The effect of metformin on senescence of T lymphocytes

Abstract

Background: Immunosenescence occurs as people age, leading to an increased incidence of age-related diseases. The number of senescent T cells also rises with age. T cell senescence and immune response dysfunction can result in a decline in immune function, especially in anti-tumor immune responses. Metformin has been shown to have various beneficial effects on health, such as lowering blood sugar levels, reducing the risk of cancer development, and slowing down the aging process. However, the immunomodulatory effects of metformin on senescent T cells still need to be investigated.

Methods: PBMCs isolation from different age population (n = 88); Flow Cytometry is applied to determine the phenotypic characterization of senescent T lymphocytes; intracellular staining is applied to determine the function of senescent T cells; Enzyme-Linked Immunosorbent Assay (ELISA) is employed to test the telomerase concentration. The RNA-seq analysis of gene expression associated with T cell senescence.

Results: The middle-aged group had the highest proportion of senescent T cells. We found that metformin could decrease the number of CD8 + senescent T cells. Metformin affects the secretion of SASP, inhibiting the secretion of IFN-γ in CD8 + senescent T cells. Furthermore, metformin treatment restrained the production of the proinflammatory cytokine IL-6 in lymphocytes. Metformin had minimal effects on Granzyme B secretion in senescent T cells, but it promoted the production of TNF-α in senescent T cells. Additionally, metformin increased the concentration of telomerase and the frequency of undifferentiated T cells. The results of RNA-seq showed that metformin promoted the expression of genes related to stemness and telomerase activity, while inhibiting the expression of DNA damage-associated genes.

Conclusion: Our findings reveal that metformin could inhibit T cell senescence in terms of cell number, effector function, telomerase content and gene expression in middle-aged individuals, which may serve as a promising approach for preventing age-related diseases in this population.

Keywords: Inhibition; Metformin; SASP; Senescent T cell.

Fig. 1

The highest frequency of CD8 + senescent T cells in middle-age population was analyzed by FACS. a Representative flow cytometric analyses of senescent T cells (CD3 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) in CD8 and CD4 T cells (top quadrant) and representative flow cytometric analyses of CD27-CD28-CD57 + KLRG1 + subsets, gated on CD3 + CD4 + /CD8 + CD45RO + CCR7-(Tem) population (bottom quadrant). The frequency of CD8 + senescent T cells (b) and CD4 + senescent T cells (c) at young group, middle-age group and elderly group. The percentage of CD57 + KLRG1 + expression on CD3 + CD8 + CD45RO + CCR7-CD27-CD28-population (d) and CD3 + CD4 + CD45RO + CCR7-CD27-CD28-population (e) at different age groups. Expressed as the mean ± SEM. *P < 0.05, **P < 0.01; Mann– Whitney test (two-tailed) and nonpaired Student’s t-test. Tem, effector memory T cell

Fig. 2

The number of CD8 + senescent T cells in middle-age group decreased significantly after metformin treatment. (a) Frequency of CD3 + CD8 + CD45RA + /CD45RO + CCR7-CD27-CD28- cells which gated on CD3 + CD8 + T cells treated with the different concentrations of metformin (0 mM, 5 mM, 10 mM or 20 mM). Quantification of the percentage of CD8 + senescent T cells derived from Teff cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) (b) and Tem cells (CD8 + CDR45RO + CCR7-CD27-CD28-CD57 + KLRG1 +) (c) at middle-age group with 20 mM metformin treatment. Expressed as the mean ± SEM. ***P < 0.001; Mann–Whitney test (two-tailed) and paired Student’s t-test. Teff, effector T cells; Tem, effector memory T cells; Met(-), control; Met( +), 20 mM metformin treatment

Fig. 3

Metformin reduces the secretion of IFN-γ in CD8 + senescent T cells. Representative flow histogram of IFN-γ production by CD8 + senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) (a) and non- senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1-) (e) in the control group and the 20 mM Met treatment group relative to the unstimulated controls from middle-age subjects. Analysis of IFN-γ-secreting CD8 + senescent T cells (b) and MFI of IFN-γ in CD8 + senescent T cells (c) with 20 mM metformin treatment. d Frequency of CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + IFN-γ + T cells in lymphocytes with Met treatment and control. f Percentage and MFI of IFN-γ in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1- T cells with Met treatment and control. g Representative flow histogram of IFN-γ secretion by CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + /CD57- population relative to the unstimulated controls from middle-age donors. h Percentage and MFI of IFN-γ in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + / CD57- population. Expressed as the mean ± SEM. **P < 0.01, ***P < 0.001; Paired t test. Met, metformin

Fig. 4

Metformin inhibited the production of proinflammatory cytokine IL-6 in CD3 negative cells. Representative flow analysis of IL-6 production and MFI level in CD3- cells (a), CD3-CD57 + cells (c) and CD3-CD57- cells (d) between 20 mM Met treatment and control for 24 h in middle age group. Representative flow analysis of IL-6 production and MFI in CD3-CD57 + and CD3-CD57- population (b). Expressed as the mean ± SEM. ***P < 0.001; Paired t test. Met, metformin

Fig. 5

The effect of Metformin on GB secretion. Representative flow histogram of GB secretion by CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + (a) and CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1- (d) in the control group and the 20 mM Met treatment group relative to the unstimulated controls group from middle-age donors. Analysis of GB production in CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + (b) / CD57-KLRG1- (e) T cells, cultured with 0 mM or 20 mM metformin for 24 h in the middle age group. The proportion of CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 + GB + (c) and CD57-KLRG1-GB + (f) T cells in lymphocytes with metformin treatment compare to control group. g Representative flow histogram of GB secretion by CD3 + CD57 + and CD3 + CD57-T cells relative to the unstimulated controls group in middle-age donors. h Percentage and MFI of GB in CD3 + CD57 + and CD3 + CD57-T cells. Expressed as the mean ± SEM. *P < 0.05; Paired t test. Met, metformin

Fig. 6

Metformin promotes the production of TNF-α in senescence T cells. Analysis of TNF-α-secreting cells from CD8 + senescent T cells (CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57 + KLRG1 +) (a) and CD3 + CD8 + CD45RA + CCR7-CD27-CD28-CD57-KLRG1-T cells (c) in the control group and the 20 mM Met treatment group in middle-age group. b The frequency of TNF-α + CD8 + senescent T cells in lymphocytes between the control and Met treatment group. d Representative flow histogram of TNF-α in CD8 + CD45RA + CCR7-CD27-CD28-CD57 + /CD57-T cell and the percentage and MFI of TNF-α in CD8 + CD45RA + CCR7-CD27-CD28-CD57 + /CD57-T cell. Expressed as the mean ± SEM. **P < 0.01; Paired t test. Met, metformin

Fig. 7

Metformin increased the telomerase concentration and the undifferentiated T cells. a The concentration of telomerase in medium (ng/ml) was analyzed in PBMCs with 20 mM Metformin treatment and control at middle age group, examined by ELISA. The flow cytometric analysis the frequency of CD3 + CD4 + CD45RA + CCR7 + CD27 + CD28 + (b) and CD3 + CD8 + CD45RA + CCR7 + CD27 + CD28 + (c) T cells in lymphocytes between the metformin treatment and control. Expressed as the mean ± SEM. *P < 0.05, **P < 0.01; Mann–Whitney test (two-tailed) and paired Student’s t-test. ELISA, Enzyme-linked immunosorbent assay. Met (-), control; Met ( +),20 mM metformin treatment

Fig. 8

The RNA -seq analysis of gene expression associated with T cell senescence with metformin treatment. a Venn diagram portraying the intersections and disjunctive unions of differentially expressed genes in metformin treatment group (green) and control group (gray). b The Volcano Plot showed the overall distribution of genes with up-regulated expression (green), down-regulated expression (dark gray) and no significant difference (light gray) in the metformin treatment group compared to the control group. c Expression heatmap of a selection of senescence- and stemness-associated genes in PBMCs from metformin treatment group and control group at middle-age subjects (n = 3). Data are represented as Z-scores. d Representative genome browser visualizations of normalized reads at indicated gene loci in PBMCs from a representative donor before and after metformin treatment. e The chart portrayed a selection of biological process, from GO functional enrichment analysis, which were up-regulated and down-regulated with metformin treatment. All the above RNA sequencing datas came from three independent donors and three independent experiments