A murine neonatal model of necrotizing enterocolitis caused by anemia and red blood cell transfusions

Krishnan Mohankumar,Samuel A Wickline,Benjamin A Torres,Byeong Jake Cha,Hua Pan,Joo-Yeun Oh,Akhil Maheshwari,Andrea Slate,Kopperuncholan Namachivayam,Rakesh P Patel,Ling He,Tanjing Song,Jeanne E Hendrickson

doi:10.1038/s41467-019-11199-5

Abstract

Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants. Clinical studies have linked NEC with antecedent red blood cell (RBC) transfusions, but the underlying mechanisms are unclear. Here we report a neonatal murine model to investigate this association. C57BL/6 mouse pups rendered anemic by timed phlebotomy and then given RBC transfusions develop NEC-like intestinal injury with prominent necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon within 12–24 h. The anemic intestine is infiltrated by inflammatory macrophages, which are activated in situ by RBC transfusions via a Toll-like receptor (TLR)-4-mediated mechanism and cause bowel injury. Chelation of RBC degradation products with haptoglobin, absence of TLR4, macrophage depletion, and inhibition of macrophage activation is protective. Intestinal injury worsens with increasing severity and the duration of anemia prior to transfusion, indicating a need for the re-evaluation of current transfusion guidelines for premature infants.

Highlights

Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants
We hypothesized that red blood cell (RBC) transfusions may trigger NEC in the presence of severe anemia; the emphasis on anemia was based on several emerging lines of evidence: (a) NEC has been reported in patients with anemia related to diverse neonatal conditions such as glucose-6-phosphate dehydrogenase deficiency, hemolytic disease of the newborn, and twin-to-twin transfusion syndrome;[27,28] (b) transfusion-associated NEC is usually a late event seen beyond 4 weeks of postnatal age, when these infants are usually anemic;[11] and (c) intestinal injury has been observed in other populations of critically ill infants such as those undergoing treatment with cardiopulmonary bypass or extracorporeal membrane oxygenation, when they receive top-up transfusions to treat severe anemia[29]
Considering the potential importance of enteric Gammaproteobacteria in NEC pathogenesis[30], we introduced a well-characterized strain of Serratia marcescens [104 colonyforming units (CFU) by gavage] in our mice on postnatal day (P) 7 to achieve fecal Gammaproteobacteria abundance similar to premature infants (Supplementary Fig. 1a)[31]

Summary

Introduction

Necrotizing enterocolitis (NEC) is an idiopathic, inflammatory bowel necrosis of premature infants. We report a neonatal murine model to investigate this association. C57BL/6 mouse pups rendered anemic by timed phlebotomy and given RBC transfusions develop NEC-like intestinal injury with prominent necrosis, inflammation, and submucosal edema/separation of the lamina propria in the ileocecal region and colon within 12–24 h. The anemic intestine is infiltrated by inflammatory macrophages, which are activated in situ by RBC transfusions via a Toll-like receptor (TLR)-4-mediated mechanism and cause bowel injury. We address these knowledge gaps by developing a neonatal murine model of transfusion-associated NEC. We show that severe anemia in the murine neonate leads to the development of a lowgrade inflammatory state in the intestine with prominent macrophage infiltration. Subsequent RBC transfusions activate these macrophages and causes NEC-like intestinal injury

Methods

Results

Conclusion