Toward clinically applicable biomarkers for asthma: An EAACI position paper.

Inflammation, structural, and functional abnormalities within the airways are key features of asthma. Although these processes are well documented, their expression varies across the heterogeneous spectrum of asthma. Type 2 inflammatory responses are characterized by increased levels of eosinophils, FeNO, and type 2 cytokines in blood and/or airways. Presently, type 2 asthma is the best-defined endotype, typically found in patients with allergic asthma, but surprisingly also in nonallergic patients with (severe) asthma. The etiology of asthma with non-type 2 inflammation is less clear. During the past decade, targeted therapies, including biologicals and small molecules, have been increasingly integrated into treatment strategies of severe asthma. These treatments block specific inflammatory pathways or single mediators. Single or composite biomarkers help to identify patients who will benefit from these treatments. So far, only a few inflammatory biomarkers have been validated for clinical application. The European Academy of Allergy & Clinical Immunology Task Force on Biomarkers in Asthma was initiated to review different biomarker sampling methods and to investigate clinical applicability of new and existing inflammatory biomarkers (point-of-care) to support diagnosis, targeted treatment, and monitoring of severe asthma. Subsequently, we discuss existing and novel targeted therapies for asthma as well as applicable biomarkers.

COVID-19 vaccination acceptance, safety and side-effects in European patients with severe asthma.

Background: Vaccination is vital for achieving population immunity to severe acute respiratory syndrome coronavirus 2, but vaccination hesitancy presents a threat to achieving widespread immunity. Vaccine acceptance in chronic potentially immunosuppressed patients is largely unclear, especially in patients with asthma. The aim of this study was to investigate the vaccination experience in people with severe asthma. Methods: Questionnaires about vaccination beliefs (including the Vaccination Attitudes Examination (VAX) scale, a measure of vaccination hesitancy-related beliefs), vaccination side-effects, asthma control and overall safety perceptions following coronavirus disease 2019 (COVID-19) vaccination were sent to patients with severe asthma in 12 European countries between May and June 2021. Results: 660 participants returned completed questionnaires (87.4% response rate). Of these, 88% stated that they had been, or intended to be, vaccinated, 9.5% were undecided/hesitant and 3% had refused vaccination. Patients who hesitated or refused vaccination had more negative beliefs towards vaccination. Most patients reported mild (48.2%) or no side-effects (43.8%). Patients reporting severe side-effects (5.7%) had more negative beliefs. Most patients (88.8%) reported no change in asthma symptoms after vaccination, while 2.4% reported an improvement, 5.3% a slight deterioration and 1.2% a considerable deterioration. Almost all vaccinated (98%) patients would recommend vaccination to other severe asthma patients. Conclusions: Uptake of vaccination in patients with severe asthma in Europe was high, with a small minority refusing vaccination. Beliefs predicted vaccination behaviour and side-effects. Vaccination had little impact on asthma control. Our findings in people with severe asthma support the broad message that COVID-19 vaccination is safe and well tolerated.

Heterogeneity in the use of biologics for severe asthma in Europe: a SHARP ERS study.

Introduction: Treatment with biologics for severe asthma is informed by international and national guidelines and defined by national regulating bodies, but how these drugs are used in real-life is unknown. Materials and methods: The European Respiratory Society (ERS) SHARP Clinical Research Collaboration conducted a three-step survey collecting information on asthma biologics use in Europe. Five geographically distant countries defined the survey questions, focusing on seven end-points: biologics availability and financial issues, prescription and administration modalities, inclusion criteria, continuation criteria, switching biologics, combining biologics and evaluation of corticosteroid toxicity. The survey was then sent to SHARP National Leads of 28 European countries. Finally, selected questions were submitted to a broad group of 263 asthma experts identified by national societies. Results: Availability of biologics varied between countries, with 17 out of 28 countries having all five existing biologics. Authorised prescribers (pulmonologists and other specialists) also differed. In-hospital administration was the preferred deliverance modality. While exacerbation rate was used as an inclusion criterion in all countries, forced expiratory volume in 1 s was used in 46%. Blood eosinophils were an inclusion criterion in all countries for interleukin-5 (IL-5)-targeted and IL-4/IL-13-targeted biologics, with varying thresholds. There were no formally established criteria for continuing biologics. Reduction in exacerbations represented the most important benchmark, followed by improvement in asthma control and quality of life. Only 73% (191 out of 263) of surveyed clinicians assessed their patients for corticosteroid-induced toxicity. Conclusion: Our study reveals important heterogeneity in the use of asthma biologics across Europe. To what extent this impacts on clinical outcomes relevant to patients and healthcare services needs further investigation.

The effect of the COVID-19 pandemic on severe asthma care in Europe: will care change for good?

Background: The coronavirus disease 2019 (COVID-19) pandemic has put pressure on healthcare services, forcing the reorganisation of traditional care pathways. We investigated how physicians taking care of severe asthma patients in Europe reorganised care, and how these changes affected patient satisfaction, asthma control and future care. Methods: In this European-wide cross-sectional study, patient surveys were sent to patients with a physician-diagnosis of severe asthma, and physician surveys to severe asthma specialists between November 2020 and May 2021. Results: 1101 patients and 268 physicians from 16 European countries contributed to the study. Common physician-reported changes in severe asthma care included use of video/phone consultations (46%), reduced availability of physicians (43%) and change to home-administered biologics (38%). Change to phone/video consultations was reported in 45% of patients, of whom 79% were satisfied or very satisfied with this change. Of 709 patients on biologics, 24% experienced changes in biologic care, of whom 92% were changed to home-administered biologics and of these 62% were satisfied or very satisfied with this change. Only 2% reported worsening asthma symptoms associated with changes in biologic care. Many physicians expect continued implementation of video/phone consultations (41%) and home administration of biologics (52%). Conclusions: Change to video/phone consultations and home administration of biologics was common in severe asthma care during the COVID-19 pandemic and was associated with high satisfaction levels in most but not all cases. Many physicians expect these changes to continue in future severe asthma care, though satisfaction levels may change after the pandemic.

Regulatory role of CD8+ T lymphocytes in bone marrow eosinophilopoiesis.

BACKGROUND: There is a growing body of evidence to suggest that CD8+ T lymphocytes contribute to local allergen-induced eosinophilic inflammation. Since bone marrow (BM) responses are intricately involved in the induction of airway eosinophilia, we hypothesized that CD8+ T lymphocytes, as well as CD4+ T lymphocytes, may be involved in this process. METHODS: Several approaches were utilized. Firstly, mice overexpressing interleukin-5 (IL-5) in CD3+ T lymphocytes (NJ.1638; CD3IL-5+ mice) were bred with gene knockout mice lacking either CD4+ T lymphocytes (CD4-/-) or CD8+ T lymphocytes (CD8-/-) to produce CD3IL-5+ knockout mice deficient in CD4+ T lymphocytes (CD3IL-5+/CD4-/-) and CD8+ T lymphocytes (CD3IL-5+/CD8-/-), respectively. Secondly, CD3+, CD4+ and CD8+ T lymphocytes from naïve CD3IL-5+ and C57BL/6 mice were adoptively transferred to immunodeficient SCID-bg mice to determine their effect on BM eosinophilia. Thirdly, CD3IL-5+, CD3IL-5+/CD8-/- and CD3IL-5+/CD4-/- mice were sensitized and allergen challenged. Bone marrow and blood samples were collected in all experiments. RESULTS: The number of BM eosinophils was significantly reduced in CD3IL-5+/CD8-/- mice compared to CD3IL-5+ mice and CD3IL-5+/CD4-/- mice. Serum IL-5 was significantly higher in CD3IL-5+/CD4-/- mice compared to CD3IL-5+ mice but there was no difference in serum IL-5 between CD3IL-5+/CD4-/- and CD3IL-5+/CD8-/- mice. Adoptive transfer of CD8+, but not CD4+ T lymphocytes from naïve CD3IL-5+ and C57BL/6 mice restored BM eosinophilia in immunodeficient SCID-bg mice. Additionally, allergen challenged CD3IL-5+/CD8-/- mice developed lower numbers of BM eosinophils compared to CD3IL-5+ mice and CD3IL-5+/CD4-/- mice. CONCLUSION: This study shows that CD8+ T lymphocytes are intricately involved in the regulation of BM eosinophilopoiesis, both in non-sensitized as well as sensitized and allergen challenged mice.

A synthetic VIP peptide analogue inhibits neutrophil recruitment in rat airways in vivo.

Currently, there is no effective pharmacotherapy against exaggerated mobilisation of neutrophils in human airway diseases such as chronic obstructive pulmonary disease and asthma. We evaluated the effect of two synthetic vasoactive intestinal peptide (VIP)-like analogues on cytokine-induced neutrophil recruitment in airways in vivo. Recombinant interleukin (IL)-1 beta was administered intratracheally (i.t.) to intubated, spontaneously breathing Sprague-Dawley rats. The rats were pretreated either with a VIP synthetic peptide analogue, a pituitary adenylate cyclase-activating peptide (PACAP)-1-27 synthetic analogue, the beta(2)-adrenoceptor agonist salbutamol or vehicle, systemically or locally. Differential cell counts were performed on bronchoalveolar lavage fluid (BALf) cytospins. Effects on mean arterial blood pressure (MAP) were monitored in separate experiments. Systemic administration of the VIP analogue, the PACAP analogue and salbutamol attenuated the cytokine-induced increase in BALf neutrophil number. Local administration of the VIP analogue and salbutamol, but not the PACAP analogue, also decreased the neutrophil number in BALf. Local administration of the VIP analogue and salbutamol caused a transient decrease in MAP. Systemic or local administration of a synthetic VIP peptide analogue inhibits cytokine-induced neutrophil recruitment in airways in vivo. This action is exerted without severe, sustained cardiovascular side effects, and deserves to be further evaluated in obstructive pulmonary diseases in human.

Fungal allergy in asthma-state of the art and research needs.

Sensitization to fungi and long term or uncontrolled fungal infection are associated with poor control of asthma, the likelihood of more severe disease and complications such as bronchiectasis and chronic pulmonary aspergillosis. Modelling suggests that >6.5 million people have severe asthma with fungal sensitizations (SAFS), up to 50% of adult asthmatics attending secondary care have fungal sensitization, and an estimated 4.8 million adults have allergic bronchopulmonary aspergillosis (ABPA). There is much uncertainty about which fungi and fungal allergens are relevant to asthma, the natural history of sensitisation to fungi, if there is an exposure response relationship for fungal allergy, and the pathogenesis and frequency of exacerbations and complications. Genetic associations have been described but only weakly linked to phenotypes. The evidence base for most management strategies in ABPA, SAFS and related conditions is weak. Yet straightforward clinical practice guidelines for management are required. The role of environmental monitoring and optimal means of controlling disease to prevent disability and complications are not yet clear. In this paper we set out the key evidence supporting the role of fungal exposure, sensitisation and infection in asthmatics, what is understood about pathogenesis and natural history and identify the numerous areas for research studies.

Mucin5B expression by lung alveolar macrophages is increased in long-term smokers.

This study investigated the expression of MUC5B by AMs in the lungs of cigarette smokers and nonsmokers. We analyzed MUC5B expression by measuring the levels of apomucin and mRNA in human BALF cells from 50 subjects (20 nonsmokers, 17 patients with CB, and 13 patients with COPD). apoMUC5B was observed in BALF mononuclear cells in 60% of all subjects, but a significantly higher frequency of apoMUC5B(+) cells was found in subjects with CB (95% CI, 4.5-24.9) or COPD (95% CI, 6.2-39.6) than in nonsmokers (95% CI, 0.5-2.5). apoMUC5B(+) mononuclear cells showed strong expression of CD163, confirming their identity as AMs. MUC5B mRNA expression was detected by ISH in AMs of subjects investigated, and real-time qPCR analysis confirmed MUC5B mRNA expression. In conclusion, MUC5B is expressed in a subset of lung AMs and long-term cigarette smoking may increase the level of MUC5B produced by these cells.

Sonic Hedgehog pathway activity in prostate cancer.

Abnormal activation of the Sonic hedgehog (Shh) signaling pathway has been demonstrated in a number of human tumors, including prostate cancer. The study aimed to assess the activity of Shh pathway components (Shh, Gli1, Gli2 and Gli3), as well as the proliferation markers FoxA1 and Notch1 during cancer progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP). We evaluated changes in respective proteins by immunohistochemistry at three time points (12, 17 and 21 weeks of age) in the tissue of TRAMP and C57Bl/6 mice. Moreover, the expression of mRNA of these proteins was assessed. The present study shows a significant age-dependent increase in the number of Shh, Gli1, Gli3 and FoxA1-positive prostate cells and a decrease in Gli2-positive cells in TRAMP. The study also supports the hypothesis that the development of prostate cancer and its metastasis is associated with activation of the Shh signaling pathway.

Smoothened agonist augments proliferation and survival of neural cells.

Sonic hedgehog signaling pathway is important in developmental processes like dorsoventral neural tube patterning, neural stem cell proliferation and neuronal and glial cell survival. Shh is also implicated in the regulation of the adult hippocampal neurogenesis. Recently, nonpeptidyl Smoothened activators of the Shh pathway have been identified. The aim of this study was to investigate the effects of chlorobenzothiophene-containing molecule, Smo agonist (SAG), which has been shown to activate Shh signaling pathway, in neurogenesis and neuronal survival in in vitro and in vivo models. Our in vitro experiments showed that SAG induces increased expression of Gli1 mRNA, transcriptional target and mediator of Shh signal. In vitro experiments also demonstrated that SAG in low-nanomolar concentrations induces proliferation of neuronal and glial precursors without affecting the differentiation pattern of newly produced cells. In contrast to Shh, SAG did not induce neurotoxicity in neuronal cultures. The SAG and Shh treatment also promoted the survival of newly generated neural cells in the dentate gyrus after their intracerebroventricular administration to adult rats. We propose that SAG and similar compounds represent attractive molecules to be developed for treatment of disorders where stimulation of the generation and survival of new neural cells would be beneficial.

Interleukin-17 as a recruitment and survival factor for airway macrophages in allergic airway inflammation.

Recent data indicate that the proinflammatory cytokine, interleukin (IL)-17, stimulates certain effector functions of human macrophages. We evaluated whether IL-17 mediates allergen-induced accumulation of airway macrophages and, if so, whether such an effect relates to the control of macrophage recruitment and survival. BALB/c mice were sensitized and challenged with ovalbumin. Three hours before challenge an anti-mouse IL-17 mAb (a-IL-17) was administered. Sampling was conducted 24 h after the allergen challenge. In vitro chemotaxis assay for blood monocytes and culture of airway macrophages, immunocytochemistry for Fas-antigen, and matrix metalloproteinase-9 (MMP-9) were used to determine the effect of IL-17 on the recruitment, survival, and activity of airway macrophages. A-IL-17 reduced the number of airway neutrophils and macrophages after allergen challenge. In vitro, recombinant IL-17 induced migration of blood monocytes and prolonged survival of airway macrophages. A-IL-17 also increased the expression of Fas-antigen in airway macrophages in vivo. Finally, the expression of MMP-9 by airway neutrophils and macrophages in vivo was downregulated by a-IL-17. This study indicates that endogenous IL-17 mediates the accumulation of macrophages during allergen-induced airway inflammation. IL-17 exerts its effects by acting directly on airway macrophages by promoting their recruitment and survival. Furthermore, IL-17 is involved in controlling the proteolytic activity of macrophages and neutrophils in allergen-induced airway inflammation.

Allergen exposure-induced differences in CD34+ cell phenotype: relationship to eosinophilopoietic responses in different compartments.

We hypothesized that the allergen-induced increased number of airway eosinophils results from increased recruitment of eosinophils from bone marrow (BM) and local development of CD34(+) cells into eosinophils. We also assumed that the phenotype of airway eosinophils depends on whether these cells have differentiated within BM or airway. C57BL/6 mice were sensitized and subsequently exposed to ovalbumin (OVA) on 5 consecutive days. Newly produced cells were labeled with a thymidine analog. Clonogenic activity and interleukin 5 (IL-5) release from bronchoalveolar lavage fluid (BALf) CD34(+) cells were evaluated by using cell-culture techniques. Allergen exposure induces increase in CD135(+) primitive myeloid progenitors within the BM CD34(+) cell population, without significant changes in total number of CD34(+) cells or newly produced CD34(+) cells. CD34(+)/IL-5R alpha(+) cells in the first stage of cell differentiation were found only in BM, arguing that early commitment of CD34(+) cells into the eosinophil lineage is restricted to the BM compartment. Allergen exposure induces a shift in differentiation of BM, blood, and BALf eosinophillineage-committed CD34(+) cells toward mature eosinophils and recruitment of these cells via blood into airway. We further demonstrate in vitro that ability to multiply persists in BALf CD34(+) cells but not CD34(-) cells, likely via autocrine IL-5 release and IL-5-induced up-regulation of IL-5R alpha.

Allergen-induced traffic of bone marrow eosinophils, neutrophils and lymphocytes to airways.

We evaluated whether bone marrow (BM) inflammatory cells have capacity to traffic into the airways following allergen exposure in a mouse model of allergen-induced airway inflammation. We also evaluated the effect of IL-5 overexpression on (i) the production of eosinophils in BM, (ii) the accumulation of eosinophils, neutrophils and lymphocytes in blood and airways and (iii) the changes in CD34+ cell numbers in BM, blood and airways. Bromodeoxyuridine (BrdU) was used to label cells produced during the exposure period. Furthermore, CD3 splenocytes were adoptively transferred to investigate the BM inflammatory response. Allergen exposure induced traffic of BM eosinophils, neutrophils and lymphocytes to the airways and increased the number of BrdU+ eosinophils, neutrophils, lymphocytes and CD34+ cells in BALf. IL-5 overexpression enhanced the eosinophilopoiesis and increased the presence of BrdU+ eosinophils and CD34+ cells in airways and enhanced the number of CD34+ cells in BM and blood after allergen exposure. Adoptive transfer of CD3 lymphocytes overexpressing IL-5 caused increased BM eosinophilia. In conclusion, allergen exposure induces traffic of not only newly produced eosinophils but also newly produced neutrophils and lymphocytes into the airways.

Intranasal fluticasone propionate inhibits allergen induced bone marrow eosinophilia in mice.

Local corticosteroids are currently the most efficient safe anti-allergic treatment, which attenuate eosinophilic tissue inflammation through several mechanisms. We evaluated the effect of local airways corticosteroid on repeated allergen exposure-induced bone marrow activation and airway eosinophilia using the number of eosinophils in bone marrow, bronchoalveolar lavage fluid (BALf) and airways tissue as study end-points. Male BALB/c mice were sensitized by intraperitoneal injections of aluminum-precipitated ovalbumin (OVA) on two different days (5 days apart). Eight days after the second sensitization, the animals were challenged intranasally with OVA or phosphate-buffered saline (PBS) on 5 consecutive days. Concomitantly with challenges mice were treated with fluticasone propionate or respective vehicle. OVA exposures induced a significant increase in eosinophil numbers in bone marrow, BALf and airways tissue (P<0.005). Treatment with fluticasone propionate significantly reduced the increase of absolute number of mature bone marrow eosinophils (P=0.014) and showed a tendency towards decrease in the immature bone marrow eosinophil number (P=0.057) compared to controls. However, fluticasone propionate had no significant effect on BALf and airways tissue eosinophils (P=0.28 and 0.07, respectively). In this murine allergy model intranasal corticosteroid reduced number of bone marrow mature eosinophils, but did not significantly affect airways cell populations.

Regulation of bone marrow and airway CD34+ eosinophils by interleukin-5.

The aim of this study was to evaluate the effect of a neutralizing anti-interleukin (IL)-5 monoclonal antibody (TRFK-5) on bone marrow and airway CD34(+) and immature eosinophils. A focus was to determine the effect of the timing of treatment. Balb/c mice were ovalbumin-sensitized and subsequently exposed to ovalbumin for 5-10 d via airway route. Animals were treated intraperitoneally with TRFK-5 or its isotype control (50 microg) once at different time points. Newly produced eosinophils were labeled using 5-bromo-2'-deoxyuridine (BrdU). BrdU(+) and CD34(+) eosinophil numbers were examined by immunocytochemistry. TRFK-5 reduced bone marrow immature eosinophils within 3 d. This effect was closely related to a reduction of BrdU(+) and CD34(+) bone marrow eosinophils, and reduced numbers of blood eosinophils. However, bronchoalveolar lavage (BAL) eosinophilia was not attenuated to the same degree. The effect of TRFK-5 was most prominent in the extended allergen-exposure protocol, where the treatment was given in the middle of the exposure, with strongly reduced bone marrow CD34(+) and immature bone marrow eosinophils, blood eosinophils as well as BAL BrdU(+) eosinophils, and BAL CD34(+) eosinophils. These data argue that anti-IL-5 downregulates eosinophilopoiesis within 3 d by action in the bone marrow, by inhibition of the early stages of eosinophil maturation from CD34(+) cells.