The effect of anti-IL5 monoclonal antibodies on regulatory and effector T cells in severe eosinophilic asthma.

INTRODUCTION: Biological treatments have redesigned the clinical management of severe eosinophilic asthmatic (SA) patients. Despite emerging evidence supporting the role of natural Killer (NK), and T regulatory cells (Treg) in the pathogenesis of asthma, no data is available on the effects of anti-IL5/IL5R therapies on these cell subsets. METHODS: We prospectively enrolled fourteen SA patients treated with benralizumab (n = 7) or mepolizumab (n = 7) and compared them with healthy controls (HC) (n = 11) and mild to moderate asthmatic (MM) patients (n = 9). Clinical parameters were collected at baseline (T0) and during follow-up. Cellular analysis, including the analysis of T/NK cell subsets, was determined through multicolor flow cytometry. RESULTS: At T0, SA patients showed higher percentages of CD4 TEM (33.3 ± 17.9 HC, 42.6 ± 16.6 MM and 66.1 ± 19.7 in SA; p < 0.0001) than HC and MM patients. With different timing, the two drugs induce a reduction of CD4 TEM ( 76 ± 19 T0; 43 ± 14 T1; 45 ± 23 T6; 62 ± 18 at T24; p < 0.0001 for mepolizumab and 55 ± 21 T0; 55 ± 22 T1; 43 ± 14 T6; 27 ± 12 at T24; p < 0.0001 for benralizumab) and an increase of Treg cells (1.2 ± 1.3 T0; 5.1 ± 2.5 T1; 6.3 ± 3.4 T6; 8.4 ± 4.6 at T24; p < 0.0001 for mepolizumab and 3.4 ± 1.7 T0; 1.9 ± 0.8 T1; 1.9 ± 1 T6; 5.1 ± 2.4 at T24; p < 0.0001 for benralizumab). The change of CD56dim PD-1+ significantly correlated with FEV1% (r = - 0.32; p < 0.01), while Treg expressing PD-1 correlates with the use of oral steroids ( r = 0.36 p = 0.0008) and ACT score (r = 0.36 p = 0.0008) p < 0.001) CONCLUSIONS: Beyond the clinical improvement, anti-IL-5 treatment induces a rebalancing of Treg and T effector cells in patients with SA.

Benralizumab affects NK cell maturation and proliferation in severe asthmatic patients.

INTRODUCTION: The mechanism of action of benralizumab is determined by its afucosylated constant fragment that binds CD16a receptors on the membrane of natural killer cells. Here we analysed changes in Natural Killer and T-cells in Severe asthmatic patients, before and after benralizumab. METHODS: Natural Killer and T-cell subsets were detected through multiparametric flow cytometry. The concentrations of serum cytokines levels were detected through multiplex assay. Functional proliferation assay was performed in follow-up samples in severe asthmatic patients. RESULTS: At baseline, severe asthmatic patients showed higher percentages of immature Natural Killer cells when compared with healthy controls. We demonstrate the proliferative capacity of these cells and their activation after benralizumab administration. Benralizumab shifted Natural Killer cell phenotypes towards maturity. Correlation between the Natural Killer cells and functional parameters and with steroid-sparing was observed. CONCLUSION: Together this data contributes to our understanding of the mechanisms of action of benralizumab in the resolution of inflammation in severe asthma patients.

NK and T Cell Immunological Signatures in Hospitalized Patients with COVID-19.

Severe acute respiratory syndrome caused by coronavirus 2 emerged in Wuhan (China) in December 2019 and has severely challenged the human population. NK and T cells are involved in the progression of COVID-19 infection through the ability of NK cells to modulate T-cell responses, and by the stimulation of cytokine release. No detailed investigation of the NK cell landscape in clinical SARS-CoV-2 infection has yet been reported. A total of 35 COVID-19 hospitalised patients were stratified for clinical severity and 17 healthy subjects were enrolled. NK cell subsets and T cell subsets were analysed with flow cytometry. Serum cytokines were detected with a bead-based multiplex assay. Fewer CD56dimCD16brightNKG2A+NK cells and a parallel increase in the CD56+CD69+NK, CD56+PD-1+NK, CD56+NKp44+NK subset were reported in COVID-19 than HC. A significantly higher adaptive/memory-like NK cell frequency in patients with severe disease than in those with mild and moderate phenotypes were reported. Moreover, adaptive/memory-like NK cell frequencies were significantly higher in patients who died than in survivors. Severe COVID-19 patients showed higher serum concentrations of IL-6 than mild and control groups. Direct correlation emerged for IL-6 and adaptive/memory-like NK. All these findings provide new insights into the immune response of patients with COVID-19. In particular, they demonstrate activation of NK through overexpression of CD69 and CD25 and show that PD-1 inhibitory signalling maintains an exhausted phenotype in NK cells. These results suggest that adaptive/memory-like NK cells could be the basis of promising targeted therapy for future viral infections.

Integrated approach to bronchoalveolar lavage cytology to distinguish interstitial lung diseases.

BACKGROUND: Broncho-alveolar lavage (BAL) is a safe diagnostic procedure, useful for differentiating fibrotic lung disorders and for excluding malignancy and infection. A recent multicenter study demonstrated a new, relatively sensitive, and specific index called Bronchoalveolar Cytology Threshold (BCT), useful for distinguishing healthy individuals from patients with lung diseases. OBJECTIVES: In our study, BCT was applied for the first time to the analysis of interstitial lung diseases (ILDs), investigating its potential for differential diagnosis. Combinations of BAL cells that improve diagnostic accuracy for ILDs were studied and are proposed. METHODS: A retrospective analysis of BAL samples was performed. We considered more than 1000 BAL samples from patients investigated for ILD, performed at Siena University Hospital. The samples enrolled for the study included 468 patients: 413 with and 55 without ILD. BAL was performed for diagnostic purposes in line with international guidelines. BCT were calculated according to available literature. RESULTS: Among ILDs, patients with fibrotic hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF) and sarcoidosis showed significantly lower BCTs than unclassified ILD. Asbestosis patients showed significantly lower BCTs than nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), connective tissue disease related ILD (CTD-ILD), sarcoidosis and unclassified ILD patients. COP patients showed significantly higher BCT than IPF, f-HP and sarcoidosis. Moreover, COP patients were best distinguished by BCT. CONCLUSION: The analysis of BAL features is currently included in the diagnostic algorithm of ILDs. BAL cell patterns and BCT index can provide useful information for distinguishing ILDs, reducing the need for invasive procedures. Integrated approaches to BAL analysis can improve the interpretation of results without further cost or loss of time.

A Comprehensive Evaluation of Mepolizumab Effectiveness in a Real-Life Setting.

INTRODUCTION: Interleukin-5 (IL-5) is the principal cytokine regulating eosinophil growth, differentiation, activation, and expression. It is a specific target of mepolizumab, an anti-IL-5 monoclonal antibody used in the treatment of severe eosinophilic asthma. This new drug can improve symptoms, reduce asthma exacerbations and steroid use. Few data are available on its efficacy for nasal symptoms. OBJECTIVE: To describe the all-round clinical impact of mepolizumab in a real-life setting, evaluating the efficacy and safety of the drug in severe eosinophilic asthma patients. POPULATION AND METHODS: We retrospectively collected the clinical and functional data on 27 patients (16 males) affected with severe eosinophilic asthma, diagnosed at the Siena Regional Referral Centre and monitored for 6 months. Clinical, immunological, and functional data at baseline and follow-up were entered in a database together with comorbidities, number of exacerbations, steroid treatment, multiple-flow exhaled nitric oxide, and validated questionnaires. RESULTS: A significant reduction in asthma exacerbations was observed in all patients after 6 months of the biological therapy (p = 0.0009), and 4/6 patients discontinued chronic oral steroids. A significant improvement in ACT, FEV1, SNOT22, and alveolar nitric oxide was observed after 1 month of mepolizumab (p = 0.003, p = 0.007, p = 0.047, and p = 0.019, respectively) and maintained after 6 months of treatment. After 6 months, FeNO 50 was reduced as well (p = 0.030). Mepolizumab was very well tolerated, and no major side effects were observed. CONCLUSIONS: Our study suggests that mepolizumab is effective in improving control of asthma, lung function parameters, exhaled biomarkers, and nasal symptoms in patients with severe eosinophilic asthma.

Pirfenidone in idiopathic pulmonary fibrosis: real-life experience in the referral centre of Siena.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia and has a median survival after diagnosis of 2-5 years. Pirfenidone is the first approved antifibrotic drug for the treatment of IPF. Here we report the functional progress, side effects and survival data of a population of patients with IPF, diagnosed at our centre and treated with pirfenidone. METHODS: We enrolled 91 patients with IPF (71 males) treated with pirfenidone. Clinical, survival and functional details were collected retrospectively at start of therapy and after 12, 24, 36 and 48 months of treatment. Lung function tests at least 12 months before starting therapy were available for 40 patients and were entered in the database, as well as side effects. RESULTS: During the observation period (922 ± 529 days), 27 patients died, 5 patients underwent lung transplant and 10 patients interrupted therapy due to adverse events or IPF progression. The median survival was 1606 days. There was a significant reduction in disease progression rate, as measured by trend of forced vital capacity, after 1 year of therapy with respect to before treatment (p = 0.0085). Forced vital capacity reduction rate was progressively higher in the subsequent years of treatment. Treatment-related side effects were reported in 25 patients and were predominantly mild. Overall, four patients discontinued therapy due to severe photosensitivity. CONCLUSIONS: Our findings confirm the efficacy of pirfenidone in reducing functional progression of IPF and its excellent safety profile in a real-life setting. This study, designed on a long-term follow up, contributes to the growing evidence on safety, tolerability and efficacy of pirfenidone in IPF. The reviews of this paper are available via the supplemental material section.

Role of BNP and echo measurement for pulmonary hypertension recognition in patients with interstitial lung disease: An algorithm application model.

BACKGROUND: This study evaluated the role of echocardiography and BNP in patients with interstitial lung disease (ILD), to identify those with PH and RV dysfunction. The aims of this study were: 1-to evaluate the accuracy of an algorithm including BNP, DLCO and echocardiographic measurements to identify PH and RV dysfunction; 2- to evaluate BNP and Echo values concordance in relation to right catheterization measurement. METHODS: We analyzed 113 patients with diagnosis of ILD. Echo examination included: Pulmonary systolic, diastolic and mean Arterial Pressure (PAPs, PAPd, PAP mean), End-Diastolic and End-Systolic right ventricle diameters, Inferior Caval Vein diameter, and Tricuspid Annular Plane Systolic Excursion (TAPSE). Patients revealing increased PAPs at echocardiography underwent to catheterization. RESULTS: Patients with PAPs > 40 mm Hg (37 patients), PAPmean ≥ 25 mm Hg (23 patients) and PAPd ≥ 20 mm Hg showed BNP increased (157 ± 96 vs 16 ± 14 pg/ml p = 0.004; 201 ± 120 vs 28 ± 17 pg/mL; 124 ± 88 vs 23 ± 18 pg/ml p < 0.001) as patients with TAPSE ≤16 mm (25 patients) (145 ± 104 vs 26 ± 21 pg/ml p < 0.001). In catheterized patients (37 patients) BNP was increased in patients with invasive PAPs > 40 mm Hg (165 ± 112 vs 29 ± 14 pg/ml p < 0.02), as well as in patients with Wedge pressure > 14 mm Hg (199 + 153 vs 54 + 39 pg/mL; p = 0.01). ROC Curve analysis showed that elevated values of BNP, PAPs, PAP mean are able to assess PH. On the other hand, lower values of DLCO (<40%) and TAPSE (≤16 mm) detect PH. Logistic regression analysis of the previous parameters, confirmed their diagnostic role in PH detection. CONCLUSIONS: In patients with ILD, an algorithm including BNP, DLCO and echocardiography could be useful for non invasive screening of PH. CLINICAL TRIAL REGISTRATION NAME AND NUMBER: ARTEMIS-HP trial; ID number: NCT00879229.