Neutrophils induce paracrine telomere dysfunction and senescence in ROS-dependent manner. (2021)
Figure 6

Fig 6. p16Ink4a-expressing cells recruit neutrophils during aging

a) RNA-sequencing data showing expression of genes associated with neutrophil recruitment during liver aging;

b, c) Quantification of b) neutrophil infiltrates (NIMP positive) and c) mean number of TAF positive hepatocytes in liver of young (3 months) and old (24 months) mice. Data are mean±S.E.M. of 3-5 mice per group ;

d) Quantification of mean number of TAF in hepatocytes as a function of the distance from a Ly6G labelled neutrophil. Data are from 5 mice aged 29 months of age, line indicates linear relationship between mean TAF and distance (linear regression and F test: R2=0.35; P<0.0001);

e) Representative micrograph showing hepatocyte from aged mouse containing a TAF located in close proximity to a neutrophil (labelled with Ly6G);

f) schematic depicting experimental design;

g) % of neutrophil migration towards conditioned media (CM) obtained from young (early passage) and replicatively senescent human fibroblasts. Media alone and fMLP were used as negative and positive controls respectively. Data are mean±S.E.M. of 3 independent experiments;

h) Schematic depicting experimental design for panels i-k;

i) Comparison between p16Ink4a mRNA levels of young (3 months old) and old INK-ATTAC mice (28-29 month old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-8 mice per age group;

j) % of TAF positive hepatocytes in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-6 mice per group;

k) Liver neutrophils (Ly6G) in old INK-ATTAC mice (28-29 months old) treated with vehicle or AP20187. Data are mean ± SEM of n = 5-7 mice per group;

l) Intrahepatic leukocyte profiling by mass CyTOF. CyTOF was performed on young (6 months) and old (24 months) INK-ATTAC mice (n=5 mice per group); 23 unique clusters of intrahepatic leukocytes were defined by a 24-cell surface marker panel using the Rphenograph clustering algorithm and were visualized on a tSNE plot (t-distributed stochastic neighbor embedding plot).;

m) (left) tSNE plots comparing the distribution of young and old intrahepatic leukocytes. Red indicates high frequency categorization of cells to a cluster while blue indicates low frequency; (right) tSNE plots comparing p16Ink4a expression in young and old intrahepatic leukocytes. Red indicates high p16Ink4a expression while blue indicates low expression. Data are from n=5 mice per group;

n) % of p16Ink4a positive cells (detected by CyTOF) in different intrahepatic leukocyte populations isolated from young and old mice. Data are mean±s.e.m of n=5 mice per group;

o) Schematic depicting experimental design for panels p and q. 3 month old wild-type mice were injected with murine Cxcl1 for 7 days;

p) Quantification of liver neutrophils (Ly6G) in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 7-10 mice per group ;

q) Quantification of the % of TAF positive hepatocytes in mice injected with vehicle or Cxcl1. Data are mean ± SEM of n = 6-7 mice per group;

r) Correlation between TAF positive hepatocytes and neutrophils (linear regression and F test: r2=0.70; p=0.0001).

Data information: Statistical analysis was performed using one‐way ANOVA (Holm-Sidak method) for multiple comparisons and two‐tailed t‐test for single comparisons. **P < 0.01; *P < 0.04.

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