SARS-CoV-2 envelope protein regulates innate immune tolerance.

Severe COVID-19 often leads to secondary infections and sepsis that contribute to long hospital stays and mortality. However, our understanding of the precise immune mechanisms driving severe complications after SARS-CoV-2 infection remains incompletely understood. Here, we provide evidence that the SARS-CoV-2 envelope (E) protein initiates innate immune inflammation, via toll-like receptor 2 signaling, and establishes a sustained state of innate immune tolerance following initial activation. Monocytes in this tolerant state exhibit reduced responsiveness to secondary stimuli, releasing lower levels of cytokines and chemokines. Mice exposed to E protein before secondary lipopolysaccharide challenge show diminished pro-inflammatory cytokine expression in the lung, indicating that E protein drives this tolerant state in vivo. These findings highlight the potential of the SARS-CoV-2 E protein to induce innate immune tolerance, contributing to long-term immune dysfunction that could lead to susceptibility to subsequent infections, and uncovers therapeutic targets aimed at restoring immune function following SARS-CoV-2 infection.

Delta like 4 regulates cerebrovascular development and endothelial integrity via DLL4-NOTCH-CLDN5 pathway and is vulnerable to neonatal hyperoxia.

The mechanisms governing brain vascularization during development remain poorly understood. A key regulator of developmental vascularization is delta like 4 (DLL4), a Notch ligand prominently expressed in endothelial cells (EC). Exposure to hyperoxia in premature infants can disrupt the development and functions of cerebral blood vessels and lead to long-term cognitive impairment. However, its role in cerebral vascular development and the impact of postnatal hyperoxia on DLL4 expression in mouse brain EC have not been explored. We determined the DLL4 expression pattern and its downstream signalling gene expression in brain EC using Dll4+/+ and Dll4+/LacZ mice. We also performed in vitro studies using human brain microvascular endothelial cells. Finally, we determined Dll4 and Cldn5 expression in mouse brain EC exposed to postnatal hyperoxia. DLL4 is expressed in various cell types, with EC being the predominant one in immature brains. Moreover, DLL4 deficiency leads to persistent abnormalities in brain microvasculature and increased vascular permeability both in vivo and in vitro. We have identified that DLL4 insufficiency compromises endothelial integrity through the NOTCH-NICD-RBPJ-CLDN5 pathway, resulting in the downregulation of the tight junction protein claudin 5 (CLDN5). Finally, exposure to neonatal hyperoxia reduces DLL4 and CLDN5 expression in developing mouse brain EC. We reveal that DLL4 is indispensable for brain vascular development and maintaining the blood-brain barrier's function and is repressed by neonatal hyperoxia. We speculate that reduced DLL4 signalling in brain EC may contribute to the impaired brain development observed in neonates exposed to hyperoxia. KEY POINTS: The role of delta like 4 (DLL4), a Notch ligand in vascular endothelial cells, in brain vascular development and functions remains unknown. We demonstrate that DLL4 is expressed at a high level during postnatal brain development in immature brains and DLL4 insufficiency leads to abnormal cerebral vasculature and increases vascular permeability both in vivo and in vitro. We identify that DLL4  regulates endothelial integrity through NOTCH-NICD-RBPJ-CLDN5 signalling. Dll4 and Cldn5 expression are decreased in mouse brain endothelial cells exposed to postnatal hyperoxia.

Butyrate suppresses experimental necrotizing enterocolitis-induced brain injury in mice.

Background: Necrotizing enterocolitis (NEC) is a devastating disease in premature infants, and 50% of infants with surgical NEC develop neurodevelopmental defects. The mechanisms by which NEC-induced cytokine release and activation of inflammatory cells in the brain mediate neuronal injury, and whether enteral immunotherapy attenuates NEC-associated brain injury remain understudied. Based on our prior work, which demonstrated that experimental NEC-like intestinal injury is attenuated by the short-chain fatty acid, butyrate, in this study, we hypothesize that NEC-induced brain injury would be suppressed by enteral butyrate supplementation. Methods: A standardized NEC mouse model [enteral formula feeding, lipopolysaccharide (LPS), and hypoxia] was used. Mice were randomized into the following groups: control, NEC, butyrate pretreated NEC, and butyrate control. NEC scoring (1-4 with 4 representing severe injury) was performed on ileal sections using a validated scoring system. Intestinal and brain lysates were used to assess inflammation, proinflammatory signaling, and apoptosis. Results: NEC-induced intestinal injury was attenuated by butyrate supplementation. NEC-induced microglial activation in the cerebral cortex and hippocampus was suppressed with butyrate. NEC increased the number of activated microglial cells but decreased the number of oligodendrocytes. Butyrate pretreatment attenuated these changes. Increased activation of proinflammatory Toll-like receptor signaling, cytokine expression, and induction of GFAP and IBA1 in the cerebral cortex observed with NEC was suppressed with butyrate. Conclusion: Experimental NEC induced inflammation and activation of microglia in several regions of the brain, most prominently in the cortex. NEC-induced neuroinflammation was suppressed with butyrate pretreatment. The addition of short-chain fatty acids to diet may be used to attenuate NEC-induced intestinal injury and neuroinflammation in preterm infants.

HDAC6 and ERK/ADAM17 Regulate VEGF-Induced NOTCH Signaling in Lung Endothelial Cells.

Angiogenesis plays a critical role in various physiological and pathological processes and is regulated by VEGF. Histone Deacetylase 6 (HDAC6) is a class IIB HDAC that regulates cytoplasmic signaling through deacetylation and is emerging as a target for modulating angiogenesis. We investigated the hypothesis that VEGF-induced endothelial cell (EC) NOTCH signaling is regulated by HDAC6 through acetylation of NOTCH intracellular cytoplasmic domain (NICD). In pulmonary endothelial cells (EC), VEGF-induced activation of the NICD transcriptional response was regulated by ERK1/2 and ADAM 17 and required DLL4. While HDAC6 inhibition induced the acetylation of NICD and stabilized NICD, it repressed NICD-SNW1 binding required for the NOTCH transcriptional responses. In vitro experiments showed that HDAC6 inhibition inhibited lung EC angiogenesis, and neonatal mice treated with a systemic HDAC6 inhibitor had significantly altered angiogenesis and alveolarization. These findings shed light on the role of HDAC6 in modulating VEGF-induced angiogenesis through acetylation and repression of the transcriptional regulators, NICD and SNW1.

The Detrimental Effects of Peripartum Antibiotics on Gut Proliferation and Formula Feeding Injury in Neonatal Mice Are Alleviated with Lactobacillus rhamnosus GG.

Peripartum antibiotics can negatively impact the developing gut microbiome and are associated with necrotizing enterocolitis (NEC). The mechanisms by which peripartum antibiotics increase the risk of NEC and strategies that can help mitigate this risk remain poorly understood. In this study, we determined mechanisms by which peripartum antibiotics increase neonatal gut injury and evaluated whether probiotics protect against gut injury potentiated by peripartum antibiotics. To accomplish this objective, we administered broad-spectrum antibiotics or sterile water to pregnant C57BL6 mice and induced neonatal gut injury to their pups with formula feeding. We found that pups exposed to antibiotics had reduced villus height, crypt depth, and intestinal olfactomedin 4 and proliferating cell nuclear antigen compared to the controls, indicating that peripartum antibiotics impaired intestinal proliferation. When formula feeding was used to induce NEC-like injury, more severe intestinal injury and apoptosis were observed in the pups exposed to antibiotics compared to the controls. Supplementation with the probiotic Lactobacillus rhamnosus GG (LGG) reduced the severity of formula-induced gut injury potentiated by antibiotics. Increased intestinal proliferating cell nuclear antigen and activation of the Gpr81-Wnt pathway were noted in the pups supplemented with LGG, suggesting partial restoration of intestinal proliferation by probiotics. We conclude that peripartum antibiotics potentiate neonatal gut injury by inhibiting intestinal proliferation. LGG supplementation decreases gut injury by activating the Gpr81-Wnt pathway and restoring intestinal proliferation impaired by peripartum antibiotics. Our results suggest that postnatal probiotics may be effective in mitigating the increased risk of NEC associated with peripartum antibiotic exposure in preterm infants.

IRF7 and UNC93B1 variants in an infant with recurrent herpes simplex virus infection.

Neonatal herpes simplex virus (HSV) infection is a devastating disease with substantial morbidity and mortality. The genetic basis of susceptibility to HSV in neonates remains undefined. We evaluated a male infant with neonatal skin/eye/mouth (SEM) HSV-1 disease, who had complete recovery after acyclovir but developed HSV-1 encephalitis at 1 year of age. An immune workup showed an anergic PBMC cytokine response to TLR3 stimulation but no other TLRs. Exome sequencing identified rare missense variants in IFN-regulatory factor 7 (IRF7) and UNC-93 homolog B1 (UNC93B1). PBMC single-cell RNA-Seq done during childhood revealed decreased expression of several innate immune genes and a repressed TLR3 pathway signature at baseline in several immune cell populations, including CD14 monocytes. Functional studies in fibroblasts and human leukemia monocytic THP1 cells showed that both variants individually suppressed TLR3-driven IRF3 transcriptional activity and the type I IFN response in vitro. Furthermore, fibroblasts expressing the IRF7 and UNC93B1 variants had higher intracellular viral titers with blunting of the type I IFN response upon HSV-1 challenge. This study reports an infant with recurrent HSV-1 disease complicated by encephalitis associated with deleterious variants in the IRF7 and UNC93B1 genes. Our results suggest that TLR3 pathway mutations may predispose neonates to recurrent, severe HSV.

The SARS-CoV-2 E protein induces Toll-like receptor 2-mediated neonatal lung injury in a model of COVID-19 viremia that is rescued by the glucocorticoid ciclesonide.

SARS-CoV-2 viremia is associated with increased acute lung injury (ALI) and mortality in children and adults. The mechanisms by which viral components in the circulation mediate ALI in COVID-19 remain unclear. We tested the hypothesis that the SARS-CoV-2 envelope (E) protein induces Toll-like receptor (TLR)-mediated ALI and lung remodeling in a model of neonatal COVID-19. Neonatal C57BL6 mice given intraperitoneal E protein injections revealed a dose-dependent increase in lung cytokines [interleukin 6 (Il6), tumor necrosis factor (Tnfα), and interleukin 1 beta (Il1ß)] and canonical proinflammatory TLR signaling. Systemic E protein induced endothelial immune activation, immune cell influx, and TGFß signaling and lung matrix remodeling inhibited alveolarization in the developing lung. E protein-mediated ALI and transforming growth factor beta (TGFß) signaling was repressed in Tlr2-/-, but not Tlr4-/- mice. A single dose of intraperitoneal E protein injection induced chronic alveolar remodeling as evidenced by a decrease in radial alveolar counts and increase in mean linear intercepts. Ciclesonide, a synthetic glucocorticoid, inhibited E protein-induced proinflammatory TLR signaling and ALI. In vitro, E protein-mediated inflammation and cell death were TLR2-dependent in human primary neonatal lung endothelial cells and were rescued by ciclesonide. This study provides insight into the pathogenesis of ALI and alveolar remodeling with SARS-CoV-2 viremia in children, whereas revealing the efficacy of steroids.NEW & NOTEWORTHY We reveal that the envelope protein of SARS-CoV-2 mediates acute lung injury (ALI) and alveolar remodeling through Toll-like receptor activation, which is rescued by the glucocorticoid, ciclesonide.

Neonatal hyperoxia induces activated pulmonary cellular states and sex-dependent transcriptomic changes in a model of experimental bronchopulmonary dysplasia.

Hyperoxia disrupts lung development in mice and causes bronchopulmonary dysplasia (BPD) in neonates. To investigate sex-dependent molecular and cellular programming involved in hyperoxia, we surveyed the mouse lung using single cell RNA sequencing (scRNA-seq), and validated our findings in human neonatal lung cells in vitro. Hyperoxia-induced inflammation in alveolar type (AT) 2 cells gave rise to damage-associated transient progenitors (DATPs). It also induced a new subpopulation of AT1 cells with reduced expression of growth factors normally secreted by AT1 cells, but increased mitochondrial gene expression. Female alveolar epithelial cells had less EMT and pulmonary fibrosis signaling in hyperoxia. In the endothelium, expansion of Car4+ EC (Cap2) was seen in hyperoxia along with an emergent subpopulation of Cap2 with repressed VEGF signaling. This regenerative response was increased in females exposed to hyperoxia. Mesenchymal cells had inflammatory signatures in hyperoxia, with a new distal interstitial fibroblast subcluster characterized by repressed lipid biosynthesis and a transcriptomic signature resembling myofibroblasts. Hyperoxia-induced gene expression signatures in human neonatal fibroblasts and alveolar epithelial cells in vitro resembled mouse scRNA-seq data. These findings suggest that neonatal exposure to hyperoxia programs distinct sex-specific stem cell progenitor and cellular reparative responses that underpin lung remodeling in BPD.

Short-chain fatty acids ameliorate necrotizing enterocolitis-like intestinal injury through enhancing Notch1-mediated single immunoglobulin interleukin-1-related receptor, toll-interacting protein, and A20 induction.

Single immunoglobulin interleukin-1-related receptor (SIGIRR), toll-interacting protein (TOLLIP), and A20 are major inhibitors of toll-like receptor (TLR) signaling induced postnatally in the neonatal intestine. Short-chain fatty acids (SCFAs), fermentation products of indigestible carbohydrates produced by symbiotic bacteria, inhibit intestinal inflammation. Herein, we investigated the mechanisms by which SCFAs regulate SIGIRR, A20, and TOLLIP expression and mitigate experimental necrotizing enterocolitis (NEC). Butyrate induced NOTCH activation by repressing sirtuin 1 (SIRT1)-mediated deacetylation of the Notch intracellular domain (NICD) in human intestinal epithelial cells (HIECs). Overexpression of NICD induced SIGIRR, A20, and TOLLIP expression. Chromatin immunoprecipitation revealed that butyrate-induced NICD binds to the SIGIRR, A20, and TOLLIP gene promoters. Notch1-shRNA suppressed butyrate-induced SIGIRR/A20 upregulation in mouse enteroids and HIEC. Flagellin (TLR5 agonist)-induced inflammation in HIEC was inhibited by butyrate in a SIGIRR-dependent manner. Neonatal mice fed butyrate had increased NICD, A20, SIGIRR, and TOLLIP expression in the ileal epithelium. Butyrate inhibited experimental NEC-induced intestinal apoptosis, cytokine expression, and histological injury. Our data suggest that SCFAs can regulate the expression of the major negative regulators of TLR signaling in the neonatal intestine through Notch1 and ameliorate experimental NEC. Enteral SCFAs supplementation in preterm infants provides a promising bacteria-free, therapeutic option for NEC.NEW & NOTEWORTHY Short-chain fatty acids (SCFAs), such as propionate and butyrate, metabolites produced by symbiotic gut bacteria are known to be anti-inflammatory, but the mechanisms by which they protect against NEC are not fully understood. In this study, we reveal that SCFAs regulate intestinal inflammation by inducing the key TLR and IL1R inhibitors, SIGIRR and A20, through activation of the pluripotent transcriptional factor NOTCH1. Butyrate-mediated SIGIRR and A20 induction represses experimental NEC in the neonatal intestine.

Effect of Various Preterm Infant Milk Formulas on NEC-Like Gut Injury in Mice.

Formula feeding is an important risk factor for the development of necrotizing enterocolitis in preterm infants. The potential harmful effects of different preterm formulas on the developing intestinal tract remain incompletely understood. Here we demonstrate that feeding newborn mouse pups with various preterm formulas resulted in differing effects on intestinal inflammation, apoptosis, and activation of the pro-inflammatory transcription factor NFκB. 16S rRNA sequencing revealed that each preterm formula resulted in significant gut microbial alterations that were different from dam-fed controls. Formula feeding with EleCare and Similac Special Care caused greater intestinal injury compared to NeoSure. Pre-treatment with Lactobacillus rhamnosus GG ameliorated severity of intestinal injury from EleCare and Similac Special Care. Our findings indicate that not all preterm formulas are the same, and different formulations can have varying effects on intestinal inflammation, apoptosis, and microbiome composition.

EPHB4 Mutation Suppresses PROX1 Expression and Disrupts Lymphatic Development in Neonatal Hydrops.

This case report highlights the importance of screening for mutations in EPHB4 and other genes that regulate lymphatic development in infants with the nonimmune hydrops fetalis.

Angiopoietin-1 protects against endotoxin-induced neonatal lung injury and alveolar simplification in mice.

BACKGROUND: Sepsis in premature newborns is a risk factor for bronchopulmonary dysplasia (BPD), but underlying mechanisms of lung injury remain unclear. Aberrant expression of endothelial cell (EC) angiopoietin 2 (ANGPT2) disrupts angiopoietin 1 (ANGPT1)/TIE2-mediated endothelial quiescence, and is implicated in sepsis-induced acute respiratory distress syndrome in adults. We hypothesized that recombinant ANGPT1 will mitigate sepsis-induced ANGPT2 expression, inflammation, acute lung injury (ALI), and alveolar remodeling in the saccular lung. METHODS: Effects of recombinant ANGPT1 on lipopolysaccharide (LPS)-induced endothelial inflammation were evaluated in human pulmonary microvascular endothelial cells (HPMEC). ALI and long-term alveolar remodeling were assessed in newborn mice exposed to intraperitoneal LPS and recombinant ANGPT1 pretreatment. RESULTS: LPS dephosphorylated EC TIE2 in association with increased ANGPT2 in vivo and in vitro. ANGPT1 suppressed LPS and ANGPT2-induced EC inflammation in HPMEC. Neonatal mice treated with LPS had increased lung cytokine expression, neutrophilic influx, and cellular apoptosis. ANGPT1 pre-treatment suppressed LPS-induced lung Toll-like receptor signaling, inflammation, and ALI. LPS-induced acute increases in metalloproteinase 9 expression and elastic fiber breaks, as well as a long-term decrease in radial alveolar counts, were mitigated by ANGPT1. CONCLUSIONS: In an experimental model of sepsis-induced BPD, ANGPT1 preserved endothelial quiescence, inhibited ALI, and suppressed alveolar simplification. IMPACT: Key message: Angiopoietin 1 inhibits LPS-induced neonatal lung injury and alveolar remodeling. Additions to existing literature: Demonstrates dysregulation of angiopoietin-TIE2 axis is important for sepsis- induced acute lung injury and alveolar simplification in experimental BPD. Establishes recombinant Angiopoietin 1 as an anti-inflammatory therapy in BPD. IMPACT: Angiopoietin 1-based interventions may represent novel therapies for mitigating sepsis-induced lung injury and BPD in premature infants.

SIGIRR Mutation in Human Necrotizing Enterocolitis (NEC) Disrupts STAT3-Dependent microRNA Expression in Neonatal Gut.

BACKGROUND & AIMS: Single immunoglobulin interleukin-1-related receptor (SIGIRR) is a major inhibitor of Toll-like receptor signaling. Our laboratory identified a novel SIGIRR stop mutation (p.Y168X) in an infant who died of severe necrotizing enterocolitis (NEC). Herein, we investigated the mechanisms by which SIGIRR mutations induce Toll-like receptor hyper-responsiveness in the neonatal gut, disrupting postnatal intestinal adaptation. METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was used to generate transgenic mice encoding the SIGIRR p.Y168X mutation. Ileal lysates, mouse intestinal epithelial cell (IEC) lysates, and intestinal sections were used to assess inflammation, signal transducer and activator of transcription 3 (STAT3) phosphorylation, microRNA (miRNA), and interleukin-1-related-associated kinase 1 (IRAK1) expression. Western blot, quantitative reverse-transcription polymerase chain reaction(qRT-PCR), and luciferase assays were performed to investigate SIGIRR-STAT3 signaling in human intestinal epithelial cells (HIEC) expressing wild-type or SIGIRR (p.Y168X) plasmids. RESULTS: SigirrTg mice showed increased intestinal inflammation and nuclear factor-κB activation concomitant with decreased IEC expression of miR-146a and miR-155. Mechanistic studies in HIECs showed that although SIGIRR induced STAT3-mediated expression of miR-146a and miR-155, the p.Y168X mutation disrupted SIGIRR-mediated STAT3-dependent miRNA expression. Chromatin immunoprecipitation and luciferase assays showed that SIGIRR activation of STAT3-induced miRNA expression is dependent on IRAK1. Both in HIECs and in the mouse intestine, decreased expression of miR-146a observed with the p.Y168X mutation increased expression of IRAK1, a protein whose down-regulation is important for postnatal gut adaptation. CONCLUSIONS: Our results uncover a novel pathway (SIGIRR-STAT3-miRNA-IRAK1 repression) by which SIGIRR regulates postnatal intestine adaptation, which is disrupted by a SIGIRR mutation identified in human NEC. These data provide new insights into how human genetic mutations in SIGIRR identified in NEC result in loss of postnatal intestinal immune tolerance.

Ciclesonide activates glucocorticoid signaling in neonatal rat lung but does not trigger adverse effects in the cortex and cerebellum.

Synthetic glucocorticoids (sGCs) such as dexamethasone (DEX), while used to mitigate inflammation and disease progression in premature infants with severe bronchopulmonary dysplasia (BPD), are also associated with significant adverse neurologic effects such as reductions in myelination and abnormalities in neuroanatomical development. Ciclesonide (CIC) is a sGC prodrug approved for asthma treatment that exhibits limited systemic side effects. Carboxylesterases enriched in the lower airways convert CIC to the glucocorticoid receptor (GR) agonist des-CIC. We therefore examined whether CIC would likewise activate GR in neonatal lung but have limited adverse extra-pulmonary effects, particularly in the developing brain. Neonatal rats were administered subcutaneous injections of CIC, DEX or vehicle from postnatal days 1-5 (PND1-PND5). Systemic effects linked to DEX exposure, including reduced body and brain weight, were not observed in CIC treated neonates. Furthermore, CIC did not trigger the long-lasting reduction in myelin basic protein expression in the cerebral cortex nor cerebellar size caused by neonatal DEX exposure. Conversely, DEX and CIC were both effective at inducing the expression of select GR target genes in neonatal lung, including those implicated in lung-protective and anti-inflammatory effects. Thus, CIC is a promising, novel candidate drug to treat or prevent BPD in neonates given its activation of GR in neonatal lung and limited adverse neurodevelopmental effects. Furthermore, since sGCs such as DEX administered to pregnant women in pre-term labor can adversely affect fetal brain development, the neurological-sparing properties of CIC, make it an attractive alternative for DEX to treat pregnant women severely ill with respiratory illness, such as with asthma exacerbations or COVID-19 infections.

FOXC2 Autoregulates Its Expression in the Pulmonary Endothelium After Endotoxin Stimulation in a Histone Acetylation-Dependent Manner.

The innate immune response of pulmonary endothelial cells (EC) to lipopolysaccharide (LPS) induces Forkhead box protein C2 (FOXC2) activation through Toll Like Receptor 4 (TLR4). The mechanisms by which FOXC2 expression is regulated in lung EC under LPS stimulation remain unclear. We postulated that FOXC2 regulates its own expression in sepsis, and its transcriptional autoregulation directs lymphatic EC cell-fate decision. Bioinformatic analysis identified potential FOXC2 binding sites in the FOXC2 promoter. In human lung EC, we verified using chromatin immunoprecipitation (ChIP) and luciferase assays that FOXC2 bound to its own promoter and stimulated its expression after LPS stimulation. Chemical inhibition of histone acetylation by garcinol repressed LPS-induced histone acetylation in the FOXC2 promoter region, and disrupted LPS-mediated FOXC2 binding and transcriptional activation. CRISPR/dCas9/gRNA directed against FOXC2-binding-element (FBE) suppressed LPS-stimulated FOXC2 binding and autoregulation by blocking FBEs in the FOXC2 promoter, and repressed expression of lymphatic EC markers. In a neonatal mouse model of sterile sepsis, LPS-induced FOXC2 binding to FBE and FOXC2 expression in lung EC was attenuated with garcinol treatment. These data reveal a new mechanism of LPS-induced histone acetylation-dependent FOXC2 autoregulation.

Delta-like 4 is required for pulmonary vascular arborization and alveolarization in the developing lung.

The molecular mechanisms by which endothelial cells (ECs) regulate pulmonary vascularization and contribute to alveolar epithelial cell development during lung morphogenesis remain unknown. We tested the hypothesis that delta-like 4 (DLL4), an EC Notch ligand, is critical for alveolarization by combining lung mapping and functional studies in human tissue and DLL4-haploinsufficient mice (Dll4+/lacz). DLL4 expressed in a PECAM-restricted manner in capillaries, arteries, and the alveolar septum from the canalicular to alveolar stage in mice and humans. Dll4 haploinsufficiency resulted in exuberant, nondirectional vascular patterning at E17.5 and P6, followed by smaller capillaries and fewer intermediate blood vessels at P14. Vascular defects coincided with polarization of lung EC expression toward JAG1-NICD-HES1 signature and decreased tip cell-like (Car4) markers. Dll4+/lacZ mice had impaired terminal bronchiole development at the canalicular stage and impaired alveolarization upon lung maturity. We discovered that alveolar type I cell (Aqp5) markers progressively decreased in Dll4+/lacZ mice after birth. Moreover, in human lung EC, DLL4 deficiency programmed a hypersprouting angiogenic phenotype cell autonomously. In conclusion, DLL4 is expressed from the canalicular to alveolar stage in mice and humans, and Dll4 haploinsufficiency programs dysmorphic microvascularization, impairing alveolarization. Our study reveals an obligate role for DLL4-regulated angiogenesis in distal lung morphogenesis.

ITGB2 (Integrin ß2) Immunomodulatory Gene Variants in Premature Infants With Necrotizing Enterocolitis.

ABSTRACT: Aberrant toll-like receptor (TLR) activation is central to necrotizing enterocolitis (NEC) pathogenesis. ß2 integrins regulate TLR signaling, and integrin ß2 (ITGB2) deficiency causes TLR hyperresponsiveness. To test the hypothesis that ITGB2 genetic variants modulate NEC susceptibility, we sequenced the exonic ITGB2 locus to compare the prevalence of deleterious variants among 221 preterm infants with and without NEC. ITGB2 variants were not associated with NEC in our entire cohort (NEC [9/56] versus controls [16/165], P = 0.19) or in extremely low birthweight infants (ELBW, controls [7.9%] versus NEC [18.2%]; P = 0.11) but were increased compared to the populace (4.5%, gnomad.broadinstitute.org). Combined annotation-dependent depletion -predicted deleterious ITGB2 variants increased proportionately with increasing NEC severity in ELBW infants (controls [6.7%] versus medical NEC [16.7%] versus surgical NEC [19%] (P = 0.03). Although ITGB2 variants were not associated with NEC in our preterm cohort, subgroup analysis showed a trend towards enrichment with NEC severity in ELBW infants.

FOSL1 is a novel mediator of endotoxin/lipopolysaccharide-induced pulmonary angiogenic signaling.

Systemic sepsis is a known risk factor for bronchopulmonary dysplasia (BPD) in premature infants, a disease characterized by dysregulated angiogenesis and impaired vascular and alveolar development. We have previoulsy reported that systemic endotoxin dysregulates pulmonary angiogenesis resulting in alveolar simplification mimicking BPD in neonatal mice, but the underlying mechanisms remain unclear. We undertook an unbiased discovery approach to identify novel signaling pathways programming sepsis-induced deviant lung angiogenesis. Pulmonary endothelial cells (EC) were isolated for RNA-Seq from newborn C57BL/6 mice treated with intraperitoneal lipopolysaccharide (LPS) to mimic systemic sepsis. LPS significantly differentially-regulated 269 genes after 6 h, and 1,934 genes after 24 h. Using bioinformatics, we linked 6 h genes previously unknown to be modulated by LPS to 24 h genes known to regulate angiogenesis/vasculogenesis to identify pathways programming deviant angiogenesis. An immortalized primary human lung EC (HPMEC-im) line was generated by SV40 transduction to facilitate mechanistic studies. RT-PCR and transcription factor binding analysis identified FOSL1 (FOS like 1) as a transcriptional regulator of LPS-induced downstream angiogenic or vasculogenic genes. Over-expression and silencing studies of FOSL1 in immortalized and primary HPMEC demonstrated that baseline and LPS-induced expression of ADAM8, CXCR2, HPX, LRG1, PROK2, and RNF213 was regulated by FOSL1. FOSL1 silencing impaired LPS-induced in vitro HPMEC angiogenesis. In conclusion, we identified FOSL1 as a novel regulator of sepsis-induced deviant angiogenic signaling in mouse lung EC and human fetal HPMEC.

NEC-like intestinal injury is ameliorated by Lactobacillus rhamnosus GG in parallel with SIGIRR and A20 induction in neonatal mice.

BACKGROUND: Exaggerated Toll-like receptor (TLR) signaling and intestinal dysbiosis are key contributors to necrotizing enterocolitis (NEC). Lactobacillus rhamnosus GG (LGG) decreases NEC in preterm infants, but underlying mechanisms of protection remain poorly understood. We hypothesized that LGG alleviates dysbiosis and upregulates TLR inhibitors to protect against TLR-mediated gut injury. METHODS: Effects of LGG (low- and high-dose) on intestinal pro-inflammatory TLR signaling and injury in neonatal mice subjected to formula feeding (FF) and NEC were determined. 16S sequencing of stool and expression of anti-TLR mediators SIGIRR (single immunoglobulin interleukin-1-related receptor) and A20 were analyzed. RESULTS: FF induced mild intestinal injury with increased expression of interleukin-1ß (IL-1ß) and Kupffer cell (KC) (mouse homolog of IL-8) compared to controls. LGG decreased IL-1ß and KC in association with attenuated TLR signaling and increased SIGIRR and A20 expression in a dose-dependent manner. Low- and high-dose LGG had varying effects on gut microbiome despite both doses providing gut protection. Subsequent experiments of LGG on NEC revealed that pro-inflammatory TLR signaling and intestinal injury were also decreased, and SIGIRR and A20 expression increased, in a dose-dependent manner with LGG pre-treatment. CONCLUSIONS: LGG protects against intestinal TLR-mediated injury by upregulating TLR inhibitors without major changes in gut microbiome composition.

Histone deacetylase 6 regulates endothelial MyD88-dependent canonical TLR signaling, lung inflammation, and alveolar remodeling in the developing lung.

Lung endothelial cell (EC) immune activation during bacterial sepsis contributes to acute lung injury and bronchopulmonary dysplasia in premature infants. The epigenetic regulators of sepsis-induced endothelial immune activation, lung inflammation, and alveolar remodeling remain unclear. Herein, we examined the role of the cytoplasmic histone deacetylase, HDAC6, in regulating EC Toll-like receptor 4 (TLR4) signaling and modulating sepsis-induced lung injury in a neonatal model of sterile sepsis. In human primary microvascular endothelial cells (HPMEC), lipopolysaccharide (LPS)-induced MAPK, IKK-ß, and p65 phosphorylation as well as inflammatory cytokine expression were exaggerated with the HDAC6 inhibitor tubastatin A, and by dominant-negative HDAC6 with a mutated catalytic domain 2. Expression of HDAC6 wild-type protein suppressed LPS-induced myeloid differentiation primary response 88 (MyD88) acetylation, p65 (Lys310) acetylation, MyD88/TNF receptor-associated factor 6 (TRAF6) coimmunoprecipitation, and proinflammatory TLR4 signaling in HPMEC. In a neonatal mouse model of sepsis, the HDAC6 inhibitor tubastatin A amplified lung EC TLR4 signaling and vascular permeability. HDAC6 inhibition augmented LPS-induced MyD88 acetylation, MyD88/TRAF6 binding, p65 acetylation, canonical TLR4 signaling, and inflammation in the developing lung. Sepsis-induced decreases in the fibroblast growth factors FGF2 and FGF7 and increase in matrix metalloproteinase-9 were worsened with HDAC6 inhibition, while elastin expression was equally suppressed. Exaggerated sepsis-induced acute lung inflammation observed with HDAC6 inhibition worsened alveolar simplification evidenced by increases in mean linear intercepts and decreased radial alveolar counts. Our studies reveal that HDAC6 is a constitutive negative regulator of cytoplasmic TLR4 signaling in EC and the developing lung. The therapeutic efficacy of augmenting HDAC6 activity in neonatal sepsis to prevent lung injury needs to be evaluated.