Interplay of leucocyte-platelet complexes and clinical response to eltrombopag in immune thrombocytopenia patients.

Eltrombopag (ELT) is a thrombopoietin-receptor agonist that stimulates platelet (PLT) production in patients with primary immune thrombocytopenia (ITP). One potential mechanism of ELT is modulating the inflammatory response by increasing PLTs binding to leucocytes. This study examined the effect of ELT on leucocyte-PLTs complexes in 38 ITP patients. Patients, predominantly females with a mean age of 59 years, underwent treatments like corticosteroids, intravenous immunoglobulin and splenectomy. Compared to healthy donors, ITP patients exhibited lower percentages of lymphocyte with bound PLTs, but similar monocyte- or neutrophil with bound PLTs. ELT treatment increased PLTs counts and all types of leucocyte with bound PLTs. Network analysis showed dynamic changes in leucocyte with bound PLTs relationships due to ELT. Machine learning indicated that higher percentages of monocytes with bound PLTs were linked to a better clinical response to ELT. A possible mechanism was an increased IL-10 production in monocytes with bound PLTs from responder patients. This study provides insights into the immunological changes in ITP patients undergoing ELT and suggests potential predictive biomarkers for treatment response and disease monitoring.

Levels of Lysozyme and SLPI in Bronchoalveolar Lavage: Exploring Their Role in Interstitial Lung Disease.

Interstitial lung diseases (ILDs) are characterized by inflammation or fibrosis of the pulmonary parenchyma. Despite the involvement of immune cells and soluble mediators in pulmonary fibrosis, the influence of antimicrobial peptides (AMPs) remains underexplored. These effector molecules display a range of activities, which include immunomodulation and wound repair. Here, we investigate the role of AMPs in the development of fibrosis in ILD. We compare the concentration of different AMPs and different cytokines in 46 fibrotic (F-ILD) and 17 non-fibrotic (NF-ILD) patients by ELISA and using peripheral blood mononuclear cells from in vitro stimulation in the presence of lysozyme or secretory leukocyte protease inhibitor (SLPI) from 10 healthy donors. We observed that bronchoalveolar lavage (BAL) levels of AMPs were decreased in F-ILD patients (lysozyme: p < 0.001; SLPI: p < 0.001; LL-37: p < 0.001; lactoferrin: p = 0.47) and were negatively correlated with levels of TGF-ß (lysozyme: p = 0.02; SLPI: p < 0.001) and IL-17 (lysozyme: p < 0.001; SLPI: p < 0.001). We observed that lysozyme increased the percentage of CD86+ macrophages (p < 0.001) and the production of TNF-α (p < 0.001). We showed that lysozyme and SLPI were associated with clinical parameters (lysozyme: p < 0.001; SLPI: p < 0.001) and disease progression (lysozyme: p < 0.001; SLPI: p = 0.01). These results suggest that AMPs may play an important role in the anti-fibrotic response, regulating the effect of pro-fibrotic cytokines. In addition, levels of lysozyme in BAL may be a potential biomarker to predict the progression in F-ILD patients.

The Effects of Interstitial Lung Diseases on Alveolar Extracellular Vesicles Profile: A Multicenter Study.

Diagnosis of interstitial lung diseases (ILD) is difficult to perform. Extracellular vesicles (EVs) facilitate cell-to-cell communication, and they are released by a variety of cells. Our goal aimed to investigate EV markers in bronchoalveolar lavage (BAL) from idiopathic pulmonary fibrosis (IPF), sarcoidosis and hypersensitivity pneumonitis (HP) cohorts. ILD patients followed at Siena, Barcelona and Foggia University Hospitals were enrolled. BAL supernatants were used to isolate the EVs. They were characterized by flow cytometry assay through MACSPlex Exsome KIT. The majority of alveolar EV markers were related to the fibrotic damage. CD56, CD105, CD142, CD31 and CD49e were exclusively expressed by alveolar samples from IPF patients, while HP showed only CD86 and CD24. Some EV markers were common between HP and sarcoidosis (CD11c, CD1c, CD209, CD4, CD40, CD44, CD8). Principal component analysis distinguished the three groups based on EV markers with total variance of 60.08%. This study has demonstrated the validity of the flow cytometric method to phenotype and characterize EV surface markers in BAL samples. The two granulomatous diseases, sarcoidosis and HP, cohorts shared alveolar EV markers not revealed in IPF patients. Our findings demonstrated the viability of the alveolar compartment allowing identification of lung-specific markers for IPF and HP.

Reduced airway levels of fatty-acid binding protein 4 in COPD: relationship with airway infection and disease severity.

BACKGROUND: For still unclear reasons, chronic airway infection often occurs in patients with Chronic Obstructive Pulmonary Disease (COPD), particularly in those with more severe airflow limitation. Fatty-acid binding protein 4 (FABP4) is an adipokine involved in the innate immune response against infection produced by alveolar macrophages (Mɸ). We hypothesized that airway levels of FABP4 may be altered in COPD patients with chronic airway infection. METHODS: In this prospective and controlled study we: (1) compared airway FABP4 levels (ELISA) in induced sputum, bronchoalveolar lavage fluid (BALF) and plasma samples in 52 clinically stable COPD patients (65.2 ± 7.9 years, FEV1 59 ± 16% predicted) and 29 healthy volunteers (55.0 ± 12.3 years, FEV1 97 ± 16% predicted); (2) explored their relationship with the presence of bacterial airway infection, defined by the presence of potentially pathogenic bacteria (PPB) at ≥103 colony-forming units/ml in BALF; (3) investigated their relationship with the quantity and proportion of Mɸ in BALF (flow cytometry); and, (4) studied their relationship with the severity of airflow limitation (FEV1), GOLD grade and level of symptoms (CAT questionnaire). RESULTS: We found that: (1) airway levels of FABP4 (but not plasma ones) were reduced in COPD patients vs. controls [219.2 (96.0-319.6) vs. 273.4 (203.1-426.7) (pg/ml)/protein, p = 0.03 in BALF]; (2) COPD patients with airway infection had lower sputum FABP4 levels [0.73 (0.35-15.3) vs. 15.6 (2.0-29.4) ng/ml, p = 0.02]; (3) in COPD patients, the number and proportion of Mɸ were positively related with FABP4 levels in BALF; (4) BALF and sputum FABP4 levels were positively related with FEV1, negatively with the CAT score, and lowest in GOLD grade D patients. CONCLUSIONS: Airway FABP4 levels are reduced in COPD patients, especially in those with airway infection and more severe disease. The relationship observed between Mɸ and airway FABP4 levels supports a role for FABP4 in the pathogenesis of airway infection and disease severity in COPD.

Antimicrobial peptides, disease severity and exacerbations in bronchiectasis.

RATIONALE: Recently a frequent exacerbator phenotype has been described in bronchiectasis, but the underlying biological mechanisms are unknown. Antimicrobial peptides (AMPs) are important in host defence against microbes but can be proinflammatory in chronic lung disease. OBJECTIVES: To determine pulmonary and systemic levels of AMP and their relationship with disease severity and future risk of exacerbations in bronchiectasis. METHODS: A total of 135 adults with bronchiectasis were prospectively enrolled at three European centres. Levels of cathelicidin LL-37, lactoferrin, lysozyme and secretory leucocyte protease inhibitor (SLPI) in serum and sputum were determined at baseline by ELISA. Patients were followed up for 12 months. We examined the ability of sputum AMP to predict future exacerbation risk. MEASUREMENTS AND MAIN RESULTS: AMP levels were higher in sputum than in serum, suggesting local AMP release. Patients with more severe disease at baseline had dysregulation of airway AMP. Higher LL-37 and lower SLPI levels were associated with Bronchiectasis Severity Index, lower FEV1 (forced expiratory volume in 1 s) and Pseudomonas aeruginosa infection. Low SLPI levels were also associated with the exacerbation frequency at baseline. During follow-up, higher LL-37 and lower SLPI levels were associated with a shorter time to the next exacerbation, whereas LL-37 alone predicted exacerbation frequency over the next 12 months. CONCLUSIONS: Patients with bronchiectasis showed dysregulated sputum AMP levels, characterised by elevated LL-37 and reduced SLPI levels in the frequent exacerbator phenotype. Elevated LL-37 and reduced SLPI levels are associated with Pseudomonas aeruginosa infection and can predict future risk of exacerbations in bronchiectasis.

A point-of-care neutrophil elastase activity assay identifies bronchiectasis severity, airway infection and risk of exacerbation.

INTRODUCTION: Neutrophil elastase activity in sputum can identify patients at high risk of airway infection and exacerbations in bronchiectasis. Application of this biomarker in clinical practice is limited, because no point-of-care test is available. We tested whether a novel semi-quantitative lateral flow device (neutrophil elastase airway test stick - NEATstik®) can stratify bronchiectasis patients according to severity, airway infection and exacerbation risk. METHODS: Sputum samples from 124 patients with stable bronchiectasis enrolled in the UK and Spain were tested using the NEATstik®, which scores neutrophil elastase concentration from 0 (<8 µg·mL-1 elastase activity) to 10 (maximum detectable neutrophil elastase activity). High neutrophil elastase activity was regarded as a NEATstik® grade >6. Severity of disease, airway infection from sputum culture and exacerbations over the 12 months were recorded. An independent validation was conducted in 50 patients from Milan, Italy. MEASUREMENTS AND MAIN RESULTS: Patients had a median age of 69 years and forced expiratory volume in 1 s (FEV1) 69%. High neutrophil elastase activity was associated with worse bronchiectasis severity using the bronchiectasis severity index (p=0.0007) and FEV1 (p=0.02). A high NEATstik® grade was associated with a significant increase in exacerbation frequency, incident rate ratio 2.75 (95% CI 1.63-4.64, p<0.001). The median time to next exacerbation for patients with a NEATstik® grade >6 was 103 days compared to 278 days. The hazard ratio was 2.59 (95% CI 1.71-3.94, p<0.001). Results were confirmed in the independent validation cohort. CONCLUSIONS: A novel lateral flow device provides assessment of neutrophil elastase activity from sputum in minutes and identifies patients at increasing risk of airway infection and future exacerbations.

Binding of Platelets to Lymphocytes: A Potential Anti-Inflammatory Therapy in Rheumatoid Arthritis.

Soluble factors released from platelets can modulate the immune response of leukocytes. We and others have recently found that T lymphocytes with bound platelets have reduced proliferation and IFN-γ and IL-17 production. Thus, we speculate that if we induce the binding of platelets to lymphocytes, we will be able to regulate the inflammatory response. When we cocultured platelets with lymphocytes at different ratios, we were able to increase the percentage of lymphocytes with bound platelets. The coculture of platelets with lymphocytes in the presence of stimulation decreased the production of IFN-γ and TNF-α, T cell proliferation, and the expression of CD25, PD-L1, and SLAM. However, this coculture increased CD39 expression. All of these effects were dependent on the dose of platelets and operated indistinctly with platelets from different healthy donors. When platelets were cocultured in the same compartment with lymphocytes, we observed less IFN-γ and TNF-α production and T lymphocyte proliferation than in cultures with platelets separated from lymphocytes by a 0.4-µm pore size filter. The binding of platelets to lymphocytes was blocked with anti-P-selectin Abs, and when this occurred we observed higher IFN-γ and TNF-α production than in nonblocked conditions. The cocultures of platelets with synovial fluid cells from rheumatoid arthritis patients reduced inflammatory cytokine production and increased IL-10 production. These results suggest that platelet binding to lymphocytes effectively regulates T lymphocyte function. This mechanism could be easily applied to reduce inflammatory responses.

Association of Platelet Binding to Lymphocytes with B Cell Abnormalities and Clinical Manifestations in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with the polyclonal activation of B lymphocytes and the production of autoantibodies that cause immune complex-related inflammation. Immunological factors derived from platelets modulate B cell function in SLE disease. However, platelets do not only modify the immune system by soluble factors. The binding of platelets to lymphocytes can modulate immune response. Thus, we speculate that the binding of platelets to lymphocytes in SLE patients may play a role in abnormal B lymphocyte response and the pathogenesis of SLE. We observed that levels of lymphocytes with bound platelets were higher in SLE patients than in healthy donors (HD). In SLE patients, the percentage of B lymphocytes with bound platelets positively correlated with plasmatic levels of IgG, IgA, IL-10, and soluble CD40L and negatively correlated with IgM levels, though not in HD. Preswitched memory B lymphocytes were the subpopulation with more bound platelets. Lymphocytes with bound platelets from both HD and SLE patients had major levels of CD86 and BAFFR and a greater production of IL-10 than lymphocytes without bound platelets. However, only B lymphocytes with bound platelets from SLE patients had increased levels of IgG and IgA on their surface. SLE patients with a suggestive renal manifestation had the highest levels of B and T lymphocytes with bound platelets. These results suggest that the binding of platelets to lymphocytes plays a role in SLE disease and that controlling this binding may be a promising therapeutic approach.

Rituximab-induced interleukin-15 reduction associated with clinical improvement in rheumatoid arthritis.

Rituximab therapy alters all aspects of B-cell participation in the disturbed immune response of rheumatoid arthritis patients. To determine the impact of B-cell depletion on other immune compartments, we analysed levels of soluble and surface interleukin-15 (IL-15) along with the frequency of IL-15-related subsets after rituximab treatment. We then studied the correlation of observed changes with clinical activity. Heparinized blood samples from 33 rheumatoid arthritis patients were collected on days 0, 30, 90 and 180 after each of three rituximab cycles. Serum cytokine levels were determined by ELISA. Interleukin-15 trans-presentation was analysed by cytometry. Flow cytometry with monoclonal antibodies was performed to analyse circulating cell subsets. Interleukin-15 was detected in the serum of 25 patients before initiating the treatment. Rituximab then progressively reduced serum IL-15 (138 ± 21 pg/ml at baseline, 48 ± 18 pg/ml after third cycle, P = 0·03) along with IL-17 (1197 ± 203 pg/ml at baseline, 623 ± 213 pg/ml after third cycle, P = 0·03) and tended to increase the frequency of circulating regulatory T cells (3·1 ± 1 cells/µl at baseline, 7·7 ± 2 cells/µl after third cycle). Rituximab also significantly decreased IL-15 trans-presentation on surface monocytes of patients negative for IL-15 serum (mean fluorescence intensity: 4·82 ± 1·30 at baseline, 1·42 ± 0·69 after third cycle P = 0·05). Reduction of serum IL-15 was associated with decrease in CD8(+)  CD45RO(+) /RA(+) ratio (1·17 ± 0·21 at baseline, 0·36 ± 0·06 at third cycle, P = 0·02). DAS28, erythrocyte sedimentation rate and C-reactive protein correlated significantly with CD8(+)  CD45RO(+) /RA(+) ratio (R = 0·323, R = 0·357, R = 0·369 respectively, P < 0·001). Our results suggest that sustained clinical improvement after rituximab treatment is associated with IL-15/memory T-cell-related mechanisms beyond circulating B cells.

Potential Role of Circulating PD-L1+ Leukocytes as a Predictor of Response to Anti-PD-(L)1 Therapy in NSCLC Patients.

PD-(L)1 inhibitors are part of the treatment strategy for non-small cell lung cancer (NSCLC) although its efficacy is limited to certain patients. Our study aimed to identify patients who might benefit from anti-PD-(L)1 inhibitors by analyzing the PD-L1 expression on circulating leukocytes and its evolution during treatment. One hundred thirteen NSCLC patients, according to their radiological response after 10-12 weeks of treatment, were classified into responders, stable, and progressive disease. Percentages of circulating PD-L1+ leukocytes, PD-L1+ platelets (PLTs), and leukocyte-PLT complexes were assessed using flow cytometry, and plasma concentrations of soluble immunomodulatory factors were quantified by ELISA. Responders exhibited significantly higher pre-treatment percentages of PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs than progressors. The percentages of these populations decreased in responders post-treatment, contrasting with stables and progressors. PLTs notably contributed to PD-L1 expression in CD14+ cells and neutrophils. Plasma cytokine analysis revealed baseline differences only in IL-17 concentration among groups, whereas network analyses highlighted distinct association patterns between plasma molecules and PD-L1+ leukocytes after 10-12 weeks of treatment. Our findings suggest that pre-treatment assessment of circulating PD-L1+ neutrophils, PD-L1+ CD14+ cells, and PD-L1+ PLTs may be helpful in identifying NSCLC patients who are potential candidates for anti-PD-(L)1 therapy.