Biomarker profiles and immune cell populations in distinct asthma endotypes

<p dir="ltr">Asthma affects 260 million individuals globally and imposes a substantial health burden. Its hallmarks include chronic airway inflammation, airway hyperresponsiveness, and structural and immune remodeling, manifesting as cough, dyspnea, and impaired lung function. Asthma comprises heterogeneous endotypes defined by underlying mechanistic pathways. Significant knowledge gaps remain in understanding clinical characteristics and pathophysiology in various endotypes, and there is an unmet need for clinically applicable biomarkers to guide diagnosis and treatment. This thesis aimed to identify clinical characteristics, blood protein biomarkers, and immune cell populations in asthma endotypes in early adulthood.</p><p dir="ltr">In Study I, we investigated clinical characteristics and inflammation-related proteins in individuals with and without asthma, stratified by blood eosinophil and neutrophil counts. The study included 2063 young adults from the BAMSE (Children, Allergy, Environment, Stockholm, Epidemiology) cohort. Ninety-two plasma proteins were quantified by Proximity Extension Assay (PEA). Clinical features were analyzed using group-wise statistics, and logistic regression identified protein biomarkers across asthma endotypes. Eosinophilic asthma showed a distinct profile with atopic comorbidities, higher body mass index (BMI), elevated fractional exhaled nitric oxide (FeNO), and impaired lung function, whereas neutrophilic asthma lacked a clear phenotype. Matrix metalloproteinase 10 (MMP10) and C-C motif chemokine ligand 4 (CCL4) emerged as potential plasma biomarkers for eosinophilic and neutrophilic asthma, respectively.</p><p dir="ltr">In Study II, we examined innate lymphoid cell (ILC) and T cell subsets in blood in individuals with and without asthma, stratified by type 2 and non-type 2 features. Eighty-six young adults from the BAMSE cohort were included and categorized based on asthma status and elevated blood eosinophil levels (≥0.3x10⁹/L) and/or IgE sensitization to inhalant allergens (≥0.35 kU/L). We performed 18-parameter flow cytometry on peripheral blood mononuclear cells to identify ILCs and CD4⁺ and CD8⁺ T cells. Data were normalized, clustered, and analyzed using logistic regression. A higher frequency of CD4⁺ CRTH2⁺ T memory cells was associated with type 2 features independent of asthma. Type 2 asthma showed increased CD62L⁺ ILC2s and reduced KLRG1⁺ central memory T cell frequencies, whereas non-type 2 asthma exhibited elevated CD45RO⁺ ILC2s and CD8⁺ memory T cells. Thus, type 2 features reflected ILC2 and Th2 cell signatures, while non-type 2 asthma was associated with CD45RO⁺ ILC2s and CD8⁺ memory T cells.</p><p dir="ltr">In Study III, we investigated inflammation-related blood proteins for classifying eosinophilic and non-eosinophilic asthma, compared protein levels across eosinophil subgroups, and evaluated their relationships with lung function. Levels of 384 proteins were quantified in dried blood spots from 762 young adults in the BAMSE cohort using PEA. Participants were stratified by asthma status and blood eosinophil count (< or ≥0.3x10⁹/L). Feature selection was performed using sparse Partial Least Squares Discriminant Analysis (sPLS-DA) and Light Gradient-Boosting Machine (LightGBM). Logistic regression examined associations between proteins and asthma endotypes, while linear regression assessed links with lung function. 18 proteins contributed to eosinophilic endotype classification; among these, Cysteine rich with EGF like domains 2 (CRELD2) and Syntaxin 8 (STX8) contributed to eosinophilic asthma, whereas Cathepsin C (CTSC) was linked to non-eosinophilic asthma. Several shortlisted proteins showed subgroup associations, and five were associated with lung function among individuals with asthma.</p><p dir="ltr">Together, the findings in this thesis indicate that</p><ul><li>Eosinophilic and type 2 asthma exhibited clear clinical phenotypes, whereas neutrophilic asthma and non-type 2 asthma lacked defining characteristics. We hypothesize that type 2 mechanisms are more readily captured systemically than non-type 2 mechanisms.</li><li>MMP10 and CCL4 emerged as potential plasma biomarkers for eosinophilic and neutrophilic asthma. Among 18 proteins contributing to eosinophilic endotype classification, CTSC was linked to non-eosinophilic asthma, while CRELD2 and STX8 were candidate biomarkers for eosinophilic asthma. Additionally, five proteins were associated with lung function in eosinophilic and non-eosinophilic asthma, warranting further evaluation.</li><li>Type 2 features, with and without asthma, were well represented by Th2 cells and CD62L⁺ ILC2s. Th2 cells were associated with type 2 features regardless of asthma status, suggesting limited specificity for type 2 asthma. Non-type 2 asthma was linked to CD45RO⁺ ILC2 and CD8⁺ memory T cell profiles.</li></ul><p dir="ltr">Our work advances understanding of asthma endotypes through integrated analyses of clinical profiles, protein biomarkers, and immune cell perturbations.</p><h3 dir="ltr">List of scientific papers</h3><p dir="ltr">I. <b>Kere M,</b> Klevebro S, Hernandez-Pacheco N, Ödling M, Ekström S, Mogensen I, Janson C, Palmberg L, van Hage M, Georgelis A, Bergström A, Kull I, Melén E, Björkander S. Exploring proteomic plasma biomarkers in eosinophilic and neutrophilic asthma. Clin Exp Allergy. 2023 Feb;53(2):186-197. <a href="https://doi.org/10.1111/cea.14229" rel="noreferrer" target="_blank">https://doi.org/10.1111/cea.14229</a></p><p dir="ltr">II. <b>Kere MM</b>, Björkander S, Merid SK, Hernandez-Pacheco N, Maier P, Merritt AS, Bergstrom A, Kull I, Daub CO, Mjösberg J, Tibbitt CA, Melen E. Distinct Phenotypes of Peripheral Innate Lymphoid Cells and T Cells in Type 2 and Non-Type 2 Asthma. Clin Transl Allergy. 2025 Sep;15(9):e70108. <a href="https://doi.org/10.1002/clt2.70108" rel="noreferrer" target="_blank">https://doi.org/10.1002/clt2.70108</a></p><p dir="ltr">III. <b>Kere MM</b>, Restuadi R, Bendes A, Merid SK, Björkander S, Gruzieva O, Merritt AS, Kull I, Bergstrom A, Schwenk J, Daub CO, Lenhard B, Melen E. Inflammation-related blood proteins associate with lung function in eosinophilic and non-eosinophilic asthma endotypes. [Manuscript]</p>