Diagnostic Performance of Magnetic Resonance Imaging for Pediatric Ovarian Neoplasms: A Multi-Institutional Review.

STUDY OBJECTIVE: To assess the diagnostic performance of MRI to predict ovarian malignancy alone and compared with other diagnostic studies. METHODS: A retrospective analysis was conducted of patients aged 2-21 years who underwent ovarian mass resection between 2009 and 2021 at 11 pediatric hospitals. Sociodemographic information, clinical and imaging findings, tumor markers, and operative and pathology details were collected. Diagnostic performance for detecting malignancy was assessed by calculating sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for MRI with other diagnostic modalities. RESULTS: One thousand and fifty-three patients, with a median age of 14.6 years, underwent resection of an ovarian mass; 10% (110/1053) had malignant disease on pathology, and 13% (136/1053) underwent preoperative MRI. MRI sensitivity, specificity, PPV, and NPV were 60%, 94%, 60%, and 94%. Ultrasound sensitivity, specificity, PPV, and NPV were 31%, 99%, 73%, and 95%. Tumor marker sensitivity, specificity, PPV, and NPV were 90%, 46%, 22%, and 96%. MRI and ultrasound concordance was 88%, with sensitivity, specificity, PPV, and NPV of 33%, 99%, 75%, and 94%. MRI sensitivity in ultrasound-discordant cases was 100%. MRI and tumor marker concordance was 88% with sensitivity, specificity, PPV, and NPV of 100%, 86%, 64%, and 100%. MRI specificity in tumor marker-discordant cases was 100%. CONCLUSION: Diagnostic modalities used to assess ovarian neoplasms in pediatric patients typically agree. In cases of disagreement, MRI is more sensitive for malignancy than ultrasound and more specific than tumor markers. Selective use of MRI with preoperative ultrasound and tumor markers may be beneficial when the risk of malignancy is uncertain. CONCISE ABSTRACT: This retrospective review of 1053 patients aged 2-21 years who underwent ovarian mass resection between 2009 and 2021 at 11 pediatric hospitals found that ultrasound, tumor markers, and MRI tend to agree on benign vs malignant, but in cases of disagreement, MRI is more sensitive for malignancy than ultrasound.

Laparoscopy versus laparotomy for pediatric ovarian dermoids.

BACKGROUND: With increased surgeon comfort using laparoscopy, we hypothesized resection of pediatric ovarian dermoids using laparoscopy would yield a shorter length of stay and no increase in morbidity or recurrence compared to laparotomy. METHODS: A retrospective review was performed amongst eleven pediatric hospitals. Patients aged 2 to 21 who underwent resection of an ovarian dermoid from 2010 to 2020 were included. Patient characteristics, operative details, and outcomes by approach were evaluated using Chi-squared and Wilcoxon-Mann tests. RESULTS: 466 patients were included, with a median age of 14.4 and median follow-up of 4.0 months. 279 patients underwent laparoscopy (60%), 139 laparotomy (30%), and 48 laparoscopy converted to laparotomy (10%). There were no differences in rates of tumor spillage by approach (p = 0.15). 65% underwent ovarian-sparing surgery and 35% underwent oophorectomy. Length of stay was significantly shorter amongst patients who underwent laparoscopy (1 day versus 2 days for laparotomy and converted, p<0.0001). There were no differences in rates of suspected recurrence or reoperation (p = 0.19 and p = 0.57, respectively). CONCLUSION: Patients who underwent laparoscopy experienced no differences in the rates of tumor spillage, recurrence, or reoperation and had a shorter length of stay compared to laparotomy. Laparoscopy is an acceptable approach for resection of pediatric ovarian dermoids.

Multi-Institutional Review of the Preoperative Diagnostic Accuracy for Pediatric Ovarian Mature Cystic Teratomas.

STUDY OBJECTIVE: To assess the preoperative imaging impression and surgeon diagnostic accuracy for pediatric ovarian mature cystic teratomas (MCTs) DESIGN: Retrospective review SETTING: Eleven pediatric hospitals PARTICIPANTS: Patients ages 2 to 21 who underwent surgical management of an ovarian neoplasm or adnexal torsion with an associated ovarian lesion INTERVENTION: None MAIN OUTCOME MEASURES: Preoperative imaging impression, surgeon diagnosis, tumor markers, and pathology RESULTS: Our cohort included 946 females. Final pathology identified 422 (45%) MCTs, 405 (43%) other benign pathologies, and 119 (12%) malignancies. Preoperative imaging impression for MCTs had a 70% sensitivity, 92% specificity, 88% positive predictive value (PPV), and 79% negative predictive value (NPV). For the preoperative surgeon diagnosis, sensitivity was 59%, specificity 96%, PPV 92%, and NPV 74%. Some measures of diagnostic accuracy were affected by the presence of torsion, size of the lesion on imaging, imaging modality, and surgeon specialty. Of the 352 masses preoperatively thought to be MCTs, 14 were malignancies (4%). Eleven patients with inaccurately diagnosed malignancies had tumor markers evaluated and 82% had at least 1 elevated tumor marker, compared with 49% of those with MCTs. CONCLUSIONS: Diagnostic accuracy for the preoperative imaging impression and surgeon diagnosis is lower than expected for pediatric ovarian MCTs. For all ovarian neoplasms, preoperative risk assessment including a panel of tumor markers and a multidisciplinary review is recommended. This process could minimize the risk of misdiagnosis and improve operative planning to maximize the use of ovarian-sparing surgery for benign lesions and allow for appropriate resection and staging for lesions suspected to be malignant.

Factors Associated with Torsion in Pediatric Patients with Ovarian Masses.

BACKGROUND: Management of ovarian torsion has evolved toward ovarian preservation regardless of ovarian appearance during surgery. However, patients with torsion and an ovarian neoplasm undergo a disproportionately high rate of oophorectomy. Our objectives were to identify factors associated with ovarian torsion among females with an ovarian mass and to determine if torsion is associated with malignancy. METHODS: A retrospective review of females aged 2-21 y who underwent an operation for an ovarian cyst or neoplasm between 2010 and 2016 at 10 children's hospitals was performed. Multivariate logistic regression was used to assess factors associated with torsion. Imaging data were assessed for sensitivity, specificity, and predictive value in identifying ovarian torsion. RESULTS: Of 814 girls with an ovarian neoplasm, 180 (22%) had torsion. In risk-adjusted analyses, patients with a younger age, mass size >5 cm, abdominal pain, and vomiting had an increased likelihood of torsion (P < 0.01 for all). Patients with a mass >5 cm had two times the odds of torsion (odds ratio: 2.1; confidence interval: 1.2, 3.6). Imaging was not reliable at identifying torsion (sensitivity 34%, positive predictive value 49%) or excluding torsion (specificity 72%, negative predictive value 87%). The rates of malignancy were lower in those with an ovarian mass and torsion than those without torsion (10% versus 17%, P = 0.01). Among the 180 girls with torsion and a mass, 48% underwent oophorectomy of which 14% (n = 12) had a malignancy. CONCLUSIONS: In females with an ovarian neoplasm, torsion is not associated with an increased risk of malignancy and ovarian preservation should be considered.

Cavitary lung lesions and pneumothorax in a healthy patient with active coronavirus-19 (COVID-19) viral pneumonia.

Severe respiratory sequelae drive morbidity-associated with coronavirus 2019 (COVID-19) disease. We report a case of COVID-19 pneumonia complicated by cavitary lesions and pneumothorax in a young healthy male. Pneumothorax management with catheter thoracostomy and rapid resolution of the cavitary lesions are described. An extensive work-up for other causes a cavitation was negative and the temporal correlation of the cavities with COVID-19 infection plus their rapid resolution suggest a direct relationship. We propose a mechanism for cavitation secondary to microangiopathy, a cause of cavitation in the vasculitides and a known feature of COVID-19.

Inter-rater Reliability of Sacral Ratio Measurements in Patients with Anorectal Malformations.

BACKGROUND: Anorectal malformations (ARMs) are a spectrum of congenital anomalies with varying prognosis for fecal continence. The sacral ratio (SR) is a measure of sacral development that has been proposed as a method to predict future fecal continence in children with ARM. The aim of this study was to quantify the inter-rater reliability (IRR) of SR calculations by radiologists at different institutions. MATERIALS AND METHODS: x-Rays in the anteroposterior (AP) and lateral planes were reviewed by a pediatric radiologist at each of six different institutions. Subsequently, images were reviewed by a single, central radiologist. The IRR was assessed by calculating Pearson correlation coefficients and intraclass correlation coefficients from linear mixed models with patient and rater-level random intercepts. RESULTS: Imaging from 263 patients was included in the study. The mean inter-rater absolute difference in the AP SR was 0.05 (interquartile range, 0.02-0.10), and in the lateral SR was 0.16 (interquartile range, 0.06-0.25). Overall, the IRR was excellent for AP SRs (intraclass correlation coefficient [ICC], 81.5%; 95% confidence interval, 75.1%-86.0%) and poor for lateral SRs (ICC, 44.0%; 95% CI, 29.5%-59.2%). For both AP and lateral SRs, ICCs were similar when examined by the type of radiograph used for calculation, severity of the ARM, presence of sacral or spinal anomalies, and age at imaging. CONCLUSIONS: Across radiologists, the reliability of SR calculations was excellent for the AP plane but poor for the lateral plane. These results suggest that better standardization of lateral SR measurements is needed if they are going to be used to counsel families of children with ARM.

Understanding the Value of Tumor Markers in Pediatric Ovarian Neoplasms.

PURPOSE: The purpose of this study was to determine the diagnostic accuracy of tumor markers for malignancy in girls with ovarian neoplasms. METHODS: A retrospective review of girls 2-21 years who presented for surgical management of an ovarian neoplasm across 10 children's hospitals between 2010 and 2016 was performed. Patients who had at least one concerning feature on imaging and had tumor marker testing were included in the study. Sensitivity, specificity, and negative and positive predictive values (PPV) of tumor markers were calculated. RESULTS: Our cohort included 401 patients; 22.4% had a malignancy. Testing for tumor markers was inconsistent. AFP had high specificity (98%) and low sensitivity (42%) with a PPV of 86%. The sensitivity, specificity, and PPV of beta-hCG was 44%, 76%, and 32%, respectively. LDH had high sensitivity (95%) and Inhibin A and Inhibin B had high specificity (97% and 92%, respectively). CONCLUSIONS: Tumor marker testing is helpful in preoperative risk stratification of ovarian neoplasms for malignancy. Given the variety of potential tumor types, no single marker provides enough reliability, and therefore a panel of tumor marker testing is recommended if there is concern for malignancy. Prospective studies may help further elucidate the predictive value of tumor markers in a pediatric ovarian neoplasm population. TYPE OF STUDY: Retrospective Cohort Review. LEVEL OF EVIDENCE: Level III.

Can fecal continence be predicted in patients born with anorectal malformations?

PURPOSE: The purpose of this study was to identify factors associated with attaining fecal continence in children with anorectal malformations (ARM). METHODS: We performed a multi-institutional cohort study of children born with ARM in 2007-2011 who had spinal and sacral imaging. Questions from the Baylor Social Continence Scale were used to assess fecal continence at the age of ≥4 years. Factors present at birth that predicted continence were identified using multivariable logistic regression. RESULTS: Among 144 ARM patients with a median age of 7 years (IQR 6-8), 58 (40%) were continent. The rate of fecal continence varied by ARM subtype (p = 0.002) with the highest rate of continence in patients with perineal fistula (60%). Spinal anomalies and the lateral sacral ratio were not associated with continence. On multivariable analysis, patients with less severe ARM subtypes (perineal fistula, recto-bulbar fistula, recto-vestibular fistula, no fistula, rectal stenosis) were more likely to be continent (OR = 7.4, p = 0.001). CONCLUSION: Type of ARM was the only factor that predicted fecal continence in children with ARM. The high degree of incontinence, even in the least severe subtypes, highlights that predicting fecal continence is difficult at birth and supports the need for long-term follow-up and bowel management programs for children with ARM. TYPE OF STUDY: Prospective Cohort Study. LEVEL OF EVIDENCE: II.

Ambient urban dust particulate matter reduces pathologic T cells in the CNS and severity of EAE.

BACKGROUND: Autoimmune diseases have increased in incidence and prevalence worldwide. While genetic predispositions play a role, environmental factors are a major contributor. Atmospheric particulate matter (PM) is a complex mixture composed of metals, nitrates, sulfates and diverse adsorbed organic compounds like polycyclic aromatic hydrocarbons (PAHs) and dioxins. Exposure to atmospheric PM aggravates autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus, among others. PAHs and dioxins are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and directs the balance between effector and regulatory T cells in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify pathways that contribute to autoimmune disease and their potential use as therapeutic targets to alleviate symptoms and the need for global immunosuppression. This study tests the hypothesis that atmospheric PM enhances effector T cell differentiation and aggravates autoimmune disease. RESULTS: An atmospheric ambient urban dust PM sample, standard reference material (SRM)1649b, was tested for its effects on autoimmunity. SRM1649b PM enhanced Th17 differentiation in an AHR-dependent manner in vitro, however intranasal treatment of SRM1649b PM delayed onset of EAE and reduced cumulative and peak clinical scores. Chronic and acute intranasal exposure of SRM1649b PM delayed onset of EAE. Chronic intranasal exposure did not reduce severity of EAE while acute intranasal exposure significantly reduced severity of disease. Acute intranasal treatment of low dose SRM1649b PM had no effect on clinical score or day of onset in EAE. Delayed onset of EAE by intranasal SRM1649b PM was AHR-dependent in vivo. Oral gavage of SRM1649b PM, in the absence of AHR ligands in the diet, had no effect on day of disease onset or severity of EAE. Day 10 analysis of T cells in the CNS after intranasal treatment of SRM1649b PM showed a reduction of pathologic T cell subsets in vivo. Moreover, MOG-specific splenocytes require AHR to generate or maintain IL-10 producing cells and reduce IFNγ producing cells in vitro. CONCLUSIONS: These results identify the AHR pathway as a potential target for driving targeted immunosuppression in the CNS in the context of atmospheric PM-mediated autoimmune disease. The effects of SRM1649b PM on EAE are dependent on route of exposure, with intranasal treatment reducing severity of EAE and delaying disease onset while oral gavage has no effect. Intranasal SRM1649b PM reduces pathologic T cells in the CNS, specifically Th1 cells and Th1Th17 double positive cells, leading to reduced severity of EAE and AHR-dependent delayed disease onset. Additionally, SRM1649b PM treatment of antigen-specific T cells leads to AHR-dependent increase in percent IL-10 positive cells in vitro. These findings may shed light on the known increase of infection after exposure to atmospheric PM and serve as the first step in identifying components of the AHR pathway responsible for Th1-mediated immunosuppression in response to atmospheric PM exposure.

Gut Symbionts Lactobacillus reuteri R2lc and 2010 Encode a Polyketide Synthase Cluster That Activates the Mammalian Aryl Hydrocarbon Receptor.

A mechanistic understanding of microbe-host interactions is critical to developing therapeutic strategies for targeted modulation of the host immune system. Different members of the gut symbiont species Lactobacillus reuteri modulate host health by, for example, reduction of intestinal inflammation. Previously, it was shown that L. reuteri activates the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that plays an important role in the mucosal immune system, by the production of tryptophan catabolites. Here, we identified a novel pathway by which L. reuteri activates AhR, which is independent of tryptophan metabolism. We screened a library of 36 L. reuteri strains and determined that R2lc and 2010, strains with a pigmented phenotype, are potent AhR activators. By whole-genome sequencing and comparative genomics, we identified genes unique to R2lc and 2010. Our analyses demonstrated that R2lc harbors two genetically distinct polyketide synthase (PKS) clusters, functionally unknown (fun) and pks, each carried by a multicopy plasmid. Inactivation of pks, but not fun, abolished the ability of R2lc to activate AhR. L. reuteri 2010 has a gene cluster homologous to the pks cluster in R2lc with an identical gene organization, which is also responsible for AhR activation. In conclusion, we identified a novel PKS pathway in L. reuteri R2lc and 2010 that is responsible for AhR activation.IMPORTANCE Temporary changes in the composition of the microbiota, for example, by oral administration of probiotics, can modulate the host immune system. However, the underlying mechanisms by which probiotics interact with the host are often unknown. Here, we show that Lactobacillus reuteri R2lc and 2010 harbor an orthologous PKS gene cluster that activates the aryl hydrocarbon receptor (AhR). AhR is a ligand-activated transcription factor that plays a key role in a variety of diseases, including amelioration of intestinal inflammation. Understanding the mechanism by which a bacterium modulates the immune system is critical for applying rational selection strategies for probiotic supplementation. Finally, heterologous and/or optimized expression of PKS is a logical next step toward the development of next-generation probiotics to prevent and treat disease.

Differential effects of diesel exhaust particles on T cell differentiation and autoimmune disease.

BACKGROUND: Exposure to particulate matter (PM) has been associated with increased incidence and severity of autoimmune disease. Diesel PM is primarily composed of an elemental carbon core and adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and contributes up to 40% of atmospheric PM. The organic fraction (OF) of PM excludes all metals and inorganics and retains most organic compounds, such as PAHs. Both PM and OF increase inflammation in vitro and aggravate autoimmune disease in humans. PAHs are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and effector function in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify whether the total mass or active components of PM are responsible for activating pathways associated with exposure to PM and autoimmune disease. This study tests the hypothesis that active components present in diesel PM and their OF enhance effector T cell differentiation and aggravate autoimmune disease. RESULTS: Two different diesel samples, each characterized for their components, were tested for their effects on autoimmunity. Both diesel PM enhanced effector T cell differentiation in an AHR-dose-dependent manner and suppressed regulatory T cell differentiation in vitro. Both diesel PM aggravated EAE in vivo. Fractionated diesel OFs exhibited the same effects as PM in vitro, but unlike PM, only one diesel OF aggravated EAE. Additionally, both synthetic PAH mixtures that represent specific PAHs found in the two diesel PM samples enhanced Th17 differentiation, however one lost this effect after metabolism and only one required the AHR. CONCLUSIONS: These findings suggest that active components of PM and not total mass are driving T cell responses in vitro, but in vivo the PM matrix and complex mixtures adsorbed to the particles, not just the OF, are contributing to the observed EAE effects. This implies that examining OF alone may not be sufficient in vivo. These data further suggest that bioavailability and metabolism of organics, especially PAHs, may have an important role in vivo.

Screening practices and associated anomalies in infants with anorectal malformations: Results from the Midwest Pediatric Surgery Consortium.

BACKGROUND: This study evaluates screening practices and the incidence of associated anomalies in infants with anorectal malformations (ARM). METHODS: We performed a multi-institutional retrospective cohort study of children born between 2007 and 2011 who underwent surgery for ARM at 10 children's hospitals. ARM type was classified based on the location of the distal rectum, and all screening studies were reviewed. RESULTS: Among 506 patients, the most common ARM subtypes were perineal fistula (40.7%), no fistula (11.5%), and vestibular fistula (10.1%). At least 1 screening test was performed in 96.6% of patients, and 11.3% of patients underwent all. The proportion of patients with ≥1 abnormal finding on any screening test varied by type of ARM (p<0.001). Screening rates varied from 15.2% for limb anomalies to 89.7% for renal anomalies. The most commonly identified anomalies by screening category were: spinal: tethered cord (20.6%); vertebral: sacral dysplasia/hemisacrum (17.8%); cardiac: patent foramen ovale (58.0%); renal: hydronephrosis (22.7%); limb: absent radius (7.9%). CONCLUSION: Screening practices and the incidence of associated anomalies varied by type of ARM. The rate of identifying at least one associated anomaly was high across all ARM subtypes. Screening for associated anomalies should be considered standard of care for all ARM patients. TYPE OF STUDY: Multi-institutional retrospective cohort study. LEVEL OF EVIDENCE: III.

Toll-like receptor 4-mediated endoplasmic reticulum stress in intestinal crypts induces necrotizing enterocolitis.

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Discovery and validation of a new class of small molecule Toll-like receptor 4 (TLR4) inhibitors.

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the ß-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.

Innate immune signaling in the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a challenging disease to treat, and caring for patients afflicted by it remains both frustrating and difficult. While NEC may develop quickly and without warning, it may also develop slowly, insidiously, and appear to take the caregiver by surprise. In seeking to understand the molecular and cellular processes that lead to NEC development, we have identified a critical role for the receptor for bacterial lipopolysaccharide (LPS) toll like receptor 4 (TLR4) in the pathogenesis of NEC, as its activation within the intestinal epithelium of the premature infant leads to mucosal injury and reduced epithelial repair. The expression and function of TLR4 were found to be particularly elevated within the intestinal mucosa of the premature as compared with the full-term infant, predisposing to NEC development. Importantly, factors within both the enterocyte itself, such as heat shock protein 70 (Hsp70), and in the extracellular environment, such as amniotic fluid, can curtail the extent of TLR4 signaling and reduce the propensity for NEC development. This review will highlight the critical TLR4-mediated steps that lead to NEC development, with a focus on the proinflammatory responses of TLR4 signaling that have such devastating consequences in the premature host.

Endothelial TLR4 activation impairs intestinal microcirculatory perfusion in necrotizing enterocolitis via eNOS-NO-nitrite signaling.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS(-/-) mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate--a precursor for enteral generation of nitrite and nitric oxide--and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate-nitrite-NO signaling.

A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) develops in response to elevated TLR4 signaling in the newborn intestinal epithelium and is characterized by TLR4-mediated inhibition of enterocyte migration and reduced mucosal healing. The downstream processes by which TLR4 impairs mucosal healing remain incompletely understood. In other systems, TLR4 induces autophagy, an adaptive response to cellular stress. We now hypothesize that TLR4 induces autophagy in enterocytes and that TLR4-induced autophagy plays a critical role in NEC development. Using mice selectively lacking TLR4 in enterocytes (TLR4(ΔIEC)) and in TLR4-deficient cultured enterocytes, we now show that TLR4 activation induces autophagy in enterocytes. Immature mouse and human intestine showed increased expression of autophagy genes compared with full-term controls, and NEC development in both mouse and human was associated with increased enterocyte autophagy. Importantly, using mice in which we selectively deleted the autophagy gene ATG7 from the intestinal epithelium (ATG7(ΔIEC)), the induction of autophagy was determined to be required for and not merely a consequence of NEC, because ATG7(ΔIEC) mice were protected from NEC development. In defining the mechanisms involved, TLR4-induced autophagy led to impaired enterocyte migration both in vitro and in vivo, which in cultured enterocytes required the induction of RhoA-mediated stress fibers. These findings depart from current dogma in the field by identifying a unique effect of TLR4-induced autophagy within the intestinal epithelium in the pathogenesis of NEC and identify that the negative consequences of autophagy on enterocyte migration play an essential role in its development.

Modeling the interactions of bacteria and Toll-like receptor-mediated inflammation in necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a severe disease of the gastrointestinal tract in premature infants, characterized by a disrupted intestinal epithelium and an exaggerated pro-inflammatory response. Since the activation of Toll-like receptor-4 (TLR4) blocks cell migration and proliferation and contributes to an uncontrolled inflammatory response within the intestine, this receptor has been identified as a key contributor to the development of NEC. Toll-like receptor-9 (TLR9) has been shown to sense bacterial genome components (CpG DNA) and to play an anti-inflammatory role in NEC. We present in vitro results demonstrating direct inhibition of TLR4 activation by CpG DNA, and we develop a mathematical model of bacteria-immune interactions within the intestine to investigate how such inhibition of TLR4 signaling might alter inflammation, associated bacterial invasion of tissue, and resulting outcomes. The model predicts that TLR9 can inhibit both the beneficial and detrimental effects of TLR4, and thus a proper balance of action by these two receptors is needed to promote intestinal health. The model results are also used to explore three interventions that could potentially prevent the development of NEC: reducing bacteria in the mucus layer, administering probiotic treatment, and blocking TLR4 activation. While the model shows that these interventions would be successful in most cases, the model is also used to identify situations in which the proposed treatments might be harmful.

Toll-like receptor 4 is expressed on intestinal stem cells and regulates their proliferation and apoptosis via the p53 up-regulated modulator of apoptosis.

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-ß (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.

Intestinal epithelial Toll-like receptor 4 regulates goblet cell development and is required for necrotizing enterocolitis in mice.

BACKGROUND & AIMS: Little is known about factors that regulate intestinal epithelial differentiation; microbial recognition receptors such as Toll-like receptor (TLR)4 might be involved. We investigated whether intestinal TLR4 regulates epithelial differentiation and is involved in development of necrotizing enterocolitis (NEC) of the immature intestine. METHODS: Mice with conditional disruption of TLR4 in the intestinal epithelium and TLR4 knockout (TLR4(-/-)) mice were generated by breeding TLR4(loxp/loxp) mice with villin-cre and Ella-cre, respectively. Enterocytes that did not express or overexpressed TLR4 were created by lentiviral or adenoviral transduction. Intestinal organoids were cultured on tissue matrices. Bile acids were measured by colorimetric assays, and microbial composition was determined by 16S pyrosequencing. NEC was induced in 7- to 10-day-old mice by induction of hypoxia twice daily for 4 days. RESULTS: TLR4(-/-) mice and mice with enterocyte-specific deletion of TLR4 were protected from NEC; epithelial differentiation into goblet cells was increased via suppressed Notch signaling in the small intestinal epithelium. TLR4 also regulates differentiation of goblet cells in intestinal organoid and enterocyte cell cultures; differentiation was increased on deletion of TLR4 and restored when TLR4 was expressed ectopically. TLR4 signaling via Notch was increased in intestinal tissue samples from patients with NEC, and numbers of goblet cells were reduced. 16S pyrosequencing revealed that wild-type and TLR4-deficient mice had similar microbial profiles; increased numbers of goblet cells were observed in mice given antibiotics. TLR4 deficiency reduced levels of luminal bile acids in vivo, and addition of bile acids to TLR4-deficient cell cultures prevented differentiation of goblet cells. CONCLUSIONS: TLR4 signaling and Notch are increased in intestinal tissues of patients with NEC and required for induction of NEC in mice. TLR4 prevents goblet cell differentiation, independently of the microbiota. Bile acids might initiate goblet cell development.