Retraction Note: Intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity in neonatal rats.

This paper has been retracted at the request of the authors.

Intermittent hypoxia during recovery from neonatal hyperoxic lung injury causes long-term impairment of alveolar development: A new rat model of BPD.

Bronchopulmonary dysplasia (BPD) is a chronic lung injury characterized by impaired alveologenesis that may persist into adulthood. Rat models of BPD using varying degrees of hyperoxia to produce injury either cause early mortality or spontaneously recover following removal of the inciting stimulus, thus limiting clinical relevance. We sought to refine an established rat model induced by exposure to 60% O2 from birth by following hyperoxia with intermittent hypoxia (IH). Rats exposed from birth to air or 60% O2 until day 14 were recovered in air with or without IH (FIO2 = 0.10 for 10 min every 6 h) until day 28 Animals exposed to 60% O2 and recovered in air had no evidence of abnormal lung morphology on day 28 or at 10-12 wk. In contrast, 60% O2-exposed animals recovered in IH had persistently increased mean chord length, more dysmorphic septal crests, and fewer peripheral arteries. Recovery in IH also increased pulmonary vascular resistance, Fulton index, and arterial wall thickness. IH-mediated abnormalities in lung structure (but not pulmonary hypertension) persisted when reexamined at 10-12 wk, accompanied by increased pulmonary vascular reactivity and decreased exercise tolerance. Increased mean chord length secondary to IH was prevented by treatment with a peroxynitrite decomposition catalyst [5,10,15,20-Tetrakis(4-sulfonatophenyl)-21H,23H-porphyrin iron (III) chloride, 30 mg/kg/day, days 14-28], an effect accompanied by fewer inflammatory cells. We conclude that IH during recovery from hyperoxia-induced injury prevents recovery of alveologenesis and leads to changes in lung and pulmonary vascular function lasting into adulthood, thus more closely mimicking contemporary BPD.

Early-Life Intranasal Colonization with Nontypeable Haemophilus influenzae Exacerbates Juvenile Airway Disease in Mice.

Accumulating evidence suggests a connection between asthma development and colonization with nontypeable Haemophilus influenzae (NTHi). Specifically, nasopharyngeal colonization of human infants with NTHi within 4 weeks of birth is associated with an increased risk of asthma development later in childhood. Monocytes derived from these infants have aberrant inflammatory responses to common upper respiratory bacterial antigens compared to those of cells derived from infants who were not colonized and do not go on to develop asthma symptoms in childhood. In this study, we hypothesized that early-life colonization with NTHi promotes immune system reprogramming and the development of atypical inflammatory responses. To address this hypothesis in a highly controlled model, we tested whether colonization of mice with NTHi on day of life 3 induced or exacerbated juvenile airway disease using an ovalbumin (OVA) allergy model of asthma. We found that animals that were colonized on day of life 3 and subjected to induction of allergy had exacerbated airway disease as juveniles, in which exacerbated airway disease was defined as increased cellular infiltration into the lung, increased amounts of inflammatory cytokines interleukin-5 (IL-5) and IL-13 in lung lavage fluid, decreased regulatory T cell-associated FOXP3 gene expression, and increased mucus production. We also found that colonization with NTHi amplified airway resistance in response to increasing doses of a bronchoconstrictor following OVA immunization and challenge. Together, the murine model provides evidence for early-life immune programming that precedes the development of juvenile airway disease and corroborates observations that have been made in human children.

Boronic acid-containing CXCR1/2 antagonists: Optimization of metabolic stability, in vivo evaluation, and a proposed receptor binding model.

Blockade of undesired neutrophil migration to sites of inflammation remains an area of substantial pharmaceutical interest. To effect this blockade, a validated therapeutic target is antagonism of the chemokine receptor CXCR2. Herein we report the discovery of 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide 6, an antagonist with activity at both CXCR1 and CXCR2 receptors (IC50 values 31 and 21 nM, respectively). Compound 6 exhibited potent inhibition of neutrophil influx in a rat model of pulmonary inflammation, and is hypothesized to interact with a unique intracellular binding site on CXCR2. Compound 6 (SX-576) is undergoing further investigation as a potential therapy for pulmonary inflammation.

Boronic acid-containing aminopyridine- and aminopyrimidinecarboxamide CXCR1/2 antagonists: Optimization of aqueous solubility and oral bioavailability.

The chemokine receptors CXCR1 and CXCR2 are important pharmaceutical targets due to their key roles in inflammatory diseases and cancer progression. We have previously identified 2-[5-(4-fluoro-phenylcarbamoyl)-pyridin-2-ylsulfanylmethyl]-phenylboronic acid (SX-517) and 6-(2-boronic acid-5-trifluoromethoxy-benzylsulfanyl)-N-(4-fluoro-phenyl)-nicotinamide (SX-576) as potent non-competitive boronic acid-containing CXCR1/2 antagonists. Herein we report the synthesis and evaluation of aminopyridine and aminopyrimidine analogs of SX-517 and SX-576, identifying (2-{(benzyl)[(5-boronic acid-2-pyridyl)methyl]amino}-5-pyrimidinyl)(4-fluorophenylamino)formaldehyde as a potent chemokine antagonist with improved aqueous solubility and oral bioavailability.

Prenatal air pollution exposure induces sexually dimorphic fetal programming of metabolic and neuroinflammatory outcomes in adult offspring.

Environmental chemical exposures during critical windows of development may contribute to the escalating prevalence of obesity. We tested the hypothesis that prenatal exposure to diesel exhaust particles (DEP), a primary component of air pollution, would prime microglia long-term, resulting in exacerbated metabolic and affective outcomes following exposure to a high-fat diet in adulthood. Time-mated mouse dams were intermittently exposed to respiratory instillations of either vehicle (VEH) or DEP throughout gestation. Adult male and female offspring were then fed either a low-fat diet (LFD) or high-fat diet (HFD) for 9 weeks. The male offspring of DEP-exposed dams exhibited exaggerated weight gain, insulin resistance, and anxiety-like behavior on HFD compared to the male offspring of VEH-exposed dams, whereas female offspring did not differ according to prenatal treatment. Furthermore, HFD induced evidence of macrophage infiltration of both adipose tissue and the brain in both sexes, but these cells were more activated specifically in DEP/HFD males. DEP/HFD males also expressed markedly higher levels of microglial/macrophage, but not astrocyte, activation markers in the hippocampus, whereas females exhibited only a suppression of astrocyte activation markers due to HFD. In a second experiment, DEP male offspring mounted an exaggerated peripheral IL-1ß response to an LPS challenge at postnatal day (P)30, whereas their central IL-1ß response did not differ from VEH male offspring, which is suggestive of macrophage priming due to prenatal DEP exposure. In sum, prenatal air pollution exposure "programs" offspring for increased susceptibility to diet-induced metabolic, behavioral, and neuroinflammatory changes in adulthood in a sexually dimorphic manner.

Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo.

BACKGROUND: Increased adhesivity of red blood cells (RBCs) to endothelial cells (ECs) may contribute to organ dysfunction in malaria, sickle cell disease, and diabetes. RBCs normally export nitric oxide (NO)-derived vascular signals, facilitating blood flow. S-nitrosothiols (SNOs) are thiol adducts formed in RBCs from precursor NO upon the oxygenation-linked allosteric transition in hemoglobin. RBCs export these vasoregulatory SNOs on demand, thereby regulating regional blood flow and preventing RBC-EC adhesion, and the large (system L) neutral amino acid transporter 1 (LAT1; SLC7A5) appears to mediate SNO export by RBCs. METHODS: To determine the role of LAT1-mediated SNO import by ECs generally and of LAT1-mediated SNO import by ECs in RBC SNO-dependent modulation of RBC sequestration and blood oxygenation in vivo, we engineered LAT1fl/fl; Cdh5-Cre+ mice, in which the putative SNO transporter LAT1 can be inducibly depleted (knocked down, KD) specifically in ECs ("LAT1ECKD"). RESULTS: We show that LAT1 in mouse lung ECs mediates cellular SNO uptake. ECs from LAT1ECKD mice (tamoxifen-induced LAT1fl/fl; Cdh5-Cre+) import SNOs poorly ex vivo compared with ECs from wild-type (tamoxifen-treated LAT1fl/fl; Cdh5-Cre-) mice. In vivo, endothelial depletion of LAT1 increased RBC sequestration in the lung and decreased blood oxygenation after RBC transfusion. CONCLUSION: This is the first study showing a role for SNO transport by LAT1 in ECs in a genetic mouse model. We provide the first direct evidence for the coordination of RBC SNO export with EC SNO import via LAT1. SNO flux via LAT1 modulates RBC-EC sequestration in lungs after transfusion, and its disruption impairs blood oxygenation by the lung.

S-nitrosothiol transport via PEPT2 mediates biological effects of nitric oxide gas exposure in macrophages.

The pharmacological effects of nitric oxide (NO) administered as a gas are dependent on the conversion to S-nitrosocysteine, and as such are largely mediated by the L-type amino-acid transporters (LATs) in several cell types. The dipeptide transporter PEPT2 has been proposed as a second route for S-nitrosothiol (SNO) transport, but this has never been demonstrated. Because NO governs important immune functions in alveolar macrophages, we exposed rat alveolar macrophages (primary and NR8383 cells) to NO gas at the air-liquid interface ± LPS stimulation in the presence of PEPT2 substrate Cys-Gly (or the LAT substrate L-Cys) ± transporter competitors. We found that SNO uptake and NO-dependent actions, such as the activation of soluble guanylyl cyclase (sGC), the augmentation of sGC-dependent filamentous actin (F-actin) polymerization, phagocytosis, and the inhibition of NF-κB activation, were significantly augmented by the addition of Cys-Gly in a manner dependent on PEPT2 transport. We found parallel (and greater) effects that were dependent on LAT transport. The contribution of cystine/cysteine shuttling via system x cystine transporter (xCT) to SNO uptake was relatively minor. The observed effects were unaffected by NO synthase inhibition. The NO gas treatment of alveolar macrophages increased SNO uptake, the activation of sGC, F-actin polymerization, and phagocytosis, and inhibited NF-κB activation, in a manner dependent on SNO transport via PEPT2, as well as via LAT.

Prenatal air pollution exposure induces neuroinflammation and predisposes offspring to weight gain in adulthood in a sex-specific manner.

Emerging evidence suggests environmental chemical exposures during critical windows of development may contribute to the escalating prevalence of obesity. We tested the hypothesis that prenatal air pollution exposure would predispose the offspring to weight gain in adulthood. Pregnant mice were exposed to filtered air (FA) or diesel exhaust (DE) on embryonic days (E) 9-17. Prenatal DE induced a significant fetal brain cytokine response at E18 (46-390% over FA). As adults, offspring were fed either a low-fat diet (LFD) or high-fat diet (HFD) for 6 wk. Adult DE male offspring weighed 12% more and were 35% less active than FA male offspring at baseline, whereas there were no differences in females. Following HFD, DE males gained weight at the same rate as FA males, whereas DE females gained 340% more weight than FA females. DE-HFD males had 450% higher endpoint insulin levels than FA-HFD males, and all males on HFD showed decreased activity and increased anxiety, whereas females showed no differences. Finally, both DE males and females fed HFD showed increased microglial activation (30-66%) within several brain regions. Thus, prenatal air pollution exposure can "program" offspring for increased susceptibility to diet-induced weight gain and neuroinflammation in adulthood in a sex-specific manner.

Intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity in neonatal rats.

BACKGROUND: We previously showed that intra-amniotic lipopolysaccharide (LPS) amplifies alveolar hypoplasia induced by postnatal hyperoxia. We determined whether the priming effect of intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity (AHR). METHODS: LPS or normal saline was injected into the amniotic cavities of pregnant rats at the 20th day of gestation. After birth, rat pups were exposed to 60% O2 or air for 14 d. On postnatal day 14, rat pups underwent forced oscillometry, which included a challenge with nebulized methacholine, and the lungs were harvested for morphological studies. RESULTS: Hyperoxia significantly increased airway reactivity and decreased compliance. Intra-amniotic LPS further increased hyperoxia-induced AHR but did not further impair respiratory system compliance. Hyperoxia-induced changes in lung parenchymal and small airway morphology were not further altered by intra-amniotic LPS. However, combined exposure to intra-amniotic LPS and hyperoxia increased the proportion of degranulating mast cells in the hilar airways. CONCLUSION: Intra-amniotic LPS amplified postnatal hyperoxia-induced AHR. This was associated with increased airway mast cell degranulation, which has previously been linked with hyperoxia-induced AHR. There were no morphologic changes of parenchyma or airways that would account for the LPS augmentation of hyperoxia-induced AHR.

2022 Year in review: Neonatal pulmonology.

This review outlines some of the major contributions to Neonatal Pulmonology published in 2022 in Pediatric Pulmonology in the areas of lung ultrasound, prevention and treatment of bronchopulmonary dysplasia, and pulmonary function outcomes of neonatal lung disease.

Maternal diesel inhalation increases airway hyperreactivity in ozone-exposed offspring.

Air pollutant exposure is linked with childhood asthma incidence and exacerbations, and maternal exposure to airborne pollutants during pregnancy increases airway hyperreactivity (AHR) in offspring. To determine if exposure to diesel exhaust (DE) during pregnancy worsened postnatal ozone-induced AHR, timed pregnant C57BL/6 mice were exposed to DE (0.5 or 2.0 mg/m(3)) 4 hours daily from Gestation Day 9-17, or received twice-weekly oropharyngeal aspirations of the collected DE particles (DEPs). Placentas and fetal lungs were harvested on Gestation Day 18 for cytokine analysis. In other litters, pups born to dams exposed to air or DE, or to dams treated with aspirated diesel particles, were exposed to filtered air or 1 ppm ozone beginning the day after birth, for 3 hours per day, 3 days per week for 4 weeks. Additional pups were monitored after a 4-week recovery period. Diesel inhalation or aspiration during pregnancy increased levels of placental and fetal lung cytokines. There were no significant effects on airway leukocytes, but prenatal diesel augmented ozone-induced elevations of bronchoalveolar lavage cytokines at 4 weeks. Mice born to the high-concentration diesel-exposed dams had worse ozone-induced AHR, which persisted in the 4-week recovery animals. Prenatal diesel exposure combined with postnatal ozone exposure also worsened secondary alveolar crest development. We conclude that maternal inhalation of DE in pregnancy provokes a fetal inflammatory response that, combined with postnatal ozone exposure, impairs alveolar development, and causes a more severe and long-lasting AHR to ozone exposure.

Transpulmonary flux of S-nitrosothiols and pulmonary vasodilation during nitric oxide inhalation: role of transport.

Inhaled nitric oxide (iNO) is used to treat pulmonary hypertension and is being investigated for prevention of bronchopulmonary dysplasia in neonates. Extrapulmonary effects of iNO are widely recognized, but the underlying chemistry and pharmacology are poorly understood. Growing evidence suggests that, in addition to acting via diffusion, NO can be converted into nitrosants capable of reacting with endogenous L-cysteine (L-Cys) in the alveolar lining fluid, forming S-nitrosothiol (SNO)-L-cysteine (CSNO). CSNO can then enter cells via the type L amino acid transporter (LAT). To determine the influence of LAT and supplemental L-Cys on the functional activity of iNO and transpulmonary movement of SNOs or other related species, we exposed C57Bl6 mice to nebulized L-Cys or D-cysteine (D-Cys) and/or LAT competitors. Isolated lungs were then perfused with physiologic buffer while effluent was collected to assay perfusate SNOs. Nebulized L-Cys, but not D-Cys, augmented the iNO-induced increase in circulating SNOs in the effluent without altering iNO-induced pulmonary vasodilation. Addition to the perfusate of either L-leucine (L-Leu) or 2-amino-2-norborane carboxylic acid, two distinct LAT competitors, inhibited appearance in the perfusate of SNOs in L-Cys-exposed lungs; a higher concentration of L-Leu significantly inhibited the iNO-induced pulmonary vasodilation as well as SNO accumulation. We conclude that iNO-induced pulmonary vasodilation and the transpulmonary movement of iNO-derived SNOs are mediated in part by formation of extracellular CSNO, uptake by alveolar epithelial LAT, and/or export by LAT from the pulmonary endothelium into the circulation. Therapies that exploit and optimize LAT-dependent SNO transport might improve the efficacy of and clinical outcomes with NO-based therapy by improving systemic SNO delivery.

Biochemical effects of ozone on asthma during postnatal development.

BACKGROUND: Ozone exposure during early life has the potential to contribute to the development of asthma as well as to exacerbate underlying allergic asthma. SCOPE OF REVIEW: Developmentally regulated aspects of sensitivity to ozone exposure and downstream biochemical and cellular responses. MAJOR CONCLUSIONS: Developmental differences in antioxidant defense responses, respiratory physiology, and vulnerabilities to cellular injury during particular developmental stages all contribute to disparities in the health effects of ozone exposure between children and adults. GENERAL SIGNIFICANCE: Ozone exposure has the capacity to affect multiple aspects of the "effector arc" of airway hyperresponsiveness, ranging from initial epithelial damage and neural excitation to neural reprogramming during infancy. This article is part of a Special Issue entitled: Biochemistry of Asthma.

Pharmacologically augmented S-nitrosylated hemoglobin improves recovery from murine subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: S-nitrosylated hemoglobin (S-nitrosohemoglobin) has been implicated in the delivery of O(2) to tissues through the regulation of microvascular blood flow. This study tested the hypothesis that enhancement of S-nitrosylated hemoglobin by ethyl nitrite inhalation improves outcome after experimental subarachnoid hemorrhage (SAH). METHODS: A preliminary dosing study identified 20 ppm ethyl nitrite as a concentration that produced a 4-fold increase in S-nitrosylated hemoglobin concentration with no increase in methemoglobin. Mice were subjected to endovascular perforation of the right anterior cerebral artery and were treated with 20 ppm ethyl nitrite in air, or air alone for 72 hours, after which neurologic function, cerebral vessel diameter, brain water content, cortical tissue Po(2), and parenchymal red blood cell flow velocity were measured. RESULTS: At 72 hours after hemorrhage, air- and ethyl nitrite-exposed mice had similarly sized blood clots. Ethyl nitrite improved neurologic score and rotarod performance; abated SAH-induced constrictions in the ipsilateral anterior, middle cerebral, and internal carotid arteries; and prevented an increase in ipsilateral brain water content. Ethyl nitrite inhalation increased red blood cell flow velocity and cortical tissue Po(2) in the ipsilateral cortex with no effect on systemic blood pressure. CONCLUSIONS: Targeted S-nitrosylation of hemoglobin improved outcome parameters, including vessel diameter, tissue blood flow, cortical tissue Po(2), and neurologic function in a murine SAH model. Augmenting endogenous Po(2)-dependent delivery of NO bioactivity to selectively dilate the compromised cerebral vasculature has significant clinical potential in the treatment of SAH.

NOS2 regulation of LPS-induced airway inflammation via S-nitrosylation of NF-{kappa}B p65.

Inducible nitric oxide synthase (NOS2) expression is increased in the airway epithelium in acute inflammatory disorders although the physiological impact remains unclear. We have previously shown that NOS2 inhibits NF-κB (p50-p65) activation in respiratory epithelial cells by inducing S-nitrosylation of the p65 monomer (SNO-p65). In addition, we have demonstrated that mouse lung SNO-p65 levels are acutely depleted in a lipopolysaccharide (LPS) model of lung injury and that augmenting SNO-p65 levels before LPS treatment results in decreased airway epithelial NF-κB activation, airway inflammation, and lung injury. We now show that aerosolized LPS induces NOS2 expression in the respiratory epithelium concomitant with an increase in lung SNO-p65 levels and a decrease in airway NF-κB activity. Genetic deletion of NOS2 results in an absence of SNO-p65 formation, persistent NF-κB activity in the respiratory epithelium, and prolonged airway inflammation. These results indicate that a primary function of LPS-induced NOS2 expression in the respiratory epithelium is to modulate the inflammatory response through deactivation of NF-κB via S-nitrosylation of p65, thereby counteracting the initial stimulus-coupled denitrosylation.

The protective effect of overexpression of extracellular superoxide dismutase on nitric oxide bioavailability in the lung after exposure to hyperoxia stress.

The objective of this study was to determine whether overexpression of human extracellular superoxide dismutase (hEC-SOD) can preserve nitric oxide (NO) bioavailability. In vitro studies examined the transient expression of hEC-SOD in mouse epithelial (C10) cells and its effect on extracellular accumulation of NO, intracellular cyclic guanosine monophosphate (cGMP), and nuclear factor kappa B (NF-κB) activation under normal and oxidative stress conditions. In vivo, newborn rabbits were treated with a plasmid containing hEC-SOD cDNA or vehicle plasmid alone, followed by exposure to hyperoxia (Fio2 = 95% for 7 days). A third group was raised under normoxic conditions. cGMP and NF-κB activation were studied. There was significantly higher NO accumulation in cells expressing hEC-SOD exposed to oxidative stress compared with nontransfected cells. Accumulation of cGMP was significantly higher in cells expressing hEC-SOD. Oxidative stress induced NF-κB activation, which was abrogated by hEC-SOD expression. In vivo, there was significantly higher cGMP accumulation in transfected neonatal rabbit lung tissue at 3 and 7 days of hyperoxic exposure. Immunostaining for NF-κB, showed a marked increase in NF-κB concentration in nontreated neonatal rabbit lung tissue compared to transfected neonatal lung with hEC-SOD and the control air group. These results show that transient EC-SOD overexpression maintains NO bioavailability, which directly leads to maintenance of cGMP activity and reduction of NF-κB activation under oxidative stress.

2020 year in review: Neonatal pulmonology.

Pediatric Pulmonology publishes original research, reviews, and case reports related to a wide range of children's respiratory disorders. This review summarizes the past year's publications in the topic area of neonatal pulmonology, in the context of selected literature from other journals relevant to the discipline.

Mast cells mediate hyperoxia-induced airway hyper-reactivity in newborn rats.

Premature infants are at increased risk of developing airway hyper-reactivity (AHR) after oxidative stress and inflammation. Mast cells contribute to AHR partly by mediator release, so we sought to determine whether blocking mast cell degranulation or recruitment prevents hyperoxia-induced AHR, mast cell accumulation, and airway smooth muscle (ASM) changes. Rats were exposed at birth to air or 60% O2 for 14 d, inducing significantly increased AHR in the latter group, induced by nebulized methacholine challenge and measured by forced oscillometry. Daily treatment (postnatal d 1-14) with intraperitoneal cromolyn prevented hyperoxia-induced AHR, as did treatment with imatinib on postnatal d 5-14, compared with vehicle treated controls. Cromolyn prevented mast cell degranulation in the trachea but not hilar airways and blocked mast cell accumulation in the hilar airways. Imatinib treatment completely blocked mast cell accumulation in tracheal/hilar airway tissues. Hyperoxia-induced AHR in neonatal rats is mediated, at least in part, via the mast cell.