Clinical and Experimental Immunology

OR I G INA L A RTI CLE

doi:10.1111/cei.12810

Regulatory T cell frequencies are increased in preterm infants with clinical early-onset sepsis

J. Pagel,*† A. Hartz,*‡ J. Figge,‡ C. Gille,§ S. Eschweiler,‡ K. Petersen,* L. Schreiter,*‡ J. Hammer,* C. M. Karsten,‡ D. Friedrich,† E. Herting,* W. G€ opel,* J. Rupp† and C. H€artel*

Summary

Pediatrics, University Hospital Schleswig

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (Tregs) are potential candidates for the ontogenetic control of immune activation and tissue damage in preterm infants. It was the aim of our study to characterize lymphocyte subsets and in particular CD41CD251forkhead box protein 3 (FoxP3)1 Tregs in peripheral blood of well-phenotyped preterm infants (n 5 117; 23 1 0 – 36 1 6 weeks of gestational age) in the first 3 days of life in comparison to term infants and adults. We demonstrated a negative correlation of Treg frequencies and gestational age. Tregs were increased in blood samples of preterm infants compared to term infants and adults. Notably, we found an increased Treg frequency in preterm infants with clinical early-onset sepsis while cause of preterm delivery, e.g. chorioamnionitis, did not affect Treg frequencies. Our data suggest that Tregs apparently play an important role in maintaining maternal-fetal tolerance, which turns into an increased sepsis risk after preterm delivery. Functional analyses are needed in order to elucidate whether Tregs have potential as future target for diagnostics and therapeutics.

Holstein, Campus L€ ubeck, Ratzeburger Allee 160, D-23538 L€ ubeck, Germany.

Keywords: amniotic infection, early-onset sepsis, FoxP3, preterm infants,

E-mail: [email protected]

regulatory T cells

*Department of Pediatrics, University Clinic Schleswig Holstein, Campus L€ ubeck, †

Department of Infectious Diseases and Microbiology, University of L€ ubeck, ‡Institute for Systemic Inflammation Research, University of L€ ubeck, L€ ubeck, and §Department of Neonatology, University of T€ ubingen, T€ ubingen,

Germany

Accepted for publication 1 May 2016 Correspondence: Julia Pagel, Department of

Introduction Newborn infants, especially preterm infants, are highly susceptible to invasive infections which are leading causes for mortality and long-term morbidity [1–3]. Despite considerable advances in neonatal intensive care, 10–25% of very low birth weight (VLBW) infants still suffer from bloodculture-proven sepsis and 30–50% from clinical sepsis within the first weeks of life [2,3]. Thus, there is an urgent need to understand the complex pathophysiology of infections in these vulnerable infants and to characterize risk profiles to optimize prevention, diagnostics and therapy. The transition from intra- to extrauterine life is a major challenge for the neonatal immune system. Whereas the function of the tolerogenic state of the semi-allogeneic fetus is adapted to prevent potentially damaging inflammation, it might predispose postnatally to infection. Conversely, the ontogenetic specificity of temporary immunosuppression may allow the establishment of microbiota without major inflammation [4]. In term infants, several cell types such as T helper type 2 (Th2) cells with a specific cytokine profile [5,6], neutrophilic myeloid-derived

suppressor cells [7], erythroid CD 711 cells [8] and regulatory T cells (Treg) [9–11] are potential mediators of this immunosuppressive state. The situation of the preterm infant is even more complex, and is characterized by ineffective responses to pathogens, significant risk for sustained inflammation and dysbiosis [12–16]. In this context, the different underlying causes of preterm birth including inflammation at the feto–maternal interface may be regarded as a first major hit to the neonatal immune response. Forkhead box protein 3 (FoxP3)-expressing Tregs play an indispensable role in immunological self-tolerance and immune homeostasis as well as the control of immune responses to pathogens [17,18]. A negative correlation of Treg levels and gestational age has been shown in several studies [10,11,19,28]. To date, these studies were restricted to the use of cord blood. Moreover, to what extent Tregs contribute to the distinct susceptibility to infection of preterm infants is as yet unknown. In the chorioamnionitis milieu, the fetus is in direct contact with inflammatory cells and cytokines from the

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amniotic fluid. It has been shown in fetal sheep models that chorioamnionitis causes inflammatory changes in many organ systems after birth and a decrease in the number of Tregs in the fetal gut due to intra-amniotic interleukin (IL)-1a [27,28]. Decreased Treg levels have also been described in human cord blood from infants with chorioamnionitis [20]. Despite the important role of Tregs in mediating immunosuppression in adult patients with severe sepsis [21,22], to what extent Tregs contribute to the distinct susceptibility to infection of preterm infants is still unknown [23,24]. In our observational study we tested the hypothesis that preterm infants have higher Treg levels than term infants and adults. Moreover, we aimed to evaluate the association of Treg levels with amniotic infection and development of early-onset infection.

Early-onset sepsis (EOS). EOS was defined as sepsis occurring within the first 72 h of life. Clinical EOS. Clinical EOS was defined as the condition when neonatologists decided to treat the infant with antibiotics within the first 72 h of life and continue for at least 5 days due to the following reasons:  two clinical signs of systemic inflammatory response: temperature > 388C or < 3658C, tachycardia > 200/min, new-onset or increased frequency of bradycardias or apneas, hyperglycaemia > 140 mg/dl, base excess < 210 mval/l, changed skin colour and increased oxygen need; and one laboratory sign: C-reactive protein > 10 mg/l, platelet count < 100/nl, immature/total neutrophil ratio > 02 and white blood cell count < 5/nl [26,27]. Blood culture-confirmed sepsis. Blood culture-confirmed sepsis was defined as clinical sepsis with proof of causative agent in the blood culture.

Materials and methods Study cohort We performed a single-centre observational study and enrolled preterm infants as part of our IRoN (Immunoregulation of Neonates) study project. Data were collected from infants born between 1 October 2014 and 30 September 2015. The inclusion criteria were as follows: preterm infants with gestational age 23 1 0 and < 37 1 0 weeks with need for medical attention. Term infants and healthy adult blood donors served as controls. In a subgroup of infants, we were able to obtain maternal blood to evaluate levels of Tregs. After written informed consent was given by the parents, infants were enrolled into IRoN by the attending physicians.

Ethics Written informed consent was obtained from parents on behalf of the infants enrolled into our study. The study parts were approved by the local committee on research in human subjects at the University of L€ ubeck. All blood samples were obtained within a medically required blood withdrawal procedure. The additional blood volume obtained for research purposes (< 1% of whole body blood volume per blood sampling) was in line with current guidelines of the European Medical Agency on the investigation of medicinal products in term and preterm infants; Committee for Medicinal Products for Human Use and Pediatric Committee (PDCO, 2009).

Definitions Gestational age. Gestational age was calculated from the best obstetric estimate based on early prenatal ultrasound and obstetric examination. Small-for-gestational age (SGA) was defined as a birth weight less than the 10th percentile 220

for gestational age according to gender-specific standards for birth weight by gestational age in Germany [25].

Antenatal antibiotics. Antenatal antibiotics were defined as maternal treatment with antibiotics within the time-frame of  24h before delivery. Cause of preterm delivery. The cause of preterm delivery was determined at the discretion of the attending obstetrician, specifically: (1) preterm labour (labour refractory to tocolytic agents) or suspected amniotic infection syndrome [AIS; labour 6 rupture of membranes, increased maternal inflammatory markers without any other cause (CRP > 10 mg/l or elevation of white blood cell count > 16 000/ml)] but no clinical signs of severe AIS. Severe AIS was defined as maternal fever ( 3808C), increased maternal inflammatory markers without any other cause (CRP > 10 mg/l or elevation of white blood cell count > 16 000/ml), labour 6 rupture of membranes, fetal or maternal tachycardia, painful uterus and foul-smelling amniotic liquor; (2) pre-eclampsia (pregnancy-induced maternal hypertension, oedema, proteinuria); (3) pathological Doppler (e.g. Arteria umbilicalis Doppler, Ductus venosus flow, Arteria cerebri media Doppler) or intrauterine growth restriction as diagnosed by the attending specialist for antenatal ultrasound; and (4) others, including placental abruption, cholestasis, etc.

White blood cell counts and lymphocyte subsets White blood cell counts including numbers of lymphocytes were assessed as part of the clinical routine laboratory evaluation [ethylenediamine tetraacetic acid (EDTA) blood] by the central laboratory of the university hospital 24 h, 7 days a week. The determination of lymphocyte subsets (CD41/ CD81/CD191/CD561) in addition to white blood cell counts was performed on weekdays, Monday–Friday from 8 a.m. to 4 p.m.

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Analysis of Tregs derived from peripheral blood (EDTA) samples was performed in our research laboratory. Due to the limits of our study design (informed parental consent, preterm delivery on weekday, sufficient blood volume), we were not able to obtain blood samples for Treg quantification on days 1 and 3 of life of all infants. When Treg data were available on both days 1 and 3, Treg frequencies on day 1 of life were included in the analyses. If day 1 was not available, data from day 3 of life were used.

Cell separation and flow cytometry of Tregs The Treg percentage was determined via flow cytometry. EDTA whole blood samples were stored at room temperature and processed within 24 h after withdrawal. A cell viability test was performed on a regular basis to control for dead cells (eBioscience, San Diego, CA, USA). A whole blood staining with fluorochrome-labelled antibodies to characterize T cell populations was performed. For staining we used cell permeabilization and fixation reagents (FoxP3/Transcription Factor Staining Buffer Set; eBioscience). For multi-colour flow cytometry analysis, cells were first stained with surface antibodies specific for CD3/ fluorescein isothiocyanate (FITC) (eBioscience), CD4/phycoerythrin (PE) (Miltenyi Biotec, Bergisch Gladbach, Germany), CD25/Pacific Blue, BV421 (BioLegend, San Diego, CA, USA) followed by intranuclear staining for FoxP3 (APC/eFluor660; eBioscience). FoxP3 intranuclear staining was performed according to the manufacturer’s protocol following cell surface staining. The fixed and stained cells were diluted in fluorescence activated cell sorter (FACS) staining buffer (eBioscience) and stored directly at 48C. Flow cytometric analysis was performed within 4 days with a BD LSR II cytometer and analysed with FACS Diva software (BD Bioscience, San Jose, CA, USA) and FlowJo (Tree Star, Ashland, OR, USA). Tregs were determined by their position in the forward-/side-scatter plot (size/granularity) and co-expression of CD3, CD4, CD25 and FoxP3 (Supporting information, Fig. S1). Single antibody stainings were used to calculate the compensations. Fluorescence minus one (FMO) controls were used to establish gating boundaries and to identify any background spread of fluorochromes. CD127 antibody was used as a control for Treg gating strategy (Supporting information, Fig. S2).

Statistical analysis Data analysis was performed using the SPSS version 22.0 data analysis package (SPSS Inc., Munich, Germany). Differences between groups were evaluated with the v2, Fisher’s exact and Mann–Whitney U-tests and correlations were evaluated using Pearson’s test. A P-value < 005 was considered as statistically significant for single tests.

Results Study cohort During the observational period, 194 preterm infants aged between  23 10 and < 37 1 0 weeks of gestational age were eligible for our study. Parents of 37 infants were not approached for study participation and parents of four infants refused consent. In 117 of the remaining 153 preterm infants frequencies of lymphocyte subsets, including CD31CD41CD251FoxP31 Tregs, were measured within the first 3 days of life. Detailed clinical data of the study cohort are described in Table 1. The preterm infants were divided into three gestational age groups: group 1:  23 1 0 – 28 1 6 weeks, group 2:  29 1 0 – 32 1 6 and group 3:  33 1 0 – 36 1 6 weeks of gestational age.

Immune cell subsets in preterm infants We compared the immune cell subset counts of our study cohort on days 1 and 3 of life (Table 2). On day 1, white blood cell counts were significantly higher in preterm infant group 3, whereas there were no significant differences on day 3 of life. The lymphocytes were significantly higher in group 3 compared to group 1 on day 1 of life without any differences on day 3. We observed no group differences in lymphocyte subsets apart from a significantly lower rate of CD191 B cells in group 3 compared to group 1 on day 3 of life (P 5 001).

Preterm infants display higher frequencies of Tregs than term infants and adults In contrast to all other immune cell subsets, the percentage of CD31CD41CD251FoxP31 Tregs was significantly higher in group 1 compared to group 3 on both days 1 and 3 of life (Table 2). There were no significant differences of Treg frequencies between day 1 compared to day 3 of life in all groups. We noted significantly higher Treg frequencies in groups 1 and 2 compared to term infants (P 5 0006, P 5 0007, respectively), while no differences were observed between term infants and late preterm infants (i.e. group 3). Across all gestational age groups, preterm infants displayed higher Treg frequencies on either days 1 or 3 of life compared to adults (Fig. 1).

Preterm infants display higher Treg frequencies than their mothers In a subgroup of 22 mother–infant pairs [maternal mean 6 standard deviation (s.d.) age: 302 6 42 years; for infant characteristics see Table 1] we observed that preterm infants display higher levels of Tregs than their mothers (Fig. 2). This phenomenon might be independent of the cause of preterm delivery, i.e. mother–infant pairs with suspected/severe AIS: P 5 008, mother–infant pairs without AIS: P 5 007. Mothers of preterm infants did not differ in

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J. Pagel et al. Table 1. Clinical characteristics of the study cohort.

No. Gestational age (mean 6 s.d., weeks) Birth weight (mean 6 s.d., g) SGA (%) Gender (male, %) Mode of delivery (%) Spontaneous Elective C/S Emergency C/S Cause of delivery (%) Preterm labour or AIS Pre-eclampsia Pathological Doppler Others Antenatal antibiotics (%) AIS (%) Clinical EOS (%)† Outcomes RDS (%) IVH (%) IVH grade, 1, 2/3 or 4 PVL (%) NEC or FIP (%) ROP (%) BPD (%) Mortality (%) BPD or mortality (%)

All preterm infants

23 1 0–28 1 6 weeks

29 1 0–32 1 6 weeks

33 1 0–36 1 6 weeks

term

Subgroup mother–infant pairs

117 310 6 33 1624 6 648

29 259 6 14 812 6 218

44 312 6 12 1559 6 398

44 340 6 07 2223 6 355

8 393 6 10 3024 6 855

22 305 6 38 1610 6 775

85 46

69 59

68 32

114 52

0 38

7 46

16 76 8

10 83 7

16 73 11

21 75 5

86 14 0

23 73 4

63 9 22 6 54 44 19

69 0 28 3 72 72 45

55 14 32 0 52 46 16

69 9 9 13 43 25 5

29* 14 29 29 29 14 14

55 0 36 9 46 41 22

872 43 26/17 0 26 0 17 34 51

100 172 103/69 0 103 0 69 138 207

100 0 0 0 0 0 0 0 0

651 0 0 0 0 0 0 0 0

SGA 5 small for gestational age (birth weight < 10th percentile); C/S 5 Caesarean section; *term labour, AIS 5 amniotic infection syndrome; EOS 5 early-onset sepsis (no case of blood-culture-proven EOS was noted); RDS 5 respiratory distress syndrome developing after birth with any need for respiratory support for > 2 h; NEC 5 necrotizing enterocolitis or FIP 5 focal intestinal perforation: surgery for NEC or FIP; ROP 5 retinopathy of prematurity:  stage 2 with need for therapy; BPD 5 bronchopulmonary dysplasia: 36 weeks corrected age with oxygen need or need for continuous positive airway pressure (CPAP)/high flow; mortality 5 mortality during primary stay in hospital; PVL 5 periventricular leucomalacia; IVH 5 intraventricular haemorrhage; s.d. 5 standard deviation. †

Treg frequencies from non-pregnant adults (n 5 33; data not shown).

Percentage of Tregs correlates with gestational age With regard to clinical parameters, we noted no differences in Treg frequencies on days 1 or 3 of life for gender [mean/ median; 95% confidence interval (CI); female: n 5 63, 66/ 62; 59–73 versus male: n 5 54, 71/65; 59–83% CD41CD251FoxP31 cells, P 5 088], SGA (SGA: n 5 10, 66/57; 50–81 versus no SGA: n 5 107, 69/64; 62–76% CD41CD251FoxP31 cells, P 5 096) and exposure to antenatal antibiotics (antenatal antibiotics: n 5 63, 70/60; 60– 80 versus no antenatal antibiotics: n 5 54, 67/67; 58– 75% CD41CD251FoxP31 cells; P 5 092). There were no remarkable differences for Treg frequencies with respect to cause of preterm delivery, specifically AIS (Fig. 3). We 222

noted a trend for lower Treg frequencies on day 1 of life in severe AIS infants (n 5 5, 46/40, 95% CI 5 22–69) compared to controls (n 5 32, 69/60, 95% CI 5 56–01, P 5 003). The gestational age had a major impact on Treg frequencies in those infants who had no signs of clinical EOS (Fig. 4a). In contrast, Treg frequencies of EOS patients appeared to be regulated on an individual basis (Fig. 4b).

Clinical EOS is associated with higher Treg frequencies Preterm infants with clinical EOS (n 5 22; gestational age: mean 6 s.d. 281 6 37 weeks; birth weight: mean 6 s.d. 1121 6 613 g) had significantly higher Treg frequencies on days 1 or 3 of life than infants without EOS (n 5 95; gestational age: mean 6 s.d. 316 6 29 weeks; birth weight: mean 6 s.d. 1741 6 601 g; P 5 0008; Fig. 5). This

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Increased Tregs in early-onset sepsis Table 2. Immune cell count of the study cohort, preterm infants (n 5 117). Gestational age group White blood count, cells/nl [mean/median (95% CI, n)] White blood cells, total Day 1 Day 3 Lymphocytes, total Day 1 Day 3 Lymphocytes, cells/nl [mean/median (95% CI, n)] CD31 Day 1 Day 3 CD41 Day 1 Day 3 CD81 Day 1 Day 3 CD191 Day 1 Day 3 CD561 Day 1 Day 3 CD4/CD8 ratio Day 1 Day 3 CD251FoxP31 (% CD41) [mean/median (95% CI, n)] Day 1 Day 3 Days 1 or 3

1

Age group 2 (29 1 0–32 1 6 weeks)

Age group 3 (33 1 0–36 1 6 weeks)

123/92 (93–150; n 5 28) 154/131 (109–199; n 5 24)

116/111 (98–134; n 5 42) 103/88 (86–120; n 5 30)

132/124 (92–144; n 5 39)† 105/107 (90–119; n 5 29)

50/47 (38–62; n 5 23) 33/32 (26–40; n 5 18)

61/62 (53–70; n 5 37) 33/30 (25–41; n 5 22)

69/65 (61–76; n 5 39)* 59/30 (23–95; n 5 25)

45/43 (0–92; n 5 3) 23/23 (17–28; n 5 17)

34/26 (16–51; n 5 8) 27/23 (21–33; n 5 23)

42/43 (30–53; n 5 7) 30/28 (25–35; n 5 28)

35/32 (0–76; n 5 3) 17/18 (13–21; n 5 17)

25/18 (12–38; n 5 8) 21/17 (16–26; n 5 22)

30/32 (22–39; n 5 7) 23/21 (19–28; n 5 28)

11/12 (04–18; n 5 3) 057/057 (043–07; n 5 17)

092/077 (03–15; n 5 8) 06/056 (043–078; n 5 22)

12/12 (07–16; n 5 7) 065/067 (033–077; n 5 28)

17/15 (0–35; n 5 3) 060/057 (039–080; n 5 17)

14/11 (04–24; n 5 8) 038/032 (027–050; n 5 22)

11/11 (07–16; n 5 7) 036/028 (025–047; n 5 27)*

16/11 (0–45; n 5 3) 025/017 (017–034; n 5 17)

13/13 (06–2; n 5 8) 019/019 (014–025; n 5 22)

16/15 (10–22; n 5 7) 023/020 (017–029; n 5 27)

30/27 (11–49; n 5 3) 32/29 (26–38; n 5 17)

34/34 (23–44; n 5 8) 39/33 (31–47; n 5 22)

28/28 (23–32; n 5 7) 39/35 (32–46; n 5 27)

82/77 (58–106; n 5 16) 89/72 (62–116; n 5 21) 87/74 (59–72; n 5 29)

60/60 (52–68; n 5 22) 64/65 (56–72; n 5 36) 65/67 (59–72; n 5 44)

58/52 (50–67; n 5 24)* 59/58 (50–68; n 5 30)* 59/53 (53–66; n 5 44)*

*Significant P-values age groups 1 versus 3; †significant P-values age groups 2 versus 3. FoxP3 5 forkhead box P3; CI 5confidence interval.

observation was explained mainly by differences on day 3 of life (mean 6 s.d. % FoxP31CD41cells, EOS: n 5 18, 91 6 64% versus no EOS: n 5 69, 62 6 24%, P 5 003) but not on day 1 [mean 6 s.d. % FoxP31CD41 cells, EOS: n 5 11, 68 6 27% versus no EOS: n 5 51, 64 6 31%, not significant (n.s.)]. In a subgroup of infants with available Treg data on days 1 and 3 of life (n 5 27) we noted no significant differences between both time-points (mean/ median; 95% CI 5 FoxP31CD41 cells; day 1: 67/60; 53– 80% versus day 3: 65/64; 53–77% FoxP31CD41 cells, P 5 07). The analysis of whole blood cell count and lymphocyte subsets in preterm infants revealed no differences between EOS and no EOS patients on days 1 and 3 apart from lower white blood cell counts on day 1 in EOS patients (mean/ median 6 s.d. number/nl; EOS, n 5 16: 121/81 6 92 versus no EOS, n 5 82: 122/116 6 45; P 5 003) and lower lymphocyte counts in EOS patients on day 1 (mean/

median 6 s.d. number/nl; EOS: 46/46 6 26 versus no EOS: 65/64 6 24, P 5 0014).

Discussion In this single-centre observational study we investigated the frequencies of CD31CD41CD251FoxP31 Tregs in peripheral blood of 117 preterm infants in the first days of life. We found that Treg frequencies correlate negatively with gestational age. In particular, extremely preterm infants < 29 weeks display higher Treg frequencies than term infants and adults. As such, preterm infants have higher Treg frequencies than their mothers directly after birth. Interestingly, infants who develop clinical EOS had an increased Treg frequency which is not affected by gestational age. In contrast, AIS as cause of preterm delivery did not affect Treg numbers in our setting.

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Fig. 1. The frequency of regulatory T cells (Tregs) is higher in preterm infants than in adults. Box-plots [median, interquartile range (IQR), 95% confidence interval (CI)] describe the frequencies of Tregs across groups of different gestational age. Two outliers [230% and 310% CD41CD251forkhead box protein 3 (FoxP31) cells] were not depicted. Preterm but not term infants display higher frequencies of Tregs compared to healthy adult blood donors (n 5 33; mean age 403 6 11 years; median 425, 95% CI 5 363–443; Tregs mean % 43 6 11, 95% CI 5 39–47).

There is a paucity of immunological studies performed with peripheral whole blood of the most susceptible population of VLBW infants. Despite the limited sample volumes obtainable, peripheral blood sampling has several advantages in the complex setting of preterm birth. The blood is taken by the attending neonatologist when a clini-

Fig. 2. Preterm infants display higher regulatory T cell (Treg) frequencies than their mothers. Maternal Treg frequencies [grey boxplots; median, interquartile range (IQR), 95% confidence interval (CI)] were available directly after birth and compared with Treg data of their infants (white box-plots).

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Fig. 3. Regulatory T cell (Treg) frequencies are not associated with clinical amniotic infection syndrome (AIS). Data are depicted as box-plots [median, interquartile range (IQR), 95% confidence interval (CI)] for infants without preterm labor/suspected AIS (white), suspected AIS (grey) and severe AIS (red).

cal blood sample is withdrawn for routine laboratory use. This blood sampling is performed even in emergency situations such as placental abruption. However, in most settings cord blood sampling is not performed by the neonatologist; cord blood might be needed for other purposes (blood group, storage, etc.) and the placenta might not be double-clamped for blood withdrawal or sent for histological examination. Moreover, the reliability of cord blood sampling (blood withdrawal from the umbilical vein rather than umbilical artery) is highly variable, which enhances the likelihood of contamination with maternal cells. To our knowledge, our large cohort of preterm infants includes this specific patient group for the first time. Although some data sets are incomplete due to limited sample volume and pre-laboratory time restrictions, our study enabled us to investigate the influence of the Treg frequency on the clinical outcomes within the first days of life. As Tregs are crucial for immune tolerance and inhibit potentially harmful proinflammatory immune responses, our findings have implications for the understanding of the immune regulation of preterm infants. The negative correlation of Treg frequency and gestational age underlines the potential role of fetal Tregs to promote self-tolerance and tolerance to maternal antigens actively [9,19,28]. Conversely, our data suggest a unique role for immunoregulation in the first days of life. During pregnancy a substantial number of maternal immune cells infiltrate the fetal blood and colonize in fetal lymphoid tissues. This maternal alloantigen stimulus induces the development of fetal Tregs that maintain maternal–fetal tolerance [28,29] and therefore explains the high percentages of Tregs in early preterm compared to full term neonates [20,30,31].

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Fig. 4. (a) Regulatory T cell (Treg) frequencies correlate negatively with gestational age in non-infected infants. The graph depicts the negative correlation between gestational age and Treg frequencies in the first 3 days of life in those infants who were not clinically infected [n 5 101; R 5 Pearson’s correlation; one outlier not depicted (23%)]. The same observation was made for Tregs on day 1 (n 5 54; R 5 2041, P 5 0002) and day 3 (n 5 72; R 5 026, P 5 002). (b) No correlation between gestational age and Treg frequencies in infants with clinical early-onset sepsis (EOS). In infants with clinical infection, no correlation between gestational age and Treg frequencies in the first 3 days of life was demonstrated [n 5 23; R 5 2024, P 5 026; one outlier not depicted (31%)]. The same observation was made for Tregs on day 1 (n 5 11; R 5 2018, P 5 06) and day 3 (n 5 19; R 5 034, P 5 015).

The main novel finding of our analysis is that infants with clinical EOS have significantly higher Treg frequencies than infants without EOS. The analysis of Tregs from days 1 and 3 of life in these patients revealed no significant difference. Interestingly, recent studies in adult patients with severe sepsis or septic shock demonstrated an increase in number and function of Tregs during the course of disease and a positive correlation between Treg level and the mor-

Fig. 5. Preterm infants with clinical early-onset sepsis (EOS) have increased regulatory T cell (Treg) frequencies. Data are depicted as box-plots [median, interquartile range (IQR), 95% confidence interval (CI); white: no EOS, grey: EOS]. Five of 22 preterm infants with EOS were born after severe amniotic infection syndrome (AIS), 10 of 22 infants due to preterm labour/suspicion of AIS and seven of 22 due to other causes.

tality rate of the patients [21,22]. We hypothesize that Tregs may cause a temporary down-regulation of central host immune functions after birth and therefore enable establishment of microbiota. Conversely, our data lead to the hypothesis that high Treg rates may cause an uncontrolled immunosuppression and therefore result in an increased sepsis risk for the individual preterm infant in the first days of life. Thus, we propose Tregs as a potential diagnostic target, especially for the most vulnerable VLBW infants during the first days of life. Animal models are needed to reveal functional relevance for neonatal sepsis. Future studies will aim at the development of an individualized diagnostic and treatment protocol for each preterm infant including, immune therapeutic agents, which ameliorate the prognosis of the patients. Currently, there are several clinical trials on immune therapy in sepsis in adult patients with diverse outcome [32]. Our data showed no difference in the Treg levels in patients with AIS compared to no AIS infants in the first days of life. This is in contrast with previous studies, which differed in methodology using cord blood instead of peripheral blood [20]. In fetal AIS sheep models, it was shown that the number of Tregs in the fetal gut, thymus and lymph nodes is decreased due to intra-amniotic IL-1a [33–37]. In the same models, these studies demonstrated that chorioamnionitis causes inflammatory changes in many organ systems after birth. We therefore argue that there is an increase of the Treg rate in the transition from the in-utero situation to the first days of life in the case of AIS. Moreover, this finding indicates that maternal rather

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than fetal immune reactions may participate in the pathogenesis of gestational-associated diseases causing prematurity. In-vivo studies are needed to test the functional relevance of this finding and whether manipulation of the Treg-induced immunological changes could ameliorate the prognosis of preterm infants with severe AIS. The major strengths of our study are the large sample size and detailed phenotypical characterization of infants. There are potential limitations, e.g. single-centre observation which do not allow extrapolating our data for multicentre approaches. Secondly, there is an ongoing controversial discussion about characterization of Tregs with different gating strategies. We used the CD41CD251FoxP31-staining approach, which may include contamination by effector T cells. Alternative Treg gating strategies include CD41CD251CD127low1/– –, CD41FoxP31CD127low1/– – or even CD41CD251FoxP31CD127low1/– – Tregs. We used the latter gating strategy as a control in five samples and found similar results to the CD41CD251FoxP31-staining approach (Supporting information, Fig. S2). Thirdly, our study is hypothesis-generating, a functional relationship of both Tregs and effector T cells, and has yet to be examined in infants affected with EOS compared to non-affected children. In addition to purely quantitative research, future studies are needed to define and evaluate the effects of Tregs on the development of EOS in preterm infants, including the interaction of neonatal Tregs with other immunoregulatory cell types of innate and adaptive immunity. In line with this, Tregs have been described to exert immunosuppression on the proliferation of naive T cells and the effector function of differentiated T cells, B cells, NK cells, macrophages and dendritic cells [38]. In studies performed with human cord blood or animal studies it has been reported recently that Tregs from preterm infants have less suppressive capacity than those from term neonates, whereas the latter were similar to those of adults [19,20,39,40]. In-vivo studies could further reveal the question whether individual manipulation of Tregs in preterm infants, either boosting or blocking regulatory responses, could be a future therapeutic option. In addition, a longitudinal study with preterm infants, especially VLBW infants, investigating the relationship between changes in Treg proportions and clinical outcomes such as development of sepsis, necrotizing enterocolitis and bronchopulmonary dysplasia, are future tasks.

Acknowledgements We are indebted to the patients and their families for their participation and the physicians of the local NICU for their generous collaboration in this study. We would like to thank Constanze Siggel and Tillman Vollbrandt for the excellent technical support with the FACS experiments. This work was supported by grants from the German Center for Infec226

tion Research (DZIF). J. P. was supported by a grant of the German Centre of Infection Research (DZIF; financed by the German Ministry of Education and Research, BMBF). A. H., L. S. and D.F. were supported by grants from the IRTG 1911 (financed by the German Society of Research, DFG).

Author contributions Experimental concept and design: J. P., C. H., C. G. and J. R. Performance/realization of experiments: J. P., A. H., K. P., J. H. and L. S. Contribution of reagents/materials/analysis tools: J. P., A. H., J. F., S. E., D. F., C. H., C. G., C. M. K., J. R., W. G. and E. H. Data analysis: J. P., C. H., J. R. and W. G. Writing of paper: J. P. and C. H.

Disclosure These authors declare no conflicts of interest.

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Supporting information Additional Supporting information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Flow cytometry analysis of CD31CD41CD251 forkhead box protein 3 (FoxP31) regulatory T cells (Tregs). The gating strategy for Tregs is presented. The numbers in the panels indicate the frequency of the gated cells in percentages (FSC 5 forward-scatter; SSC 5 sidescatter). Fig. S2. Flow cytometry analysis of CD31CD41CD251 forkhead box protein 3 (FoxP31)CD127– regulatory T cells (Tregs). The gating strategy for FoxP31 and CD127– Tregs is presented. The numbers in the panels indicate the frequency of the gated cells in percentages (FSC 5 forward-scatter; SSC 5 side-scatter).

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Regulatory T cell frequencies are increased in preterm infants with clinical early-onset sepsis.

The predisposition of preterm neonates to invasive infection is, as yet, incompletely understood. Regulatory T cells (Tregs ) are potential candidates...
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