Metabolic immaturity of newborns and breast milk bile acid metabolites are the central determinants of heightened neonatal vulnerability to norovirus diarrhea.

Noroviruses are the leading global cause of acute gastroenteritis, responsible for 685 million annual cases. While all age groups are susceptible to noroviruses, children are vulnerable to more severe infections than adults, underscored by 200 million pediatric cases and up to 200,000 deaths in children annually. Understanding the basis for the increased vulnerability of young hosts is critical to developing effective treatments. The pathogenic outcome of any enteric virus infection is governed by a complex interplay between the virus, intestinal microbiota, and host immune factors. A central mediator in these complex relationships are host- and microbiota-derived metabolites. Noroviruses bind a specific class of metabolites, bile acids, which are produced by the host and then modified by commensal bacterial enzymes. Paradoxically, bile acids can have both proviral and antiviral roles during norovirus infections. Considering these opposing effects, the microbiota-regulated balance of the bile acid pool may be a key determinant of the pathogenic outcome of a norovirus infection. The bile acid pool in newborns is unique due to immaturity of host metabolic pathways and developing gut microbiota, which could underlie the vulnerability of these hosts to severe norovirus infections. Supporting this concept, we demonstrate herein that microbiota and their bile acid metabolites protect from severe norovirus diarrhea whereas host-derived bile acids promote disease. Remarkably, we also report that maternal bile acid metabolism determines neonatal susceptibility to norovirus diarrhea during breastfeeding by delivering proviral bile acids to the newborn. Finally, directed targeting of maternal and neonatal bile acid metabolism can protect the neonatal host from norovirus disease. Altogether, these data support the conclusion that metabolic immaturity in newborns and ingestion of proviral maternal metabolites in breast milk are the central determinants of heightened neonatal vulnerability to norovirus disease.

Scattered Crypt Intestinal Epithelial Cell Apoptosis Induces Necrotizing Enterocolitis Via Intricate Mechanisms.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is a life-threatening disease affecting mostly the ileum of preemies. Intestinal epithelial cell (IEC) apoptosis contributes to NEC pathogenesis. However, how scattered crypt IEC apoptosis leads to NEC with excessive villus epithelial necrosis remains unclear. METHODS: A novel triple-transgenic mouse model, namely, 3xTg-iAPcIEC (inducible apoptosis phenotype in crypt-IEC), was developed to induce IEC-specific overexpression of Fasl transgene using doxycycline (Dox)-inducible tetO-rtTA system and villin-cre technology. The 3-days-old neonatal 3xTg-iAPcIEC mice and their littermate controls were subcutaneously (s.c.) challenged with a single dose of Dox. Intestinal tissues were processed at different time points to examine scattered crypt IEC apoptosis-mediated NEC development. Gene knockout technology, antibody-mediated cell depletion, and antibiotic-facilitated Gram-positive bacteria depletion were used to study mechanisms. RESULTS: Treatment of 3xTg-iAPcIEC mouse pups with Dox induces scattered crypt IEC apoptosis followed by crypt inflammation and excessive villous necrosis resembling NEC. This progression correlated with elevated Ifng, Rip3, CD8+ T cells, and Gram-positive bacteria in the ileum. Mechanistically, IFN-γ and RIP3-activated signals mediate the effect of scattered crypt IEC apoptosis on the induction of intestinal crypt inflammation and villous necrosis. Meanwhile, pathophysiological events of CD8+ T cell infiltration and dysbiosis with Gram-positive bacteria primarily contribute to excessive villous inflammation and necrosis. Notably, blocking any of these events protects against NEC development in 3xTg-iAPcIEC mouse pups, underlining their central roles in NEC pathogenesis. CONCLUSIONS: Scattered crypt IEC apoptosis induces NEC in mouse pups via IFN-γ, RIP3, CD8+ T cells, and Gram-positive bacteria-mediated comprehensive pathophysiological events. Our findings may advance knowledge in the prevention and treatment of NEC.

Recombinant IGF-1/BP3 protects against intestinal injury in a neonatal mouse NEC model.

BACKGROUND: Recombinant human IGF-1/binding protein-3 (rhIGF-1/BP3) is currently being tested in phase II clinical trials in premature infants to prevent bronchopulmonary dysplasia, but its impact on the neonatal intestine remains unclear. The aim of this study was to determine whether rhIGF-1/BP3 protects against necrotizing enterocolitis (NEC) in mice and to investigate the mechanisms involved. METHODS: Neonatal mice were dam fed or injected intraperitoneally with rhIGF-1/BP3 (or vehicle) and submitted to an experimental NEC model. Serum IGF-1 was assessed by ELISA and intestinal vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) expression by Western blot. Intestinal endothelial cell proliferation, and enterocyte proliferation and migration were examined by immunofluorescence. Pup survival and histological intestinal injury were determined. RESULTS: In pups exposed to experimental NEC, serum IBP3-bound IGF-1 level was decreased. Exogenous rhIGF-1/BP3 preserved VEGF and VEGFR2 protein expression, decreased vascular permeability, and preserved endothelial cell proliferation in the small intestine. Furthermore, rhIGF-1/BP3 promoted enterocyte proliferation and migration, which effects were attenuated by inhibiting VEGFR2 signaling, decreased enterocyte apoptosis and decreased systemic and intestinal inflammation. rhIGF-1/BP3 improved survival and reduced the incidence of severe intestinal injury in experimental NEC. CONCLUSIONS: Exogenous rhIGF-1/BP3 protects neonatal mice against experimental NEC via multiple mechanisms. IMPACT: Exogenous rhIGF-1/BP3 preserves intestinal microvascular development and integrity, promotes enterocyte proliferation and migration, decreases local and systemic inflammation, and protects neonatal mice against NEC. The article adds pre-clinical evidence of a protective role for rhIGF-1/BP3 on the premature gut. It provides evidence supporting the use of rhIGF1/BP3 in premature neonates to protect against NEC.

Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans: report of an expert meeting.

The present report summarizes the United States Department of Veterans Affairs (VA) field-based meeting titled "Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans." Our Veteran patient population experiences a high incidence of service-related chronic physical and mental health problems, such as infection, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), various forms of hematological and non-hematological malignancies, neurologic conditions, end-stage organ failure, requiring transplantation, and posttraumatic stress disorder (PTSD). We report the views of a group of scientists who focus on the current state of scientific knowledge elucidating the mechanisms underlying the aforementioned disorders, novel therapeutic targets, and development of new approaches for clinical intervention. In conclusion, we dovetailed on four research areas of interest: 1) microbiome interaction with immune cells after hematopoietic cell and/or solid organ transplantation, graft-versus-host disease (GVHD) and graft rejection, 2) intestinal inflammation and its modification in IBD and cancer, 3) microbiome-neuron-immunity interplay in mental and physical health, and 4) microbiome-micronutrient-immune interactions during homeostasis and infectious diseases. At this VA field-based meeting, we proposed to explore a multi-disciplinary, multi-institutional, collaborative strategy to initiate a roadmap, specifically focusing on host microbiome-immune interactions among those with service-related chronic diseases to potentially identify novel and translatable therapeutic targets.

B Cell Lymphocytosis in Juvenile Dermatomyositis.

In this study, we determined if B lymphocytosis may serve as a JDM biomarker for disease activity. Children with untreated JDM were divided into two groups based on age-adjusted B cell percentage (determined through flow cytometry): 90 JDM in the normal B cell group and 45 in the high B cell group. We compared through T-testing the age, sex, ethnicity, duration of untreated disease (DUD), disease activity scores for skin (sDAS), muscle (mDAS), total (tDAS), CMAS, and neopterin between these two groups. The patients in the high B cell group had a higher tDAS (p = 0.009), mDAS (p = 0.021), and neopterin (p = 0.0365). Secondary analyses included B cell values over time and BAFF levels in matched patients with JM (juvenile myositis) and concurrent interstitial lung disease (ILD); JM alone and healthy controls Patient B cell percentage and number was significantly higher after 3-6 months of therapy and then significantly lower on completion of therapy (p =< 0.0001). The JM groups had higher BAFF levels than controls 1304 vs. 692 ng/mL (p = 0.0124). This study supports B cell lymphocytosis as a JDM disease-activity biomarker and bolsters the basis for B cell-directed therapies in JDM.

Severe gut mucosal injury induces profound systemic inflammation and spleen-associated lymphoid organ response.

Clinical evidence indicates a connection between gut injuries, infections, inflammation, and an increased susceptibility to systemic inflammation. Nevertheless, the animal models designed to replicate this progression are inadequate, and the fundamental mechanisms are still largely unknown. This research explores the relationship between gut injuries and systemic inflammation using a Dextran Sulfate Sodium (DSS)-induced colonic mucosal injury mouse model. Continuous treatment of adult mice with 4% DSS drinking water yielded a remarkable mortality rate by day 7, alongside intensified gut injury and detectable peripheral inflammation. Moreover, RNAscope in situ hybridization with 16S rRNA probe noted bacterial penetration into deeper colon compartments of the mice following treatment with DSS for 7 days. Histological analysis revealed inflammation in the liver and lung tissues of DSS-treated mice. In addition, we found that DSS-treated mice exhibited elevation of Alanine transaminase (ALT) and Aspartate transaminase (AST) in peripheral blood and pro-inflammatory cytokine levels in the liver. Notably, the DSS-treated mice displayed a dampened metabolic profile, reduced CD45 marker expression, and an increase in apoptosis within the lymphoid organ such as spleen. These findings suggest that high-dose DSS-induced gut injury gives rise to sepsis-like systemic inflammation characterized by multiple organ injury and profound splenocyte apoptosis and dysfunction of CD45+ cells in the spleen, indicating the role of the spleen in the pathogenesis of gut-derived systemic inflammation. Together, the severe colonic mucosal injury model facilitates research into gut damage and associated peripheral immune responses, providing a vital framework for investigating mechanisms related to clinically relevant, gut-derived systemic inflammation.

The expression and function of growth hormone secretagogue receptor in immune cells: A current perspective.

The orexigenic hormone ghrelin and its receptor, growth hormone secretagogue receptor (GHS-R), have been extensively studied in the last two decades, revealing that ghrelin signaling has important implications in health and disease. Metabolic diseases, such as obesity and diabetes, are often accompanied by low-grade chronic inflammation, that has been coined as "meta-inflammation." Immune cells are key cellular mediators of meta-inflammation, controlling both initiation and resolution of inflammation. Immune cells exhibit dynamic changes in cellular characteristics and functional output in response to the stimuli/insults from their surrounding microenvironment. Emerging evidence shows that ghrelin has an important effect on inflammation, in addition to its well-known effects on metabolism. However, the cellular/molecular mechanism of ghrelin signaling in immunity is largely unknown because the knowledge in regard to the expression and function of GHS-R in immune cells is currently sparse. In this review, we have accumulated the recent findings related to the expression and functions of GHS-R in various immune cells under different physiological and pathological states. This review aims to inspire further investigation of the immunological roles of ghrelin signaling and advance the therapeutic applications of ghrelin signaling in meta-inflammation.

Feeding mode influences dynamic gut microbiota signatures and affects susceptibility to anti-CD3 mAb-induced intestinal injury in neonatal mice.

Feeding modes influence the gut microbiome, immune system, and intestinal barrier homeostasis in neonates; how feeding modes impact susceptibility to neonatal gastrointestinal (GI) diseases is still uncertain. Here, we investigated the impact of dam feeding (DF) and formula feeding (FF) on features of the gut microbiome and physiological inflammation during the first 2 days of postnatal development and on the susceptibility to intestinal injury related to the inflammatory state in neonatal mouse pups. 16S rRNA sequencing data revealed microbiome changes, lower α-diversity, and a distinct pattern of ß-diversity including expansion of f_Enterobacteriaceae and f_Enterococcaceae in the ileum of FF pups compared with DF pups by postnatal day (P)2. Together with gut dysbiosis, the FF cohort also had greater ileal mucosa physiological inflammatory activity compared with DF pups by P2 but maintained normal histological features. Interestingly, FF but not DF mouse pups developed necrotizing enterocolitis (NEC)-like intestinal injury within 24 h after anti-CD3 mAb treatment, suggesting that FF influences the susceptibility to intestinal injury in neonates. We further found that NEC-like incidence in anti-CD3 mAb-treated FF neonatal pups was attenuated by antibiotic treatment. Collectively, our data suggest that FF predisposes mouse pups to anti-CD3 mAb-induced intestinal injury due to abnormal f_Enterobacteriaceae and f_Enterococcaceae colonization. These findings advance our understanding of FF-associated microbial colonization and intestinal inflammation, which may help inform the development of new therapeutic approaches to GI diseases like NEC in infants.NEW & NOTEWORTHY This report shows that a feeding mode profoundly affects gut colonization in neonatal mice. Furthermore, our results demonstrate that formula feeding predisposes mouse pups to anti-CD3 mAb-induced necrotizing enterocolitis (NEC)-like intestinal injury upon inadequate microbial colonization. The study suggests the role of the combined presence of formula feeding-associated dysbiosis and mucosal inflammation in the pathogenesis of NEC and provides a new mouse model to study this disease.

Macrophage-derived IGF-1 protects the neonatal intestine against necrotizing enterocolitis by promoting microvascular development.

Necrotizing enterocolitis (NEC) is a deadly bowel necrotic disease of premature infants. Low levels of plasma IGF-1 predispose premature infants to NEC. While increasing evidence suggests that defective perinatal intestinal microvascular development plays a role in NEC, the involved mechanism remains incompletely understood. We report here that serum and intestinal IGF-1 are developmentally regulated during the perinatal period in mice and decrease during experimental NEC. Neonatal intestinal macrophages produce IGF-1 and promote endothelial cell sprouting in vitro via IGF-1 signaling. In vivo, in the neonatal intestine, macrophage-derived IGF-1 promotes VEGF expression and endothelial cell proliferation and protects against experimental NEC. Exogenous IGF-1 preserves intestinal microvascular density and protects against experimental NEC. In human NEC tissues, villous endothelial cell proliferation and IGF-1- producing macrophages are decreased compared to controls. Together, our results suggest that defective IGF-1-production by neonatal macrophages impairs neonatal intestinal microvascular development and predisposes the intestine to necrotizing enterocolitis.

Novel Role of Ghrelin Receptor in Gut Dysbiosis and Experimental Colitis in Aging.

Chronic low-grade inflammation is a hallmark of aging, which is now coined as inflamm-aging. Inflamm-aging contributes to many age-associated diseases such as obesity, type 2 diabetes, cardiovascular disease, and inflammatory bowel disease (IBD). We have shown that gut hormone ghrelin, via its receptor growth hormone secretagogue receptor (GHS-R), regulates energy metabolism and inflammation in aging. Emerging evidence suggests that gut microbiome has a critical role in intestinal immunity of the host. To determine whether microbiome is an integral driving force of GHS-R mediated immune-metabolic homeostasis in aging, we assessed the gut microbiome profiles of young and old GHS-R global knockout (KO) mice. While young GHS-R KO mice showed marginal changes in Bacteroidetes and Firmicutes, aged GHS-R KO mice exhibited reduced Bacteroidetes and increased Firmicutes, featuring a disease-susceptible microbiome profile. To further study the role of GHS-R in intestinal inflammation in aging, we induced acute colitis in young and aged GHS-R KO mice using dextran sulfate sodium (DSS). The GHS-R KO mice showed more severe disease activity scores, higher proinflammatory cytokine expression, and decreased expression of tight junction markers. These results suggest that GHS-R plays an important role in microbiome homeostasis and gut inflammation during aging; GHS-R suppression exacerbates intestinal inflammation in aging and increases vulnerability to colitis. Collectively, our finding reveals for the first time that GHS-R is an important regulator of intestinal health in aging; targeting GHS-R may present a novel therapeutic strategy for prevention/treatment of aging leaky gut and inflammatory bowel disease.

SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells.

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a disease that involves significant lung tissue damage. How SARS-CoV-2 infection leads to lung injury remains elusive. The open reading frame 8 (ORF8) protein of SARS-CoV-2 (ORF8SARS-CoV-2) is a unique accessory protein, yet little is known about its cellular function. We examined the cellular distribution of ORF8SARS-CoV-2 and its role in the regulation of human lung epithelial cell proliferation and antiviral immunity. Using live imaging and immunofluorescent staining analyses, we found that ectopically expressed ORF8SARS-CoV-2 forms aggregates in the cytosol and nuclear compartments of lung epithelial cells. Using in silico bioinformatic analysis, we found that ORF8SARS-CoV-2 possesses an intrinsic aggregation characteristic at its N-terminal residues 1-18. Cell culture did not reveal any effects of ORF8SARS-CoV-2 expression on lung epithelial cell proliferation and cell cycle progression, suggesting that ORF8SARS-CoV-2 aggregates do not affect these cellular processes. Interestingly, ectopic expression of ORF8SARS-CoV-2 in lung epithelial cells suppressed basal expression of several antiviral molecules, including DHX58, ZBP1, MX1, and MX2. In addition, expression of ORF8SARS-CoV-2 attenuated the induction of antiviral molecules by IFNγ but not by IFNß in lung epithelial cells. Taken together, ORF8SARS-CoV-2 is a unique viral accessory protein that forms aggregates when expressing in lung epithelial cells. It potently inhibits the expression of lung cellular anti-viral proteins at baseline and in response to IFNγ in lung epithelial cells, which may facilitate SARS-CoV-2 escape from the host antiviral innate immune response during early viral infection. In addition, it seems that formation of ORF8SARS-CoV-2 aggregate is independent from the viral infection. Thus, it would be interesting to examine whether any COVID-19 patients exhibit persistent ORF8 SARS-CoV-2 expression after recovering from SARS-CoV-2 infection. If so, the pathogenic effect of prolonged ORF8SARS-CoV-2 expression and its association with post-COVID symptoms warrant investigation in the future.

MFG-E8 Plays an Important Role in Attenuating Cerulein-Induced Acute Pancreatitis in Mice.

Milk fat globule-EGF factor 8 (MFG-E8) is a secreted glycoprotein that regulates tissue homeostasis, possesses potent anti-inflammatory properties, and protects against tissue injury. The human pancreas expresses MFG-E8; however, the role of MFG-E8 in the pancreas remains unclear. We examined the expression of MFG-E8 in the pancreas at baseline and during cerulein-induced acute pancreatitis in mice and determined whether MFG-E8 attenuates the progression of pancreatitis, a serious inflammatory condition that can be life-threatening. We administered cerulein to wild-type (WT) and Mfge8 knockout (KO) mice to induce pancreatitis. Immunoblot analysis showed that MFG-E8 is constitutively expressed in the murine pancreas and is increased in mice with cerulein-induced acute pancreatitis. In situ hybridization revealed that ductal epithelial cells in the mouse pancreas express Mfge8 transcripts at baseline. During pancreatitis, Mfge8 transcripts were abundantly expressed in acinar cells and endothelial cells in addition to ductal epithelial cells. Knocking out Mfge8 in mice exacerbated the severity of cerulein-induced acute pancreatitis and delayed its resolution. In contrast, administration of recombinant MFG-E8 attenuated cerulein-induced acute pancreatitis and promoted repair of pancreatic injury in Mfge8 KO mice. Taken together, our study suggests that MFG-E8 protects the pancreas against inflammatory injury and promotes pancreatic tissue repair. MFG-E8 may represent a novel therapeutic target in acute pancreatitis.

Cell death of intestinal epithelial cells in intestinal diseases.

Gut injury continues to be the devastating and unpredictable critical illness associated with increased cell death of intestinal epithelial cells (IECs). The IECs, immune system and microbiome are the interrelated entities to maintain normal intestinal homeostasis and barrier integrity. In response to microbial invasion, IEC cell death occurs to maintain intestinal epithelium function and retain the continuous renewal and tissue homeostasis. But the imbalance of IEC cell death results in increased intestinal permeability and barrier dysfunction that leads to several acute and chronic intestinal diseases, such as intestinal ischemia/reperfusion (I/R), sepsis, inflammatory bowel diseases (IBD), necrotizing enterocolitis (NEC), etc. During the pathophysiological state, the excessive IEC apoptotic cell death leads to a chronic inflammatory condition, later switches to necroptotic cell death mechanism that induces more pathological features than apoptosis and may also induce other lytic cell death mechanisms like pyroptosis and ferroptosis to increase the pathogenesis of the intestinal diseases. But still, there remains gaps in the fundamental knowledge about the IEC cell death mechanisms in chronic intestinal diseases. Together, a deep understanding of the specific cell death mechanisms underlying chronic intestinal diseases, including sepsis, IBD, NEC, and intestinal I/R, is desperately needed to develop emerging novel promising therapeutic strategies. This review aims to show how the acute and critical illness in the gut are driven by IEC cell death mechanism, such as apoptosis, necrosis, necroptosis, pyroptosis, and ferroptosis.

Interferon-γ inhibits sirtuin 6 gene expression in intestinal epithelial cells through a microRNA-92b-dependent mechanism.

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3'-untranslated region (UTR) contains a putative binding site for miR-92b. Luciferase assay showed that Ifnγ inhibited Sirt6 3'-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3'-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b-mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation.

Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis.

Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17. Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development.

Blocking NF-κB Activation in Ly6c+ Monocytes Attenuates Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants with intestinal inflammation and necrosis. The neonatal intestinal inflammatory response is rich in macrophages, and blood monocyte count is low in human NEC. We previously found that NF-κB mediates the intestinal injury in experimental NEC. However, the role of NF-κB in myeloid cells during NEC remains unclear. Herein, inhibitor of kappaB kinase ß (IKKß), a critical kinase mediating NF-κB activation, was deleted in lysozyme M (Lysm)-expressing cells, which were found to be Cd11b+Ly6c+ monocytes but not Cd11b+Ly6c- macrophages in the dam-fed neonatal mouse intestine. NEC induced differentiation of monocytes into intestinal macrophages and up-regulation of monocyte recruitment genes (eg, L-selectin) in the macrophage compartment in wild-type mice, but not in pups with IKKß deletion in Lysm+ cells. Thus, NF-κB is required for NEC-induced monocyte activation, recruitment, and differentiation in neonatal intestines. Furthermore, pups with Lysm-IKKß deletion had improved survival and decreased incidence of severe NEC compared with littermate controls. Decreased NEC severity was not associated with an improved intestinal barrier. In contrast, NEC was unabated in mice with IKKß deletion in intestinal epithelial cells. Together, these data suggest that recruitment of Ly6c+ monocytes into the intestine, NF-κB activation in these cells, and differentiation of Ly6c+ monocytes into macrophages are critical cellular and molecular events in NEC development to promote disease.

In Inflamed Intestinal Tissues and Epithelial Cells, Interleukin 22 Signaling Increases Expression of H19 Long Noncoding RNA, Which Promotes Mucosal Regeneration.

BACKGROUND & AIMS: Inflammation affects regeneration of the intestinal epithelia; long noncoding RNAs (lncRNAs) regulate cell functions, such as proliferation, differentiation, and migration. We investigated the mechanisms by which the lncRNA H19, imprinted maternally expressed transcript (H19) regulates regeneration of intestinal epithelium using cell cultures and mouse models of inflammation. METHODS: We performed RNA-sequencing transcriptome analyses of intestinal tissues from mice with lipopolysaccharide (LPS)-induced sepsis to identify lncRNAs associated with inflammation; findings were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization analyses of intestinal tissues from mice with sepsis or dextran sulfate sodium (DSS)-induced mucosal wound healing and patients with ulcerative colitis compared to healthy individuals (controls). We screened cytokines for their ability to induce expression of H19 in HT-29 cells and intestinal epithelial cells (IECs), and confirmed findings in crypt epithelial organoids derived from mouse small intestine. IECs were incubated with different signal transduction inhibitors and effects on H19 lncRNA levels were measured. We assessed intestinal epithelial proliferation or regeneration in H19ΔEx1/+ mice given LPS or DSS vs wild-type littermates (control mice). H19 was overexpressed in IECs using lentiviral vectors and cell proliferation was measured. We performed RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays to study functions of H19 in IECs. RESULTS: In RNA-sequencing transcriptome analysis of lncRNA expression in intestinal tissues from mice, we found that levels of H19 lncRNA changed significantly with LPS exposure. Levels of H19 lncRNA increased in intestinal tissues of patients with ulcerative colitis, mice with LPS-induced and polymicrobial sepsis, or mice with DSS-induced colitis, compared with controls. Increased H19 lncRNA localized to epithelial cells in the intestine, regardless of Lgr5 messenger RNA expression. Exposure of IECs to interleukin 22 (IL22) increased levels of H19 lncRNA with time and dose, which required STAT3 and protein kinase A activity. IL22 induced expression of H19 in mouse intestinal epithelial organoids within 6 hours. Exposure to IL22 increased growth of intestinal epithelial organoids derived from control mice, but not H19ΔEx1/+ mice. Overexpression of H19 in HT-29 cells increased their proliferation. Intestinal mucosa healed more slowly after withdrawal of DSS from H19ΔEx1/+ mice vs control mice. Crypt epithelial cells from H19ΔEx1/+ mice proliferated more slowly than those from control mice after exposure to LPS. H19 lncRNA bound to p53 and microRNAs that inhibit cell proliferation, including microRNA 34a and let-7; H19 lncRNA binding blocked their function, leading to increased expression of genes that promote regeneration of the epithelium. CONCLUSIONS: The level of lncRNA H19 is increased in inflamed intestinal tissues from mice and patients. The inflammatory cytokine IL22 induces expression of H19 in IECs, which is required for intestinal epithelial proliferation and mucosal healing. H19 lncRNA appears to inhibit p53 protein and microRNA 34a and let-7 to promote proliferation of IECs and epithelial regeneration.

Genome-wide association study of maternal genetic effects and parent-of-origin effects on food allergy.

Previous genetic studies of food allergy (FA) have mainly focused on inherited genotypic effects. The role of parental genotypic effects remains largely unexplored. Leveraging existing genome-wide association study (GWAS) data generated from the Chicago Food Allergy Study, we examined maternal genotypic and parent-of-origin (PO) effects using multinomial likelihood ratio tests in 588 complete and incomplete Caucasian FA trios. We identified 1 single nucleotide polymorphism with significant (P < 5×10) maternal effect on any FA (rs4235235), which is located in a noncoding RNA (LOC101927947) with unknown function. We also identified 3 suggestive (P < 5×10) loci with maternal genetic effects: 1 for any FA (rs976078, in a gene desert region on 13q31.1) and 2 for egg allergy (rs1343795 and rs4572450, in the ZNF652 gene, where genetic variants have been associated with atopic dermatitis). Three suggestive loci with PO effect were observed: 1 for peanut allergy (rs4896888 in the ADGB gene) and 2 for any FA in boys only (rs1036504 and rs2917750 in the IQCE gene). Findings from this family-based GWAS of FA provided some preliminary evidence on maternal genotypic or PO effects on FA. Additional family-based studies are needed to confirm our findings and gain new insight into maternal and paternal genetic contribution to FA.