Immune system development and age-dependent maintenance in Klotho-hypomorphic mice

Abstract

Circulating Klotho peptide hormone has anti-aging activity and affects tissue maintenance. Hypomorphic mutant Klotho [kl/kl] mice on C57BL/6xC3H, BALB/c and 129 genetic backgrounds, show decreased Klotho expression that correlate with accelerated aging including pre-mature death due to abnormally high levels of serum vitamin D. These mice also show multiple impairments in the immune system. However, it remains unresolved if the defects in the immune system stem from decreased Klotho expression or high vitamin D levels in the serum. Transfer of the kl/kl allele to pure C57BL/6 genetic background [B6-kl/kl] significantly reduced expression of Klotho at all ages. Surprisingly, B6-kl/kl mice showed normalized serum vitamin D levels, amelioration of severe aging-related phenotypes and normal lifespan. This paper reports a detailed analysis of the immune system in B6-kl/kl mice in the absence of detrimental levels of serum vitamin D. Remarkably, the data reveal that in the absence of overt systemic stress, such as abnormally high vitamin D levels, reduced expression of Klotho does not have a major effect on the generation and maintenance of the immune system.

Keywords: C57BL/6 background; aging; immune cells; klotho hypomorphic allele; vitamin D3.

Figure 1

B6-kl/kl mice maintain an overall lower body weight than C57BL/6 as they age. (A) A graphical representation of the kl gene. Black arrows indicate the primer sets used to amplify the secreted and membrane forms by quantitative PCR. (B) Secreted kl mRNA and (C) membrane kl mRNA expression in kidney normalized to gapdh at 1-3 months, 4-7 months, and 8+ months of age (n=2 per group). Bars represent standard error mean. Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: ** p ≤ 0.01, *** p ≤ 0.001, and *** p ≤ 0.0001. Weights of C57BL/6 and B6-kl/kl (D) male and (E) female mice at 1-3 months, 4-7 months, and 8+ months of age. For male mice C57BL/6 1-3 months n=34, 4-7 months n=8, and 8⁺ months n=11 and B6-kl/kl 1-3 months n=21, 4-7 months n=14, and 8+ months n=11. For female mice C57BL/6 1-3 months n=38, 4-7 months n=8, and 8+ months n=13 and B6-kl/kl 1-3months n=17, 4-7 months n=19, and 8⁺ months n=19. Statistical significance determined by multiple t tests: * p ≤ 0.05, ** p ≤ 0.001, and **** p ≤ 0.0001.

Figure 2

Immune cells in the bone marrow have a similar composition in C57BL/6 and B6-kl/kl mice. (A) Total bone marrow (BM) cells in C57BL/6 and B6-kl/kl at 4-19 weeks (C57BL/6 n=15 and B6-kl/kl n=18) of age or 20+ weeks (C57BL/6 n=4 and B6-kl/kl n=18) from pooled male and female mice. Statistical significance determined by multiple t tests: **** p ≤ 0.0001. (B) Representative flow cytometry plot and (C) frequency of MPP and (D) HSC of the LSK (Lin^-cKit⁺Sca1⁺) cells. C57BL/6 n=15, 4-19 weeks; n=4, 20+ weeks. B6-kl/kl n=18, 4-19 weeks; n=18, 20+ weeks. Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: *** p≤ 0.001 and **** p≤ 0.0001. (E) Representative flow cytometry plot of CLP (Lin^-CD127⁺AA4.1⁺Sca1^low), and (F) frequency of CLP in of Lin^-CD127⁺ cells. C57BL/6 n=15, 4-19 weeks; n=4, 20+ weeks. B6-kl/kl n=18, 4-19 weeks; n=18, 20+ weeks. Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: **** p ≤ 0.0001. (G) Representative flow cytometry plot of Myeloid cells (CD11b⁺CD11c^-) and (H) frequency of CD11b⁺CD11c^- cells of B220^-CD19^- cells. C57BL/6 n=10, 4-19 weeks; n=4, 20+ weeks. B6-kl/kl n=15, 4-19 weeks; n=15, 20+ weeks. (I) Representative flow cytometry plot for Pre B cells (B220^lowCD43^-), Pro B cells (B220⁺CD43⁺) and new B cells (B220⁺IgM⁺) and (J) frequency of B cell subsets from BM. C57BL/6 n=15, 4-19 weeks; n=4, 20+ weeks. B6-kl/kl n=18, 4-19 weeks; n=16, 20+ weeks. Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: **** p ≤ 0.0001.

Figure 3

C57BL/6 and B6-kl/kl mice have similar thymocyte and thymic epithelial cell (TEC) populations. (A) Total thymocytes in C57BL/6 and B6-kl/kl at 4-19 weeks (C57BL/6 n=30 and B6-kl/kl n=22) of age or 20+ weeks (C57BL/6 n=25 and B6-kl/kl n=27) from pooled male and female mice. (B) Representative flow cytometry plot of thymocyte populations and (C) frequency of developing thymocyte populations based on CD4 and CD8 expression. C57BL/6 n=30, 4-19 weeks; n=25, 20+ weeks. B6-kl/kl n=19, 4-19 weeks; n=15, 20+ weeks. (D) Representative flow cytometry plots of cortical TEC (cTEC; CD45.2^-EpCAM⁺Ly51⁺) and medullary TEC (mTEC; CD45.2^-EpCAM⁺UEA-1⁺). (E) Total thymic epithelial cells (TECs) in C57BL/6 and B6-kl/kl at 4-19 weeks (C57BL/6 n=30 and B6-kl/kl n=22) of age or 20+ weeks (C57BL/6 n=25 and B6-kl/kl n=27) from pooled male and female mice. (F) Frequency of cTEC and mTEC of CD45.2^-EpCAM⁺ cells. C57BL/6 n=27, 4-19 weeks; n=25, 20+ weeks. B6-kl/kl n=16, 4-19 weeks; n=15, 20+ weeks Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: * p ≤ 0.05, ** p ≤ 0.01, and **** p ≤ 0.0001. (G) Frequency of hi and lo mTEC. C57BL/6 n=27, 4-19 weeks; n=25, 20+ weeks. B6-kl/kl n=16, 4-19 weeks; n=15, 20+ weeks.

Figure 4

Innate immune cell composition in the spleen is similar in C57BL/6 and B6-kl/kl mice. (A) Total splenocytes in C57BL/6 and B6-kl/kl mice at 4-19 weeks (C57BL/6 n=25 and B6-kl/kl n=19) and 20+ weeks (C57BL/6 n=25 and B6-kl/kl n=27) of age from pooled male and female mice. Statistical significance determined by multiple t tests: * p ≤ 0.05. (B) Representative flow cytometry plots of NKT cells (CD19^-CD1d⁺TCRβ⁺), Neutrophils (Neut) (CD19^-CD1d^-TCRβ-^-NK1.1^-Ly6G⁺), NK cells (CD19-CD1d^-TCRβ^-NK1.1⁺Ly6G^-), dendritic cells (DC) (CD19^-CD1d^-TCRβ-NK1.1^-Ly6G^-CD11c⁺), and macrophages (Mac) (CD19^-CD1d^-TCRβ-NK1.1^-Ly6G^-CD11c^-F4/80⁺). Frequency of (C) NKT, (D) neutrophils, (E) NK cells, (F) dendritic cells, and (G) macrophages. C57BL/6 n=13, 4-19 weeks; n=19, 20+ weeks. B6-kl/kl n=11, 4-19 weeks; n=16, 20⁺ weeks. Bars represent the standard error mean.

Figure 5

Adaptive immune cell composition in the spleen is similar in C57BL/6 and B6-kl/kl mice. (A) Representative flow cytometry plots of splenic B cells. Frequency of (B) total B cells and (C) IgM⁺ or IgD⁺ B cells in the spleen of C57BL/6 and B6-kl/kl mice at 4-19 weeks (C57BL/6 n=18 and B6-kl/kl n=18) and 20+ weeks (C57BL/6 n=16 and B6-kl/kl n=23) of age from pooled male and female mice. Bars represent the standard error mean. Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: * p ≤ 0.05. (D) Representative flow cytometry plots of splenic naïve (Tn; CD62L⁺CD44^lo), effector-phenotype (Teff; CD44^hiCD62^lo), and memory-phenotype (Tmem; CD44^hiCD62⁺) CD4 T cells (CD19^-CD4⁺) and CD8 T cells (CD19^-CD8⁺). (E) Frequency of CD4 and CD8 T cells. C57BL/6 n=24, 4-19 weeks; n=25, 20+ weeks. B6-kl/kl n=19, 4-19 weeks; n=19, 20+ weeks. Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: * p ≤ 0.05 and ** p ≤ 0.001. Bars represent the standard error mean. Frequency of naïve, effector-phenotype, and memory-phenotype of (F) CD4 T cells and (G) CD8 T cells. C57BL/6 n=24, 4-19 weeks; n=25, 20+ weeks. B6-kl/kl n=19, 4-19 weeks; n=19, 20+ weeks. Statistical significance determined by 2way ANOVA and Tukey’s multiple comparison test: *** p ≤ 0.001 and **** p ≤ 0.0001. Bars represent the standard error mean.