Neonatal infections are a cause of significant morbidity and mortality in preterm neonates during their hospitalization in the NICU (2). It is known that preterm neonates exposed to chorioamnionitis have an increased risk of developing early-onset sepsis (blood stream infection that occurs within the first 72 hours of life) (21, 24, 34). It is unclear if this infection risk is due to a common pathogen causing both conditions or alterations in the neonatal immune response following chorioamnionitis exposure, or both. Multiple studies have shown that exposure to chorioamnionitis impacts the neonatal immune system by altering gene transcription and innate immune responses (20–22). These altered immune responses include dampened pro-inflammatory cytokine expression when a second pathogen is encountered (21, 22). Appropriate pro-inflammatory cytokine expression is necessary for the clearance of microorganisms, so these chorioamnionitis-induced changes to neonatal immune responses are thought to be at least partially responsible for this increased risk of infection. However, it is unclear how long chorioamnionitis-induced dampened cytokine expression persists, as studies are conflicting about whether chorioamnionitis exposure protects against or increases the risk for developing late onset sepsis (blood stream infection that presents after 72 hours of life) (24, 35–37).
To assess the persistence of chorioamnionitis-induced dampened pro-inflammatory cytokine expression in preterm neonates, we performed longitudinal cytokine and chemokine profiling in very preterm neonates from birth to NICU discharge. We chose a panel of cytokines and chemokines known to be significant contributors to neonatal immune responses. Neonates primarily rely upon the innate immune system early in life to protect against infections due to limited antigen exposure in utero and major deficiencies in adaptive immune responses (38, 39). Innate immune cells, including monocytes, macrophages and neutrophils, require signaling from cellular messengers such as cytokines and chemokines in order to mount a coordinated response to an infectious pathogen (40). CCL2 and CCL3 are chemokines that recruits monocytes, macrophages and neutrophils to local sites of infection and are necessary for prominent signaling pathways in the neonatal immune system (33). IL-8 shows similar chemotactic affinity for neutrophils and stimulates bacterial phagocytosis (32). IL-6, IL-1b and TNF-a are pro-inflammatory cytokines important to the acute phase response necessary to assist in the clearance of microorganisms (29, 30). IL-10 is an immunoregulatory cytokine important for immune homeostasis that also suppresses autoinflammation (31). We believe this panel of cytokines and chemokines provides a broad overview of neonatal innate immune reactivity.
In this study, we used a novel method of cytokine and chemokine evaluation, using each preterm neonate as its own matched control to compare levels at different chronologic ages. While this method has previously been used to demonstrate a significant decline in IL-1b, IL-6 and TNF-a from DOL 1 to DOL 40 in term neonates, we are the first to use it to evaluate changes in cytokine and chemokine levels over time in preterm neonates (13, 14). We found that in our population of preterm neonates, levels of IL-6, IL-8, CCL2 and CCL3 decreased between the first and second weeks of life. Non-chorioamnionitis exposed preterm neonates had a consistent decrease in levels of IL-1b, IL-6, IL-8, IL-10, TNF-a and CCL2 over the first month of life, reaching what appeared to be baseline levels around three weeks after birth. This is in contrast to chorioamnionitis-exposed preterm neonates, whose cytokine and chemokine levels demonstrated differences over the first month of life without a consistent pattern based on chronologic age. We additionally found that the trajectory of IL-10 and TNF-a serum levels differed between chorioamnionitis-exposed and unexposed preterm neonates. These findings are important as most of these cytokines and chemokines have been proposed as biomarkers to diagnose or predict prematurity-based complications, including sepsis, necrotizing enterocolitis and bronchopulmonary dysplasia (8–11, 41, 42). Our findings suggest that chronologic age and chorioamnionitis-exposure should be taken into consideration when using cytokines and chemokines as biomarkers in premature neonates.
The altered cytokine and chemokine responses seen in the chorioamnionitis-exposed preterm neonates is in line with previous reports demonstrating altered cytokine responses from chorioamnionitis-exposed umbilical cord blood monocytes following stimulation with either LPS or Staphylococcus epidermidis (21, 22). This chorioamnionitis-induced immune dysregulation may provide insight into immune-related complications experienced by chorioamnionitis-exposed neonates, including late onset sepsis, persistent wheezing and asthma (37, 43). These findings suggest that exposure to early life inflammation has long-lasting consequences for preterm neonates that increases their risk for immune-related diseases well beyond the neonatal period.
Our 7-plex cytokine microring resonator assay was robustly validated for all targets simultaneously to ensure reproducible results across all samples analyzed. Each assay was 38 minutes to result, creating a quick method for analyzing important clinical samples. Using this multiplexed immunoassay, we were able to collect large amounts of immunological data quickly and with little starting sample volume. This technology has the potential to provide clinically relevant information quickly for the most vulnerable patients, which could impact bedside patient care.
This study has several limitations. All samples were collected from clinically indicated laboratory tests, so the timing of sample collection varied between patients and was not standardized. There were differences between the exposure groups, and chorioamnionitis-exposed subjects were more likely to be born earlier, African American and by vaginal delivery than unexposed subjects. It is unclear if these differences impacted cytokine and chemokine expression. Degree of prematurity and mode of delivery have been shown to impact immune responses in prior studies, so these factors were accounted for in out statistical evaluation (44–46). Samples were excluded from subjects who had a suspected or confirmed infection and were receiving antibiotics at the time of sample collection. However, samples were included from these patients later during their NICU course once the infection was treated. It is unclear if the suspected or confirmed infections influenced future cytokine and chemokine expression. Furthermore, corrections were not made for clinical differences such as mode of respiratory support, presence of BPD, steroid administration, or PDA treatment. Consistent with previous reports, chorioamnionitis-exposed preterm neonates in this study had an increased incidence of early onset sepsis (21, 24, 34). It is unclear what impact this had on subsequent cytokine or chemokine responses and if the presence of early onset sepsis further compounded dampened cytokine and chemokine expression. The numbers in this study are not large enough to directly address this, but future studies containing more subjects would be of benefit.