Gene Expressional Profile of Neonatal Neutrophils in Response to Bacterial Lipopolysaccharide (LPS) and Possible Mechanism of Transcription Factor NR4A2 on the Immune Signal Cascade

Gram-negative bacterial infection is a serious condition in neonates which could lead to septicemic shock, disseminated intravascular coagulation and death. LPS, the cell wall component of Gram-negative bacteria is a potent stimulator of the host immune response system, mediated by binding to toll-like receptor (TLR)-4 and CD14 receptors. Neutrophils are the first line of innate defense which possess antimicrobial and acute inflammatory activities. Neonatal neutrophils, though incompletely characterized, are suggested to be functionally immature and compromised in their bactericidal capacity compared with adults.

We performed a comparative genome-wide expression array analysis on purified (>95%) human cord blood (term delivery) neutrophils upon challenge by LPS for 4 hours at 100 ng/mL. With the criteria of 2-fold differences and statistical significance (P<0.01), 1236 transcripts were increased and 1633 transcripts were decreased in LPS-treated neutrophils (n=3).  A panel of 20 target genes was selected by their magnitude of regulation, novelty and relevant immune functions and validated by qPCR.  Eleven of these genes could be linked upstream or downstream of the novel transcription factor, nuclear receptor subfamily-4, group-A, member-2 (NR4A2) by pathway analysis (Metacore). We thus proposed a regulatory network mediated by binding of LPS to TLR4/CD14 receptors, leading to NR4A2 upregulation, thereby promoting a cascade of downstream effector genes associated with antibacterial and/or inflammatory activities in neutrophils.

To validate the upstream signal cascade of LPS-induced regulation of NR4A2, neonatal neutrophils were pretreated with specific TLR-4, p38MAPK or NF-κB inhibitors, and stimulated with LPS, followed by quantification of target gene expression by qPCR (n=6 in each group). Our results showed that LPS-induced NR4A2 expression was significantly decreased (all P<0.05) after pretreatment with TLR-4 antibody (0.47 fold), p38MAPK inhibitor SB203580 (0.27 fold), and NF-κB inhibitors BAY-117082 (0.14 fold) and pyrrolidine dithiocarbamate (0.07 fold). These results suggested that NR4A2 could be upregulated by LPS-receptor-mediated p38MAPK and NF-κB signals.

To compare the capacity and developmental regulation of LPS-induced responses of NR4A2-associated signals, purified cord blood neutrophils from preterm (gestational age 33-36 weeks) and term (37-40 weeks) neonates, and peripheral blood from healthy male adult (age  25-30 years old) were evaluated (n=10 in each group). LPS-induced expression of NR4A2 and associated molecular signals (EGR1, PPARG, NF-κB, ATOH8, G0S2, GPR84, IL-8, PTGS2, TNF-α, CAMK2G and GCH1) were measured by qPCR. Our result showed that expressions of these target genes were significantly up- or downregulated in LPS-activated neutrophils of neonates and adults. However, at the basal level, expressions of EGR1, GOS2, IL-8 and  PTSG2 were significantly lower (P<0.05) in preterm or term neonatal neutrophils compare with those in adults, showing that there might be compromised levels in neonates at the unstimulated status. Upon stimulation with LPS, expressions of GOS2 and IL-8 were significantly lower (P<0.05) in preterm or term neonates than in adult, indicating that there could be developmental regulation of NR4A2 downstream genes in neutrophils secondary to LPS stimulations. Correlation analysis showed that the expressions of EGR1 and PTGS2 were positively correlated with NR4A2 in LPS-induced neutrophils from both term (EGR1: R=0.805, P<0.05; PTGS2: R=0.912, P<0.05) and preterm neonates (EGR1: R=0.636, P<0.05; PTGS2: R=0.830, P<0.05), but not in adult peripheral neutrophils. These results indicated that there could be developmental differences in the regulation of the NR4A2 immune cascade involving apoptosis (EGR1, GOS2)/ inflammatory cytokine (IL-8) and prostaglandin synthesis (PTGS2) between adult and neonatal neutrophils at basal and/or LPS-activation states.

In summary, we reported the expressional profile and deregulated target genes in neonatal neutrophils upon stimulation by LPS and identified the downstream NR4A2 immune cascade that could be regulated by LPS, p38MAPK and NF-κB signals, as well as the developmental status.