Valentina Sorbello

IFN-γ inhibits the proliferation of allergen-activated T lymphocytes from atopic, asthmatic patients by inducing Fas/FasL-mediated apoptosis

The defect in interferon‐γ (IFN‐γ) production that results in a T helper cell type 2‐dominated response may be responsible for a decrease in the apoptosis of allergen‐activated T cells in asthma. We investigated the effect of recombinant IFN‐γ on proliferation, Fas/Fas ligand (FasL) expression, and apoptosis in allergen‐stimulated peripheral blood mononuclear cells obtained from atopic, asthmatic patients and nonatopic, control subjects. The addition of IFN‐γ at the start of cultures markedly inhibited the proliferative response to a specific allergen in cells from all asthmatic patients, whereas no change was observed in cells from nonatopic, control subjects. IFN‐γ induced an increase in the expression of Fas and FasL by allergen‐stimulated CD4+ T cells from asthmatic patients and caused the apoptosis of these cells. A Fas‐blocking monoclonal antibody prevented the inhibitory effect of IFN‐γ on allergen‐induced proliferation. These results suggest that IFN‐γ inhibits the proliferation of allergen‐stimulated CD4+ T cells from atopic, asthmatic patients by inducing the surface expression of Fas and FasL, which in turn triggers their apoptotic program. The defect in IFN‐γ production involved in the allergic, immune response may therefore be responsible for a decrease in apoptosis of allergen‐activated T lymphocytes in the airways of atopic, asthmatic patients.

Identification of IL-17F/frequent exacerbator endotype in asthma

Background Severe asthma might be associated with overexpression of Th17 cytokines, which induce neutrophil recruitment via neutrophil‐mobilizing cytokines in airways. Objective To study IL‐17–related cytokines in nasal/bronchial biopsies from controls and mild asthmatics (MAs) to severe asthmatics (SAs) in relation to exacerbation rate. Methods Inflammatory cells and IL‐17A+, IL‐17F+, IL‐21+, IL‐22+, and IL‐23+ cells were examined by immunohistochemistry in cryostat sections of bronchial/nasal biopsies obtained from 33 SAs (21 frequent exacerbators [FEs]), 31 MAs (3 FEs), and 14 controls. IL‐17F protein was also measured by ELISA in bronchial/nasal lysates and by immunohistochemistry in bronchial tissue obtained from subjects who died because of fatal asthma. Immunofluorescence/confocal microscopy was used for IL‐17F colocalization. Results Higher number (P < .05) of neutrophils, IL‐17A+, IL‐17F+, and IL‐21+ cells in bronchial biopsies and higher numbers (P < .01) of IL‐17F+ and IL‐21+ cells in nasal biopsies were observed in SAs compared with MAs. Bronchial IL‐17F+ cells correlated with bronchial neutrophils (r = 0.54), exacerbation rate (r = 0.41), and FEV1 (r = −0.46). Nasal IL‐17F+ cells correlated with bronchial IL‐17F (r = 0.35), exacerbation rate (r = 0.47), and FEV1 (r = −0.61). FEs showed increased number of bronchial neutrophils/eosinophils/CD4+/CD8+ cells and bronchial/nasal IL‐17F+ cells. Receiver operating characteristic curve analysis evidenced predictive cutoff values of bronchial neutrophils and nasal/bronchial IL‐17F for discriminating between asthmatics and controls, between MAs and SAs and between FEs and non‐FEs. IL‐17F protein increased in bronchial/nasal lysates of SAs and FEs and in bronchial tissue of fatal asthma. IL‐17F colocalized in CD4+/CD8+ cells. Conclusions IL‐17–related cytokines expression was amplified in bronchial/nasal mucosa of neutrophilic asthma prone to exacerbation, suggesting a pathogenic role of IL‐17F in FEs. Graphical abstract Figure. No Caption available.

Nasal IL‐17F is related to bronchial IL‐17F/neutrophilia and exacerbations in stable atopic severe asthma

Severe asthma (SA) is associated with neutrophil recruitment and T helper (TH)17 chemokine overexpression in bronchial biopsies. We aimed to evaluate IL‐17A and IL‐17F expression in nasal/bronchial lamina propria of atopic mild‐to‐severe asthmatics and controls in relation to neutrophilia and asthma exacerbations. Cryostat sections of nasal/bronchial biopsies obtained from 14 SA and 14 mild asthma (MA) stable atopic patients with rhinitis, and seven healthy controls were analyzed by immunohistochemistry for neutrophils, IL‐17A and IL‐17F expression. Atopic SA showed an increase in asthma exacerbations number, IL‐17F and IL‐17A expression in nasal/bronchial lamina propria compared to MA and controls, and a higher expression of bronchial neutrophils in SA compared to MA and controls. In all asthmatics, significant relationships were found between bronchial IL‐17F and neutrophils/FEV1, nasal IL‐17F and bronchial neutrophil/IL‐17 markers and between the latter and exacerbations, suggesting that nasal IL‐17F might be informative on bronchial IL17‐driven neutrophilia in atopic SA.

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.

Expression of vascular remodelling markers in relation to bradykinin receptors in asthma and COPD

Background Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD. Methods Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD. Results In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A+ cell numbers were higher than old control non-smokers (p<0.05). Angiogenin+, B2R+ and B1R+ cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p<0.01). Angiogenin+ cell numbers were higher in patients with COPD than both old control groups (p<0.05). In all patients with asthma the number of B2R+ cells was positively related to the numbers of B1R+ (rs=0.43), angiogenin+ (rs=0.42) and CD31 cells (rs=0.46) (p<0.01). Angiogenin+ cell numbers were negatively related to forced expiratory volume in 1 s (rs=−0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10−6M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD. Conclusions Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

Bradykinin-induced asthmatic fibroblast/myofibroblast activities via bradykinin B2 receptor and different MAPK pathways

Bradykinin drives normal lung fibroblasts into myofibroblasts, induces fibroblast proliferation and activates mitogen activated protein kinase pathways (MAPK) but its effects on bronchial fibroblasts from asthmatics (HBAFb) have not been yet studied. We studied bradykinin-induced fibroblast proliferation and differentiation and the related intracellular mechanisms in HBAFb compared to normal bronchial fibroblasts (HNBFb). Bradykinin-stimulated HBAFb and HNBFb were used to assess: bradykinin B2 receptor expression by Western blot analysis; cell proliferation by [(3)H] thymidine incorporation; α-smooth muscle actin (SMA) expression/polymerization by Western blot and immunofluorescence; epidermal growth factor (EGF) receptor, extracellular-regulated kinase (ERK) 1/2 and p38 MAPK activation by immunoprecipitation and Western blot, respectively. Constitutive bradykinin B2 receptor and α-SMA expression was higher in HBAFb as compared to HNBFb. Bradykinin increased bradykinin B2 receptor expression in HBAFb. Bradykinin, via bradykinin B2 receptor, significantly increased fibroblast proliferation at lower concentration (10(-11)M) and α-SMA expression/polymerization at higher concentration (10(-6)M) in both cells. Bradykinin increased ERK1/2 and p38 phosphorylation via bradykinin B2 receptor; EGF receptor inhibitor AG1478 and panmetalloproteinase inhibitor GM6001 blocked bradykinin-induced ERK1/2 activation but not p38 phosphorylation. Bradykinin, via bradykinin B2 receptor, induced EGF receptor phosphorylation that was suppressed by AG1478. In HBAFb AG1478, GM6001, the ERK1/2-inhibitor U0126 and the p38 inhibitor SB203580 suppressed bradykinin-induced cell proliferation, but only SB203580 reduced myofibroblast differentiation. These data indicate that bradykinin is actively involved in asthmatic bronchial fibroblast proliferation and differentiation, through MAPK pathways and EGF receptor transactivation, by which bradykinin may contribute to airway remodeling in asthma, opening new horizons for potential therapeutic implications in asthmatic patients.

Bradykinin B2 receptor expression in the bronchial mucosa of allergic asthmatics: The role of NF-kB

Bradykinin (BK) mediates acute allergic asthma and airway remodelling. Nuclear factor‐kappa B (NF‐kB) is potentially involved in BK B2 receptor (B2R) regulation.

Phospho-p38 MAPK Expression in COPD Patients and Asthmatics and in Challenged Bronchial Epithelium

Background: The role of mitogen-activated protein kinases (MAPK) in regulating the inflammatory response in the airways of patients with chronic obstructive pulmonary disease (COPD) and asthmatic patients is unclear. Objectives: To investigate the expression of activated MAPK in lungs of COPD patients and in bronchial biopsies of asthmatic patients and to study MAPK expression in bronchial epithelial cells in response to oxidative and inflammatory stimuli. Methods: Immunohistochemical expression of phospho (p)-p38 MAPK, p-JNK1 and p-ERK1/2 was measured in bronchial mucosa in patients with mild/moderate (n = 17), severe/very severe (n = 16) stable COPD, control smokers (n = 16), control non-smokers (n = 9), in mild asthma (n = 9) and in peripheral airways from COPD patients (n = 15) and control smokers (n = 15). Interleukin (IL)-8 and MAPK mRNA was measured in stimulated 16HBE cells. Results: No significant differences in p-p38 MAPK, p-JNK or p-ERK1/2 expression were seen in bronchial biopsies and peripheral airways between COPD and control subjects. Asthmatics showed increased submucosal p-p38 MAPK expression compared to COPD patients (p < 0.003) and control non-smokers (p < 0.05). Hydrogen peroxide (H2O2), cytomix (tumour necrosis factor-α + IL-1β + interferon-γ) and lipopolysaccharide (LPS) upregulated IL-8 mRNA at 1 or 2 h. p38 MAPKα mRNA was significantly increased after H2O2 and LPS treatment. JNK1 and ERK1 mRNA were unchanged after H2O2, cytomix or LPS treatments. Conclusion: p-p38 MAPK expression is similar in stable COPD and control subjects but increased in the bronchi of mild asthmatics compared to stable COPD patients. p38 MAPK mRNA is increased after bronchial epithelial challenges in vitro. These data together suggest a potential role for this MAPK in Th2 inflammation and possibly during COPD exacerbations.

Effect of Ambroxol and Beclomethasone on Lipopolysaccharide-Induced Nitrosative Stress in Bronchial Epithelial Cells

Background: Nitrosative stress is involved in different airway diseases. Lipopolysaccharide (LPS) induces neutrophil-related cytokine release and nitrosative stress in human bronchial epithelial (BEAS-2B) cells alone or with human polymorphonuclear neutrophils (PMNs). Ambroxol protects against oxidative stress, and beclomethasone dipropionate is an anti-inflammatory drug. Objectives: We evaluated the ability of ambroxol and/or beclomethasone dipropionate to inhibit LPS-induced expression/release of RANTES, IL-8, inducible NO synthase (iNOS), myeloperoxidase (MPO) and 3-nitrotyrosine (3-NT: nitrosative stress biomarker) in BEAS-2B ± PMNs stimulated with LPS (1 μg/ml). Methods: The effect of ambroxol and/or beclomethasone dipropionate on IL-8, RANTES and iNOS levels was assessed by Western blot analysis; IL-8, MPO and 3-NT levels were measured by ELISA. Cell viability was assessed by the trypan blue exclusion test. Results: In BEAS-2B alone, LPS (at 12 h) increased RANTES/iNOS expression and IL-8 levels (p < 0.001). Ambroxol suppressed LPS-induced RANTES expression and IL-8 release (p < 0.001), whilst inhibiting iNOS expression (p < 0.05). Beclomethasone dipropionate had no effect on RANTES but halved iNOS expression and IL-8 release. Coculture of BEAS-2B with PMNs stimulated IL-8, MPO and 3-NT production (p < 0.001), potentiated by LPS (p < 0.001). Ambroxol and beclomethasone dipropionate inhibited LPS-stimulated IL-8, MPO and 3-NT release (p < 0.05). Ambroxol/beclomethasone dipropionate combination potentiated the inhibition of IL-8 and 3-NT production in BEAS-2B with PMNs (p < 0.05 and p < 0.01, respectively). Ambroxol and/or beclomethasone dipropionate inhibited nitrosative stress and the release of neutrophilic inflammatory products in vitro. Conclusion: The additive effect of ambroxol and beclomethasone dipropionate on IL-8 and 3-NT inhibition suggests new therapeutic options in the treatment of neutrophil-related respiratory diseases such as chronic obstructive pulmonary disease and respiratory infections.

Th17 polarization and upper airways: new insights

It is well known that allergic rhinitis (AR) is a very common disorder affecting upper airways. There is wide evidence that AR is characterized by an inflammatory reaction driven by complex events, involving several immune-competent cells. Liu et al. [1] reviewed the role of Th17 response in upper airway diseases. At present, a robust body of evidence underlines the role played by Th17 response in upper airway inflammation. In this regard, the authors considered several aspects of Th17 involvement and modulation. However, we would like to remark some points that could emphasize the clinical relevance of Th17-mediated inflammation in upper airway disorders, mainly concerning AR. In this regard, formal demonstration of Th17 role has been provided by the Florence school that demonstrated by elegant experiments the presence of memory T cell subsets producing both IL-4 and IL17 [2]. This report followed some previous studies which reported that high serum IL-17A levels may identify patients with severe allergy both during and outside the birch pollen season [3, 4]. Noteworthy, it was proposed that IL-17 could be considered a reliable biomarker of severe allergy. This concept was proposed on the basis of these reports and the very strong relationship between serum IL-17 levels and visual analogue scale values in patients with AR [5]. In fact, patients with severe allergy to birch had the highest serum IL-17 levels. Moreover, it has been reported that sublingual immunotherapy (SLIT) was capable of decreasing serum IL-17 in allergic children [6]. This outcome was confirmed also in adult patients, particularly a significant positive association was achieved between serum IL-17 and TGF-b changes as well as with symptom improvement [7]. In addition, it has been recently reported that there are different ex vivo peripheral CD4 T subsets that express both IL-17 and IFN-c in patients with allergic rhinitis [8]. This study also reported that the functional analysis after allergen-specific CD4 expansion demonstrated that allergic patients had higher frequencies of IL-17-producing T cells than healthy non-allergic subjects. The study also provided new, interesting evidence that there is a subset coexpressing IL-17 and IFN-c for both CD4 and CD8 cells [8]. Similar findings were reported in a paediatric population [9]. Recently, it has been reported that CD161 is a highly upregulated gene in Th17 clones and all IL-17-producing cells are contained in CD161 T cells. It was demonstrated that allergic patients had a significantly higher frequency of CD3CD161, CD4CD161 and CD8CD161 cells than healthy non-allergic subjects [10]. Moreover, the expression of CD161 cells was significantly related to clinical severity. Very recently, we also reported that atopic patients with severe asthma had an increase in asthma exacerbations number, IL-17F and IL-17A expression in both nasal and bronchial lamina propria compared to mild asthmatics and controls [11]. In addition, a significant relationship was found between nasal IL-17F and bronchial neutrophil/IL-17 markers and between the latter and exacerbations, suggesting that nasal IL-17F might be informative on bronchial IL17-driven neutrophilia in atopic and severe asthmatics. This finding could reinforce the concept that IL-17 might be considered a