Cyclic GMP-AMP synthase contributes to epithelial homeostasis in intestinal inflammation via Beclin-1-mediated autophagy.

Inflammatory bowel disease (IBD) represents a set of idiopathic and chronic inflammatory diseases of the gastrointestinal tract. Central to the pathogenesis of IBD is a dysregulation of normal intestinal epithelial homeostasis. cGAS is a DNA-sensing receptor demonstrated to promote autophagy, a mechanism that removes dysfunctional cellular components. Beclin-1 is a crucial protein involved in the initiation of autophagy. We hypothesized that cGAS plays a key role in intestinal homeostasis by upregulating Beclin-1-mediated autophagy. We evaluated intestinal cGAS levels in humans with IBD and in murine colonic tissue after performing a 2% dextran sulfate sodium (DSS) colitis model. Autophagy and cell death mechanisms were studied in cGAS KO and WT mice via qPCR, WB analysis, H&E, IF, and TUNEL staining. Autophagy was measured in stimulated intestinal epithelial cells (IECs) via WB analysis. Our data demonstrates cGAS to be upregulated during human and murine colitis. Furthermore, cGAS deficiency leads to worsened colitis and decreased levels of autophagy proteins including Beclin-1 and LC3-II. Co-IP demonstrates a direct binding between cGAS and Beclin-1 in IECs. Transfection of cGAS in stimulated HCT-116 cells leads to increased autophagy. IECs isolated from cGAS KO have diminished autophagic flux. cGAS KO mice subjected to DSS have increased cell death and cleaved caspase-3. Lastly, treatment of cGAS KO mice with rapamycin decreased the severity of colitis. Our data suggest that cGAS maintains intestinal epithelial homeostasis during human IBD and murine colitis by upregulating Beclin-1-mediated autophagy and preventing IEC death. Rescue of autophagy can attenuate the severity of colitis associated with cGAS deficiency.

Management and Outcome of Pediatric Patients With Transient Small Bowel-Small Bowel Intussusception.

OBJECTIVE: The aim of this study was to assess the management and outcomes of healthy pediatric patients diagnosed radiologically with transient and benign small bowel-small bowel intussusception (SB-SBI). METHODS: Retrospective cohort study of healthy patients 0 to 18 years of age who presented to a children's hospital emergency department from January 1, 2005, to June 30, 2015, and had transient and benign SB-SBI characterized by spontaneous resolution (ie, transient), diameter of less than 2.5 cm, no lead point, normal bowel wall thickness, nondilated proximal small bowel, and no colonic involvement (ie, benign radiographic features). Charts were reviewed for demographics, clinical presentation, radiologic studies obtained, outcomes, and further management. Medical and radiologic records were also reviewed for 1 year after presentation for any subsequent pathologic diagnoses. RESULTS: Sixty-eight patients were included in our study, with a total of 87 episodes of transient and benign SB-SBI on initial or follow-up examination. Overall, 39 patients (57%) were admitted to the hospital, and 38 patients (56%) had a surgical consultation. Twenty-four patients (35%) had further radiologic studies obtained, including computed tomography scans, esophagogastroduodenoscopy, Meckel's scan, barium swallow studies, and magnetic resonance imaging. All studies were negative for concerning pathology including apparent lead points. None of the patients required surgical intervention or had any complications. CONCLUSIONS: Transient and benign SB-SBIs with reassuring radiologic and clinical features diagnosed in healthy pediatric patients are likely incidentally found and are unlikely to be associated with a pathologic lead point.

Calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation contributes to the pathogenesis of experimental colitis via inhibition of intestinal epithelial cell proliferation.

The incidence and prevalence of inflammatory bowel disease (IBD) are increasing worldwide. IBD is known to be multifactorial, but inflammatory signaling within the intestinal epithelium and a subsequent failure of the intestinal epithelial barrier have been shown to play essential roles in disease pathogenesis. CaMKIV is a multifunctional protein kinase associated with inflammation and cell cycle regulation. CaMKIV has been extensively studied in autoimmune diseases, but a role in idiopathic intestinal inflammation has not been described. In this study, active CaMKIV was highly expressed within the intestinal epithelium of humans with ulcerative colitis and wild-type (WT) mice with experimental induced colitis. Clinical disease severity directly correlates with CaMKIV activation, as does expression of proinflammatory cytokines and histologic features of colitis. In WT mice, CaMKIV activation is associated with increases in expression of 2 cell cycle proarrest signals: p53 and p21. Cell cycle arrest inhibits proliferation of the intestinal epithelium and ultimately results in compromised intestinal epithelial barrier integrity, further perpetuating intestinal inflammation during experimental colitis. Using a CaMKIV null mutant mouse, we demonstrate that a loss of CaMKIV protects against murine DSS colitis. Small molecules targeting CaMKIV activation may provide therapeutic benefit for patients with IBD.-Cunningham, K. E., Novak, E. A., Vincent, G., Siow, V. S., Griffith, B. D., Ranganathan, S., Rosengart, M. R., Piganelli, J. D., Mollen, K. P. Calcium/calmodulin-dependent protein kinase IV (CaMKIV) activation contributes to the pathogenesis of experimental colitis via inhibition of intestinal epithelial cell proliferation.

The Pediatric Surgeon-Scientist: Succeeding in Today's Academic Environment.

BACKGROUND: Pediatric surgeons have long been advocates of basic science research. However, new challenges facing the scientific community have threatened the success of academic surgeons pursuing basic science careers. The purpose of this study was to compare academic pediatric surgeons' perceptions of their ability to effectively conduct basic science research to those of other surgical subspecialties. METHODS: An online survey was distributed to all members of the Association for Academic Surgery and Society of University Surgeons. A total of 1033 members (41%) responded, and 137 (13.3%) were pediatric surgeons. Comparisons were made between the five most-represented surgical subspecialties. Data are presented as reporting percentage and P values by Student's t-test. RESULTS: Among the specialists studied, pediatric surgeons are those most likely to believe that surgeons can succeed as basic scientists in today's research environment. Pediatric surgery reported the highest rates of National Institutes of Health funding of all surgical specialties and the lowest rates of perceived external pressures related to clinical demands, hospital administrative duties, and work-life balance concerns than their surgical peers. CONCLUSIONS: Pediatric surgeons have a more optimistic perspective on the state of basic science research in surgery while exhibiting an enhanced ability to overcome the challenges that surgeon-scientists currently face. Our findings suggest that pediatric surgery may provide a model for succeeding in basic science in today's challenging surgical research environment.

Peroxisome Proliferator-activated Receptor-γ Coactivator 1-α (PGC1α) Protects against Experimental Murine Colitis.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is the primary regulator of mitochondrial biogenesis and was recently found to be highly expressed within the intestinal epithelium. PGC1α is decreased in the intestinal epithelium of patients with inflammatory bowel disease, but its role in pathogenesis is uncertain. We now hypothesize that PGC1α protects against the development of colitis and helps to maintain the integrity of the intestinal barrier. We selectively deleted PGC1α from the intestinal epithelium of mice by breeding a PGC1α(loxP/loxP) mouse with a villin-cre mouse. Their progeny (PGC1α(ΔIEC) mice) were subjected to 2% dextran sodium sulfate (DSS) colitis for 7 days. The SIRT1 agonist SRT1720 was used to enhance PGC1α activation in wild-type mice during DSS exposure. Mice lacking PGC1α within the intestinal epithelium were more susceptible to DSS colitis than their wild-type littermates. Pharmacologic activation of PGC1α successfully ameliorated disease and restored mitochondrial integrity. These findings suggest that a depletion of PGC1α in the intestinal epithelium contributes to inflammatory changes through a failure of mitochondrial structure and function as well as a breakdown of the intestinal barrier, which leads to increased bacterial translocation. PGC1α induction helps to maintain mitochondrial integrity, enhance intestinal barrier function, and decrease inflammation.

Red blood cell transfusion in premature infants leads to worse necrotizing enterocolitis outcomes.

BACKGROUND: Necrotizing enterocolitis (NEC) is a severe intestinal disease of premature infants with high mortality. Studies suggest a causative relationship between red blood cell (RBC) transfusion and NEC; however, whether RBC transfusion leads to worse outcomes in NEC is unknown. We sought to determine whether RBC transfusion was associated with an increased risk of surgical NEC and mortality. METHODS: In this retrospective study, 115 patients were enrolled with NEC Bell's stage 2A or greater from 2010-2015. Patients were classified based on the timing of RBC transfusion before NEC: ≤72 h, >72 h, and no transfusion. Variables including gestational age (GA), birth weight (BW), feedings, and hematocrit levels were analyzed. Outcomes were surgical intervention for NEC following RBC transfusion and mortality. RESULTS: Twenty-three (20%) infants developed NEC ≤ 72 h after RBC transfusion, 16 (69.6%) required surgery with a mortality rate of 21.7% (n = 5). Seventeen (15%) infants developed NEC > 72 h after RBC transfusion, 12 (70.6%) required surgery with a mortality rate of 23.5% (n = 4). 75 (65%) patients developed NEC without RBC transfusion, 17 (22.7%) required surgery with a mortality rate of 4% (n = 3). Lower GA and BW were significantly associated with RBC transfusion and the need for surgical intervention. RBC transfusion ≤72 h before NEC was associated with surgical NEC (pairwise adjusted P < 0.001) and mortality (pairwise adjusted P = 0.048). However, multivariable logistic regression analysis revealed RBC transfusion is not an independent risk factor for surgical NEC. CONCLUSIONS: Infants of lower GA and BW were more likely to receive an RBC transfusion before NEC, which was significantly associated with surgical intervention and an increasing risk of mortality. Judicious use of transfusions in premature infants may improve NEC outcomes.

Ouch, my pouch! a clinician's guide to pouchitis.

Pouchitis is defined as inflammation of the ileal pouch created during a restorative proctocolectomy with ileal pouch-anal anastomosis. Although the incidence of this inflammatory condition is high, the exact etiology often remains unclear and the management challenging. In this review, we summarize the clinical presentation, pathogenesis, diagnosis, and management of this common complication.

Signaling of high mobility group box 1 (HMGB1) through toll-like receptor 4 in macrophages requires CD14.

High mobility group box 1 (HMGB1) is a DNA-binding protein that possesses cytokinelike, proinflammatory properties when released extracellularly in the C23-C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with toll-like receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/myeloid differentiation protein 2 (MD2) complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the coreceptor CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW264.7 macrophages after stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates tumor necrosis factor (TNF)-α release in WT but not in TLR4(-/-), CD14(-/-), TIR domain-containing adapter-inducing interferon-ß (TRIF)(-/-) or myeloid differentiation primary response protein 88 (MyD88)(-/-) macrophages. HMGB1 induces the release of monocyte chemotactic protein 1 (MCP-1), interferon gamma-induced protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in a TLR4- and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Validity of the American College of Radiology Thyroid Imaging Reporting and Data System in Children.

OBJECTIVE: To assess the validity of the American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) for evaluating thyroid nodules in children. METHODS: Patients aged <19 years with thyroid nodule(s) evaluated by ultrasound (US) from 2007-2018 at a tertiary children's hospital were included. Two radiologists scored de-identified thyroid US images using ACR TI-RADS (from 1, "benign" to 5, "highly suspicious"). The radiologists recorded size and rated vascularity for each nodule. Ultrasound findings were compared to pathology results (operative cases, n = 91) and clinical follow-up without disease progression (non-operative cases, n = 15). RESULTS: Thyroid images from 115 patients were reviewed. Nine patients were excluded due to the absence of an evaluable nodule. Forty-seven benign and 59 malignant nodules were included. Median age at ultrasound was 15 years (range 0.9-18 years). Twenty (18.9%) patients were male. There was moderate agreement between TI-RADS levels assigned by the two raters (kappa = 0.57, p < 0.001). When the raters' levels were averaged, >3 as the threshold for malignancy correctly categorized the greatest percentage of nodules (68.9%). Eleven (18.6%) malignant nodules received a TI-RADS level of 2 (n = 3) or 3 (n = 8). Sensitivity, specificity, and positive and negative predictive values were 81.4%, 53.2%, 68.6%, and 69.4%, respectively. Although not part of TI-RADS, vascularity was similar between benign and malignant nodules (p = 0.56). CONCLUSION: In a pediatric population, TI-RADS can help distinguish between benign and malignant nodules with comparable sensitivity and specificity to adults. However, the positive and negative predictive values suggest TI-RADS alone cannot eliminate the need for FNA. LEVEL OF EVIDENCE: 3 Laryngoscope, 133:2394-2401, 2023.

Epithelial NAD+ depletion drives mitochondrial dysfunction and contributes to intestinal inflammation.

Introduction: We have previously demonstrated that a pathologic downregulation of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1α) within the intestinal epithelium contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanism underlying downregulation of PGC1α expression and activity during IBD is not yet clear. Methods: Mice (male; C57Bl/6, Villincre/+;Pgc1afl/fl mice, and Pgc1afl/fl) were subjected to experimental colitis and treated with nicotinamide riboside. Western blot, high-resolution respirometry, nicotinamide adenine dinucleotide (NAD+) quantification, and immunoprecipitation were used to in this study. Results: We demonstrate a significant depletion in the NAD+ levels within the intestinal epithelium of mice undergoing experimental colitis, as well as humans with ulcerative colitis. While we found no decrease in the levels of NAD+-synthesizing enzymes within the intestinal epithelium of mice undergoing experimental colitis, we did find an increase in the mRNA level, as well as the enzymatic activity, of the NAD+-consuming enzyme poly(ADP-ribose) polymerase-1 (PARP1). Treatment of mice undergoing experimental colitis with an NAD+ precursor reduced the severity of colitis, restored mitochondrial function, and increased active PGC1α levels; however, NAD+ repletion did not benefit transgenic mice that lack PGC1α within the intestinal epithelium, suggesting that the therapeutic effects require an intact PGC1α axis. Discussion: Our results emphasize the importance of PGC1α expression to both mitochondrial health and homeostasis within the intestinal epithelium and suggest a novel therapeutic approach for disease management. These findings also provide a mechanistic basis for clinical trials of nicotinamide riboside in IBD patients.

Excess Dietary Sugar Alters Colonocyte Metabolism and Impairs the Proliferative Response to Damage.

BACKGROUND & AIMS: The colonic epithelium requires continuous renewal by crypt resident intestinal stem cells (ISCs) and transit-amplifying (TA) cells to maintain barrier integrity, especially after inflammatory damage. The diet of high-income countries contains increasing amounts of sugar, such as sucrose. ISCs and TA cells are sensitive to dietary metabolites, but whether excess sugar affects their function directly is unknown. METHODS: Here, we used a combination of 3-dimensional colonoids and a mouse model of colon damage/repair (dextran sodium sulfate colitis) to show the direct effect of sugar on the transcriptional, metabolic, and regenerative functions of crypt ISCs and TA cells. RESULTS: We show that high-sugar conditions directly limit murine and human colonoid development, which is associated with a reduction in the expression of proliferative genes, adenosine triphosphate levels, and the accumulation of pyruvate. Treatment of colonoids with dichloroacetate, which forces pyruvate into the tricarboxylic acid cycle, restored their growth. In concert, dextran sodium sulfate treatment of mice fed a high-sugar diet led to massive irreparable damage that was independent of the colonic microbiota and its metabolites. Analyses on crypt cells from high-sucrose-fed mice showed a reduction in the expression of ISC genes, impeded proliferative potential, and increased glycolytic potential without a commensurate increase in aerobic respiration. CONCLUSIONS: Taken together, our results indicate that short-term, excess dietary sucrose can directly modulate intestinal crypt cell metabolism and inhibit ISC/TA cell regenerative proliferation. This knowledge may inform diets that better support the treatment of acute intestinal injury.

Unique Molecular Signatures Are Associated with Aggressive Histology in Pediatric Differentiated Thyroid Cancer.

Background: Molecular testing (MT) enhances the diagnostic accuracy of thyroid fine-needle aspiration biopsy, reducing the need for diagnostic lobectomy in adult patients with indeterminate nodules (Bethesda class III/IV). However, little is known about genetic alterations in pediatric thyroid carcinoma (TC). Our aim was to analyze MT results of pediatric differentiated TC (DTC) cases to determine associations with histological and clinical features. Methods: A retrospective review identified all patients (aged <19 years) diagnosed with DTC from 2001 to 2017 at the University of Pittsburgh Medical Center. Histology was rereviewed to confirm diagnosis and identify tissue for MT using next-generation sequencing (ThyroSeq, version 3, TSv3). Correlation with histological and clinical features was analyzed using regression analysis. Results: Of 71 patients with MT results, 62 (87%) patients had papillary TC. All patients were alive at a median follow-up of 6 years (range 18 days to 18 years). Genetic alterations were identified in 65 (92%) patients. These alterations were grouped as BRAF-like point mutations or fusions (39, 55%), RAS-like mutations or fusions (21, 30%), or copy number alterations (5, 7%). On multiple regression analysis accounting for patient sex and tumor size in patients with papillary TC, increased tumor stage (ß: 0.234, p < 0.001), multifocal disease (odds ratio [OR]: 3.60, p = 0.042), and lymph node metastases (OR: 6.13, p = 0.044) were associated with BRAF-like gene fusions. When considering individual mutations, ETV6/NTRK3 fusions were associated with increased tumor stage (ß: 2.07, p = 0.023) and BRAF-like point mutations were associated with increased likelihood of surgery for recurrence over time (hazard ratio: 19.5, p = 0.004). Conclusions: Among our cohort of pediatric TC patients who underwent comprehensive MT, >90% had an identifiable genetic alteration. Aggressive features were primarily associated with BRAF-like gene fusions. Preoperative MT results may be useful in guiding the extent of the initial operation in pediatric patients (aged <19 years) with TC.

Inclusion and representation in the pediatric surgery workforce: Strategies to mitigate bias in the fellowship application process.

Diverse perspectives are critical components of effective teams in every industry. Underrepresentation of minorities in medicine leads to worse outcomes for minority patients, and efforts to increase diversity in the health care workforce are critical. Presently, about 70% of the pediatric surgery workforce is white, and pediatric surgeons at large do not reflect the racial or ethnic diversity of the populations they serve. Pediatric surgery fellowship training programs are the gateway to the field, and fellow selection processes should be optimized to support diversity and inclusion. The Association of Pediatric Surgery Training Program Directors (APSTPD) Diversity Equity and Inclusion subcommittee compiled best practices for bias mitigation during fellow selection, drawing from published literature and personal experiences in our own programs. A list of concrete recommendations was compiled, which can be implemented in every phase from applicant screening to rank list creation. We present these as a position statement that has been endorsed by the executive committee of the APSTPD. Pediatric surgery fellowship programs can utilize this focused review of best practices to mitigate bias and support diverse applicants.

Eicosatetraynoic Acid and Butyrate Regulate Human Intestinal Organoid Mitochondrial and Extracellular Matrix Pathways Implicated in Crohn's Disease Strictures.

BACKGROUND: Perturbagen analysis of Crohn's disease (CD) ileal gene expression data identified small molecules including eicosatetraynoic acid (ETYA), which may exert an antifibrotic effect. We developed a patient-specific human intestinal organoid (HIO) model system to test small molecule regulation of mitochondrial and wound-healing functions implicated in stricturing behavior. METHODS: HIOs were made from CD induced pluripotent stem cells with and without a loss-of-function haplotype in the DUOX2 gene implicated in ileal homeostasis and characterized under basal conditions and following exposure to butyrate and ETYA using RNA sequencing, flow cytometry, and immunofluorescent and polarized light microscopy. Mitochondrial activity was measured using high-resolution respirometry and tissue stiffness using atomic force microscopy. RESULTS: HIOs expressed core mitochondrial and extracellular matrix (ECM) genes and enriched biologic functions implicated in CD ileal strictures; ECM gene expression was suppressed by both butyrate and ETYA, with butyrate also suppressing genes regulating epithelial proliferation. Consistent with this, butyrate, but not ETYA, exerted a profound effect on HIO epithelial mitochondrial function, reactive oxygen species production, and cellular abundance. Butyrate and ETYA suppressed HIO expression of alpha smooth muscle actin expressed by myofibroblasts, type I collagen, and collagen protein abundance. HIOs exhibited tissue stiffness comparable to normal human ileum; this was reduced by chronic ETYA exposure in HIOs carrying the DUOX2 loss-of-function haplotype. CONCLUSIONS: ETYA regulates ECM genes implicated in strictures and suppresses collagen content and tissue stiffness in an HIO model. HIOs provide a platform to test personalized therapeutics, including small molecules prioritized by perturbagen analysis.

A subset of pediatric Crohn's disease patients develop intestinal strictures requiring surgery. The microbial metabolite butyrate and eicosatetraynoic acid regulate pathways implicated in stricture formation in a human intestinal organoid model system, which may be used to test new therapies.

Inactivation of RIP3 kinase sensitizes to 15LOX/PEBP1-mediated ferroptotic death.

Ferroptosis and necroptosis are two pro-inflammatory cell death programs contributing to major pathologies and their inhibition has gained attention to treat a wide range of disease states. Necroptosis relies on activation of RIP1 and RIP3 kinases. Ferroptosis is triggered by oxidation of polyunsaturated phosphatidylethanolamines (PUFA-PE) by complexes of 15-Lipoxygenase (15LOX) with phosphatidylethanolamine-binding protein 1 (PEBP1). The latter, also known as RAF kinase inhibitory protein, displays promiscuity towards multiple proteins. In this study we show that RIP3 K51A kinase inactive mice have increased ferroptotic burden and worse outcome after irradiation and brain trauma rescued by anti-ferroptotic compounds Liproxstatin-1 and Ferrostatin 16-86. Given structural homology between RAF and RIP3, we hypothesized that PEBP1 acts as a necroptosis-to-ferroptosis switch interacting with either RIP3 or 15LOX. Using genetic, biochemical, redox lipidomics and computational approaches, we uncovered that PEBP1 complexes with RIP3 and inhibits necroptosis. Elevated expression combined with higher affinity enables 15LOX to pilfer PEBP1 from RIP3, thereby promoting PUFA-PE oxidation and ferroptosis which sensitizes Rip3K51A/K51A kinase-deficient mice to total body irradiation and brain trauma. This newly unearthed PEBP1/15LOX-driven mechanism, along with previously established switch between necroptosis and apoptosis, can serve multiple and diverse cell death regulatory functions across various human disease states.

Nitrite potently inhibits hypoxic and inflammatory pulmonary arterial hypertension and smooth muscle proliferation via xanthine oxidoreductase-dependent nitric oxide generation.

BACKGROUND: Pulmonary arterial hypertension is a progressive proliferative vasculopathy of the small pulmonary arteries that is characterized by a primary failure of the endothelial nitric oxide and prostacyclin vasodilator pathways, coupled with dysregulated cellular proliferation. We have recently discovered that the endogenous anion salt nitrite is converted to nitric oxide in the setting of physiological and pathological hypoxia. Considering the fact that nitric oxide exhibits vasoprotective properties, we examined the effects of nitrite on experimental pulmonary arterial hypertension. METHODS AND RESULTS: We exposed mice and rats with hypoxia or monocrotaline-induced pulmonary arterial hypertension to low doses of nebulized nitrite (1.5 mg/min) 1 or 3 times a week. This dose minimally increased plasma and lung nitrite levels yet completely prevented or reversed pulmonary arterial hypertension and pathological right ventricular hypertrophy and failure. In vitro and in vivo studies revealed that nitrite in the lung was metabolized directly to nitric oxide in a process significantly enhanced under hypoxia and found to be dependent on the enzymatic action of xanthine oxidoreductase. Additionally, physiological levels of nitrite inhibited hypoxia-induced proliferation of cultured pulmonary artery smooth muscle cells via the nitric oxide-dependent induction of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). The therapeutic effect of nitrite on hypoxia-induced pulmonary hypertension was significantly reduced in the p21-knockout mouse; however, nitrite still reduced pressures and right ventricular pathological remodeling, indicating the existence of p21-independent effects as well. CONCLUSIONS: These studies reveal a potent effect of inhaled nitrite that limits pathological pulmonary arterial hypertrophy and cellular proliferation in the setting of experimental pulmonary arterial hypertension.

Nix-Mediated Mitophagy Modulates Mitochondrial Damage During Intestinal Inflammation.

Aims: Mitochondrial stress and dysfunction within the intestinal epithelium are known to contribute to the pathogenesis of inflammatory bowel disease (IBD). However, the importance of mitophagy during intestinal inflammation remains poorly understood. The primary aim of this study was to investigate how the mitophagy protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L/NIX) mitigates mitochondrial damage during intestinal inflammation in the hopes that these data will allow us to target mitochondrial health in the intestinal epithelium as an adjunct to immune-based treatment strategies. Results: In the intestinal epithelium of patients with ulcerative colitis, we found that NIX was upregulated and targeted to the mitochondria. We obtained similar findings in wild-type mice undergoing experimental colitis. An increase in NIX expression was found to depend on stabilization of hypoxia-inducible factor-1 alpha (HIF1α), which binds to the Nix promoter region. Using the reactive oxygen species (ROS) scavenger MitoTEMPO, we were able to attenuate disease and inhibit both HIF1α stabilization and subsequent NIX expression, suggesting that mitochondrially derived ROS are crucial to initiating the mitophagic response during intestinal inflammation. We subjected a global Nix-/- mouse to dextran sodium sulfate colitis and found that these mice developed worse disease. In addition, Nix-/- mice were found to exhibit increased mitochondrial mass, likely due to the inability to clear damaged or dysfunctional mitochondria. Innovation: These results demonstrate the importance of mitophagy within the intestinal epithelium during IBD pathogenesis. Conclusion: NIX-mediated mitophagy is required to maintain intestinal homeostasis during inflammation, highlighting the impact of mitochondrial damage on IBD progression.

Depletion of gut microbiota is associated with improved neurologic outcome following traumatic brain injury.

Signaling between intestinal microbiota and the brain influences neurologic outcome in multiple forms of brain injury. The impact of gut microbiota following traumatic brain injury (TBI) has not been well established. Our objective was to compare TBI outcomes in specific pathogen-free mice with or without depletion of intestinal bacteria. Adult male C57BL6/J SPF mice (n = 6/group) were randomized to standard drinking water or ampicillin (1 g/L), metronidazole (1 g/L), neomycin (1 g/L), and vancomycin (0.5 g/L) (AMNV) containing drinking water 14 days prior to controlled cortical impact (CCI) model of TBI. 16S rRNA gene sequencing of fecal pellets was performed and alpha and beta diversity determined. Hippocampal neuronal density and microglial activation was assessed 72 h post-injury by immunohistochemistry. In addition, mice (n = 8-12/group) were randomized to AMNV or no treatment initiated immediately after CCI and memory acquisition (fear conditioning) and lesion volume assessed. Mice receiving AMNV had significantly reduced alpha diversity (p < 0.05) and altered microbiota community composition compared to untreated mice (PERMANOVA: p < 0.01). Mice receiving AMNV prior to TBI had increased CA1 hippocampal neuronal density (15.2 ± 1.4 vs. 8.8 ± 2.1 cells/0.1 mm; p < 0.05) and a 26.6 ± 6.6% reduction in Iba-1 positive cells (p < 0.05) at 72 h. Mice randomized to AMNV immediately after CCI had attenuated associative learning deficit on fear conditioning test (%freeze Cue: 63.7 ± 2.7% vs. 41.0 ± 5.1%, p < 0.05) and decreased lesion volume (27.2 ± 0.8 vs. 24.6 ± 0.7 mm3, p < 0.05). In conclusion, depletion of intestinal microbiota was consistent with a neuroprotective effect whether initiated before or after injury in a murine model of TBI. Further investigations of the role of gut microbiota in TBI are warranted.

Early events in the recognition of danger signals after tissue injury.

The systemic inflammatory response observed in the setting of overwhelming infection bears striking similarities to that observed in the setting of severe traumatic injury from a clinical and physiologic standpoint. Recent observations have demonstrated that these disparate clinical entities share common mediators on a molecular level. TLRs, specifically TLR4, and the endogenous molecule high-mobility group box 1 are among the mediators that are known to play a role in inflammation in the setting of sepsis. Evidence is accumulating that demonstrates that these mediators also play a role in the host response to tissue injury. Here, we highlight findings from the 7th World Conference on Trauma, Shock, Inflammation and Sepsis in Munich, Germany, in the context of this growing body of literature.