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

The decrease in Klotho expression results in lower body weight in the B6-kl/kl mice

Klotho-hypormorphic C57BL/6xC3H-kl/kl mice [19] were backcrossed to C57BL/6J mice for 10 generations (B6-kl/kl). The purity of the genetic B6-kl/kl background was confirmed by typing ten mice for 96 C57BL/6 microsatellite markers using DNA extracted from the tails. All mice were homozygous for at least 94 out of the 96 markers analyzed (background ‘purity’ >98.96%) (Table 1). These data demonstrate that B6-kl/kl mice were syngenic with C57BL/6 mice.

In the kidney of mutant C57BL/6xC3H-kl/kl mice the level of kl mRNA expression was shown to be significantly decreased [19]. To confirm that our mice retained this feature on the C57BL/6 background, expression of secreted and membrane kl mRNAs in kidney were analyzed by qPCR (Figure 1A). The data show that B6-kl/kl mice express significantly lower levels of secreted and membrane forms of kl mRNA (Figure 1B and C). The immune system is likely to be most affected by the expression of the soluble form, which was also expressed at significantly lower levels in B6-kl/kl mice (Figure 1B). These data show that the hypomorphic allele [kl/kl] was appropriately transferred to the C57BL/6 background in B6-kl/kl mice.

The C57BL/6xC3H-kl/kl mice lived a short lifespan and had reproductive organ defects [19]. In this study, B6-kl/kl mice lived a lifespan that was indistinguishable from littermates that were not mutant, and showed no abnormality in reproductive organs or impaired reproductive capability (data not shown). The C57BL/6xC3H-kl/kl mice were significantly smaller in size [19]. Likewise, both male and female B6-kl/kl mice in this study showed significantly lower body weight (Figure 1D and E) as was previously reported [24]. These results show that the effects of decreased Klotho expression on lifespan and the premature aging-related phenotypes were largely alleviated on a C57BL/6 background.

Frequency of immune cells in the Bone Marrow (BM) of B6-kl/kl mice was comparable to age-matched C57BL/6 mice

We sought to determine the effect of Klotho on immune cell development and maintenance in adult mice. We divided adult mice into young-adult (young-4-19 weeks) and older-adult (old-20-54 weeks) group. Total number of BM cells recovered from B6-kl/kl mice were significantly reduced in young-adult and older-adult B6-kl/kl mice compared to age-matched C57BL/6 mice (Figure 2A). This reduction in cell number is most likely due to the overall smaller size of the B6-kl/kl mice (Figure 1B and C). As mice age the frequency of multi-potential progenitors (MPP) and short-term HSCs increase [2]. These age-related changes were not affected by decreased Klotho expression as B6-kl/kl mice did not show differences in the frequency of MPPs or HSCs compared to age-matched C57Bl/6 mice (Figure 2B, C and D). Importantly, the comparable age-associated loss of MPPs and gain of HSCs in both C57Bl/6 and B6-kl/kl mice indicate normal maintenance with age in adult mice. Common lymphoid progenitors (CLPs) were also present in similar frequencies in the B6-kl/kl mice compared to the C57Bl/6 mice and showed an analogous and significant loss with age (Figure 2E and F). B6-kl/kl mice showed comparable myeloid cells in the bone marrow compared to C57Bl/6 mice (Figure 2G and H). There was a reduction in the frequency of pre-B cells in the young B6-kl/kl mice, but this did not affect B cell development as B cells, pro B cells and new B cell frequencies were not reduced (Figure 2I and J). Overall, in the B6-kl/kl BM, immune cells developed and were maintained at comparable frequencies to age-matched C57BL/6 mice.

T cell development, maintenance of thymic epithelial cells and age-related thymic involution in B6-kl/kl mice was comparable to age-matched C57BL/6 mice

To evaluate the effect of Klotho paucity during T cell development, thymocytes from B6-kl/kl mice at different age ranges were analyzed. Total thymocyte numbers in B6-kl/kl mice were comparable to age-matched C57BL/6 mice as were the frequencies of double positive (DP), single positive CD4 (CD4 SP) and single positive CD8 (CD8 SP) cells in the B6-kl/kl and C57BL/6 mice (Figure 3A-C). Thus, Klotho deficiency does not affect T cell development in the thymus.

Age-associated thymic involution is defined as an irreversible decrease in thymocyte numbers orchestrated by the decline in thymic epithelial cells (TECs) and the thymic environment [26]. To determine if TECs were affected by hypomorphic expression of Klotho in the C57BL/6 background, total numbers and subpopulations of TECs were analyzed and found to be comparable in age-matched B6-kl/kl and C57BL/6 mice (Figure 3D-G). Furthermore, all significant age-dependent changes noted in TECs were comparable in age-matched B6-kl/kl and C57BL/6 mice. Medullary TECs can be further differentiated into mTEC low (mTEClo) and mTEC high (mTEChi) based on expression level of major histocompatibility molecule II (MHCII). The frequency of these cells was also comparable between the two groups (Figure 3G). Together, this detailed analysis of the thymus shows that hypomorphic expression of Klotho did not impair T cell development and that age-related thymic atrophy occurred normally in B6-kl/kl mice compared to C57BL/6 mice.

Innate and adaptive immune cell maintenance in the spleen of B6-kl/kl mice

The spleens from B6-kl/kl mice were neither visibly abnormal nor hypercellular and demonstrated no enhanced erythropoiesis as noted in Klotho-deficient 129 mice [23] (Figure 4A). In the young B6-kl/kl there was a small reduction in the number of splenocytes compared to the C57Bl/6 mice, which was ameliorated with age and therefore did not represent a defect in maintenance of immune cells (Figure 4A). The overall assessment of the innate immune cells in the spleen included natural killer T cells (NKT), neutrophils (Neut), natural killer cells (NK), dendritic cells (DC), and macrophages (Mac) (Figure 4B). The frequency of these cell populations was comparable in the young-adult and older-adult B6-kl/kl mice compared to C57BL/6 mice (Figure 4C-G). There was a small but significant reduction in the frequency of B cells in the spleens of older-adult B6-kl/kl mice compared to C57BL/6 mice (Figure 5A and B), but no differences in frequency of IgD⁺ or IgM⁺ B cells (Figure 5C). B6-kl/kl had comparable frequency of naïve (Tn), effector-phenotype (Teff), and memory-phenotype (Tmem) T cells in both CD4 and CD8 T cell compartments compared to C57BL/6 mice (Figure 5D-G). Together these data demonstrate that adaptive and innate immune systems remained unaffected by decreased Klotho expression in the spleen of B6-kl/kl mice.

Mice

Klotho-hypormorphic kl/kl mice on C3H/Jx C57BL/6J mixed background were provided by Dr. Makoto Kuro-o [19] and backcrossed for 10 generations to C57BL/6J mice obtained from The Jackson Laboratory to generate B6-kl/kl mice. B6-kl/kl mice were screened by conventional PCR immediately after weaning using the primer forward: 5’-tggagattggaagtggac-3’ together with the primers reverse: 5’-caaggaccagttcatcatcg-3’ and reverse: 5’-ttaaggactcctgcatctgc-3’ for the Klotho hypomorphic and for the WT genotype, respectively. All mice were bred and maintained in an animal facility at the National Institute of Aging (NIA) in a 12-hour light-dark cycle and fed ad libitum with a diet containing 0.7% phosphorus (0.4% non-phytate phosphorus). The studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (NRC, 2010). The protocol was approved by the Animal Care and Use Committee of the NIA Intramural Research Program, NIH.

C57BL/6 background genotyping for B6-kl/kl mice

Tail samples from B6-kl/kl mice were sent to the Animal Molecular Diagnostics Laboratory at Frederick National Laboratory for Cancer Research to verify congenicity with C57BL/6 strain. Briefly, 1cm tails from 10 mutant mice were digested and DNA extracted using DNeasy® Blood & Tissue Kit (Qiagen, Venlo, Netherlands). DNA samples were then quantified using a nanodrop (BMG Labtech, Offenberg, Germany) and screened for 96 C57BL/6 microsatellite markers distributed over 19 chromosomes using an ABI 3130 Genetic Analyzer.

Tissue collection

Thymus, spleen and bone marrow were harvested and processed into single cell suspensions for flow cytometry analysis by following standard protocols. Thymic epithelial cells were obtained by gently teasing thymii to release the thymocytes and subsequently digested with 5mg/mL Liberase TH (Roche, Basel, Switerland) and 50mg/mL DNase I (Roche) at 37 °C, as previously described [34,35]. Bone marrow cells were harvested from the femur and tibia of the mice and processed accordingly to standard protocols.

Flow cytometry analysis

Cells were incubated with Fc Block (CD16/32, BD Biosciences, San Jose, CA), stained with antibodies and then fixed with 2% paraformaldehyde. Samples were acquired on the FACS Canto II (BD Biosciences) and analyzed using FlowJo (TreeStar, Ashland, OR). Dead cells were excluded using the eBioscience Fixable Viability Dye eFluor® 506 (ThermoFisher Scientific, Waltham, MA). The following antibodies conjugated to biotin, FITC, PE, PerCP-Cy5.5, PE-Cy5, PE-Cy7, APC, APC-Cy7 or Pacific Blue were used (ThermoFisher Scientific, BD Biosciences, BioLegend, San Diego, CA): anti-CD4 (GK1.5), anti-CD8 (53-6.7), anti-TCR-β (H57-597), anti-CD19 (6D5), anti-CD44 (IM7), anti-B220 (RA3-6B2), anti-IgM (RMM-1), anti-IgD (11-26c.2a), anti-NK1.1 (PK136), anti-F4/80 (BM8), anti-Ly6G (1A8), anti-CD11c (HL3), anti-CD62L (MEL-14), anti-EpCAM (G8.8), anti-CD45.2 (104), anti-Ly51 (BP-1), anti-MHCII (M5/114.15.2), anti-UEA-1 (B-1065, Vector Labs), anti-Sca-1 (E13-161.7), anti-cKit (2B8), anti-Flt3/Flk2 (A2F10), anti-CD127 (A7R34), anti-AA4.1 (AA4.1), anti-CD43 (S7), anti-CD11b (M1/70), and Streptavidin. The LIN cocktail contained anti-NK1.1, anti-CD11b, anti-GR1 (RB6-8C5), anti-TER119 (TER-119), anti-CD3e (145-2C11), anti-CD19, and anti-B220. APC- conjugated mouse CD1d tetramers loaded with glycolipid PBS-57 (CD1d-tet) and an unloaded tetramer comprised of only the glycolipid PBS57 were obtained from the tetramer facility of the US National Institutes of Health.

Gene expression analysis

Kidneys from C57BL/6 and B6-kl/kl mice were collected for RNA extraction and qPCR analysis. Briefly, kidneys were disrupted and homogenized using a TissueRuptor II, and RNA was extracted from lysates using the RNeasy mini kit (Qiagen). DNA was eliminated from the samples by incubating with DNase (Qiagen). First strand cDNA synthesis was performed by using 1μg of total RNA together with oligo(dT)_12–18 and the Invitrogen SuperScript II Reverse Transcriptase (Thermo Fisher Scientific), accordingly to the manufacturer's instructions. Quantification of klotho mRNA expression was conducted using real-time qPCR performed on an Applied Biosystems ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific). Primers were designed to amplify specific amplicons of the membrane kl (F:5’-ggctctgaaagcctacgtgttg-3’; R:5’-gggagctgagcgatcactaagt-3’), secreted kl (F:5’- tgctggctttcctctaggtcat-3’; R:5’-ttaggcgttctgatgctgtca-3’) and gapdh (F:5’-gtcgtggagtctactggtgtc; R:5’cagaaggggcggagatgatg-3’) genes. Each cDNA sample was diluted 5-fold and then 5μL of dilutions added to 5pmol of each primer and SYBR Green Master (Roche). The cycling parameters were: 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Quantification of gene expression was performed by the E^^− ΔCt method using gapdh as the normalizer gene (where E stands for primer amplification efficiency). Each sample was quantified in triplicate and primer amplification efficiencies were calculated and validated with the standard curves obtained through the amplification of cDNA serial dilution.

Statistical analysis

Data is presented as mean ± standard error mean. Significance was determined using 2way ANOVA, Tukey’s multiple comparison tests, and multiple t-tests with GraphPad Prism 6.0 (GraphPad, San Diego, CA) software.