Severe neurological impairment and immune function: altered neutrophils, monocytes, T lymphocytes, and inflammasome activation

Abstract

Background: Infections cause significant morbidity and mortality in children with Severe Neurological Impairment (SNI). Alterations in immune cell numbers and function in children with neurodisability have been reported. We aimed to characterise neutrophil, monocyte and lymphocyte proportions and activation, at baseline and in response to stimulation with lipopolysaccharide, in children with SNI compared to healthy controls.

Methods: Whole blood samples of children with SNI and controls were incubated in the presence or absence of lipopolysaccharide (10 ng/ml). Monocyte and neutrophil function (Cluster of Differentiation (CD)11b, (TLR)-4 and CD66b expression) and lymphocytes were assessed by flow cytometry. Expression of genes involved in the inflammasome (NLR Family Pyrin Domain Containing(NLRP)-3, Apoptosis-Associated Speck-like protein (ASC) and Interleukin(IL)1β) were assessed by PCR.

Results: Monocytes and CD8+ T cells were lower in children with SNI (n = 14). CD66b, was hyporesponsive and monocyte TLR4 was hyperresponsive to lipopolysaccharide in children with SNI compared to controls (n = 14). NLRP3 expression was higher at baseline and IL1β expression was not upregulated in response to lipopolysaccharide in children with SNI in contrast to controls.

Conclusion: We have found significant differences in immune regulation in children with SNI compared to controls which may provide a useful therapeutic target in the future.

Impact: Children with SNI have reduced monocyte and CD8+ T cells. Neutrophils and monocytes in children with SNI show altered markers of activation in response to lipopolysaccharide. Expression of NLRP3 at the RNA level was higher at baseline in children with SNI. This study adds to the existing literature that children with neurological impairment have altered inflammatory and immune cell responses. This may provide a useful therapeutic target to reduce infection-related morbidity and mortality, and tertiary neurological injury in the future.

Fig. 1. Percentage change in expression of markers of activation in monocytes, monocyte subsets and neutrophils following LPS.

Whole blood samples were processed for flow cytometry and expression of CD66b, TLR4 and CD11b on neutrophils and monocytes was quantified at baseline and following LPS stimulation. Values are shown as percentage change in expression of CD66b, TLR4 and CD11b following incubation with LPS (10 ng/ml) for 1 h. Mann-Whitney test (median, 95%CI). Control (Con, n = 14); Severe Neurological Impairment (SNI, n = 14); *p ≤ 0.05; **p ≤ 0.01.

Fig. 2. Proportion of T cells and CD8+ cells as a percentage of the total lymphocyte count are significantly lower in children with SNI compared to controls.

T cells (a), NK cells (b), B cells (c), CD4+ (d), CD8+ (e), V delta 1 (f), V delta 2 (g). Samples were processed for flow cytometry. Values are expressed as percentages of total lymphocytes or T cells. Mann–Whitney test (median, 95% CI). Control (Con, n = 14); Severe Neurological Impairment (SNI, n = 14); NK natural killer cells, *p ≤ 0.05; **p ≤ 0.01.

Fig. 3. Expression of genes from the Nucleotide-binding and oligomerization domain (NOD)-like receptor protein 3 (NLRP-3) inflammasome at baseline and following LPS.

Gene expression of NLRP3, Apoptosis-associated speck-like protein containing a CARD (ASC) and Interleukin 1β (IL1β), was quantified by real-time polymerase chain reaction (PCR) at baseline and following LPS. Values are expressed as fold changes in gene expression following incubation with LPS (10 ng/ml) for 1 h. Two way ANOVA (mean ± SD); Control (Con, n = 10); Severe Neurological Impairment (SNI, n = 5); Veh, Vehicle; *p ≤ 0.05; ****p ≤ 0.0001.