Cord blood antimicrobial peptide LL37 levels in preterm neonates and association with preterm complications

Cathelicidin/LL-37, the only known human cathelicidin, belongs to the most important human antimicrobial peptides families and plays a significant role in the human immune defense reaction, including regulation of innate immunity and inflammation-induced tissue repair [1‐7]. The role of LL37 in inflammation associated diseases is controversial. Previous studies in adults showed decreased LL-37 levels could be high risk factor for patients in developing severe chronic obstructive pulmonary disease (COPD) [8], but also contributed to arthritis [9]. Exposure to inflammatory mechanisms constitutes the cornerstone of the complications associated with prematurity [10‐12]. Among the preterm complications, bronchopulmonary dysplasia (BPD) is still the primary major complication of preterm birth causing neonatal mortality and morbidity [10‐12]. Persistent prenatal and postnatal inflammation plays a crucial role in the pathogenesis of bronchopulmonary dysplasia (BPD) [12]. In adult COPD study, it was found the lower LL37 lever was associated with higher NF-κB activation, therefore contributing to chronic inflammatory response [8]. Previous study showed a significant correlation was observed between maternal and cord plasma LL37 levels [13]. LL37 concentration in preterm infants was lower in tracheal aspirates and breast milk as compared to term infants [14, 15], and LL37 level was elevated in tracheal aspirates during respiratory infection [15]. However, little is known about human cord blood LL37 in preterm neonates and the association with preterm complications, as well as the underlying mechanisms. This study was designed to investigate the concentration of LL37 in cord blood of preterm infants and the correlation with the development of preterm complications.

Preterm neonates were often exposed to insults like pulmonary infection, invasive mechanical ventilation and hyperoxia, which causes aberrant regulation of lung inflammation [19‐22]. Antimicrobial peptides LL37 was the only known human cathelicidin [1‐3]. It was an effector molecule of the inflammatory response system of the lung [23]. This current study firstly investigated the cord blood level of LL37 in preterm neonates and the association with preterm complications. The results showed (1) Cord blood LL37 level increased during gestation; (2) Prenatal steroid usage reduced cord blood LL37; (3) LL37 was an independent protective factor for developing BPD. Very preterm infants with higher cord blood LL37 also showed less risk to develop severer BPD, the protection may be associated with an regulating effect on inflammatory response.

In human beings, the cathelicidin LL-37 was regarded as the major microbicidal peptides, as it provided a first line of defense against infection and played a vital role in innate immunity regulation in newborn infants, especially in preterm neonates [4, 24, 25]. The level of LL-37 in neonates was evaluated in several previous studies [1‐5, 13‐15]. Previous study showed the average LL37 level in neutrophils derived from cord blood of full-term newborns has been demonstrated to be much lower than in adult blood [4]. Susan Schaller demonstrated preterm infants exhibited reduced LL37 in tracheal aspirates as compared to term infants, and an up-regulation of bronchoalveolar lavage fluid LL37 in response to infection probably represented an effector molecule of the respiratory defense system [15]. G. Velázquez-Sámano showed increased LL-37 in the sputum was associated with less respiratory infections [26]. However, less investigation was conducted in preterm neonates among whom the LL37 played a more important role in their less mature immunity system [3, 4, 27]. Our study firstly showed cord blood LL37 level increased during gestation. The LL37 levels in preterm infants were significantly lower than term babies. Considering the immunomodulation and antimicrobial effect of LL37 [1‐4, 13‐15], the lower LL37 may contribute to higher susceptibility to sepsis and excessive inflammatory response in preterm neonates. And its increasing can be considered as a response to the acute inflammatory or infectious insults.

In the further multiple regression analysis investigating the factors affecting LL37 level, we found antenatal steroid use could reduce the cord blood level of LL37, but did not affect the serum LL37 level after 48–72 hours. The serum LL37 level after 48–72 hours was higher than that in the cord blood. Previous study showed LL37 could be induced after LPS exposure [5]. The increased of LL37 level later after birth might be attributed to regulation by pro-inflammatory extrauterine micro-environment and acted as a favorable feedback for preterm neonates.

Inflammation induced injury were regarded to be the common denominator in all these multi-factorial origins resulting in preterm complications [6‐8]. Neonatal immature immune system, particularly in preterm infants, showed a stronger pro-inflammatory signature after injurious exposure [27, 28]. A study in adult showed decreased LL-37 levels could be high risk for patients in develop severe COPD which disease was also characterized as chronic lung inflammation [6]. The underlying mechanism might be that LL37 could inhibit nuclear factor-kappa B thus alleviating expression of inflammatory cytokines networks. In other studies of adults, low LL-37 was found to be associated with poor prognosis in several conditions including chronic dialysis, sepsis, and tuberculosis [29‐31]. However, this may not the same condition in neonates because of the difference of immunity system compared with adults. No research was ever conducted in BPD patients yet. Since LL37 level reduced as gestational age (GA) decreased and very preterm infants were at higher risk for preterm associated complications, we compared the cord LL37 level in different complications to access the LL37 correlation with preterm outcomes. We found these very preterm neonates later developed BPD had lower cord blood LL37, and multiple regression analysis showed higher LL37 is an independent protective factor for developing BPD as well as severer BPD. Compared with children with mild or moderate BPD, those with severe BPD had more in-hospital mortality, poorer prognosis of respiratory system and long-term neurodevelopment outcome [32]. This indicated LL37 could promote preterm pulmonary development, which laid foundation for translational application of LL37 in preventing preterm BPD.

The association between inflammatory responses and aberrant lung development have been well established [10, 19]. Inflammatory mediators, which are increased during lung injurious processes after invasive mechanical ventilation, exposure to hyperoxia, and sepsis, appear to be a significant component of the pathogenesis of BPD [20, 32]. The immature immune system of preterm neonates leaded to aberrant regulation of lung inflammation [10, 33]. Therefore, we tested the relationship between cord blood LL37 and concentration of two main pro-inflammatory cytokines - IL-6 and TNF-a. Our study showed IL-6 level but not TNF-α was negatively associated with cord blood LL37. Damaged lung tissue releases chemotactic factors and inflammatory cytokines, such as interleukin IL-6 [33‐35]. Multiple studies have shown that IL-6 was elevated very early in the respiratory course of the human preterm population that ultimately develop BPD [33‐35]. Studies also indicated crosstalk between mesenchyme cells and alveolar epithelium cells may be disturbed by interleukin IL-6, therefore interrupt the pulmonary repairing process after injury [34]. The possible regulation of IL-6 by LL37 may be the underlying mechanism of LL37 for improving respiratory outcomes of very preterm infants. Further research on the mechanisms of LL37 regulation on IL-6 was needed.

There are several limitations of this study. Firstly, among the enrolled 112 very preterm infants less than 32 weeks, only 102 had available LL37 level results, 10 infants failed to test the LL37 level in cord blood. Therefore, when we analyzed the association between LL37 level and outcomes, only 102 infants with available LL37 results were included. As we used abandoned blood from routine blood tests 48–72 hours after birth for enzyme-linked immunosorbent assay test, and blood samples were collected only when it was clinically required during routine care. Thus, not all infants enrolled had available LL37 level results. Secondly, among 112 infants with a GA at birth < 32 weeks, only 14 infants had a GA at birth < 28 weeks. Further study should include more extremely preterm infants who are at greater risk of BPD and complications in general. Thirdly, elevated LL37 was found in respiratory infection condition, which is an acute inflammatory response process [15], however, BPD or COPD in adults are chronic lung inflammatory process. We found serum LL37 48–72 hours after birth had no association with BPD. Further study is needed to measure LL37 on different time points after birth and analyze the association of LL37 and chronic lung inflammation.

The cord blood LL37 levels increased during gestation and decreased after perinatal steroid usage. Higher cord blood LL37 is an independent protective factor for developing BPD and severer BPD. The protective effect of LL37 on BPD and its severity may be mediated by an inhibiting function of pro-inflammatory cytokine IL-6. These results laid clinical foundation for the translational application of LL37 in preterm BPD.