Giulia Anzalone

Th17 Immunity in Children with Allergic Asthma and Rhinitis: A Pharmacological Approach

Th17 cells and IL-17A play a role in the development and progression of allergic diseases. We analyzed the IL-17A levels in sputum supernatants (Ss), nasal wash (NW) and plasma (P) from Healthy Controls (HC) and children with Asthma/Rhinitis. We tested the expression of IL-17A, RORγ(t) and FOXP3 in peripheral blood T-lymphocytes from intermittent and mild-moderate asthma. The effect of Budesonide and Formoterol was tested “in vitro” on IL-17A, RORγ(t) and FOXP3 expression in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis patients, and on nasal and bronchial epithelial cells stimulated with NW and Ss from mild-moderate asthma/persistent rhinitis. Further, the effect of 12 weeks of treatment with Budesonide and Formoterol was tested “in vivo” in T-lymphocytes from mild-moderate asthma/persistent rhinitis patients. IL-17A was increased in Ss, NW and P from children with mild-moderate asthma compared with intermittent and HC. In cultured T-lymphocytes IL-17A and RORγ(t) expression were higher in mild-moderate asthma/persistent rhinitis than in mild-moderate asthma/intermittent rhinitis, while FOXP3 was reduced. Budesonide with Formoterol reduced IL-17A and RORγ(t), while increased FOXP3 in cultured T-lymphocytes from mild-moderate asthma/persistent rhinitis, and reduced the IL-8 release mediated by IL-17A present in NW and Ss from mild-moderate asthma/persistent rhinitis in nasal and bronchial epithelial cells. Finally, Budesonide with Formoterol reduced IL-17A levels in P and Ss, CD4+IL-17A+T-cells, in naïve children with mild-moderate asthma/persistent rhinitis after 12 weeks of treatment. Th17 mediated immunity may be involved in the airway disease of children with allergic asthma and allergic rhinitis. Budesonide with Formoterol might be a useful tool for its therapeutic control.

Increased levels of Th17 cells are associated with non-neuronal acetylcholine in COPD patients

T-lymphocytes, including Th17-cells and T-cells expressing acetylcholine (ACh), are key components of systemic inflammation in chronic obstructive pulmonary disease (COPD). We investigated whether ACh promotes Th17 cells in COPD. ACh, IL-17A, IL-22, RORγt, FOXP3 expression and AChIL-17A, AChIL-22, AChRORγt coexpression was evaluated in peripheral blood mononuclear cells (PBMC) from COPD patients (n=16), healthy smokers (HS) (n=12) and healthy control subjects (HC) (n=13) (cultured for 48 h with PMA) by flow cytometry. Furthermore, we studied the effect of Tiotropium (Spiriva®) (100 nM) and Olodaterol (1nM) alone or in combination, and of hemicholinium-3 (50 μM) on AChIL-17A, AChIL-22, AChRORγt, and FOXP3 expression in CD3+PBT-cells of PBMC from COPD patients (n=6) cultured for 48 h with PMA. CD3+PBT-cells expressing ACh, IL-17A, IL-22 and RORγt together with CD3+PBT-cells co-expressing AChIL-17A, AChIL-22 and AChRORγt were significantly increased in COPD patients compared to HC and HS subjects with higher levels in HS than in HC without a significant difference. CD3+FOXP3+PBT-cells were increased in HS than in HC and COPD. Tiotropium and Olodaterol reduced the percentage of CD3+PBT-cells co-expressing AChIL-17A, AChIL-22, and AChRORγt while increased the CD3+FOXP3+PBT-cells in PBMC from COPD patients, cultured in vitro for 48 h, with an additive effect when used in combination. Hemicholnium-3 reduced the percentage of ACh+IL-17A+, ACh+IL-22+, and ACh+RORγt+ while it did not affect FOXP3+ expression in CD3+PBT-cells from cultured PBMC from COPD patients. We concluded that ACh might promote the increased levels of Th17-cells in systemic inflammation of COPD. Long-acting β2-agonists and anticholinergic drugs might contribute to control this event.

Cigarette smoke affects IL-17A, IL-17F and IL-17 receptor expression in the lung tissue: Ex vivo and in vitro studies.

Cigarette smoke is a risk factor for Chronic Obstructive Pulmonary Disease (COPD). Th-17 cytokines are involved in the pathogenesis of COPD. We aimed to evaluate the role of cigarette smoke on the expression of IL-17A, IL-17F and IL-17R in airways of COPD patients. Epithelial and subepithelial immunoreactivity for IL-17A, IL-17F and IL-17R was assessed in surgical specimens from COPD patients (n=15) and from healthy subjects (HC) (n=10) by immunohistochemistry. In vitro, human epithelial cell line 16HBE and A549 as well as PBMC from normal donors were stimulated with cigarette smoke extract (CSE) (0%, 2.5%, 5%, 10%) to evaluate the IL-17A, IL-17F and IL-17R expression by flow cytometry. Furthermore, rhIL-17A and CSE stimulation was evaluated on proliferation and apoptosis in 16HBE and in A549. In central and distal airways immunoreactivity for IL-17A, IL-17F and IL-17R significantly increased in the epithelium and IL-17A in the subepithelium from COPD than in HC. In distal airway, immunoreactivity for IL-17F increased in the subepithelium of COPD than in HC. IL-17A immunoreactivity positively correlate with IL-17R and total pack years in the epithelium from central and distal airways of COPD patients. In vitro, CSE stimulation significantly increased IL-17F and IL-17R in 16HBE (2.5%) and A549 (5%) while IL-17A and IL-17F in PBMC (10%). IL-17A and CSE stimulation, rather than CSE or rhIL-17A alone, significantly increased proliferation in 16HBE and apoptosis in A549. Cigarette smoke increases Th17 immunity in lung tissue of COPD patients, promoting the mechanism of proliferation and apoptosis in airway epithelial cells.

Acetylcholine leads to signal transducer and activator of transcription 1 (STAT-1) mediated oxidative/nitrosative stress in human bronchial epithelial cell line

The induction of nitric oxide synthase (iNOS) expression via the signal transducer and activator of transcription 1 (STAT-1) is involved in the mechanism of oxidative/nitrosative stress. We investigated whether acetylcholine (ACh) generates oxidative/nitrosative stress in bronchial epithelial cells during airway inflammation of COPD and evaluated the effects of Tiotropium, a once-daily antimuscarinic drug, and Olodaterol, a long-acting β2-agonist on these mechanisms. Human bronchial epithelial cells (16-HBE) were stimulated (4h, 37°C) with induced sputum supernatants (ISSs) from healthy controls (HC) (n=10), healthy smokers (HS) (n=10) or COPD patients (n=10), as well as with ACh (from 1μM to 100μM). The activation of STAT-1 pathway (STAT-1Ser727 and STAT-1Tyr701) and iNOS was evaluated in the cell lysates by Western blot analysis as well as nitrotyrosine levels by ELISA, while reactive oxygen species (ROS) were evaluated by flow cytometry. Finally, the effect of Tiotropium (Spiriva®) (100nM), alone or in combination with Olodaterol (1nM), was tested in this model. ISSs from COPD patients significantly increased the phosphorylation of STAT-1Ser727 and STAT-1Tyr701, iNOS and ROS/Nitrotyrosine when compared with ISSs from HC or HS subjects in 16-HBE cells. Furthermore, synthetic ACh increased all these parameters in stimulated 16HBE when compared with untreated cells. Tiotropium and Olodaterol reduced the oxidative/nitrosative stress generated by ACh and ISSs. We concluded that ACh mediated the oxidative/nitrosative stress involving the STAT-1 pathway activation in human bronchial epithelial cells during COPD. β2-Long acting and antimuscarinic drugs, normally used in the treatment of COPD as bronchodilator, might be able to control these cellular events.

Beclomethasone dipropionate and formoterol reduce oxidative/nitrosative stress generated by cigarette smoke extracts and IL-17A in human bronchial epithelial cells

Interleukin-17A (IL-17A), cigarette smoke and oxidative/nitrosative stress are involved in inflammatory airway diseases, and the mechanisms behind these processes are still poorly understood. We investigated whether recombinant human IL-17A (rhIL-17A), in combination with cigarette smoke extracts (CSE), increases the levels of inducibile nitric oxide synthase (iNOS), reactive oxygen species, nitrotyrosine (NT) and the activation of signal transducer and activator of transcription 1 (STAT-1) in normal human bronchial epithelial cells (16HBE). The effect of beclomethasone dipropionate (BDP), formoterol and their combination was also evaluated. We demonstrated that rhIL-17A or CSE alone increases iNOS expression, reactive oxygen species and NT production and STAT-1 downstream signalling activation in terms of STAT-1ser727 and STAT-1tyr701 phosphorylation. The combination of both stimuli further increased iNOS, ROS, NT and STAT-1ser727 phosphorylation. The silencing of STAT-1 expression partially reduced the levels of iNOS, reactive oxygen species and NT generated by rhIL-17A and inhibited the effect of CSE alone in 16HBE cells. The treatment of the cells with the MEK1/2 inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto butadiene) abolished the expression of iNOS and STAT-1ser727 phosphorylation generated by rhIL-17A. 16HBE treated with BDP or formoterol alone partially suppressed the effect of IL-17A or CSE on ROS, NT, and STAT-1 activation. Furthermore the use of the drugs in combination showed an additive effect in 16HBE. Our findings demonstrate that IL-17A increases oxidative/nitrosative markers, likely via ERK1/2 downstream signalling and STAT-1 pathway activation in human bronchial epithelial cells. BDP and formoterol treatment reduces this effect showing an additive effect used in combination.

Cigarette smoke alters non-neuronal cholinergic system components inducing MUC5AC production in the H292 cell line.

Cigarette smoke extract (CSE) affects the expression of Choline Acetyl-Transferase (ChAT), muscarinic acetylcholine receptors, and mucin production in bronchial epithelial cells. Mucin 5AC (MUC5AC), muscarinic acetylcholine receptor M3, ChAT expression, acetylcholine levels and acetylcholine binding were measured in a human pulmonary mucoepidermoid carcinoma cell line (H292) stimulated with CSE. We performed ChAT/RNA interference experiments in H292 cells stimulated with CSE to study the role of ChAT/acetylcholine in MUC5AC production. The effects of Hemicholinium-3 (HCh-3) (50 μM) (a potent and selective choline uptake blocker) and Tiotropium bromide (Spiriva(®)) (100 nM), alone or in combination with Salmeterol (SL) and Fluticasone propionate (FP), were tested in this model. MUC5AC, muscarinic acetylcholine receptor M3, ChAT, acetylcholine expression and acetylcholine binding significantly increased in H292 cells stimulated with CSE (5%) compared to untreated cells. HCh-3 reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. ChAT/RNA interference eliminated the effect of CSE on MUC5AC production. FP reduced ChAT and acetylcholine binding in unstimulated cells, while showing a partial effect in CSE stimulated cells. SL increased the ChAT expression and acetylcholine binding in H292 cells stimulated with or without CSE. Tiotropium, alone or together with FP and SL, reduced acetylcholine binding and MUC5AC production in H292 cells stimulated with CSE. CSE affects the ChAT/acetylcholine expression, increasing MUC5AC production in H292 cells. Pharmacological treatment with anticholinergic drugs reduces the secretion of MUC5AC generated by autocrine acetylcholine activity in airway epithelial cells.

Prostaglandin E2 possesses different potencies in inducing Vascular Endothelial Growth Factor and Interleukin-8 production in COPD human lung fibroblasts

We studied the role of PGE2, its biosynthetic enzymes and its receptors, in regulating the functions of lung fibroblasts through the production of Vascular Endothelial Growth Factor (VEGF) and Interleukin-8 (IL-8) in COPD subjects. Lung fibroblasts from Control (C) (n=6), Smoker (HS) (n=6) and COPD patients (n=8) were cultured, and basal PGE2, VEGF, and IL-8 measured in supernatants by ELISA. COX-1/COX-2 and EP receptors expression were assessed by western blot and by RT-PCR. Release of VEGF and IL-8 by human fetal lung fibroblasts (HFL-1; lung, diploid, human) was evaluated under different conditions. PGE2, VEGF, and IL-8 levels, COX-2, EP2, and EP4 protein expression and mRNA were increased in COPD when compared to Controls. Low concentrations of synthetic PGE2 increased the release of VEGF in HFL-1, but higher concentrations were needed to induce the release of IL-8. This effect was mimicked by an EP2 agonist and modulated by an EP4 antagonist. In the airways of COPD subjects, fibroblast-derived PGE2 may regulate angiogenesis and inflammation through the production of VEGF and IL-8 respectively, suggesting that the increase in expression of COX-2, EP2 and EP4 observed in COPD fibroblasts may contribute to steering the role of PGE2 from homeostatic to pro-inflammatory.

Autocrine Acetylcholine, Induced by IL-17A via NFκB and ERK1/2 Pathway Activation, Promotes MUC5AC and IL-8 Synthesis in Bronchial Epithelial Cells

IL-17A is overexpressed in the lung during acute neutrophilic inflammation. Acetylcholine (ACh) increases IL-8 and Muc5AC production in airway epithelial cells. We aimed to characterize the involvement of nonneuronal components of cholinergic system on IL-8 and Muc5AC production in bronchial epithelial cells stimulated with IL-17A. Bronchial epithelial cells were stimulated with recombinant human IL-17A (rhIL-17A) to evaluate the ChAT expression, the ACh binding and production, the IL-8 release, and the Muc5AC production. Furthermore, the effectiveness of PD098,059 (inhibitor of MAPKK activation), Bay11-7082 (inhibitor of IkBα phosphorylation), Hemicholinium-3 (HCh-3) (choline uptake blocker), and Tiotropium bromide (Spiriva®) (anticholinergic drug) was tested in our in vitro model. We showed that rhIL-17A increased the expression of ChAT, the levels of ACh binding and production, and the IL-8 and Muc5AC production in stimulated bronchial epithelial cells compared with untreated cells. The pretreatment of the cells with PD098,059 and Bay11-7082 decreased the ChAT expression and the ACh production/binding, while HCh-3 and Tiotropium decreased the IL-8 and Muc5AC synthesis in bronchial epithelial cells stimulated with rhIL-17A. IL-17A is involved in the IL-8 and Muc5AC production promoting, via NFκB and ERK1/2 pathway activation, the synthesis of ChAT, and the related activity of autocrine ACh in bronchial epithelial cells.

IL-17A induces chromatin remodeling promoting IL-8 release in bronchial epithelial cells: Effect of Tiotropium.

AIMS IL-17A plays a key role in the persistence of airway inflammation, oxidative stress, and reduction of steroid-sensitivity in COPD. We studied the effect of IL-17A on chromatin remodeling and IL-8 production. MAIN METHODS We measured the levels of IL-8 and IL-17A in induced sputum supernatants (ISS) from healthy controls (HCs), healthy smokers (HSs), and COPD patients by enzyme-linked immunosorbent assay (ELISA). A human bronchial epithelial cell line (16HBE) was stimulated with ISS from HCs, HSs, or COPD subjects. IL-8 was evaluated in 16HBE by Western blot and real-time polymerase chain reaction (PCR). Histone deacetylase 2 (HDAC2), acetyl histone H3 (Ac-His H3) (k9) and inhibitor kappa kinase alpha (IKKα) levels were evaluated in the nuclear extract by Western blot. Finally, we evaluated the effect of IL-17A depletion in ISS, the silencing of IKKα, and the anti-inflammatory effects of Tiotropium Spiriva® (100nM) on 16HBE. KEY FINDINGS IL-8 and IL-17A levels were higher in ISS from COPD patients and HSs than from HCs. IL-8 protein and messenger RNA (mRNA) levels were increased in 16HBE stimulated with ISS from COPD patients compared with untreated cells. Furthermore, ISS from COPD patients reduced the nuclear levels of HDAC2 while increasing the activity of both Ac-His H3 (k9) and IKKα in stimulated 16HBE. IL-17A depletion in ISS and the IKKα silencing in 16HBE significantly increased the nuclear levels of HDAC2, reduced Ac-His H3 (k9), and promoted IL-8 synthesis in stimulated 16HBE. Tiotropium controls the proinflammatory activity generated by ISS from COPD patients in 16HBE. SIGNIFICANCE IL-17A present in the airway of COPD patients, which induces chromatin remodeling, promotes the release of IL-8 in the bronchial epithelium. Tiotropium is able to control this proinflammatory activity.

Crosstalk between mAChRM3 and β2AR, via acetylcholine PI3/PKC/PBEP1/Raf-1 MEK1/2/ERK1/2 pathway activation, in human bronchial epithelial cells after long-term cigarette smoke exposure

Background: Cigarette smoke extract (CSE) affects the expression of non‐neuronal components of cholinergic system in bronchial epithelial cells and, as PEBP1/Raf‐mediated MAPK1/2 and ERK1/2 pathway, promotes inflammation and oxidative stress. Aims: We studied whether Acetylcholine (ACh) is involved in the mechanism of crosstalk between mAChRM3 and &bgr;2Adrenergic receptors (&bgr;2AR) promoting, via PI3/PKC/PBEP1/Raf/MEK1/2/ERK1/2 activation, &bgr;2AR desensitization, inflammation and, oxidative stress in a bronchial epithelial cell line (16HBE) after long‐term exposure to cigarette smoke extract (LECSE). Methods: We evaluated mAChRM3 and Choline Acetyltransferase (ChAT) expression, ACh production, PEBP1, ERk1/2, and &bgr;2AR phosphorylation, as well as NOX‐4, ROS production and IL‐8 release in 16HBE after LECSE. The inhibitory activity of Hemicholinium (HCh‐3) (a potent choline uptake blocker), LY294002 (a highly selective inhibitor of PI3 kinase), Tiotropium (Spiriva®) (anticholinergic drug) and Olodaterol (&bgr;2AR agonist), were tested in 16HBE after LECSE. Results: mAChRM3, ChAT, ACh activity, pPEBP1, p&bgr;2AR, pERK1/2, ROS, NOX‐4 and IL‐8 increased after LECSE in 16HBE LECSE compared to untreated cells. HCh‐3 and LY294002 (alone or in combination) as well as Tiotropium (Spiriva®) or Olodaterol (alone or in combination) all reduced the levels of pPEBP1, p&bgr;2AR, pERK1/2, ROS, NOX‐4, and IL‐8 in 16HBE LECSE compared to untreated cells. Conclusions: LECSE promotes ACh production which enhances PI3/PKC/PEBP1/Raf‐ERK1/2 pathway activation, heterologous &bgr;2AR desensitization, as well as release of inflammatory and oxidative mediators in bronchial epithelial cells. The use of anticholinergic drugs and long‐acting &bgr;2‐agonists, alone or in combination may be dampen these inflammatory mechanisms when used in combination in some epithelial cell types. Graphical abstract: Figure. No caption available.