Hemopexin reverses activation of lung eIF2α and decreases mitochondrial injury in chlorine-exposed mice.

We assessed the mechanisms by which nonencapsulated heme, released in the plasma of mice after exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult male and female C57BL/6 mice to Cl2 (500 ppm for 30 min), returned them to room air, and injected them intramuscularly with either human hemopexin (hHPX; 5 µg/g BW in 50-µL saline) or vehicle at 1 h post-exposure. Upon return to room air, Cl2-exposed mice, injected with vehicle, developed respiratory acidosis, increased concentrations of plasma proteins in the alveolar space, lung mitochondrial DNA injury, increased levels of free plasma heme, and major alterations of their lung proteome. hHPX injection mice mitigated the onset and development of lung and mitochondrial injury and the increase of plasma heme, reversed the Cl2-induced changes in 83 of 237 proteins in the lung proteome at 24 h post-exposure, and improved survival at 15 days post-exposure. Systems biology analysis of the lung global proteomics data showed that hHPX reversed changes in a number of key pathways including elF2 signaling, verified by Western blotting measurements. Recombinant human hemopexin, generated in tobacco plants, injected at 1 h post-Cl2 exposure, was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and the therapeutic effects of hemopexin.NEW & NOTEWORTHY Herein, we demonstrate that exposure of mice to chlorine gas causes significant changes in the lung proteome 24 h post-exposure. Systems biology analysis of the proteomic data is consistent with damage to mitochondria and activation of eIF2, the master regulator of transcription and protein translation. Post-exposure injection of hemopexin, which scavenges free heme, attenuated mtDNA injury, eIF2α phosphorylation, decreased lung injury, and increased survival.

The SARS-CoV-2 spike S1 protein induces global proteomic changes in ATII-like rat L2 cells that are attenuated by hyaluronan.

The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants, COVID-19 has led to 651,918,402 confirmed cases and 6,656,601 deaths worldwide (as of December 27, 2022; https://covid19.who.int/). Despite advances in our understanding of COVID-19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in the proteome of alveolar type II (ATII)-like rat L2 cells that lack ACE2 receptors. Systems biology analysis revealed that the S1-induced proteomics changes were associated with three significant network hubs: E2F1, CREB1/RelA, and ROCK2/RhoA. We also found that pretreatment of L2 cells with high molecular weight hyaluronan (HMW-HA) greatly attenuated the S1 effects on the proteome. Western blotting analysis and cell cycle measurements confirmed the S1 upregulation of E2F1 and ROCK2/RhoA in L2 cells and the protective effects of HMW-HA. Taken as a whole, our studies revealed profound and novel biological changes that contribute to our current understanding of both S1 and hyaluronan biology. These data show that the S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV-2-induced cell injury.

Halogen-Induced Chemical Injury to the Mammalian Cardiopulmonary Systems.

The halogens chlorine (Cl2) and bromine (Br2) are highly reactive oxidizing elements with widespread industrial applications and a history of development and use as chemical weapons. When inhaled, depending on the dose and duration of exposure, they cause acute and chronic injury to both the lungs and systemic organs that may result in the development of chronic changes (such as fibrosis) and death from cardiopulmonary failure. A number of conditions, such as viral infections, coexposure to other toxic gases, and pregnancy increase susceptibility to halogens significantly. Herein we review their danger to public health, their mechanisms of action, and the development of pharmacological agents that when administered post-exposure decrease morbidity and mortality.

AICAR decreases acute lung injury by phosphorylating AMPK and upregulating heme oxygenase-1.

AIM: We investigated the mechanisms by which N1-(ß-d-ribofuranosyl)-5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase (AMPK), decreases lung injury and mortality when administered to mice post exposure to bromine gas (Br2). METHODS: We exposed male C57BL/6 mice and heme oxygenase-1 (HO-1)-deficient (HO-1-/-) and corresponding wild-type (WT) littermate mice to Br2 (600 ppm for 45 or 30 min, respectively) in environmental chambers and returned them to room air. AICAR was administered 6 h post exposure (10 mg·kg-1, intraperitoneal). We assessed survival, indices of lung injury, high mobility group box 1 (HMGB1) in the plasma, HO-1 levels in lung tissues and phosphorylation of AMPK and its upstream liver kinase B1 (LKB1). Rat alveolar type II epithelial (L2) cells and human club-like epithelial (H441) cells were also exposed to Br2 (100 ppm for 10 min). After 24 h we measured apoptosis and necrosis, AMPK and LKB1 phosphorylation, and HO-1 expression. RESULTS: There was a marked downregulation of phosphorylated AMPK and LKB1 in lung tissues and in L2 and H441 cells post exposure. AICAR increased survival in C57BL/6 but not in HO-1-/- mice. In WT mice, AICAR decreased lung injury and restored phosphorylated AMPK and phosphorylated LKB1 to control levels and increased HO-1 levels in both lung tissues and cells exposed to Br2. Treatment of L2 and H441 cells with small interfering RNAs against nuclear factor erythroid 2-related factor 2 or HO-1 abrogated the protective effects of AICAR. CONCLUSIONS: Our data indicate that the primary mechanism for the protective action of AICAR in toxic gas injury is the upregulation of lung HO-1 levels.

Halogen gas exposure: toxic effects on the parturient.

The elemental halogens include chlorine, bromine, and phosgene. Halogen gas can be directly weaponized and employed in warfare or terrorism. Industrial stockpiles or halogen transport can provide targets for terrorist attack as well as an origin for accidental release creating a risk for potential mass-casualty incidents. Pregnant and post-partum women represent a substantial and vulnerable subset of the population who may be at particular risk during an attack or accidental exposure. We review the effects of halogen exposure on the parturient with a focus on bromine toxicity. Bromine is the most extensively studied agent in the context of pregnancy and to-date murine models form the basis for the majority of current knowledge. Pregnancy potentiates the acute lung injury after halogen exposure. In addition, halogen exposure precipitates a preeclamptic-like syndrome in mice. This phenotype is characterized by systemic and pulmonary hypertension, endothelial dysfunction, decreased cardiac output, placental injury and fetal growth restriction. This constellation contributes to increased maternal and fetal mortality observed after bromine exposure. Angiogenic imbalance is noted with overexpression of the soluble fms-like tyrosine kinase-1 (sFlt-1) form of the vascular endothelial growth factor receptor 1 reminiscent of human preeclampsia. Additional research is needed to further explore the effect of halogen gas exposure in pregnancy and to develop therapeutic interventions to mitigate risk to this unique population.

Effects of hyperoxia on alveolar and pulmonary vascular development in germ-free mice.

Airway microbial dysbiosis is associated with subsequent bronchopulmonary dysplasia (BPD) development in very preterm infants. However, the relationship of airway microbiome in normal pulmonary development has not been defined. To better understand the role of the airway microbiome, we compared normal and abnormal alveolar and pulmonary vascular development in mice with or without a microbiome. We hypothesized that the lungs of germ-free (GF) mice would have an exaggerated phenotypic response to hyperoxia compared with non-germ-free (NGF) mice. With the use of a novel gnotobiotic hyperoxia chamber, GF and NGF mice were exposed to either normoxia or hyperoxia. Alveolar morphometry, pulmonary mechanics, echocardiograms, inflammatory markers, and measures of pulmonary hypertension were studied. GF and NGF mice in normoxia showed no difference, whereas GF mice in hyperoxia showed protected lung structure and mechanics and decreased markers of inflammation compared with NGF mice. We speculate that an increase in abundance of pathogenic bacteria in NGF mice may play a role in BPD pathogenesis by regulating the proinflammatory signaling and neutrophilic inflammation in lungs. Manipulation of the airway microbiome may be a potential therapeutic intervention in BPD and other lung diseases.

Mitochondrial DNA variation modulates alveolar development in newborn mice exposed to hyperoxia.

Hyperoxia-induced oxidant stress contributes to the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. Mitochondrial functional differences due to mitochondrial DNA (mtDNA) variations are important modifiers of oxidant stress responses. The objective of this study was to determine whether mtDNA variation independently modifies lung development and mechanical dysfunction in newborn mice exposed to hyperoxia. Newborn C57BL6 wild type (C57n/C57mt, C57WT) and C3H/HeN wild type (C3Hn/C3Hmt, C3HWT) mice and novel Mitochondrial-nuclear eXchange (MNX) strains with nuclear DNA (nDNA) from their parent strain and mtDNA from the other-C57MNX (C57n/C3Hmt) and C3HMNX (C3Hn/C57mt)-were exposed to 21% or 85% O2 from birth to postnatal day 14 (P14). Lung mechanics and histopathology were examined on P15. Neonatal mouse lung fibroblast (NMLF) bioenergetics and mitochondrial superoxide (O2-) generation were measured. Pulmonary resistance and mitochondrial O2- generation were increased while alveolarization, compliance, and NMLF basal and maximal oxygen consumption rate were decreased in hyperoxia-exposed C57WT mice (C57n/C57mt) versus C57MNX mice (C57n/C3Hmt) and in hyperoxia-exposed C3HMNX mice (C3Hn/C57mt) versus C3HWT (C3Hn/C3Hmt) mice. Our study suggests that neonatal C57 mtDNA-carrying strains have increased hyperoxia-induced hypoalveolarization, pulmonary mechanical dysfunction, and mitochondrial bioenergetic and redox dysfunction versus C3H mtDNA strains. Therefore, mtDNA haplogroup variation-induced differences in mitochondrial function could modify neonatal alveolar development and BPD susceptibility.

The Effects of Gestational Psychological Stress on Neonatal Mouse Intestinal Development.

BACKGROUND: Psychological stress during pregnancy has been shown to cause subsequent harm to the fetus and newborn. Many studies focus on neurodevelopmental outcomes, but little is known about the effect of gestational stress on intestinal immunity and development. The purpose of this study was to determine the effect of psychological stress during pregnancy on intestinal architecture and growth in newborns. METHODS: Eight-week-old C57BL6 littermates underwent timed breeding. Pregnant dams were subjected to 1 h of daily psychological stress by using a well-established restraint model during days E7-E14. The distal ileum of 2-wk-old offspring of stressed mothers and nonstressed controls was harvested for histologic analysis. Slides were blinded to measure villus height and crypt depth and surface area. Serum was obtained to measure serum corticosterone levels. An explant model was used to measure corticosterone on the intestinal stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and growth factors epidermal growth factor receptor and insulin-like growth factor-1. RESULTS: The villus height, crypt depth, and surface area were significantly decreased in newborn exposed to stress during gestation. In addition, corticosterone levels were elevated in 2-wk-old mice exposed to stress. Real-time polymerase chain reaction revealed that explants exposed to corticosterone had a decrease in LGR5 compared with controls and an increase in epidermal growth factor receptor. CONCLUSIONS: Here, we establish that neonatal mice from mothers that were subjected to psychological stress during pregnancy have significantly shorter villi and crypts compared with controls. In addition, pups from stressed mothers have decreased expression levels of the intestinal stem cell marker LGR5. These findings will aid in determining the effect of gestational psychological stress on intestinal development and stem cell plasticity.

Exposure of neonatal mice to bromine impairs their alveolar development and lung function.

The halogen bromine (Br2) is used extensively in industry and stored and transported in large quantities. Its accidental or malicious release into the atmosphere has resulted in significant casualties. The pathophysiology of Br2-induced lung injury has been studied in adult animals, but the consequences of Br2 exposure to the developing lung are completely unknown. We exposed neonatal mouse littermates on postnatal day 3 (P3) to either Br2 at 400 ppm for 30 min (400/30), to Br2 at 600 ppm for 30 min (600/30), or to room air, then returned them to their dams and observed until P14. Mice exposed to Br2 had decreased survival (S) and had decreased weight (W) at P14 in the 400/30 group (S = 63.5%, W = 6.67 ± 0.08) and in the 600/30 group (S = 36.1%, W = 5.13 ± 0.67) as compared with air breathing mice (S = 100%, W = 7.96 ± 0.30). Alveolar development was impaired, as evidenced by increased mean linear intercept at P14. At P14, Br2 exposed mice also exhibited a decrease of arterial partial pressure of oxygen, decreased quasi-static lung compliance, as well as increased alpha smooth muscle actin mRNA and protein and increased mRNA for IL-1ß, IL-6, CXCL1, and TNFα. Global gene expression, evaluated by RNA sequencing and Ingenuity Pathway Analysis, revealed persistent abnormalities in gene expression profiles at P14 involving pathways of "formation of lung" and "pulmonary development." The data indicate that Br2 inhalation injury early in life results in severe lung developmental consequences, wherein persistent inflammation and global altered developmental gene expression are likely mechanistic contributors.

Aurothioglucose does not improve alveolarization or elicit sustained Nrf2 activation in C57BL/6 models of bronchopulmonary dysplasia.

We previously showed that the thioredoxin reductase-1 (TrxR1) inhibitor aurothioglucose (ATG) improves alveolarization in hyperoxia-exposed newborn C3H/HeN mice. Our data supported a mechanism by which the protective effects of ATG are mediated via sustained nuclear factor E2-related factor 2 (Nrf2) activation in hyperoxia-exposed C3H/HeN mice 72 h after ATG administration. Given that inbred mouse strains have differential sensitivity and endogenous Nrf2 activation by hyperoxia, the present studies utilized two C57BL/6 exposure models to evaluate the effects of ATG on lung development and Nrf2 activation. The first model (0-14 days) was used in our C3H/HeN studies and the 2nd model (4-14 days) is well characterized in C57BL/6 mice. ATG significantly inhibited lung TrxR1 activity in both models; however, there was no effect on parameters of alveolarization in C57BL/6 mice. In sharp contrast to C3H/HeN mice, there was no effect of ATG on pulmonary NADPH quinone oxidoreductase-1 ( Nqo1) and heme oxygenase-1 ( Hmox1) at 72 h in either C57BL/6 model. In conclusion, although ATG inhibited TrxR1 activity in the lungs of newborn C57BL/6 mice, effects on lung development and sustained Nrf2-dependent pulmonary responses were blunted. These findings also highlight the importance of strain-dependent hyperoxic sensitivity in evaluation of potential novel therapies.

Surgical necrotizing enterocolitis in extremely premature neonates is associated with genetic variations in an intergenic region of chromosome 8.

BackgroundTwin studies suggest that genetic factors may account for up to 50% increased risk for necrotizing enterocolitis (NEC), but genome-wide association studies for NEC are lacking.MethodsGenotyping was done on Illumina BeadChip, followed by analysis using PLINK with logistic regression under an additive model.ResultsAmong 751 extremely-low-birth-weight (<1,000 g, >401 g) neonates, 30 had surgical NEC. Two hundred and sixty-one single-nucleotide polymorphisms (SNPs) showed association with NEC at P<0.05, of which 35 were significant at P<10-7. Minor allele(s) in a cluster of SNPs spanning a 43-kb region of chromosome 8 (8q23.3) conferred an odds ratio of 4.72 (95% confidence interval (CI): 2.51-8.88) for elevated risk of NEC. Two smaller clusters on chromosome 14 and chromosome 11 exhibited P values of 10-7-10-8. The chromosome 8 cluster is in an intergenic region between CUB and Sushi multiple domains 3 (-1.43 Mb) and trichorhinophalangeal syndrome I (+542 kb). RNA sequencing in this region identified a potential novel open-reading frame corresponding to a long interspersed element-1 retrotransposable element.ConclusionGenetic variation in an intergenic region of chromosome 8 is associated with increased risk for NEC with a mechanism that is yet to be identified.

Hyperoxia Induces Intracellular Acidification in Neonatal Mouse Lung Fibroblasts: Real-Time Investigation Using Plasmonically Enhanced Raman Spectroscopy.

It is important to understand the molecular mechanisms underlying oxygen toxicity, which contributes to multiple human disorders. The archetype model of oxygen toxicity is neonatal lung injury induced by hyperoxia exposure. Here, we utilized plasmonically enhanced Raman spectroscopy (PERS) in combination with fluorescence and proteomic analysis to provide comprehensive information on hyperoxia-induced biomolecular modifications in neonatal mouse lung fibroblasts (nMLFs). During this study, we made the novel observation that hyperoxia induces intracellular acidification in nMLF, which we probed in real-time using label-free PERS. We found that intracellular acidification induces conformational modifications in proteins followed by significant changes in Raman vibrations corresponding to aromatic amino acids such as phenylalanine and tryptophan as well as cysteine moieties. Hyperoxia-induced intracellular pH changes and subsequent modifications in protein expression and associated post-translational modifications within the cells were further validated by fluorescence and proteomic analysis. These new insights may help identifying unique oxidant stress-induced mechanisms in disease processes and may guide the development of more efficient therapeutic strategies.

Pathogenesis of necrotizing enterocolitis: modeling the innate immune response.

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. The pathophysiology is likely secondary to innate immune responses to intestinal microbiota by the premature infant's intestinal tract, leading to inflammation and injury. This review provides an updated summary of the components of the innate immune system involved in NEC pathogenesis. In addition, we evaluate the animal models that have been used to study NEC with regard to the involvement of innate immune factors and histopathological changes as compared to those seen in infants with NEC. Finally, we discuss new approaches to studying NEC, including mathematical models of intestinal injury and the use of humanized mice.

Platelet-activating factor: a role in preterm delivery and an essential interaction with Toll-like receptor signaling in mice.

Platelet-activating factor (PAF), a potent phospholipid activator of inflammation that signals through its cognate receptor (platelet-activating factor receptor, PTAFR), has been shown to induce preterm delivery in mice. Toll-like receptors (TLRs) are transmembrane receptors that mediate innate immunity. We have shown previously that Escherichia coli-induced preterm delivery in mice requires TLR signaling via the adaptor protein myeloid differentiation primary response gene 88 (MyD88), but not an alternative adaptor, Toll/IL-1 receptor domain-containing adapter protein-inducing interferon-beta (TRIF). In the present work, we analyzed the role of endogenously produced PAF in labor using mice lacking (knockout [KO]) PAF acetylhydrolase (PAF-AH; the key degrading enzyme for PAF). PAF-AH KO mice are more susceptible to E. coli-induced preterm delivery and inflammation than controls. In peritoneal macrophages, the PTAFR agonist carbamyl PAF induces production of inflammatory markers previously demonstrated to be upregulated during bacterially induced labor, including: inducible nitric oxide synthase (Nos2), the chemokine Ccl5 (RANTES), tumor necrosis factor (Tnf), and level of their end-products (NO, CCL5, TNF) in a process dependent upon both IkappaB kinase and calcium/calmodulin-dependent protein kinase II. Interestingly, this induced expression was completely eliminated not only in macrophages deficient in PTAFR, but also in those lacking either TLR4, MyD88, or TRIF. The dependence of PAF effects upon TLR pathways appears to be related to production of PTAFR itself: PAF-induced expression of Ptafr mRNA was eliminated completely in TLR4 KO and partially in MyD88 and TRIF KO macrophages. We conclude that PAF signaling plays an important role in bacterially induced preterm delivery. Furthermore, in addition to its cognate receptor, PAF signaling in peritoneal macrophages requires TLR4, MyD88, and TRIF.

Maternal breast milk transforming growth factor-beta and feeding intolerance in preterm infants.

BACKGROUND: Feeding intolerance (FI) occurs commonly in the neonatal intensive care unit. Breast milk contains a large pool of transforming growth factor-beta (TGF-ß). Few studies describe TGF-ß levels in preterm milk, and the relationship to FI remains unexplored. We measured TGF-ß levels in preterm breast milk to investigate a correlation with FI in preterm infants. METHODS: Prospective observational trial of 100 mother-infant pairs, enrolling infants born below 32 wk gestation and less than 1,500 g, and mothers who planned to provide breast milk. TGF-ß levels were measured using enzyme-linked immunosorbent assay. Infant charts were reviewed for outcomes. RESULTS: TGF-ß declined postnatally, most elevated in colostrum (P < 0.01). TGF-ß2 levels were higher than TGF-ß1 at all time points (P < 0.01). Colostrum TGF-ß levels correlated inversely with birth weight (P < 0.01) and gestational age (P < 0.05). One-week TGF-ß2 levels were reduced in growth-restricted infants with FI (P < 0.01). Of infants with necrotizing enterocolitis (NEC), TGF-ß2 levels appeared to be low, but small sample size precluded meaningful statistical comparisons. CONCLUSION: TGF-ß levels decline temporally in preterm milk. TGF-ß1 colostrum levels correlate inversely with birth weight and gestational age. TGF-ß2 may play a role in FI in growth-restricted infants. The relationship of TGF-ß2 and NEC merits future investigation.

Inflammatory signals that regulate intestinal epithelial renewal, differentiation, migration and cell death: Implications for necrotizing enterocolitis.

Necrotizing enterocolitis is a disease entity with multiple proposed pathways of pathogenesis. Various combinations of these risk factors, perhaps based on genetic predisposition, possibly lead to the mucosal and epithelial injury that is the hallmark of NEC. Intestinal epithelial integrity is controlled by a tightly regulated balance between proliferation and differentiation of epithelium from intestinal epithelial stem cells and cellular loss by apoptosis. various signaling pathways play a key role in creating and maintaining this balance. The aim of this review article is to outline intestinal epithelial barrier development and structure and the impact of these inflammatory signaling and regulatory pathways as they pertain to the pathogenesis of NEC.

Impact of toll-like receptor 4 deficiency on the response to uterine ischemia/reperfusion in mice.

Our objective was to determine the role of toll-like receptor 4 (TLR4) in uterine ischemia/reperfusion (I/R)-induced fetal growth restriction (FGR). Pregnant TLR4-deficient and wild-type mice were subjected to I/R or a sham procedure. Fetal and placental weights were recorded and tissues were collected. Pep-1 (inhibits low-molecular-weight hyaluronan (LMW-HA) binding to TLR4) was used to determine whether LMW-HA-TLR4 interaction has a role in FGR. TLR4-deficient mice exhibited significantly lower baseline fetal weights compared with wild-type mice (P<0.05), along with extensive placental calcification that was not present in wild-type mice. Following I/R, fetal and placental weights were significantly reduced in wild-type (P<0.05) but not in TLR4-deficient mice. However, I/R increased fetal loss (P<0.05) only in TLR4-deficient mice. Corresponding with the reduced fetal weights, uterine myeloperoxidase activity increased in wild-type mice (P<0.001), indicating an inflammatory response, which was absent in TLR4-deficient mice. TLR4 was shown to have a regulatory role for two anti-inflammatory cytokines: interferon-B1 decreased only in wild-type mice (P<0.01) and interleukin-10 increased only in TLR4-deficient mice (P<0.001), in response to I/R. Pep-1 completely prevented I/R-induced FGR (P<0.001), indicating a potential role for the endogenous TLR4 ligand LMW-HA in I/R-induced FGR. In conclusion, uterine I/R in pregnancy produces FGR that is dependent on TLR4 and endogenous ligand(s), including breakdown products of HA. In addition, TLR4 may play a role in preventing pregnancy loss after uterine I/R.

Platelet Activating Factor Activity Modulates Hyperoxic Neonatal Lung Injury Severity.

Hyperoxia-induced inflammation contributes significantly to developmental lung injury and bronchopulmonary dysplasia (BPD) in preterm infants. Platelet activating factor (PAF) is known to be a major driver of inflammation in lung diseases such as asthma and pulmonary fibrosis, but its role in BPD has not been previously investigated. Therefore, to determine whether PAF signaling independently modulates neonatal hyperoxic lung injury and BPD pathogenesis, lung structure was assessed in 14 day-old C57BL/6 wild-type (WT) and PAF receptor knockout (PTAFR KO) mice that were exposed to 21% (normoxia) or 85% O 2 (hyperoxia) from postnatal day 4. Lung morphometry showed that PTAFR KO mice had attenuated hyperoxia-induced alveolar simplification when compared to WT mice. Functional analysis of gene expression data from hyperoxia-exposed vs. normoxia-exposed lungs of WT and PTAFR KO showed that the most upregulated pathways were the hypercytokinemia/hyperchemokinemia pathway in WT mice, NAD signaling pathway in PTAFR KO mice, and agranulocyte adhesion and diapedesis as well as other pro-fibrotic pathways such as tumor microenvironment and oncostatin-M signaling in both mice strains, indicating that PAF signaling may contribute to inflammation but may not be a significant mediator of fibrotic processes during hyperoxic neonatal lung injury. Gene expression analysis also indicated increased expression of pro-inflammatory genes such as CXCL1, CCL2 and IL-6 in the lungs of hyperoxia-exposed WT mice and metabolic regulators such as HMGCS2 and SIRT3 in the lungs of PTAFR KO mice, suggesting that PAF signaling may modulate BPD risk through changes in pulmonary inflammation and/or metabolic reprogramming in preterm infants.

Mitochondrial DNA Variations Modulate Alveolar Epithelial Mitochondrial Function and Oxidative Stress in Newborn Mice Exposed to Hyperoxia.

Oxidative stress is an important contributor to bronchopulmonary dysplasia (BPD), a form of chronic lung disease that is the most common morbidity in very preterm infants. Mitochondrial functional differences due to inherited and acquired mutations influence the pathogenesis of disorders in which oxidative stress plays a critical role. We previously showed using mitochondrial-nuclear exchange (MNX) mice that mitochondrial DNA (mtDNA) variations modulate hyperoxia-induced lung injury severity in a model of BPD. In this study, we studied the effects of mtDNA variations on mitochondrial function including mitophagy in alveolar epithelial cells (AT2) from MNX mice. We also investigated oxidant and inflammatory stress as well as transcriptomic profiles in lung tissue in mice and expression of proteins such as PINK1, Parkin and SIRT3 in infants with BPD. Our results indicate that AT2 from mice with C57 mtDNA had decreased mitochondrial bioenergetic function and inner membrane potential, increased mitochondrial membrane permeability and were exposed to higher levels of oxidant stress during hyperoxia compared to AT2 from mice with C3H mtDNA. Lungs from hyperoxia-exposed mice with C57 mtDNA also had higher levels of pro-inflammatory cytokines compared to lungs from mice with C3H mtDNA. We also noted changes in KEGG pathways related to inflammation, PPAR and glutamatergic signaling, and mitophagy in mice with certain mito-nuclear combinations but not others. Mitophagy was decreased by hyperoxia in all mice strains, but to a greater degree in AT2 and neonatal mice lung fibroblasts from hyperoxia-exposed mice with C57 mtDNA compared to C3H mtDNA. Finally, mtDNA haplogroups vary with ethnicity, and Black infants with BPD had lower levels of PINK1, Parkin and SIRT3 expression in HUVEC at birth and tracheal aspirates at 28 days of life when compared to White infants with BPD. These results indicate that predisposition to neonatal lung injury may be modulated by variations in mtDNA and mito-nuclear interactions need to be investigated to discover novel pathogenic mechanisms for BPD.

Hemopexin Reverses Activation of Lung eIF2a and Decreases Mitochondrial Injury in Chlorine Exposed Mice.

We assessed the mechanisms by which non-encapsulated heme, released in the plasma of mice post exposure to chlorine (Cl 2 ) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult C57BL/6 male and female to Cl 2 (500 ppm for 30 min) in environmental chambers and returned them to room air and injected them intramuscularly with a single dose of human hemopexin (hHPX; 5 µg/ g BW), the most efficient scavenger of heme, 30-60 min post exposure. Concentrations of hHPX in plasma of air and Cl 2 exposed mice were 9081±900 vs. 1879± 293 at 6 h and 2966±463 vs. 1555±250 at 50 h post injection (ng/ml; X±1 SEM=3; p<0.01). Cl 2 exposed mice developed progressive acute lung injury post exposure characterized by increased concentrations of plasma heme, marked inflammatory response, respiratory acidosis and increased concentrations of plasma proteins in the alveolar space. Injection of hHPX decreased the onset of acute lung injury at 24 h post exposure; mean survival, for the saline and hHPX groups were 40 vs. 80% (P<0.001) at 15 d post exposure. Non-supervised global proteomics analysis of mouse lungs at 24 h post exposure, revealed the upregulation of 92 and downregulation of 145 lung proteins. Injection of hHPX at one h post exposure moderated the Cl 2 induced changes in eighty-three of these 237 lung proteins. System biology analysis of the global proteomics data showed that hHPX reversed changes in mitochondrial dysfunction and elF2 and integrin signaling. Western blot analysis of lung tissue showed significant increase of phosphorylated elF2 at 24 h post exposure in vehicle treated mice but normal levels in those injected with hHPX. Similarly, RT-PCR analysis of lung tissue showed that hHPX reversed the onset of mtDNA lesions. A form of recombinant human hemopexin generated in tobacco plants was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl 2 results in acute lung injury and to the therapeutic effects of hemopexin.