Determination of accelerated ageing in critical illness

Sullivan, Jack Alexander (2023). Determination of accelerated ageing in critical illness. University of Birmingham. Ph.D.

Preview

Sullivan2023PhD.pdf
Text - Accepted Version
Available under License All rights reserved.
Download (11MB) | Preview

Abstract

Aims: The primary aim of this research was to test the hypothesis that injury or critical illness is sufficient to cause an acceleration of the ageing process. This hypothesis is to be tested by quantifying the immune, cellular, and epigenetic changes which occur following injury, such as a severe burn or following illness such as COVID-19 or sepsis. A secondary aim was to see if cellular senescence could be reversed.

Methods: Absolute telomere length was measured via qPCR in PBMCs taken from burns patients following admission and again at 6 months post injury (n = 33) and compared with the telomere length of healthy controls (n=28). Flow cytometry was used to profile the immune system of burn patients 6 months post injury (n = 31) and healthy controls (n = 27) to look for signs of immunosenescence. Immunohistochemistry was used to see how the percentage of p16INK4A+ senescent cells changed in injured and non-injured skin taken from burns patients over a long period (n = 7). A compound library was used in 48-hour exposures in an x-ray induced senescence hTert-BJ fibroblast model, as well as in non-senescent hTert-BJ control fibroblasts to identify compounds with a senescence modulating ability. Changes in cell viability and SA-βGal activity were measured. Patients had their methylomes profiled using either the illumina 450k or EPIC array. Burns patients were profiled following admission (n = 53) and again 6 months post injury (n = 34) as were a cohort of septic (n = 66) and non-septic (n = 68) ICU patients and patients with severe (n = 38) or moderate (n = 25) COVID-19 infections. Their epigenetic ages were calculated using the Horvath, Hannum and DNAm PhenoAge clocks. Differential methylation gene and pathways analysis was also performed. Total RNA sequencing was performed on RNA isolated from the PBMCs of burn injury patients who survived “unsurvivable” burn injury and patients who died following severe burn injury. The sequencing platform was the illumina HiSeq Xten.

Results: There was no significant difference between the mean absolute telomere length of PMBCs of burns patients taken at admission to hospital or 6 months post injury (p = 0.35). There was also no significant difference between the patient’s absolute telomere lengths at admission (p = 0.85) or 6 months post (p = 0.35) injury when compared to healthy controls. Signs of immune ageing were observed in burn injury survivors 6 months post injury in comparison to a healthy control population. Burn patients showed a lower proportion of naïve CD4+ T cells (p = 5.6e-07) and recent thymic emigrants (p = 0.00024) as well as a greater proportion of total CD4+ memory cells (p = 1.1e-07) than the control cohort. Immune remodelling was also observed in CD8 T cell and B cell sub-populations. Epigenetic clocks showed mixed results, with burns and COVID-19 patients only showing epigenetic age acceleration according to the Horvath clock, and septic and non-septic ICU patients showing significant epigenetic age acceleration according to the Hannum and DNAm PhenoAge clocks. Burns patients had significantly greater epigenetic ages following admission compared with 6 months post injury according to all clocks. ICU patients who died had significantly greater epigenetic age acceleration than those who survived, with septic patients having the greatest epigenetic age acceleration. Patients with severe COVID-19 had significantly greater epigenetic age residuals than those with moderate infections. Multiple pathways which relate to long term patient outcomes such as cell cycle and signalling, chromatin remodelling, metabolic pathways, and pathways regulating cell death and apoptosis were significantly differentially methylated between groups. No significant differences were observed in the RNA expression profiles of patients who survived severe burn injuries considered “unsurvivable” compared with patients who died.

Conclusion: Not all, but most areas examined showed evidence that suggests injury and severe infection are associated with accelerated ageing.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):