The tumor necrosis factor family molecules LIGHT and lymphotoxins in sinus mucosa of patients with chronic rhinosinusitis with or without nasal polyps.

BACKGROUND: Little is known about the role of lymphotoxins (LTs) family in the sinonasal mucosa of patients with chronic rhinosinusitis (CRS). This study aims at investigating the expression of LIGHT, LTα, LTß, and their receptors, LTßR and HVEM in normal and inflammatory sinus mucosa, and the effect of LIGHT and LTalpha1beta2 on chemokine secretion in epithelial cells, epithelial permeability, and leukocyte migration. MATERIAL AND METHODS: The expression of LTs family in sinonasal mucosa was evaluated with real-time PCR, immunohistochemistry, and western blot. In LTßR, HVEM siRNA, or control siRNA-transfected epithelial cells treated with LIGHT or LTalpha1beta2, the expression of chemokines, the epithelial permeability, and the expression of junctional complex proteins were evaluated using real-time PCR, ELISA, western blot, confocal microscopy, and FITC-dextran. In cultured endothelial cells treated with LIGHT or LTalpha1beta2, the expression of ICAM-1 and VCAM-1, and leukocyte migration were elucidated. RESULTS: LTs family was expressed in normal mucosa and their levels were increased in inflammatory mucosa of CRS patients. Recombinant LIGHT and LTalpha1beta2 induced chemokine secretion, increased epithelial permeability, and promoted leukocyte migration. However, the activity of LIGHT and LTalpha1beta2 was attenuated in cells transfected with LTßR and HVEM siRNA. CONCLUSIONS: LIGHT and LTs may participate in the ongoing process of chronic inflammation, inducing chemokine secretion, leukocyte migration, and dysregulated epithelial barrier through LTßR and HVEM in sinonasal mucosa.

Does central sensitization affect hyperalgesia after staged bilateral total knee arthroplasty? A randomized controlled trial.

OBJECTIVE: Osteoarthritis (OA) patients who undergo staged bilateral total knee arthroplasty (TKA) feel postoperative hyperalgesia in the second operated knee compared with the first knee. Ketamine is an important drug for central temporal summation and inhibition of secondary mechanical hyperalgesia. This study investigated whether central sensitization has a significant effect on hyperalgesia after consecutive operations. METHODS: Seventy-one of 80 OA patients were randomly allocated to the ketamine or saline group. A bolus of ketamine (group K) or saline (group C) (0.5 mg/kg) was injected before induction and at an infusion rate of 3 µg/kg/minute during surgery. A visual analog scale (VAS) was used to assess resting and moving pain and opioid consumption on postoperative days 1, 2, and 3. RESULTS: The difference in the VAS score between stages 1 and 2 (DV2-V1) was higher in the ketamine compared with the saline group. DV2-V1 for movement between the two groups was not inferior for all periods. Ketamine did not show a large analgesic effect on second-operated knee hyperalgesia in staged bilateral TKAs. CONCLUSIONS: We could not confirm that hyperalgesia was only related to central sensitization with low-dose ketamine. Other factors might be also associated with the hyperexcitability of nociceptive stimuli.Clinical Research Information Service (CRIS) trial registry no: KCT0001481.

Batch-to-Batch Consistency of SB4 and SB2, Etanercept and Infliximab Biosimilars.

BACKGROUND: Biosimilars must meet stringent regulatory requirements, both at the time of authorization and during their lifecycle. Yet it has been suggested that divergence in quality attributes over time may lead to clinically meaningful differences between two versions of a biologic. Therefore, this study investigated the batch-to-batch consistency across a range of parameters for released batches of the etanercept biosimilar (SB4) and infliximab biosimilar (SB2). METHODS: SB4 (Benepali®) and SB2 (Flixabi®) were both developed by Samsung Bioepis and are manufactured in Europe by Biogen at their facility in Hillerød, Denmark. A total of 120 batches of SB4 and 25 batches of SB2 were assessed for consistency and compliance with specified release parameters, including purity, post-translational glycosylation (SB4 only), protein concentration, and biological activity. RESULTS: The protein concentration, purity, tumor necrosis factor-α (TNF-α) binding, and TNF-α neutralization of all batches of SB4 and SB2 were within the strict specification limits set by regulatory agencies, as was the total sialic acid (TSA) content of all batches of SB4. CONCLUSIONS: Quality attributes of SB4 and SB2 batches showed little variation and were consistently within the rigorous specifications defined by regulatory agencies.

Decreased expression of type I (IFN-ß) and type III (IFN-λ) interferons and interferon-stimulated genes in patients with chronic rhinosinusitis with and without nasal polyps.

BACKGROUND: Little is known about antiviral responses in the sinonasal mucosal tissue of patients with chronic rhinosinusitis (CRS). OBJECTIVE: we investigated the presence of virus and the expression of Toll-like receptor (TLR) 3, TLR7, and interferon and interferon-stimulated genes (ISGs) in healthy mucosal tissue of control subjects and the inflammatory sinus mucosal tissue of CRS patients, and evaluated whether levels of interferons and ISGs might be affected by CRS-related cytokines and by treatment with macrolides, dexamethasone, or TLR3 and TLR7 agonists. METHODS: The presence of virus in the sinonasal mucosa was evaluated with real-time PCR. The expression of interferons and ISGs in the sinonasal mucosa and in cultured epithelial cells treated with TH1 and TH2 cytokines, macrolides, dexamethasone, or TLR3 and TLR7 agonists were evaluated with real-time PCR and Western blotting. The expression of TLR3 and TLR7 in the sinonasal mucosa were evaluated with immunohistochemistry. RESULTS: Respiratory viruses were detected in 15% of samples. Interferons and ISGs are expressed in normal mucosa, but their levels were decreased in patients with CRS. Interferon and ISG levels were upregulated in cells treated with macrolides, dexamethasone, or TLR3 agonist, but some were decreased in cytokine-treated cells. TLR3 and TLR7 levels showed no significant difference between normal and inflammatory sinus mucosal tissue. CONCLUSION: These results suggest that decreased levels of interferons and ISGs in patients with CRS might contribute to impairment of the antiviral innate response in inflammatory sinonasal epithelial cells. Macrolides and glucocorticoids might provide positive effects on the treatment of CRS by upregulating interferon and ISG expression.

The Dual Inhibition of Met and EGFR by ME22S, a Novel Met/EGFR Bispecific Monoclonal Antibody, Suppresses the Proliferation and Invasion of Laryngeal Cancer.

PURPOSE: It has been reported that the abnormal activation of receptor tyrosine kinases is associated with the development of many human carcinomas and the high activation of EGFR and Met mediates the tumorigenicity of laryngeal carcinoma. In this study, we have done the therapeutic efficacy of ME22S (a novel EGFR/Met bispecific antibody) in laryngeal carcinoma in vitro and in vivo was thoroughly evaluated. METHODS: The effects of ME22S on cell viability was assessed through MTT assays, and then Western blotting and immunocytochemistry were used to determine the expression of EGFR and Met. Also, wound healing and invasion assays were performed to observe the inhibitory effects of ME22S. RESULTS: We found the ability of ME22S reducing the expression of both EGFR and Met and significantly inhibiting the cell migration, invasion, and proliferation of SNU899 and HN3 in vitro. Also, the notably reduced levels of p-Met, p-ERK, and p-AKT were found when the cells were treated with only ME22S alone or with HGF together. Meanwhile, ME22S, interestingly enough, caused caspase-3-dependent apoptotic cell death when HN3 cells were treated with ME22S for 72 h, decreased the HGF-induced Slug expression, and also inhibited the tumor growth of HN3 cells in a xenograft model in vivo. CONCLUSIONS: Taken together, our findings suggest that the dual inhibition of EGFR and Met through ME22S largely suppresses the invasion and growth of laryngeal carcinoma both in vitro and in vivo, hence, can be a practical approach as a novel therapeutic strategy for the treatment of laryngeal carcinoma.

Influenza A virus-dependent remodeling of pulmonary clock function in a mouse model of COPD.

Daily oscillations of pulmonary function depend on the rhythmic activity of the circadian timing system. Environmental tobacco/cigarette smoke (CS) disrupts circadian clock leading to enhanced inflammatory responses. Infection with influenza A virus (IAV) increases hospitalization rates and death in susceptible individuals, including patients with Chronic Obstructive Pulmonary Disease (COPD). We hypothesized that molecular clock disruption is enhanced by IAV infection, altering cellular and lung function, leading to severity in airway disease phenotypes. C57BL/6J mice exposed to chronic CS, BMAL1 knockout (KO) mice and wild-type littermates were infected with IAV. Following infection, we measured diurnal rhythms of clock gene expression in the lung, locomotor activity, pulmonary function, inflammatory, pro-fibrotic and emphysematous responses. Chronic CS exposure combined with IAV infection altered the timing of clock gene expression and reduced locomotor activity in parallel with increased lung inflammation, disrupted rhythms of pulmonary function, and emphysema. BMAL1 KO mice infected with IAV showed pronounced detriments in behavior and survival, and increased lung inflammatory and pro-fibrotic responses. This suggests that remodeling of lung clock function following IAV infection alters clock-dependent gene expression and normal rhythms of lung function, enhanced emphysematous and injurious responses. This may have implications for the pathobiology of respiratory virus-induced airway disease severity and exacerbations.

The effectiveness of pregabalin for post-tonsillectomy pain control: a randomized controlled trial.

BACKGROUND: Although various analgesics have been used, postoperative pain remains one of the most troublesome aspects of tonsillectomy for patients. OBJECTIVE: The aim of the present study was to evaluate the effectiveness of premedication using pregabalin compared with placebo (diazepam) on postoperative pain control in patients undergoing tonsillectomy. METHODS: Forty-eight adult patients were randomly divided into a control group and a pregabalin group. Preoperatively, patients in the control group received 4 mg diazepam orally as placebo, whereas those in the pregabalin group received 300 mg pregabalin orally. All participants were provided with patient-controlled analgesia using fentanyl for 24 hours after surgery. Postoperative pain treatment included acetaminophen 650 mg three times daily for 8 postoperative days. The primary outcome measure was the total amount of patient-controlled fentanyl consumption after tonsillectomy. Secondary outcome measures were the number of injections of ketorolac tromethamine (each 30 mg) requested by patients, pain scores, overall satisfaction scores, drowsiness, nausea, dizziness, headache, and vomiting after the surgery. P < 0.05 was considered statistically significant. RESULTS: The total amount of fentanyl demanded decreased significantly in the pregabalin group (P < 0.001). There were no significant differences in the number of ketorolac tromethamine injections, pain scores, overall satisfaction scores, drowsiness, nausea, dizziness, headache, and vomiting between the two groups. CONCLUSION: Administration of 300 mg pregabalin prior to tonsillectomy decreases fentanyl consumption compared with that after 4 mg diazepam, without an increased incidence of adverse effects. TRIAL REGISTRATION: KCT0001215.

Endogenous production of hydrogen sulfide in human sinus mucosa and its expression levels are altered in patients with chronic rhinosinusitis with and without nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNPs) or CRS without nasal polyps (CRSsNPs) is characterized by persistent inflammation of sinonasal mucosa. No one causative factor fully explains for the pathological manifestations of CRS. Endogenous hydrogen sulfide (H2S) has been shown to participate in inflammatory diseases, functioning as an inflammatory mediator in various organs. We analyzed the contents and synthesis activity of H2S, the expression and distribution pattern of H2S-generating enzymes, cystathione ß-synthase (CBS), and cystathione γ-lyase (CSE) in CRSwNPs and CRSsNPs. The effects of H2S on the expression of CRS-relevant cytokines and the effects of cytokines on the expression of CBS and CSE were assessed in an in vitro experiment. METHODS: The contents and synthesis activity of H2S and the expression and distribution pattern of CBS and CSE in sinus mucosa were evaluated using spectrophotometry, real-time polymerase chain reaction, Western blot, and immunohistochemistry. Cultured epithelial cells were used to elucidate the effects of H2S donor, sodium hydrosulfide (NaHS), on the expression of CRS-relevant cytokines and the effects of cytokines on H2S-generating enzymes expression. RESULTS: The contents and synthesis activity of H2S were increased in CRSwNPs and CRSsNPs. CBS and CSE were localized to the superficial epithelium and submucosal glands, but CSE was also found in vascular endothelium. NaHS induced increased expression of IL-4, IL-5, interferon γ, and TNF-α. CBS and CSE expression in cultured cells was up-regulated by CRS-relevant cytokines. CONCLUSION: H2S levels are increased in CRS, contributing to increased production of cytokines. These results suggest that H2S may function as inflammatory mediator in CRS.

Circadian clock function is disrupted by environmental tobacco/cigarette smoke, leading to lung inflammation and injury via a SIRT1-BMAL1 pathway.

Patients with obstructive lung diseases display abnormal circadian rhythms in lung function. We determined the mechanism whereby environmental tobacco/cigarette smoke (CS) modulates expression of the core clock gene BMAL1, through Sirtuin1 (SIRT1) deacetylase during lung inflammatory and injurious responses. Adult C57BL6/J and various mice mutant for SIRT1 and BMAL1 were exposed to both chronic (6 mo) and acute (3 and 10 d) CS, and we measured the rhythmic expression of clock genes, circadian rhythms of locomotor activity, lung function, and inflammatory and emphysematous responses in the lungs. CS exposure (100-300 mg/m(3) particulates) altered clock gene expression and reduced locomotor activity by disrupting the central and peripheral clocks and increased lung inflammation, causing emphysema in mice. BMAL1 was acetylated and degraded in the lungs of mice exposed to CS and in patients with chronic obstructive pulmonary disease (COPD), compared with lungs of the nonsmoking controls, linking it mechanistically to CS-induced reduction of SIRT1. Targeted deletion of Bmal1 in lung epithelium augmented inflammation in response to CS, which was not attenuated by the selective SIRT1 activator SRT1720 (EC50=0.16 µM) in these mice. Thus, the circadian clock, specifically the enhancer BMAL1 in epithelium, plays a pivotal role, mediated by SIRT1-dependent BMAL1, in the regulation of CS-induced lung inflammatory and injurious responses.

SIRT1 redresses the imbalance of tissue inhibitor of matrix metalloproteinase-1 and matrix metalloproteinase-9 in the development of mouse emphysema and human COPD.

Sirtuin1 (SIRT1), a protein/histone deacetylase, protects against the development of pulmonary emphysema. However, the molecular mechanisms underlying this observation remain elusive. The imbalance of tissue inhibitor of matrix metalloproteinases (TIMPs)/matrix metalloproteinases (MMPs) plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that SIRT1 protects against emphysema by redressing the imbalance between MMPs and TIMPs. To test this hypothesis, SIRT1-deficient and overexpressing/transgenic mice were exposed to cigarette smoke (CS). The protein level and activity of MMP-9 were increased in lungs of SIRT1-deficient mice exposed to CS compared with wild-type (WT) littermates, and these effects were attenuated by SIRT1 overexpression. SIRT1 deficiency decreased the level of TIMP-1, which was augmented in SIRT1 transgenic mice compared with WT littermates by CS. However, the level of MMP-2, MMP-12, TIMP-2, TIMP-3, or TIMP-4 was not altered by SIRT1 in response to CS exposure. SIRT1 reduction was associated with imbalance of TIMP-1 and MMP-9 in lungs of smokers and COPD patients. Mass spectrometry and immunoprecipitation analyses revealed that TIMP-1 acetylation on specific lysine residues was increased, whereas its interaction with SIRT1 and MMP-9 was reduced in mouse lungs with emphysema, as well as in lungs of smokers and COPD patients. SIRT1 deficiency increased CS-induced TIMP-1 acetylation, and these effects were attenuated by SIRT1 overexpression. These results suggest that SIRT1 protects against COPD/emphysema, in part, via redressing the TIMP-1/MMP-9 imbalance involving TIMP-1 deacetylation. Thus redressing the TIMP-1/MMP-9 imbalance by pharmacological activation of SIRT1 is an attractive approach in the intervention of COPD.

Redox regulation of SIRT1 in inflammation and cellular senescence.

Sirtuin 1 (SIRT1) regulates inflammation, aging (life span and health span), calorie restriction/energetics, mitochondrial biogenesis, stress resistance, cellular senescence, endothelial functions, apoptosis/autophagy, and circadian rhythms through deacetylation of transcription factors and histones. SIRT1 level and activity are decreased in chronic inflammatory conditions and aging, in which oxidative stress occurs. SIRT1 is regulated by a NAD(+)-dependent DNA repair enzyme, poly(ADP-ribose) polymerase-1 (PARP1), and subsequent NAD(+) depletion by oxidative stress may have consequent effects on inflammatory and stress responses as well as cellular senescence. SIRT1 has been shown to undergo covalent oxidative modifications by cigarette smoke-derived oxidants/aldehydes, leading to posttranslational modifications, inactivation, and protein degradation. Furthermore, oxidant/carbonyl stress-mediated reduction of SIRT1 leads to the loss of its control on acetylation of target proteins including p53, RelA/p65, and FOXO3, thereby enhancing the inflammatory, prosenescent, and apoptotic responses, as well as endothelial dysfunction. In this review, the mechanisms of cigarette smoke/oxidant-mediated redox posttranslational modifications of SIRT1 and its roles in PARP1 and NF-κB activation, and FOXO3 and eNOS regulation, as well as chromatin remodeling/histone modifications during inflammaging, are discussed. Furthermore, we have also discussed various novel ways to activate SIRT1 either directly or indirectly, which may have therapeutic potential in attenuating inflammation and premature senescence involved in chronic lung diseases.

Paeonol attenuates inflammation-mediated neurotoxicity and microglial activation.

Chronic activation of microglial cells endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. The root of Paeonia lactiflora Pall has been considered useful for the treatment of various disorders in traditional oriental medicine. Paeonol, found in the root of Paeonia lactiflora Pall, has a wide range of pharmacological functions, including anti-oxidative, anti-inflammatory and neuroprotective activities. The objective of this study was to examine the efficacy of paeonol in the repression of inflammation-induced neurotoxicity and microglial cell activation. Organotypic hippocampal slice cultures and primary microglial cells from rat brain were stimulated with bacterial lipopolysaccharide. Paeonol pretreatment was performed for 30 minutes prior to lipopolysaccharide addition. Cell viability and nitrite (the production of nitric oxide), tumor necrosis factor-alpha and interleukin-1beta products were measured after lipopolysaccharide treatment. In organotypic hippocampal slice cultures, paeonol blocked lipopolysaccharide-related hippocampal cell death and inhibited the release of nitrite and interleukin-1beta. Paeonol was effective in inhibiting nitric oxide release from primary microglial cells. It also reduced the lipopolysaccharide-stimulated release of tumor necrosis factor-alpha and interleukin-1ß from microglial cells. Paeonol possesses neuroprotective activity in a model of inflammation-induced neurotoxicity and reduces the release of neurotoxic and proinflammatory factors in activated microglial cells.

High aldehyde dehydrogenase activity enhances stem cell features in breast cancer cells by activating hypoxia-inducible factor-2α.

High aldehyde dehydrogenase (ALDH) activity has been recognized as a marker of cancer stem cells (CSCs) in breast cancer. In this study, we examined whether inhibition of ALDH activity suppresses stem-like cell properties in a 4T1 syngeneic mouse model of breast cancer. We found that ALDH-positive 4T1 cells showed stem cell-like properties in vitro and in vivo. Blockade of ALDH activity reduced the growth of CSCs in breast cancer cell lines. Treatment of mice with the ALDH inhibitor diethylaminobenzaldehyde (DEAB) significantly suppressed 4T1 cell metastasis to the lung. Recent evidence suggests that ALDH affects the response of stem cells to hypoxia; therefore, we examined a possible link between ALDH and hypoxia signaling in breast cancer. Hypoxia-inducible factor-2α (HIF-2α) was highly dysregulated in ALDH-positive 4T1 cells. We observed that ALDH was highly correlated with the HIF-2α expression in breast cancer cell lines and tissues. DEAB treatment of breast cancer cells reduced the expression of HIF-2α in vitro. In addition, reduction of HIF-2α expression suppressed in vitro self-renewal ability and in vivo tumor initiation in ALDH-positive 4T1 cells. Therefore, our findings may provide the evidence necessary for exploring a new strategy in the treatment of breast cancer.

SIRT1 protects against emphysema via FOXO3-mediated reduction of premature senescence in mice.

Chronic obstructive pulmonary disease/emphysema (COPD/emphysema) is characterized by chronic inflammation and premature lung aging. Anti-aging sirtuin 1 (SIRT1), a NAD+-dependent protein/histone deacetylase, is reduced in lungs of patients with COPD. However, the molecular signals underlying the premature aging in lungs, and whether SIRT1 protects against cellular senescence and various pathophysiological alterations in emphysema, remain unknown. Here, we showed increased cellular senescence in lungs of COPD patients. SIRT1 activation by both genetic overexpression and a selective pharmacological activator, SRT1720, attenuated stress-induced premature cellular senescence and protected against emphysema induced by cigarette smoke and elastase in mice. Ablation of Sirt1 in airway epithelium, but not in myeloid cells, aggravated airspace enlargement, impaired lung function, and reduced exercise tolerance. These effects were due to the ability of SIRT1 to deacetylate the FOXO3 transcription factor, since Foxo3 deficiency diminished the protective effect of SRT1720 on cellular senescence and emphysematous changes. Inhibition of lung inflammation by an NF-κB/IKK2 inhibitor did not have any beneficial effect on emphysema. Thus, SIRT1 protects against emphysema through FOXO3-mediated reduction of cellular senescence, independently of inflammation. Activation of SIRT1 may be an attractive therapeutic strategy in COPD/emphysema.

Mitogen- and stress-activated kinase 1 (MSK1) regulates cigarette smoke-induced histone modifications on NF-κB-dependent genes.

Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis of COPD.

NF-κB inducing kinase, NIK mediates cigarette smoke/TNFα-induced histone acetylation and inflammation through differential activation of IKKs.

BACKGROUND: Nuclear factor (NF)-κB inducing kinase (NIK) is a central player in the non-canonical NF κB pathway, which phosphorylates IκB kinase α (IKKα) resulting in enhancement of target gene expression. We have recently shown that IKKα responds to a variety of stimuli including oxidants and cigarette smoke (CS) regulating the histone modification in addition to its role in NF-κB activation. However, the primary signaling mechanism linking CS-mediated oxidative stress and TNFα with histone acetylation and pro-inflammatory gene transcription is not well understood. We hypothesized that CS and TNFα increase NIK levels causing phosphorylation of IKKα, which leads to histone acetylation. METHODOLOGY: To test this hypothesis, we investigated whether NIK mediates effects of CS and TNFα on histone acetylation in human lung epithelial cells in vitro and in lungs of mouse exposed to CS in vivo. CS increased the phosphorylation levels of IKKα/NIK in lung epithelial cells and mouse lungs. NIK is accumulated in the nuclear compartment, and is recruited to the promoters of pro-inflammatory genes, to induce posttranslational acetylation of histones in response to CS and TNFα. Cells in which NIK is knocked down using siRNA showed partial attenuation of CSE- and TNFα-induced acetylation of histone H3 on pro-inflammatory gene promoters. Additional study to determine the role of IKKß/NF-κB pathway in CS-induced histone acetylation suggests that the canonical pathway does not play a role in histone acetylation particularly in response to CS in mouse lungs. CONCLUSIONS: Overall, our findings provide a novel role for NIK in CS- and TNFα-induced histone acetylation, especially on histone H3K9.

FOXO3 deficiency leads to increased susceptibility to cigarette smoke-induced inflammation, airspace enlargement, and chronic obstructive pulmonary disease.

Forkhead box class O 3a (FOXO3) is a member of the FoxO transcription factor subfamily, which regulates the expression of target genes not only through DNA binding as a transcription factor, but also through protein-protein interaction. Although FoxO3 is a well-known transcription factor involved in diverse biological processes, the role of FoxO3 in cigarette smoke (CS)-induced lung inflammation and injury has not been studied. It is, therefore, hypothesized that deficiency of FoxO3 leads to increased susceptibility to CS-induced lung inflammatory response and airspace enlargement. In this article, we show that the levels of FOXO3 are significantly decreased in lungs of smokers and patients with chronic obstructive pulmonary disease, as well as in lungs of mice exposed to CS. Genetic ablation of FoxO3 led to pulmonary emphysema and exaggerated inflammatory response in lungs of mice exposed to CS. We further showed that CS induced the translocation of FoxO3 into the nucleus where FoxO3 interacted with NF-κB and disrupted NF-κB DNA-binding ability, leading to inhibition of its activity. Targeted disruption of FoxO3 also resulted in downregulation of antioxidant genes in mouse lungs in response to CS exposure. These results suggest that FoxO3 plays a pivotal role in regulation of lung inflammatory response and antioxidant genes, and deficiency of FoxO3 results in development of chronic obstructive pulmonary disease/emphysema.

Peroxiredoxin 6 differentially regulates acute and chronic cigarette smoke–mediated lung inflammatory response and injury.

Peroxiredoxin 6 (Prdx6) exerts its protective role through peroxidase activity against H2O2 and phospholipid hydroperoxides. We hypothesized that targeted disruption of Prdx6 would lead to enhanced susceptibility to cigarette smoke (CS)-mediated lung inflammation and/or emphysema in mouse lung. Prdx6 null (Prdx6⁻/⁻mice exposed to acute CS showed no significant increase of inflammatory cell influx or any alterations in lung levels of proinflammatory cytokines compared to wild-type (WT) mice. Lung levels of antioxidant enzymes were significantly increased in acute CS-exposed Prdx6⁻/⁻ compared to WT mice. Overexpressing (Prdx6⁻/⁻) mice exposed to acute CS showed significant decrease in lung antioxidant enzymes associated with increased inflammatory response compared to CS-exposed WT mice or air-exposed Prdx6⁻/⁻ mice. However, chronic 6 months of CS exposure resulted in increased lung inflammatory response, mean linear intercept (Lm), and alteration in lung mechanical properties in Prdx6⁻/⁻ when compared to WT mice exposed to CS. These data show that targeted disruption of Prdx6 does not lead to increased lung inflammatory response but is associated with increased antioxidants, suggesting a critical role of lung Prdx6 and several compensatory mechanisms during acute CS-induced adaptive response, whereas this protection is lost in chronic CS exposure leading to emphysema.

Extracellular superoxide dismutase protects against pulmonary emphysema by attenuating oxidative fragmentation of ECM.

Extracellular superoxide dismutase (ECSOD or SOD3) is highly expressed in lungs and functions as a scavenger of O(2)(*-). ECM fragmentation, which can be triggered by oxidative stress, participates in the pathogenesis of chronic obstructive pulmonary disease (COPD) through attracting inflammatory cells into the lungs. The level of SOD3 is significantly decreased in lungs of patients with COPD. However, the role of endogenous SOD3 in the development/progression of emphysema is unknown. We hypothesized that SOD3 protects against emphysema by attenuating oxidative fragmentation of ECM in mice. To test this hypothesis, SOD3-deficient, SOD3-transgenic, and WT C57BL/6J mice were exposed to cigarette smoke (CS) for 3 d (300 mg total particulate matter/m(3)) to 6 mo (100 mg/m(3) total particulate matter) or by intratracheal elastase injection. Airspace enlargement, lung inflammation, lung mechanical properties, and exercise tolerance were determined at different time points during CS exposure or after elastase administration. CS exposure and elastase administration caused airspace enlargement as well as impaired lung function and exercise capacity in SOD3-null mice, which were improved in mice overexpressing SOD3 and by pharmacological SOD mimetic. These phenomena were associated with SOD3-mediated protection against oxidative fragmentation of ECM, such as heparin sulfate and elastin, thereby attenuating lung inflammatory response. In conclusion, SOD3 attenuates emphysema and reduces oxidative fragmentation of ECM in mouse lung. Thus, pharmacological augmentation of SOD3 in the lung may have a therapeutic potential in the intervention of COPD/emphysema.

Emphysema is associated with increased inflammation in lungs of atherosclerosis-prone mice by cigarette smoke: implications in comorbidities of COPD.

BACKGROUND: Chronic obstructive pulmonary disease is associated with numerous vascular effects including endothelial dysfunction, arterial stiffness and atherogenesis. It is also known that a decline in lung function is associated with increased cardiovascular comorbidity in smokers. The mechanism of this cardiopulmonary dual risk by cigarette smoke (CS) is not known. We studied the molecular mechanisms involved in development of emphysema in atherosclerosis-prone apolipoprotein E-deficient (ApoE-/-) mice in response to CS exposure. METHODS: Adult male and female wild-type (WT) mice of genetic background C57BL/6J and ApoE-/- mice were exposed to CS, and lung inflammatory responses, oxidative stress (lipid peroxidation products), mechanical properties as well as airspace enlargement were assessed. RESULTS AND DISCUSSION: The lungs of ApoE-/- mice showed augmented inflammatory response and increased oxidative stress with development of distal airspace enlargement which was accompanied with decline in lung function. Interestingly, the levels and activities of matrix metalloproteinases (MMP-9 and MMP-12) were increased, whereas the level of eNOS was decreased in lungs of CS-exposed ApoE-/- mice as compared to air-exposed ApoE-/- mice or CS-exposed WT mice. CONCLUSION: These findings suggest that CS causes premature emphysema and a decline of lung function in mice susceptible to cardiovascular abnormalities via abnormal lung inflammation, increased oxidative stress and alterations in levels of MMPs and eNOS.