702 - Restoring MIC-1/GDF-15 Signaling in Macrophages Attenuates NEC Injury in Murine Neonates

Publication Number: 702.6509

Megan M. Ferris, University of Nebraska College of Medicine, Omaha, NE, United States; Balamurugan Ramatchandirin, University of Nebraska Medical Center, Omaha, NE, United States; Marie Amalie Balamurugan, University of Nebraska Medical Center, Omaha, NE, United States; Arjun S. Subrramanya, University of Nebraska Medical Center, Omaha, NE, United States; Juanitaa George Raj, University of Nebraska College of Medicine, Omaha, NE, United States; Zainab D. Lawal, University of Nebraska College of Medicine, OMAHA, NE, United States; Liza Konnikova, Yale School of Medicine, North Haven, CT, United States; Mohan Krishnan, University of Nebraska Medical Center, Omaha, NE, United States

Background: Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis that is characterized by infiltration of inflammatory macrophages and smad7 signaling interrupts TGF-B signaling thus promoting inflammatory activation of thesis cells during NEC. The macrophage inhibitory cytokine-1 (MIC-1; growth differentiation factor-15 [GDF-15]) is an important down-regulator of inflammatory signals and studying its role in NEC will develop a novel therapeutic approach.

Objective: Investigating the anti-inflammatory action of GDF-15, which is inhibited by Smad7 signaling in NEC macrophages.

Design/Methods: C57BL/6 mouse pups were studied in two groups: (1) naïve controls; (2) NEC. NEC-like injury was induced on P10 with 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) by gavage and rectal installation. Blood samples were collected and utilized for plasma detection of GDF-15 with ELISA. On P11, both groups were sacrificed, and intestinal tissue was utilized for the following: RT-qPCR to analyze GDF-15, TGFB receptors, and Smads; western blotting to confirm TGFBRII expression, and immunohistochemistry to visualize co-localization of GDF-15, TGFBRII, Ly6C, and F4/80. De-identified human NEC tissue samples were visualized using GDF-15, CD14, and HAM56. Smad7 knockdown in a murine macrophage cell line was used to investigate Smad7’s role in attenuating GDF-15 action via RT-qPCR and western blotting.

Results: Mouse pups with TNBS-induced NEC showed significantly higher plasma concentrations of GDF-15 than control by ELISA (2,185 pg/mL), increased intestinal mRNA expression of GDF-15 (6.762.16; fold change of mRNA vs 18s), and fervent co-localization of GDF-15 with macrophage markers Ly6C and F4/80 in intestinal tissue. Moreover, one of GDF-15’s receptors, TGFBRII showed significantly higher expression in NEC macrophages, indicating that GDF-15 was not exerting its expected anti-inflammatory actions on macrophages by Smad7 signaling. Consistently, Smad7 knockdown in murine macrophage cells displayed M2 macrophage phenotype, suggesting anti-inflammatory actions of GDF-15 might be recovered by Smad7 inhibition. Finally, strong immunoreactivity for CD14 and HAM56 macrophages with GDF-15 was observed in human NEC tissue, validating the defect of GDF-15 signaling in intestinal macrophages.

Conclusion(s): The role of GDF-15 in the neonatal NEC inflammatory response remains unknown. Our study shows increased GDF-15 in the macrophage population of the neonatal NEC intestine and suggests that GDF-15 anti-inflammatory actions via TGFBRII are inhibited by Smad7.