Towards precision medicine in asthmaimmune cell subsets and small extracellular vesicles associated biomarkers for phenotype and severity assessment

Resumo

Asthma is a chronic inflammatory syndrome that encompasses a heterogeneous group of diseases. Although common pathological dysfunctions are shared by all of them such as airway remodelling, hyperresponsiveness, inflammation, and exaggerated mucus production the causative immunopathological pathways differ. Hence, the overall aim of this thesis was to discover cell and molecular biomarkers associated with different phenotypes and severities to improve asthma diagnosis, prognosis, and treatment. Thus, given the involvement of various immune cell types throughout the disease's development like eosinophils, neutrophils, and lymphocytes our study focused specifically on these leukocytes and their subsets. Regarding molecular biomarkers, the is research also focused on the study of exosomes/small extracellular vesicles (sEVs) produced by these immune cells and their cargo (e.g., proteins and miRNAs) as a potential source of biomarkers to identify different subtypes or severities in asthma. Additionally, it is also focused on CD26, a cell surface protease whose levels distinguish naïve and different subsets of memory CD4+ T lymphocytes. Memory T cells play an essential role in asthma, as they perpetuate the recurrent infiltration of lung tissues in response to different stimuli (e.g. the presence of allergens) and the appearance of flare-ups over time. To our knowledge, this is the first time that circulating effector memory cell ratios have been used together with clinical data to identify different asthma subtypes. Extracellular vesicles, particularly sEVs or exosomes, are lipid particles secreted by all cell types that carry signalling molecules (e.g., miRNAs and proteins), participating in cell-to-cell communication. sEVs can be found in various biofluids such as serum, and therefore can serve as a source of molecular markers. In the first two chapters of the present thesis, we have focused on both miRNAs and protein content of sEVs delivered by immune cell populations involved in asthma pathology. In the first chapter, we have analysed preselected miRNAs associated with asthma or classic effector Th populations (Th1, Th2, Th17, Treg) by qRT-PCR in sEVs isolated from serum samples of patients (N=119) with different asthma phenotypes and disease severities and healthy donors (N=30). We have found four of the preselected miRNAs (hsa miR-21-5p, hsa miR-126-3p, hsa-miR146a-5p, and hsa-miR-215-5p) upregulated in moderate-severe (MSA) compared to mild (MA) asthma. In addition, a logistic regression model including these miRNAs was capable of discriminating MSA and MA individuals, computing a ROC curve AUC of 0.896 (0.830-0.961). We have also found increased levels in T2high atopic patients of certain miRNAs (hsa-miR-21-5p and hsa-miR-126-3p) related to the Th1/Th2 balance, and diminished levels of hsa-miR-21-5p, hsa-miR-126-3p, and has miR 146a-5p in patients with markers of systemic inflammation (obese MSA with high IL 6 and TNF). Finally, this study found variations in miRNA sEV cargo associated with seasonal changes, with increased levels during autumn. Therefore, these immune related miRNAs can be used as molecular biomarkers of asthma phenotype and severity. On the other hand, in the second chapter of the thesis, we have focused on another group of biomolecules comprising the sEV cargo: the proteins. In this chapter, we have selected a two-stage method to underscore the challenge of studying proteins from sEVs isolated from serum, given that these samples yield preparations with high content in contaminating serum proteins, such as albumin, which hinders the study of low-abundant proteins with non-targeted proteomic techniques. Thus, we performed: 1) a non-targeted qualitative and quantitative proteomic analysis (LC-MS/MS) to characterise sEVs in vitro delivered by different immune cell populations involved in asthma pathogenesis (eosinophils, neutrophils, Th1, Th2 and Th17) to identify specific protein markers; and 2) a preliminary study to validate the applicability of these identified protein biomarkers through their targeted quantification in serum sEV samples from asthma patients with different phenotypes and severities. As a result, we have found proteomic differences among the sEVs delivered by each cell group, especially between granulocytes (eosinophils and neutrophils) and Th cells (Th1, Th2, and Th17). Those from granulocytes were enriched in antimicrobial proteins (e.g., LCN2, LTF, MPO), while T delivered sEVs presented higher levels of ribosomal proteins (RPL and RPS proteins). Additionally, we found differentially abundant proteins between neutrophil and eosinophil sEVs (e.g., ILF2, LTF, LCN2) and among Th subsets (e.g., ISG15, ITGA4, ITGB2 or NAMPT). Finally, preliminary results using sEVs isolated from pooled serum samples of asthma individuals validated the capability of Th2-related markers (e.g., ITGA4 and ITGB2) to differentiate T2high from T2low asthma Therefore, these immune linked biomarkers could potentially serve as indicators of the underlying type of inflammation in asthma, even though a deeper analysis is needed. In the third chapter, we have shifted the focus to cellular biomarkers. Specifically, we have investigated the proportion of different memory CD4+ T lymphocyte subsets segregated based on CD26 surface levels. To do so, we have measured the expression of this protease by flow cytometry in CD4+ T effector (Teff) cells, total CD4- lymphocytes, -T cells, NK, and NKT cells in the same adult asthma cohort from chapters I and II. Our results have confirmed the expansion of CD4+CD26-/lo lymphocytes in asthma, especially in atopic patients, previously described by our group. This population was further characterised by LC-MS/MS, which revealed the upregulation of atypical proteins for a lymphocyte population. Particularly, this population expressed increased levels of proteins involved in innate immunity (e.g., MPO and RNASE2) and proteins related to cytoskeleton/extracellular matrix (e.g., MMP9, ACTN1). This finding is consistent with the initial description of this CD4+CD26-/lo subpopulation by our group as effector memory (TEM) or terminally differentiated effector memory (TEMRA) cells. Additionally, a higher proportion of cells with abnormal flower-likecloverleaf nuclei was also observed within the CD4+CD26-/lo subset. Finally, a clustering analysis using both clinical and immunophenotyping data resulted in 4 groups of patients. Interestingly, an eosinophilic and a neutrophilic-prone group were identified with different patterns of CD26 levels in all studied lymphocytes populations. Therefore, our study demonstrates an association between asthma phenotypes and CD26 levels in different lymphocyte populations, and especially a potential role of a highly differentiated effector-memory T cell population (CD4+CD26-/lo T cells) in the development of this disease. Altogether, these findings offer potential pathways for personalised therapeutic interventions and complete our understanding of the complex immunological landscape in asthma.