The Cytokines, Exhaled Nitric Oxide are Risk Factors in Preterm Infants for Bronchopulmonary Dysplasia

Background Bronchopulmonary dysplasia (BPD) remains the most frequently complication of extreme preterm infants. Multiple clinical factors and inflammatory markers have all been associated with BPD. Therefore, this study targeted to detect cytokines and fractional exhaled nitric oxide(FeNO) to evaluate their mechanism and possible predicted significance for BPD. Preterm infants born at gestational age ≤ 32 weeks were recruited between January 2018 and October 2019. The clinical data of infant characteristics and maternal characteristics were collected. Our study detected a total of ten cytokines include IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8 and TNF-α on day 1–3, day 7–14, and day 21–28 after birth via Meso Scale Discovery (MSD) technology. FeNO levels were measured when the infants met discharge criteria.


Abstract Background
Bronchopulmonary dysplasia (BPD) remains the most frequently complication of extreme preterm infants. Multiple clinical factors and inflammatory markers have all been associated with BPD.
Therefore, this study targeted to detect cytokines and fractional exhaled nitric oxide(FeNO) to evaluate their mechanism and possible predicted significance for BPD.
FeNO levels were measured when the infants met discharge criteria.

Results
A total of 46 preterm infants were enrolled in this study, including 14 infants in BPD group and 32 infants in control group. The gestational age (27.5 ± 1.3) vs. (29.9 ± 1.3) weeks and birth weight (1021 ± 261)g vs. (1489 ± 357)g of BPD group were lower than those of control group.
Multivariate logistic regression analysis showed that gestational age < 30 weeks, birth weight < 1000 g, PDA, longer mechanical ventilation and invasive ventilation duration were high risk factors for BPD. The cytokines of IL-6, IL-8 on day 7-14 and IL-4, IL-6, IL-8, TNF-α on day 21-28 were also the high risk factors for BPD. Other risk factors for BPD included elevated Eosnophils on day 21-28 and FeNO.

Conclusion
The preterm infants with PDA, prolonged mechanical ventilation tended to develop BPD. The FeNO, Eosnophils, cytokines such as IL-4, IL-6, IL-8, TNF-α were high risk factors for BPD. Our study speculate that NO was related to BPD though Th2-cell-mediatedinflammatory responses such as IL-4.
The cytokines may provide a certain predictive value for the occurrence of BPD.

Background
Bronchopulmonary dysplasia (BPD) remains the most frequently complication of extreme preterm birth. Currently, it's recognized as the results of an aberrant respiratory distress response to the developing lungs. BPD is a major form of chronic lung diseases in preterm infants and is primarily due to respiratory distress syndrome (RDS). Arguably, mechanical ventilation and long-term use of oxygen may contribute to BPD onset [1]. However, the incidence of BPD has not decreased as multiple strategies to provide ventilation and oxygen therapy. Although great advances have been made in animal BPD models, clinical research to explore the pathogenesis and treatment of BPD is imperative.
In preterm infants, episodes of sepsis and the ongoing use of supplemental oxygen, mechanical ventilation, and parenteral nutrition have all been associated with systemic inflammatory responses.
Fractional exhaled nitric oxide (FeNO) is one of the most promising biomarkers for airway inflammation, and there is a good relationship between FeNO levels and eosinophilic airway inflammation. In 1993, a study was conducted to detect airway inflammation by measuring FeNO levels, and it was believed that it is significantly positively related to the severity of inflammation [3].In 2009, the American Thoracic Society / European Respiratory Society (ATS / ERS) joint statement issued affirmed FeNO Clinical application as a biological indicator of airway inflammation [4]. In 2011, ATS published clinical guidelines for FeNO [5]. FeNO testing is simple, reliable, and reproducible, making it an ideal method for non-invasive evaluation of airway inflammatory diseases. Therefore, this study targeted preterm infants to detect cytokines and FeNO to evaluate their mechanism and possible predicted significance for BPD.

Methods
We conducted a prospective study, and the study was approved by the Ethics Institutional Review Board of Peking Union Medical College Hospital. The informed consents were signed when infants were admitted to the NICU if parents agreed to use the residual serum from routine testing for detecting the cytokines. Infants diagnosed with BPD were assigned to BPD group, and infants without BPD were assigned to control group. BPD was diagnosed as oxygen dependency >28 days after birth, according to the workshop definition by the National Institutes of Child Health, the Human Development/National Heart, Lung, and Blood Institute and the Office of Rare Diseases [6].

Data collection and Analysis
Clinical data were prospectively collected by a single investigator and verified by another. The baseline data of infant characteristics were collected, including gestational age, birth weight, Apgar score, Neonatal Respiratory Distress Syndrome(NRDS), anemia, patent ductus arteriosus(PDA), eosinophils, duration of mechanical Ventilation(days), duration of invasive ventilation(days),et al.
Eosinophilia was defined as an absolute eosinophil count more than 700/mm 3 [7]. The baseline data of maternal characteristics were collected, including delivery mode, antenatal steroids, premature rupture of membrane(PROM),et al.

Inflammation Biomarkers
To avoid a sample selection bias, parents or legal guardians of eligible infants were approached as early as possible after birth (usually <72 hours). Infants were sampled blood between birth and 30 days of age according to the clinical needs for routine clinical testing weekly. To limit invasiveness, analyses for the research were carried out only from residual (leftover) serum of routine testing from the hospital laboratory. So the inflammation biomarkers were tested once a week. The cytokines of serum were detected on day 1-3, day 7-14, and day 21-28 after birth. Our study detected a total of ten cytokines include IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL

Detection of FeNO levels
FeNO levels were measured when the infants met discharge criteria (weight>2000g, Corrected gestational age>34w, Fulloral feeding). No bronchodilators or nebulized inhaled hormones were used 12 h before the test. The study subject was in a quiet sleep state, lying on his back, with a cushion height of about 2 cm under his or her shoulders, taking the nasal inhalation position, covering nose and mouth with a mask, tightening the mask along the breath. The nitric oxide filter guaranteed the inhalation of nitric oxide gas without impurities. FeNO was measured using the NIOX Flex (Aerocrine, Sweden), based on the principle of ozone-NO2-based chemiluminescence. Calibrations were performed in accordance to manufacturer instructions. Before FeNO samples were obtained, samples of ambient NO in the room were collected. When ambient NO was below5 parts per billion (ppb), measurements were conducted. Adequate ventilation of the room was assured. Analyses were conducted immediately after collection.

Statistics
Statistical analysis was performed using SPSS 25.0. The Quantitative data of normal distribution was described by the mean ± standard error of the mean( ). The data between two groups were compared using the t-test. The Quantitative data that did not meet the normal distribution was described by M(P25-P75), and the comparison between groups was tested by rank sum test. The qualitative data was described by frequency and rate, the Pearson chi-square test or fisher's precise test was performed. Logistic regression model was used to analyze the independent risk factors of BPD. P <0.05 was considered statistically significant.

Baseline Data
From January 2018 to October 2019, 52 preterm infants were born at gestational age ≤32 weeks.
Four preterm infants were excluded due to insufficient residual serum of routine testing for cytokines detect. Two preterm infants were excluded due to death from severe infection. A total of 46 preterm infants were enrolled in this study, including 14 infants in BPD group and 32 infants in control group.
A total of 138 blood samples were collected. The incidence of BPD was 30% in our study. The (1489±357)g P=0.000 of BPD group were lower than those of control group. As shown in Table 1-1 to Table 1-3. The incidence of PDA, anemia and NRDS was higher in BPD group, duration with mechanical ventilation, invasive ventilation, parenteral nutrition, length of hospital stay, however, were significantly longer in BPD group.

Inflammatory Cytokine Profile of the BPD Patients
The number of cytokine data points over time confirmed similar distribution between infants with or without BPD in the first 4 weeks. A total of 1380 times of cytokines were detected. The results of ten cytokines include IFN-γ, IL-10, IL-12p70, IL-13, IL-1β, IL-2, IL-4, IL-6, IL-8 and TNF-α showed in Table 2.
On day 1-3, IL-10 was significantly decreased in the BPD group. On day 7-14, IL-6, IL-8 and TNF-α were significantly increased in the BPD group while the IL-10 was decreased. On day 21-28, IL-1β, IL-4, IL-6, IL-8 and TNF-α were significantly increased in the BPD group while the IL-10 was decreased.
The comparison of cytokines between BPD and Non-BPD group was showed in the Figure 1-6.

Comparison of FeNO and Eosnophils between two groups
All the enrolled preterm infants were tested for FeNO before they discharged, there was no significant  1-3). The eosinophilia in the infants of the BPD group was significantly increased at 21-28 days after birth. The differences were statistically significant (P values <0.05).

Multivariate logistic regression of BPD influencing factors
Multivariate logistic regression analysis was conducted in this study. The statistically significant factors in the univariate analysis were used as independent variables and BPD as the dependent variables( Table 3 The gestational age and birth weight are the most important factors affecting the occurrence of BPD.
The incidence of BPD in preterm infants with a gestational age of 30 weeks is 12%; the incidence with a gestational age of 25 to 26 weeks is 30-40% [8].In this study, the gestational age and birth weight of the BPD group were low, and multivariate analysis showed that birth gestational age < 30 weeks and birth weight < 1000 g were risk factors for BPD. This was consistent to the previous research results.
Other known risk factors for BPD included maternal preeclampsia [9], chorioamnionitis [10], sepsis [11] and fetal growth restriction [12], and early mechanical ventilation for > 7 days [13]. In our study, prolonged mechanical ventilation and invasive ventilation duration were risk factors for BPD, this was consistent to the previous research results. Recent meta-analysis suggested that strategies to avoid endotracheal ventilation had significant effects in preventing BPD [14].
In this study, although the proportion of patients with NRDS in BPD group was higher than that in non-BPD group, but multivariate analysis did not suggest that respiratory distress syndrome was a risk factor for BPD. It was considered to be related to the fact that the research subjects were all premature babies, most of them received prenatal corticosteroid and the pulmonary surfactant, prenatal corticosteroid and surfactant treatment in the BPD group improved the respiratory symptoms quickly.
Our study found that PDA was a high risk factor for BPD. We thought the reason was that PDA could result. In the previous study, IL-6 had been directly implicated in a murine hyperoxia model of BPD [15]. The results from clinical trials were consistent with this. In the BPD infants, the inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-10 levels were altered in the amniotic fluid [16], cord blood [17], and tracheal aspirate samples [18]. TNF-α single nucleotide polymorphisms could predict BPD onset and severity in preterm neonates [19]. Harald Ehrhardt et al. reported that TNF-α in tracheal aspirate samples are associated with BPD severity [20]. Therefore, we speculated that these cytokines may play a direct role in the lung injury that leaded to BPD in premature infants. In our study, we found Because lung inflammation could impede lung maturation, FeNO is elevated due to ventilator-assisted breathing and inhaled oxygen. FeNO was a marker of airway eosinophil inflammation in adults and older children [21]. In infants with BPD, bronchial hyperresponsiveness may appear later in life, which was related to tracheal intubation and ventilator-assisted breathing in early life. May. C had shown that exhaled nitric oxide levels were raised in infants with established BPD, particularly in those developing moderate or severe BPD, and may reflect ongoing inflammation [22]. Our study found that the FeNO and eosinophils of BPD group were higher than that of non-BPD group. The multivariate analysis showed that FeNO and elevated eosnophils on Day 21-28 were high risk factors for BPD. All these results showed that BPD was closely related to eosinophilic airway inflammation. cytokines such as IL-4, IL-5, IL-13 [23]. On the contrary, IL-4 could also increase the expression of iNOS [24]. IL-4 is known to induce the alternative activation pathway in M2 macrophages, have been shown to regulate NO production by inducing arginase I, which catalyzes the hydrolysis of L-arginine, a common substrate of iNOS, and thereby regulates NO synthesis by competing with the substrate [25]. Expression of iNOS can be observed in epithelial, endothelial, neural, and smooth muscle cells, eosinophils, neutrophils, and alveolar macrophages in the lung after its induction by IL-4, and/or IL-13 [26] [27].In our study, we found that both IL-4 and FeNO were increased and related to BPD by univariate analysis and multifactor analysis. We speculate that NO was related to BPD though

NO is generated by NO synthase (NOS). NO is involved in the
Th2-cell-mediatedinflammatory responses such as IL-4. However, excessive NO often induces immune and pathological processes and mediates the production of some inflammatory mediators, resulting in tissue damage [28].
One limitation of our study is that serum cytokine concentrations may not accurately reflect