A long noncoding RNA antisense to ICAM-1 is involved in allergic asthma associated hyperreactive response of airway epithelial cells.

Epithelial cells of the conducting airways are a pivotal first line of defense against airborne pathogens and allergens that orchestrate inflammatory responses and mucociliary clearance. Nonetheless, the molecular mechanisms responsible for epithelial hyperreactivity associated with allergic asthma are not completely understood. Transcriptomic analysis of human airway epithelial cells (HAECs), differentiated in-vitro at air-liquid interface (ALI), showed 725 differentially expressed immediate-early transcripts, including putative long noncoding RNAs (lncRNAs). A novel lncRNA on the antisense strand of ICAM-1 or LASI was identified, which was induced in LPS-primed HAECs along with mucin MUC5AC and its transcriptional regulator SPDEF. LPS-primed expression of LASI, MUC5AC, and SPDEF transcripts were higher in ex-vivo cultured asthmatic HAECs that were further augmented by LPS treatment. Airway sections from asthmatics with increased mucus load showed higher LASI expression in MUC5AC+ goblet cells following multi-fluorescent in-situ hybridization and immunostaining. LPS- or IL-13-induced LASI transcripts were mostly enriched in the nuclear/perinuclear region and were associated with increased ICAM-1, IL-6, and CXCL-8 expression. Blocking LASI expression reduced the LPS or IL-13-induced epithelial inflammatory factors and MUC5AC expression, suggesting that the novel lncRNA LASI could play a key role in LPS-primed trained airway epithelial responses that are dysregulated in allergic asthma.

Characterization of long G4-rich enhancer-associated genomic regions engaging in a novel loop:loop 'G4 Kissing' interaction.

Mammalian antibody switch regions (∼1500 bp) are composed of a series of closely neighboring G4-capable sequences. Whereas numerous structural and genome-wide analyses of roles for minimal G4s in transcriptional regulation have been reported, Long G4-capable regions (LG4s)-like those at antibody switch regions-remain virtually unexplored. Using a novel computational approach we have identified 301 LG4s in the human genome and find LG4s prone to mutation and significantly associated with chromosomal rearrangements in malignancy. Strikingly, 217 LG4s overlap annotated enhancers, and we find the promoters regulated by these enhancers markedly enriched in G4-capable sequences suggesting G4s facilitate promoter-enhancer interactions. Finally, and much to our surprise, we also find single-stranded loops of minimal G4s within individual LG4 loci are frequently highly complementary to one another with 178 LG4 loci averaging >35 internal loop:loop complements of >8 bp. As such, we hypothesized (then experimentally confirmed) that G4 loops within individual LG4 loci directly basepair with one another (similar to characterized stem-loop kissing interactions) forming a hitherto undescribed, higher-order, G4-based secondary structure we term a 'G4 Kiss or G4K'. In conclusion, LG4s adopt novel, higher-order, composite G4 structures directly contributing to the inherent instability, regulatory capacity, and maintenance of these conspicuous genomic regions.

Long Noncoding Transcriptome in Chronic Obstructive Pulmonary Disease.

Chronic airway inflammation from recurring exposures to noxious environmental stimuli results in a progressive and irreversible airflow limitation and the lung parenchymal damage that characterizes chronic obstructive pulmonary disease (COPD). The large variability observed in the onset and progression of COPD is primarily driven by complex gene-environment interactions. The transcriptomic and epigenetic memory potential of lung epithelial and innate immune cells drive responses, such as mucus hyperreactivity and airway remodeling, that are tightly regulated by various molecular mechanisms, for which several candidate susceptibility genes have been described. However, the recently described noncoding RNA species, in particular the long noncoding RNAs, may also have an important role in modulating pulmonary responses to chronic inhalation of toxic substances and the development of COPD. This review outlines the features of long noncoding RNAs that have been implicated in regulating the airway inflammatory responses to cigarette smoke exposure and their possible association with COPD pathogenesis. As COPD continues to debilitate the increasingly aging population and contribute to higher morbidity and mortality rates worldwide, the search for better biomarkers and alternative therapeutic options is pivotal.

Gestational Exposure to Sidestream (Secondhand) Cigarette Smoke Promotes Transgenerational Epigenetic Transmission of Exacerbated Allergic Asthma and Bronchopulmonary Dysplasia.

Embryonic development is highly sensitive to xenobiotic toxicity and in utero exposure to environmental toxins affects physiological responses of the progeny. In the United States, the prevalence of allergic asthma (AA) is inexplicably rising and in utero exposure to cigarette smoke increases the risk of AA and bronchopulmonary dysplasia (BPD) in children and animal models. We reported that gestational exposure to sidestream cigarette smoke (SS), or secondhand smoke, promoted nicotinic acetylcholine receptor-dependent exacerbation of AA and BPD in mice. Recently, perinatal nicotine injections in rats were reported to induce peroxisome proliferator-activated receptor γ-dependent transgenerational transmission of asthma. Herein, we show that first generation and second generation progeny from gestationally SS-exposed mice exhibit exacerbated AA and BPD that is not dependent on the decrease in peroxisome proliferator-activated receptor γ levels. Lungs from these mice show strong eosinophilic infiltration, excessive Th2 polarization, marked airway hyperresponsiveness, alveolar simplification, decreased lung compliance, and decreased lung angiogenesis. At the molecular level, these changes are associated with increased RUNX3 expression, alveolar cell apoptosis, and the antiangiogenic factor GAX, and decreased expression of HIF-1α and proangiogenic factors NF-κB and VEGFR2 in the 7-d first generation and second generation lungs. Moreover, the lungs from these mice exhibit lower levels of microRNA (miR)-130a and increased levels of miR-16 and miR-221. These miRs regulate HIF-1α-regulated apoptotic, angiogenic, and immune pathways. Thus the intergenerational effects of gestational SS involve epigenetic regulation of HIF-1α through specific miRs contributing to increased incidence of AA and BPD in the progenies.

Human genetic and metabolite variation reveals that methylthioadenosine is a prognostic biomarker and an inflammatory regulator in sepsis.

Sepsis is a deleterious inflammatory response to infection with high mortality. Reliable sepsis biomarkers could improve diagnosis, prognosis, and treatment. Integration of human genetics, patient metabolite and cytokine measurements, and testing in a mouse model demonstrate that the methionine salvage pathway is a regulator of sepsis that can accurately predict prognosis in patients. Pathway-based genome-wide association analysis of nontyphoidal Salmonella bacteremia showed a strong enrichment for single-nucleotide polymorphisms near the components of the methionine salvage pathway. Measurement of the pathway's substrate, methylthioadenosine (MTA), in two cohorts of sepsis patients demonstrated increased plasma MTA in nonsurvivors. Plasma MTA was correlated with levels of inflammatory cytokines, indicating that elevated MTA marks a subset of patients with excessive inflammation. A machine-learning model combining MTA and other variables yielded approximately 80% accuracy (area under the curve) in predicting death. Furthermore, mice infected with Salmonella had prolonged survival when MTA was administered before infection, suggesting that manipulating MTA levels could regulate the severity of the inflammatory response. Our results demonstrate how combining genetic data, biomolecule measurements, and animal models can shape our understanding of disease and lead to new biomarkers for patient stratification and potential therapeutic targeting.

Whole exome sequencing identifies novel candidate genes that modify chronic obstructive pulmonary disease susceptibility.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by an irreversible airflow limitation in response to inhalation of noxious stimuli, such as cigarette smoke. However, only 15-20 % smokers manifest COPD, suggesting a role for genetic predisposition. Although genome-wide association studies have identified common genetic variants that are associated with susceptibility to COPD, effect sizes of the identified variants are modest, as is the total heritability accounted for by these variants. In this study, an extreme phenotype exome sequencing study was combined with in vitro modeling to identify COPD candidate genes. RESULTS: We performed whole exome sequencing of 62 highly susceptible smokers and 30 exceptionally resistant smokers to identify rare variants that may contribute to disease risk or resistance to COPD. This was a cross-sectional case-control study without therapeutic intervention or longitudinal follow-up information. We identified candidate genes based on rare variant analyses and evaluated exonic variants to pinpoint individual genes whose function was computationally established to be significantly different between susceptible and resistant smokers. Top scoring candidate genes from these analyses were further filtered by requiring that each gene be expressed in human bronchial epithelial cells (HBECs). A total of 81 candidate genes were thus selected for in vitro functional testing in cigarette smoke extract (CSE)-exposed HBECs. Using small interfering RNA (siRNA)-mediated gene silencing experiments, we showed that silencing of several candidate genes augmented CSE-induced cytotoxicity in vitro. CONCLUSIONS: Our integrative analysis through both genetic and functional approaches identified two candidate genes (TACC2 and MYO1E) that augment cigarette smoke (CS)-induced cytotoxicity and, potentially, COPD susceptibility.

HIF-1α Plays a Critical Role in the Gestational Sidestream Smoke-Induced Bronchopulmonary Dysplasia in Mice.

RATIONALE: Smoking during pregnancy increases the risk of bronchopulmonary dysplasia (BPD) and, in mice, gestational exposure to sidestream cigarette smoke (SS) induces BPD-like condition characterized by alveolar simplification, impaired angiogenesis, and suppressed surfactant protein production. Normal fetal development occurs in a hypoxic environment and nicotinic acetylcholine receptors (nAChRs) regulate the hypoxia-inducible factor (HIF)-1α that controls apoptosis and angiogenesis. To understand SS-induced BPD, we hypothesized that gestational SS affected alveolar development through HIF-1α. METHODS: Pregnant BALB/c mice were exposed to air (control) or SS throughout the gestational period and the 7-day-old lungs of the progeny were examined. RESULTS: Gestational SS increased apoptosis of alveolar and airway epithelial cells. This response was associated with increased alveolar volumes, higher levels of proapoptotic factors (FOXO3a, HIPK2, p53, BIM, BIK, and BAX) and the antiangiogenic factor (GAX), and lower levels of antiapoptotic factors (Akt-PI3K, NF-κB, HIF-1α, and Bcl-2) in the lung. Although gestational SS increased the cells containing the proangiogenic bombesin-like-peptide, it markedly decreased the expression of its receptor GRPR in the lung. The effects of SS on apoptosis were attenuated by the nAChR antagonist mecamylamine. CONCLUSIONS: Gestational SS-induced BPD is potentially regulated by nAChRs and associated with downregulation of HIF-1α, increased apoptosis of epithelial cells, and increased alveolar volumes. Thus, in mice, exposure to sidestream tobacco smoke during pregnancy promotes BPD-like condition that is potentially mediated through the nAChR/HIF-1α pathway.

HIV gp120 induces mucus formation in human bronchial epithelial cells through CXCR4/α7-nicotinic acetylcholine receptors.

Lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, and lung infections are major causes of morbidity and mortality among HIV-infected patients even in the era of antiretroviral therapy (ART). Many of these diseases are strongly associated with smoking and smoking is more common among HIV-infected than uninfected people; however, HIV is an independent risk factor for chronic bronchitis, COPD, and asthma. The mechanism by which HIV promotes these diseases is unclear. Excessive airway mucus formation is a characteristic of these diseases and contributes to airway obstruction and lung infections. HIV gp120 plays a critical role in several HIV-related pathologies and we investigated whether HIV gp120 promoted airway mucus formation in normal human bronchial epithelial (NHBE) cells. We found that NHBE cells expressed the HIV-coreceptor CXCR4 but not CCR5 and produced mucus in response to CXCR4-tropic gp120. The gp120-induced mucus formation was blocked by the inhibitors of CXCR4, α7-nicotinic acetylcholine receptor (α7-nAChR), and γ-aminobutyric acid (GABA)AR but not the antagonists of CCR5 and epithelial growth factor receptor (EGFR). These results identify two distinct pathways (α7-nAChR-GABAAR and EGFR) for airway mucus formation and demonstrate for the first time that HIV-gp120 induces and regulates mucus formation in the airway epithelial cells through the CXCR4-α7-nAChR-GABAAR pathway. Interestingly, lung sections from HIV ± ART and simian immunodeficiency virus (SIV) ± ART have significantly more mucus and gp120-immunoreactivity than control lung sections from humans and macaques, respectively. Thus, even after ART, lungs from HIV-infected patients contain significant amounts of gp120 and mucus that may contribute to the higher incidence of obstructive pulmonary diseases in this population.

Gestational exposure of mice to secondhand cigarette smoke causes bronchopulmonary dysplasia blocked by the nicotinic receptor antagonist mecamylamine.

BACKGROUND: Cigarette smoke (CS) exposure during gestation may increase the risk of bronchopulmonary dysplasia (BPD)-a developmental lung condition primarily seen in neonates that is characterized by hypoalveolarization, decreased angiogenesis, and diminished surfactant protein production and may increase the risk of chronic obstructive pulmonary disease. OBJECTIVE: We investigated whether gestational exposure to secondhand CS (SS) induced BPD and sought to ascertain the role of nicotinic acetylcholine receptors (nAChRs) in this response. METHODS: We exposed BALB/c and C57BL/6 mice to filtered air (control) or SS throughout the gestation period or postnatally up to 10 weeks. Lungs were examined at 7 days, 10 weeks, and 8 months after birth. RESULTS: Gestational but not postnatal exposure to SS caused a typical BPD-like condition: suppressed angiogenesis [decreased vascular endothelial growth factor (VEGF), VEGF receptor, and CD34/CD31 (hematopoietic progenitor cell marker/endothelial cell marker)], irreversible hypoalveolarization, and significantly decreased levels of Clara cells, Clara cell secretory protein, and surfactant proteins B and C, without affecting airway ciliated cells. Importantly, concomitant exposure to SS and the nAChR antagonist mecamylamine during gestation blocked the development of BPD. CONCLUSIONS: Gestational exposure to SS irreversibly disrupts lung development leading to a BPD-like condition with hypoalveolarization, decreased angiogenesis, and diminished lung secretory function. Nicotinic receptors are critical in the induction of gestational SS-induced BPD, and the use of nAChR antagonists during pregnancy may block CS-induced BPD.

Role of nicotinic receptors and acetylcholine in mucous cell metaplasia, hyperplasia, and airway mucus formation in vitro and in vivo.

BACKGROUND: Airway mucus hypersecretion is a key pathophysiologic feature in a number of lung diseases. Cigarette smoke/nicotine and allergens are strong stimulators of airway mucus; however, the mechanism of mucus modulation is unclear. OBJECTIVES: We sought to characterize the pathway by which cigarette smoke/nicotine regulates airway mucus and identify agents that decrease airway mucus. METHODS: IL-13 and γ-aminobutyric acid type A receptors (GABA(A)Rs) are implicated in airway mucus. We examined the role of IL-13 and GABA(A)Rs in nicotine-induced mucus formation in normal human bronchial epithelial (NHBE) and A549 cells and secondhand cigarette smoke-induced, ovalbumin-induced, or both mucus formation in vivo. RESULTS: Nicotine promotes mucus formation in NHBE cells; however, the nicotine-induced mucus formation is independent of IL-13 but sensitive to the GABA(A)R antagonist picrotoxin. Airway epithelial cells express α7-, α9-, and α10-nicotinic acetylcholine receptors (nAChRs), and specific inhibition or knockdown of α7- but not α9/α10-nAChRs abrogates mucus formation in response to nicotine and IL-13. Moreover, addition of acetylcholine or inhibition of its degradation increases mucus in NHBE cells. Nicotinic but not muscarinic receptor antagonists block allergen- or nicotine/cigarette smoke-induced airway mucus formation in NHBE cells, murine airways, or both. CONCLUSIONS: Nicotine-induced airway mucus formation is independent of IL-13, and α7-nAChRs are critical in airway mucous cell metaplasia/hyperplasia and mucus production in response to various promucoid agents, including IL-13. In the absence of nicotine, acetylcholine might be the biological ligand for α7-nAChRs to trigger airway mucus formation. α7-nAChRs are downstream of IL-13 but upstream of GABA(A)Rα2 in the MUC5AC pathway. Acetylcholine and α7-nAChRs might serve as therapeutic targets to control airway mucus.

Inhalation of sulfur mustard causes long-term T cell-dependent inflammation: possible role of Th17 cells in chronic lung pathology.

Sulfur mustard (SM) is a highly toxic chemical warfare agent that remains a threat to human health. The immediate symptoms of pulmonary distress may develop into chronic lung injury characterized by progressive lung fibrosis, the major cause of morbidity among the surviving SM victims. Although SM has been intensely investigated, little is known about the mechanism(s) by which SM induces chronic lung pathology. Increasing evidence suggests that IL-17(+) cells are critical in fibrosis, including lung fibrotic diseases. In this study we exposed F344 rats and cynomolgus monkeys to SM via inhalation and determined the molecular and cellular milieu in their lungs at various times after SM exposure. In rats, SM induced a burst of pro-inflammatory cytokines/chemokines within 72 h, including IL-1ß, TNF-α, IL-2, IL-6, CCL2, CCL3, CCL11, and CXCL1 that was associated with neutrophilic infiltration into the lung. At 2 wks and beyond (chronic phase), lymphocytic infiltration and continued elevated expression of cytokines/chemokines were sustained. TGF-ß, which was undetectable in the acute phase, was strongly upregulated in the chronic phase; these conditions persisted until the animals were sacrificed. The chronic phase was also associated with myofibroblast proliferation, collagen deposition, and presence of IL-17(+) cells. At ≥30 days, SM inhalation promoted the accumulation of IL-17(+) cells in the inflamed areas of monkey lungs. Thus, SM inhalation causes acute and chronic inflammatory responses; the latter is characterized by the presence of TGF-ß, fibrosis, and IL-17(+) cells in the lung. IL-17(+) cells likely play an important role in the pathogenesis of SM-induced lung injury.

Discriminative value of inflammatory biomarkers for suspected sepsis.

BACKGROUND: Circulating biomarkers can facilitate sepsis diagnosis, enabling early management and improved outcomes. Procalcitonin (PCT) has been suggested to have superior diagnostic utility compared to other biomarkers. STUDY OBJECTIVES: To define the discriminative value of PCT, interleukin-6 (IL-6), and C-reactive protein (CRP) for suspected sepsis. METHODS: PCT, CRP, and IL-6 were correlated with infection likelihood, sepsis severity, and septicemia. Multivariable models were constructed for length-of-stay and discharge to a higher level of care. RESULTS: Of 336 enrolled subjects, 60% had definite infection, 13% possible infection, and 27% no infection. Of those with infection, 202 presented with sepsis, 28 with severe sepsis, and 17 with septic shock. Overall, 21% of subjects were septicemic. PCT, IL6, and CRP levels were higher in septicemia (median PCT 2.3 vs. 0.2 ng/mL; IL-6 178 vs. 72 pg/mL; CRP 106 vs. 62 mg/dL; p < 0.001). Biomarker concentrations increased with likelihood of infection and sepsis severity. Using receiver operating characteristic analysis, PCT best predicted septicemia (0.78 vs. IL-6 0.70 and CRP 0.67), but CRP better identified clinical infection (0.75 vs. PCT 0.71 and IL-6 0.69). A PCT cutoff of 0.5 ng/mL had 72.6% sensitivity and 69.5% specificity for bacteremia, as well as 40.7% sensitivity and 87.2% specificity for diagnosing infection. A combined clinical-biomarker model revealed that CRP was marginally associated with length of stay (p = 0.015), but no biomarker independently predicted discharge to a higher level of care. CONCLUSIONS: In adult emergency department patients with suspected sepsis, PCT, IL-6, and CRP highly correlate with several infection parameters, but are inadequately discriminating to be used independently as diagnostic tools.

Prenatal secondhand cigarette smoke promotes Th2 polarization and impairs goblet cell differentiation and airway mucus formation.

Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8-10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.

Fibrogenic and redox-related but not proinflammatory genes are upregulated in Lewis rat model of chronic silicosis.

Silicosis, a fibrotic granulomatous lung disease, may occur through accidental high-dose or occupational inhalation of silica, leading to acute/accelerated and chronic silicosis, respectively. While chronic silicosis has a long asymptomatic latency, lung inflammation and apoptosis are hallmarks of acute silicosis. In animal models, histiocytic granulomas develop within days after high-dose intratracheal (IT) silica instillation. However, following chronic inhalation of occupationally relevant doses of silica, discrete granulomas resembling human silicosis arise months after the final exposure without significant lung inflammation/apoptosis. To identify molecular events associated with chronic silicosis, lung RNA samples from controls or subchronic silica-exposed rats were analyzed by Affymetrix at 28 wk after silica exposures. Results suggested a significant upregulation of 144 genes and downregulation of 7 genes. The upregulated genes included complement cascade, chemokines/chemokine receptors, G-protein signaling components, metalloproteases, and genes associated with oxidative stress. To examine the kinetics of gene expression relevant to silicosis, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), Luminex-bead assays, Western blotting, and/or zymography were performed on lung tissues from 4 d, 28 wk, and intermediate times after subchronic silica exposure and compared with 14-d acute silicosis samples. Results indicated that genes regulating fibrosis (secreted phosphoprotein-1, Ccl2, and Ccl7), redox enzymes (superoxide dismutase-2 and arginase-1), and the enzymatic activities of matrix metalloproteinases 2 and 9 were upregulated in acute and chronic silicosis models. However, proinflammatory cytokines were strongly upregulated only in acute silicosis. Thus, inflammatory cytokines are associated with acute but not chronic silicosis. Data suggest that genes regulating fibrosis, oxidative stress, and metalloproteases may contribute to both acute and chronic silicosis.

The role of IL-1ß in nicotine-induced immunosuppression and neuroimmune communication.

Although a number of inflammatory cytokines are increased during sepsis, the clinical trials aimed at down-regulating these mediators have not improved the outcome. These paradoxical results are attributed to loss of the "tolerance" phase that normally follows the proinflammatory response. Chronic nicotine (NT) suppresses both adaptive and innate immune responses, and the effects are partly mediated by the nicotinic acetylcholine receptors in the brain; however, the mechanism of neuroimmune communication is not clear. Here, we present evidence that, in rats and mice, NT initially increases IL-1ß in the brain, but the expression is downregulated within 1-2 week of chronic exposure, and the animals become resistant to proinflammatory/pyrogenic stimuli. To examine the relationship between NT, IL-1ß, and immunosuppression, we hypothesized that NT induces IL-1ß in the brain, and its constant presence produces immunological "tolerance". Indeed, unlike wild-type C57BL/6 mice, chronic NT failed to induce immunosuppression or downregulation of IL-1ß expression in IL-1ß-receptor knockout mice. Moreover, while acute intracerebroventricular administration of IL-1ß in Lewis (LEW) rats activated Fyn and protein tyrosine kinase activities in the spleen, chronic administration of low levels of IL-1ß progressively diminished the pyrogenic and T cell proliferative responses of treated animals. Thus, IL-1ß may play a critical role in the perception of inflammation by the CNS and the induction of an immunologic "tolerant" state. Moreover, the immunosuppressive effects of NT might be at least partly mediated through its effects on the brain IL-1ß. This represents a novel mechanism for neuroimmune communication.

Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors.

Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC(4). Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcepsilonRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using alpha-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) alpha7, alpha9, and alpha10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for > or = 8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A(2) activity and the PI3K/ERK/NF-kappaB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kappaB. The suppressive effect of nicotine on the late-phase response was blocked by the alpha7/alpha9-nAChR antagonists methyllycaconitine and alpha-bungarotoxin, as well as by small interfering RNA knockdown of alpha7-, alpha9-, or alpha10-nAChRs, suggesting a functional interaction between alpha7-, alpha9-, and alpha10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

Granuloma formation induced by low-dose chronic silica inhalation is associated with an anti-apoptotic response in Lewis rats.

Chronic human silicosis results primarily from continued occupational exposure to silica and exhibits a long asymptomatic latency. Similarly, continued exposure of Lewis rats to low doses of silica is known to cause delayed granuloma formation with limited lung inflammation and injury. On the other hand, intratracheal exposure to large doses of silica induces acute silicosis characterized by granuloma-like formations in the lung associated with apoptosis, severe alveolitis, and alveolar lipoproteinosis. To ascertain similarities/differences between acute and chronic silicosis, in this communication, we compared cellular and molecular changes in established rat models of acute and chronic silicosis. In Lewis rats, acute silicosis was induced by intratracheal instillation of 35 mg silica, and chronic silicosis through inhalation of aerosolized silica (6.2 mg/m(3), 5 d/wk for 6 wk). Animals exposed to acute high-dose silica were sacrificed at 14 d after silica instillation while chronically silica-treated animals were sacrificed between 4 d and 28 wk after silica exposure. The lung granulomas formation in acute silicosis was associated with strong inflammation, presence of TUNEL-positive cells, and increases in caspase-3 activity and other molecular markers of apoptosis. On the other hand, lungs from chronically silica-exposed animals exhibited limited inflammation and increased expression of anti-apoptotic markers, including dramatic increases in Bcl-2 and procaspase-3, and lower caspase-3 activity. Moreover, chronic silicotic lungs were TUNEL-negative and overexpressed Bcl-3 and NF-kappaB-p50 but not NF-kappaB-p65 subunits. These results suggest that, unlike acute silicosis, chronic exposures to occupationally relevant doses of silica cause significantly lower lung inflammation and elevated expression of anti-apoptotic rather than proapoptotic markers in the lung that might result from interaction between NF-kappaB-p50 and Bcl-3.

Nicotine primarily suppresses lung Th2 but not goblet cell and muscle cell responses to allergens.

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C(4); and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.

Early postnatal exposure to cigarette smoke impairs the antigen-specific T-cell responses in the spleen.

Annually, approximately two million babies are exposed to cigarette smoke in utero and postnatally through cigarette smoking of their mothers. Exposure to mainstream cigarette smoke is known to impair both innate and adaptive immunities, and it has been hypothesized that the effects of in utero exposure to cigarette smoke on children's health might primarily stem from the adverse effects of cigarette smoke on the immune system. To simulate the environment that babies from smoking mothers encounter, we examined the effects of prenatal mainstream and postnatal sidestream cigarette smoke on spleen cell responses. Results show that postnatal exposure of newborn Balb/c mouse pups to sidestream cigarette smoke through the first 6 weeks of life strongly suppresses the antibody response of spleen cells to the T-cell-dependent antigen, sheep red blood cells. The reduction in the antibody response seen within 6 weeks of postnatal smoke exposure is much quicker than the published data on the time 25 weeks) required to establish reproducible immunosuppression in adult rats and mice. Moreover, the immunosuppression is not associated with significant changes in T-cell numbers or subset distribution. While the postnatal exposure to cigarette smoke did not affect the mitogenic response of T and B cells, the exposure inhibited the T cell receptor-mediated rise in the intracellular calcium concentration. These results suggest that the early postnatal period is highly sensitive to the immunosuppressive effects of environmental tobacco smoke, and the effects are causally associated with impaired antigen-mediated signaling in T cells.

Chronic nicotine inhibits inflammation and promotes influenza infection.

Epidemiological data suggest an association between smoking, respiratory infections, and impaired wound healing. Inflammation is critical in the body's defense against pathogens and in the wound-healing process. Although nicotine is used to treat some inflammatory conditions, the mechanism of this action is largely unknown. To determine how nicotine affects inflammation, rats and mice were exposed to nicotine via miniosmotic pumps, and the inflammatory response to turpentine or influenza virus was assessed. Results showed that while nicotine suppressed the migration of leukocytes to the inflammation/infection site, it increased the influenza titer in the lung. The decreased inflammation correlated with lower chemotaxis/chemokinesis of peripheral blood mononuclear cells (PBMC) toward formyl-methionyl-leucyl-phenylalanine and monocyte chemoattractant protein-1 without affecting the density of their respective receptors. However, nicotine suppressed the chemokine-induced Ca(2+) response in PBMC, indicating impaired chemokine signaling. Thus, because nicotine suppresses leukocyte migration, it might contribute to the delayed wound healing and increased incidence of respiratory infections among smokers.