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HLA-DR Expression on Monocyte Subsets in Critically Ill Children

Boeddha, Navin P. MD*,†; Kerklaan, Dorian MD, PhD*; Dunbar, Albert MSc; van Puffelen, Esther MD*; Nagtzaam, Nicole M. A. BSc; Vanhorebeek, Ilse PhD§; Van den Berghe, Greet MD, PhD§; Hazelzet, Jan A. MD, PhD; Joosten, Koen F. MD, PhD*; Verbruggen, Sascha C. MD, PhD*; Dik, Willem A. PhD; Driessen, Gertjan J. MD, PhD†,‖

The Pediatric Infectious Disease Journal: October 2018 - Volume 37 - Issue 10 - p 1034–1040
doi: 10.1097/INF.0000000000001990
Pathogenesis and Host Response

Background: To longitudinally study blood monocyte subset distribution and human leukocyte antigen-DR (HLA-DR) expression on monocyte subsets in children with sepsis, post-surgery and trauma in relation to nosocomial infections and mortality.

Methods: In 37 healthy children and 37 critically ill children (12 sepsis, 11 post-surgery, 10 trauma and 4 admitted for other reasons)—participating in a randomized controlled trial on early versus late initiation of parenteral nutrition—monocyte subset distribution and HLA-DR expression on monocyte subsets were measured by flow cytometry upon admission and on days 2, 3 and 4 of pediatric intensive care unit (PICU) stay.

Results: Upon PICU admission, critically ill children had a higher proportion of classical monocytes (CD14++CD16−) than healthy children [PICU 95% (interquartile range [IQR] 88%–98%); controls, 87% (IQR 85%–90%), P < 0.001]. HLA-DR expression was significantly decreased within all monocyte subsets and at all time points, being most manifest on classical monocytes and in patients with sepsis. Percentage of HLA-DR expressing classical monocytes [upon PICU admission 67% (IQR 44%–88%); controls 95% (IQR 92%–98%), P < 0.001], as well as the HLA-DR mean fluorescence intensity [upon PICU admission 3219 (IQR 2650–4211); controls 6545 (IQR 5558–7647), P < 0.001], decreased during PICU stay. Patients who developed nosocomial infections (n = 13) or who died (n = 6) had lower HLA-DR expression on classical monocytes at day 2 (P = 0.002) and day 3 (P = 0.04), respectively.

Conclusions: Monocytic HLA-DR expression decreased during PICU stay and was lower compared with controls on all examined time points, especially on classical monocytes and in children admitted for sepsis. Low HLA-DR expression on classical monocytes was associated with nosocomial infections and death.

From the *Intensive Care and Department of Pediatric Surgery

Department of Pediatrics, Division of Pediatric Infectious Diseases & Immunology, Erasmus MC-Sophia Children’s Hospital, University Medical Center Rotterdam, Rotterdam, The Netherlands

Department of Immunology, Laboratory Medical Immunology

Department of Public Health, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

§Department of Cellular and Molecular Medicine, Clinical Division and Laboratory of Intensive Care Medicine, KU Leuven University Hospital, Leuven, Belgium

Department of Paediatrics, Juliana Children’s Hospital/Haga Teaching Hospital, The Hague, The Netherlands.

Accepted for publication February 28, 2018.

W. A. D. and G. J. D. have contributed equally to the work.

The authors declare no conflicts of interest. Research leading to these results has received funding from the European Union’s seventh Framework program under EC-GA No. 279185 (EUCLIDS, to J. A. H.), Fonds NutsOhra (to S. C. V.), Erasmus MC Cost-Effectiveness Research Grant (Mrace, to S. C. V.), Erasmus Trustfonds via Erasmus University Rotterdam (to S. C. V.) and a grant from the Flemish Agency for Innovation through Science and Technology (IWT-TBM110685, to G. V. d. B.).

Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (

Address for correspondence: Gertjan J. Driessen, MD, PhD, Department of Paediatrics, Juliana Children’s Hospital/Haga Teaching Hospital, The Hague, The Netherlands. E-mail:

Infection, major surgery and severe injury are frequently associated with a period of immunosuppression.1–3 Immunosuppression in its most severe form is often referred to as immunoparalysis and is associated with increased risk of nosocomial infections and death.1–4 Therefore, there is a growing interest in immunostimulatory therapy, such as interferon-gamma and granulocyte–macrophage colony-stimulating factor, as potential treatment options to improve outcome in severely immunosuppressed patients.4–8 Laboratory markers that guide the need for immunostimulatory therapy are needed.

Human blood monocytes are a heterogeneous cell population and are subdivided into 3 main subsets based on expression of cell surface markers: (1) classical monocytes (CD14++CD16− monocytes), which represent the major monocyte subset, (2) intermediate monocytes (CD14++CD16+ monocytes) and (3) nonclassical monocytes (CD14+/-CD16++ monocytes).9,10 Classical monocytes migrate from bone marrow to peripheral blood circulation and differentiate into intermediate monocytes and sequentially into nonclassical monocytes.11 In different types of diseases, mostly in infection or inflammatory conditions, expansion of intermediate and/or nonclassical monocytes has been described.12

Human leukocyte antigen-DR (HLA-DR) is an MHC class II cell surface molecule that is expressed, among others, by monocytes to present antigens to T cells. Numerous studies, mainly in adults, have identified decreased monocytic HLA-DR (mHLA-DR) expression as marker for immunosuppression and showed that low mHLA-DR expression is associated with nosocomial infections and death.8,13–16 In addition, measurement of mHLA-DR expression has been proven effective to identify septic adults eligible for immunostimulatory therapy.17–19

Studies on blood monocyte subset distribution and mHLA-DR expression in critically ill children are limited so far, especially studies focusing on longitudinal monitoring of mHLA-DR expression during the course of disease.13–15,20–22 In addition, previous studies were restricted to 1 specific patient category; thus, studies comparing mHLA-DR expression between children admitted for different clinical reasons are lacking. The primary objective of this study was to longitudinally monitor monocyte subset distribution and mHLA-DR expression in children with sepsis, post-surgery and trauma in relation to nosocomial infections and mortality.

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Patients and Controls

Critically ill children, from term newborns to 17 years of age, who had been enrolled to a randomized controlled trial on macronutrient management in the pediatric intensive care unit (PICU) of Erasmus MC-Sophia Children’s Hospital, were eligible for this study.23,24 Patients had been randomized to early (within 24 hours) supplementation of parenteral nutrition (PN) or late (not before day 8) supplementation of PN when enteral nutrition was insufficient to reach caloric targets. We obtained 0.5 mL whole blood in ethylenediaminetetraacetic acid tubes on admission to the PICU and on the mornings of day 2, day 3, and day 4. Subsequently, samples were stored at 4°C until analysis, which was performed within 4 hours after blood sampling. The number of patients who could be included was limited because of blood sample volume restrictions and logistic reasons at our hospital.

For comparison, blood from 37 healthy controls was sampled immediately after placement of an intravenous catheter before minor elective surgery.

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Laboratory Analysis

Monocyte subsets (classical monocytes: CD14++CD16−, intermediate monocytes: CD14++CD16+, nonclassical monocytes: CD14+/-CD16++) and mHLA-DR expression were determined by flow cytometry. The following antibodies were used: CD45 PO (Invitrogen, clone HI30), CD14 APC-H7 (Becton Dickinson, clone MO-P9), CD16 PeC7 (Becton Dickinson, clone 3G8) and HLA-DR PB (Biolegend, clone L243). Antibodies were added to 50 μl of whole blood and incubated for 15 minutes at room temperature in the dark. Subsequently, 500 μl NH4Cl was added for 10 minutes at room temperature in the dark to lyse erythrocytes. Eventually, the sample was measured on a flow cytometer (FACSCanto II machine; Becton Dickinson) that was calibrated according to a standardized instrument setting described in detail in the EuroFlow protocols.25 Analysis was performed with Infinicyt 1.7 flow cytometry software. Monocytes, lymphocytes and granulocytes were identified on the basis of CD45 expression along with side-scatter characteristics (SSC). Monocytes were analyzed for CD14 and CD16 expression, and the 3 monocyte subsets were identified on the basis of CD14 and CD16 expression levels.

HLA-DR expression was determined as percentage of HLA-DR-positive cells within the monocyte subsets, using HLA-DR-negative lymphocytes (T cells) and HLA-DR-positive lymphocytes (B cells) as an internal control,26 as well as the mean fluorescence intensity (MFI) within each monocyte subset (Fig., Supplemental Digital Content 1, Absolute counts of monocytes and monocyte subsets were calculated based on the total white blood cell count. Total white blood cell count in the peripheral blood was determined by a standard hematology analyzer (Sysmex XP-300).

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Clinical Data Collection

The presence of infection on admission to the PICU and the identification of nosocomial infections was determined by consensus opinion of 2 infectious disease specialists, who made their decision on the basis of study protocol guidelines.23,24 Infectious disease specialists selected all patients receiving antimicrobial agents for more than 48 hours, after excluding all patients who received prophylaxis. Each patient who fulfilled the criteria for infection, as well as the type of infection, was identified as such based on thorough review of the medical record.27 Patients for whom antimicrobial agents were initiated before PICU admission or within the first 48 hours of admission, while the criteria for infection were fulfilled, were classified in the “sepsis” subgroup. When antimicrobial agents were initiated after randomization and beyond the first 48 hours in the PICU and were given for more than 48 hours while the criteria for infection were fulfilled, the patient was labeled as having acquired a “nosocomial infection.”23,27 The Pediatric logistic organ dysfunction (PELOD) score indicated illness severity.28 We defined death as death within 90 days after admission to PICU.

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Statistical Analysis

Variables are presented as numbers and proportions, means and standard deviations or medians and interquartile ranges (IQRs). Characteristics of patients from different diagnostic categories were compared by χ2 tests or by Kruskal–Wallis tests, including post hoc Mann–Whitney U tests. In controls, the distribution of cells between 5 age categories was tested by the Kruskal–Wallis test. Spearman correlation coefficient was used to analyze the correlation between monocyte subsets and age. Monocyte subsets and HLA-DR expression in patients were compared with controls using the Mann–Whitney U test. The relation between mHLA-DR expression and nosocomial infections, death and the effect of early versus late PN was analyzed by Mann–Whitney U tests. Statistical analyses were performed with SPSS version 21. Graphs were created with GraphPad Prism 7.00. Two-sided P values <0.05 were considered to indicate statistical significance.

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Ethical Aspects

This study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. Protocols were approved by the Central Committee on Research Involving Human Subjects and our institutional ethical review board.

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We enrolled 37 patients of which 12 patients were admitted for sepsis, 11 post-surgery, 10 after trauma, 3 for severe asthma and 1 patient for renal failure. Baseline characteristics are presented in Table 1; patients with sepsis included a lower percentage of males (33%) compared with patients post-surgery (82%, P = 0.019). Also, patients with sepsis had higher illness severity compared with patients post-surgery, as reflected by higher Pediatric logistic organ dysfunction (PELOD) score at day 1 (P = 0.011) and more days on ventilator (P = 0.008). Patients admitted after trauma were older than patients from the 2 other categories (P = 0.025 compared with sepsis, P = 0.024 compared with post-surgery). None of the enrolled patients had a primary immunodeficiency.



Furthermore, we enrolled 37 healthy controls who underwent a minor elective surgical procedure [21 males; median age, 3 year (IQR, 11 months to 8 year)].

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Effect of Age in Controls

In Figure 1A, we show that the absolute number of white blood cells decreased significantly (P = 0.001) with age, from 7580 cells/μl in infants to 3099 cells/μl in children 12 to 18 years of age. No differences were observed between males and females (data not shown). The absolute number of total monocytes and the percentage total monocytes did not differ between age groups (Fig. 1B). The relative distribution of the 3 monocyte subsets showed no correlation with age (classical monocytes, r = 0.11, P = 0.51; intermediate monocytes, r = −0.09, P = 0.60; nonclassical monocytes, r = −0.14, P = 0.42; Fig. 1C). However, the absolute numbers of classical monocytes and nonclassical monocytes were significantly and negatively correlated to age (classical monocytes, r = −0.44, P = 0.007; nonclassical monocytes, r = −0.41, P = 0.01; Fig. 1D). Therefore, for further analyses, the relative proportion of monocyte subsets of the control group could be compared with that of patients.



Reference values for absolute numbers and relative distribution of blood monocyte subsets and HLA-DR expression are derived from our control cohort (Table, Supplemental Digital Content 2, The percentage of HLA-DR-expressing monocytes within the 3 monocyte subsets and the HLA-DR MFI within the monocyte subsets did not differ between age groups; thus, for further analysis, we did not have to adjust for age.

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Monocyte Subset Distribution in Critically Ill Children

Figures, Supplemental Digital Content 3, 4, and 5,;; depict monocyte subset distributions according to the main diagnostic categories (sepsis, post-surgery and trauma) over a period of 4 days after admission to the PICU. On admission, for the whole group of patients, the percentage of classical monocytes was significantly (P < 0.001) higher (95%, IQR 88%–98%) compared with controls (87%, IQR 85%–90%), while the percentage of nonclassical monocytes was significantly lower in patients (patients 2%, IQR 1%–5%; controls 9%, IQR 7%–11%; P < 0.001).

The most prominent differences in monocyte subset distribution were found in patients admitted for sepsis. In the sepsis group, the percentage of classical monocytes tend (P = 0.09) to be lower (82%, IQR 73%–88%) compared with that of controls (87%, IQR 85%–90%). The percentage of intermediate monocytes at admission in sepsis patients (8%, IQR 5%–14%) was significantly (P = 0.003) higher compared with that of controls (4%, IQR 3%–5%) and increased even further to 14% (IQR 5%–28%) at day 2 (P = 0.001 compared with controls).

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HLA-DR Expression on Monocyte Subsets

For the total group of patients, HLA-DR expression was significantly decreased within all monocyte subsets and at all time points, being most manifest on classical monocytes where 67% (IQR 44%–88%) expressed HLA-DR on admission compared with 95% (IQR 92%–98%) of this population in controls (P < 0.001; Fig. 2A). Also the MFI of HLA-DR expression on classical monocytes, presented in Figure 2B, was significantly (P < 0.001) lower in patients at PICU admission (MFI: 3219, IQR 2650–4211) compared with controls (MFI: 6545, IQR 5558–7647). A further drop in both the percentage of HLA-DR expressing classical monocytes and HLA-DR MFI on this population was evident during the following 3 days after PICU admission.



The HLA-DR decrease on classical monocytes was most prominent in the patients with sepsis (Fig. 2C–H). Therefore, we focused our analysis on this monocyte subset. Intermediate and nonclassical monocytes have a largely comparable decrease of HLA-DR expression, as shown in Figures, Supplemental Digital Content 6 and 7,;

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HLA-DR Expression in Relation to Nosocomial Infections and Death

Thirteen patients (35%) acquired at least 1 nosocomial infection during stay at PICU. At day 2 after PICU admission, the percentage of HLA-DR expressing classical monocytes was found to be significantly (P = 0.002) lower in patients who acquired nosocomial infections (42%, IQR 16%–66%) than in patients who did not acquire infections (78%, IQR 52%–89%; Fig. 3A). Patients who died (n = 6, 16%) had a significantly (P = 0.04) lower percentage of HLA-DR expressing classical monocytes at day 3 after PICU admission (33%, IQR 23%–40%) compared with survivors (63%, IQR 33%–77%; Fig. 3C). Clinical characteristics of patients with complications are summarized in Table, Supplemental Digital Content 8, One patient admitted with sepsis acquired an infection caused by an opportunistic pathogen (Candida albicans). In 54% of the infections, no pathogen could be identified.



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HLA-DR Expression in Relation to Parenteral Nutrition

Patients participated in a randomized controlled trial on early versus late supplementation of PN. Patients still in the PICU on day 4 and exposed to early PN had a lower proportion of classical monocytes on day 4 compared with children who did not receive PN (Early PN: n = 5, median 81%, IQR 44%-88%; Late PN: n = 9, median 93%, IQR 90%-96%; P = 0.007; Fig., Supplemental Digital Content 9, HLA-DR expression within all monocyte subsets and at all time points did not differ between the 2 randomization groups (Fig., Supplemental Digital Content 10,

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The percentage of HLA-DR-expressing cells within all monocyte subsets and the HLA-DR MFI within the monocyte subsets in critically ill children was lower compared with that in controls and decreased further during PICU stay. These findings were most pronounced for classical monocytes and in patients admitted for sepsis. In addition, low HLA-DR on classical monocytes on day 2 and day 3 after PICU admission was significantly associated with the occurrence of nosocomial infections and with mortality, respectively.

Our study confirms previous findings, mainly in adults, of decreased HLA-DR on monocytes after admission for sepsis, trauma, burns or surgery, and that such a decreased mHLA-DR expression is associated with nosocomial infections and death.13–16,20,21,29,30 However, our study clearly demonstrates for the first time that the decrease in HLA-DR expression is most prominent on classical monocytes. It remains unclear whether this decrease results from downregulation of HLA-DR expression on the monocyte or from emergency myelopoiesis, that is, increased recruitment of immature, low HLA-DR-expressing cells from bone marrow in a proinflammatory state, including myeloid-derived suppressor cells.31–34

The decreased mHLA-DR was most prominent in children admitted for sepsis. Although clinical outcome did not differ significantly between diagnostic groups, patients with sepsis had higher illness severity at admission to PICU (compared with post-surgery patients), which could partly explain decreased mHLA-DR.15,16,30 Prolonged sepsis-induced immunosuppression is considered to contribute to long-term morbidity and mortality in sepsis survivors.2,35,36 An explorative study in 8 adult long-term sepsis survivors hinted toward recurrent infections for months to years after surviving sepsis and also infections caused by opportunistic infections.37 However, laboratory analysis in that study showed no substantial differences in mHLA-DR expression between sepsis survivors and controls. Further studies in larger cohorts are therefore needed to draw firm conclusions on the relation between mHLA-DR expression and long-term complications in sepsis survivors. Furthermore, future studies have to determine whether associations are present between mHLA-DR expression and specific clinical infectious syndromes or specific pathogens.

In addition to decreased mHLA-DR expression, our study revealed shifts in monocyte subset distribution in critically ill children. For the total group of 37 patients, on admission to PICU, a shift toward increased classical monocytes and decreased nonclassical monocytes was observed. However, we observed a different pattern in sepsis patients compared with post-surgery patients and trauma patients as a considerable increase in the percentage of intermediate monocytes occurred in the sepsis patients. However, because sepsis patients usually develop symptoms days before admission to PICU, these patients may reflect a progressed course of disease compared with patients post-surgery or trauma, who usually are admitted to PICU at short notice. In adults, an increase in intermediate monocytes has been observed in a variety of infections, including bacterial and viral infections.12,38 Also in children with sepsis, a higher proportion of intermediate monocytes has been reported compared with healthy controls.22 However, in 30 children and young adults after hematopoietic stem cell transplantation, 11 of these patients developed sepsis, but no shift in monocyte subset distribution was observed.20 Thus, although we do not understand the clinical significance of monocyte subsets, these subsets might have specific functions and alterations in monocyte subset distribution could be disease-specific.12

Parenteral nutrition, in particular fatty acids and lipids, may adversely affect immune function, resulting in lower mHLA-DR expression.39–41 Patients included in this study participated in a randomized controlled trial on early versus late initiation of PN.23 At day 4 of PICU stay, early supplementation of PN was associated with a lower proportion of classical monocytes compared with withholding PN. We did not find significant differences in mHLA-DR expression between the randomization groups. However, this substudy was not aimed and thus not sufficiently powered to detect such differences; thus, the lower incidence of nosocomial infections reported in patients with late initiation of PN23 could potentially be associated with mHLA-DR expression.

We generated a small reference cohort for blood monocyte subset distribution and mHLA-DR expression. Previous studies in healthy individuals suggested dynamic changes of monocyte subsets and mHLA-DR expression in neonates,42 children21 and adults.43 In our cohort, the relative proportion of monocyte subsets and the percentage as well as MFI of HLA-DR-expressing monocytes did not vary with age. Also, no impact of gender on monocyte subset or mHLA-DR expression was observed. Despite the small number of controls, our data could be used for future studies because reference values of monocyte subsets and their HLA-DR expression in children are scarce.

This is the first study that examined HLA-DR expression on all 3 major blood monocyte subsets in a population of critically ill children, allowing us to compare children admitted for sepsis, post-surgery and after trauma. So far, a few other studies in children examined these major blood monocyte subsets in a longitudinal way but were all limited to 1 specific patient group.14,15,20,22 Our study is limited by the small number of patients included (resulting in high standard deviations) and missing values in a not-normally distributed dataset of repeated measures. Therefore, we unfortunately could not study alterations in mHLA-DR expression between different time points during PICU stay. However, we observed a clear trend toward decreasing mHLA-DR expression during PICU stay. For this same limitation, our findings on shifts in monocyte subset distribution need to be interpreted with caution. We found relatively small changes in monocyte subset distribution, which need to be validated in larger cohorts. A second important limitation is that we only examined 4 time points. For the total group of patients, nadir mHLA-DR expression was detected on day 4, but the declining trend was still ongoing. Therefore, the validity of our findings is limited by our inability to show a time to recovery of mHLA-DR expression. It is essential that future studies monitor mHLA-DR for a longer period of time. Prolonged decrease in mHLA-DR expression might be relevant for outcome and could be useful in predicting long-term complications. Also, we determined the proportion of HLA-DR expressing monocytes—which has a potential of interobserver variability—as well as the HLA-DR MFI on monocytes—that may depend on the type of flow cytometer and instrument settings used—making comparison between different laboratories troublesome. Therefore, a more standardized flow cytometry protocol, based on the generation of a calibration curve and an anti-HLA-DR antibody, conjugated 1:1 with a fluorochrome, which allows measurement of bound HLA-DR molecules per cell independently of the type of flow cytometer and instrument settings used, would allow better comparison of data between centers and studies.44,45 Finally, our data need to be interpreted with caution because we did not specifically exclude patients on immunosuppressive therapy, which might have influenced HLA-DR expression. Also, we found a significant association between low HLA-DR expression on classical monocytes and clinical complications only at days 2 and 3 and only in the percentage of HLA-DR-positive cells.

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In critically ill children, HLA-DR expression on all monocyte subsets decreased the first 4 days of PICU stay and was lower compared with controls on all examined time points, especially on classical monocytes and in children admitted for sepsis. Low HLA-DR expression on classical monocytes was associated with nosocomial infections and death. Future studies should include a larger cohort of children, including subgroups of different clinical infectious syndromes and different pathogens, and more time points to study the utility of mHLA-DR expression as a prognostic marker and a marker to guide future immunomodulatory therapy trials.

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HLA-DR antigens; antigens; CD14, infection/immunology; systemic inflammatory response syndrome; flow cytometry

Supplemental Digital Content

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