Rejuvenation of leukocyte trafficking in aged mice through PEPITEM intervention

Abstract

Inflammageing leads to uncontrolled leukocyte trafficking in response to inflammatory insults. Here, we used a zymosan-induced peritonitis mouse model on inflammation to investigate the role of the PEPITEM pathway on leukocyte migration in ageing. We then analysed whether PEPITEM could modulate leukocyte migration in older adults. We observed a loss of functionality in the PEPITEM pathway, which normally controls leukocyte trafficking in response to inflammation, in older adults and aged mice and show that this can be rescued by supplementation with PEPITEM. Thus, leading to the exciting possibility that PEPITEM supplementation may represent a potential pre-habilitation geroprotective agent to rejuvenate immune functions.

Fig. 1. PEPITEM regulates T-cell trafficking in young and old mice.

A Schematic representation of zymosan-induced peritonitis (blue) for young (3-month; white) and aged (21-month; grey) mice treated without (black) or with PEPITEM (red) (created by biorender.com). The total number of peritoneal B CD45⁺ leukocytes, C CD3⁺ T-cells, D CD3⁺CD4⁺ T-cells, E CD3⁺CD8⁺ T-cells and F CD3⁺KLRG1⁺ T-cells were quantified using flow cytometry. Data are mean ± SEM for n = 2 independent experiments, using n = 7 and n = 5–6 mice per group for young and old mice. *p < 0.05 and **p < 0.01 by Bonferroni post-test.

Fig. 2. PEPITEM regulates infiltration of T- and B-cell subsets in young and old mice.

T- and B-cell subpopulations were measured in the peritoneal lavage fluid of zymosan-induced peritonitis (blue) from young (3-month; white) and aged (21-month; grey) mice treated without (black) or with PEPITEM (red). Total number of peritoneal CD3⁺ A CD4⁺CD62L⁺CD44^- naive, B CD8⁺CD62L⁺CD44^- naive, C CD4⁺CD62L⁺CD44⁺ central memory, D CD8⁺CD62L⁺CD44⁺ central memory, E CD4⁺CD62L^-CD44⁺ effector memory and F CD8⁺CD62L^-CD44⁺ effector memory T-cells, G CD19⁺ B-cells and H CD19⁺CD21^-CD93^-CD23^-CD43^- age-associated B-cells were quantified using flow cytometry. Data are mean ± SEM for n = 2 independent experiments, using n = 7 and n = 5-6 mice per group for young and old mice. *p < 0.05 and **p < 0.01 by Bonferroni post-test.

Fig. 3. PEPITEM restores endogenous regulation of lymphocyte migration in older adults.

PBL from young (black, n ≥ 9) and older (red, n ≥ 9) donors were left untreated (control, C) or treated with adiponectin (AQ) or PEPITEM (P) prior to addition to cytokine-stimulated endothelium. PBL A adhesion or B transmigration expressed as the number of cells/mm2/number of cells added or the percentage of adhered cells that had transmigrated, respectively. Adiponectin receptor 1 C–D and E–F 2 analysed as C, E frequency of B-cells positive for expression or D–F median fluorescence intensity (MFI; n = 9). G Representative histograms of AdipoR1 and 2 expression on young and older B-cells. H–J B-cells from young (black) and older (red) donors were left C-G, I untreated or H, J treated with adiponectin for 15 minutes. H APPL-1 protein expression was assessed by western blot in B-cell lysates and I normalised to actin loading control (n = 5). J Fold-change in 14-3-3ζ abundance in adiponectin-treated B-cells normalised to β-actin (n = 5). Data are mean ± SEM. *p < 0.05, **p ≤ 0.01 and ***p ≤ 0.001 by (A, B) Dunnett’s post-test or (C–D, G–H) unpaired t-test.