Colitis-Induced Small Intestinal Hypomotility Is Dependent on Enteroendocrine Cell Loss in Mice.

BACKGROUND & AIMS: The abdominal discomfort experienced by patients with colitis may be attributable in part to the presence of small intestinal dysmotility, yet mechanisms linking colonic inflammation with small-bowel motility remain largely unexplored. We hypothesize that colitis results in small intestinal hypomotility owing to a loss of enteroendocrine cells (EECs) within the small intestine that can be rescued using serotonergic-modulating agents. METHODS: Male C57BL/6J mice, as well as mice that overexpress (EECOVER) or lack (EECDEL) NeuroD1+ enteroendocrine cells, were exposed to dextran sulfate sodium (DSS) colitis (2.5% or 5% for 7 days) and small intestinal motility was assessed by 70-kilodalton fluorescein isothiocyanate-dextran fluorescence transit. EEC number and differentiation were evaluated by immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, and quantitative reverse-transcriptase polymerase chain reaction. Mice were treated with the 5-hydroxytryptamine receptor 4 agonist prucalopride (5 mg/kg orally, daily) to restore serotonin signaling. RESULTS: DSS-induced colitis was associated with a significant small-bowel hypomotility that developed in the absence of significant inflammation in the small intestine and was associated with a significant reduction in EEC density. EEC loss occurred in conjunction with alterations in the expression of key serotonin synthesis and transporter genes, including Tph1, Ddc, and Slc6a4. Importantly, mice overexpressing EECs revealed improved small intestinal motility, whereas mice lacking EECs had worse intestinal motility when exposed to DSS. Finally, treatment of DSS-exposed mice with the 5-hydroxytryptamine receptor 4 agonist prucalopride restored small intestinal motility and attenuated colitis. CONCLUSIONS: Experimental DSS colitis induces significant small-bowel dysmotility in mice owing to enteroendocrine loss that can be reversed by genetic modulation of EEC or administering serotonin analogs, suggesting novel therapeutic approaches for patients with symptomatic colitis.

Ccr2 Dependent Monocytes Exacerbate Intestinal Inflammation and Modulate Gut Serotonergic Signaling Following Traumatic Brain Injury.

BACKGROUND: Traumatic brain injury (TBI) leads to acute gastrointestinal dysfunction and mucosal damage, resulting in feeding intolerance. Ccr2+ monocytes are crucial immune cells that regulate the gut's inflammatory response via the brain-gut axis. Using CCR2KO mice, we investigated the intricate interplay between these cells to better elucidate the role of systemic inflammation after TBI. METHODS: A murine-controlled cortical impact model was utilized, and results were analyzed on post-injury days (PID) 1 and 3. The experimental groups included (1) Sham C57Bl/6 wild-type (WT), (2) TBI WT, (3) Sham CCR2KO and (4) TBI CCR2KO. Mice were euthanized on PID 1 and 3 to harvest the ileum and study intestinal dysfunction and serotonergic signaling using a combination of quantitative real-time PCR (qRT-PCR), immunohistochemistry, FITC-dextran motility assays, and flow cytometry. Student's t-test and one-way ANOVA were used for statistical analysis, with significance achieved when p < 0.05. RESULTS: TBI resulted in severe dysfunction and dysmotility of the small intestine in WT mice as established by significant upregulation of inflammatory cytokines iNOS, Lcn2, TNFα, and IL1ß and the innate immunity receptor toll-like receptor 4 (Tlr4). This was accompanied by disruption of genes related to serotonin synthesis and degradation. Notably, CCR2KO mice subjected to TBI showed substantial improvements in intestinal pathology. TBI CCR2KO groups demonstrated reduced expression of inflammatory mediators (iNOS, Lcn2, IL1ß, and Tlr4) and improvement in serotonin synthesis genes, including tryptophan hydroxylase 1 (Tph1) and dopa decarboxylase (Ddc). CONCLUSION: Our study reveals a critical role for Ccr2+ monocytes in modulating intestinal homeostasis after TBI. Ccr2+ monocytes aggravate intestinal inflammation and alter gut-derived serotonergic signaling. Therefore, targeting Ccr2+ monocyte-dependent responses could provide a better understanding of TBI-induced gut inflammation. Further studies are required to elucidate the impact of these changes on brain neuroinflammation and cognitive outcomes. STUDY TYPE: Original Article (Basic Science, level of evidence N/A).

Human milk oligosaccharides reduce necrotizing enterocolitis-induced neuroinflammation and cognitive impairment in mice.

Necrotizing enterocolitis (NEC) is the leading cause of morbidity and mortality in premature infants. One of the most devastating complications of NEC is the development of NEC-induced brain injury, which manifests as impaired cognition that persists beyond infancy and which represents a proinflammatory activation of the gut-brain axis. Given that oral administration of the human milk oligosaccharides (HMOs) 2'-fucosyllactose (2'-FL) and 6'-sialyslactose (6'-SL) significantly reduced intestinal inflammation in mice, we hypothesized that oral administration of these HMOs would reduce NEC-induced brain injury and sought to determine the mechanisms involved. We now show that the administration of either 2'-FL or 6'-SL significantly attenuated NEC-induced brain injury, reversed myelin loss in the corpus callosum and midbrain of newborn mice, and prevented the impaired cognition observed in mice with NEC-induced brain injury. In seeking to define the mechanisms involved, 2'-FL or 6'-SL administration resulted in a restoration of the blood-brain barrier in newborn mice and also had a direct anti-inflammatory effect on the brain as revealed through the study of brain organoids. Metabolites of 2'-FL were detected in the infant mouse brain by nuclear magnetic resonance (NMR), whereas intact 2'-FL was not. Strikingly, the beneficial effects of 2'-FL or 6'-SL against NEC-induced brain injury required the release of the neurotrophic factor brain-derived neurotrophic factor (BDNF), as mice lacking BDNF were not protected by these HMOs from the development of NEC-induced brain injury. Taken in aggregate, these findings reveal that the HMOs 2'-FL and 6'-SL interrupt the gut-brain inflammatory axis and reduce the risk of NEC-induced brain injury.NEW & NOTEWORTHY This study reveals that the administration of human milk oligosaccharides, which are present in human breast milk, can interfere with the proinflammatory gut-brain axis and prevent neuroinflammation in the setting of necrotizing enterocolitis, a major intestinal disorder seen in premature infants.

Pharmacologic Toll-like receptor 4 inhibition skews toward a favorable A1/A2 astrocytic ratio improving neurocognitive outcomes following traumatic brain injury.

BACKGROUND: Astrocytes are critical neuroimmune cells that modulate the neuroinflammatory response following traumatic brain injury (TBI) because of their ability to acquire neurotoxic (A1) or neuroprotective (A2) phenotypes. Using C34, a novel pharmacologic Toll-like receptor (TLR) 4 inhibitor, we explored their respective polarization states after TBI. METHODS: A murine controlled cortical impact model was used, and the results were analyzed on postinjury days (PIDs) 1, 7, and 28. The experimental groups are as follows: (1) sham, (2) sham + C34, (3) TBI, and (4) TBI + C34. Quantitative real-time polymerase chain reaction was used to quantify gene expression associated with proinflammatory (A1) and anti-inflammatory (A2) phenotypes. Morris water maze was used to assess neurocognitive outcomes. Fixed frozen cortical samples were sectioned, stained for myelin basic protein and 4',6-diamidino-2-phenylindole, and then imaged. Student t test and one-way analysis of variance were used for statistical analysis with significance achieved when p < 0.05. RESULTS: On quantitative real-time polymerase chain reaction, C34-treated groups showed a significant decrease in the expression of A1 markers such as Gbp2 and a significant increase in the expression of A2 markers such as Emp1 when compared with untreated groups on PID 1. On PIDs 7 and 28, the expression of most A1 and A2 markers was also significantly decreased in the C34-treated groups. On immunohistochemistry, C34-treated groups demonstrated increased myelin basic protein staining into the lesion by PID 28. C34-treated groups showed more platform entries on Morris water maze when compared with untreated groups on PID 7 and PID 28. CONCLUSION: Following TBI, early TLR4 blockade modulates astrocytic function and shifts its polarization toward the anti-inflammatory A2-like phenotype. This is accompanied by an increase in myelin regeneration, providing better neuroprotection and improved neurocognitive outcomes. Targeting A1/A2 balance with TLR4 inhibition provides a potential therapeutic target to improve neurobehavioral outcomes in the setting of TBI.

Infiltrating anti-inflammatory monocytes modulate microglial activation through toll-like receptor 4/interferon-dependent pathways following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is the leading cause of morbidity and mortality in the pediatric population. Microglia and infiltrating monocyte-derived macrophages are crucial immune cells that modulate the neuroinflammatory response following TBI. Using C34, a novel pharmacologic toll-like receptor 4 inhibitor, we investigated the intricate interactions between these cells in a murine TBI model. METHODS: A murine controlled cortical impact model was used, and the results were analyzed on postinjury days 1, 7, 28, and 35. The experimental groups are as follows: (1) sham C57BL/6 wild-type (WT), (2) TBI WT, (3) sham WT + C34, and (4) TBI WT + C34. Quantitative real-time polymerase chain reaction was used to quantify gene expression associated with microglial activation, apoptotic pathways, and type 1 interferon pathway. Flow cytometry was used to isolate microglia and infiltrating monocytes. Brain lesion volumes were assessed using magnetic resonance imaging. Last, neurocognitive outcomes were evaluated using the Morris Water Maze test. Student's t test and one-way analysis of variance were used for statistical analysis with significance achieved when p < 0.05. RESULTS: Toll-like receptor 4 inhibition leads to improved neurological sequela post-TBI, possibly because of an increase in infiltrating anti-inflammatory monocytes and a decrease in IFN regulatory factor 7 during acute inflammation, followed by a reduction in apoptosis and M2 microglial expression during chronic inflammation. CONCLUSION: Toll-like receptor 4 inhibition with C34 skews infiltrating monocytes toward an anti-inflammatory phenotype, leading to enhanced neurocognitive outcomes. Moreover, although M2 microglia have been consistently shown as inducers of neuroprotection, our results clearly demonstrate their detrimental role during the chronic phases of healing post-TBI.

The administration of amnion-derived multipotent cell secretome ST266 protects against necrotizing enterocolitis in mice and piglets.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is steadily rising in frequency. Patients who develop NEC have a very high mortality, illustrating the importance of developing novel prevention or treatment approaches. We and others have shown that NEC arises in part from exaggerated signaling via the bacterial receptor, Toll-like receptor 4 (TLR4) on the intestinal epithelium, leading to widespread intestinal inflammation and intestinal ischemia. Strategies that limit the extent of TLR4 signaling, including the administration of amniotic fluid, can reduce NEC development in mouse and piglet models. We now seek to test the hypothesis that a secretome derived from amnion-derived cells can prevent or treat NEC in preclinical models of this disease via a process involving TLR4 inhibition. In support of this hypothesis, we show that the administration of this secretome, named ST266, to mice or piglets can prevent and treat experimental NEC. The protective effects of ST266 occurred in the presence of marked TLR4 inhibition in the intestinal epithelium of cultured epithelial cells, intestinal organoids, and human intestinal samples ex vivo, independent of epidermal growth factor. Strikingly, RNA-seq analysis of the intestinal epithelium in mice reveals that the ST266 upregulates critical genes associated with gut remodeling, intestinal immunity, gut differentiation. and energy metabolism. These findings show that the amnion-derived secretome ST266 can prevent and treat NEC, suggesting the possibility of novel therapeutic approaches for patients with this devastating disease.NEW & NOTEWORTHY This work provides hope for children who develop NEC, a devastating disease of premature infants that is often fatal, by revealing that the secreted product of amniotic progenitor cells (called ST266) can prevent or treat NEC in mice, piglet, and "NEC-in-a-dish" models of this disease. Mechanistically, ST266 prevented bacterial signaling, and a detailed transcriptomic analysis revealed effects on gut differentiation, immunity, and metabolism. Thus, an amniotic secretome may offer novel approaches for NEC.

The administration of a pre-digested fat-enriched formula prevents necrotising enterocolitis-induced lung injury in mice.

Necrotising enterocolitis (NEC) is a devastating gastrointestinal disease of prematurity that typically develops after the administration of infant formula, suggesting a link between nutritional components and disease development. One of the most significant complications that develops in patients with NEC is severe lung injury. We have previously shown that the administration of a nutritional formula that is enriched in pre-digested Triacylglyceride that do not require lipase action can significantly reduce the severity of NEC in a mouse model. We now hypothesise that this 'pre-digested fat (PDF) system' may reduce NEC-associated lung injury. In support of this hypothesis, we now show that rearing newborn mice on a nutritional formula based on the 'PDF system' promotes lung development, as evidenced by increased tight junctions and surfactant protein expression. Mice that were administered this 'PDF system' were significantly less vulnerable to the development of NEC-induced lung inflammation, and the administration of the 'PDF system' conferred lung protection. In seeking to define the mechanisms involved, the administration of the 'PDF system' significantly enhanced lung maturation and reduced the production of reactive oxygen species (ROS). These findings suggest that the PDF system protects the development of NEC-induced lung injury through effects on lung maturation and reduced ROS in the lung and also increases lung maturation in non-NEC mice.

Toll-like receptor 4-mediated enteric glia loss is critical for the development of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants, whose pathogenesis remains incompletely understood, although activation of the Gram-negative bacterial receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium plays a critical role. Patients with NEC typically display gastrointestinal dysmotility before systemic disease is manifest, suggesting that dysmotility could drive NEC development. Both intestinal motility and inflammation are governed by the enteric nervous system, a network of enteric neurons and glia. We hypothesized here that enteric glia loss in the premature intestine could lead to dysmotility, exaggerated TLR4 signaling, and NEC development. We found that intestinal motility is reduced early in NEC in mice, preceding the onset of intestinal inflammation, whereas pharmacologic restoration of intestinal motility reduced NEC severity. Ileal samples from mouse, piglet, and human NEC revealed enteric glia depletion, and glia-deficient mice (Plp1ΔDTR, Sox10ΔDTR, and BdnfΔDTR) showed increased NEC severity compared with wild-type mice. Mice lacking TLR4 on enteric glia (Sox10-Tlr4ko) did not show NEC-induced enteric glia depletion and were protected from NEC. Mechanistically, brain-derived neurotrophic factor (BDNF) from enteric glia restrained TLR4 signaling on the intestine to prevent NEC. BDNF was reduced in mouse and human NEC, and BDNF administration reduced both TLR4 signaling and NEC severity in enteric glia­deficient mice. Last, we identified an agent (J11) that enhanced enteric glial BDNF release, inhibited intestinal TLR4, restored motility, and prevented NEC in mice. Thus, enteric glia loss might contribute to NEC through intestinal dysmotility and increased TLR4 activation, suggesting enteric glia therapies for this disorder.

Maternal aryl hydrocarbon receptor activation protects newborns against necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a disease of premature infants characterized by acute intestinal necrosis. Current dogma suggests that NEC develops in response to post-natal dietary and bacterial factors, and so a potential role for in utero factors in NEC remains unexplored. We now show that during pregnancy, administration of a diet rich in the aryl hydrocarbon receptor (AHR) ligand indole-3-carbinole (I3C), or of breast milk, activates AHR and prevents NEC in newborn mice by reducing Toll-like receptor 4 (TLR4) signaling in the newborn gut. Protection from NEC requires activation of AHR in the intestinal epithelium which is reduced in mouse and human NEC, and is independent of leukocyte activation. Finally, we identify an AHR ligand ("A18") that limits TLR4 signaling in mouse and human intestine, and prevents NEC in mice when administered during pregnancy. In summary, AHR signaling is critical in NEC development, and maternally-delivered, AHR-based therapies may alleviate NEC.

Necrotizing enterocolitis induces T lymphocyte-mediated injury in the developing mammalian brain.

Necrotizing enterocolitis (NEC) causes acute intestinal necrosis in premature infants and is associated with severe neurological impairment. In NEC, Toll-like receptor 4 is activated in the intestinal epithelium, and NEC-associated brain injury is characterized by microglial activation and white matter loss through mechanisms that remain unclear. We now show that the brains of mice and humans with NEC contained CD4+ T lymphocytes that were required for the development of brain injury. Inhibition of T lymphocyte influx into the brains of neonatal mice with NEC reduced inflammation and prevented myelin loss. Adoptive intracerebroventricular delivery of gut T lymphocytes from mice with NEC into Rag1 -/- recipient mice lacking CD4+ T cells resulted in brain injury. Brain organoids derived from mice with or without NEC and from human neuronal progenitor cells revealed that IFN-γ release by CD4+ T lymphocytes induced microglial activation and myelin loss in the organoids. IFN-γ knockdown in CD4+ T cells derived from mice with NEC abrogated the induction of NEC-associated brain injury after adoptive transfer to naïve Rag1 -/- recipient mice. T cell receptor sequencing revealed that NEC mouse brain-derived T lymphocytes shared homology with gut T lymphocytes from NEC mice. Intraperitoneal injection of NEC gut-derived CD4+ T lymphocytes into naïve Rag1 -/- recipient mice induced brain injury, suggesting that gut-derived T lymphocytes could mediate neuroinflammation in NEC. These findings indicate that NEC-associated brain injury may be induced by gut-derived IFN-γ-releasing CD4+ T cells, suggesting that early management of intestinal inflammation in children with NEC could improve neurological outcomes.

The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.

BACKGROUND: Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can reduce NEC through inhibition of TLR4 signaling. METHODS: NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2'-FL, 6'-SL, or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, and human tissue explants and via in silico modeling. RESULTS: Supplementation of infant formula with either 2'-FL and/or 6'-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2'-FL and 6'-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2'-FL and 6'-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling. CONCLUSIONS: 2'-FL and 6'-SL, but not lactose, prevent NEC in mice and piglet models and attenuate NEC inflammation in the human ileum, in part through TLR4 inhibition. IMPACT: Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants that occurs in the setting of bacterial colonization of the gut and administration of formula feeds and activation by the innate immune receptor toll-like receptor 4 (TLR4). Breast milk prevents NEC through unclear mechanisms. We now show that breast milk-enriched human milk oligosaccharides (HMOs) that are derived from lactose prevent NEC through inhibition of TLR4. The human milk oligosaccharides 2'-FL and 6'-SL, but not the backbone sugar lactose, prevent NEC in mice and piglets. 2'-FL and 6'-SL but not lactose inhibited TLR4 signaling in cultured enterocytes, in enteroids derived from mouse intestine, and in human intestinal explants obtained at the time of surgical resection for patients with NEC. In seeking the mechanisms involved, 2'-FL and 6'-SL but not lactose were found to directly bind to TLR4, explaining the inhibition and protection against NEC. These findings may impact clinical practice by suggesting that administration of HMOs could serve as a preventive strategy for premature infants at risk for NEC development.

A Novel Role for Necroptosis in the Pathogenesis of Necrotizing Enterocolitis.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by Toll-like receptor 4 (TLR4)-dependent intestinal inflammation and enterocyte death. Given that necroptosis is a proinflammatory cell death process that is linked to bacterial signaling, we investigated its potential role in NEC, and the mechanisms involved. METHODS: Human and mouse NEC intestine were analyzed for necroptosis gene expression (ie, RIPK1, RIPK3, and MLKL), and protein activation (phosphorylated RIPK3). To evaluate a potential role for necroptosis in NEC, the effects of genetic (ie, Ripk3 knockout or Mlkl knockout) or pharmacologic (ie, Nec1s) inhibition of intestinal inflammation were assessed in a mouse NEC model, and a possible upstream role of TLR4 was assessed in Tlr4-deficient mice. The NEC-protective effects of human breast milk and its constituent milk oligosaccharides on necroptosis were assessed in a NEC-in-a-dish model, in which mouse intestinal organoids were cultured as either undifferentiated or differentiated epithelium in the presence of NEC bacteria and hypoxia. RESULTS: Necroptosis was activated in the intestines of human and mouse NEC in a TLR4-dependent manner, and was up-regulated specifically in differentiated epithelium of the immature ileum. Inhibition of necroptosis genetically and pharmacologically reduced intestinal-epithelial cell death and mucosal inflammation in experimental NEC, and ex vivo in the NEC-in-a-dish system. Strikingly, the addition of human breast milk, or the human milk oligosaccharide 2 fucosyllactose in the ex vivo system, reduced necroptosis and inflammation. CONCLUSIONS: Necroptosis is activated in the intestinal epithelium upon TLR4 signaling and is required for NEC development, and explains in part the protective effects of breast milk.

A Dynamic Variation of Pulmonary ACE2 Is Required to Modulate Neutrophilic Inflammation in Response to Pseudomonas aeruginosa Lung Infection in Mice.

Angiotensin-converting enzyme 2 (ACE2) is a potent negative regulator capable of restraining overactivation of the renin-angiotensin system, which contributes to exuberant inflammation after bacterial infection. However, the mechanism through which ACE2 modulates this inflammatory response is not well understood. Accumulating evidence indicates that infectious insults perturb ACE2 activity, allowing for uncontrolled inflammation. In the current study, we demonstrate that pulmonary ACE2 levels are dynamically varied during bacterial lung infection, and the fluctuation is critical in determining the severity of bacterial pneumonia. Specifically, we found that a pre-existing and persistent deficiency of active ACE2 led to excessive neutrophil accumulation in mouse lungs subjected to bacterial infection, resulting in a hyperinflammatory response and lung damage. In contrast, pre-existing and persistent increased ACE2 activity reduces neutrophil infiltration and compromises host defense, leading to overwhelming bacterial infection. Further, we found that the interruption of pulmonary ACE2 restitution in the model of bacterial lung infection delays the recovery process from neutrophilic lung inflammation. We observed the beneficial effects of recombinant ACE2 when administered to bacterially infected mouse lungs following an initial inflammatory response. In seeking to elucidate the mechanisms involved, we discovered that ACE2 inhibits neutrophil infiltration and lung inflammation by limiting IL-17 signaling by reducing the activity of the STAT3 pathway. The results suggest that the alteration of active ACE2 is not only a consequence of bacterial lung infection but also a critical component of host defense through modulation of the innate immune response to bacterial lung infection by regulating neutrophil influx.

Development of Intestinal Scaffolds that Mimic Native Mammalian Intestinal Tissue.

IMPACT STATEMENT: This study is significant because it demonstrates an attempt to design a scaffold specifically for small intestine using a novel fabrication method, resulting in an architecture that resembles intestinal villi. In addition, we use the versatile polymer poly(glycerol sebacate) (PGS) for artificial intestine, which has tunable mechanical and degradation properties that can be harnessed for further fine-tuning of scaffold design. Moreover, the utilization of PGS allows for future development of growth factor and drug delivery from the scaffolds to promote artificial intestine formation.

Toll Like Receptor 4 Mediated Lymphocyte Imbalance Induces Nec-Induced Lung Injury.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants, and is associated with the development of severe lung inflammation. The pathogenesis of NEC-induced lung injury remains unknown, yet infiltrating immune cells may play a role. In support of this possibility, we now show that NEC in mice and humans was associated with the development of profound lung injury that was characterized by an influx of Th17 cells and a reduction in T regulatory lymphocytes (Tregs). Importantly, the adoptive transfer of CD4 T cells isolated from lungs of mice with NEC into the lungs of immune incompetent mice (Rag1 mice) induced profound inflammation in the lung, while the depletion of Tregs exacerbated NEC induced lung injury, demonstrating that imbalance of Th17/Treg in the lung is required for the induction of injury. In seeking to define the mechanisms involved, the selective deletion of toll-like receptor 4 (TLR4) from the Sftpc1 pulmonary epithelial cells reversed lung injury, while TLR4 activation induced the Th17 recruiting chemokine (C-C motif) ligand 25 (CCL25) in the lungs of mice with NEC. Strikingly, the aerosolized inhibition of both CCL25 and TLR4 and the administration of all trans retinoic acid restored Tregs attenuated NEC-induced lung injury. In summary, we show that TLR4 activation in Surfactant protein C-1 (Sftpc1) cells disrupts the Treg/Th17 balance in the lung via CCL25 leading to lung injury after NEC and reveal that inhibition of TLR4 and stabilization of Th17/Treg balance in the neonatal lung may prevent this devastating complication of NEC.

Cognitive impairments induced by necrotizing enterocolitis can be prevented by inhibiting microglial activation in mouse brain.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease of the premature infant. One of the most important long-term complications observed in children who survive NEC early in life is the development of profound neurological impairments. However, the pathways leading to NEC-associated neurological impairments remain unknown, thus limiting the development of prevention strategies. We have recently shown that NEC development is dependent on the expression of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) on the intestinal epithelium, whose activation by bacteria in the newborn gut leads to mucosal inflammation. Here, we hypothesized that damage-induced production of TLR4 endogenous ligands in the intestine might lead to activation of microglial cells in the brain and promote cognitive impairments. We identified a gut-brain signaling axis in an NEC mouse model in which activation of intestinal TLR4 signaling led to release of high-mobility group box 1 in the intestine that, in turn, promoted microglial activation in the brain and neurological dysfunction. We further demonstrated that an orally administered dendrimer-based nanotherapeutic approach to targeting activated microglia could prevent NEC-associated neurological dysfunction in neonatal mice. These findings shed light on the molecular pathways leading to the development of NEC-associated brain injury, provide a rationale for early removal of diseased intestine in NEC, and indicate the potential of targeted therapies that protect the developing brain in the treatment of NEC in early childhood.

Fat composition in infant formula contributes to the severity of necrotising enterocolitis.

Necrotising enterocolitis (NEC) is a devastating disease that typically affects formula-fed premature infants, suggesting that dietary components may influence disease pathogenesis. TAG are the major fat components of infant formula, and their digestion requires pancreatic lipases, which may be naturally deficient in premature neonates. We hypothesise that NEC develops partly from the accumulation of incompletely digested long-chain TAG-containing unsaturated fatty acids within the intestinal epithelial cells, leading to oxidative stress and enterocyte damage. We further hypothesise that the administration of a formula that contains reduced TAG ('pre-digested fat') that do not require lipase action may reduce NEC severity. To test these hypotheses, we induced NEC in neonatal mice using three different fat formulations, namely 'standard fat', 'pre-digested fat' or 'very low fat', and determined that mice fed 'standard fat' developed severe NEC, which was significantly reduced in mice fed 'pre-digested fat' or 'very low fat'. The expression level of the critical fat-digesting enzyme carboxyl ester lipase was significantly lower in the newborn compared with older pups, leading to impaired fat digestion. The accumulation of mal-digested fat resulted in the significant accumulation of fat droplets within the intestinal epithelium of the distal ileum, resulting in the generation of reactive oxygen species and intestinal inflammation. Strikingly, these changes were prevented in pups fed 'pre-digested fat' or 'very low fat' formulas. These findings suggest that nutritional formula containing a pre-digested fat system may overcome the natural lipase deficiency of the premature gut, and serve as a novel approach to prevent NEC.

The Development of Newborn Porcine Models for Evaluation of Tissue-Engineered Small Intestine.

Short bowel syndrome (SBS) is a major cause of morbidity and mortality in the pediatric population, for which treatment options are limited. To develop novel approaches for the treatment of SBS, we now focus on the development of a tissue-engineered intestine (also known as an "artificial intestine"), in which intestinal stem cells are cultured onto an absorbable bioscaffold, followed by implantation into the host. To enhance the translational potential of these preclinical studies, we have developed three clinically relevant models in neonatal piglets, which approximate the size of the human infant and were evaluated after implantation and establishment of intestinal continuity over the long term. The models included (1) a staged, multioperation approach; (2) a single operation with a de-functionalized loop of small intestine; and (3) a single operation with an intestinal bypass. The first model had complications related to multiple operations in a short time period, including surgical site infections and wound hernias. The second model avoided wound complications, but was associated with high ostomy output, local skin breakdown, and systemic dehydration with associated electrolyte imbalances. The third model was the most effective, although resulted in stoma prolapse. In summary, we have now developed and evaluated three operative methods for the long-term evaluation of the artificial intestine in the piglet, and conclude that a single operation with a de-functionalized loop of small intestine may be an optimal approach for evaluation over the long term.

Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg9 bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.