Biomarkers in asthma, potential for therapeutic intervention.

Asthma is a heterogeneous disease characterized by multiple phenotypes with varying risk factors and therapeutic responses. This Commentary describes research on biomarkers for T2-"high" and T2-"low" inflammation, a hallmark of the disease. Patients with asthma who exhibit an increase in airway T2 inflammation are classified as having T2-high asthma. In this endotype, Type 2 cytokines interleukins (IL)-4, IL-5, and IL-13, plus other inflammatory mediators, lead to increased eosinophilic inflammation and elevated fractional exhaled nitric oxide (FeNO). In contrast, T2-low asthma has no clear definition. Biomarkers are considered valuable tools as they can help identify various phenotypes and endotypes, as well as treatment response to standard treatment or potential therapeutic targets, particularly for biologics. As our knowledge of phenotypes and endotypes expands, biologics are increasingly integrated into treatment strategies for severe asthma. These treatments block specific inflammatory pathways or single mediators. While single or composite biomarkers may help to identify subsets of patients who might benefit from these treatments, only a few inflammatory biomarkers have been validated for clinical application. One example is sputum eosinophilia, a particularly useful biomarker, as it may suggest corticosteroid responsiveness or reflect non-compliance to inhaled corticosteroids. As knowledge develops, a meaningful goal would be to provide individualized care to patients with asthma.

Reliability of Total Serum IgE Levels to Define Type 2 High and Low Asthma Phenotypes.

Background: High total IgE levels are weak predictors of T2High and have been reported in nonallergic asthma. Therefore, the role of total serum IgE (IgE) in the T2High phenotype is still debated. Objective: This study investigated the reliability of stratifying asthmatics into IgEHigh and IgELow within the T2High and T2Low phenotypes. Methods: This cross-sectional single-center study investigated the association of clinical, functional, and bio-humoral parameters in a large asthmatic population stratified by IgE ≥ 100 kU/L, allergen sensitization, B-EOS ≥ 300/µL, and FENO ≥ 30 ppb. Results: Combining T2 biomarkers and IgE identifies (1) T2Low-IgELow (15.5%); (2) T2Low-IgEHigh (5.1%); (3) T2High-IgELow (33.6%); and T2High-IgEHigh (45.7%). T2Low-IgELow patients have more frequent cardiovascular and metabolic comorbidities, a higher prevalence of emphysema, and higher LAMA use than the two T2High subgroups. Higher exacerbation rates, rhinitis, and anxiety/depression syndrome characterize the T2Low-IgEHigh phenotype vs. the T2Low-IgELow phenotype. Within the T2High, low IgE was associated with female sex, obesity, and anxiety/depression. Conclusions: High IgE in T2Low patients is associated with a peculiar clinical phenotype, similar to T2High in terms of disease severity and nasal comorbidities, while retaining the T2Low features. IgE may represent an additional biomarker for clustering asthma in both T2High and T2Low phenotypes rather than a predictor of T2High asthma "per se".

Blood transcriptomic signature in type-2 biomarker-low severe asthma and asthma control.

BACKGROUND: Patients with type-2 (T2) cytokine-low severe asthma often have persistent symptoms despite suppression of T2 inflammation with corticosteroids. OBJECTIVES: We sought to analyze whole blood transcriptome from 738 samples in T2-biomarker-high/-low patients with severe asthma to relate transcriptomic signatures to T2 biomarkers and asthma symptom scores. METHODS: Bulk RNA-seq data were generated for blood samples (baseline, week 24, week 48) from 301 participants recruited to a randomized clinical trial of corticosteroid optimization in severe asthma. Unsupervised clustering, differential gene expression analysis, and pathway analysis were performed. Patients were grouped by T2-biomarker status and symptoms. Associations between clinical characteristics and differentially expressed genes (DEGs) associated with biomarker and symptom levels were investigated. RESULTS: Unsupervised clustering identified 2 clusters; cluster 2 patients were blood eosinophil-low/symptom-high and more likely to be receiving oral corticosteroids (OCSs). Differential gene expression analysis of these clusters, with and without stratification for OCSs, identified 2960 and 4162 DEGs, respectively. Six hundred twenty-seven of 2960 genes remained after adjusting for OCSs by subtracting OCS signature genes. Pathway analysis identified dolichyl-diphosphooligosaccharide biosynthesis and assembly of RNA polymerase I complex as significantly enriched pathways. No stable DEGs were associated with high symptoms in T2-biomarker-low patients, but numerous associated with elevated T2 biomarkers, including 15 that were upregulated at all time points irrespective of symptom level. CONCLUSIONS: OCSs have a considerable effect on whole blood transcriptome. Differential gene expression analysis demonstrates a clear T2-biomarker transcriptomic signature, but no signature was found in association with T2-biomarker-low patients, including those with a high symptom burden.

Mitochondria dysfunction in airway epithelial cells is associated with type 2-low asthma.

Background: Type 2 (T2)-low asthma can be severe and corticosteroid-resistant. Airway epithelial cells play a pivotal role in the development of asthma, and mitochondria dysfunction is involved in the pathogenesis of asthma. However, the role of epithelial mitochondria dysfunction in T2-low asthma remains unknown. Methods: Differentially expressed genes (DEGs) were identified using gene expression omnibus (GEO) dataset GSE4302, which is originated from airway epithelial brushings from T2-high (n = 22) and T2-low asthma patients (n = 20). Gene set enrichment analysis (GSEA) was implemented to analyze the potential biological pathway involved between T2-low and T2-high asthma. T2-low asthma related genes were identified using weighted gene co-expression network analysis (WGCNA). The mitochondria-related genes (Mito-RGs) were referred to the Molecular Signatures Database (MSigDB). T2-low asthma related mitochondria (T2-low-Mito) DEGs were obtained by intersecting the DEGs, T2-low asthma related genes, and Mito-RGs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) was performed to further explore the potential function of the T2-low-Mito DEGs. In addition, the hub genes were further identified by protein-protein interaction (PPI), and the expressions of hub genes were verified in another GEO dataset GSE67472 and bronchial brushings from patients recruited at Tongji Hospital. Results: Six hundred and ninety-two DEGs, including 107 downregulated genes and 585 upregulated genes were identified in airway epithelial brushings from T2-high and T2-low asthma patients included in GSE4302 dataset. GSEA showed that mitochondrial ATP synthesis coupled electron transport is involved in T2-low asthma. Nine hundred and four T2-low asthma related genes were identified using WGCNA. Twenty-two T2-low-Mito DEGs were obtained by intersecting the DEGs, T2-low asthma and Mito-RGs. The GO enrichment analysis of the T2-low-Mito DEGs showed significant enrichment of mitochondrial respiratory chain complex assembly, and respiratory electron transport chain. PPI network was constructed using 22 T2-low-Mito DEGs, and five hub genes, ATP5G1, UQCR10, NDUFA3, TIMM10, and NDUFAB1, were identified. Moreover, the expression of these hub genes was validated in another GEO dataset, and our cohort of asthma patients. Conclusion: This study suggests that mitochondria dysfunction contributes to T2-low asthma.

T2-Low Asthma: A Discussed but Still Orphan Disease.

Asthma affects 10% of the worldwide population; about 5% of cases are severe with the need for target therapies such as biologics. All the biologics approved for asthma hit the T2 pathway of inflammation. T2-high asthma is classified as allergic and non-allergic, whereas T2-low asthma can be further defined as paucigranulocytic asthma, Type 1 and Type-17 inflammation and the neutrophilic form that accounts for 20-30% of all patients with asthma. Neutrophilic asthma's prevalence is even higher in patients with severe or refractory asthma. We searched Medline and PubMed archives from the past ten years for articles with the subsequent titles: "neutrophilic asthma", "non-type 2 asthma" and "paucigranulocytic asthma". We identified 177 articles; 49 were considered relevant by the title and 33 by the reading of the abstract. Most of these articles are reviews (n = 19); only 6 are clinical trials. No study identified an effective treatment. We used the literature reported by these articles to search for further biologic treatments that target pathways different from T2. We identified 177 articles, 93 of which were considered relevant for the review and included in the present article. In conclusion, T2-low asthma remains poorly investigated in terms of biomarkers, especially as a therapeutic orphan disease.

Comprehensive Characterization of Difficult-to-Treat Asthma Reveals Near Absence of T2-Low Status.

BACKGROUND: Asthma is conventionally stratified as type 2 inflammation (T2)-high or T2-low disease. Identifying T2 status has therapeutic implications for patient management, but a real-world understanding of this T2 paradigm in difficult-to-treat and severe asthma remains limited. OBJECTIVES: To identify the prevalence of T2-high status in difficult-to-treat asthma patients using a multicomponent definition and compare clinical and pathophysiologic characteristics between patients classified as T2-high and T2-low. METHODS: We evaluated 388 biologic-naive patients from the Wessex Asthma Cohort of difficult asthma (WATCH) study in the United Kingdom. Type 2-high asthma was defined as 20 parts per billion or greater FeNO , 150 cells/µL or greater peripheral blood eosinophils, the need for maintenance oral corticosteroids, and/or clinically allergy-driven asthma. RESULTS: This multicomponent assessment identified T2-high asthma in 93% of patients (360 of 388). Body mass index, inhaled corticosteroid dose, asthma exacerbations, and common comorbidities did not differ by T2 status. Significantly worse airflow limitation was found in T2-high compared with T2-low patients (FEV1/FVC 65.9% vs 74.6%). Moreover, 75% of patients defined as having T2-low asthma had raised peripheral blood eosinophils within the preceding 10 years, which left only seven patients (1.8%) who had never had T2 signals. Incorporation of sputum eosinophilia 2% or greater into the multicomponent definition in a subset of 117 patients with induced sputum data similarly found that 96% (112 of 117) met criteria for T2-high asthma, 50% of whom (56 of 112) had sputum eosinophils 2% or greater. CONCLUSIONS: Almost all patients with difficult-to-treat asthma have T2-high disease; less than 2% of patients never display T2-defining criteria. This highlights a need to assess T2 status comprehensively in clinical practice before labeling a patient with difficult-to-treat asthma as T2-low.

Clinical features of asthma with connective tissue diseases.

BACKGROUND: The clinical features of asthma with connective tissue diseases (CTDs) are not well-known. This study therefore aimed to investigate the clinical characteristics of asthma with CTDs. METHODS: We retrospectively examined the records of adults (≥18 years old) with asthma followed up between January 2010 and December 2019. We then compared the clinical features of asthma with and without CTDs. RESULTS: Among 568 subjects with asthma, 42 subjects (7.4%) had CTDs. The most frequent concomitant CTD was rheumatoid arthritis (n = 23, 54.8%), followed by systemic lupus erythematosus (n = 6, 14.3%). The proportion of women (with vs. without CTDs, 85.7% vs. 56.5%, p < 0.001) and Global Initiative for Asthma step were higher (Step 4 or 5, with vs. without CTDs, 81.0% vs. 62.0%, p = 0.01) in asthma with CTDs, whereas frequency of allergic rhinitis was higher in asthma without CTDs (with vs. without CTDs, 7.1% vs. 26.1%, p = 0.005). Eosinophil ratio (with vs. without CTDs, 2.1% vs. 3.5%, p = 0.009) and total immunoglobulin E level (with vs. without CTDs, 43 IU/mL vs. 237 IU/mL, p = 0.002) were lower in asthma with CTDs. In terms of lung function, percentage predicted forced vital capacity (with vs. without CTDs, 86.7% vs. 99.7%, p = 0.008) and percentage predicted forced expiratory volume in 1 s (%FEV1) (with vs. without CTDs, 77.2% vs. 88.4%, p = 0.02) were all lower in asthma with CTDs. With multivariable analysis, CTDs (odds ratio [OR] 2.8, 95%CI 1.3-6.0; p = 0.008), chronic obstructive pulmonary disease (OR 3.8, 95%CI 2.1-6.7; p < 0.001) and asthma onset at <20 years old (OR 1.8, 95%CI 1.1-3.2; p = 0.03) were associated with low FEV1 (defined as %FEV1 < 80%) in asthma. CONCLUSIONS: Asthma with CTDs was related to lower lung function and low-T2 inflammation asthma.

Evaluating the effects of vitamin D Level on airway obstruction in two asthma endotypes in humans and in two mouse models with different intake of vitamin D during early-life.

Introduction: Asthma is primarily divided into two categories: type 2 (T2-high) and non-type 2 (T2-low). A relationship between asthma severity and vitamin D deficiency has been identified, but its impact on each asthma endotype remains unknown. Methods: We clinically examined the influence of vitamin D on patients with T2-high (n = 60) or T2-low asthma (n = 36) compared with controls (n = 40). Serum 25(OH)D levels, inflammatory cytokines and spirometry were measured. Mouse models were then used to further analyze the effects of vitamin D on both asthmatic endotypes. BALB/c mice were fed with vitamin D-deficient (LVD), -sufficient (NVD), or -supplemented diets (HVD) throughout lactation and offspring followed the same diet after weaning. Offspring were sensitized/challenged with ovalbumin (OVA) to establish "T2-high" asthma or OVA combined with ozone exposure (OVA + ozone) to induce "T2-low" asthma. Spirometry and serum, bronchoalveolar lavage fluid (BALF), and lung tissues were analyzed. Results: Serum 25(OH)D levels were decreased in asthmatic patients compared with controls. Patients with vitamin D deficiency (Lo) had varying degrees of elevation of the pro-inflammatory cytokines IL-5, IL-6, and IL-17A, decreased expression of the anti-inflammatory cytokine IL-10, and altered forced expiratory volume in the first second as a percentage of predicted value (FEV1%pred) in both asthmatic endotypes. Vitamin D status had a stronger correlation with FEV1%pred in T2-low asthma than T2-high asthma, and 25(OH)D level was only positively linked to maximal mid-expiratory flow as a percentage of predicted value (MMEF%pred) in the T2-low group. Inflammation, hyperresponsiveness, and airway resistance (RL) was increased in both asthma models compared with controls while vitamin D deficiency further increased airway inflammation and airway obstruction. These findings were particularly prominent in T2-low asthma. Discussion: The potential function and mechanisms of vitamin D and both asthma endotypes should be studied individually, and further analysis of the potential signaling pathways involved with vitamin D on T2-low asthma is warranted.

Club cell 10-kDa protein (CC10) as a surrogate for identifying type 2 asthma phenotypes.

Club cell 10-kDa protein (CC10) is a documented biomarker for airway obstructive diseases. Primarily produced by nonciliated club cells in the distal airway and in nasal epithelial cells, CC10 suppresses Th2 cell differentiation and Th2 cytokine production. In this study, we aimed to determine whether CC10 can also be used as an alternative biomarker for identifying Type 2 (T2) asthma.74 patients with asthma, and 24 healthy controls were enrolled in the study. T2-high asthma was defined as elevation in two or more biomarkers, such as sputum eosinophilia ≥ 3%, high blood eosinophils ≥ 300/µL, or high FeNO ≥ 30 ppb. T2-low asthma was defined as no elevation in biomarkers. Enzyme-linked immunosorbent assay (ELISA) was used to assess the CC10 levels in plasma.The plasma CC10 level in patients with T2-high asthma was lower than that of patients with T2-low asthma and healthy controls (P < 0.05). To distinguish between T2-high and T2-low phenotype in patients with asthma, a receiver-operating characteristic (ROC) analysis was performed. It showed a sensitivity of 58.1% and specificity of 78.0% when using 22.74 ng/ml of plasma CC10. Correlation analysis indicated that the plasma CC10 level was inversely correlated with sputum eosinophil, blood eosinophil, and FeNO, and positively correlated with log PD20. However, no correlation with sputum neutrophil percentages, macrophage percentages, IgE, or lung function was found.Plasma CC10 is potentially useful in predicting T2-high and T2-low asthma. Lower plasma CC10 was associated with enhanced airway hyperresponsiveness, and Type 2 inflammation.

Asthma Management in Adults.

Management of asthma in adults has advanced in the past 10 years. Central to these advances has been further clarification of type (T) 2 mechanisms of airway inflammation and utilization of T2 biomarkers, that is, eosinophils and fractional exhaled nitric oxide. In addition, epithelial cells are emerging as significant contributors to inflammation through generation of alarmins to initiate local injury as well as downstream pathways. Five new biologics, mepolizumab, benralizumab, reslizumab, dupilumab, and tezepelumab, were approved to join omalizumab and revolutionize severe asthma treatment. These biologics significantly prevent exacerbations to spare systemic corticosteroids use and their side effects. Guidelines attest to the effectiveness of inhaled corticosteroids/long-acting ß-agonists (formoterol) for both maintenance and rescue therapy. Focused updates to the Expert Panel Report addressed limited but specific questions relevant to asthma control. Future guidelines should include phenotype/endotype-directed therapeutics to gain more precision-directed treatment.

Exacerbation Profile and Risk Factors in a Type-2-Low Enriched Severe Asthma Cohort: A Clinical Trial to Assess Asthma Exacerbation Phenotypes.

Rationale: The past 25 years have seen huge progress in understanding of the pathobiology of type-2 (T2) asthma, identification of measurable biomarkers, and the emergence of novel monoclonal antibody treatments. Although present in a minority of patients with severe asthma, very little is known about the mechanisms underlying T2-low asthma, making it a significant unmet need in asthma research. Objectives: The objective of this study was to explore the differences between study exacerbators and nonexacerbators, to describe physiological changes at exacerbation in those who are T2HIGH and T2LOW at the time of exacerbation, and to evaluate the stability of inflammatory phenotypes when stable and at exacerbation. Methods: Exacerbation assessment was a prespecified secondary analysis of data from a 48-week, multicenter, randomized controlled clinical study comparing the use of biomarkers and symptoms to adjust steroid treatment in a T2-low severe asthma-enriched cohort. Participants were phenotyped as T2LOW (fractional exhaled nitric oxide ⩽ 20 ppb and blood eosinophil count ⩽ 150 cells/µl) or T2HIGH (fractional exhaled nitric oxide > 20 or blood eosinophil count > 150) at study enrollment and at each exacerbation. Here, we report the findings of the exacerbation analyses, including comparison of exacerbators and nonexacerbators, the physiological changes at exacerbation in those who had evidence of T2 biology at exacerbation versus those that did not, and the stability of inflammatory phenotypes when stable and at exacerbation. Measurements and Main Results: Of the 301 participants, 60.8% (183) had one or more self-reported exacerbations (total of 390). Exacerbators were more likely to be female, have a higher body mass index, and have more exacerbations requiring oral corticosteroid and unscheduled primary care attendances for exacerbations. At enrollment, 23.6% (71) were T2LOW and 76.4% (230) T2HIGH. The T2LOW group had more asthma primary care attendances, were more likely to have a previous admission to HDU (high dependency unit)/ICU and to be receiving maintenance oral corticosteroids. At exacerbation, the T2LOW events were indistinguishable from T2HIGH exacerbations in terms of lung function (mean fall in T2LOW FEV1, 200 [400] ml vs. T2HIGH 200 [300] ml; P = 0.93) and symptom increase (ACQ5: T2LOW, 1.4 [0.8] vs. T2HIGH, 1.3 [0.8]; P = 0.72), with no increase in T2 biomarkers from stable to exacerbation state in the T2LOW exacerbations. The inflammatory phenotype within individual patients was dynamic; inflammatory phenotype at study entry did not have a significant association with exacerbation phenotype. Conclusions: Asthma exacerbations demonstrating a T2LOW phenotype were physiologically and symptomatically similar to T2HIGH exacerbations. T2LOW asthma was an unstable phenotype, suggesting that exacerbation phenotyping should occur at the time of exacerbation. The clinically significant exacerbations in participants without evidence of T2 biology at the time of exacerbation highlight the unmet and pressing need to further understand the mechanisms at play in non-T2 asthma. Clinical trial registered with www.clinicaltrials.gov (NCT02717689).

Genome-wide association study identifies kallikrein 5 in type 2 inflammation-low asthma.

BACKGROUND: Clinical studies of type 2 (T2) cytokine-related neutralizing antibodies in asthma have identified a substantial subset of patients with low levels of T2 inflammation who do not benefit from T2 cytokine neutralizing antibody treatment. Non-T2 mechanisms are poorly understood in asthma but represent a redefined unmet medical need. OBJECTIVE: We sought to gain a better understanding of genetic contributions to T2-low asthma. METHODS: We utilized an unbiased genome-wide association study of patients with moderate to severe asthma stratified by T2 serum biomarker periostin. We also performed additional expression and biological analysis for the top genetic hits. RESULTS: We identified a novel protective single nucleotide polymorphism at chr19q13.41, which is selectively associated with T2-low asthma and establishes Kallikrein-related peptidase 5 (KLK5) as the causal gene mediating this association. Heterozygous carriers of the single nucleotide polymorphisms have reduced KLK5 expression. KLK5 is secreted by human bronchial epithelial cells and elevated in asthma bronchial alveolar lavage. T2 cytokines IL-4 and IL-13 downregulate KLK5 in human bronchial epithelial cells. KLK5, dependent on its catalytic function, induces epithelial chemokine/cytokine expression. Finally, overexpression of KLK5 in airway or lack of an endogenous KLK5 inhibitor, SPINK5, leads to spontaneous airway neutrophilic inflammation. CONCLUSION: Our data identify KLK5 to be the causal gene at a novel locus at chr19q13.41 associated with T2-low asthma.

STOP: an open label crossover trial to study ICS withdrawal in patients with a combination of obesity and low-inflammatory asthma and evaluate its effect on asthma control and quality of life.

BACKGROUND: Asthma patients with obesity often have a high disease burden, despite the use of high-dose inhaled corticosteroids (ICS). In contrast to asthmatics with normal weight, the efficacy of ICS in patients with obesity and asthma is often relatively low. Meanwhile, patients do suffer from side effects, such as weight gain, development of diabetes, cataract, or high blood pressure. The relatively poor response to ICS might be explained by the low prevalence of type 2 inflammatory patterns (T2-low) in patients with asthma and obesity. T2-low inflammation is characterized by low eosinophilic count, low Fractional exhaled NO (FeNO), no clinically allergy-driven asthma, and no need for maintenance oral corticosteroids (OCS). We aim to study whether ICS can be safely withdrawn in patients with T2-low asthma and obesity while maintaining an equal level of asthma control. Secondary outcomes focus on the prevalence of 'false-negative' T2-low phenotypes (i.e. T2-hidden) and the effect of ICS withdrawal on parameters of the metabolic syndrome. This study will lead to a better understanding of this poorly understood subgroup and might find new treatable traits. METHODS: The STOP trial is an investigator-initiated, multicenter, non-inferiority, open-label, crossover study aiming to assess whether ICS can be safely withdrawn in adults aged 17-75 years with T2-low asthma and obesity (body mass index (BMI) ≥ 30 kg/m2). Patients will be randomly divided into two arms (both n = 60). One arm will start with fixed-dose ICS (control group) and one arm will taper and subsequently stop ICS (intervention group). Patients in the intervention group will remain ICS naïve for ten weeks. After a washout of 4 weeks, patients will crossover to the other study arm. The crossover study takes 36 weeks to complete. Patients will be asked to participate in the extension study, to investigate the long-term metabolic benefits of ICS withdrawal. DISCUSSION: This study yields valuable data on ICS tapering in patients with T2-low asthma and obesity. It informs future guidelines and committees on corticosteroid-sparing algorithms in these patients. Trial registration Netherlands Trial Register, NL8759, registered 2020-07-06, https://www.trialregister.nl/trial/8759 . Protocol version and date: version 2.1, 20 November 2020.

Ruxolitinib Ameliorates Airway Hyperresponsiveness and Lung Inflammation in a Corticosteroid-Resistant Murine Model of Severe Asthma.

Asthma prevalence has increased considerably over the decades and it is now considered as one of the most common chronic disorders in the world. While the current anti-asthmatic therapies are effective for most asthma patients, there are 5-10% subjects whose disease is not controlled by such agents and they account for about 50% of the asthma-associated healthcare costs. Such patients develop severe asthma (SA), a condition characterized by a dominant Th1/Th17 cytokine response that is accompanied by Type 2 (T2)-low endotype. As JAK (Janus Kinase) signaling is very important for the activation of several cytokine pathways, we examined whether inhibition of JAKs might lessen the clinical and laboratory manifestations of SA. To that end, we employed a recently described murine model that recapitulates the complex immune response identified in the airways of human SA patients. To induce SA, mice were sensitized with house dust mite extract (HDME) and cyclic (c)-di-GMP and then subsequently challenged with HDME and a lower dose of c-di-GMP. In this model, treatment with the JAK inhibitor, Ruxolitinib, significantly ameliorated all the features of SA, including airway hyperresponsiveness and lung inflammation as well as total IgE antibody titers. Thus, these studies highlight JAKs as critical targets for mitigating the hyper-inflammation that occurs in SA and provide the framework for their incorporation into future clinical trials for patients that have severe or difficult-to manage asthma.

STAT3 and IL-6 Contribute to Corticosteroid Resistance in an OVA and Ozone-induced Asthma Model with Neutrophil Infiltration.

Exposure to high levels of ozone contributes to insensitivity to glucocorticoids in asthma treatment, but the underlying mechanisms are not known. We built two asthma models: a "T2-high" asthma model was established by ovalbumin (OVA) sensitization/challenge and OVA sensitization/challenge combined with ozone exposure (OVA + ozone) was used to induce airway inflammation with increased numbers of neutrophils to simulate "T2-low" asthma. The expression of T-helper (Th)1/2/17-related cytokines was measured by cytokine antibody arrays. Bronchial provocation tests were carried out to evaluate the lung resistance of mice. Hematoxylin and eosin staining, periodic acid-Schiff staining, and immunohistochemical (IHC) analyses of alpha-smooth muscle actin were undertaken to observe morphology changes in lungs. The expression of glucocorticoid receptors (GRs) and phosphorylated-GR (p-GR) was measured by western blotting. Nr3c1 mRNA was quantified by RT-qPCR. Protein expression of proinflammatory cytokines, signal transducer and activator of transcription 3 (STAT3), suppressor of cytokine signaling 3 (SOCS3), and CXCL1 was measured through ELISAs, western blotting, or IHC analyses. Resected lung tissue from seven asthma patients and 10 healthy controls undergoing thoracotomy for pulmonary nodules was evaluated by IHC analyses and ELISAs. In both asthma models, mucus hypersecretion, as well as inflammation, hyperresponsiveness, and remodeling of the airways, was present compared with the control group, whereas the OVA + ozone group showed severe neutrophil infiltration. The expression of Th17-related cytokines (interleukin (IL)-6, IL-17A, IL-21), GR protein, and CXCL1 increased in the OVA + ozone group, whereas the expression of p-GR decreased. Dexamethasone (Dex) could not totally reverse the expression of p-GR and histone deacetylase-2 in the OVA + ozone group. STAT3 expression increased in the OVA + ozone group and could not be completely reversed by Dex, and nor could IL-6 expression. A positive correlation between IL-6 or IL-17A and STAT3 and negative correlation between SOCS3 and STAT3 were shown, suggesting that the IL-6/STAT3 pathway may be involved in OVA + ozone-induced corticosteroid-resistant airway inflammation. In clinical samples, IL-17A expression in lung tissue was positively correlated with percent STAT3-positive area and negatively correlated with SOCS3 expression. The IL-6/STAT3 pathway may contribute to corticosteroid insensitivity in OVA + ozone-induced neutrophilic airway inflammation through regulation of Th17 cells and could provide new targets for individual treatment of corticosteroid resistance in asthma.

Characterization of T2-Low and T2-High Asthma Phenotypes in Real-Life.

Asthma is a heterogeneous and complex condition characterized by chronic airway inflammation, which may be clinically stratified into three main phenotypes: type 2 (T2) low, T2-high allergic, and T2-high non-allergic asthma. This real-world study investigated whether phenotyping patients with asthma using non-invasive parameters could be feasible to characterize the T2-low and T2-high asthma phenotypes in clinical practice. This cross-sectional observational study involved asthmatic outpatients (n = 503) referring to the Severe Asthma Centre of the San Luigi Gonzaga University Hospital. Participants were stratified according to the patterns of T2 inflammation and atopic sensitization. Among outpatients, 98 (19.5%) patients had T2-low asthma, 127 (25.2%) T2-high non-allergic, and 278 (55.3%) had T2-high allergic phenotype. In comparison to T2-low, allergic patients were younger (OR 0.945, p < 0.001) and thinner (OR 0.913, p < 0.001), had lower smoke exposure (OR 0.975, p < 0.001) and RV/TLC% (OR 0.950, p < 0.001), higher prevalence of asthma severity grade 5 (OR 2.236, p < 0.05), more frequent rhinitis (OR 3.491, p < 0.001) and chronic rhinosinusitis with (OR 2.650, p < 0.001) or without (OR 1.919, p < 0.05) nasal polyps, but less common arterial hypertension (OR 0.331, p < 0.001). T2-high non-allergic patients had intermediate characteristics. Non-invasive phenotyping of asthmatic patients is possible in clinical practice. Identifying characteristics in the three main asthma phenotypes could pave the way for further investigations on useful biomarkers for precision medicine.

Innate Lymphoid Cells in Airway Inflammation.

Airways are constantly exposed to antigens and various pathogens. Immune cells in the airways act as first line defense system against these pathogens, involving both innate and acquired immunity. There is accumulating evidence that innate lymphoid cells, newly identified lymphoid lineage cells, play a critical role in regulating tissue homeostasis and in the pathogenesis of inflammation. Cytokines produced by other cells activate innate lymphoid cells, which in turn produce large amount of cytokines that result in inflammation. Type 2 innate lymphoid cells (ILC2s) are recognized as key component of T helper type 2 (Th2) inflammation, and are known to be elevated in type 2 (T2) human airway diseases (asthma). Th2 cytokines produced by ILC2s amplify inflammation via activation of eosinophils, B cells, mast cell, and macrophages. "T2 high asthma" has an increased Th2 response triggered by elevation of IL-4, IL-5 and IL-13 and other inflammatory mediators, leading to increased eosinophilic inflammation. The growing evidence of ILC2 contribution in the induction and maintenance of allergic inflammation suggests that targeting upstream mediators may affect both the innate and adaptive immune responses and all disease phenotypes. Blocking ILC2 activators, activation of inhibitory pathways of ILC2, or suppression of ILC2-mediated pathways may be therapeutic strategies for the type 2 airway diseases.

Tezepelumab: a novel biological therapy for the treatment of severe uncontrolled asthma.

Introduction: Thymic stromal lymphopoietin (TSLP) is overexpressed in the airways of severe asthmatics and is an upstream cytokine that orchestrates inflammatory responses in asthma. TSLP exerts its effects by binding to a high affinity heteromeric receptor complex composed of TSLPR and IL-7Rα. An association of polymorphisms in TSLP with airway hyperresponsiveness, IgE, eosinophilia and asthma has been documented. TSLP has been implicated in asthma pathophysiology. Tezepelumab is a first-in-class human monoclonal antibody that binds to TSLP, thus inhibiting its interaction with TSLP receptor complex. Tezepelumab given as an add-on-therapy to patients with severe uncontrolled asthma has shown safety, tolerability and efficacy. Several trials are evaluating the long-term safety and the efficacy of tezepelumab in adults and adolescents with severe uncontrolled asthma.Areas covered: We provide an overview of the monoclonal antibody therapeutics market for severe uncontrolled asthma, examine the underlying pathophysiology that drives TSLP and discuss the use of tezepelumab for the treatment of severe uncontrolled asthma,Expert opinion: TSLP is a promising target for T2-high and perhaps some patients with T2-low asthma. The results of preliminary clinical trials are encouraging. Several unanswered questions concerning basic pathophysiological aspects of TSLP variants, the long-term safety and efficacy of tezepelumab with different phenotypes/endotypes of asthma should be addressed.

A randomised pragmatic trial of corticosteroid optimization in severe asthma using a composite biomarker algorithm to adjust corticosteroid dose versus standard care: study protocol for a randomised trial.

BACKGROUND: Patients with difficult-to-control asthma consume 50-60% of healthcare costs attributed to asthma and cost approximately five-times more than patients with mild stable disease. Recent evidence demonstrates that not all patients with asthma have a typical type 2 (T2)-driven eosinophilic inflammation. These asthmatics have been called 'T2-low asthma' and have a minimal response to corticosteroid therapy. Adjustment of corticosteroid treatment using sputum eosinophil counts from induced sputum has demonstrated reduced severe exacerbation rates and optimized corticosteroid dose. However, it has been challenging to move induced sputum into the clinical setting. There is therefore a need to examine novel algorithms to target appropriate levels of corticosteroid treatment in difficult asthma, particularly in T2-low asthmatics. This study examines whether a composite non-invasive biomarker algorithm predicts exacerbation risk in patients with asthma on high-dose inhaled corticosteroids (ICS) (± long-acting beta agonist) treatment, and evaluates the utility of this composite score to facilitate personalized biomarker-specific titration of corticosteroid therapy. METHODS/DESIGN: Patients recruited to this pragmatic, multi-centre, single-blinded randomised controlled trial are randomly allocated into either a biomarker controlled treatment advisory algorithm or usual care group in a ratio of 4:1. The primary outcome measure is the proportion of patients with any reduction in ICS or oral corticosteroid dose from baseline to week 48. Secondary outcomes include the rate of protocol-defined severe exacerbations per patient per year, time to first severe exacerbation from randomisation, dose of inhaled steroid at the end of the study, cumulative dose of inhaled corticosteroid during the study, proportion of patients on oral corticosteroids at the end of the study, proportion of patients who decline to progress to oral corticosteroids despite composite biomarker score of 2, frequency of hospital admission for asthma, change in the 7-item Asthma Control Questionnaire (ACQ-7), Asthma Quality of Life Questionnaire (AQLQ), forced expiratory volume in 1 s (FEV1), exhaled nitric oxide, blood eosinophil count, and periostin levels from baseline to week 48. Blood will also be taken for whole blood gene expression; serum, plasma, and urine will be stored for validation of additional biomarkers. DISCUSSION: Multi-centre trials present numerous logistical issues that have been addressed to ensure minimal bias and robustness of study conduct. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02717689 . Registered on 16 March 2016.

New approaches for identifying and testing potential new anti-asthma agents.

INTRODUCTION: Asthma is a chronic disease with significant heterogeneity in clinical features, disease severity, pattern of underlying disease mechanisms, and responsiveness to specific treatments. While the majority of asthmatic patients are controlled by standard pharmacological strategies, a significant subgroup has limited therapeutic options representing a major unmet need. Ongoing asthma research aims to better characterize distinct clinical phenotypes, molecular endotypes, associated reliable biomarkers, and also to develop a series of new effective targeted treatment modalities. Areas covered: The expanding knowledge on the pathogenetic mechanisms of asthma has allowed researchers to investigate a range of new treatment options matched to patient profiles. The aim of this review is to provide a comprehensive and updated overview of the currently available, new and developing approaches for identifying and testing potential treatment options for asthma management. Expert opinion: Future therapeutic strategies for asthma require the identification of reliable biomarkers that can help with diagnosis and endotyping, in order to determine the most effective drug for the right patient phenotype. Furthermore, in addition to the identification of clinical and inflammatory phenotypes, it is expected that a better understanding of the mechanisms of airway remodeling will likely optimize asthma targeted treatment.