Invariant Natural Killer T cells coordinate removal of senescent cells

Abstract

Background: The failure of immune surveillance to remove senescent cells drive age-related diseases. Here, we target an endogenous immune surveillance mechanism that can promote elimination of senescent cells and reverse disease progression.

Methods: We identify a class of lipid-activated T cells, invariant natural killer T cells (iNKTs) are involved in the removal of pathologic senescent cells. We use two disease models in which senescent cells accumulate to test whether activation of iNKT cells was sufficient to eliminate senescent cells in vivo.

Findings: Senescent preadipocytes accumulate in white adipose tissue of chronic high-fat diet (HFD) fed mice, and activation of iNKT cells with the prototypical glycolipid antigen alpha-galactosylceramide (αGalCer) led to a reduction of these cells with improved glucose control. Similarly, senescent cells accumulate within the lungs of mice injured by inhalational bleomycin, and αGalCer-induced activation of iNKT cells greatly limited this accumulation, decreased the lung fibrosis and improved survival. Furthermore, co-culture experiments showed that the preferential cytotoxic activity of iNKT cells to senescent cells is conserved in human cells.

Conclusions: These results uncover a senolytic capacity of tissue-resident iNKT cells and pave the way for anti-senescence therapies that target these cells and their mechanism of activation.

Keywords: Fibrosis; IPF; Metabolic dysfunction; Senescence; iNKT cells; senolytic.

Figure 1.. Senescent preadipocytes are defined by high SA-βgal activity and accumulate in white adipose tissue of HFD mice.

(a) eWAT SVF cells was isolated from chow or HFD mice (16 weeks on HFD) and depleted of CD45⁺ cells leaving a CD45^– SVF population (CD45^–) for X-gal staining to detect SA-βgal and qRT-PCR. (b) X-gal staining for SA-βgal activity on CD45-depleted eWAT SVF cells isolated from chow and HFD mice. (c) qRT-PCR of senescence markers on CD45-depleted eWAT SVF cells from chow and HFD mice. Data are mean ± SEM from n = 6 mice for each group. (d) CD1d expression on the eWAT SVF isolated from chow and HFD mice. The cells were stained with antibodies for CD45 and CD31 (to gate out immune and endothelial cells) along with the fluorogenic substrate C₁₂FDG and anti-CD1d antibody. The senescent preadipocytes (DAPI-CD45⁻CD31⁻ C12FDG^Hi) were then gated for relative CD1d expression. Quantification of CD1d expression from the CD45⁻ CD31⁻ C12FDG^Hi subset from chow and HFD mice is shown in the right panel. Data are represented as mean ± SD from n=4 per experiment. (e) eWAT SVF was isolated from chow and HFD mice and stained with antibodies for CD45 and CD31 (to gate out immune and endothelial cells) along with the fluorogenic substrate C₁₂FDG to detect SA-βgal activity. (f) Representative dot-plot showing the cell size (FSC-A) and percent of cells with the highest C₁₂FDG expression in chow and HFD mice in the CD45⁻, CD31⁻ subpopulation. The representative histogram shows C₁₂FDG staining and gating on the subset with the highest fluorescence (C₁₂FDG⁺) in the same CD45⁻, CD31⁻ subpopulation. (g) Quantification of C₁₂FDG⁺ cells from chow or HFD mice. Data are mean ± SD from n = 4 mice per group. (h) Quantification of the C₁₂FDG MFI of the entire CD45⁻CD31⁻ population in the same chow and HFD mice as in (g). Data are mean ± SD from n = 4 mice per group. *p < 0.05, **p < 0.005, ***p< 0.0005, two-tailed T-tests.

Figure 2.. Senescent preadipocytes with high SA-βgal activity express SASP genes and are preferentially eliminated with senolytic treatment.

(a) FACS was performed to isolate the CD45⁻, C₁₂FDG^Lo and C₁₂FDG^Hi subpopulations from eWAT SVF of HFD mice for qRT-PCR. Shown is a representative histogram of CD45⁻ population showing the C₁₂FDG subpopulations sorted. (b) C₁₂FDG MFI of the C₁₂FDG^Lo and C₁₂FDG^Hi populations as shown from (g). Data are mean ± SD from n = 3 mice per group. (c) qRT-PCR of senescence markers in C₁₂FDG^Hi relative to C₁₂FDG^Lo cells from the same HFD mice. Data are mean ± SD from n = 3 mice per group. (d) HFD mice were treated with ABT-737 or vehicle for 1 week prior to harvest of eWAT SVF for the C₁₂FDG assay. Shown is a representative histogram of CD45⁻, CD31⁻ population C₁₂FDG staining. (e) Quantification of the positive cells in the C₁₂FDG^Hi and C₁₂FDG^Lo subsets from vehicle and ABT-737 treated HFD mice. Data are mean ± SD from n = 3 mice per group. *p < 0.05, **p < 0.005, ***p <0.0005, two-tailed T-tests.

Figure 3.. Activation of iNKT cells leads to clearance of senescent cells in white adipose tissue of HFD mice.

(a) eWAT was Isolated and the SVF prepared from age-matched Chow, HFD untreated controls (HFD Ctrl), or HFD treated with α-GalCer on day 4 (HFD+GC) and senescent cells were enumerated with the C₁₂FDG SA-βgal assay while iNKT cells were enumerated with GC loaded CD1d Tetramer and anti-CD3. (b) Representative dot plots of iNKT cell percentages from total live eWAT SVF cells, defined as GC loaded CD1d-Tetramer (GC-Tetramer)⁺/CD3⁺ cells In representative Chow, HFD Ctrl and HFD+GC mice. (c) Quantification of the iNKT cell percentages in Chow (n=3), HFD Ctrl (n=4) and HFD+GC (n=5) mice per group. Data are mean ± SD. (d) Representative histograms of C₁₂FDG fluorescence and quantification of the C₁₂FDG^Hi subpopulation of CD45⁻ cells from Chow (n = 4), HFD Ctrl (n=5) and HFD+GC (n=5) mice per group. Data are mean ± SD. (e) Effects of α-GalCer treatment of HFD-fed mice on metabolic parameters 10 days after treatment. (f) Glucose tolerance test (GTT) was performed at 10 days after α-GalCer injection. Mice were administered 2g/kg glucose via i.p. injection after a 14 hr fast. Blood glucose was measured at 0, 15, 30, 60, and 120 after glucose injection. Data are mean ± SEM from n = 8–9 mice per group. (g) HOMA-IR was assessed at 10 days after α-GalCer injection based on the fasting blood glucose and fasting insulin concentration using the equation, HOMA-IR= (glucose in mmol/L x insulin In mIU/mL)/22.5. Data are mean ± SEM from n = 8–9 mice per group. (h) Adoptive transfer of GalCer-activated eWAT iNKT cells from HFD mice. On day 3 after α-GalCer treatment, iNKT cells were sort purified from eWAT SVF (~90% purity) of donor HFD mice and transferred by i.p. injection into recipient HFD mice (HFD+iNKT), alongside untreated HFD Ctrl or α-GalCer-treated HFD mice (HFD+GC). On day 4 post-transfer or post α-GalCer, eWAT SVF was isolated for C₁₂FDG SA-βgal assay. (i) Representative histograms of C₁₂FDG fluorescence and gating for HFD Ctrl, HFD+iNKT and HFD+GC mice. (j) Quantification of the percent of C₁₂FDG^Hi subpopulation from the CD45⁻ CD31⁻ subset for each group of mice. Data are mean ± SD from n = 2 experiments. *p < 0.05, **p < 0.005, ***p< 0.0005, one-way ANOVA.

Figure 4.. Activation of iNKT cells preferentially eliminates senescent cells in a murine lung injury model and in vitro using human cells

(a) Bleomycin was used to induce lung injury and a separate group of mice remained uninjured as controls. On day 10 post-injury α-GalCer was administered, or mice remained untreated, and control mice were administered vehicle. In cohort #1, day 14 post-injury mice lungs were harvested for analysis of senescent cells by C₁₂FDG assay and qRT-PCR and enumeration of iNKTs. In cohort #2, mice were used for analysis of fibrosis on day 21. (b) Representative histogram of C₁₂FDG fluorescence and quantifications of mean±SD from n = 3 mice per group in CD45⁻ lung parenchymal cells. (c) qRT-PCR of senescence and SASP in CD45-depleted lung cells, showing mean ± SD of n = 3 mice per group, *p < 0.05, one-way ANOVA. (d) Percentages of iNKT cells as a proportion of T cells from the same mice as (b), mean ± SD of n = 2 mice per group. (e) Quantification of fibrosis by hydroxyproline (HP) concentrations in Control (n = 5), Bleo (n = 10), and Bleo+GC (n = 10) mice per group, error bars are SD. ***p < 0.0005, *p < 0.05, one-way ANOVA Krusak-Wallis test. (f) Kaplan Meier survival curve for mice injured with intratracheal bleomycin (4 U/kg) and injected with α-GalCer or vehicle at day 5 (arrow). N=10 mice per group. Significance calculated by Log-rank (Mantel-Cox) test. (g) In vitro co-culture assay to monitor cytotoxicity using human iNKT cells isolated from donor PBMCs and WI-38 primary human lung fibroblasts. Dendritic cells differentiated from donor PBMCs were loaded with α-GalCer, while iNKTs were isolated from the PBMCs. WI-38 female lung fibroblasts were induced to senescence with etoposide or remained proliferative as a control. WI-38 cells were incubated with varying ratios of purified human iNKT cells. Changes in impedance were monitored with the XCelligence assay system and software was used to calculate percent cytolysis. (h) Time course of XCelligence assay showing mean percent of cytolysis over 18 hours of the assay for the indicated samples. Each time-point measurement was from n = 3 biological replicates for each sample. (i) Mean percent cytolysis at 8 h and 18 h for indicated 1:2 target-effector ratios (n = 3 biological replicates each), error bars are SD. *p < 0.05, ***p < 0.0005 one-way ANOVA.