726 - Targeted RNA sequencing identified gene expression profiles linked to survivors and non-survivors in preterm infants with surgical necrotizing enterocolitis.

Publication Number: 726.6658

Lavanya Challagundla, University of Mississippi School of Medicine, Brandon, MS, United States; Padma Garg, University of Mississippi School of Medicine, Jackson, MS, United States; Seth Lirette, University of Mississippi Medical Center, Jackson, MS, United States; Michael Garrett, University of Mississippi Medical center, Jackson, MS, United States; Parvesh Mohan Garg, Wake Forest School of Medicine of Wake Forest Baptist Medical Center, winston salem, NC, United States

Background: Genetic factors and mechanisms affecting necrotizing enterocolitis (NEC) development in neonates are not fully understood.

Objective: To identify immune gene expression changes that occur in survivors and non-survivors in neonates with surgical necrotizing enterocolitis.

Design/Methods: RNA was isolated from NEC formalin-fixed, paraffin-embedded (FFPE) human intestine specimens (N=36).Targeted RNA sequencing using the AmpliSeq Illumina Immune Response Panel was utilized for targeted quantitative expression of genes linked to immune system interactions which included 400 genes.DeSeq2 was used to analyze differential expression in survivors and the non -survivors while accounting for other covariates such as necrosis.

Results: Differential expression analysis showed 6 genes being significantly differentially expressed (FDR adjusted p < 0.05) between survivors and the non -survivors. Genes related to chemokines attracting neutrophils (CXCL1), GBP1( Guanylate binding protein -1, role in cell-autonomous immunity) and PTGS-2 ( prostaglandin-endoperoxide synthase 2 gene, codes for the cyclooxygenase-2 (COX-2) enzyme, needed for prostanoid biosynthesis involved in inflammation and mitogenesis),CXCL11( cellular response to lipopolysaccharide; chemokine-mediated signaling pathway; and neutrophil chemotaxis) ,CXCL9 ( Leukocyte chemotaxis, recruitment and differentiation) ,and CXCL10( recruits immune cells like macrophages, DCs, NK cells, and Th1 cells to sites of inflammation) were all upregulated in the non -survivors group ( Figure 1). When accounting for the necrosis severity and outcomes , 26 genes were up- or downregulated in survivors and non-survivors. Genes involved in epithelial cell structure, related to altered host defense, immune cell signaling and development (IGJ, GZMA, KLRB1, KRT5, CD160), were all downregulated in patients with severe necrosis suggesting a suppressed immune response (Figure 2). On the other hand, expression of leukocytes antigens (ITGAM, ITGAX); cytokines and chemokines receptors (such as IL1B, CCL2, CCL3, CCL4) were upregulated in patients with mild necrosis (figure 3).

Conclusion(s): Survivors and non-survivors with surgical NEC show distinct immune gene expression profiles, with non-survivors exhibiting increased inflammation and chemotactic signaling. Survivors have upregulation of leukocyte antigens and cytokine receptors, indicating a more active immune response. Degree of necrosis impacts additional genes, emphasizing its role in immune regulation differences. Targeting excessive inflammation and understanding these pathogenic pathways may improve outcomes.

Figure 1: Gene expression Profile in survivor's and non -survivor's neonates with surgical NEC.
Figure 1: Gene expression Profile in survivor's and non -survivor's neonates with surgical NEC.

Figure 2: Gene expression Profile in survivor's and non -survivor's neonates with surgical NEC when controlled for necrosis.
Figure 2: Gene expression Profile in survivor's and non -survivor's neonates with surgical NEC when controlled for necrosis.

Figure 3: Gene expression Profile with respect to mild vs. severe necrosis
Figure 3: Gene expression Profile with respect to mild vs. severe necrosis