The role of PAF, TLR, and the inflammatory response in neonatal necrotizing enterocolitis

Seminars in Pediatric Surgery (2005) 14, 145-151

The role of PAF, TLR, and the inflammatory response in neonatal necrotizing enterocolitis Michael S. Caplan, MD, Dyan Simon, MD, and Tamas Jilling, MD From the Department of Pediatrics, Evanston Northwestern Healthcare and The Evanston Northwestern Healthcare Research Institute, Northwestern University, Feinberg School of Medicine, Evanston, Illinois. INDEX WORDS Neonatal necrotizing enterocolitis, NEC; Platelet activating factor, PAF; Bacteria; Human toll-like receptor, TLR

The pathogenesis of neonatal necrotizing enterocolitis remains poorly understood. Recent evidence suggests that PAF (platelet activating factor) and human toll-like receptors (TLRs) contribute to the pro-inflammatory response that is characteristic of NEC pathology. Understanding the regulation of these molecular interactions may provide new approaches for prevention or treatment of this dreaded condition. © 2005 Elsevier Inc. All rights reserved.

The pathophysiology of necrotizing enterocolitis (NEC) has been difficult to ascertain, but accumulating evidence suggests that an imbalance between an activated proinflammatory response with inadequate antiinflammatory protection results in the hallmark findings of intestinal necrosis and NEC. This chapter will review the data supporting the importance of: (1) inflammation in NEC, (2) platelet activating factor (PAF) and other cytokines on intestinal mucosal injury, and the mechanisms of action, and (3) interaction of bacteria and intestinal human toll-like receptors (TLRs) on neonatal NEC.

Role of inflammation in NEC Necrotizing enterocolitis was first described many years ago, and autopsy specimens demonstrated bland necrosis of affected bowel segments in preterm neonates with this Address reprint requests and correspondence: Michael S. Caplan, MD, Department of Pediatrics, Evanston Northwestern Healthcare, 2650 Ridge Avenue, Evanston, IL 60201. E-mail: [email protected].

1055-8586/$ -see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.sempedsurg.2005.05.002

unique condition. Subsequent studies with more rigorous investigation revealed an inflammatory response in the acute presentation, with influx of neutrophils and macrophages into lamina propria and submucosal portions of affected intestine.1 In clinical studies measuring systemic activation of the inflammatory response, samples from NEC patients showed elevated levels of various inflammatory mediators compared with gestational-age matched controls. For example, elevations of urinary thromboxane A2, and serum IL-1, IL-6, tumor necrosis factor (TNF), and PAF have been associated with the presence of NEC in the acute stages of disease.2-4 Moreover, stool analyses to identify local perturbations in the inflammatory response have revealed increased PAF concentrations compared with nonNEC patients.5 In animal models, clinical risk factors of NEC (bacterial colonization, intestinal ischemia, and formula feeding) stimulate the inflammatory response that ultimately leads to intestinal injury. These findings suggest that the inflammatory response is activated in this condition, and clinical signs and symptoms of capillary leak, hypotension, DIC, respiratory failure, renal failure, and the emerging evidence of brain injury with neurodevelopmental disability in the human condition support this hypothesis.

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Role of PAF in neonatal NEC PAF biochemistry and physiology PAF is an endogenous phospholipid inflammatory mediator with potent biological effects.6,7 PAF formation occurs in most cells and tissues beginning with the conversion of a phosphatidylcholine precursor to a biologically inactive intermediate, lysoPAF, under the influence of a specific phospholipase A2 called phospholipase A2-II. Subsequent acetylation of lysoPAF at the n-2 position by acetyltransferase completes PAF synthesis. This PAF molecule has a very short half-life as it is rapidly converted back to lysoPAF under the control of PAF-acetylhydrolase (PAF-AH).8 PAF binds to a specific PAF receptor (PAFR) that is present on most cells, and is a member of the G protein-coupled 7 transmembrane domain receptor family (GPCR). Similar to other GPCRs, ligand binding to the PAFR elicits both G protein-dependent and G protein-independent signaling as well as receptor internalization. Downstream signaling includes elevation of cytoplasmic free [Ca2⫹], stimulation of protein kinase-C, MAPK, MEK, and ERK.9 This signaling cascade activates NF␬B and STAT-3 synthesis, resulting in their translocation into the nucleus and transcriptional activation of additional inflammatory molecules, including iNOS, TNF, endothelin-1 (ET-1), IL-1, IL-6, IL-8, and a potent inflammatory response. While in some cells, such as leukocytes, PAFR activation results in cell proliferation and survival in others, including enterocytes, PAF activates pathways that result in caspase activation and apoptosis. In various experimental models, PAF has been shown to cause capillary leak, myocardial dysfunction, airway hyperreactivity, renal dysfunction, neutropenia, thrombocytopenia, and hypotension. While the specific pathomechanisms are not completely defined, exposure of intestinal mucosa to high local PAF concentrations results in apoptosis, mucosal permeability, intestinal inflammation, activation of the secondary inflammatory response, and bowel necrosis.

PAF in intestinal necrosis Initial studies in adult rats utilizing an acute model have shown that: (1) exogenous PAF given intravenously results in ischemic bowel necrosis,10 (2) endotoxin, hypoxia, or TNF-induced intestinal injury can be prevented by PAF receptor antagonists,11-13 and (3) endotoxin and hypoxia stress increases intestinal PAF content.11,14 Further evaluation of PAF-induced intestinal necrosis has identified the contribution of oxygen free radicals and reactive nitrogen species, endothelin-1, cyclooxygenase, and leukotriene activation, and complement in this pathophysiology.15,16

Figure 1 Examples of histologic findings from the experimental neonatal rat model of NEC. Representative sections show (a) mother-fed, healthy intestine; (b) moderate intestinal injury with villus necrosis and inflammation; and (c) transmural necrosis (H&E, ⫻100 with insets ⫻400 magnification). (Color version of figure is available online.)

Role of PAF in NEC Additional experiments have evaluated the importance of PAF in neonatal rats using the typical risk factors of NEC, including asphyxia and formula feeding (Figure 1).17 In our

neonatal model, asphyxia and formula feeding are both necessary to initiate NEC, and these risk factors together activate abnormal intestinal gene expression of the PAF synthesizing enzyme PLA2-II and the PAF receptor on

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intestinal epithelium. Furthermore, using this model, we have shown that PAF receptor blockade reduces the incidence of NEC,18 and that the PAF-degrading enzyme PAFacetylhydrolase (PAF-AH) given with enteral feedings blocks the initiation of disease.19 Studies from adult rats indicate that PAF-receptor gene is constitutively expressed at the highest concentration in ileum compared with lung, kidney, and other organs.20 This fact suggests that intestine may be at particular risk for PAF-induced pathophysiology, thus leading to NEC. Furthermore, PAF and LPS in a rat model stimulates PAF-receptor mRNA synthesis within 30 minutes of exposure,20 and PAF appears to increase its own synthesis via PLA2 expression. While human analyses of intestinal tissue are difficult to obtain, serum from NEC patients demonstrate higher PAF levels and TNF activity compared with age-matched controls, and stool from NEC patients have much higher PAF content then in patients without intestinal necrosis. Of interest, human neonates have low or absent circulating PAF-AH activity,21 and breast milk (thought to reduce the risk for NEC) contains significant quantities of PAF-AH.22 Of particular interest, PAF-AH given enterally to neonatal rats is not absorbed into the systemic circulation, but remains functionally active and present in the intestinal epithelium.19 These results imply that changes in local PAF metabolism in the intestinal microenvironment may play a key role in the initiation of neonatal NEC.

PAF activates signaling cascade and apoptosis The PAF-receptor is a member of the GPCR superfamily.23 As discussed above, activation of PAF-receptor leads to stimulation of several signal transduction pathways culminating in pathophysiologic changes of vasoconstriction and/or vasodilatation, leukocyte stimulation and migration, synthesis and activation of cell adhesion molecules, increased capillary permeability, production of reactive oxygen and nitrogen species, and alterations in intestinal mucosal permeability.6,24 Our recent studies have shown that PAF receptor signals through the dephosphorylation of Akt, a molecular event that can be quantitatively measured, and used to evaluate PAF receptor function and signal transduction in cell culture monolayers and in vivo systems. Furthermore, in transfected COS-7 cells and in HUVEC cultures, PAFR signals via tyrosine phosphorylation of Tyk2 kinase and subsequent activation of STAT, leading to gene expression of multiple inflammatory mediators, including nitric oxide, phospholipase A2, and endothelin-1.25,26 Several lines of evidence suggest that PAF activates apoptosis, or programmed cell death of intestinal epithelial cells, and that this process is an early initiator of experimental NEC. We have shown that accelerated enterocyte apoptosis precedes histological damage in the neonatal rat model of NEC, and that caspase inhibition reduces the incidence of experimental NEC (Figure 2).27 Furthermore,

Figure 2 TUNEL-stained sections of neonatal rat small bowel specimens from (a) mother-fed control animals; (b) formula-fed, asphyxia stressed at 24 hours; and (c) formula-fed, asphyxia stressed at 72 hours. Images are results of pseudocolored overlays of corresponding TUNEL-FITC (shown in green) and Hoechst nuclear counter stain (shown in red). (Color version of figure is available online.)

in vitro studies have shown that PAF can initiate enterocyte apoptosis via a mechanism involving Bax translocation to mitochondria, a collapse of mitochondrial membrane potential, and subsequent caspase 3 activation, and that heterologous and inducible over-expression of Bcl-2, an antiapoptotic protein, can block this cascade and protect enterocytes from apoptosis.28 Utilizing Ussing chamber analyses, we have shown that PAF regulates transepithelial ion transport in colonic epithelial cells and that PAFR localizes exclusively to the apical plasma membrane of these cells.29 In these studies, PAF stimulates changes in chloride transport that result in intracellular acidification and presumed enzyme dysfunction. Increased apoptosis of intestinal epithelial cells results in exaggerated mucosal permeability, loss of tight junctional integrity, and the likelihood of bacterial translocation that has been implicated as an important event in the pathophysiologic cascade. Whether the presence of bacteria on the mucosal surface is enough to initiate intestinal inflammation and injury, or whether translocation across mucosa is required, remains to be determined.

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Bacteria stimulate intestinal inflammation Bacteria and/or bacterial cell wall products, including endotoxin (LPS, lipopolysaccharide derived from Gram-negative organisms) and lipoteichoic acid (LTA, found in Gram-positive bacteria), have been implicated in the pathogenesis of NEC. Following normal vaginal delivery and initiation of breast-milk feeding, the neonate rapidly develops intestinal colonization by multiple bacterial strains including aerobic and anaerobic organisms.30 In the premature newborn cared for in the NICU environment, patterns of bacterial colonization are abnormal with decreased species diversity and a paucity of anaerobes.31 It has been suggested that this altered colonization pattern influences the incidence of NEC in this high-risk population. While some studies have identified specific pathogens in epidemic outbreaks of NEC, most cases occur endemically, and appear to be unrelated to a specific organism. Nonetheless, the presence of enteric bacteria is a prerequisite for disease, as inferred from the observation that antenatal intestinal ischemia, in the face of a sterile intestinal environment, leads to intestinal atresia rather than NEC. Of interest, experimental studies suggest that PAF-induced intestinal injury is dependent on normal gut colonization; germ-free rats demonstrate attenuated responses in this important model.32 Analyses from our laboratory have shown that PAF receptor expression in intestinal epithelium is increased with bacterial exposure and that this response may correlate with NEC. Nonetheless, the specific role of bacteria in this complex pathophysiologic process remains poorly understood. Using our experimental model of NEC in neonatal rats, we investigated the influence of bacteria on intestinal necrosis.17 In the past, the incidence of intestinal injury following the protocol has varied between 20% and 70%, and due to this significant fluctuation, we investigated the effect of sanitization versus bacterial contamination in the model. By carefully controlling the bacterial load, we were able to induce a high incidence of NEC reproducibly in either rats or mice. We found that exposure to exogenous bacteria, given enterally via the orogastric feeding tube, increased the incidence of NEC in neonatal rats. In animals that were fed with colonized catheters, a 53% incidence of NEC was similar to the frequency described for this model previously. In animals that were fed with sanitized catheters (rinsed with alcohol after each usage, every 3 hours), the incidence of experimental NEC was dramatically decreased to 19%. To further identify bacteria involved in catheter contamination, we analyzed the catheter water in which the feeding catheters were maintained between feeds. Three predominant organisms, Klebsiella pneumoniae, Serratia marcescens, and Streptococcus viridans, were isolated, grown in pure cultures, and then each bacterial strain was fed to various groups to distinguish their independent contribution to NEC, and compared with the sanitized groups. We found that there was an independent increase in NEC scores for all bacteria species isolated. S. marcescens, K. pneumoniae,

Seminars in Pediatric Surgery, Vol 14, No 3, August 2005 and S. viridans had a 55.5%, 40.7%, and 44.4% incidence of histologic NEC, respectively, when compared with the sanitized group of 18.5%. Of note, these findings mirror the experience in the NICU, where colonization of feeding catheters positively correlated with feeding intolerance and NEC.33 These findings confirm that intestinal bacterial colonization is required to initiate NEC, but the mechanisms responsible for this response remain undefined.

Role of TLRs in neonatal NEC Human toll-like receptors (TLRs) represent a large family of transmembrane molecules homologous to Drosophila Toll protein that recognize specific repetitive patterns contained in bacterial products.34 For example, LPS has been shown to bind to and activate TLR-4, whereas LTA specifically binds to TLR-2. Additional bacterial product ligands and TLRs have been defined, including dsRNA with TLR3, flagellin with TLR-5, and CpG/bacterial DNA with TLR-9. TLR activation results in signal transduction involving a complex cascade with IL-1R-associated protein kinases (IRAKs), TGF-␤-activated kinase (TAK1), TAK1-binding proteins (TAB1 and TAB2), and TNF receptor-associated factor 6 (TRAF6).35 Activation and phosphorylation of these molecules activates IKK, allowing degradation of I␬B and release and activation of NF␬B, subsequent cytokine production, and a robust inflammatory response.36,37 Studies have shown that adult human intestine expresses low levels of TLR-2 and TLR-4, and this downregulation may serve to limit intestinal inflammation and injury in the face of normal intraluminal bacterial exposure.38 In intestinal epithelial cells, cytokines such as IFN and TNF appear to increase TLR4 expression, thereby rendering them more sensitive to LPS-induced activation.39 Additional studies have shown that expression of MD-2, an LPS coreceptor linked with TLR4, is regulated by the JAK/STAT pathway and is blocked by SOCS3, a STAT inhibitor.40 In adult intestine, it has been shown that TLRs (using Myd88 knockout mice, an adapter protein important for TLR signal transduction) function to control intestinal epithelial homeostasis and as bacteria-recognition receptors to protect against gut injury. Although the data show that TLRs protect against inflammatory bowel disease in adult mouse models, little is known about the development of TLR responses in the neonate during ontogeny and during the critical period of bacterial colonization. Of interest, C3H/HeJ mice that harbor a mutation in their TLR-4 gene rendering them effectively TLR-4-deficient, are resistant to PAF-induced intestinal injury, suggesting that TLR-4 dysfunction may have the opposite effect from the adult IBD models and protect against neonatal NEC.41 Bacterial colonization of the intestinal tract develops in the neonate following birth during the first several days of life. During this important process, interaction between bacterial ligands and intestinal epithelium initiates a series of

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immunomodulatory events. If TLRs were expressed on epithelial cells during this sensitive time, activation of the inflammatory response may occur and pose danger for the developing intestine. To evaluate these events, we focused our investigation on TLR4, which is a prototypical member of the family, senses one of the best known and most common bacterial-derived noxious agent (lipopolysaccharide), and ample genetic tools are available for its investigation, including TLR4⫺/⫺ mice. Our data indicate that there is a proximal-to-distal increasing gradient of TLR4 expression level in normal neonatal rat intestinal tract and that in mother-fed neonatal rats TLR4 expression is developmentally downregulated in the first 72 hours of life (Figure 3). However, in formula-fed stressed animals, we observed a dramatic increase of TLR4 expression at 48 and 72 hours of life along the intestinal tract (Figure 3). To determine the localization of increased TLR4 expression, we performed laser capture microdissection from intestinal frozen sections collected at 72 hours of life. Real-time PCR analysis of these samples revealed that TLR4 expression in the submucosa was unchanged in formula-fed stressed animals, but epithelial TLR4 expression was significantly increased both in the crypts and in the villi. These findings suggest that neonatal intestinal expression of TLR following stress is abnormally upregulated, and together with an altered bacterial colonization pattern would place the neonate at risk for a proinflammatory response and NEC. To better understand the importance of TLR4 in neonatal NEC, we developed a mouse model of disease analogous to the neonatal rat model described above to easily study gene knockouts. In the first series of 50 neonatal mice stressed with asphyxia and formula feeding, we observed gross and histologic evidence of NEC in 66% of animals by the 4th day of life with typical clinical signs of abdominal disten-

Figure 3 Effect of formula feeding with cold asphyxia stress (FFCAS) compared with mother-fed controls on whole intestinal TLR4 mRNA (normalized as percent to GAPDH) synthesis over time.

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Figure 4 Dose-dependent effect of PAF on TLR4 gene expression (normalized to GAPDH) in IEC-6 cells after 5 hours of incubation. *, P ⬍ 0.05 compared with vehicle incubations.

tion, bloody stools, and respiratory distress. Following development of the model with consistent results, we obtained endotoxin-resistant mice that harbor a mutation in the TLR4 gene (C3H/HeJ) conferring dysfunction of TLR4, and compared their incidence of NEC to an appropriate control strain (C3H/FeJ). Histologically, there was a dramatic decrease in NEC in the TLR-4 deficient mice when compared with controls: 5/43 (12%) C3H/HeJ versus 16/26 (62%) C3H/FeJ controls. Together with the increased intestinal TLR4 expression in formula-fed animals described above, these data strongly suggest that TLR4 plays a critical role in the development of NEC. Since PAF plays a major role in the pathogenesis of experimental NEC in neonatal rats exposed to asphyxia and formula feeding, we evaluated whether PAF influenced TLR2 and TLR4 expression in enterocytes. Treatment of IEC-6 cells with PAF results in a threefold induction of TLR2 and TLR4 mRNA within 5 hours of treatment (Figure 4). It is notable that PAF induces high levels of TLR expression at concentrations when PAF-induced direct proapoptotic effect on enterocytes is almost undetectable.42,43 These data support our hypothesis that PAF influences TLR expression in enterocytes, and that this might be yet another mechanism whereby PAF could initiate intestinal inflammation and necrosis. Because we detected PAF-induced increased TLR4 expression, since the TLR4 promoter region has STAT and NF␬Bresponsive elements, and PAF was shown to activate STAT-s, we performed preliminary experiments evaluating the effects of PAF on STAT translocation to the nuclei using a panel of STAT antibodies, including anti-STAT1, -2, -3, -4, -5, and -6. We found that PAF induces phosphorylation and translocation

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Figure 5 Proposed mechanism for the interaction between PAFrelated signaling, toll-like receptors, and bacteria in the pathogenesis of NEC.

of STAT3 to the nuclei of IEC-6 cells. Similarly, PAF induced a translocation of STAT3 into the nuclei of COS-7 cells transfected with the human PAFR. These results begin to delineate the mechanisms involved in PAF-induced TLR expression in epithelial cells, but additional work is necessary to clarify these complex mechanisms. Nonetheless, these findings provide additional support for a dysregulation between PAF and TLR in neonatal NEC.

Summary In summary, NEC is a complex pathophysiologic process characterized by the activation of the intestinal inflammatory response. Based on experimental data and human analyses, PAF is an endogenous mediator that plays a critical role in this process. Bacterial colonization is a prerequisite for this disease; these organisms stimulate PAF production and signaling via specific recognition receptors called human toll-like receptors (TLRs). Experimental evidence suggests that abnormal TLR activation, perhaps via the influence of PAF, in the developing neonate increases the likelihood of developing NEC (Figure 5). Human analyses are needed to corroborate these most interesting findings.

Acknowledgments This work was supported in part by the March of Dimes, The National Institutes of Health, Mead Johnson Nutritionals, and the Jessica Jacobi Golder Endowment.

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