IL-1/inhibitory κB kinase ε-induced glycolysis augment epithelial effector function and promote allergic airways disease.

BACKGROUND: Emerging studies suggest that enhanced glycolysis accompanies inflammatory responses. Virtually nothing is known about the relevance of glycolysis in patients with allergic asthma. OBJECTIVES: We sought to determine whether glycolysis is altered in patients with allergic asthma and to address its importance in the pathogenesis of allergic asthma. METHODS: We examined alterations in glycolysis in sputum samples from asthmatic patients and primary human nasal cells and used murine models of allergic asthma, as well as primary mouse tracheal epithelial cells, to evaluate the relevance of glycolysis. RESULTS: In a murine model of allergic asthma, glycolysis was induced in the lungs in an IL-1-dependent manner. Furthermore, administration of IL-1ß into the airways stimulated lactate production and expression of glycolytic enzymes, with notable expression of lactate dehydrogenase A occurring in the airway epithelium. Indeed, exposure of mouse tracheal epithelial cells to IL-1ß or IL-1α resulted in increased glycolytic flux, glucose use, expression of glycolysis genes, and lactate production. Enhanced glycolysis was required for IL-1ß- or IL-1α-mediated proinflammatory responses and the stimulatory effects of IL-1ß on house dust mite (HDM)-induced release of thymic stromal lymphopoietin and GM-CSF from tracheal epithelial cells. Inhibitor of κB kinase ε was downstream of HDM or IL-1ß and required for HDM-induced glycolysis and pathogenesis of allergic airways disease. Small interfering RNA ablation of lactate dehydrogenase A attenuated HDM-induced increases in lactate levels and attenuated HDM-induced disease. Primary nasal epithelial cells from asthmatic patients intrinsically produced more lactate compared with cells from healthy subjects. Lactate content was significantly higher in sputum supernatants from asthmatic patients, notably those with greater than 61% neutrophils. A positive correlation was observed between sputum lactate and IL-1ß levels, and lactate content correlated negatively with lung function. CONCLUSIONS: Collectively, these findings demonstrate that IL-1ß/inhibitory κB kinase ε signaling plays an important role in HDM-induced glycolysis and pathogenesis of allergic airways disease.

Arousal from sleep in response to intermittent hypoxia in rat pups is modulated by medullary raphe GABAergic mechanisms.

Arousal is an important defense against hypoxia during sleep. Rat pups exhibit progressive arousal impairment (habituation) with multiple hypoxia exposures. The mechanisms are unknown. The medullary raphe (MR) is involved in autonomic functions, including sleep, and receives abundant GABAergic inputs. We hypothesized that inhibiting MR neurons with muscimol, a GABA(A) receptor agonist, or preventing GABA reuptake with nipecotic acid, would impair arousal and enhance arousal habituation and that blocking GABA(A) receptors with bicuculline would enhance arousal and attenuate habituation. Postnatal day 15 (P15) to P25 rat pups were briefly anesthetized, and microinjections with aCSF, muscimol, bicuculline, or nipecotic acid were made into the MR. After a ∼30-min recovery, pups were exposed to four 3-min episodes of hypoxia separated by 6 min of normoxia. The time to arousal from the onset of hypoxia (latency) was determined for each trial. Latency progressively increased across trials (habituation) in all groups. The overall latency was greater after muscimol and nipecotic acid compared with aCSF, bicuculline, or noninjected controls. Arousal habituation was reduced after bicuculline compared with aCSF, muscimol, nipecotic acid, or noninjected pups. Increases in latency were mirrored by decreases in chamber [O2] and oxyhemoglobin saturation. Heart rate increased during hypoxia and was greatest in muscimol-injected pups. Our results indicate that the MR plays an important, not previously described, role in arousal and arousal habituation during hypoxia and that these phenomena are modulated by GABAergic mechanisms. Arousal habituation may contribute to sudden infant death syndrome, which is associated with MR serotonergic and GABAergic receptor dysfunction.

Eliminating medullary 5-HT neurons delays arousal and decreases the respiratory response to repeated episodes of hypoxia in neonatal rat pups.

Arousal from sleep is a critical defense mechanism when infants are exposed to hypoxia, and an arousal deficit has been postulated as contributing to the etiology of the sudden infant death syndrome (SIDS). The brainstems of SIDS infants are deficient in serotonin (5-HT) and tryptophan hydroxylase (TPH) and have decreased binding to 5-HT receptors. This study explores a possible connection between medullary 5-HT neuronal activity and arousal from sleep in response to hypoxia. Medullary raphe 5-HT neurons were eliminated from neonatal rat pups with intracisterna magna (CM) injections of 5,7-dihydroxytryptamine (DHT) at P2-P3. Each pup was then exposed to four episodes of hypoxia during sleep at three developmental ages (P5, P15, and P25) to produce an arousal response. Arousal, heart rate, and respiratory rate responses of DHT-injected pups were compared with pups that received CM artificial cerebrospinal fluid (aCSF) and those that received DHT but did not have a significant reduction in medullary 5-HT neurons. During each hypoxia exposure, the time to arousal from the onset of hypoxia (latency) was measured together with continuous measurements of heart and respiratory rates, oxyhemoglobin saturation, and chamber oxygen concentration. DHT-injected pups with significant losses of medullary 5-HT neurons exhibited significantly longer arousal latencies and decreased respiratory rate responses to hypoxia compared with controls. These results support the hypothesis that in newborn and young rat pups, 5-HT neurons located in the medullary raphe contribute to the arousal response to hypoxia. Thus alterations medullary 5-HT mechanisms might contribute to an arousal deficit and contribute to death in SIDS infants.

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells.

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKß), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKß and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKß. SiRNA-mediated knockdown of GSTP decreased IKKß-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKß-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor.

Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione-S-transferase π.

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-S-transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFß-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp-/- mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFß-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF.

Impaired arousal in rat pups with prenatal alcohol exposure is modulated by GABAergic mechanisms.

Prenatal alcohol exposure (PAE) increases the risk for The Sudden Infant Death Syndrome (SIDS) in human infants. In rat pups, the arousal response to hypoxia is modulated by medullary raphe GABAergic mechanisms. We hypothesized that arousal to hypoxia is impaired by PAE, and is associated with an increase in medullary GABA and enhanced GABAergic activity. Pregnant dams received an ethanol liquid diet (ETOH), an iso-caloric pair fed diet (PF) or a standard chow diet (CHOW). We first measured the time to arousal (latency), during four episodes of hypoxia in P5, P15, and P21 CHOW, PF, and ETOH pups. We also measured brainstem GABA concentration in the same groups of pups. Finally, we injected artificial cerebrospinal fluid (aCSF), nipecotic acid (NIP) or gabazine into the medullary raphe of P15 and P21 pups receiving the three diets. For statistical analysis, the PF and CHOW groups were combined into a single CONTROL group. Our main finding was that compared to CONTROL, arousal latency to hypoxia is increased in ETOH pups at P15 and P21, and the concentration of brainstem GABA is elevated at P21. NIP administration in CONTROL pups led to arousal latencies similar in magnitude to those in ETOH pups after aCSF injection. NIP injected ETOH pups had no further increases in arousal latency. We conclude that PAE impairs arousal latency and this is mediated or modulated by medullary GABAergic mechanisms.