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 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 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).

New insights into the pathogenesis of necrotizing enterocolitis and the dawn of potential therapeutics.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disorder in premature infants that causes significant morbidity and mortality. Research efforts into the pathogenesis of NEC have discovered a pivotal role for the gram-negative bacterial receptor, Toll-like receptor 4 (TLR4), in its development. TLR4 is activated by dysbiotic microbes within the intestinal lumen, which leads to an exaggerated inflammatory response within the developing intestine, resulting in mucosal injury. More recently, studies have identified that the impaired intestinal motility that occurs early in NEC has a causative role in disease development, as strategies to enhance intestinal motility can reverse NEC in preclinical models. There has also been broad appreciation that NEC also contributes to significant neuroinflammation, which we have linked to the effects of gut-derived pro-inflammatory molecules and immune cells which activate microglia in the developing brain, resulting in white matter injury. These findings suggest that the management of the intestinal inflammation may secondarily be neuroprotective. Importantly, despite the significant burden of NEC on premature infants, these and other studies have provided a strong rationale for the development of small molecules with the capability of reducing NEC severity in pre-clinical models, thus guiding the development of specific anti-NEC therapies. This review summarizes the roles of TLR4 signaling in the premature gut in the pathogenesis of NEC, and provides insights into optimal clinical management strategies based upon findings from laboratory studies.

Necrotizing enterocolitis, gut microbes, and sepsis.

Necrotizing enterocolitis (NEC) is a devastating disease in premature infants and the leading cause of death and disability from gastrointestinal disease in this vulnerable population. Although the pathophysiology of NEC remains incompletely understood, current thinking indicates that the disease develops in response to dietary and bacterial factors in the setting of a vulnerable host. As NEC progresses, intestinal perforation can result in serious infection with the development of overwhelming sepsis. In seeking to understand the mechanisms by which bacterial signaling on the intestinal epithelium can lead to NEC, we have shown that the gram-negative bacterial receptor toll-like receptor 4 is a critical regulator of NEC development, a finding that has been confirmed by many other groups. This review article provides recent findings on the interaction of microbial signaling, the immature immune system, intestinal ischemia, and systemic inflammation in the pathogenesis of NEC and the development of sepsis. We will also review promising therapeutic approaches that show efficacy in pre-clinical studies.

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.

Models of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of death and disability from gastrointestinal disease in premature infants. The mortality of patients with NEC is approximately 30%, a figure that has not changed in many decades, reflecting the need for a greater understanding of its pathogenesis. Progress towards understanding the cellular and molecular mechanisms underlying NEC requires the study of highly translational animal models. Such animal models must mimic the biology and physiology of premature infants, while still allowing for safe experimental manipulation of environmental and microbial factors thought to be associated with the risk and severity of NEC. Findings from animal models have yielded insights into the interactions between the host, the colonizing microbes, and the innate immune receptor Toll-like Receptor 4 (TLR4) in driving disease development. This review discusses the relative strengths and weaknesses of available in vivo, in vitro, and NEC-in-a-dish models of this disease. We also highlight the unique contributions that each model has made to our understanding of the complex interactions between enterocytes, microbiota, and immune cells in the pathogenesis of NEC. The overall purpose of this review is to provide a menu of options regarding currently available animal models of NEC, while in parallel hopefully reducing the potential uncertainty and confusion regarding NEC models to assist those who wish to enter this field from other disciplines.

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.

In vivo phenotyping of the microvasculature in necrotizing enterocolitis with multicontrast optical imaging.

OBJECTIVE: Necrotizing enterocolitis (NEC) is the most prevalent gastrointestinal emergency in premature infants and is characterized by a dysfunctional gut microcirculation. Therefore, there is a dire need for in vivo methods to characterize NEC-induced changes in the structure and function of the gut microcirculation, that is, its vascular phenotype. Since in vivo gut imaging methods are often slow and employ a single-contrast mechanism, we developed a rapid multicontrast imaging technique and a novel analyses pipeline for phenotyping the gut microcirculation. METHODS: Using an experimental NEC model, we acquired in vivo images of the gut microvasculature and blood flow over a 5000 × 7000 µm2 field of view at 5 µm resolution via the following two endogenous contrast mechanisms: intrinsic optical signals and laser speckles. Next, we transformed intestinal images into rectilinear "flat maps," and delineated 1A/V gut microvessels and their perfusion territories as "intestinal vascular units" (IVUs). Employing IVUs, we quantified and visualized NEC-induced changes to the gut vascular phenotype. RESULTS: In vivo imaging required 60-100 s per animal. Relative to the healthy gut, NEC intestines showed a significant overall decrease (i.e. 64-72%) in perfusion, accompanied by vasoconstriction (i.e. 9-12%) and a reduction in perfusion entropy (19%)within sections of the vascular bed. CONCLUSIONS: Multicontrast imaging coupled with IVU-based in vivo vascular phenotyping is a powerful new tool for elucidating NEC pathogenesis.

Bench to bedside - new insights into the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is the leading cause of death and disability from gastrointestinal disease in premature infants. Recent discoveries have shed light on a unifying theorem to explain the pathogenesis of NEC, suggesting that specific treatments might finally be forthcoming. A variety of experiments have highlighted how the interaction between bacterial signalling receptors on the premature intestine and an abnormal gut microbiota incites a pro-inflammatory response in the intestinal mucosa and its underlying endothelium that leads to NEC. Central amongst the bacterial signalling receptors implicated in NEC development is the lipopolysaccharide receptor Toll-like receptor 4 (TLR4), which is expressed at higher levels in the premature gut than in the full-term gut. The high prenatal intestinal expression of TLR4 reflects the role of TLR4 in the regulation of normal gut development, and supports additional studies indicating that NEC develops in response to signalling events that occur in utero. This Review provides new evidence explaining the pathogenesis of NEC, explores new findings indicating that NEC development has origins before birth, and discusses future questions and opportunities for discovery in this field.

Acute Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Pregnancy Is Associated with Placental Angiotensin-Converting Enzyme 2 Shedding.

While the human placenta may be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the rate of fetal transmission is low, suggesting a barrier at the maternal-fetal interface. Angiotensin-converting enzyme (ACE)2, the main receptor for SARS-CoV-2, is regulated by a metalloprotease cleavage enzyme, a disintegrin and metalloprotease domain 17 (ADAM17). ACE2 is expressed in the human placenta, but its regulation in relation to maternal SARS-CoV-2 infection in pregnancy is not well understood. This study evaluated ACE2 expression, ADAM17 activity, and serum ACE2 abundance in a cohort of matched villous placental and maternal serum samples from control pregnancies (SARS-CoV-2 negative, n = 8) and pregnancies affected by symptomatic maternal SARS-CoV-2 infections in the second trimester [2nd Tri coronavirus disease (COVID), n = 8] and third trimester (3rd Tri COVID, n = 8). In 3rd Tri COVID compared with control and 2nd Tri COVID villous placental tissues, ACE2 mRNA expression was remarkably elevated; however, ACE2 protein expression was significantly decreased with a parallel increase in ADAM17 activity. Soluble ACE2 was also significantly increased in the maternal serum from 3rd Tri COVID infections compared with control and 2nd Tri COVID pregnancies. These data suggest that in acute maternal SARS-CoV-2 infections, decreased placental ACE2 protein may be the result of ACE2 shedding and highlights the importance of ACE2 for studies on SARS-CoV-2 responses at the maternal-fetal interface.

Galectin-4 as a Novel Biomarker of Neonatal Intestinal Injury.

BACKGROUND: Neonates are at risk of gastrointestinal emergencies including necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP). Identifying biomarkers to aid in diagnosis is imperative. We hypothesized that circulating intestinal-specific protein concentrations would distinguish infants with intestinal injury from controls. AIMS: To identify serum concentrations of intestinal-specific protein(s) in infants with intestinal injury and controls. METHODS: We used an in silico approach to identify intestinal-specific proteins. We collected serum from control infants and infants with NEC or SIP and measured protein concentrations using ELISA. If baseline concentrations were near the detection limit in initial control assays, we proceeded to assess concentrations in a larger cohort of controls and infants with injury. Control infants were frequency matched to infants with injury and compared with nonparametric and mixed-effects models analysis. RESULTS: We evaluated four proteins with high intestinal expression: Galectin-4 (Gal-4), S100G, Trefoil Factor-3, and alanyl aminopeptidase. Only Gal-4 demonstrated consistent results near the lower limit of quantification in controls and was studied in the larger cohorts. Gal-4 concentration was low in 111 control infants (median 0.012 ng/ml). By contrast, Gal-4 was significantly increased at diagnosis in infants with surgical NEC and SIP (n = 14, p ≤ 0.001 and n = 8, p = 0.031) compared to matched controls, but not in infants with medical NEC (n = 32, p = 0.10). CONCLUSIONS: Of the intestinal-specific proteins evaluated, circulating Gal-4 concentrations were at the assay detection limit in control infants. Gal-4 concentrations were significantly elevated in infants with surgical NEC or SIP, suggesting that Gal-4 may serve as a biomarker for neonatal intestinal injury.

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.

Acute SARS-CoV-2 infection in pregnancy is associated with placental ACE-2 shedding.

Human placental tissues have variable rates of SARS-CoV-2 invasion resulting in consistently low rates of fetal transmission suggesting a unique physiologic blockade against SARS-CoV-2. Angiotensin-converting enzyme (ACE)-2, the main receptor for SARS-CoV-2, is expressed as cell surface and soluble forms regulated by a metalloprotease cleavage enzyme, ADAM17. ACE-2 is expressed in the human placenta, but the regulation of placental ACE-2 expression in relation to timing of maternal SARS-CoV-2 infection in pregnancy is not well understood. In this study, we evaluated ACE-2 expression, ADAM17 activity and serum ACE-2 abundance in a cohort of matched villous placental and maternal serum samples from Control pregnancies (SARS-CoV-2 negative, n=8) and pregnancies affected by symptomatic maternal SARS-CoV-2 infections in the 2 nd trimester ("2 nd Tri COVID", n=8) and 3rd trimester ("3 rd Tri COVID", n=8). In 3 rd Tri COVID as compared to control and 2 nd Tri-COVID villous placental tissues ACE-2 mRNA expression was remarkably elevated, however, ACE-2 protein expression was significantly decreased with a parallel increase in ADAM17 activity. Soluble ACE-2 was also significantly increased in the maternal serum from 3 rd Tri COVID infections as compared to control and 2 nd Tri-COVID pregnancies. These data suggest that in acute maternal SARS-CoV-2 infections, decreased placental ACE-2 protein may be the result of ACE-2 shedding. Overall, this work highlights the importance of ACE-2 for ongoing studies on SARS-CoV-2 responses at the maternal-fetal interface.

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.

Age-dependent regulation of SARS-CoV-2 cell entry genes and cell death programs correlates with COVID-19 severity.

Novel coronavirus disease 2019 (COVID-19) severity is highly variable, with pediatric patients typically experiencing less severe infection than adults and especially the elderly. The basis for this difference is unclear. We find that mRNA and protein expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, increases with advancing age in distal lung epithelial cells. However, in humans, ACE2 expression exhibits high levels of intra- and interindividual heterogeneity. Further, cells infected with SARS-CoV-2 experience endoplasmic reticulum stress, triggering an unfolded protein response and caspase-mediated apoptosis, a natural host defense system that halts virion production. Apoptosis of infected cells can be selectively induced by treatment with apoptosis-modulating BH3 mimetic drugs. Notably, epithelial cells within young lungs and airways are more primed to undergo apoptosis than those in adults, which may naturally hinder virion production and support milder COVID-19 severity.

Prenatal Immunity and Influences on Necrotizing Enterocolitis and Associated Neonatal Disorders.

Prior to birth, the neonate has limited exposure to pathogens. The transition from the intra-uterine to the postnatal environment initiates a series of complex interactions between the newborn host and a variety of potential pathogens that persist over the first few weeks of life. This transition is particularly complex in the case of the premature and very low birth weight infant, who may be susceptible to many disorders as a result of an immature and underdeveloped immune system. Chief amongst these disorders is necrotizing enterocolitis (NEC), an acute inflammatory disorder that leads to necrosis of the intestine, and which can affect multiple systems and have the potential to result in long term effects if the infant is to survive. Here, we examine what is known about the interplay of the immune system with the maternal uterine environment, microbes, nutritional and other factors in the pathogenesis of neonatal pathologies such as NEC, while also taking into consideration the effects on the long-term health of affected children.

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.