Differential Effects of Benralizumab and Mepolizumab on Pro‐Resolving Mediators

Asthma is a chronic airway inflammation resulting from repeated stimuli and inadequate pro-resolving pathway engagement 1. Fatty acids and their metabolites regulate inflammation persistence and resolution by pro-inflammatory eicosanoids and specialized pro-resolving mediators (SPM) (resolvins, lipoxins, protectins, and maresins) 2. Currently, biological therapies have revolutionized asthma management; however, the mechanisms underlying disease resolution remain incompletely understood. Therefore, we aim to evaluate the production of resolvins in severe asthmatic (SA) patients treated with mepolizumab or benralizumab over 1 year. We conducted this prospective study involving 6 healthy individuals, 18 mild–moderate asthmatic patients, 29 SA patients treated with benralizumab (n = 16) or mepolizumab (n = 13). Asthma was diagnosed in accordance with the Spanish Guidelines for Asthma Management version 5. 5 (GEMA 5. 5) 3. It was also used to guide the administration of biological therapy, considering blood eosinophil counts and lung function. Biologic treatments consisted of subcutaneous administration of mepolizumab (100 mg every 4 weeks) or benralizumab (30 mg every 4 weeks for the first three doses, followed by 30 mg every 8 weeks). Peripheral blood, serum, and induced sputum samples from healthy and asthmatics individuals were collected at baseline (pretreated), 6 months, and 1 year after treatment initiation, recording clinical and demographic variables (Table 1 and Table S1). All biological samples were collected during scheduled treatment visits, and they were not obtained during medical consultations related to acute asthma exacerbations or respiratory infections. Human Resolvin E3 (RvE3) (MyBioSource Inc. , San Diego, CA, USA) and Protectin D1 (PD1) (FineTest, Palm Coast, FL, USA) were analyzed in serum and sputum supernatant by ELISA. A panel of cytokines was evaluated using Luminex (TGFβ1, CXCL10, IFNγ, IL-2, IL-4, IL-5, IL-10, IL-12 (p70), IL-13, IL-17A and TNFα; Merck KGaA, Darmstadt, Germany) in both types of samples. The study protocol was approved by the Ethics Committees (PIC002-19FJD) and conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all study subjects. Healthy vs Mild–moderate asthma (p = 0. 0239) Healthy vs Benra (p = 0. 0007) Healthy vs Mepo (p = 0. 0006) Mild–moderate asthma vs Benra (p = 0. 0006) Mild–moderate asthma vs Mepo (p < 0. 0001) Mild–moderate asthma vs Benra (p = 0. 0013) Mild–moderate asthma vs Mepo (p < 0. 0001) Benra vs Mepo (p = 0. 0073) Mild–moderate asthma vs Benra (p = 0. 0003) Mild–moderate asthma vs Mepo (p = 0. 0176) Healthy vs Mepo (p = 0. 005) Mild–moderate asthma vs Mepo (p = 0. 0002) Mild–moderate asthma vs Benra (p = 0. 009) Mild–moderate asthma vs Mepo (p = 0. 0197) Mild–moderate asthma vs Benra (p < 0. 0001) Mild–moderate asthma vs Mepo (p = 0. 0002) In benralizumab-treated patients, asthma exacerbations were eliminated after 1 year, with improvements in ACT, FEV1%, and FVC% already evident at 6 months (Figure S1; Table 1; Tables S1 and S2). Sputum eosinophils and basophils decreased significantly, while total IgE (tIgE), FeNO, and other sputum cell populations remained unchanged (Figure S1; Table 1; Tables S1 and S2). In mepolizumab-treated patients, although asthma exacerbations were significantly higher at baseline compared with the benralizumab-treated patients and accompanied by lower ACT scores (Table 1), asthma exacerbations declined significantly over 1 year, similar to treatment with benralizumab, accompanied by marked improvements in ACT, FEV1%, and FVC%, as well as a significant reduction in sputum eosinophils (Figure S1; Table 1; Tables S1 and S2). Moreover, at baseline, SA patients assigned to benralizumab treatment exhibited greater peripheral eosinophilia and lower FEV1/FVC ratio compared with those assigned to mepolizumab (Table 1). These differences may reflect the typical clinical profile of patients selected for each therapy, as benralizumab is often indicated for patients with high blood eosinophil counts and worse lung function. Also, we analyzed the distribution of T2-related comorbidities (atopy, nasal polyposis and allergic rhinitis) and BMI, between treatment groups, with no significant differences observed (Table 1). Serum SPM analysis showed that PD1 levels were significantly lower in patients with SA than in both healthy individuals and those with mild-to-moderate asthma (Figure 1A; Table S3). A similar decrease was observed for RvE3, but only when comparing SA with the mild-to-moderate asthma group (Figure 1C and Table S3). In benralizumab-treated patients, PD1 serum levels increased slightly after treatment and were significantly higher than in those receiving mepolizumab at 6 months (Figure 1B; Table S3) ; between groups, no differences were observed for RvE3 levels; however, a significant decrease from 6 months to 1 year was noted in the mepolizumab group (Figure 1D; Table S3). Notably, baseline SPM levels in SA treated with mepolizumab positively correlated with improvements in lung function and negatively with inflammatory parameters (Table S4), suggesting an association with a more favorable clinical response. Moreover, we observed a moderate negative correlation between PD1 levels and the percentage of sputum eosinophils (Spearman r = −0. 544, p = 0. 0469; Table S4) in the benralizumab group at 6 months after treatment. Serum pro-inflammatory analysis revealed significantly lower CXCL10 and higher IL-17A levels in patients with SA compared with both healthy individuals and those with mild-to-moderate asthma (Figure 1E, G; Table S3). At baseline and at 1 year, CXCL10 levels were significantly higher in the mepolizumab group (Figure 1F; Table S3). IL-17A levels were significantly lower in this group at 1 year, with no baseline differences (Figure 1H). Across all time points, patients with SA treated with benralizumab showed higher TGFβ1 levels than those receiving mepolizumab, with a decreasing trend at 6 months in the mepolizumab group (Figure 1J). No other pro-inflammatory mediators showed significant changes, except for TNF-α at baseline between treatment groups (Figure 1; Table S3). In sputum supernatants (Figure S2), RvE3 levels were significantly lower in patients with SA than in those with mild-to-moderate asthma (Figure S2C; Table S5). No significant differences were observed for PD1 or other pro-inflammatory mediators across groups (healthy, mild-to-moderate asthma, and SA; Figure S2; Table S5). PD1 levels showed an increasing trend after treatment with mepolizumab, while RvE3 tended to increase in patients receiving benralizumab (Figure S2B, D; Table S5). Additionally, TGFβ1 levels significantly decreased after treatment in benralizumab-treated patients, with no changes in the other mediators (Figure S2J; Table S5). Given that the biosynthesis of SPM depends on the activity of specific immune cell populations, we explored whether changes in circulating blood cells could be associated with the observed differences in PD1 and RvE3 levels (Table S6). In the benralizumab-treated group, increased serum PD1 levels at 6 months were accompanied by higher monocyte counts (Figure 1B; Table S6). Considering that PD1 has been reported to promote macrophage polarization toward non-inflammatory phenotypes 4, this finding may suggest a potential mechanism contributing to the resolution of inflammation in these patients. Another explanation could be that increased circulating monocytes reflect enhanced monocyte-to-macrophage trafficking to the airways, consistent with ADCC-driven macrophage expansion induced by benralizumab 5. In contrast, although RvE3 levels decreased over time in the mepolizumab-treated group (Figure 1D), no parallel changes in circulating immune cell populations were observed, except for reductions in total leukocytes and lymphocytes at specific time points (Table S6). These findings could indicate that the relationship between circulating immune cells and SPM levels is likely multifactorial and may be influenced by multiple factors, not only by peripheral blood cells. This study provides the first evidence that SPMs are modulated by biological therapies. PD1 and RvE3 are key mediators involved in the resolution of inflammation. PD1, detected in the airways of patients with asthma, reduces eosinophilia, inflammatory mediator production, and airway hyperresponsiveness supporting its role in resolving allergic airway inflammation 6. Its inverse correlation with sputum eosinophils supports the hypothesis that complete eosinophil clearance may trigger resolution pathways, aligning with the idea that asthma control requires not only inflammation suppression but also active resolution 7, 8, consistent with lung-function improvements observed with benralizumab. In addition, alterations in its biosynthesis have been reported in eosinophils from patients with SA, demonstrating that activated human eosinophils represent an important source of PD1 and may act as a self-resolving mechanism in eosinophilic inflammation; however, its production appears to be impaired in SA 9. Similarly, RvE3 has been associated with the active resolution of allergic airway inflammation through the regulation of leukocyte recruitment and macrophage-mediated clearance of inflammatory cells 10. Furthermore, RvE3 has been shown to inhibit the chemotaxis of polymorphonuclear leukocytes and reduce the production of pro-inflammatory cytokines, supporting its role in the resolution of inflammatory responses 1. Additionally, both mediators can be detected in systemic circulation and have been proposed as biomarkers reflecting systemic resolution pathways 11. Our findings suggest that, beyond eosinophil depletion, benralizumab and mepolizumab may promote resolution pathways and help restore airway homeostasis, pointing to potential mechanistic differences between IL-5 and IL-5Rα blockade. Baseline differences in CXCL10 and TGFβ1 levels between treatment groups likely reflect distinct patient profiles rather than treatment effects. Patients receiving benralizumab typically present higher eosinophil counts and poorer lung function 12, as also observed in our cohort. These biological differences may help explain the divergent patterns observed in CXCL10 and TGFβ1. CXCL10 is a chemokine commonly associated with the maintenance of T1 immune responses in severe asthma 13. The higher levels of serum CXCL10 observed in the mepolizumab-treated group at baseline may indicate a relatively stronger T1 component, whereas lower levels in the benralizumab group could be consistent with a more pronounced T2/eosinophilic inflammatory phenotype 14, 15. In contrast, TGFβ1 is a key mediator of airway remodeling and fibrosis 16. The higher levels observed in serum and sputum in the benralizumab group may be associated with structural airway changes, contributing to airflow limitation and lower FEV1/FVC ratios. This is consistent with previous evidence linking increased TGFβ1 expression to airway fibrosis and impaired lung function in SA 16. Overall, these differences in CXCL10 and TGFβ1 likely reflect distinct inflammatory and remodeling profiles between treatment groups, rather than a direct pharmacological effect. This study has several limitations. The relatively small cohort size may limit the generalizability of the findings, and the observational design precludes causal interferences. In addition, SPM are typically present at very low concentrations in biological fluids, which may affect the specificity and sensitivity of their quantification, particularly when using ELISA 17. Although all measurements were within the assay's detection range, ELISA-based results should ideally be confirmed using more sensitive and specific techniques, such as mass spectrometry 18. Therefore, larger cohorts and comprehensive lipidomic analyses are needed to validate these findings and better define the role of SPM in biological therapy for SA. Nonetheless, the consistent patterns observed across analyses provide preliminary evidence supporting the involvement of these mediators in SA and its treatment with biological drugs. Summarizing, treatment with mepolizumab and benralizumab was associated with significant clinical improvement in patients with SA, including enhanced lung function and disease control. Our findings also indicate that both biological drugs may differentially modulate SPM and pro-inflammatory mediators profiles at both systemic and pulmonary levels, suggesting distinct mechanisms of action. Further studies in larger cohorts are needed to validate the role of SPM as biomarkers and potential therapeutic targets in SA. V. P. conceived of the manuscript and designed the study. J. B. , J. A. C. , Á. A. -F. , J. M. R. -M. , D. R. -G. , A. S. -S. , G. G. -S. , and A. M. F. performed experiments; J. B. , J. A. C. , Á. A. -F. , J. M. R. -M. , A. M. F. , and V. P. performed data analysis. J. B. , M. V. -M. , D. B. , E. J. P. -R. , C. R. -B. , M. J. R. -N. , and J. S. included patients and retrieved clinical data. J. B. , J. A. C. , and V. P. edited and wrote the manuscript. All authors contributed to the article and approved the submitted version. The authors would like to thank all the patients for their voluntary participation as well as all technical and nursing staff involved in the project (Manuela Garcia del Potro, Erica Aguado Wakui, Esther Gamella Álvarez, María Remedios Marquina Valero, Almudena Batanero Rodríguez and Alba Sevillano Nava). Declaration of Artificial Intelligence generated content: During the preparation of this manuscript, the authors used ChatGPT for the purposes of improving the grammar and readability of the text. This work was supported by ISCIII—Instituto de Salud Carlos III and co-funded by the European Union, FIS (Fondo de Investigación Sanitaria—Spanish Health Research Fund) grant numbers PI21/00896 and PI24/00024; Miguel Servet Program grant number CP23/00017; Contratos PFIS grant number FI25/00057; Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Respiratorias (CIBERES) ; research support grants provided by the Spanish Society of Allergology and Clinical Immunology (SEAIC) Foundation grant number 24B₀2; Conchita Rábago Foundation (FCR) ; FEDER funds (Fondo Europeo de Desarrollo Regional). This work has been supported with the collaboration of the “Programa de la Fundación AstraZeneca Premios Jóvenes Investigadores”. The study protocol was approved by the Ethics Committee of the involved hospitals, and the study was conducted in accordance with the principles set forth in the Declaration of Helsinki. J. A. C. , J. M. R. -M. and D. R. -G. reports receiving payments for lectures and/or educational events form Astra Zeneca and/or GSK. J. A. C. and M. J. R. -N. reports receiving research grant support from AstraZeneca. M. J. R. -N. has received payments for lectures from AstraZeneca and SANOFI. M. V. -M. reports receiving payments for lectures and support for attending meetings and/or travel by AstraZeneca, GSK, Gebro, and Organon S. A. ; as well as having received grant support for research from Organon S. A. V. P. has received honoraria (advisory board, speaker) and/or institutional grant/research support from AstraZeneca and GSK and has held an unpaid leadership or fiduciary role in committees belonging to the EAACI. The rest of authors declare no conflicts of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1: all70356-sup-0001-Supinfo. docx. Figure S1: Longitudinal monitoring of clinical and lung function outcomes in severe asthmatic patients treated with benralizumab or mepolizumab. Panels display changes from baseline over a 1-year period for (A) exacerbation rates, (B) asthma control test (ACT score), and lung function parameters: (C, D) FEV1, (E, F) FVC, (G) FEV1/FVC and (H) FeNO. Bars represent mean values at each specified time point. Percentage change from baseline is showed numerically with directional arrows. Figure S2: Modulation of sputum SPM and inflammatory mediators following anti–IL-5/IL-5Rα treatment in patients with SA. (A, C, E, G, I) Baseline sputum levels of PD1, RvE3, CXCL10, IL-17A, and TGFβ1 in healthy individuals, patients with mild-to-moderate asthma, and patients with severe asthma. (B, D, F, H, J) Longitudinal changes in sputum PD1, RvE3, CXCL10, IL-17A, and TGFβ1 of patients with severe asthma treated with benralizumab or mepolizumab at baseline and after the treatment (Post-Tx). Data are presented as mean ± SEM with individual values. Table S1: Demographic and clinical characteristics of severe asthmatic patients recruited to the study after biologic treatment. Table S2: Changes in clinical and inflammatory parameters from baseline to 6 and 12 months in benralizumab- and mepolizumab-treated patients. Table S3: Specialized pro resolving and pro-inflammatory mediators measured in serum. Table S4: Correlations between serum pro-inflammatory mediators and clinical inflammatory markers in biologic-treated severe asthmatics patients. Table S5: Specialized pro resolving and pro-inflammatory mediators measured in sputum supernatant. Table S6: Changes in circulating blood cell populations at baseline, 6 months, and 12 months in benralizumab- and mepolizumab-treated patients. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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