M1 to M2 macrophage polarization in heparin-binding epidermal growth factor-like growth factor therapy for necrotizing enterocolitis

Abstract

Background Macrophages can be polarized into pro-inflammatory (M1) and anti-inflammatory (M2) subtypes. However, whether macrophage polarization plays a role in necrotizing enterocolitis (NEC) remains unknown. Materials and methods Macrophages were derived from the THP-1 human monocyte cell line. Apoptosis of human fetal small intestinal epithelial FHs-74 cells was determined by Annexin V/PI flow cytometry and by Western blotting to detect cleaved caspase-3. The effect of heparin-binding EGF-like growth factor (HB-EGF) on macrophage polarization was determined by flow cytometry with M1/ M2 markers and real time PCR. In vivo, experimental NEC was induced in mouse pups by repeated exposure to hypoxia, hypothermia and hypertonic feedings. Intestinal histologic sections were subjected to immunohistochemical staining for the detection of M1 and M2 macrophages. Results In vitro, FHs-74 cell apoptosis was increased after co-culture with macrophages and LPS. This apoptosis was increased by exposure to M1 conditioned medium (CM) and suppressed by exposure to M2 CM. HB-EGF significantly decreased LPS-induced M1 polarization and promoted M2 polarization via STAT3 activation. Addition of HB-EGF to LPS-stimulated macrophages suppressed the pro-apoptotic effects of the macrophages on FHs-74 cells. In vivo, we found enhanced intestinal macrophage infiltration in pups subjected to NEC, the majority of which were M1 macrophages. HB-EGF treatment of pups subjected to experimental NEC significantly reduced M1 and increased M2 polarization, and protected the intestines from NEC. Conclusions M1 macrophages promote NEC by increasing intestinal epithelial apoptosis. HB-EGF protects the intestines from NEC by preventing M1 and promoting M2 polarization.

Pathogenesis of NEC: Role of the innate and adaptive immune response

“…EGF promotes IEC proliferation and differentiation 80 , IEC restitution after injury 81 , and reduces IEC autophagy 82 . EGF may also act by increasing production of helpful mucus by increasing goblet cells and their production of MUC2 (for more detailed discussion on the importance of intestinal mucus layers, see below); by improving intestinal barrier function through increased tight junction protein expression (occludin and claudin) 79,83 ; by reducing TLR-4 signaling 84 ; and by promoting anti-inflammatory macrophages and reducing pro-inflammatory macrophages 85 (for more detailed discussion of the role of TLR-4 and macrophages in NEC, see below). EGF is supplied by amniotic fluid throughout pregnancy and by colostrum in human milk.…”

“…Macrophage products such as the proinflammatory chemokines IL-6, IL-8, and TNF-α have been found to be greatly elevated in infants with surgical NEC as compared to other preterm intestinal injury 238 . Activated M1 macrophages can further potentiate intestinal damage during NEC by augmenting intestinal epithelial cell apoptosis 85 . Thus, inhibiting the differentiation and/or effector functions of M1 macrophages has been considered as an approach to limit the dysregulated inflammatory response in the NEC intestine.…”

Cytokines and growth factors in the developing intestine and during necrotizing enterocolitis

“…54 Enterally administered HB-EGF can prevent NEC-like injury in rodent models by decreasing adhesion molecule expression and neutrophil infiltration; promoting alternative activation of macrophages (M2) to attenuate the inflammatory responses; suppressing inducible nitric oxide (NO) synthase, and the generation of NO and reactive oxygen species; improving gut microvascular perfusion; preserving intestinal stem cells; maintaining crypt cell proliferation; and protecting the intestinal barrier function. 46,55–58 …”

MSCS in Scenarios of Infection and Inflammation: Focus on Neonatal Diseases

“…This confirms the notion that MSCs are most effective when administered in the context of inflammation. Accordingly, the combined administration of both ↑ in murine HI cerebral tissue [37] ↑ Th1 and Th17 in murine HI cerebral tissue [34] ↓ Treg in murine HI cerebral tissue [34] BPD ↓ M1 macrophages in murine lung [38] ↓M2 macrophages in murine lung [38] ↓ activated DCs in murine and human lung [39,40] Unchanged in murine lung [38] ↓ Th1 and Th17 in peripheral blood during the first week of life in BPD patients [41] ↓ Treg in cord blood in BPD patients [36] NEC ↓ M1 macrophages in murine NEC tissue [42] Unknown ↓ Th17 in human NEC tissue [35] ↓Treg in human NEC tissue [35] Periventricular leukomalacia (PVL), Hypoxic ischemic encephalopathy (HIE), Bronchopulmonary dysplasia (BPD), Necrotising enterocolitis (NEC), Lymphocytes T helper (Th), cerebrospinal fluid (CSF), Denditric cells (DCs), Hypoxia-ischemia (HI)…”

“…In experimental NEC, an increased number of intestinal macrophages, mostly M1 macrophages, were found. M1 macrophages promote NEC by increasing intestinal epithelial apoptosis, whereas M2 polarization protects the intestine from NEC [42] ( Table 1).…”