Search for biomarkers related to rhinitis and different asthma phenotypes by serum proteomics and immunoassays

Resumo

Asthma is a heterogeneous disease with several clinical phenotypes and molecular endotypes. However, the specific connection between asthma phenotypes and the underlying pathological features is difficult to explain. Thus, the overall aim of the present thesis was to search for biomarkers associated with rhinitis and different phenotypes (allergic and non-allergic) and severities (intermittent-mild and moderate-severe) of asthma, which could have an application in the diagnosis, prognosis or treatment of this disease. The mechanisms underlying asthma are multiple and complex, but the immune system plays a key role in the pathophysiology of this disease. Therefore, we decided to further study the role of the immune system in different phenotypes or severities of asthma through the analysis of certain biomarkers previously related to this disease: CD14 (innate immune system) and CD26/CD126 (adaptive immune system). CD14 is a receptor mainly expressed on monocytes, which participates in the lipopolysaccharide (LPS) signalling. Our results show that both the expression of CD14 on monocytes and the normalized levels of soluble CD14 (sCD14) in serum are reduced in allergic asthma vs. healthy controls. This reduction can be explained by the expansion of CD14low cells, probably non-classical monocytes with a high capacity to differentiate into M2 macrophages. In addition, sCD14 levels are associated with the promoter SNP of CD14 (-159 C/T). Thus, subjects with the C allele and CC genotype have lower levels of sCD14, as well as increased risk of allergic asthma. On the other hand, CD26 is a peptidase mainly expressed by helper T (TH) lymphocytes. Our results evidence a high correlation between the expression of CD26 on those cells and DPP4 activity (~ soluble CD26/sCD26) in vitro. Moreover, the expression of this molecule differentiates subtypes of T cells (TH17>>TH1>TH2>Treg), as well as discriminates between cells with different stages of differentiation: central-memory T cells (TCM, CD26high, CD45RA-CCR7+CD28+), naïve T cells (TN, CD26int, CD45RA+CCR7+CD28+), and terminally differentiated or effector-memory T cells (TEM or TEMRA, CD26low, CD45RA+/-CCR7-CD28-). In addition, there is an expansion of different subpopulations with low expression of CD26 in asthma: Tlow cells (CD25lowCD26lowCD127low) in allergic asthma and CD26- γδ-T cells in non-allergic asthma. This expansion could explain the decreased levels of sCD26 in serum in both allergic and non-allergic asthma. The reduction of CD26 levels in asthmatics could lead to a higher proliferation capacity of their lymphocytes, as well as to increased migration to inflammatory sites. The expression of CD26 and CD126 in CD4- T lymphocytes is highly correlated. Thus, the population of TEM or TEMRA cells increased in non-allergic asthma is also CD126-. CD126 is also related to asthma severity, since patients with a moderate-severe profile displayed reduced levels of this molecule on the surface of monocytes, neutrophils and lymphocytes, compared to intermittent-mild asthmatics. This could imply the role of trans-signalling (activation of CD126-cells by sCD126) in the severity of asthma. Finally, a non-target study aimed to identify new biomarkers associated with different phenotypes of asthma was also performed. Firstly, a new methodology for the analysis of the low abundance proteins based on the reduction of serum proteome complexity was created. This implies the use of combinatorial peptide ligand libraries (CPLLs) and the identification and relative quantification of serum proteins by iTRAQ and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Through this methodology, 26 proteins with differential changes between groups were described, many of them possible biomarkers of allergic asthma (IGFALS, protein AMBP, or HSPG2) or non-allergic (CFI, CFH, or MASP1) and severities (e.g., IGFALS in moderate-severe allergic asthma).