We found a persistently abnormal cytokine response in children at school age following NE. GM-CSF, TNF-β, IL-2 IL-6, and IL-8, were significantly higher in children with NE compared to controls at baseline. GM-CSF and IL-8 rose significantly in children with NE in response to LPS stimulation compared to age-matched controls. Children at school-age following NE also had relative LPS hypo responsiveness in several cytokines demonstrating an altered immune phenotype compared with age-matched controls.
IL-8 was significantly elevated in school children with NE when compared with controls, and after in vitro treatment with an endotoxin (LPS) levels increased further. IL-8 was noted to be high in infants with NE when compared to normal infants and higher in those with severe encephalopathy and abnormal neuroimaging, compared to those with mild NE (3). IL-8 measured before 96 hours of age, to be a potent biomarker predicting death and abnormal neurological outcome in survivors of NE.(9). Elevated IL-8 levels in the group of children with NE, in our study compared to normal children demonstrates a persistently altered immune response at school age.
GM-CSF was also significantly elevated in children with NE compared to the controls. Interestingly increased response was also noted in the neonatal period in the same cohort of children with NE. GM-CSF activates neutrophils and macrophages and stimulates the production of other pro-inflammatory cytokines. GM-CSF production is stimulated by lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-23. GM-CSF has been reported to high in infants with NE who were eligible for TH and in non-survivors at day 1 of life compared to survivors (3). We also noted a similar response at school age in children with NE who underwent TH in the neonatal period. However, GM-CSF crosses the blood brain barrier and may be neuroprotective and reduced brain damage has been reported in previous experimental models of stroke.(17)
IL-6 was noted be significantly high on LPS stimulation in children with NE II/III who received TH and had abnormal neurodevelopmental outcome. A study demonstrated that high levels of serum Il-6 at 24-48 hours in a group of infants who received TH, were associated with abnormal MRI brain (18). A meta-analysis identified serum IL-6 and serum IL-1β measured before 96 hours of age as potential biomarkers in predicting abnormal outcome in infants with NE (9). Elevated IL-6 in newborn with NE who received TH was associated with death and abnormal neurodevelopmental outcome at 12 months of age (19).
Tumor Necrosis factor (TNF-β) is another pro-inflammatory cytokine which acts on different cells. It promotes the proliferation of fibroblasts and induces the synthesis of GM-CSF, G- CSF, IL-1, and prostaglandin-E2 in fibroblasts (20). We demonstrated high levels of TNF-β in response to LPS stimulation in school age children with NE. We found an association of high TNF-β levels in school age children with NE with low gross motor scores on developmental assessment. Previous studies have demonstrated an association between neonatal serum cytokine levels of IL-8, Il-12, TNFα and TNF-β in extremely low birth weight infants and Cerebral Palsy (21). But there are no studies to our knowledge, showing an association of poor neurodevelopmental outcome and abnormally elevated cytokine levels at school age in children with NE.
Interleukin-10 (IL-10) is an anti-inflammatory cytokine, due to its ability to inhibit macrophage activation. It also has a stimulatory function on B cells and mast cells. Increased Il-10 levels were noted in children with NE compared to controls. High levels of IL-10 were associated with severity of NE and in those who received therapeutic hypothermia (TH). IL-10 also has neuroprotective properties demonstrated in animal studies (22).Correlation of high levels of IL-10 with multiorgan dysfunction & mortality has been demonstrated in children in paediatric intensive care (23). Neonates with sepsis, pneumonia and necrotising enterocolitis demonstrated high levels of Il-10 (24). Increased levels of IL-10 at 72 hrs of age were associated with prolonged NICU stay in neonates with Transposition of great arteries who underwent the arterial switch procedure (25).
Erythropoietin (Epo) is a glycoprotein cytokine secreted by the kidney in response to cellular hypoxia. It stimulates the production of red blood cells from the bone marrow. Apart from being an antioxidant, it also has anti-apoptotic and anti-inflammatory actions. Elevated Epo levels were demonstrated in children with NE compared to controls, especially in response to LPS. Significantly high levels of Epo have been demonstrated in infants with increased severity of NE and who underwent TH at day 2-4 of life. NE grade II/III and death were associated with elevated Epo levels on Day 3 of life (2, 18). High levels of serum Epo may represent markers of severity of encephalopathy and brain injury, this theory can be supported by previous studies reported in preterm infants.(26)
TNF-α is a proinflammatory cytokine which plays a critical role in the local inflammatory response and in initiation of the cytokine cascade (27). TNF-α is an important cytokine involved in sepsis and inflammation and is a potential marker in the diagnosis of early and late onset neonatal sepsis. Increased serum level of TNF-α has been reported to be associated with mortality and abnormal neuroimaging in neonates with NE (18). In our study we demonstrated significantly increased TNF-α in response to LPS in school age children in the NE and the control groups.
We have shown in our study that school age children with NE demonstrated vigorous systemic innate immune response compared to children with normal development. Periventricular leukomalacia (PVL) or related inflammation events or both during the perinatal and postnatal period may have a programming effect, causing altered inflammatory responses in preterm children with CP (28). In their study they demonstrated that preterm school-age children with periventricular leukomalacia (PVL) induced (CP) had significantly higher levels of tumour necrosis factor (TNFα) and elevated TLR4 mRNA in peripheral blood mononuclear cells (PBMC’s), in comparison to preterm term school-age control group children.
Lipopolysaccharide (LPS) when introduced in vitro in healthy individuals not only induces the cascade of inflammatory pathways (29) but also initiates a transient refractory state, referred to as LPS hypo-responsiveness or LPS tolerance (30). This LPS tolerance state is associated with decreased capacity of whole blood and peripheral mononuclear cells to produce proinflammatory cytokines in response to LPS stimulation. Decreased levels of proinflammatory cytokines IL-6 and TNF-α in the serum and in the peripheral mononuclear cells (PBMC) supernatant are found in response to LPS stimulation in critically ill patients in ICU in comparison to healthy individuals (31). Similar results were seen in our study, the level of TNF-α rose with LPS stimulation to a lesser degree than in children with NE compared to age-matched controls. In addition, LPS hypo responsiveness was found in children with NE in cytokines IL-6 and TNF-β. Similarly, children in the TH group demonstrated decreased response to LPS in cytokines IL-6, IL-8, IL- 1α, Il-1β, TNF-α and VEGF, which correlated with severity of NE.
There are very few studies in the literature demonstrating the above finding in school age children with NE, we do find studies done in the neonatal period but not at school age. However further research studies and large randomised control trials are required to validate these findings.