On epigenetics and telomeres and how 'environmental' factors contribute to the long-term legacy of pediatric critical illness

Critically ill patients are patients who, due to a variety of triggering medical conditions, require vital organ support to avoid imminent death. Despite major progress in intensive care the patients still face a high mortality risk. Moreover, critical illness is hallmarked by features of accelerated aging where many patients experience an adverse legacy of the illness long after hospital discharge. This is mostly described for adults and includes muscle weakness, chronic renal failure despite resolution of acute renal damage, bone loss and fracture risk, neurocognitive impairment and post-traumatic stress disorders. Children, treated in the paediatric intensive care unit (PICU) during crucial developmental phases, show impaired long-term physical and neurocognitive/psychological development and reduced quality of life. The degree to which the developmental impairment was already present or predestined at PICU admission, how much of the legacy was evoked by critical illness and its management, and what the underlying biological mechanisms are, remained unclear. The main objective of this doctoral thesis was thus to gain more insight in the underlying biological processes and modifiable risk factors with regard to the long-term developmental deficit faced by critically ill children. We hypothesised that two intensive care-related interventions during PICU stay could potentially affect the long-term outcome of these critically ill children. These are the exposure to phthalates, which are toxic plasticisers shown to leach from indwelling medical devices, and the nutritional management early during the course of critical illness. We further hypothesised that this legacy of critical illness and its management could be brought about by damage-related changes in DNA methylation or by telomere length alterations. In a first part, we documented the impact of exposure to circulating phthalates during paediatric critical illness on long-term neurocognitive outcome of critically ill children. We demonstrated that plasma concentrations of several phthalate metabolites, which were virtually undetectable in healthy children, were extremely high in critically ill children upon PICU admission. They decreased rapidly, but remained markedly elevated at PICU discharge. Moreover, exposure to circulating phthalate metabolites was independently and robustly associated with the attention deficit observed years after PICU admission. These data underscore the importance to use, whenever possible, medical devices composed of alternative, safer plasticisers or with low phthalate release potential. In a second part, we focused on the nutritional management during PICU stay. Providing artificial nutritional support is thought to be of major importance in an intensive care setting, as the vast majority of critically ill patients are unable to eat normally, particularly while being mechanically ventilated. Observational data have associated malnutrition during critical illness with muscle wasting, weakness and delayed recovery. Consequently, many clinicians have assumed that artificial nutrition via the parenteral route is beneficial for patients' outcome. However, a large randomised controlled clinical study (the "PEPaNIC" trial) showed that withholding of such supplemental parenteral nutrition during the first week of paediatric intensive care reduced the incidence of new infections and accelerated recovery, allowing an earlier PICU discharge. Despite these beneficial short-term effects, concerns have been raised about long-term developmental consequences, possibly mediated by DNA methylation changes or telomere length alterations, of tolerating such a macronutrient deficit. Hence, assessment of the impact on long-term outcome of the children was crucial. In this second part we therefore documented the impact of critical illness and of early initiation of supplemental parenteral nutrition on telomere length alterations and DNA methylation changes during PICU stay, and on long-term health risks, and physical and neurocognitive outcome of PICU survivors. As accelerated telomere shortening could theoretically explain part of the legacy of paediatric critical illness, we quantified telomere length in repeated white blood cell DNA harvested in PICU from patients who were enrolled in the PEPaNIC-trial, as compared with healthy children who never needed intensive care. We found that shorter than normal leukocyte telomeres are present in critically ill children admitted to the PICU. Duration of stay in PICU and early initiation of parenteral nutrition further shortened telomeres, effects that were independent of other determinants and which could predispose these children to adverse long-term health sequelae. As DNA methylation is essential for diverse biological processes such as development and cognition, and nutrition may have a major impact on the epigenome, we investigated whether DNA methylation changes arise during the course of PICU stay and remain present until PICU discharge, and whether these could be affected by the nutritional management strategy in PICU. After adjusting for pre-admission DNA methylation changes and critical illness-induced changes in cell type composition, several genes of potentially high relevance to the long-term legacy of critical illness, such as genes involved in neuronal migration, differentiation and growth, brain development and signaling, processing of amyloid-beta precursor protein, transcriptional regulation, energy metabolism and multiple cellular signalling pathways, were found to be differentially methylated between patients upon PICU discharge and matched controls. Severity of illness and early initiation of parenteral nutrition, independent of its slowing effect on recovery, were found to be significantly associated with a large proportion of the aberrant DNA methylation changes that arose during PICU stay. In a last study, we investigated whether withholding parenteral nutrition during the first week in PICU, an intervention that clearly improved short-term PICU outcome, has an impact on long-term health risks and physical and neurocognitive development. Two years after inclusion in the PEPaNIC randomised controlled trial, we assessed health risks, and physical and neurocognitive development of 786 PICU-survivors who were randomly allocated to late or early initiation of parenteral nutrition in the PICU, in comparison with 405 matched healthy children. Late initiation of parenteral nutrition did not adversely affect survival, physical and neurocognitive development, but improved overall executive functioning, more specifically inhibition, working memory, and meta-cognition. Also externalising behavioural problems and visual-motor integration were improved. Moreover, we found that omitting supplemental parenteral nutrition early during PICU stay partially normalised the long-term neurocognitive legacy of paediatric critical illness. The protective effect of withholding parenteral nutrition was most pronounced in the youngest children. In conclusion, we demonstrated that exposure to phthalates leaching from indwelling plastic medical devices could induce an attention deficit in PICU survivors. Secondly, we showed that telomeres shortened and DNA methylation changes in genes important for neurocognitive development arose during PICU stay, partly explained by early initiation of parenteral nutrition. These changes may predispose critically ill children to adverse long-term health sequelae. Finally, we found that withholding parenteral nutrition during the first week in the PICU did not negatively affect survival, growth or health status 2 years later, and significantly improved several domains of neurocognitive development, hereby partially normalising the long-term neurocognitive legacy of paediatric critical illness. Hence, these data increase our understanding of the factors and the underlying biological processes that may contribute to the impaired development of critically ill children. They will hopefully lead to a change in legislation concerning the use of plasticisers in medical devices and to a change in clinical guidelines in favour of withholding early use of PN during paediatric critical illness, both in order to improve long-term developmental outcomes of millions of PICU survivors worldwide.status: publishe