694 - Elevation of TLR4, LY96, and MYD88 in the Blood Predicts Necrotizing Enterocolitis: Evidence from Preterm Infants and a Mouse Model

Publication Number: 1671.694

Maggie L. Aucoin, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Brenna J. Servantes, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Haniyeh Koochak, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Sepideh Saroukhani, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Michael L. Chang, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States; Joseph L. Alcorn, UT Medical School at Houston, Houston, TX, United States; Tina O. Findley, McGovern Medical School at the University of Texas Health Science Center at Houston, Houston, TX, United States

Background: Necrotizing enterocolitis (NEC) is a life-threatening condition in preterm infants, with delayed detection increasing morbidity and mortality. NEC occurs via upregulation of Toll-like receptor 4 (TLR4) and its co-receptor myeloid differentiation protein 2 (MD2 or LY96), triggering inflammation through myeloid differentiation factor 88 (MYD88). TLR4 signaling is elevated in intestinal tissue from NEC infants, yet its detection in blood prior to clinical diagnosis remains unexplored.

Objective: We hypothesized that blood expression of TLR4, LY96, and MYD88 is elevated before NEC diagnosis in preterm infants and that this temporal relationship is reproducible in a mouse model of experimental NEC.

Design/Methods: In this retrospective case-control study, infants with NEC (n=6) were compared to gestational-age matched controls (n=6). NEC infants (Bell's stage II-III) were included if blood was collected within three days before diagnosis. RNA was extracted from blood samples and analyzed by NanoString nCounter Inflammation Panel (249 genes), using nSolver 4.0 for differential expression and pathway analysis with p-values adjusted for multiple comparisons. In a mouse NEC model, pups were separated from dams on postnatal days 5-8 and underwent a five-day NEC protocol (formula ± hypoxia/hypothermia). Daily blood and distal ileum samples were collected; Tlr4, Ly96, and Myd88 expression was measured by qPCR. Temporal changes were assessed by linear regression. Ileum histology was graded 0-3 by four blinded reviewers. Gene expression levels were compared across histologic grades using the Kruskal-Wallis test.

Results: Differential expression analysis identified significant upregulation of TLR4 (p=0.01), IL15 (p=0.02), CD163 (p=0.02) and C3AR1 (p=0.01) in NEC infants compared to controls (Fig 1). LY96 (p=0.073) and MYD88 (p=0.067) showed an upward trend. Pathway analysis revealed increased activation of inflammatory and immune signaling pathways in NEC infants. Among mouse pups (n=65), the blood expression of Tlr4, Ly96, and Myd88 showed a non-significant upward trend over the five-day NEC protocol (Fig 2). Myd88 was higher on day five vs day two (2.17 vs 1.64, p=0.04). Histological Grades 0-3 correlated with blood expression of Tlr4 (p < 0.01), Ly96 (p=0.07), and Myd88 (p < 0.01) (Fig 3).

Conclusion(s): Increased TLR4, LY96, and MYD88 expression in blood was detectable before the diagnosis of NEC in preterm infants. This was recapitulated in a mouse model, where increased expression correlated with greater intestinal injury. These results support the diagnostic potential of TLR4 pathway markers for early NEC detection.

Nanostring Inflammation Panel: Differential Gene Expression in NEC Infants vs baseline of Controls
Figure 1. Volcano plot of differential gene expression in NEC infants vs. controls. NanoString analysis demonstrated significant upregulation of innate immune and inflammatory pathway genes. Yellow squares indicate genes associated with the MHC I pathway, while gray circles represent other single genes from the panel. The horizontal dashed line marks the statistical significance threshold (adjusted p-value < 0.05), and vertical blue lines denote the log₂(fold change) cutoffs (log2 fold change ≥ ±1).

Tlr4, Ly96, and Myd88 blood expression across a five-day mouse model of Experimental NEC
Figure 2. Tlr4, Ly96, and Myd88 expression increased across the five-day protocol, peaking on the final day, in pups exposed to formula/hypoxia/hypothermia compared to formula alone. The overall trend was not significant, except for Myd88, which was higher on day five vs day two (mean difference 0.87, 95% CI 0.05-1.69, p=0.04). FF, formula-fed; FH, formula-fed/hypothermia+/hypoxia.

Intestinal Histologic Injury Correlation to blood expression of Tlr4, Ly96 and Myd88
Figure 3. In the mouse model of experimental NEC, distal ileum histologic score was significantly associated with blood expression of Tlr4 (p < 0.01), Ly96 (p=0.07), and Myd88 (p < 0.01) . Grade 0, normal intestine, Grade 1, epithelial cell lifting or separation, Grade 2, sloughing of epithelial cells to the mid villous level and Grade 3, necrosis of the entire villous.