Functional analysis of granulocyte and monocyte subpopulations in neonates

Healthy term neonates were recruited between July 2016 to March 2017 and May 2018 to September 2018 at the University Medicine Greifswald. Healthy young adults served as controls.

Monocyte and granulocyte subpopulations were analysed in 11 healthy term neonates (mean gestational age = 39 weeks + 2 days (SD, 1 weeks + 2 days); male = 7, female = 4; birth mode: spontaneous = 1; primary caesarean section = 9; secondary caesarean section = 1) and 10 young healthy adults (mean age = 23.5 years (SD = 4.9 years), male = 2, female = 8).

For ROS production experiments samples of 13 healthy term neonates (mean gestational age, 38 weeks + 4 days (SD, 0 weeks + 6 days); male = 5, female = 8; birth mode: spontaneous = 5; primary caesarean section = 8; secondary caesarean section = 0) and 11 young healthy adults (mean age, 25 years (SD, 6.4 years); male = 5, female = 6) were used.

Exclusion criteria were severe congenital malformations, chromosomal aberrations, perinatal infection or chorioamnionitis and lack of written consent (see Additional file 1: Table S1 for details).

EDTA cord blood was sampled at birth and processed within 2 h to examine neonatal monocytes and granulocytes. After a red blood cell lysis using ACK lysing buffer (155 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) to induce cell swelling and rupture of membranes, cells were stained by Zombie NIR™ Fixable Viability Kit (BioLegend™) on ice for 15 min to distinguish dead and alive cells, followed by a second staining with the different cell surface antibodies for 10 min on ice. Cells were fixed in 1% paraformaldehyde (PFA). Immune cell subpopulations of monocytes and granulocytes were analysed by flow cytometry (BD LSR II) as defined by anti-HLA-DR Alexa Flour 488, anti-CD11b Brilliant Violet 421, anti-CD14 PerCP/Cy5.5, anti-CD16 Brilliant Violet 650, anti-CD62L PE-Cy7, anti-TLR-2 Alexa 647 and anti-CD32-PE (BioLegend). The results were evaluated using FlowJo Software 7.6.5 (Tree Star Inc., Ashland, OR, USA). Results are shown as percentages of granulocytes, monocytes or their subpopulations. To display the expression of activation markers, mean fluorescence intensity (MFI) was used. For the differentiation of monocytes and granulocyte subpopulation as well as activation marker, fluorescence minus one controls (FMO) were used. CD14^dim monocyte and CD16^dim neutrophil population was distinguished by gating the 25th percentile of main monocyte and neutrophil population, respectively (Additional file 2: Figure S1A/B).

ROS production was quantified by flow cytometry using the Phagoburst Kit (Glycotope Biotechnology GmbH) according to manufacturer’s instructions. Briefly, heparinized cord blood was incubated on ice with anti-CD14 APC/Cy7, anti-CD16 APC and anti-CD62L Brilliant Violet 421 antibodies (Biolegend). For detailed gating strategy see Additional file 3: Figure S2. Cells either remained unstimulated or were incubated with unlabeled opsonized E. coli (0.9–1.8 × 10⁸/ml), phorbol 12-myristate 13-acetate (PMA) (0.74 μM), or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (0.45 μM) as stimulants for 10 min at 37 °C; subsequently, DHR was added for 10 min which—by ROS-dependent conversion into rhodamine 123—allowed the quantification of reactive oxidants and determination of the percentage of phagocytes that produced ROS. The ROS production per cell was quantified by MFI. Kit-included DNA-Dye was used after red blood cell lysis using PFA-containing BD FACS™ Lysing Solution to differentiate between E. coli and cells. The flow cytometry results were evaluated with FlowJo Software 10.3 (Tree Star Inc., Ashland, OR). Gating of subpopulation was done as described above (Additional file 2: Figure S1 A/B; Additional file 3: Figure S2).

Compared with adult controls neonates showed a reduced percentage of classical granulocytes CD16⁺CD62L⁺cells while proinflammatory CD16^dimCD62L⁺cells were increased. Anti-inflammatory CD16⁺CD62L⁻ cells were not altered in comparison with controls (Fig. 1a–d).

To analyse the activation profile of granulocytes and their subpopulations in comparison with controls, we analysed the percentage of CD11b, CD32 and TLR-2 expressing cells as well as the amount of activation marker per cell by MFI.

Reduced percentages of CD11b positive granulocytes and CD32 positive granulocytes were detected in neonates in comparison with controls. Neonates showed a higher percentage of TLR-2 on all granulocyte subpopulations compared with controls (Table 1). The amount of TLR-2 expression on proinflammatory CD16^dimCD62L⁺ granulocytes was decreased in neonatal cord blood (Fig. 2).

Percentage of ROS-producing granulocyte subpopulation and ROS amount per cell (MFI) was quantified (Fig. 3). Neonates showed enhanced ROS amount per cell for classical CD16⁺CD62L⁺ and anti-inflammatory CD16⁺CD62L⁻ subpopulation in unstimulated and fMLP-stimulated cells. In addition, ROS amount per cell was upregulated in proinflammatory CD16^dimCD62L⁺cells after PMA stimulation. In E. coli–stimulated samples all neonatal granulocytes as well as their subpopulations showed a higher ROS production per cell in comparison with controls. A lower percentage of ROS-producing proinflammatory CD16^dimCD62L⁺cells was measured for unstimulated and fMLP-stimulated cells in neonates. PMA stimulation induced a reduced percentage of ROS-producing anti-inflammatory CD16⁺CD62L⁻. An explorative subanalysis of sex showed no significant differences (p ≥ 0.9999; data not shown).

Monocyte subpopulations were differentially regulated in neonates compared with controls. Classical CD14⁺CD16⁺ cells were enhanced in neonatal cord blood in comparison with lower number of anti-inflammatory CD14⁺CD16⁻ cells and unaltered proinflammatory CD14^dimCD16⁺ cells (Fig. 1e–h).

To analyse the activation profile of monocytes in comparison with healthy controls, percentages of HLA-DR, CD11b, CD62L and TLR-2 bearing cells as well as the amount per cell (MFI) were analysed.

Percentage of CD11b, CD62L, HLA-DR and TLR-2 bearing monocytes was reduced in neonatal compared with adult monocytes. The amount of CD62L and HLA-DR was significantly decreased on neonatal monocytes (Table 1).

Percentages of CD32, TLR-2 and CD11b positive classical CD14⁺CD16⁻ monocytes were decreased in neonates compared with controls. Neonatal CD14⁺CD16⁻ monocytes showed a significant decrease in CD62L positive cells and CD62L amount on cell surface. In anti-inflammatory CD14⁺CD16⁺ cells CD62L amount was reduced (Table 1). The decrease of HLA-DR positive cells in neonates as well as the decrease of HLA-DR amount was measured for all three monocyte subpopulations (Fig. 4).

Three different granulocyte subpopulations, classical CD16⁺CD62L⁺, anti-inflammatory CD16⁺CD62L⁻ and proinflammatory CD16^dimCD62L⁺, were identified in adult blood [8] (Fig. 1a–c). In neonates, this study is the first to show that classical granulocytes are downregulated, while proinflammatory granulocytes are enhanced in neonates compared with adults.

Percentage of TLR-2 was enhanced on all granulocytes and their subpopulations while the TLR-2 expression was only reduced on proinflammatory granulocytes (Fig. 2e–g). TLR-2 recognizes a large number of ligands, especially on gram-positive bacteria [17, 18]. Apart from the well-characterized TLR-2-mediated inflammation, data exist supporting the notion that TLR-2 signalling can lead to the production of the anti-inflammatory cytokine Interleukin 10 [19, 20]. Therefore, differently regulated TLR-2 might be partly responsible for the diminished inflammatory neonatal immune response. Of note, expression of soluble factors in cord blood impairs TLR-4-mediated IL-12p70 production and enhances TLR-4-mediated IL-10 production over the first weeks of life [21].

Although percentages of CD11b and CD32 were downregulated on granulocytes in general, we could not detect these alterations in granulocyte subpopulations (Table 1). The reduction of CD11b in neonates is in line with the finding that a downregulation of CR3 complexes (CD11b/CD18) leads to an impaired recognition of gram-negative bacteria dependant on gestational age [12].

Our results indicate that neutrophils of term infants are partly diminished in their ROS production, especially in proinflammatory granulocytes (Fig. 4a–f). The reduced ROS production is in line with Usmani et al. [13]. Gessler et al. measured no difference in the percentage of neutrophils undergoing respiratory burst after being stimulated by fMLP or E. coli [14]. Similarly, we did not observe any alterations for granulocytes in general stimulated by fMLP and E. coli in ROS production. Nevertheless, granulocyte subpopulations showed an enhanced ROS production per cell especially after E. coli stimulation. Also Shigeoka et al. reported an enhanced ROS response, especially in stressed neutrophils [16]. The different findings within these studies might be due to divergent experimental setups in choice of stimulants and ROS production readouts. In our study DHR conversion by superoxide radicals into rhodamine is quantified [22]. These radicals are generated by NADPH oxidase which quantitatively differs between neonates and adults [23]. The enhanced NAPDH oxidase activity per cell in contrast to the diminished ROS response of proinflammatory granulocytes might be an indicator of balance between neonatal deficits on the one hand and intact immune responses to fight pathogens effectively on the other hand.

Our data showed an enhanced percentage of anti-inflammatory monocytes while classical monocytes were decreased compared with adults (Fig. 1e–g). Others did not find those differences [24‐26]. This might be due to the different classification of monocyte subpopulations since Sohlberg et al. as well as Murphy et al. only distinguished into two major subpopulations, CD14⁺⁺CD16⁻ and CD14⁺CD16⁺ cells. Wisgrill et al. analysed counts of cells while our data compare subpopulation percentages. Especially the CD14⁺CD16⁺ monocytes have anti-inflammatory properties by the secretion of Interleukin 10 [27]. In addition to the lower frequencies of classical monocytes which are confirmed by the study of Sharma et al. in our cohort, these alterations might protect the host from overshooting proinflammatory immune responses facing invading pathogens [28]. In contrast to Sharma et al. we show an increase of anti-inflammatory monocytes without an alteration of inflammatory monocytes, which might be due to differences in gestational age and number of analysed subjects.

We found reduced monocytic HLA-DR expression resembling result by Nguyen et al. and Wisgrill et al. [25, 29]. Our study can expand this knowledge, since our data show a reduced percentage of HLA-DR positive cells in all monocyte subpopulations (Fig. 4a–f). Especially the reduction of CD62L on classical and anti-inflammatory monocytes seems to be a unique neonatal finding in our data since data from adult blood monocytes show CD62L expression especially on these both cell subpopulations but not in proinflammatory monocytes (Table 1) [30, 31].

This study only considers the production of ROS in vitro, but not its effectiveness in the killing of pathogens. E. coli as bacterial stimulus of ROS production has been described to play a role in the induction of Escherichia coli early-onset sepsis [32] as an important cause of mortality and morbidity in neonates. However our data cannot specify whether E. coli–induced ROS productions influence the clinical outcome of children. Also, only a limited amount of stimuli could be applied. Since Droussou et al. show an impaired respiratory burst in neonates challenged by sepsis, clinical or experimental ‘stress’ [33], the exact connection between differently regulated monocytes and granulocyte subpopulation, their activation state and the risk of infection still has to be revealed.

Our experiments analysed relative changes of immune cell subpopulations in neonates in comparison with healthy adults, but not absolute counts. Since we cannot provide any tendency for the influence of gestational age, birth weight and the incidence of infection, larger cohort particularly including preterm infants is needed.