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. 2017 Jun;16(3):480-487.
doi: 10.1111/acel.12571. Epub 2017 Feb 23.

Expansion of myeloid-derived suppressor cells with aging in the bone marrow of mice through a NF-κB-dependent mechanism

Rafael R Flores  1 , Cheryl L Clauson  2 , Joonseok Cho  2   3 , Byeong-Chel Lee  2   3 , Sara J McGowan  1 , Darren J Baker  4 , Laura J Niedernhofer  1 , Paul D Robbins  1
Affiliations

Expansion of myeloid-derived suppressor cells with aging in the bone marrow of mice through a NF-κB-dependent mechanism

Rafael R Flores et al. Aging Cell. 2017 Jun.

Abstract

With aging, there is progressive loss of tissue homeostasis and functional reserve, leading to an impaired response to stress and an increased risk of morbidity and mortality. A key mediator of the cellular response to damage and stress is the transcription factor NF-κB. We demonstrated previously that NF-κB transcriptional activity is upregulated in tissues from both natural aged mice and in a mouse model of a human progeroid syndrome caused by defective repair of DNA damage (ERCC1-deficient mice). We also demonstrated that genetic reduction in the level of the NF-κB subunit p65(RelA) in the Ercc1-/∆ progeroid mouse model of accelerated aging delayed the onset of age-related pathology including muscle wasting, osteoporosis, and intervertebral disk degeneration. Here, we report that the largest fraction of NF-κB -expressing cells in the bone marrow (BM) of aged (>2 year old) mice (C57BL/6-NF-κBEGFP reporter mice) are Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs). There was a significant increase in the overall percentage of MDSC present in the BM of aged animals compared with young, a trend also observed in the spleen. However, the function of these cells appears not to be compromised in aged mice. A similar increase of MDSC was observed in BM of progeroid Ercc1-/∆ and BubR1H/H mice. The increase in MDSC in Ercc1-/∆ mice was abrogated by heterozygosity in the p65/RelA subunit of NF-κB. These results suggest that NF-κB activation with aging, at least in part, drives an increase in the percentage of MDSCs, a cell type able to suppress immune cell responses.

Keywords: NF-κB; myeloid-derived suppressor cell; senescence.

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Figures

Figure 1
Figure 1
NF‐κB is highly activated in CD11b+ cells of BM with age. BM was extracted from the femur and tibia of the hind legs of mice. RBCs were depleted and the BM was washed extensively prior to flow cytometry. As shown in histogram (1), NF‐κBEGFP‐positive cells were selected. From the NF‐κBEGFP+ cells, CD11b+ cells were gated against B220+ cells (dot plot 2). The majority of the NF‐κBEGFP+ cells were CD11b+ in the BM (A) and in spleen (B). The remaining NF‐κBEGFP‐positive cells (i.e., CD11b B220 double negative cells) were CD3+ T cells in the BM (A) (dot plot 3) and particularly in the spleen (B). (C) The percentage of EGFP+CD11b+ cells in the BM and SPL of naturally aged mice is shown. The results shown represent three young mice and three old mice. The data were analyzed using nonparametric two‐way ANOVA (Mann–Whitney) ± SEM (***P < 0.001).
Figure 2
Figure 2
Expansion of NF‐κBEGFP myeloid suppressor cells in the BM of naturally aged mice. BM was extracted from the femur and tibia of the hind legs of mice. RBCs were depleted and the BM was washed extensively prior to flow cytometry. Dot plots of Gr‐1+CD11b+ cells from young (A) and old (B) mice are shown. The expression of EGFP in Gr‐1+CD11b+ cells (dash) compared with Gr‐1 (solid) and CD11b (dotted) single positive cells is shown in adjacent histograms. (C) The expression of EGFP by Gr‐1+CD11b+ cells from old and young mice is directly compared. (D) Quantitative analysis comparing the frequency of CD11b+EGFP+ and Gr‐1+CD11b+ between old and young mice. (E) The cellularity of the BM is shown. (F) The percent of MΦ (CD11b+F4/80+) and DC (CD11b+CD11c+) in the BM is shown. The results shown represent four young mice and eight aged mice. The data were analyzed using nonparametric two‐way ANOVA (Mann–Whitney) ± SEM (*P < 0.05, ****P < 0.0001).
Figure 3
Figure 3
Significant expansion of NF‐κBEGFP myeloid suppressor cells in the spleens of naturally aged mice. Splenocytes were depleted of RBC and washed extensively prior to flow cytometry. Gr‐1+CD11b+ cells from young (A) and old (B) mice are shown in dot plots. The expression of EGFP in Gr‐1+CD11b+ cells (dash) was compared to Gr‐1 (solid) and CD11b (dotted) single positive cells and shown in adjacent histograms. (C) The expression of EGFP by Gr‐1+CD11b+ cells from old and young mice is directly compared. (D) Quantitative analysis comparing the frequency of CD11b+EGFP+ and Gr‐1+CD11b+ cells in splenic (SPL) tissue from old and young mice. (E) The cellularity of the SPL is shown. (F) The percent of MΦ (CD11b+F4/80+) and DC (CD11b+CD11c+) in the SPL is shown. The results shown represent four young mice and eight naturally aged mice. The data were analyzed using nonparametric two‐way ANOVA (Mann–Whitney) ± SEM (**P < 0.01).
Figure 4
Figure 4
The activation of BM or splenic MDSCs from young and old mice is similar. To test for ROS production by MDSCs, 5 × 105 BM and splenic cells were cultured with 2.5 μm DC‐FDA ± PMA (15 μg mL−1) for 30 min. Afterward, the cells were counterstained with anti‐Gr‐1 and anti‐CD11b mAb and analyzed by flow cytometry. (A) The mean fluorescent intensity (MFI) of DC‐FDA in MDSC is shown (U/T = untreated). To determine whether BM or splenic cells could achieve full activation, and their capacity to produce nitric oxide or cytokines, 5 × 105 cells were stimulated with LPS (1 μg mL−1) for 24 h. (B) The activation status of MDSCs was assess by measuring the expression of CD86 and shown as the MFI. The supernatants were analyzed for nitric oxide (C) using a Griess reagent‐based kit and cytokine production by ELISA (D). The data shown are from three mice for (A) and (C) while for (B) and (D) the results represent five mice. The data were analyzed using nonparametric two‐way ANOVA (Mann–Whitney) ± SEM (*P < 0.05).
Figure 5
Figure 5
Expansion of myeloid suppressor cells in the BM of the Ercc1 −/∆ mouse model of a human progeroid syndrome. BM was extracted from the femur and tibia of the hind legs of mice. RBCs were depleted and the BM was washed extensively and prepared for FACS analysis. (A) The percent of NF‐κBEGFP+CD11b+ cells for both the BM and SPL from Ercc1 −/∆ NF‐κBEGFP reporter mice (n = 5) is shown compared with WT control mice (n = 5). (B) The frequency of MDSCs, T cells, and B cells is compared between WT (n = 9), Ercc1 −/∆ (n = 6), and p65+/− Ercc1 −/∆ (n = 7) mice. The data were analyzed using nonparametric two‐way ANOVA (Mann–Whitney) ± SEM (*P < 0.05, **P < 0.01).
Figure 6
Figure 6
A significant increase in the percent MDSCs detected in the BM and SPL from progeroid Bubr1H/H hypomorphic mice. BM and splenic cells were depleted of RBCs and washed extensively prior to FACS. The percentage of MDSCs and its subsets in the BM (A) and SPL (B) from Bubr1H/H mice is shown in comparison with WT mice. The mice were analyzed at 12 (n = 3) and 20 (n = 3) weeks of age. The data were analyzed using nonparametric two‐way ANOVA (Mann–Whitney) ± SEM (*P < 0.05, **P < 0.01).
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