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.

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.

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.

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.

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.

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.

Endothelial immune activation programmes cell-fate decisions and angiogenesis by inducing angiogenesis regulator DLL4 through TLR4-ERK-FOXC2 signalling.

KEY POINTS: The mechanisms by which bacteria alter endothelial cell phenotypes and programme inflammatory angiogenesis remain unclear. In lung endothelial cells, we demonstrate that toll-like receptor 4 (TLR4) signalling induces activation of forkhead box protein C2 (FOXC2), a transcriptional factor implicated in lymphangiogenesis and endothelial specification, in an extracellular signal-regulated kinase (ERK)-dependent manner. TLR4-ERK-FOXC2 signalling regulates expression of the Notch ligand DLL4 and signals inflammatory angiogenesis in vivo and in vitro. Our work reveals a novel link between endothelial immune signalling (TLR pathway) and a vascular transcription factor, FOXC2, that regulates embryonic vascular development. This mechanism is likely to be relevant to pathological angiogenesis complicating inflammatory diseases in humans. ABSTRACT: Endothelial cells (ECs) mediate a specific and robust immune response to bacteria in sepsis through the activation of toll-like receptor (TLR) signalling. The mechanisms by which bacterial ligands released during sepsis programme EC specification and altered angiogenesis remain unclear. We postulated that the forkhead box protein C2 (FOXC2) transcriptional factor directs EC cell-fate decisions and angiogenesis during TLR signalling. In human lung ECs, lipopolysaccharide (LPS) induced ERK phosphorylation, FOXC2, and delta-like 4 (DLL4, the master regulator of sprouting angiogenesis expression) in a TLR4-dependent manner. LPS-mediated ERK phosphorylation resulted in FOXC2-ERK protein ligation, ERK-dependent FOXC2 serine and threonine phosphorylation, and subsequent activation of DLL4 gene expression. Chemical inhibition of ERK or ERK-2 dominant negative transfection disrupted LPS-mediated FOXC2 phosphorylation and transcriptional activation of FOXC2. FOXC2-siRNA or ERK-inhibition attenuated LPS-induced DLL4 expression and angiogenic sprouting in vitro. In vivo, intraperitoneal LPS induced ERK and FOXC2 phosphorylation, FOXC2 binding to DLL4 promoter, and FOXC2/DLL4 expression in the lung. ERK-inhibition suppressed LPS-induced FOXC2 phosphorylation, FOXC2-DLL4 promoter binding, and induction of FOXC2 and DLL4 in mouse lung ECs. LPS induced aberrant retinal angiogenesis and DLL4 expression in neonatal mice, which was attenuated with ERK inhibition. FOXC2+/- mice treated with LPS showed a mitigated increase in FOXC2 and DLL4 compared to FOXC2+/+ mice. These data reveal a new mechanism (TLR4-ERK-FOXC2-DLL4) by which sepsis-induced EC TLR signalling programmes EC specification and altered angiogenesis.

Single-Immunoglobulin Interleukin-1-Related Receptor regulates vulnerability to TLR4-mediated necrotizing enterocolitis in a mouse model.

BackgroundThe mechanisms underlying aberrant activation of intestinal Toll-like receptor 4 (TLR4) signaling in necrotizing enterocolitis (NEC) remain unclear. In this study, we examined the role of single-immunoglobulin interleukin-1 receptor-related molecule (SIGIRR), an inhibitor of TLR signaling, in modulating experimental NEC vulnerability in mice.MethodsExperimental NEC was induced in neonatal wild-type and SIGIRR-/- mice using hypoxia, formula-feeding, and lipopolysaccharide administration. Intestinal TLR canonical signaling, inflammation, apoptosis, and severity of experimental NEC were examined at baseline and after NEC induction in mice.ResultsSIGIRR is developmentally regulated in the neonatal intestine with a restricted expression after birth and a gradual increase by day 8. At baseline, breast-fed SIGIRR-/- mouse pups exhibited low-grade inflammation and TLR pathway activation compared with SIGIRR+/+ pups. With experimental NEC, SIGIRR-/- mice had significantly more intestinal interleukin (IL)-1ß, KC (mouse homolog to IL-8), intercellular adhesion molecule-1 (ICAM-1), and interferon-beta (IFN-ß) expression in association with the amplified TLR pathway activation. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, cleaved caspase 3, and severity of intestinal injury with NEC were worse in SIGIRR-/- mice in comparison with SIGIRR+/+ mice.ConclusionSIGIRR is a negative regulator of TLR4 signaling in the developing intestine, and its insufficiency results in native intestinal TLR hyper-responsiveness conducive to the development of severe experimental NEC in mice.

A functional ATG16L1 (T300A) variant is associated with necrotizing enterocolitis in premature infants.

BACKGROUND: The genetic basis of dysfunctional immune responses in necrotizing enterocolitis (NEC) remains unknown. We hypothesized that variants in nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) and autophagy (ATG) genes modulate vulnerability to NEC. METHODS: We genotyped a multi-center cohort of premature infants with and without NEC for NOD1, NOD2, ATG16L1, CARD8, and NLRP3 variants. Chi-square tests and logistic regression were used for statistical analysis. RESULTS: In our primary cohort (n = 1,015), 86 (8.5%) infants developed NEC. The A allele of the ATG16L1 (Thr300Ala) variant was associated with increased NEC (AA vs. AG vs. GG; 11.3 vs. 8.4 vs. 4.8%, P = 0.009). In regression models for NEC that adjusted for epidemiological confounders, GA (P = 0.033) and the AA genotype (P = 0.038) of ATG16L1 variant were associated with NEC. The association between the A allele of the ATG16L1 variant and NEC remained significant among Caucasian infants (P = 0.02). In a replication cohort (n = 259), NEC rates were highest among infants with the AA genotype but did not reach statistical significance. CONCLUSION: We report a novel association between a hypomorphic variant in an autophagy gene (ATG16L1) and NEC in premature infants. Our data suggest that decreased autophagy arising from genetic variants may confer protection against NEC.

Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 Regulates LPS-Induced Inflammation and Alveolar Remodeling in the Developing Lung.

In premature infants, sepsis is associated with alveolar simplification manifesting as bronchopulmonary dysplasia. The redox-dependent mechanisms underlying sepsis-induced inflammation and alveolar remodeling in the immature lung remain unclear. We developed a neonatal mouse model of sepsis-induced lung injury to investigate whether nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) regulates Toll-like receptor (TLR)-mediated inflammation and alveolar remodeling. Six-day-old NOX2+/+ and NOX2-/- mice were injected with intraperitoneal LPS to induce sepsis. Lung inflammation and canonical TLR signaling were assessed 24 hours after LPS. Alveolar development was examined in 15-day-old mice after LPS on Day 6. The in vivo efficacy of a NOX2 inhibitor (NOX2-I) on NOX2 complex assembly and sepsis-induced lung inflammation were examined. Lung cytokine expression and neutrophil influx induced with sepsis in NOX2+/+ mice was decreased by >50% in NOX2-/- mice. LPS-induced TLR4 signaling evident by inhibitor of NF-κB kinase-ß and mitogen-activated protein kinase phosphorylation, and nuclear factor-κB/AP-1 translocation were attenuated in NOX2-/- mice. LPS increased matrix metalloproteinase 9 while decreasing elastin and keratinocyte growth factor levels in NOX2+/+ mice. An LPS-induced increase in matrix metalloproteinase 9 and decrease in fibroblast growth factor 7 and elastin were not evident in NOX2-/- mice. An LPS-induced reduction in radial alveolar counts and increased mean linear intercepts were attenuated in NOX2-/- mice. LPS-induced NOX2 assembly evident by p67phox/gp91phox coimmunoprecipitation was disrupted with NOX2-I. NOX2-I also mitigated LPS-induced cytokine expression, TLR pathway signaling, and alveolar simplification. In a mouse model of neonatal sepsis, NOX2 regulates proinflammatory TLR signaling and alveolar remodeling induced by a single dose of LPS. Our results provide mechanistic insight into the regulation of sepsis-induced alveolar remodeling in the developing lung.

Lipopolysaccharide (LPS)-mediated angiopoietin-2-dependent autocrine angiogenesis is regulated by NADPH oxidase 2 (Nox2) in human pulmonary microvascular endothelial cells.

Sepsis-mediated endothelial Angiopoeitin-2 (Ang2) signaling may contribute to microvascular remodeling in the developing lung. The mechanisms by which bacterial cell wall components such as LPS mediate Ang2 signaling in human pulmonary microvascular endothelial cells (HPMECs) remain understudied. In HPMEC, LPS-induced Ang2, Tie2, and VEGF-A protein expression was preceded by increased superoxide formation. NADPH oxidase 2 (Nox2) inhibition, but not Nox4 or Nox1 inhibition, attenuated LPS-induced superoxide formation and Ang2, Tie2, and VEGF-A expression. Nox2 silencing, but not Nox4 or Nox1 silencing, inhibited LPS-mediated inhibitor of κ-B kinase ß (IKKß) and p38 phosphorylation and nuclear translocation of NF-κB and AP-1. In HPMECs, LPS increased the number of angiogenic tube and network formations in Matrigel by >3-fold. Conditioned media from LPS-treated cells also induced angiogenic tube and network formation in the presence of Toll-like receptor 4 blockade but not in the presence of Ang2 and VEGF blockade. Nox2 inhibition or conditioned media from Nox2-silenced cells attenuated LPS-induced tube and network formation. Ang2 and VEGF-A treatment rescued angiogenesis in Nox2-silenced cells. We propose that Nox2 regulates LPS-mediated Ang2-dependent autocrine angiogenesis in HPMECs through the IKKß/NF-κB and MAPK/AP-1 pathways.

Necrotizing Enterocolitis Is Not Associated With Sequence Variants in Antioxidant Response Genes in Premature Infants.

Reactive oxygen species mediate intestinal injury in necrotizing enterocolitis (NEC), and yet the contribution of antioxidant response (ARE) gene polymorphisms to NEC risk remains unknown. Premature infants recruited in a multicenter study were genotyped for 6 ARE variants. Among 637 infants, 52 had NEC, and 22 developed surgical NEC. Gestational age <28 weeks (P < 0.02) and African American race (P = 0.03) were associated with NEC. The NFE2L2 (rs6721961), SOD2 (rs4880), GSTP1 (rs1695), NQO1 (rs1800566), GCLC (rs17883901), and HMOX1 (rs2071747) variants were not associated with medical or surgical NEC. This study does not support a role for common deleterious ARE variants in NEC.

Chemical-Biological Properties of Zinc Sensors TSQ and Zinquin: Formation of Sensor-Zn-Protein Adducts versus Zn(Sensor)2 Complexes.

Fluorescent zinc sensors are the most commonly used tool to study the intracellular mobile zinc status within cellular systems. Previously, we have shown that the quinoline-based sensors Zinquin and 6-methoxy-8-p-toluenesulfonamido-quinoline (TSQ) predominantly form ternary adducts with members of the Zn-proteome. Here, the chemistries of these sensors are further characterized, including how Zn(sensor)2 complexes may react in an intracellular environment. We demonstrate that these sensors are typically used in higher concentrations than needed to obtain maximum signal. Exposing cells to either Zn(Zinquin)2 or Zn(TSQ)2 resulted in efficient cellular uptake and the formation of sensor-Zn-protein adducts as evidenced by both a fluorescence spectral shift toward that of ternary adducts and the localization of the fluorescence signal within the proteome after gel filtration of cellular lysates. Likewise, reacting Zn(sensor)2 with the Zn-proteome from LLC-PK1 cells resulted in the formation of sensor-Zn-protein ternary adducts that could be inhibited by first saturating the Zn- proteome with excess sensor. Further, a native SDS-PAGE analysis of the Zn-proteome reacted with either the sensor or the Zn(sensor)2 complex revealed that both reactions result in the formation of a similar set of sensor-Zn-protein fluorescent products. The results of this experiment also demonstrated that TSQ and Zinquin react with different members of the Zn-proteome. Reactions with the model apo-Zn-protein bovine serum albumin showed that both Zn(TSQ)2 and Zn(Zinquin)2 reacted to form ternary adducts with its apo-Zn-binding site. Moreover, incubating Zn(sensor)2 complexes with non-zinc binding proteins failed to elicit a spectral shift in the fluorescence spectrum, supporting the premise that blue-shifted emission spectra are due to sensor-Zn-protein ternary adducts. It was concluded that Zn(sensors)2 species do not play a significant role in the overall reaction between these sensors and intact cells. In turn, this study further supports the formation of sensor-Zn-protein adducts as the principal observed fluorescent product during experiments employing these two sensors.

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.

LPS-mediated endothelial activation in pulmonary endothelial cells: role of Nox2-dependent IKK-ß phosphorylation.

Lipopolysaccharide (LPS)-mediated endothelial activation contributes to lung inflammation and alveolar remodeling seen in premature infants with bronchopulmonary dysplasia (BPD). The mechanisms underlying LPS-mediated oxidative stress and proinflammatory signaling in human pulmonary microvascular endothelial cells (HPMEC) remain unclear. We hypothesized that NADPH oxidase (Nox) mediates LPS-induced endothelial activation in HPMEC by regulating phosphorylation of Toll-like receptor (TLR) pathway proteins. LPS-induced expression of intercellular adhesion molecule 1 (ICAM-1) was associated with increased 2-OH-E(+) (marker for superoxide formation) levels and was attenuated by apocynin and the Nox inhibitor, VAS2870. LPS triggered membrane translocation of p67phox, suggesting activation of Nox2. Silencing Nox2, but not Nox4, suppressed LPS-induced ICAM-1 expression in HPMEC. Immunoprecipitation studies showed that inhibitor of κ-B kinase-ß (IKK-ß) serine phosphorylation induced by LPS was inhibited by Nox2 silencing. We examined whether Nox2-dependent, LPS-mediated IKK-ß phosphorylation was regulated by protein phosphatase 2A (PP2A) or TGF-ß associated kinase-1 (TAK1) in HPMEC. LPS increased PP2A activity in HPMEC, and inhibition of PP2A did not alter LPS-mediated ICAM-1 expression but attenuated IKK-ß phosphorylation. TAK1 inhibition decreased LPS-induced ICAM-1 expression in HPMEC, and Nox2 silencing attenuated LPS-mediated TAK1 phosphorylation (Thr184/187). We demonstrate that Nox2 regulates LPS-mediated endothelial activation in pulmonary endothelial cells by modulating phosphorylation of key kinases in the TLR signaling cascade. Our data support a novel mechanism by which Nox-dependent signaling regulates proinflammatory signaling in pulmonary endothelial cells. Inhibition of vascular Nox may potentially limit lung injury and alveolar remodeling caused by infections in BPD.

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.

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.