Association of single nucleotide polymorphisms in the F2RL1 gene with clinical and inflammatory characteristics of patients with asthma.

BACKGROUND: Proteinase-activated receptor 2 (PAR-2) is a G-protein coupled receptor associated with many inflammatory diseases, including asthma. We have shown an association between PAR-2 expression in peripheral blood monocytes and asthma severity as well as blood PAR-2 mRNA level and lung function. Since F2RL1 (the gene encoding PAR-2) polymorphisms affect PAR-2 expression, we hypothesize they may affect asthma severity. METHODS: We recruited 76 subjects with asthma of varying severity and collected clinical (FEV1 [% predicted], FEV1/FVC, IgE) and immunological (PAR-2 mRNA, blood eosinophils) disease parameters. We also genotyped these individuals for 3 F2RL1 SNPs (-45C/T, -149C/G, c.621C/T). RESULTS: We found that the F2RL1 SNP "C" allele of -45C/T (rs1529505) was associated with PAR-2 mRNA and blood eosinophils. F2RL1 SNP c.621C/T (rs631465) was associated with PAR-2 mRNA. The F2RL1 SNP -149C/G (rs2242991) had no association with any of the parameters studied. This study identified one F2RL1 SNP rs1529505 is associated with parameters of asthma, but not asthma severity. CONCLUSION: Larger studies are needed to further elucidate the role of PAR-2 in the pathophysiology of asthma and the influence of genetic variation.

Dual activation of estrogen receptor alpha and glucocorticoid receptor upregulate CRTh2-mediated type 2 inflammation; mechanism driving asthma severity in women?

BACKGROUND: Type 2-high asthma is characterized by elevated levels of circulating Th2 cells and eosinophils, cells that express chemoattractant-homologous receptor expressed on Th2 cells (CRTh2). Severe asthma is more common in women than men; however, the underlying mechanism(s) remain elusive. Here we examined whether the relationship between severe asthma and type 2 inflammation differs by sex and if estrogen influences Th2 cell response to glucocorticoid (GC). METHODS: Type 2 inflammation and the proportion of blood Th2 cells (CD4+ CRTh2+ ) were assessed in whole blood from subjects with asthma (n = 66). The effects of GC and estrogen receptor alpha (ERα) agonist on in vitro differentiated Th2 cells were examined. Expression of CRTh2, type 2 cytokines and degree of apoptosis (Annexin V+ , 7-AAD) were determined by flow cytometry, qRT-PCR, western blot and ELISA. RESULTS: In severe asthma, the proportion of circulating Th2 cells and hospitalizations were higher in women than men. Women with severe asthma also had more Th2 cells and serum IL-13 than women with mild/moderate asthma. Th2 cells, eosinophils and CRTh2 mRNA correlated with clinical characteristics associated with asthma control in women but not men. In vitro, GC and ERα agonist treated Th2 cells exhibited less apoptosis, more CRTh2 as well as IL-5 and IL-13 following CRTh2 activation than Th2 cells treated with GC alone. CONCLUSION: Women with severe asthma had higher levels of circulating Th2 cells than men, which may be due to estrogen modifying the effects of GC, enhancing Th2 cell survival and type 2 cytokine production.

The CRTh2 polymorphism rs533116 G > A associates with asthma severity in older females.

Background: CRTh2 is G protein coupled receptor for prostaglandin D2 (PGD)2 expressed by immune cells that drive type 2 inflammation such as CD4+ T cells (Th2), eosinophils and group 2 innate lymphoid cells (ILC2) as well as structural cells including smooth muscle and epithelium. CRTh2-expressing cells are increased in the blood and airways of asthmatics and severe asthma is characterized by increased activity of the PGD2-CRTh2 pathway. The CRTh2 single nucleotide polymorphism (SNP) rs533116 G > A is associated with development of asthma and increased Th2 cell differentiation. Objective: To examine whether CRTh2 rs533116G > A associates with asthma severity. Since severe asthma is more common in females than males, we performed a sex-stratified analysis. Methods: Clinical data from asthmatics (n = 170) were obtained from clinic visits and chart review. Asthma severity was assessed according to ERS/ATS guidelines. Peripheral blood cells were characterized by flow cytometry and qRT-PCR. Genotyping was performed by TaqMan assay. Results: Older females (≥45 years) homozygous for minor A allele of rs533116 were more likely to have severe asthma, lower FEV1, a higher prescribed dose of inhaled corticosteroid and more type 2 inflammation than females carrying GA or GG genotypes. Comparing females and males with the AA genotype also revealed that women had more type 2 inflammation. Conclusions and significance: The polymorphism CRTh2 rs533116 G > A associates with severe asthma and type 2 inflammation in older females. This study reveals a gene-sex-aging interaction influencing the effect of CRTh2 on asthma severity.

Protease-activated receptor-2: Role in asthma pathogenesis and utility as a biomarker of disease severity.

PAR2, a receptor activated by serine proteases, has primarily pro-inflammatory roles in the airways and may play a role in asthma pathogenesis. PAR2 exerts its effects in the lungs through activation of a variety of airway cells, but also activation of circulating immune cells. There is evidence that PAR2 expression increases in asthma and other inflammatory diseases, although the regulation of PAR2 expression is not fully understood. Here we review the available literature on the potential role of PAR2 in asthma pathogenesis and propose a model of PAR2-mediated development of allergic sensitization. We also propose, based on our previous work, that PAR2 expression on peripheral blood monocyte subsets has the potential to serve as a biomarker of asthma severity and/or control.

Th2 cell markers in peripheral blood increase during an acute asthma exacerbation.

BACKGROUND: Allergic asthma is characterized by type 2 inflammation. We have shown the presence of increased type 2 inflammation in patients with severe asthma and those with frequent exacerbations. However, it is not known whether increased type 2 inflammation drives asthma exacerbations. This study aims to determine Th2 immune parameters in patients presenting to the emergency department (ED) with an acute asthma exacerbation and correlate these parameters with clinical and physiological measures of asthma. METHODS: Sixteen adults presenting to the ED with acute asthma exacerbations were recruited after giving informed consent. Ten patients returned 2 weeks later for follow-up. Physiological parameters, asthma control (ACQ6), asthma quality of life (AQLQ) questionnaires, and venous blood were collected during both visits. An immune cell profiling was performed by whole blood flow cytometry: CD4+ T cells, Th2 cells (CD4+ CRTh2+ T cells and % of CD4+ T cells expressing CRTh2), eosinophils and innate lymphoid cells (ILC2). RESULTS: During exacerbation, peripheral blood Th2 cell numbers correlated with ACQ6 and AQLQ scores, while ILC2 and eosinophil numbers did not. Subjects had higher % of CD4+ T cells expressing CRTh2 and worse FEV1 during exacerbation compared with the follow-up. The decrease in the % of CD4+ T cells expressing CRTh2 seen during the follow-up visit correlated with the improvement in lung function. CONCLUSIONS: These data suggest that Th2 cells in peripheral blood may be a sensitive measure of increasing symptoms in patients with asthma exacerbations and may serve as a biomarker of an asthma exacerbation.

Stability of peripheral blood immune markers in patients with asthma.

BACKGROUND: Asthma is a complex disease with variable course. Efforts to identify biomarkers to predict asthma severity, the course of disease and response to treatment have not been very successful so far. We have previous suggested that PAR-2 and CRTh2 expression on specific peripheral blood cell subtypes may be biomarkers of asthma severity. We reasoned that parameters that remain stable when asthma symptoms are controlled would be the most appropriate to evaluate for their utility to predict loss of asthma control and/or severity of the disease. METHODS: Nineteen stable asthmatics were recruited from the University of Alberta Asthma clinic and followed in clinic every 3 months for a total of 4 visits. Patients had spirometry and completed the ACQ questionnaire in every visit. Blood was drawn in every visit and analyzed for a number of immune parameters by flow cytometry. These parameters included PAR-2 and CRTh2 expression on monocyte subgroups and T lymphocytes respectively, as well as numbers of eosinophils, innate lymphoid type-2 cells (ILC2) and dendritic cells. Within person stability of immune and physiological parameters was calculated using the intraclass correlation (ICC) using R version 3.4.0. RESULTS: FEV1 (% predicted), FEV1/FVC ratio, ACQ5 and ACQ7 did not differ significantly over the 4 visits, as would be expected for patients with stable asthma. Peripheral blood eosinophil numbers by Kimura stain and by flow cytometry showed ICC scores of 0.44 and 0.52 respectively, indicating moderate stability. The % of ILC2 cells in peripheral blood also showed moderate stability [ICC score of 0.45 (0.14-0.67)]. The stability for all other immune parameters was poor. CONCLUSION: Among the peripheral blood immune parameters we studied, only numbers of eosinophils and ILC2 in peripheral blood were moderately stable over a year in stable asthmatics. Further studies are required to understand the reasons for the variability of the other cell types.

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma.

BACKGROUND: Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied. METHODS: We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry. RESULTS: Subjects with severe asthma had higher PAR-2 expression on CD14++CD16+ monocytes (intermediate monocytes) and also higher percentage of CD14++CD16+PAR-2+ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14++CD16+PAR-2+ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14++CD16+ PAR-2+ cells in peripheral blood compared to subjects without exacerbations. CONCLUSIONS: PAR-2 expression is increased on CD14++CD16+ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14++CD16+ monocytes may play a role in the pathogenesis of severe asthma.

Interleukin-25 initiates Th2 differentiation of human CD4(+) T cells and influences expression of its own receptor.

Human CRTh2(+) Th2 cells express IL-25 receptor (IL-25R) and IL-25 has been shown to potentiate production of Th2 cytokines. However, regulation of IL-25R and whether it participates in Th2 differentiation of human cells have not been examined. We sought to characterize IL-25R expression on CD4(+) T cells and determine whether IL-25 plays a role in Th2 differentiation. Naïve human CD4(+) T cells were activated in the presence of IL-25, IL-4 (Th2 conditions) or both cytokines to assess their relative influence on Th2 differentiation. For experiments with differentiated Th2 cells, CRTh2-expressing cells were isolated from differentiating cultures. IL-25R, GATA3, CRTh2 and Th2 cytokine expression were assessed by flow cytometry, qRT-PCR and ELISA. Expression of surface IL-25R was induced early during Th2 differentiation (2 days). Addition of IL-25 to naïve CD4(+) T cells revealed that it induces expression of its own receptor, more strongly than IL-4. IL-25 also increased the proportions of IL-4-, GATA3- and CRTh2-expressing cells and expression of IL-5 and IL-13. Activation of differentiated CRTh2(+) Th2 cells through the TCR or by CRTh2 agonist increased surface expression of IL-25R, though re-expression of CRTh2 following TCR downregulation was impeded by IL-25. These data suggest that IL-25 may play various roles in Th2 mediated immunity. We establish here it regulates expression of its own receptor and can initiate Th2 differentiation, though not as strongly as IL-4.

Genetic mechanisms in aspirin-exacerbated respiratory disease.

Aspirin-exacerbated respiratory disease (AERD) refers to the development of bronchoconstriction in asthmatics following the exposure to aspirin or other nonsteroidal anti-inflammatory drugs. The key pathogenic mechanisms associated with AERD are the overproduction of cysteinyl leukotrienes (CysLTs) and increased CysLTR1 expression in the airway mucosa and decreased lipoxin and PGE2 synthesis. Genetic studies have suggested a role for variability of genes in disease susceptibility and the response to medication. Potential genetic biomarkers contributing to the AERD phenotype include HLA-DPB1, LTC4S, ALOX5, CYSLT, PGE2, TBXA2R, TBX21, MS4A2, IL10, ACE, IL13, KIF3A, SLC22A2, CEP68, PTGER, and CRTH2 and a four-locus SNP set composed of B2ADR, CCR3, CysLTR1, and FCER1B. Future areas of investigation need to focus on comprehensive approaches to identifying biomarkers for early diagnosis.