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The ErbB4 Ligand Neuregulin-4 Protects against Experimental Necrotizing Enterocolitis

2014; Elsevier BV; Volume: 184; Issue: 10 Linguagem: Inglês

10.1016/j.ajpath.2014.06.015

1525-2191

Steven J. McElroy, Shannon L. Castle, Jessica K. Bernard, Dana Almohazey, Catherine J. Hunter, Brandon Bell, Denise Al Alam, Larry Wang, Henri R. Ford, Mark R. Frey,

Neonatal Respiratory Health Research

Necrotizing enterocolitis (NEC) affects up to 10% of premature infants, has a mortality of 30%, and can leave surviving patients with significant morbidity. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes epithelial cell survival. Thus, this pathway could be protective in diseases such as NEC, in which epithelial cell death is a major pathologic feature. We sought to determine whether NRG4-ErbB4 signaling is protective in experimental NEC. NRG4 was used i) in the newborn rat formula feeding/hypoxia model; ii) in a recently developed model in which 14- to 16-day-old mice are injected with dithizone to induce Paneth cell loss, followed by Klebsiella pneumoniae infection to induce intestinal injury; and iii) in bacterially infected IEC-6 cells in vitro. NRG4 reduced NEC incidence and severity in the formula feed/hypoxia rat model. It also reduced Paneth cell ablation–induced NEC and prevented dithizone-induced Paneth cell loss in mice. In vitro, cultured ErbB4−/− ileal epithelial enteroids had reduced Paneth cell markers and were highly sensitive to inflammatory cytokines. Furthermore, NRG4 blocked, through a Src-dependent pathway, Cronobacter muytjensii–induced IEC-6 cell apoptosis. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 are present in human breast milk and developing human intestine, respectively. Thus, NRG4-ErbB4 signaling may be a novel pathway for therapeutic intervention or prevention in NEC. Necrotizing enterocolitis (NEC) affects up to 10% of premature infants, has a mortality of 30%, and can leave surviving patients with significant morbidity. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes epithelial cell survival. Thus, this pathway could be protective in diseases such as NEC, in which epithelial cell death is a major pathologic feature. We sought to determine whether NRG4-ErbB4 signaling is protective in experimental NEC. NRG4 was used i) in the newborn rat formula feeding/hypoxia model; ii) in a recently developed model in which 14- to 16-day-old mice are injected with dithizone to induce Paneth cell loss, followed by Klebsiella pneumoniae infection to induce intestinal injury; and iii) in bacterially infected IEC-6 cells in vitro. NRG4 reduced NEC incidence and severity in the formula feed/hypoxia rat model. It also reduced Paneth cell ablation–induced NEC and prevented dithizone-induced Paneth cell loss in mice. In vitro, cultured ErbB4−/− ileal epithelial enteroids had reduced Paneth cell markers and were highly sensitive to inflammatory cytokines. Furthermore, NRG4 blocked, through a Src-dependent pathway, Cronobacter muytjensii–induced IEC-6 cell apoptosis. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 are present in human breast milk and developing human intestine, respectively. Thus, NRG4-ErbB4 signaling may be a novel pathway for therapeutic intervention or prevention in NEC. Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting premature infants. In the United States, NEC afflicts 7% of infants weighing <1500 g.1Hunter C.J. Upperman J.S. Ford H.R. Camerini V. Understanding the susceptibility of the premature infant to necrotizing enterocolitis (NEC).Pediatr Res. 2008; 63: 117-123Crossref PubMed Scopus (185) Google Scholar In addition to prematurity, risk factors include hypoxia, bacterial colonization of the intestine, and formula feeding.2Emami C.N. Petrosyan M. Giuliani S. Williams M. Hunter C. Prasadarao N.V. Ford H.R. Role of the host defense system and intestinal microbial flora in the pathogenesis of necrotizing enterocolitis.Surg Infect (Larchmt). 2009; 10: 407-417Crossref PubMed Scopus (53) Google Scholar The development of NEC seems to be multifactorial, and patients may have any combination of risk factors at the time of presentation. The current disease model is that the immature gut barrier, along with defects in endogenous antimicrobial activity,3McElroy S.J. Underwood M.A. Sherman M.P. Paneth cells and necrotizing enterocolitis: a novel hypothesis for disease pathogenesis.Neonatology. 2013; 103: 10-20Crossref PubMed Scopus (70) Google Scholar allows bacterial translocation across the epithelium, triggering an inflammatory response that further worsens gut barrier function. Pathogenic bacteria,4Hunter C.J. Petrosyan M. Ford H.R. Prasadarao N.V. Enterobacter sakazakii: an emerging pathogen in infants and neonates.Surg Infect (Larchmt). 2008; 9: 533-539Crossref PubMed Scopus (77) Google Scholar, 5Hunter C.J. Singamsetty V.K. Chokshi N.K. Boyle P. Camerini V. Grishin A.V. Upperman J.S. Ford H.R. Prasadarao N.V. Enterobacter sakazakii enhances epithelial cell injury by inducing apoptosis in a rat model of necrotizing enterocolitis.J Infect Dis. 2008; 198: 586-593Crossref PubMed Scopus (87) Google Scholar inflammatory cytokines such as tumor necrosis factor (TNF),6Caplan M.S. Hsueh W. Necrotizing enterocolitis: role of platelet activating factor, endotoxin, and tumor necrosis factor.J Pediatr. 1990; 117: S47-S51Abstract Full Text PDF PubMed Scopus (76) Google Scholar, 7Halpern M.D. Clark J.A. Saunders T.A. Doelle S.M. Hosseini D.M. Stagner A.M. Dvorak B. Reduction of experimental necrotizing enterocolitis with anti-TNF-alpha.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G757-G764Crossref PubMed Scopus (76) Google Scholar, 8Seitz G. Warmann S.W. Guglielmetti A. Heitmann H. Ruck P. Kreis M.E. Fuchs J. Protective effect of tumor necrosis factor alpha antibody on experimental necrotizing enterocolitis in the rat.J Pediatr Surg. 2005; 40: 1440-1445Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar and Paneth cell dropout3McElroy S.J. Underwood M.A. Sherman M.P. Paneth cells and necrotizing enterocolitis: a novel hypothesis for disease pathogenesis.Neonatology. 2013; 103: 10-20Crossref PubMed Scopus (70) Google Scholar have all been associated with human NEC and contribute to NEC-like injury in animal models. Available therapy for either prevention or treatment of NEC is limited, and patients currently face a mortality rate of approximately 30%.9Blakely M.L. Lally K.P. McDonald S. Brown R.L. Barnhart D.C. Ricketts R.R. Thompson W.R. Scherer L.R. Klein M.D. Letton R.W. Chwals W.J. Touloukian R.J. Kurkchubasche A.G. Skinner M.A. Moss R.L. Hilfiker M.L. Network NECSotNNRPostoperative outcomes of extremely low birth-weight infants with necrotizing enterocolitis or isolated intestinal perforation: a prospective cohort study by the NICHD Neonatal Research Network.Ann Surg. 2005; 241 (discussion 9–94): 984-989Crossref PubMed Scopus (217) Google Scholar, 10Fitzgibbons S.C. Ching Y. Yu D. Carpenter J. Kenny M. Weldon C. Lillehei C. Valim C. Horbar J.D. Jaksic T. Mortality of necrotizing enterocolitis expressed by birth weight categories.J Pediatr Surg. 2009; 44 (discussion 5–6): 1072-1075Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 11Rowe M.I. Reblock K.K. Kurkchubasche A.G. Healey P.J. Necrotizing enterocolitis in the extremely low birth weight infant.J Pediatr Surg. 1994; 29 (discussion 90–91): 987-990Abstract Full Text PDF PubMed Scopus (113) Google Scholar Breast-fed infants have a lower risk of NEC than their formula-fed peers,12Lucas A. Cole T.J. Breast milk and neonatal necrotising enterocolitis.Lancet. 1990; 336: 1519-1523Abstract PubMed Scopus (1245) Google Scholar, 13Schanler R.J. Shulman R.J. Lau C. Feeding strategies for premature infants: beneficial outcomes of feeding fortified human milk versus preterm formula.Pediatrics. 1999; 103: 1150-1157Crossref PubMed Scopus (550) Google Scholar and a variety of studies have attempted to identify and characterize factors in human milk that confer this protection. Candidate protective molecules to date include immunoglobulins, oligosaccharides, lactoferrin, and soluble growth factors, such as epidermal growth factor (EGF)14Warner B.W. NEC, EGF, milk, and spit.Gastroenterology. 2002; 123 (discussion 4): 383-384Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar and heparin-binding EGF-like growth factor (HB-EGF).15Michalsky M.P. Lara-Marquez M. Chun L. Besner G.E. Heparin-binding EGF-like growth factor is present in human amniotic fluid and breast milk.J Pediatr Surg. 2002; 37: 1-6Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar In rat and mouse models, enteral administration of either EGF16Nair R.R. Warner B.B. Warner B.W. Role of epidermal growth factor and other growth factors in the prevention of necrotizing enterocolitis.Semin Perinatol. 2008; 32: 107-113Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 17Dvorak B. Halpern M.D. Holubec H. Williams C.S. McWilliam D.L. Dominguez J.A. Stepankova R. Payne C.M. McCuskey R.S. Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model.Am J Physiol Gastrointest Liver Physiol. 2002; 282: G156-G164Crossref PubMed Scopus (214) Google Scholar or HB-EGF18Feng J. El-Assal O.N. Besner G.E. Heparin-binding epidermal growth factor-like growth factor reduces intestinal apoptosis in neonatal rats with necrotizing enterocolitis.J Pediatr Surg. 2006; 41 (discussion -7): 742-747Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar decreases the incidence and severity of NEC. The primary receptor for both EGF and HB-EGF is EGF receptor (EGFR), the prototypic member of the ErbB receptor tyrosine kinase family. However, HB-EGF also activates ErbB4, a member of the ErbB family whose potential role in the developing gut and NEC is not known. ErbB4 has unique biochemical properties distinguishing it from other ErbB family members. Compared with EGFR, ErbB2, or ErbB3, it recognizes a broader collection of ligands, including the EGF-like growth factors HB-EGF and betacellulin as well as the heregulin/neuregulin molecules.19Carpenter G. ErbB-4: mechanism of action and biology.Exp Cell Res. 2003; 284: 66-77Crossref PubMed Scopus (205) Google Scholar At the same time, the ErbB4 c-terminus contains a distinct and somewhat restricted set of functional docking sites for downstream effectors20Kaushansky A. Gordus A. Budnik B.A. Lane W.S. Rush J. MacBeath G. System-wide investigation of ErbB4 reveals 19 sites of Tyr phosphorylation that are unusually selective in their recruitment properties.Chem Biol. 2008; 15: 808-817Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar and is thus predicted to elicit divergent cellular effects on activation versus other family members. In fact, we recently demonstrated that neuregulin-4 (NRG4), an ErbB4-specific ligand that does not bind or activate other family members, including EGFR,21Harari D. Tzahar E. Romano J. Shelly M. Pierce J.H. Andrews G.C. Yarden Y. Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase.Oncogene. 1999; 18: 2681-2689Crossref PubMed Scopus (255) Google Scholar specifically promotes survival but not migration or proliferation of mouse colon epithelial cells.22Bernard J.K. McCann S.P. Bhardwaj V. Washington M.K. Frey M.R. Neuregulin-4 is a survival factor for colon epithelial cells both in culture and in vivo.J Biol Chem. 2012; 287: 39850-39858Crossref PubMed Scopus (38) Google Scholar Thus, ErbB4 is a potentially unique and selective target for therapeutic protection in diseases in which intestinal epithelial cell death is a major pathologic feature. We previously reported that ErbB4 is up-regulated in adult human and murine colon inflammation in vivo23Frey M.R. Edelblum K.L. Mullane M.T. Liang D. Polk D.B. The ErbB4 growth factor receptor is required for colon epithelial cell survival in the presence of TNF.Gastroenterology. 2009; 136: 217-226Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar and that ErbB4 overexpression protects cultured colonocytes from cytokine-induced apoptosis in a ligand-dependent manner.24Hilliard V.C. Frey M.R. Dempsey P.J. Peek Jr., R.M. Polk D.B. TNF-alpha converting enzyme-mediated ErbB4 transactivation by TNF promotes colonic epithelial cell survival.Am J Physiol Gastrointest Liver Physiol. 2011; 301: G338-G346Crossref PubMed Scopus (23) Google Scholar Furthermore, i.p. NRG4 administration reduces the severity of acute murine dextran sulfate sodium colitis.22Bernard J.K. McCann S.P. Bhardwaj V. Washington M.K. Frey M.R. Neuregulin-4 is a survival factor for colon epithelial cells both in culture and in vivo.J Biol Chem. 2012; 287: 39850-39858Crossref PubMed Scopus (38) Google Scholar Thus, it seems that ErbB4 induction is a natural compensatory response meant to preserve the epithelium rather than part of disease pathology and that ErbB4 activation with exogenous ligand is protective against induced inflammation. However, the role of this signaling pathway in the small intestine, or during development, has not been described. We hypothesized that ErbB4 and its ligands have a protective role in the small bowel during postnatal development, particularly in the setting of NEC-associated acute injury and inflammation. To advance our understanding of ErbB4 biology in intestinal homeostasis and disease, we tested the hypothesis that NRG4-ErbB4 signaling is protective in experimental NEC. For the animal experiments, all the studies were approved and monitored by the Institutional Animal Care and Use and Biosafety committees of Children's Hospital Los Angeles (Vertebrate Animals Welfare Assurance No. A3276-01) or the University of Iowa (Vertebrate Animals Welfare Assurance No. A3021-01), as appropriate, and were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals.25Committee for the Update of the Guide for the Care and Use of Laboratory Animals; National Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar For the study of human milk, the Committee on Clinical Investigations (Institutional Review Board for research ethics) of Children's Hospital Los Angeles reviewed and approved the entire study, including recruitment and consenting practices, before the recruitment of study participants and the collection of samples. Recruitment flyers were placed in the hospital day care, and potential participants contacted researchers by telephone if they were interested in donating. Participants were given a study information sheet and were allowed to review this information at home before milk donation. Because the study represented negligible risk and no identifying information was collected, verbal consent was obtained from participants at the time of donation to minimize the participant paperwork burden. Consent, date, and volume of donation were recorded in the collecting researcher's notebook. No participant information was recorded; milk samples were labeled only with the date of collection and were placed in frozen storage. For human NEC specimens, after Institutional Review Board approval and parental consent were obtained, human tissue samples were collected from infants undergoing bowel resection at the Ann and Robert H. Lurie Children's Hospital (Chicago, IL). Clinically indicated specimens were removed. No additional samples were taken for the purposes of this study, and neither did study participants incur additional surgical risk. The type of tissue obtained, the reason for surgery, and the corrected gestational age were recorded. The intestinal samples were categorized into one of three groups: active NEC (intestine removed during surgery for perforation and sepsis), a history of NEC with ongoing complications (eg, intestine removed after a history of NEC due to sequelae of illness, such as NEC stricture), or a history of NEC but no current disease (eg, ostomy takedown, ≥12 weeks after NEC). Tissue was collected, divided into parts, and fixed and processed into paraffin blocks or preserved in Allprotect reagent (Qiagen Inc., Valencia, CA) and stored at −80°C before protein isolation for Western blot analysis. NEC was induced in newborn rats by formula feeding/hypoxia (FFH) as previously described.26Nadler E.P. Dickinson E. Knisely A. Zhang X.R. Boyle P. Beer-Stolz D. Watkins S.C. Ford H.R. Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis.J Surg Res. 2000; 92: 71-77Abstract Full Text PDF PubMed Scopus (204) Google Scholar Newborn Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) delivered at term were immediately separated from the mothers into one of three treatment groups: i) kept with mothers and breast-fed; ii) fed thrice daily by gavage of 0.2 mL of clean formula consisting of 15 g of Similac 60/40 (Abbott Nutritional, Columbus, OH) in 75 mL of Esbilac canine milk replacement (PetAg Inc., Hampshire, IL) with three times (10 minutes each) daily hypoxia exposure with 5% O2 and 95% N2 and with 200 ng per feed of recombinant NRG4 (saturating dose in a formula feed for ErbB4 phosphorylation, as defined by a concentration curve of 25 to 250 ng administered followed by Western blot analysis for phospho-ErbB4 on ileal homogenates; data not shown) added to the formula; or iii) same as group ii) but with an equal volume (2 μL) of phosphate-buffered saline vehicle instead of NRG4 added to formula. Animals were examined frequently and were euthanized if signs of severe distress were noted. On day 4 (84 hours after birth), rats were euthanized. Tissue was fixed, paraffin embedded, and hematoxylin and eosin stained. Morphologic changes in the intestinal epithelium were graded microscopically, by a pathologist blinded to groups, from grade 0 (normal) to grade 4 (severe) based on pathologic manifestations, including submucosal edema, epithelial sloughing/obliteration, neutrophil infiltration, intestinal perforation, epithelial separation from submucosa, and necrosis. CD-1 mice (14 to 16 days old) were separated from their mothers and maintained without feeds in a temperature- and humidity-controlled chamber throughout the experiment and then were subjected to an experimental NEC protocol as previously described.27Zhang C. Sherman M.P. Prince L.S. Bader D. Weitkamp J.H. Slaughter J.C. McElroy S.J. Paneth cell ablation in the presence of Klebsiella pneumoniae induces necrotizing enterocolitis (NEC)-like injury in the small intestine of immature mice.Dis Model Mech. 2012; 5: 522-532Crossref PubMed Scopus (83) Google Scholar Pups were given 75 mg/kg of the zinc chelator dithizone (Sigma-Aldrich, St. Louis, MO) or an equivalent volume of lithium carbonate vehicle alone as control by i.p. injection. This ablates Paneth cells, which are highly sensitive to zinc levels.28Sawada M. Takahashi K. Sawada S. Midorikawa O. Selective killing of Paneth cells by intravenous administration of dithizone in rats.Int J Exp Pathol. 1991; 72: 407-421PubMed Google Scholar Dithizone was given with 250 μg/kg NRG4 (saturating dose for ErbB4 phosphorylation as determined by a concentration curve of 31.25 to 500 μg/kg administered i.p. followed by Western blot analysis for phospho-ErbB4 on ileal mucosal scrapings; data not shown) or an equivalent volume of phosphate-buffered saline vehicle alone. Six hours after injection, mice were enterally infected by gastric gavage with 1 × 1011 CFU/kg Klebsiella pneumoniae (ATCC, Manassas, VA) or were given a gavage of an equivalent volume of sterile media (nutrient broth; ATCC). Mice were monitored for 10 hours after gavage and then were sacrificed for analysis. Mucosal injury was evaluated by a single blinded investigator and was graded on a 5-point scale: grade 0, no injury; grade 1, mild separation of lamina propria; grade 2, moderate separation of submucosa; grade 3, severe separation and/or edema in submucosa; and grade 4, transmural injury. Intestinal crypts were isolated from intestines of young adult (6- to 8-week-old) ErbB4flox/flox and ErbB4flox/flox;Villin-Cre mice29Long W. Wagner K.U. Lloyd K.C. Binart N. Shillingford J.M. Hennighausen L. Jones F.E. Impaired differentiation and lactational failure of Erbb4-deficient mammary glands identify ERBB4 as an obligate mediator of STAT5.Development. 2003; 130: 5257-5268Crossref PubMed Scopus (138) Google Scholar by calcium chelation (2 mmol/L EDTA in phosphate-buffered saline) and mechanical agitation (2 minutes of shaking by hand in phosphate-buffered saline containing 43.3 mmol/L sucrose and 54.9 mmol/L sorbitol) and then were embedded in Matrigel (BD Biosciences, Franklin Lakes, NJ) containing 50 ng/mL of EGF, 100 ng/mL of Noggin, and 500 ng/mL of R-spondin. Cultured crypts were established as enteroids essentially as previously described30Sato T. Stange D.E. Ferrante M. Vries R.G. Van Es J.H. Van den Brink S. Van Houdt W.J. Pronk A. Van Gorp J. Siersema P.D. Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (2093) Google Scholar and were passaged once before being used in experiments. Cronobacter muytjensii (ATCC 51329; formerly known as Cronobacter or Enterobacter sakazakii31Iversen C. Mullane N. McCardell B. Tall B.D. Lehner A. Fanning S. Stephan R. Joosten H. Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov.Int J Syst Evol Microbiol. 2008; 58: 1442-1447Crossref PubMed Scopus (452) Google Scholar) and IEC-6 rat ileal epithelial cells were obtained from ATCC. Bacteria were grown in tryptic soy broth. IEC-6 cells (passages 20 to 26) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 U/mL of penicillin G, and 100 U/L of streptomycin C. Twenty-four hours before the experiments, the cells were washed and then resupplied with medium containing no antibiotics. Rates of apoptosis on histologic specimens were determined by in situ oligo ligation DNA fragmentation assay (EMD Millipore, Billerica, MA), as we have previously done.22Bernard J.K. McCann S.P. Bhardwaj V. Washington M.K. Frey M.R. Neuregulin-4 is a survival factor for colon epithelial cells both in culture and in vivo.J Biol Chem. 2012; 287: 39850-39858Crossref PubMed Scopus (38) Google Scholar Relative apoptosis in IEC-6 cells was measured by adding a luminescent caspase substrate (Caspase-Glo 3/7; Promega Corp., Madison, WI) to cultures, then determining luminescence with a Victor3McElroy S.J. Underwood M.A. Sherman M.P. Paneth cells and necrotizing enterocolitis: a novel hypothesis for disease pathogenesis.Neonatology. 2013; 103: 10-20Crossref PubMed Scopus (70) Google Scholar multilabel plate reader (Perkin-Elmer, Waltham, MA) after 60 minutes. Restitution (migration) assays were performed using a drill press wound assay as previously described.22Bernard J.K. McCann S.P. Bhardwaj V. Washington M.K. Frey M.R. Neuregulin-4 is a survival factor for colon epithelial cells both in culture and in vivo.J Biol Chem. 2012; 287: 39850-39858Crossref PubMed Scopus (38) Google Scholar Briefly, a drill-mounted rotating silicone tip was used to make multiple approximately 1.5 mm2 wounds in confluent IEC-6 cultures. Wounds were photographed over time, area was determined using ImageJ software version 1.49d (NIH, Bethesda, MD), and percentage closure was determined. IEC-6 cultures were serum deprived (0.5% fetal bovine serum) for 48 hours and then were stimulated with growth factors for 24 hours. Cultures were labeled with 5-ethynyl-2′-deoxyuridine (EdU) for the last 2 hours of incubation and were fixed. Percentage of EdU-labeled cells was determined using a Click-iT EdU proliferation kit (Life Technologies, Grand Island, NY). The antibodies used were polyclonal anti-ErbB4 (c-18) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Src, anti–phospho-Tyr416-Src, and anti–phospho-Tyr1284-ErbB4 (Cell Signaling Technology Inc., Danvers, MA); anti-actin (Sigma-Aldrich); IRDye-conjugated secondary antibodies (Li-Cor Biosciences, Lincoln, NE); and Alexa Fluor 555–conjugated anti-rabbit (Invitrogen, Carlsbad, CA). Recombinant HRG, Noggin, and R-spondin were purchased from R&D Systems (Minneapolis, MN), EGF from PeproTech (Rocky Hill, NJ), and NRG4 from Reprokine Ltd. (Valley Cottage, NY). PP2 was purchased from Sigma-Aldrich. Breast milk was obtained from healthy volunteers. Milk was collected in standard lactation bags, deidentified, and frozen at −70°C. Samples were defrosted and centrifuged at 14,000 rpm for 15 minutes to remove the lipid layer. The remaining protein layers were resuspended, mixed, and solubilized in Laemmli buffer for Western blot analysis. Data were analyzed by analysis of variance using the Tukey post-test, U-test, or Kruskal-Wallis test, as appropriate. ErbB4 expression is induced by injury and inflammation in the adult colon23Frey M.R. Edelblum K.L. Mullane M.T. Liang D. Polk D.B. The ErbB4 growth factor receptor is required for colon epithelial cell survival in the presence of TNF.Gastroenterology. 2009; 136: 217-226Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar as a compensatory response to damage, and NRG4 reduces TNF-induced apoptosis in the adult mouse colon.22Bernard J.K. McCann S.P. Bhardwaj V. Washington M.K. Frey M.R. Neuregulin-4 is a survival factor for colon epithelial cells both in culture and in vivo.J Biol Chem. 2012; 287: 39850-39858Crossref PubMed Scopus (38) Google Scholar To test whether ErbB4 activation is protective in the developing small intestine, we used the well-established FFH model of rat NEC. In this highly penetrant model,18Feng J. El-Assal O.N. Besner G.E. Heparin-binding epidermal growth factor-like growth factor reduces intestinal apoptosis in neonatal rats with necrotizing enterocolitis.J Pediatr Surg. 2006; 41 (discussion -7): 742-747Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 26Nadler E.P. Dickinson E. Knisely A. Zhang X.R. Boyle P. Beer-Stolz D. Watkins S.C. Ford H.R. Expression of inducible nitric oxide synthase and interleukin-12 in experimental necrotizing enterocolitis.J Surg Res. 2000; 92: 71-77Abstract Full Text PDF PubMed Scopus (204) Google Scholar the combination of hypertonic formula feeding combined with hypoxia produces a clinical picture and histologic features similar to human NEC. FFH pups had extensive epithelial damage, whereas pups given NRG4 to activate ErbB4 (FFH/NRG4) had histologic features similar to those of dam-fed controls (Figure 1A). Pathologic scoring identified a significant protective effect, with mean ± SD damage scores of 1.50 ± 0.23 in FFH pups and 0.69 ± 0.21 in the FFH/NRG4 group (Figure 1B). FFH/NRG4 pups had a reduced incidence of disease, defined as the percentage of animals with histologic damage scores ≥2 at sacrifice on day 4 (15% in FFH/NRG4 versus 43% in FFH). The number of apoptotic cells in the remaining epithelial villi was also markedly reduced in NRG4-treated animals (Figure 1C). Because inflammation during development may not be the same as in adult disease, we also tested whether injury-associated ErbB4 induction occurs in experimental NEC. Immunostaining for ErbB4 showed an increase in receptor expression in FFH versus dam-fed pups (Figure 1D). To test NRG4-ErbB4 signaling in a second model of NEC, we used the recently described dithizone/Klebsiella (DK) model27Zhang C. Sherman M.P. Prince L.S. Bader D. Weitkamp J.H. Slaughter J.C. McElroy S.J. Paneth cell ablation in the presence of Klebsiella pneumoniae induces necrotizing enterocolitis (NEC)-like injury in the small intestine of immature mice.Dis Model Mech. 2012; 5: 522-532Crossref PubMed Scopus (83) Google Scholar in 2-week-old mice. In this approach, Paneth cells are ablated by dithizone injection (which chelates zinc required by Paneth cells), followed 6 hours later by K. pneumoniae gavage, resulting in severe NEC-like symptoms within 24 hours (Figure 2A). NRG4 given by i.p. injection simultaneously with dithizone administration was protective in this model, completely blocking DK-induced histologic damage (Figure 2B) (mean ± SD damage score: 1.75 ± 0.37 in DK mice versus 0.6 ± 0.16 in DK + NRG4 mice). Similar to the rat model, injury in the DK mice was associated with increased ErbB4 immunostaining (Figure 2C). In the DK model of mouse NEC, dithizone ablates the Paneth cell population and, thus, sensitizes the intestine to Klebsiella-induced damage. Human NEC has been associated with loss of Paneth cells in the ileum,3McElroy S.J. Underwood M.A. Sherman M.P. Paneth cells and necrotizing enterocolitis: a novel hypothesis for disease pathogenesis.Neonatology. 2013; 103: 10-20Crossref PubMed Scopus (70) Google Scholar suggesting that this part of the model may be especially informative for the development of clinical applications. To test whether ErbB4 signaling is affecting the Paneth cell compartment specifically, we stained ileums from control, DK-, or DK + NRG4–treated mice with Alcian blue/periodic acid-Schiff stain or by immunohistochemical analysis for lysozyme to identify and count Paneth cells. NRG4 blocked Paneth cell loss in the DK model (Figure 3) (mean ± SD Paneth cells per crypt: 2.80 ± 0.26 control, 1.35 ± 0.44 DK, 3.00 ± 0.25 DK + NRG4), suggesting that protection of this specific population, or of Paneth cell progenitors, may be part of the mechanism of protection against NEC. To further investigate the role of ErbB4 in Panet