The neonates, particularly small-for-gestational-age (SGA) ones, are susceptible to various microbial infections. Natural killer (NK) cells are critical components of host innate immunity system and the main source of the inflammatory cytokines, which provide critical protection during the early phase of viral infections before the development of an appropriate adaptive immune response. However, little is known about the antiviral effects of NK cells in neonates especially the SGA population. Herein, a prospective descriptive study was performed to determine the differences of NK cell immunity among adults, appropriate-for gestational-age (AGA) and SGA neonates. Adults have much higher NK cell number in peripheral blood than that in cord blood from neonates. In response to influenza virus stimulation, neonatal NK cells, especially SGA baby cells, expressed significantly lower antiviral cytokines including perforin, interferon (IFN)-γ and tumor-necrosis factor (TNF)-α responses than adult NK cells. In addition, the antiviral cytokine responses of NK cells were positively correlated with neonatal birth weight. Our data suggested that the depressed antiviral activity and less frequency of NK cells are likely to be responsible for the high susceptibility to microbial infection in neonates, at least in part. Improving the function of innate immunity may provide a new way to defend virus infection.
Infection has large contribution to perinatal mortality and morbidity in neonates, especially small-for-gestational-age (SGA) ones.1 The susceptibility to various infection is thought to be caused by immaturity of the innate immunity, which is dependent on gestational age and birth weight.2,3 The innate immune system mount an efficient antiviral response in the early life, which includes monocytes, phagocytes, natural killer (NK) cells, Vγ9Vδ2-T cells and other recognition and effector elements.4 NK cells are key effector cells in the innate immunity, which play a critical role in the first line of host defense against acute viral infections by directly killing infected cells and inhibiting virus replication through secretion of antiviral cytokines.5,6
NK cells are broadly reactive against different kinds of virus, such as influenza virus, herpes viruses, hepatitis B virus and Marburg virus, indicating a general role of the cells in antiviral immune responses.6,7,8 Antiviral cytokine expression is a main mechanism to inhibit virus replication and eliminate virus. Previous studies have demonstrated that NK cells from the peripheral blood had obvious secretion of interferon (IFN)-γ and tumor-necrosis factor (TNF)-α against virus but low response in appropriate-for-gestational-age (AGA) neonates.9,10,11,12,13 However, little is known about the antiviral function of NK cells in SGA neonates, neither about the relationship of NK cell function with neonatal birth weight.
In this study, we investigated the antiviral response of NK cells in cord blood. It was found that in response to influenza virus stimulation, NK cells of SGA neonates expressed much lower antiviral cytokines than adult NK cells. A positive correlation between neonatal birth weight and antiviral cytokine expression of NK cells was indicated.
Materials and methods
The cord blood was collected at the Division of Neonatology, Department of Pediatrics, Second Hospital of Sichuan University, from January 2010 to March 2012. The informed consent was obtained from the parents of the newborns. The peripheral blood was collected from healthy adult volunteers at the same time. The research protocol was approved by the Institutional Review Board of the University Hospital of Sichuan University. Detailed information and clinical features of the recruited subjects were obtained on general sociodemographic habits (education, marital status, maternal age), personal characteristics (weight, Head circumference, intake of vitamin A/D), gynaecological and obstetric history, and smoking consumption in pregnancy. No sign of distress, infection during intrauterine life or evident congenital anomalies was shown. All blood samples were collected into sodium heparin tubes.
Cell preparation and influenza virus stimulation
Peripheral blood mononuclear cells (PBMCs) and cord blood mononuclear cells (CBMCs) were isolated with Ficoll-Hypaque (Pharmacia, Sweden) gradient centrifugation as we described before.14 Human influenza A virus, A/PR/8/34 was kindly provided by the Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, and cultured in Madin–Darby canine kidney cells. The virus titer was determined by daily observation of cytopathic effect, and the median tissue culture infective dose (TCID50) was calculated according to the Reed–Muench formula. PBMCs or CBMCs of same number were infected with influenza A virus at a multiplicity of infection (MOI) of 2 and cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS). The brefeldin A (Sigma-Aldrich, St Louis, MO, USA) was added to final concentration of 10 µg/ml 4 h before the end of culture. Cells and supernatants were then collected after 24 h of culture, and the perforin, IFN-γ and TNF-α-secreting cells were examined by intracellular cytokine staining as we described before.15
Cytometric bead array analysis
PBMCs or CBMCs were infected with influenza A virus at a multiplicity of infection (MOI) of 2 and cultured in RPMI-1640 media supplemented with 10% fetal bovine serum for 24 h. The culture supernatants were collected and cytokine (TNF-α and IFN-γ) concentrations were measured using cytometric bead array (Becton & Dickinson, New Jersey, USA) as we described previously.16 Data were acquired by flow cytometry and analysed with cytometric bead array software. Standard curves for each cytokine were generated to calculate their concentration in the tested samples.
Flow cytometric analysis
Cells were stained for cell surface markers with the following antibodies: mouse anti-human CD56-PE to identify NK cells. In some experiments, the cells were further fixed with lysis buffer (eBioscience, California, USA) for 30 min, permeabilized with permeabilization buffer (eBioscience, California, USA) for 30 min and then stained with anti-human Perforin-FITC, anti-human TNF-α-APC and anti-human IFN-γ-PE-Cy7 (BD Biosciences, New Jersey, USA) as we did before.17 All samples were acquired on a Gallios (Beckman Coulter, California, USA) and analysed by FlowJo software (version 7.6.5; Tree Star, California, USA). Forward scatter and side scatter were used to indentify lymphocytes, which was further used to define NK cells and intracellular expressions of perforin, TNF-α and INF-γ.
Data were expressed as means±standard errors. Differences between the normal and stimulated cells were analysed by paired t-tests. ANOVA tests were used to compare the various fold difference among the groups. Pearson r test was used to estimate the correlation between the birth weight and NK response. P<0.05 was considered to be significant.
The clinical samples
A total of 41 neonates and 17 healthy adults (PB) were enrolled in this study. There were 20 SGA (term and near term >35-week gestation, birth weight in the lowest 10th percentile) infants and 21 AGA ones. Table 1 presents descriptive statistics of maternal and paternal variables considered in this investigation. Based on the selection criteria, the means of birth weight and head circumference were significantly lower in the SGA neonates, compared with the AGA neonates. Between the two groups, there was a marked difference in maternal age of the mothers. The mothers of AGA neonates exhibited more adequate prenatal care and enough application of the vitamin A/D. The smoking status and pregnancy-induced hypertension were more frequent in SGA pregnant group than these in AGA pregnant group (P<0.05). The gender distribution, Apgar score and parental education levels between SGA and AGA neonates were comparable (Table 1).
Impaired antiviral cytokine production of CBMCs from SGA neonates
After PBMCs and CBMCs were stimulated with influenza virus for 24 h, the supernatants were collected and examined for antiviral cytokine expression. As shown in Figure 1a, in SGA neonates, the IFN-γ concentration (121.63±5.56 pg/ml) was significantly lower than that in AGA (276.66±10.97 pg/ml) and adult (542.91±40.19 pg/ml). The TNF-α production in SGA 192.37±8.81 pg/ml and AGA (215.05±12.33 pg/ml) neonates was comparable, but both of them were much lower than that produced in adults (375.58±17.95 pg/ml) (Figure 1b).
Lower percentage of resting NK cells and less frequency of perforin expression in SGA neonates
In order to determine the possible reason of the lower concentration of the antiviral cytokines in supernatants, we firstly examined the quantity of resting NK cells in the total lymphocytes of different groups by flow cytometry. The neonates had lower percentage of NK cells in the blood than adults. The perforin expression in resting NK cells was also significantly higher in adults than that in AGA or SGA neonates. There were no differences between AGA and SGA neonates in terms of the percentages of resting NK cells and their perforin expressions (Figure 2a and b). Furthermore, the expressions of antiviral cytokines IFN-γ and TNF-α in resting NK cells were comparable among these three groups (Figure 2c and d).
Reduced cytolytic granule expression in NK cells from SGA neonates upon influenza virus stimulation
To determine the cytolytic granule expression of NK cells upon influenza virus stimulation, CBMC and PBMC were stimulated with influenza H1N1 virus, and the perforin expression in NK cells was analysed by FACS. As shown in Figure 3a and c, the influenza virus significantly increased perforin expression in NK cells from all adults, AGA and SGA neonates. However, the increase fold of perforin in SGA neonates was significantly lower than those in both adult and AGA neonates after influenza virus stimulation (Figure 3b).
Decreased antiviral cytokine response in NK cells from SGA neonates upon influenza virus stimulation
We further determined IFN-γ and TNF-α responses of NK cells induced by influenza virus stimulation. After 24 h of stimulation, the expressions of IFN-γ and TNF-α were significantly enhanced in NK cells of all adults, AGA and SGA neonates (Figure 4a c and e). The fold increase of either IFN-γ or TNF-α in adults was much higher than that in neonates. In addition, among the neonates, AGA neonates showed significantly higher fold increase of these two antiviral cytokines than SGA neonates (Figure 4b and d).
Correlation of antiviral cytokines secreted by NK cells with birth weight
The relationship of neonatal birth weight with NK cell function was further examined. CBMCs of neonates with distinct birth weight were stimulated with influenza virus. The expressions of perforin, IFN-γ and TNF-α were examined by flow cytometry. As shown in Figure 5, with the increase of neonatal birth weight, the fold increases of perforin, IFN-γ and TNF-α expressions within NK cells were also increased, which indicated a positive correlation of influenza virus-induced NK cell response with neonatal birth weight.
SGA neonates, defined as those with birth weight below the 10th percentile for gestational age, are mainly caused by intrauterine growth retardation.2 Intrauterine growth retardation creates an adverse intrauterine environment to the fetus, which leads to retarded development in organs including the immune system.18,19 The SGA neonates are highly susceptible to life-threatening infection with a high rate of perinatal morbidity and mortality due to their juvenility of adaptive and innate immune system.2,3 A series of studies have already showed the impaired adaptive immunity in SGA neonates.18,20,21,22,23,24,25 Innate immune system plays an important role during the first month after birth.18 Previous studies demonstrate that the innate immune system of neonates is immature.12,13,18,20,24,26,27,28,29,30 These impaired immune functions may be attributed to the alteration of the innate immunity genes which are related to the gestation and uterus environment.23,29,30
NK cells are key effector cells because they are playing a critical role in the first line of host defense against viral infections by killing infected cells without prior antigen stimulation.31,32 The cytotoxicity and antiviral cytokine expression of NK cells are major histocompatibility complex-unrestricted and are independent from the types of invading virus.33,34 Influenza virus is a major causative pathogen of acute respiratory diseases worldwide and accounts for substantial morbidity and mortality of neonates, particularly in SGA infants.35 Previous studies showed that adult NK cells have potent anti-influenza effects6,35 and also have high expression of NKG2D.36 In this study, the antiviral function of neonatal NK cells was examined.
We first showed that in response to influenza virus stimulation, neonatal cells, especially those of SGA babies, produced much less antiviral cytokines including TNF-α and IFN-γ than adults. The lower secretion of cytokines may be due to the immature antiviral response of NK cells in cord blood from the neonates. Therefore we next examined NK cell function and found that the percentage of NK cells in cord blood is lower than that in adults, consistent with the results of Juretic.37 In addition, perforin expression in neonatal resting NK cells was also significantly lower than that in adult NK cells. Perforin is a pore-forming molecule, and it allows apoptotic molecules such as granulysin to enter cells and cause osmotic lysis of target cells.26 It was shown that the cytotoxicity of human cord blood NK cells is significantly low.38 The depressed perforin expression detected herein may explain the low cytotoxicity of cord blood NK cells. In addition to killing virus-infected cells through cytolytic granules, NK cells also produce cytokines such as IFN-γ and TNF-α to exert direct and indirect antiviral effects. In this study, we demonstrated that neonatal NK cells expressed significantly less antiviral cytokines than adult cells upon influenza virus stimulation. Moreover, in neonates, SGA babies showed even much less IFN-γ and TNF-α expressions of NK cells than AGA babies, which suggests that the functional maturity of NK cells in neonates may be related to the birth weight of babies. This was also supported by our further findings that the expressions of cytolytic granules and antiviral cytokines in NK cells were significantly enhanced along with the increase of neonatal birth weight.
In summary, here we demonstrated that neonates, especially those with low birth weight, were born with functionally immature and low number of NK cells. In response to influenza virus challenge, neonatal NK cells had weaker antiviral responses as compared to adult cells. Depressed antiviral activity and a lower frequency of NK cells likely contribute to the high susceptibility to microbial infection in neonates, particularly SGA babies. This report, to the best of our knowledge, is the first to describe the impaired antiviral activity of NK cells against influenza virus in SGA neonates. Deciphering NK cell immunity of neonates may provide clinicians a new angle for better understanding and managing the increased susceptibility to life-threatening infections and sepsis in SGA infants.
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This work was supported by the National Natural Science Foundation of China (Nos. 30973235 and 81170606), Science and Technology project of Sichuan Science and Technology Department (2010SZ0110), General Research Fund, Research Grants Council of Hong Kong (HKU 781211M) and the Area of Excellence Scheme of the University Grants Committee, Hong Kong SAR, China (AoE/M-12/06).
The authors declare no potential conflicts of interests.
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