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Endothelial TLR4 Activation Impairs Intestinal Microcirculatory Perfusion in Necrotizing Enterocolitis Via Enos–NO–Nitrite Signaling

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Endothelial TLR4 Activation Impairs Intestinal Microcirculatory Perfusion in Necrotizing Enterocolitis Via Enos–NO–Nitrite Signaling Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS–NO–nitrite signaling Ibrahim Yazjia,b, Chhinder P. Sodhia,b, Elizabeth K. Leea,b, Misty Goodc,d, Charlotte E. Egana,b, Amin Afrazia,b, Matthew D. Neala,b, Hongpeng Jiaa,b, Joyce Lina,b, Congrong Maa,b, Maria F. Brancaa,b, Thomas Prindlea,b, Ward M. Richardsonb, John Ozoleke,f, Timothy R. Billiarb, David G. Biniong, Mark T. Gladwinh,i, and David J. Hackama,b,1 Divisions of aPediatric Surgery, cNewborn Medicine, and ePathology, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15224; and Departments of bSurgery, dPediatrics, and fPathology and Divisions of gGastroenterology and hPulmonary, Allergy, and Critical Care Medicine, and iVascular Medicine Institute, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved April 12, 2013 (received for review November 16, 2012) Necrotizing enterocolitis (NEC) is a devastating disease of pre- (NO), which is generated through the activity of endothelial NO mature infants characterized by severe intestinal necrosis and for synthase (eNOS) (6). Recent studies have shown that NO can which breast milk represents the most effective protective strat- also be generated through conversion from sodium nitrite egy. Previous studies have revealed a critical role for the lipopoly- within the diet through the activity of commensal microbes (7). saccharide receptor toll-like receptor 4 (TLR4) in NEC development Given that TLR4 is expressed on endothelial cells (8), we now through its induction of mucosal injury, yet the reasons for which hypothesize that TLR4 signaling within the endothelium plays intestinal ischemia in NEC occurs in the first place remain unknown. an essential role in the pathogenesis of NEC by reducing blood We hypothesize that TLR4 signaling within the endothelium plays flow to the small intestine through impaired eNOS signaling. an essential role in NEC development by regulating perfusion to the We further hypothesize that breast milk protects against NEC small intestine via the vasodilatory molecule endothelial nitric development by augmenting eNOS signaling, enhancing intes- oxide synthase (eNOS). Using a unique mouse system in which we tinal perfusion, and thus limiting the deleterious effects of en- MEDICAL SCIENCES selectively deleted TLR4 from the endothelium, we now show dothelial TLR4 activation and reducing NEC severity. that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion Results without effects on other organs and reduces eNOS expression via TLR4 Signaling in Endothelium Is Required for Development of activation of myeloid differentiation primary response gene 88. Experimental NEC. To evaluate directly whether TLR4 signaling − − NEC severity was significantly increased in eNOS / mice and de- within the endothelium is important in NEC pathogenesis, we creased upon administration of the phosphodiesterase inhibitor generated mice selectively lacking TLR4 on endothelial cells, sildenafil, which augments eNOS function. Strikingly, compared by first generating Tlr4loxP mice (9), which were then bred with with formula, human and mouse breast milk were enriched in Angiopoietin receptor, (Tie2) mice transgene insertion 12, sodium nitrate—a precursor for enteral generation of nitrite and Richard A Flavell (B6.Cg–Tg[endothelial specific receptor tyro- nitric oxide—and repletion of formula with sodium nitrate/nitrite sine kinase (Tek)–cre]12Flv/J) to generate the homozygous Δ Δ restored intestinal perfusion, reversed the deleterious effects of TLR4 endoth strain. TLR4 endoth mice were found to be healthy endothelial TLR4 signaling, and reduced NEC severity. These data and fertile and to produce offspring in accordance with expected identify that endothelial TLR4 critically regulates intestinal perfu- Mendelian genetics. In parallel, we also generated mice in which sion leading to NEC and reveal that the protective properties of TLR4 had been deleted from the intestinal epithelium, as we breast milk involve enhanced intestinal microcirculatory integrity have recently described (9). via augmentation of nitrate–nitrite–NO signaling. Because reliable anti-TLR4 antibodies are not available for immunohistochemistry, we confirmed that TLR4 was selec- neonatal inflammation | prematurity | infant formula | tively removed from endothelial cells using alternate approaches. Δ neonatal nutrition | sepsis First, we generated a reporter strain, Tlr4 endoth;R26endoth-mG mice, which indicated by GFP fluorescence that TLR4 had ecrotizing enterocolitis (NEC) is the leading cause of death been deleted from endothelial cells. We first bred Gene trap Nfrom gastrointestinal disease in the premature infant and is ROSA 26, Philippe Soriano; targeted mutation 4, Liqun Luo gradually increasing in frequency (1). The defining pathological (Gt)(ROSA)26Sortm4(ACTB-tdTomato,-EGFP) mice with Tlr4loxP mice feature of NEC is the presence of patchy areas of ischemia and to generate homozygous Tlr4loxP;R26mT/mG mice, which we then Δ necrosis of the small and large intestine (2). Although prema- bred with Tie2–cre mice to generate the Tlr4 endoth;R26endoth-mG turity is the leading risk factor for NEC development, breast milk administration has been identified as the most important pro- tective strategy (3). Importantly, the mechanisms that lead to the Author contributions: I.Y., C.P.S., E.K.L., C.E.E., A.A., M.D.N., H.J., J.L., C.M., M.F.B., W.M.R., acute development of intestinal necrosis in the premature intes- D.G.B., M.T.G., and D.J.H. designed research; I.Y., C.P.S., E.K.L., M.G., C.E.E., A.A., M.D.N., tine and factors within breast milk that may prevent NEC remain H.J., J.L., C.M., M.F.B., T.P., W.M.R., M.T.G., and D.J.H. performed research; T.R.B. and M.T.G. contributed new reagents/analytic tools; C.P.S., E.K.L., M.G., C.E.E., A.A., M.D.N., largely unexplored. C.M., M.F.B., T.P., W.M.R., J.O., T.R.B., D.G.B., M.T.G., and D.J.H. analyzed data; and I.Y., In seeking to understand the underlying biological mechanisms M.T.G., and D.J.H. wrote the paper. that lead to NEC, we and others have identified a critical role for Conflict of interest statement: M.T.G. holds a patent for the use of nitrite salts in cardio- the innate immune receptor toll-like receptor 4 (TLR4) in NEC vascular diseases and consults with Aires Pharmaceuticals. pathogenesis, because mice deficient in TLR4 showed reduced This article is a PNAS Direct Submission. mucosal inflammation and reduced intestinal necrosis in exper- 1To whom correspondence should be addressed. E-mail: [email protected]. imental NEC (4, 5). Microcirculatory perfusion of the premature This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. intestine is primarily regulated by the vasodilator nitric oxide 1073/pnas.1219997110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1219997110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Δ strain. In the Tlr4 endoth;R26endoth-mG strain, all endothelial cells (LPS) (Fig. 1 A, vii), confirming that TLR4 is selectively deleted lacking TLR4 express green fluorescence (green indicates from endothelial cells and is preserved in macrophages. staining of TLR4-deficient endothelial cells; Fig. 1 A, i and ii), To evaluate the role of endothelial TLR4 in the pathogenesis Δ which by whole-mount immunoconfocal analysis revealed a re- of NEC, we next subjected wild-type and TLR4 endoth mice to an ticular network within the lamina propria of each villus. In established model of NEC, consisting of 4 d of hypoxia and a control experiment, we bred the Tlr4loxP;R26mT/mG mice with formula gavage (11). As shown in Fig. 1 B and C, NEC severity fi Δendoth villin–cre mice [B6.SJL–Tg(Vil–cre)997Gum/J]todeleteTLR4 was signi cantly reduced in TLR4 mice compared with Δ from enterocytes [Tlr4 villin(enterocytes);R26villin-mG(enterocytes)], and we wild-type mice, as manifested by the relative preservation of the – observed a markedly different pattern of GFP expression, mucosal architecture (Fig. 1 B, i iv), a reduction in the degree of – which corresponded to the smooth outlines of the enterocyte intestinal ischemia and necrosis (Fig. 1 B, v viii), reduced path- membranes (yellow indicates staining of TLR4-deficient enter- ological severity score (Fig. 1C), decreased enterocyte apoptosis ocytes; Fig. 1 A, iii and iv). We next purified mouse intestinal as determined by reduced expression of cleaved caspase 3 (Fig. D fl microvascular endothelial cells (MIMECs) according to the tech- 1 ), and reduced expression of the proin ammatory cytokine IL-6 − − within the intestinal mucosa (Fig. 1E). Having identified a require- nique of Ogawa et al. (10) from wild-type, TLR4 / ,and Δ ment for TLR4 signaling in the endothelium in NEC pathogenesis, TLR4 endoth mice, along with peritoneal macrophages, and we next sought to evaluate the mechanisms involved. evaluated the expression of TLR4 on single cell suspensions by fl RT-PCR and ow cytometry. As shown in Fig. 1 A, v,both TLR4 Activation Within the Endothelium Impairs Perfusion of the endothelial cells and peritoneal macrophages that were har- Newborn Intestine in the Pathogenesis of NEC. We next sought to vested from wild-type mice expressed TLR4, yet neither cell investigate whether TLR4 activation in the endothelium could −/− type expressed TLR4 when harvested from TLR4 mice. directly regulate blood flow into the intestinal microcirculation. To Importantly, macrophages, and not endothelial cells, that were do so, we first performed intestinal microangiography in newborn Δ harvested from TLR4 endoth mice were found to express TLR4 mice, in which the distribution of a fluorescent tracer (tomato by flow cytometry (Fig. 1 A, vi). Macrophages obtained from lectin) was assessed by whole-mount confocal microscopy on Δ TLR4 endoth mice also demonstrated a robust proinflammatory freshly harvested sections of the terminal ileum 5 min after in- response after exposure to the TLR4 ligand lipopolysaccharide tracardiac injection of tomato lectin (12).
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