Exploring CD26-/lo subpopulations of lymphocytes in asthma phenotype and severity: A novel CD4+ T cell subset expressing archetypical granulocyte proteins.

BACKGROUND: Asthma pathology may induce changes in naïve/memory lymphocyte proportions assessable through the evaluation of surface CD26 (dipeptidyl peptidase 4/DPP4) levels. Our aim was to investigate the association of asthma phenotype/severity with the relative frequency of CD26-/lo, CD26int and CD26hi subsets within different lymphocyte populations. METHODS: The proportion of CD26-/lo, CD26int and CD26hi subsets within CD4+ effector T cells (Teff), total CD4- lymphocytes, γδ-T cells, NK cells and NKT cells was measured in peripheral blood samples from healthy (N = 30) and asthma (N = 119) donors with different phenotypes/severities by flow cytometry. We performed K-means clustering analysis and further characterised the CD4+CD26-/lo Teff cell subset by LC-MS/MS and immunofluorescence. RESULTS: Cluster analysis including clinical and flow cytometry data resulted in four groups, two of them with opposite inflammatory profiles (neutrophilic vs. eosinophilic). Neutrophilic asthma presented reduced CD4-CD26hi cells, which negatively correlated with systemic inflammation. Eosinophilic asthma displayed a general expansion of CD26-/lo subsets. Specifically, CD4+CD26-/lo Teff expansion was confirmed in asthma, especially in atopic patients. Proteomic characterisation of this subset with a TEM/TEMRA phenotype revealed upregulated levels of innate (e.g. MPO and RNASE2) and cytoskeleton/extracellular matrix (e.g. MMP9 and ACTN1) proteins. Immunofluorescence assays confirmed the presence of atypical proteins for CD4+ T cells, and an enrichment in 'flower-like' nuclei and MMP9/RNASE2 levels in CD4+CD26-/lo Teff compared to CD4+ T lymphocytes. CONCLUSION: There is an association between CD26 levels in different lymphocyte subsets and asthma phenotype/severity. CD4+CD26-/loTEMRA cells expressing innate proteins specific to eosinophils/neutrophils could be determinant in sustaining long-term inflammation in adult allergic asthma.

Allergen Immunotherapy Enhances Airway Epithelial Antiviral Immunity in Patients with Allergic Asthma (VITAL Study): A Double-Blind Randomized Controlled Trial.

Rationale: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. Objectives: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. Methods: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. Measurements and Main Results: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid-induced expression of IFN-ß at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. Conclusions: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT04100902).

Serum exosome inflamma-miRs are surrogate biomarkers for asthma phenotype and severity.

BACKGROUND: Asthma is a heterogeneous disease with several phenotypes, endotypes and severity degrees, in which different T-cell subpopulations are involved. These cells express specific miRNAs (i.e. inflamma-miRs) that can be released to serum in exosomes after activation and be used as biomarkers of underlying inflammation. Thus, we aim to evaluate specific T-cell miRNA signatures in serum exosomes from different subgroups of asthmatic patients. METHODS: Samples from healthy donors (N = 30) and patients (N = 119) with different asthma endotypes (T2high -Atopic/T2high -Non-atopic/T2low ) and severity degrees (mild/MA and moderate-severe/MSA) were used. Demographic, clinical, haematological and biochemical characteristics were collected. Twelve miRNAs previously associated with different Th subsets were preselected and their levels in serum exosome samples were measured using RTqPCR. RESULTS: We detected five miRNAs with high confidence in serum exosomes: miR-16-5p, miR-21-5p, miR-126-3p, miR146a-5p and miR-215-5p. All of them, except miR-16-5p were upregulated in MSA patients compared to MA. A logistic regression model including each of these miRNAs was created to discriminate both conditions, rendering a ROC curve AUC of 0.896 (0.830-0.961). miR-21-5p and miR-126-3p, both involved in Th1/Th2 differentiation, were specifically augmented in T2high -Atopic patients. Of note, all these changes were found in samples collected in autumn. On the contrary, IL-6high patients with MSA, which were more obese, older, with higher neutrophil and basophil counts and TNF levels, displayed a decrease of miR-21-5p, miR-126-3p and miR-146a-5p. CONCLUSION: Immune-related miRNAs, including miR-21-5p, miR-126-3p, miR-146a-5p and miR-215-5p, can be used as clinically relevant non-invasive biomarkers of the phenotype/endotype and severity of asthma.