Altered endotoxin responsiveness in healthy children with Down syndrome

Down syndrome (DS) is caused by an extra copy of genetic material from chromosome 21, and is the most prevalent chromosomal abnormality, affecting approximately 1 in 550 births in Ireland [1], and 1 in 700 births in the USA [2]. Co-morbidities associated with DS include developmental disability, congenital heart disease (CHD), gastrointestinal tract anomalies, and an increased risk of haematological malignancy [3]. In addition, it is the most common genetic syndrome associated with abnormal immune function and immune defects [4]. There is significant evidence of immune dysregulation in Down syndrome including T-cell and B-cell lymphopenia due to impaired expansion of these cell lines in infancy [5], a smaller thymus gland with reduced naïve Tcell and regulatory T-cell numbers [6], suboptimal antibody responses to vaccination [7‐10], and abnormal levels of serum cytokines [11‐13].

Children with Down syndrome are, therefore, at increased risk of infection, especially in early childhood, particularly respiratory tract infections [14]. Hilton et al. [15] reported a higher risk of admission to hospital and intensive care with respiratory tract infections (RTIs) in children with DS. Mortality from sepsis is 30% greater in patients with DS in comparison to children without DS who also had sepsis [16].

It is challenging to attribute causation to a specific deficit of the immune system with the increased incidence of infections and sepsis seen in this cohort. A normal innate immune system is crucial in providing first line defence against infection. Neutrophils and monocytes are crucial cellular components of the innate immune system. Defective phagocytic activity and neutrophil chemotaxis have previously been reported in DS [17, 18]. Monocyte function in DS is poorly described. Increased numbers of the non-classical (CD14dim/CD16+) monocyte pro-inflammatory sub-type have been described in DS in comparison to controls [19]. This monocyte population has previously been implicated in sepsis and chronic disease [20].

CD11b is a cell surface marker involved in mediating neutrophil and monocyte adhesion and diapedesis [21] and is an indicator of activation. Dysfunction in neutrophil adherence and migration has been shown to increase the risk of infection in adults and neonates [22]. Toll-like receptor 4 (TLR-4) is the key receptor involved in lipopolysaccharide (LPS) endotoxin recognition and activation of the innate immune system [23], and has also been implicated in the pathogenesis of autoimmune conditions such as systemic lupus erythematosus (SLE) and rheumatoid arthritis [24].

Immunomodulators can alter responses to infection, alleviate autoimmunity and ultimately improve patient care. Melatonin is an endogenous hormone which mediates its anti-inflammatory effects by modulating pro-inflammatory cytokines and inflammasome de-activation, thereby ameliorating results in endotoxaemia [25, 26]. Melatonin has a very good safety profile and is used in paediatrics in sleep management [27]. Clinical trials in adults and neonates with sepsis have demonstrated improved clinical outcomes [28, 29].

We hypothesized that children with DS have altered neutrophil and monocyte function which contributes to their increased susceptibility to infection and increased mortality from sepsis. We aimed to evaluate the in vitro effect of LPS endotoxin, and the anti-inflammatory melatonin on CD11b and TLR4 expression on neutrophils and monocytes in children with DS.

Neutrophil CD11b expression at baseline was significantly lower in children with DS compared with controls. Following LPS treatment children with DS upregulated CD11b, and this was significantly greater than controls. Novo et al. [18] reported that, at baseline, CD11b expression on neutrophils was not significantly different between children with DS (n = 12) and controls, although the smaller numbers and older population in this study may contribute to these findings. Our research suggests that although the level of CD11b may be lower under normal conditions, after contact with endotoxin there is an increased ability to activate and mobilise neutrophils in response to this stimulus. Neutrophils in children with DS may be hyper-responsive to endotoxin, which may have detrimental effects in the setting of sepsis. Adults with sepsis and renal injury in the absence of hypotension, have been shown to have increased activation of neutrophils with upregulation of CD11b [31], worsening prognosis. Furthermore, neutrophil mediated lung injury in sepsis, and multi-organ dysfunction (MODS) have been associated with increased CD11b expression on these cells [32, 33]. A blockade of this receptor could have potential benefits in these clinical contexts [34]. In paediatric studies LPS hyper-responsiveness has been demonstrated through increased CD11b expression on neutrophils and monocytes of neonates with encephalopathy [35, 36], these infants having developed significant immune dysregulation. Zhou et al. [37] examined TLR4 signalling and the CD11b response on polymorphonuclear cells in mice. The authors concluded that TLR4 mediates CD11b upregulation and is key for PMN activation in response to LPS. Further correlation between CD11b and TLR4 has been described by Guang et al. [38] who reported that CD11b mediates TLR4 signalling and trafficking in a cell specific manner in dendritic cells and macrophages, having a crucial role in balancing the innate and adaptive response to LPS. It appears that the two receptors are inter-linked and have important regulatory roles on one another in initiating the innate immune response.

Zhang et al. [39] demonstrated that mice deficient in CD11b exposed to Mycobacterium tuberculosis developed more severe granulomas, higher leucocyte recruitment and elevated pro-inflammatory cytokines. This demonstrates the immunomodulatory effect neutrophil CD11b expression exerts on the host response to infection. A persistent inflammatory response can be seen in autoimmunity and there is a higher prevalence in DS, recent studies suggest that reduced CD11b is associated with chronic inflammation in SLE and lupus nephritis [40, 41]. Neutrophil CD11b is also decreased in septic shock and correlated with poorer outcomes [42, 43]. In this context, the increased incidence of both autoimmunity and sepsis in DS is particularly noteworthy [16, 44]. We demonstrated that melatonin caused a predominant decrease in CD11b expression in both cohorts; Fig. 4. We also showed that children with DS exhibited a hyper-responsive CD11b response to LPS in neutrophils Fig. 4a. In the acute setting of sepsis/SIRS an upregulation of CD11b may be associated with deleterious effects [45], furthermore, a positive correlation between CD11b expression and the degree of systemic inflammation has been described [46], making melatonin a potential adjunct in acute sepsis/SIRS.

The classical monocyte (CD14+/CD16-) accounts for the largest proportion of monocytes (80–85%) and its main functions include antigen presentation and phagocytosis [47]. We found classical monocytes exhibited significantly higher CD11b expression at baseline, and greater fold increases in CD11b after LPS than other monocyte sub-populations in both groups. This sub-group also displayed the largest rise in TLR-4 after LPS compared with other monocyte subpopulations in both cohorts. This suggests that classical monocytes are significantly pro-inflammatory with the largest CD11b and TLR4 response to LPS than any other sub-population. Regarding differential CD11b expression on monocyte subsets Tak et al. [48] reported no significant differences, whereas another study examining differential in vivo activation of monocyte subsets reported the most significant rise in CD11b on the intermediate monocyte [49]. However, these studies [48, 49] characterised CD11b expression after lower doses of LPS with longer incubations in an adult in vivo setting as compared to our study which was undertaken in a paediatric cohort. Monocyte CD11b was highest on classical and intermediate monocytes [50].

Intermediate monocytes (CD14+/CD16+) are elevated in the setting of acute illness such as sepsis in children [51]. In our study, there was a significant rise in CD11b expression after LPS stimulation in children with DS but not in controls on intermediate monocytes. This adds to the evidence that there are hyper-responsive elements to the innate immune system in children with DS. Indeed, intermediate monocytes produce significant quantities of TNF-α once activated [50]. Previous studies have demonstrated elevated levels of TNF-α in patients with DS compared with healthy controls [13] at baseline. Intermediate monocytes demonstrated the greatest TLR-4 at baseline compared with other monocytes in both groups which has also been demonstrated in adults [20].

Non-classical monocytes (CD14dim/16+) have been implicated in both acute and chronic disease and have a pro-inflammatory phenotype with increased production of IL-1β and TNF-α [47]. This monocyte sub-group had significantly lower CD11b and TLR-4 expression in both groups at baseline. Furthermore, non-classical monocytes demonstrated a relative hypo-responsiveness to LPS versus the other sub-populations. Boyette et al. [50] assessed the phenotype, function, and differentiation monocyte subsets, and reported that non-classical monocytes had the lowest CD11b MFI and that there was the smallest response in this subset following TLR-4 stimulation. We found baseline TLR-4 expression was significantly raised in children with DS versus controls. The TLR-4 response plays a significant role in fighting infection but may also be responsible for the dysregulated inflammation seen in septic shock [52]. Williams et al. noted an increased mortality in mice with polymicrobial sepsis who exhibited early up-regulation of TLR-4, and improved survival in those with suppressed TLR gene expression [53]. Suppression of TLR-4 activation, pro-inflammatory cytokine release, and developing endotoxin tolerance is important in limiting the adverse effects of sepsis. Furthermore, a failure of this protective negative feedback process may contribute to increased mortality in sepsis [54].

We demonstrated that melatonin has an anti-inflammatory influence on innate immune function by reducing CD11b expression on neutrophils and total monocytes in children with DS and controls, thereby inhibiting neutrophil and monocyte activation and migration. Although there is a paucity of literature on the effect of melatonin on CD11b, Alvarez-Sanchez et al. reported a reduction in CD11b in melatonin-treated mice [55]. A significant reduction in TLR-4 expression only occurred in total monocyte populations. Melatonin may act as a TLR-4 antagonist and may be modulated via TLR-4 mediated inflammatory genes through molecule myeloid differentiation factor 88 (MyD88)-dependent and TRIF-dependent signalling pathways [56], thereby attenuating inflammation.

Melatonin has beneficial immunomodulatory effects in the setting of sepsis by inhibiting mitochondrial dysfunction and inflammation, reducing nitrosative and oxidative stress [29]. Melatonin has a robust antioxidant or free radical scavenging activity of [57, 58] and melatonin administration also impairs NF-κB transcriptional activity, reducing pro-inflammatory cytokine (IL-1β, TNF-α, IFN-γ) release and inhibiting activation of the NLRP3 inflammasome [59]. Melatonin improved survival and clinical outcomes in neonates versus controls in sepsis [28, 60, 61]. We have demonstrated that the immunomodulatory effects of melatonin in sepsis can also be broadened to include reducing neutrophil and monocyte activation.

Melatonin increased CD11b expression on non-classical monocytes and to a significant level in the children with DS. However, it has been shown that melatonin can have pro-inflammatory actions in response to endotoxaemia. Effenberger et al. [62] reported that melatonin enhanced the general immune response following LPS treatment. Melatonin may have differing actions on distinct cell lines, with the pro-inflammatory non-classical monocyte being preferentially activated. Further evaluation of the immunomodulatory properties of melatonin in children with DS will allow assessment of its potential as a therapeutic agent.

This research highlights important differences in the innate immunity of children with DS versus age-matched controls. To our knowledge this has not been studied previously in this population. Children with DS have an increased response to LPS in neutrophils and intermediate monocytes, while also having elevated TLR-4 expression on non-classical monocytes compared to controls. These variations may be a contributory factor in a heightened/dysregulated innate immune response, which may have deleterious effects, leading to the worse outcomes seen in sepsis in these children. Lastly, melatonin could represent a useful clinical adjunct in the treatment of sepsis as an immunomodulator and our study suggests its anti-inflammatory effects also influence neutrophil and monocyte function.