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.

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.

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.

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.

TRIP-1 via AKT modulation drives lung fibroblast/myofibroblast trans-differentiation.

BACKGROUND: Myofibroblasts are the critical effector cells in the pathogenesis of pulmonary fibrosis which carries a high degree of morbidity and mortality. We have previously identified Type II TGFß receptor interacting protein 1 (TRIP-1), through proteomic analysis, as a key regulator of collagen contraction in primary human lung fibroblasts--a functional characteristic of myofibroblasts, and the last, but critical step in the process of fibrosis. However, whether or not TRIP-1 modulates fibroblast trans-differentiation to myofibroblasts is not known. METHODS: TRIP-1 expression was altered in primary human lung fibroblasts by siRNA and plasmid transfection. Transfected fibroblasts were then analyzed for myofibroblast features and function such as α-SMA expression, collagen contraction ability, and resistance to apoptosis. RESULTS: The down-regulation of TRIP-1 expression in primary human lung fibroblasts induces α-SMA expression and enhances resistance to apoptosis and collagen contraction ability. In contrast, TRIP-1 over-expression inhibits α-SMA expression. Remarkably, the effects of the loss of TRIP-1 are not abrogated by blockage of TGFß ligand activation of the Smad3 pathway or by Smad3 knockdown. Rather, a TRIP-1 mediated enhancement of AKT phosphorylation is the implicated pathway. In TRIP-1 knockdown fibroblasts, AKT inhibition prevents α-SMA induction, and transfection with a constitutively active AKT construct drives collagen contraction and decreases apoptosis. CONCLUSIONS: TRIP-1 regulates fibroblast acquisition of phenotype and function associated with myofibroblasts. The importance of this finding is it suggests TRIP-1 expression could be a potential target in therapeutic strategy aimed against pathological fibrosis.

TRIP-1 regulates TGF-ß1-induced epithelial-mesenchymal transition of human lung epithelial cell line A549.

Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells undergo conversion to a mesenchymal phenotype contributing to wound repair by fibrosis and to cancer cell acquisition of invasive ability. Recently, we showed that type II TGF-ß receptor interacting protein-1 (TRIP-1), a protein identified as a phosphorylation target of the TGF-ß type II receptor kinase and as a functional component of eukaryotic translation initiator factor 3 (eiF3) multiprotein complex, is a novel modulator of fibroblast collagen contraction, an important step in wound repair stimulated by TGF-ß1 action. TGF-ß1 drives EMT, but it is not known whether TRIP-1 expression influences EMT induction. To investigate whether TRIP-1 plays a role in EMT induction we studied the effect of downregulating TRIP-1 expression in the well-characterized A549 model of TGF-ß1 induction of EMT. Here we report that short hairpin RNA (shRNA)-mediated depletion of TRIP-1 gene transcripts in A549 cells promotes EMT as assessed by changes in phenotypic markers, morphology, and migrative ability. Knockdown of TRIP-1 dramatically increased A549 responsiveness to TGF-ß1 induction of EMT. Mechanistically, a pathway involving increased TGF-ß type II receptor level, enhanced Smad3 phosphorylation, and the transcription factor SLUG is implicated. Altogether, the findings point to regulation of endogenous TRIP-1 protein expression as a potential strategy to target EMT, and related invasive behavior, in cancer cells.

Responses of pulmonary platelet-derived growth factor peptides and receptors to hyperoxia and nitric oxide in piglet lungs.

The peptides platelet-derived growth factor-A (PDGF-A) and especially -B have important roles in lung development. The effect of hyperoxic exposure with and without inhaled nitric oxide (iNO) on lung expression of PDGF and its receptors is unknown. We hypothesized that hyperoxia exposure would suppress mRNA expression and protein production of these ligands and their receptors. The addition of iNO to hyperoxia may further aggravate the effects of hyperoxia. Thirteen-day-old piglets were randomized to breathe 1) room air (RA); 2) 0.96 fraction of inspired oxygen (O2), or 3) 0.96 fraction of inspired oxygen plus 50 ppm of NO (O2+NO), for 5 d. Lungs were preserved for mRNA, Western immunoblot, and immunohistochemical analyses for PDGF-A and -B and their receptors PDGFR-alpha and -beta. PDGF-B mRNA expression was greater than that of PDGF-A or PDGFR-alpha and -beta in RA piglet lungs (p<0.05). Hyperoxia with or without iNO reduced lung PDGF-B mRNA and protein expression relative to the RA group lungs (p<0.01). PDGF-B immunostain intensity was significantly increased in the alveolar macrophages, which were present in greater numbers in the hyperoxia-exposed piglet lungs, with or without NO (p<0.01). PDGFR-beta immunostaining was significantly increased in airway epithelial cells in O2- and O2+NO-exposed piglets. PDGF-A and PDGFR-alpha immunostain intensity and distribution pattern were unchanged relative to the RA group. Sublethal hyperoxia decreases PDGF-B mRNA and protein expression but not PDGF-A or their receptors in piglet lungs. iNO neither aggravates nor ameliorates this effect.

Collagen scaffolding during development and its deformation with chronic lung disease.

OBJECTIVE: Infants with chronic lung disease (CLD) have an arrest of primary and secondary septation. We hypothesized that this may be related to damage or abnormal development of lung collagen secondary to positive pressure ventilation. Our aims were to identify the sites and quantity of collagen in control infants 22 to 72 weeks' postconceptional age and compare these with infants with various degrees of severity of CLD. METHODS: The controls were 22 to 42 weeks' gestation (n = 30), received minimal ventilator care, and died within 48 hours of birth, plus 5 term infants who died at 43 to 72 weeks' postconceptional age from nonpulmonary causes. Infants who were 23 to 30 weeks' gestation, were at risk for CLD, and lived 5 to 94 days (n = 33) were separated into 3 groups on the basis of respiratory score (score group; the integrated area under the curve of the average daily fraction of inspired oxygen x mean airway pressure [cm H2O] over the number of days lived). The score groups, <20, 20 to 69, and 70 to 500, related clinically to mild to moderate and severe lung disease. The lungs were tracheally perfused and formalin fixed. Total lung volume was determined by water displacement. The paraffin-embedded lung blocks were sectioned 5 micro m thick, stained with Gomori's reticulum stain, hematoxylin and eosin, and immunohistochemically for collagen IV. The parenchyma was point-counted, and the volume density of collagen was measured. The chord diameter of the peripheral airway saccules and alveoli was measured. Descriptive collagen data were assessed on en face 40- micro m-thick sections through the alveolar or saccular walls on all infants at risk for CLD and in selected controls. RESULTS: In the controls, the volume density of collagen decreased from a maximum of 9% at 22 weeks to 5% at term and 72 weeks. With Scores < or =69, the fraction of collagen was similar to controls, but in infants with scores 70 to 500, it was increased relative to controls. However, when collagen was expressed as the volume density of interstitial tissue, ie, excluding parenchymal air space, it increased from a low of 5% at 22 weeks to 25% at 72 weeks. In infants with scores 70 to 500, 79% of infants had collagens greater than controls. Saccular and alveolar diameter increased from 40 micro m at 23 weeks to 100 microm at 72 weeks. Most infants with severe CLD (scores > or =70) had diameters more than twice that of controls at the same age. The total lung parenchymal collagen had a similar pattern as the volume density of collagen in interstitial tissue, increasing from 0.4 cm3 at 23 weeks to 9.7 cm3 at 72 weeks in the controls. Eighty-five percent of infants with scores 70 to 500 had total parenchymal collagen greater than the 95% confidence interval of the controls. With en face sections, a fine collagen mesh was seen at 23 weeks, which progressively increased in fiber size and quantity until 72 weeks. With severe CLD, the secondary collagen fibers in the saccular wall were thickened, tortuous, and disorganized relative to same-aged controls. Under 30 weeks, in the controls, the interstitium contained a wide, delicate network of interconnected collagen fibers. After positive pressure ventilation, some saccules markedly increased their diameter, which compressed and obliterated the interstitial network. In contrast with severe CLD, the interstitium was wide, with coarse wavy collagen fibers. CONCLUSIONS: Parenchymal collagen increases throughout development. Before 30 weeks, there is a delicate complex interstitial collagen network, which may be important for primary septation and subsequent normal development. Positive pressure ventilation, if excessive, and depending on lung maturity and disease state, over a short time can severely compress the interstitium and damage this collagen network and prevent normal primary septation and arrest or distort future lung development. With severe CLD, distal air space diameter increases. There is a failure of primary and secondary septation, arrested lung development and remodeling, with thickened cnt and remodeling, with thickened collagenous saccular walls, and a wide interstitium with increased quantity and size of collagen fibers that can affect the mechanics of ventilation. We conclude that normal lung development is dependent on a normal interstitium and, perhaps, collagen architecture and that origins of CLD begin early in the course of positive pressure ventilation.