generated naïve B cells that secrete small amounts of TNF-α, resulting in a decline in the plasma level of TNF-α. The reduced TNF-α is followed by a decrease in IGFBP-1 and a consequential increase in IGF-1 and reactivation of B-cell lymphopoiesis in the BM.