Cases Versus Controls
Intestinal Permeability and Circulating Endotoxin
Plasma endotoxin concentrations were significantly higher in cases compared with controls (median 0.24 [interquartile range (IQR): 0.02, 0.54] vs. 0.02 (IQR: 0.01, 0.10) EU/mL, respectively; P = 0.028; Fig. 2A). Cases compared with controls also had increased intestinal permeability as measured by the LMR [median 0.09 (IQR: 0.03, 0.19) vs. 0.03 (IQR: 0.02, 0.09), respectively; P = 0.042; Fig. 2B]. This was due to both a decrease in mannitol recovery and an increase in lactulose recovery among cases (data not shown). There was a weak positive correlation between intestinal permeability and circulating endotoxin concentrations (Spearman coefficient r = 0.38, P = 0.086).
Monocyte and Neutrophil Phenotype
There were no significant differences between the ratios of monocytes to lymphocytes between cases and controls (Table 1). Examining the peripheral blood monocyte population as a whole (CD66a/c/e− CD14+ cells), cases compared with controls had a significantly lower percentage expressing HLA-DR (median 87.1% vs. 96.4%; P = 0.001), and a higher percentage expressing CD64 (median 99.6% vs. 97.6%; P = 0.041, Fig. 3); there was no significant difference in the percentage of monocytes expressing CD86 or TLR4.
Among the monocyte subpopulations, there were no significant differences between cases and controls in proportions of classical [70.8% (IQR: 46.8, 78.0) vs. 72.6% (IQR: 58.4, 78.9), P = 0.56], intermediate [25.4% (IQR: 16.5, 43.8) vs. 20.2% (IQR: 16.2, 38.3), P = 0.74], or nonclassical [4.6% (IQR: 3.8 - 10.2) vs. 5.75% (4.6% - 7.5%), P = 1.00] monocytes. The HLA-DR percentage expression was significantly lower in cases compared with controls in both the classical [78.8% (IQR: 70.5, 93.0) vs. 95.9% (IQR: 92.0, 97.7), respectively; P = 0.001] and intermediate [91.4% (IQR: 76.3, 95.9) vs. 97.8% (IQR: 94.9, 98.8), P = 0.004] subpopulations but not the nonclassical (94.3% vs. 95.7%, P = 0.96) subpopulation. CD64 percentage expression on the nonclassical subpopulation was higher in cases than controls (88.9% vs. 67.0%, P = 0.014), but there was no evidence of difference between groups in CD64 expression on the classical (99.9% vs. 99.9%, P = 0.66) and intermediate (99.9% vs. 99.6%, P = 0.26) subpopulations (Fig. 3).
Finally, among the neutrophil population (CD66a/c/e+ CD16+), the percentage expression of CD64 was significantly higher in cases than controls [median 85.2% (IQR: 32.7, 94.7) vs. 26.9% (IQR: 9.81, 48.2); P = 0.025].
Twenty-four-hour Whole Blood Stimulation
Concentrations of TNF-α, IL-1β and IL-6 measured in 24-hour whole blood culture supernatants are shown in Figure 4A. In unstimulated cultures, levels of IL-1β, but not IL-6 or TNF-α, were higher in cases versus controls. Stimulation with either 0.5 EU/mL of LPS or 0.5 µg/mL of zymosan resulted in significantly higher levels of all cytokines compared with cultures with no stimulation. Following stimulation with zymosan, cases versus controls produced significantly less TNF-α [median 2342 (IQR: 995, 3961) vs. 5130 (IQR: 2400, 6312) pg/mL, respectively; P = 0.031] and IL-1β [median 1101 (IQR: 564, 2313) vs. 2604 (IQR: 1339, 3528) pg/mL, P = 0.048], but similar IL-6 [median 36,716 (IQR: 18,830, 51,771) vs. 39,653 (IQR: 28,220, 52,695) pg/mL; P = 0.595]. Following stimulation with LPS, there was no evidence of a difference in cytokine production between cases and controls (Fig. 4A).
Percentage expression of cell surface markers on monocytes following 24 hours of culture is shown in Figure 4B. Because CD64 was universally expressed on monocytes following stimulation, the MFI was used to evaluate the level of CD64 expression on each cell. After 24-hour culture without stimulation, CD64 expression levels were higher among cases compared with controls [MFI 2570 (IQR: 1186, 2978) vs. 1591 (IQR: 950, 2173), respectively; P = 0.048; Fig. 4B], with no evidence of differences in other markers. Following stimulation with LPS, there was no evidence of differences between groups in the percentage of monocytes expressing any of the activation markers (Fig. 4B). Following stimulation with zymosan, the percentage of monocytes expressing HLA-DR in cases was significantly lower compared with controls [median 89.6% (IQR: 68.5, 97.0) vs. 99.0% (IQR: 97.4, 99.6), respectively; P = 0.009; Fig. 4B]. As has previously been observed,1 unstimulated monocyte HLA-DR expression across both groups was positively correlated with zymosan-stimulated TNF-α production (Spearman coefficient = 0.51, P = 0.006), and IL-1β (Spearman coefficient = 0.39, P = 0.041).
As expected, following 24-hour stimulation, there was a noticeable shift in the monocyte subpopulation proportions consistent with activation and maturation-specific shifts in CD14 and CD16 expression.21 The classical population was 13.6% (IQR: 4.8, 43.1) across all groups, the intermediate population was 81.3% (IQR: 57.1, 91.8) and the nonclassical was 2.6% (IQR: 0.7, 3.9). There was no significant difference in these shifts between cases and controls (data not shown).
LOW VERSUS HIGH ENDOTOXIN
To evaluate the impact of circulating endotoxin on monocyte immunophenotype, we combined cases and controls and categorized children into 2 groups: those with high circulating endotoxin (defined as ≥0.1 EU/mL), and those with low circulating endotoxin (<0.1 EU/mL). This categorization was based on a previous study, in which 0.1 EU/mL was identified as the upper normal limit in children, using the limulus amebocyte lysate assay.32 There were 11 children in the high endotoxin group [6 cases and 5 controls; median 0.18 (IQR: 0.11, 0.39) EU/mL endotoxin] and 17 in the low endotoxin group [3 cases and 14 controls; median 0.01 (IQR: 0.01, 0.025) EU/mL endotoxin].
In children with high compared with low endotoxin, the percentage of monocytes expressing the T-cell costimulatory ligand CD86 was significantly lower [69.8% (IQR: 58.8, 79.9) vs. 92.4% (IQR: 79.1, 96.4), P = 0.003; Fig. 5A]. There was no evidence of differences between groups in other markers (CD64, HLA-DR and TLR4). The lower CD86 expression on total monocytes among higher versus lower endotoxin was primarily due to lower expression on the classical monocytes [median 65.4% (IQR: 53.1, 78.3) vs. 87.2% (IQR: 76.1, 86.4); P = 0.08], and the intermediate monocytes [83.1% (IQR: 72.2, 92.0) vs. 96.6% (IQR: 84.8, 98.2); P = 0.13], but not on nonclassical monocytes [95.5% (IQR: 91.1, 97.9) vs. 96.0% (IQR: 86.3, 96.9); P = 0.41].
Twenty-four-hour Whole Blood Stimulation
When comparing high and low endotoxin groups, there was no evidence of differences in elaboration of any cytokine in response to zymosan or LPS stimulation (data not shown). After stimulation with zymosan, children in the high compared with the low endotoxin group expressed significantly less CD64 [MFI 1514 (IQR: 1170, 1822) vs. 2196 (IQR: 1353, 2857), P = 0.022] and CD86 [80.3% (IQR: 57.2, 88.4) vs. 92.2% (IQR: 79.0, 96.0); P = 0.037] on total monocytes (Fig. 5B), with no evidence of differences in other markers.
In this study, we investigated whether the increased gut permeability and immunoparalysis that characterize critical illnesses also occur in children with less severe illnesses. We show that children with noncritical infections have higher intestinal permeability, increased endotoxemia and changes indicative of immunoparalysis, with reduced monocyte HLA-DR expression, increased CD64 expression and reduced IL-1β and TNF-α production upon challenge of innate immune cells with the beta-glucan, zymosan. In contrast to previous studies, children in this study were not critically unwell. Furthermore, we excluded children with any gastrointestinal symptoms to ensure that our findings were not due to altered intestinal permeability resulting from symptomatic intestinal infections. Endotoxemia as a result of increased intestinal permeability is thought to be a mediator of immunoparalysis in critically ill patients. In our cohort, children with higher circulating endotoxin levels had lower basal monocyte CD86 expression and less upregulation of CD64 and CD86 upon stimulation, indicative of an impaired capacity to costimulate T-cells and engage with antibody Fc components for enhanced specificity and recognition of pathogens. Collectively, these findings suggest that children hospitalized for noncritical infections experience changes in the intestinal barrier and circulating innate immune cell function normally associated with more critical illness, which could leave them vulnerable to subsequent infections.
The pathogenesis of increased intestinal permeability is likely multifactorial. In the context of gastrointestinal infections, there is mechanical damage to the epithelium, loss of tight junction integrity and villous atrophy arising from local inflammatory processes that cause cytokine-dependent changes in the enterocyte cytoskeleton.33–35 In critical illness or sepsis, there is hypoperfusion of the gut, which causes epithelial hypoxic injury, acidosis and disarrangement of the mucosal cytoskeleton.35 In noncritical infections outside the gut, where there is adequate gut perfusion, intestinal permeability is poorly described. However, increased intestinal permeability has been demonstrated in murine models of pneumonia,36 and in children with measles,18 although the presence of diarrhea in 30% of measles cases37 means the mechanism underlying this observation is difficult to ascertain. This study therefore adds to our understanding of gut function during intercurrent illness, because increased intestinal permeability was seen in young children with noncritical infections (predominantly in the skin, respiratory and urinary tract), and no gastrointestinal symptoms. In light of recent observations after experimental intravenous administration of LPS to healthy adult donors, which demonstrate a dramatic change in circulating monocyte phenotype and function,19 our data elaborate the association between intestinal permeability and monocyte function in children with noncritical illness, raising the possibility that their innate immune defences may also be compromised in ways previously thought to occur only in more severe illness.
Altered intestinal permeability can impair nutrient absorption, compromise mucosal immune responses15,38 and allow microbial products (such as endotoxin) and viable bacteria to translocate to the systemic circulation.36 Microbial translocation during critical illness was demonstrated clearly in an adult intensive care study: although only 4% of patients had Gram-negative infections, over half had endotoxin levels more than 2 standard deviations above healthy control levels.5 Although we cannot define the source of endotoxin in our study, it is likely that it arose from translocation of bacterial products across the intestinal barrier. This is an assumption made in other studies,39,40 confirmed in animal models36 and supported by the positive correlation between circulating endotoxin and intestinal permeability in our study. The weak positive relationship that we observed between LMR and circulating endotoxin has been found in a previous study,41 although others have found no correlation,42,43 or a relationship with the percentage lactulose recovery but not the LMR,44 albeit in very different populations. This heterogeneity may have several explanations: first, there may be a nonlinear relationship between permeability and translocation; second, it may reflect the fact that lactulose–mannitol testing primarily measures permeability of the small intestine, while the greatest Gram-negative bacterial load is in the colon45 and third, it may indicate that LPS is not the only translocated material that is likely to be immunogenic.
LPS-driven inflammatory responses are implicated in the pathogenesis of multiorgan dysfunction in systemic inflammatory response syndrome,46 and LPS exposure in monocytes is associated with epigenetic reprogramming leading to impaired responses on restimulation.3,24 Following LPS exposure in healthy adults, there is a rapid reduction in classical monocytes as these cells either die or likely mature into intermediate monocytes21 that have enhanced antigen presenting and stimulatory ability with increased HLA-DR47 and CD86 expression.48 The significance of this for our study is unclear: HLA-DR and CD64 percentage expression was associated with clinical illness rather than higher endotoxemia. This may reflect that the endotoxemia measured at the time cases were recruited may not reflect previous endotoxemia during their illness, and once exposed to LPS in vivo effects can last weeks, as opposed to the 24 hours seen in vitro models.20
Cases had lower HLA-DR and higher CD64 percentage expression than controls, but this was not due to altered numbers of intermediate monocytes; rather, cases had reduced HLA-DR expression on both classical and intermediate monocytes. The consequence of this reduced HLA-DR continued following 24 hours of culture with another stimulant, zymosan. There was a positive correlation between basal monocyte HLA-DR expression and overall TNF-α and IL-1β production following zymosan 24-hour culture, and both cytokines were significantly reduced in cases. This was not seen in IL-6 that is primarily produced by classical monocytes,49 which were significantly reduced relative to basal proportions by the end of the 24-hour culture. This suggests that any initial changes in circulating cell phenotype persist as monocytes mature from the classical to the predominantly intermediate subtypes in vitro. LPS-stimulated TNF-α production was modest and not significantly different between groups. This may reflect the fact that at 0.5 EU/mL, the concentration of LPS used for stimulation was not substantially higher than that detected in plasma of many of the patients. We did not see reversal of some of these changes, such as HLA-DR percentage, following culture with zymosan, as has previously been seen with beta-glucan stimulation of LPS-trained monocytes in vitro.50 Lower HLA-DR and CD64 expression on monocytes have been associated with increased mortality following sepsis,29 and along with reduced stimulated cytokine production, mirror the changes seen in postsepsis immunoparalysis.
In children with higher endotoxemia at the time of venepuncture, monocytes had significantly lower expression of CD86, which is a costimulatory molecule required for T-cell priming. Low CD86 expression on monocytes has been associated with more severe sepsis, as demonstrated by an increased Sequential Organ Failure Assessment score,51 and increased mortality in mouse models of sepsis.52 Monocytes from children with high plasma endotoxin in our study exhibited lower expression of CD64 and CD86 following stimulation with zymosan compared with monocytes from children with lower levels of circulating endotoxin, indicating that exposure to circulating LPS alters monocyte responses to subsequent pathogen challenge. These findings have been associated with mortality and poorer outcomes in children with sepsis.29,52
Limitations of this study include the small sample size, along with the heterogeneity of the etiology of the illnesses involved. This was intended to be a pilot study investigating responses to systemic infection; given some of the diagnostic uncertainty present in diagnosis in young children, we were able to show these changes across a variety of different types of infections. Because of different health-seeking behaviors of parents, cases presented at different timepoints in their illnesses, so the timing of blood tests was not standardized. Although it has been shown that monocyte markers do change over the course of an illness,53 in this study, the infected children were different to the controls regardless of illness timing. Finally, although the controls in this study did not have a febrile illness, they were still recruited from hospital, so would not necessarily be classed as “healthy.”
In summary, we show that children with noncritical infections and no gastrointestinal symptoms display similar gut and innate immune cell alterations as children who have severe sepsis or organ failure. In the setting of severe sepsis, these changes are associated with increased morbidity and mortality, and susceptibility to future infections. Future studies should investigate the molecular triggers of these observed changes, how long they persist and the clinical implications during and after hospitalization. Such studies are warranted because children with noncritical infections represent a much larger population of children presenting to hospital than children with critical illness. Whether there is a refractory period of immunoparalysis following common childhood infections, and whether interventions could improve immune function during this potential window of vulnerability, is worthy of further investigation.
The authors thank Dr. Anna Caldwell at King’s College London for undertaking mass spectrometry to measure LMRs. The authors would also like to thank the participants and their families.
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child; gut permeability; innate immunity; monocytes; lipopolysaccharide
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