Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients

mRNA expression levels of target genes in resected intestinal tissues of NEC and SIP infants.

<p>Expression levels of IL-6, Ang-2, ErbB3, IL1-RII, and uPAR in resected intestinal tissues from NEC (n = 7) and SIP (n = 6) infants were quantified by qPCR and compared with surgical control tissues (n = 6). Results showed that IL-6, IL1-RII and uPAR were significantly higher (<i>P</i><0.01) in NEC tissues, compared with surgical control tissues (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036977#pone-0036977-g003" target="_blank">Figure 3A, D and E</a>). Results are presented as median, interquartile range and range of expression levels relative to β-actin.</p

Clinical characteristics of NEC, SIP and surgical control patients recruited for mRNA analysis.

<p>Results are expressed as % or median (interquartile range).</p><p>Note: All surgical specimens were of ileal origin, except 1 SIP specimen was from the descending colon.</p

Clinical characteristics of NEC and SIP patients recruited for plasma analyses.

<p>Results are expressed as % or median (interquartile range).</p><p>Note: N/A = not applicable.</p

mRNA expression levels of target genes in FHs-74 Int cell line in response to LPS and PAF.

<p>FHs-74 Int cells were cultured in the presence of LPS (100 ng/mL) or PAF (25 mM) for 6 h individually or in combination. qPCR analysis of target mRNA expressions showed that combined treatment with LPS and PAF significantly increased levels of Ang-2, IL1-RII and uPAR (n = 4; <i>P</i><0.05), whereas single stimulant, LPS or PAF, did not alter the expression levels. Results are presented as mean and SEM of expression levels relative to β-actin.</p

Comparison of plasma levels of target proteins in NEC, SIP and respective control infants by ELISA.

<p>Levels of IL-6, Ang-2, sErbB3, sIL1-RII, and suPAR in NEC (n = 13) and SIP (n = 8) infants were quantified by ELISA and compared with those in respective gestational age-matched control (CTL) infants (NEC CTL, n = 13; SIP CTL, n = 8). Levels of IL-6, Ang-2, sIL1-RII and suPAR were significantly higher in NEC infants compared with NEC-CTL (<i>P</i><0.01) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036977#pone-0036977-g002" target="_blank">Figure 2A, B, D, E</a>) and SIP infants had significantly higher level of sErbB3 compared with NEC infants (P<0.05) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036977#pone-0036977-g002" target="_blank">Figure 2C</a>). Results are presented as median, interquartile range and range.</p