Why Cell Biology of Asthma Matters

Cell types responsible for the major pathology in asthma:
1. Epithelial cells – initiate airway inflammation mucus, and
2. Smooth muscle cells – contract excessively to cause airway narrowing.

The clinical manifestations of asthma are caused by obstruction of the conducting airways of the lung. Two airway cell types are critical for asthma pathogenesis: epithelial cells and smooth muscle cells. Airway epithelial cells, which are the first line of defense against inhaled pathogens and particles, initiate airway inflammation and produce mucus, an important contributor to airway obstruction. The other main cause of airway obstruction is contraction of airway smooth muscle. Complementary experimental approaches involving cultured cells, animal models, and human clinical studies have provided many insights into diverse mechanisms that contribute to airway epithelial and smooth muscle cell pathology in this complex disease. Continued attention to the study of the cell biology of asthma will be crucial for generating new ideas for asthma prevention and treatment based on normalizing epithelial and smooth muscle function.

Note from the WAF editorial board: We wish to acknowledge and thank David J. Erle and Dean Sheppard, Lung Biology Center and Department of Medicine, University of California, San Francisco for their support for Asthma research and education.

Asthma is a common disease that affects up to 8% of children in the United States (Moorman et al., 2007) and is a major cause of morbidity worldwide. The principal clinical manifestations of asthma are repeated episodes of shortness of breath and wheezing that are at least partially reversible, recurrent cough, and excess airway mucus production. Because asthma involves an integrated response in the conducting airways of the lung to known or unknown triggers, it is a multicellular disease, involving abnormal responses of many different cell types in the lung (Locksley, 2010). Here we focus on the two cell types that are ultimately responsible for the major symptomatic pathology in asthma—epithelial cells that initiate airway inflammation in asthma and are the source of excess airway mucus, and smooth muscle cells that contract excessively to cause symptomatic airway narrowing. The current thinking about cell–cell communications that drive asthma (Fig. 1) is that known and unknown inhaled stimuli (i.e., proteases and other constituents of inhaled allergens, respiratory viruses, and air pollutants) stimulate airway epithelial cells to secrete the cytokines TSLP, interleukin (IL)-25, and IL-33, which act on subepithelial dendritic cells, mast cells, and innate lymphoid cells (iLCs) to recruit both innate and adaptive hematopoietic cells and initiate the release of T helper 2 (Th2) cytokines (principally IL-5 and IL-13; Locksley, 2010; Scanlon and McKenzie, 2012; Bando et al., 2013; Barlow et al., 2013; Nussbaum et al., 2013). Environmental stimuli also activate afferent nerves in the airway epithelium that can themselves release biologically active peptide mediators and also trigger reflex release of acetylcholine from efferent fibers in the vagus nerve. This initial response is amplified by the recruitment and differentiation of subsets of T cells that sustain secretion of these cytokines and in some cases secrete another cytokine, IL-17, at specific strategic sites in the airway wall. The released cytokines act on epithelial cells and smooth muscle cells and drive the pathological responses of these cells that contribute to symptomatic disease. The cell biology underlying the responses of the relevant hematopoietic lineages is not specific to asthma and has been discussed elsewhere (Locksley, 2010; Scanlon and McKenzie, 2012). We focus our discussion on the contributions of epithelial cells and airway smooth muscle cells.
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Figure 1.

Cell–cell communication in the airway wall in asthma. Environmental triggers concurrently act on airway afferent nerves (which both release their own peptide mediators and stimulate reflex release of the bronchoconstrictor acetylcholine) and airway epithelial cells to initiate responses in multiple cell types that contribute to the mucous metaplasia and airway smooth muscle contraction that characterize asthma. Epithelial cells release TSLP and IL-33, which act on airway dendritic cells, and IL-25, which together with IL-33 acts on mast cells, basophils, and innate type 2 lymphocytes (iLC2). These secreted products stimulate dendritic cell maturation that facilitates the generation of effector T cells and triggers the release of both direct bronchoconstrictors and Th2 cytokines from innate immune cells, which feed back on both the epithelium and airway smooth muscle and further facilitate amplification of airway inflammation through subsequent adaptive T cell responses.

Cell biology of airway epithelium

The airway is covered with a continuous sheet of epithelial cells (Crystal et al., 2008; Ganesan et al., 2013). Two major airway cell types, ciliated and secretory cells, establish and maintain the mucociliary apparatus, which is critical for preserving airway patency and defending against inhaled pathogens and allergens. The apparatus consists of a mucus gel layer and an underlying periciliary layer. Ciliated cells each project ?300 motile cilia into the periciliary layer that are critical for propelling the mucus layer up the airway. In addition, cilia are coated with membrane-spanning mucins and tethered mucopolysaccharides that exclude mucus from the periciliary space and promote formation of a distinct mucus layer (Button et al., 2012). Secretory cells produce a different class of mucins, the polymeric gel-forming mucins. The two major airway gel-forming mucins are MUC5AC and MUC5B. Some secretory cells, known as mucous or goblet cells, produce mucins and store them within easily visualized collections of mucin granules, whereas other cells produce and secrete mucins (especially MUC5B) but lack prominent granules. Gel-forming mucins are secreted into the airway lumen and are responsible for the characteristic viscoelastic properties of the mucus gel layer.
Airway epithelial injury and remodeling in asthma

A variety of structural changes in the epithelium and other portions of the airway, termed “airway remodeling,” is frequently seen in individuals with asthma (Elias et al., 1999). These changes include airway wall thickening, epithelial hypertrophy and mucous metaplasia, subepithelial fibrosis, myofibroblast hyperplasia, and smooth muscle cell hyperplasia and hypertrophy. Airway remodeling is thought to represent a response to ongoing tissue injury caused by infectious agents, allergens, or inhaled particulates and by the host responses to these stimuli. Signs of frank epithelial injury, including loss of epithelial integrity, disruption of tight junctions, impairment of barrier function, and cell death, have been identified in some studies and may correlate with asthma severity (Laitinen et al., 1985; Jeffery et al., 1989; Barbato et al., 2006; Holgate, 2007). However, in many individuals asthma symptoms and features of airway remodeling, including mucous metaplasia and subepithelial fibrosis, are seen in the absence of signs of active airway infection or overt tissue injury (Ordoñez et al., 2000), suggesting that other processes account for the persistence of asthma in these individuals. Substantial evidence suggests that the persistence of asthma is driven by ongoing host immune responses that generate mediators driving airway remodeling and airway dysfunction. The epithelium is both a site of production of these mediators and a source of cells that respond to mediators produced by immune cells and other cells within the airway. How airway epithelial cells recognize and respond to viruses, allergens, and other stimuli has been comprehensively reviewed elsewhere (Lambrecht and Hammad, 2012). Here we will focus on the contribution of the epithelium to production of and responses to Th2 cytokines.
Airway epithelial contributions to Th2 responses.

Th2 cytokines, especially IL-13, play critical roles in asthma. Multiple cytokines, including TSLP, GM-CSF, IL-1, IL-25, and IL-33, are produced by the epithelium and promote production of Th2 cytokines by immune cells (Cates et al., 2004; Hammad et al., 2009; Locksley, 2010; Nagarkar et al., 2012). Genome-wide association studies implicate multiple Th2-related genes, including IL13, IL33, and TSLP, in asthma (Moffatt et al., 2010; Torgerson et al., 2011). IL-13 is produced by innate lymphoid cells (Neill et al., 2010; Price et al., 2010; Saenz et al., 2010; Hasnain et al., 2011) and Th2 cells (Grünig et al., 1998; Wills-Karp et al., 1998) during allergic inflammation and by macrophages in a mouse model of virus-induced airway disease (Kim et al., 2008). IL-13 induces characteristic changes in airway epithelial mRNA (Kuperman et al., 2005b; Woodruff et al., 2007; Zhen et al., 2007) and miRNA (Solberg et al., 2012) expression patterns in airway epithelial cells. The IL-13 transcriptional “signature” can be used to identify individuals with “Th2 high” and “Th2 low” asthma (Woodruff et al., 2009). The IL-13–induced protein periostin is secreted basally from airway epithelial cells and can be used as a biomarker for Th2 high asthma (Jia et al., 2012; Parulekar et al., 2014). Roughly half of individuals with asthma are Th2 high, and these individuals have better responses to treatment with inhaled corticosteroids (Woodruff et al., 2009) or anti–IL-13 antibody (Corren et al., 2011). The key drivers of Th2 low asthma remain poorly understood, although Th17 family cytokines may be important (Newcomb and Peebles, 2013).

Mucous metaplasia.

Although mucus is critical for host defense, pathological mucus production is an important contributor to asthma morbidity and mortality. In fatal asthma, airways are often plugged with tenacious mucus plugs that obstruct movement of gas (Kuyper et al., 2003). This catastrophic phenomenon likely reflects increased mucin production and secretion as well as changes in mucin cross-linking, mucus gel hydration, and mucus clearance. Abnormalities in mucus are not limited to severe asthma exacerbations because an increase in intracellular mucin stores (mucous metaplasia) is seen even in individuals with stable, mild to moderate asthma (Ordoñez et al., 2001). In mouse allergic airway disease models of asthma, mucous metaplasia results from increased production and storage of mucins (especially MUC5AC) in preexisting secretory cells, including club cells (Evans et al., 2004), rather than transdifferentiation of ciliated cells (Pardo-Saganta et al., 2013). However, in virus-driven models of asthma mucous cells might arise from transdifferentiation of ciliated cells (Tyner et al., 2006). A variety of stimuli and signaling pathways have been shown to regulate mucin production and secretion in airway epithelial cells.
IL-13 stimulates mucin production in Th2 high asthma.

Direct effects of IL-13 on airway epithelial cells induce mucous metaplasia in human airway epithelial cells in culture (Laoukili et al., 2001; Zhen et al., 2007) and in mouse airway epithelial cells in vivo (Kuperman et al., 2002). IL-13 is necessary for mucous metaplasia in many mouse asthma models (Grünig et al., 1998; Wills-Karp et al., 1998; Tyner et al., 2006). Individuals with Th2 high asthma have elevated levels of bronchial epithelial cell MUC5AC mRNA compared with healthy controls or individuals with Th2 low asthma (Woodruff et al., 2009). Recent transgenic mouse studies demonstrate roles for MUC5AC in clearance of enteric nematode infections (Hasnain et al., 2011) and protection against influenza infection (Ehre et al., 2012). Increased MUC5AC expression is therefore part of an integrated immune response that contributes to host defense against pathogens or inhaled particulates. A less well-recognized feature of Th2-high asthma is the substantial decrease in expression of MUC5B (Woodruff et al., 2009). The recent discovery that MUC5B is required for normal mucociliary clearance and defense against airway infection (Roy et al., 2014) suggests further attention should be directed to the possibility that a reduction in MUC5B may be an important contributor to airway dysfunction in asthma.

IL-13 is recognized by cell surface receptors expressed on almost all cell types, including airway epithelial cells (Fig. 2). The airway epithelial cell IL-13 receptor that is critical for mucous metaplasia is a heterodimer composed of IL-13R?1 and IL-4R?. Removal of this receptor in airway epithelial secretory cells (driven by the CCSP promoter) prevented mucous metaplasia in an allergic asthma model (Kuperman et al., 2005a). IL-13 binding leads to activation of Jak kinases associated with the receptor cytoplasmic domain and subsequent phosphorylation of signal transducer and activator of transcription 6 (STAT6). STAT6 activation is required for IL-13–induced mucous metaplasia (Kuperman et al., 2002).
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Figure 2.

Mechanisms of IL-13–induced mucous metaplasia. IL-13 binds to its receptor on the surface of mucous cell progenitors (e.g., club cells) leading to phosphorylation of STAT6 and translocation of STAT6 heterodimers to the nucleus, where they bind to promoters of STAT6-responsive genes. STAT6-dependent processes that contribute to mucous metaplasia include a CLCA1-dependent pathway, a Serpin-dependent pathway, and a 15-lipoxygenase-1–dependent pathway. The transcription factor SPDEF is a master regulator of mucous cell differentiation. It inhibits FOXA2, which represses mucous cell differentiation, and activates transcription of other genes that are expressed in mucous cells.

The series of events that link STAT6 activation to mucous metaplasia are only partly understood. STAT6 does not appear to directly regulate MUC5AC transcription (Young et al., 2007) and the critical direct targets of STAT6 have not been determined. One pathway that depends upon STAT6 activation involves the protein calcium-activated chloride channel 1 (CLCA1). CLCA1 is among the most highly induced genes in airway epithelial cells from individuals with asthma (Hoshino et al., 2002; Toda et al., 2002). Despite its name, CLCA1 does not appear to function as an ion channel but instead undergoes extracellular secretion and cleavage. Extracellular CLCA1 can induce MUC5AC expression via activation of the MAP kinase MAPK13 (p38?-MAPK; Alevy et al., 2012), although the presumed CLCA1 receptor and the relevant MAPK13 targets have not yet been identified. A second pathway involves the protease inhibitor Serpin3a, the mouse orthologue of human SERPINB3 and SERPINB4. These serpins are induced by IL-13 in a STAT6-dependent fashion (Ray et al., 2005). After allergen challenge, Serpin3a?/? mice had less mucous metaplasia than wild-type mice (Sivaprasad et al., 2011), despite an intact inflammatory response. These results suggest that serpins inhibit proteases that normally degrade one or more proteins required for mucous metaplasia, although the relevant proteases and their protein substrates are not yet known. Another IL-13–induced pathway involves the enzyme 15-lipoxygenase-1 (15-LO-1; Zhao et al., 2009). 15-LO-1 converts arachidonic acid to 15-hydroxyeicosatetraenoic acid, which was shown to enhance MUC5AC expression in human airway epithelial cells.

IL-13– and STAT6-mediated mucous metaplasia depends upon changes in the activity of a network of transcription factors. Allergen-induced IL-13–mediated STAT6 activation leads to increased expression of the SAM-pointed domain–containing Ets-like factor (SPDEF; Park et al., 2007; Chen et al., 2009). The induction of SPDEF depends at least in part on FOXM1, a member of the Forkhead box (FOX) family of transcription factors (Ren et al., 2013). The SPDEF program is also important for mucous metaplasia triggered by other stimuli, including rhinoviruses (Korfhagen et al., 2012). Although SPDEF does not appear to directly regulate mucin gene transcription, SPDEF initiates a transcriptional program that is necessary and sufficient to induce mucous metaplasia. One of the effects of SPDEF is inhibition of the expression of another FOX family gene, FOXA2. In mice, deletion of Foxa2 in mucous cell precursors is sufficient to induce mucous metaplasia, and overexpression of FOXA2 inhibits allergen-induced mucous metaplasia (Zhen et al., 2007; G. Chen et al., 2010). The relationship between IL-13 and FOXA2 is complex. IL-13 inhibits expression of FOXA2, which contributes to mucous metaplasia. However, deletion of Foxa2 in airway epithelial cells during fetal development resulted in Th2 inflammation and production of IL-13 in the airway (G. Chen et al., 2010). The direct targets that are responsible for these effects of FOXA2 are not yet known.
The EGFR pathway induces mucin gene expression and mucous metaplasia.

Epidermal growth factor receptor (EGFR) binds multiple ligands including EGF, TGF-?, heparin-binding EGF, amphiregulin, ?-cellulin, and epiregulin. Ligand binding activates the EGFR kinase domain, initiating signaling cascades that are central to many fundamental biological processes, including cell proliferation, differentiation, survival, and migration. EGFR ligands induce expression of MUC5AC in human airway epithelial cell lines and a tyrosine kinase inhibitor that inhibits EGFR kinase prevents mucous metaplasia induced either by an EGFR ligand or by allergen challenge (Takeyama et al., 1999). Subsequent studies showed that bronchial epithelial EGFR levels are increased in asthma and correlate with disease severity (Takeyama et al., 2001a), and that epithelial EGFR signaling contributes to mucous metaplasia in a chronic asthma model (Le Cras et al., 2011).

Various stimuli, including bacterial products (Kohri et al., 2002; Lemjabbar and Basbaum, 2002;Koff et al., 2008), viruses (Tyner et al., 2006; Zhu et al., 2009; Barbier et al., 2012), cigarette smoke (Takeyama et al., 2001b; Basbaum et al., 2002), and inflammatory cell products (Burgel et al., 2001) can activate the EGFR pathway in airway epithelial cells. Some stimuli have been shown to initiate the EGFR signaling cascade by activating the PKC isoforms PKC ? and PKC ?, leading to recruitment of the NADPH oxidase subunits p47phox and p67phox to membrane-associated dual oxidase-1 and the generation of reactive oxygen species (ROS) at the cell surface (Shao and Nadel, 2005). ROS in turn activate latent TGF-?–converting enzyme resulting in cleavage of surface EGFR pro-ligands (Shao et al., 2003). EGFR ligand binding leads to activation of the Ras–Raf–MEK1/2–ERK1/2 pathway and MUC5AC transcriptional induction, which depends upon the Sp1 transcription factor and Sp1-binding sites within the MUC5AC promoter (Takeyama et al., 2000; Perrais et al., 2002). The IL-13 and EGFR pathways make critical but distinct contributions to gene regulation in airway epithelial cells (Zhen et al., 2007). Both pathways inhibit expression of FOXA2, suggesting that this transcription factor may represent a final common pathway for IL-13– and EGFR-induced mucous metaplasia.

Notch signaling regulates mucous cell differentiation.

Notch signaling is also important for mucous metaplasia (Tsao et al., 2011). Notch is a transmembrane receptor that binds to cell-surface ligands in the Delta-like and Jagged families. Ligand binding activates ?-secretase–mediated proteolytic cleavage and liberates the Notch intracellular domain, which enters the nucleus, associates with transcription factors, and drives expression of downstream Notch genes. Genetic manipulation of Notch signaling in mice has different effects depending on the developmental stage. In explanted embryonic lungs, addition of Notch ligand or expression of a constitutively active form of Notch increased MUC5AC-containing mucous cells, whereas a ?-secretase inhibitor reduced mucous cells (Guseh et al., 2009). Notch-induced mucous metaplasia did not require STAT6 activation, suggesting that the Notch and STAT6 pathways may operate in parallel. In contrast, in postnatal mouse lung, disruptions of Notch signaling induced mucous metaplasia (Tsao et al., 2011), a process that principally depends on the Notch ligand Jagged1 (Zhang et al., 2013). The Notch target Hes1 appears to be critical for inhibition of mucous metaplasia and MUC5AC transcription, although inactivation of Hes1 was not sufficient to induce mucous metaplasia (Ou-Yang et al., 2013). The observation that a ?-secretase inhibitor reduced IL-13–induced mucous metaplasia in cultured human airway epithelial cells (Guseh et al., 2009) suggests that further attention to the role of epithelial Notch signaling in asthma is warranted.

The secretory pathway in mucous cells

Mucin monomers are large (?5,000 amino acid residue) proteins that require extensive processing in the ER and Golgi. Each mucin monomer contains ?200 cysteine residues that can potentially participate in intra- and intermolecular disulfide bonds. The ER of mucous cells contains specialized molecules that are not widely expressed in other cell types and are required for efficient processing of mucins. One of these is anterior gradient 2 (AGR2) homologue, a member of the protein disulfide isomerase family. An active site cysteine residue in AGR2 forms mixed disulfide bonds with mucins in the ER and mice deficient in AGR2 have profound defects in intestinal mucin production (Park et al., 2009). In a mouse model of allergic asthma, AGR2-deficient mice had reduced mucus production compared with allergen-challenged wild-type mice (Schroeder et al., 2012). The reduction in mucus production was associated with activation of the unfolded protein response, a characteristic response to ER stress (Walter and Ron, 2011). AGR2 may therefore either have a direct role in mucin folding or another function necessary for maintaining normal function of the mucous cell ER. Another molecule found in the mucous cell ER is inositol-requiring enzyme 1? (IRE1?), a transmembrane ER stress sensor. IRE1? is found in mucus-producing cells in the intestine and the airways, but not in other cells. IRE1? regulates AGR2 transcription, and mice deficient in IRE1? had reduced AGR2 expression and impaired airway mucin production in an allergic asthma model (Martino et al., 2013). AGR2 and IRE1? have apparently evolved to meet the unusual demands posed by the need to produce large amounts of mucins.

ORMDL3, a member of the Orm family of transmembrane ER proteins, has also been implicated in asthma. Genetic polymorphisms at loci close to ORMDL3 were strongly associated with asthma in multiple genome-wide association studies (Moffatt et al., 2007; Galanter et al., 2008). Allergen challenge induced ORMDL3 expression in airway epithelial cells in a STAT6-dependent fashion, although ORMDL3 does not appear to be a direct target of STAT6 (Miller et al., 2012). Studies involving overexpression or knockdown of ORDML3 in HEK293 cells indicate that ORMDL3 is involved in regulating ER stress responses and ER-mediated calcium signaling (Cantero-Recasens et al., 2010). In addition, Orm proteins form complexes with serine palmitoyl-CoA transferase (SPT), the first and rate-limiting enzyme in sphingolipid production, and may thereby help coordinate lipid metabolism in the secretory pathway (Breslow et al., 2010). Genetic and pharmacologic reductions in SPT activity induced airway hyperresponsiveness in the absence of inflammation or mucous metaplasia (Worgall et al., 2013). Further studies are required to determine whether ORMDL3’s role in modulating sphingolipid production, ER stress, calcium signaling, or other ER functions in airway epithelial cells or other cells is important in asthma.

Mucins travel from the ER to the Golgi and then are packaged into large granules for secretion. In the Golgi, mucins are extensively O-glycosylated and undergo further multimerization before being released from the cell by regulated exocytosis. Throughout the airways of normal mice and in distal (smaller) airways of humans, basal secretion accounts for most mucin release, and mucin-producing cells retain too little mucin to detect using histological stains. However, mucous cells found in larger airways of humans and allergen-challenged mice contain readily detectable accumulations of mucin-containing granules that can be released by various stimuli, including the P2Y2 receptor ligands ATP and UTP and proteases that cleave protease-activated receptors. Mice lacking the exocytic priming protein Munc13-2 accumulate mucin in secretory cells that normally have minimal intracellular mucin (club cells) but can secrete mucin in response to stimulation (Zhu et al., 2008). In contrast, allergen-challenged mice lacking the low affinity calcium sensor synaptotagmin-2 have a severe defect in acute agonist-stimulated airway mucin secretion, but have preserved basal secretion and do not accumulate mucins in club cells (Tuvim et al., 2009). Agonist-stimulated secretion also depends upon the IL-13–inducible calcium-activated chloride channel TMEM16A, which is increased in mucous cells from individuals with asthma (Huang et al., 2012). Because increased production of MUC5AC via transgenic overexpression was not in itself sufficient to cause airway obstruction (Ehre et al., 2012), it seems likely that qualitative defects in mucin processing, secretion, or hydration that affect the physicochemical properties of mucus contribute to airway obstruction in asthma. Epithelial transport of water and ions, including H+ and bicarbonate, is important in maintaining the normal properties of mucus (E. Chen et al., 2010; Paisley et al., 2010; Garland et al., 2013). Rapid secretion of stored mucin, which is not fully hydrated, may result in the formation of concentrated, rubbery mucus that cannot be cleared normally by cilia or by coughing (Fahy and Dickey, 2010). Hence, IL-13 (Danahay et al., 2002; Nakagami et al., 2008) and other asthma mediators that affect airway epithelial cell water and ion transport could contribute to airway obstruction by altering the physicochemical properties of mucus.
Ciliated cell structure and function in asthma

In comparison with the extensive asthma literature regarding mucous cells, relatively few reports have focused on ciliated cells. One study of epithelial cell strips obtained by endobronchial brushing found decreased ciliary beat frequency and increases in abnormal ciliary beating patterns and ciliary ultrastructural defects in individuals with asthma compared with healthy controls (Thomas et al., 2010). These abnormalities were more pronounced in severe asthma. Ciliary abnormalities were accompanied by increases in the numbers of dead cells and evidence of loss of epithelial structural integrity, which suggests that ciliary dysfunction may be a consequence of a generalized epithelial injury. In any case, these results suggest that ciliary dysfunction might be an important contributor to impaired mucociliary clearance in asthma.
Cell biology of airway smooth muscle in asthma

The excessive airway narrowing that can lead to severe shortness of breath, respiratory failure, and death from asthma is largely due to contraction of the bands of smooth muscle present in the walls of large- and medium-sized conducting airways in the lung. In the large central airways of humans, these bands of muscle are present in the posterior portion of the airways and attach to the anterior airway cartilage rings, but in more peripheral airways smooth muscle is present circumferentially around the airways. In both locations, contraction of smooth muscle, which can be physiologically induced by release of acetylcholine from efferent parasympathetic nerves or by release of histamine and cysteinyl leukotrienes from mast cells and basophils, causes airway narrowing, with the most extensive narrowing in medium-sized airways. In healthy mammals, including humans, physiological responses to release of acetylcholine from efferent nerves or release of histamine and leukotrienes from mast cells and basophils causes only mild and generally asymptomatic airway narrowing. Normal mammals are also generally resistant to marked airway narrowing in response to pharmacologic administration of high concentrations of these contractile agonists directly into the airways. However, people with asthma have a marked increase in sensitivity to all of these agonists that can readily be demonstrated by dramatic increases in airway resistance and associated drops in maximal expiratory airflow rates during forced expiratory maneuvers (Boushey et al., 1980). Recent comparisons between responses to inhaled allergens in allergic asthmatic subjects and other subjects with similarly severe cutaneous immune responses to allergens makes it clear that all allergic humans release largely similar amounts of bronchoconstrictors into the airways (i.e., histamine and leukotrienes), but only asthmatics develop exaggerated airway narrowing in response to these mediators (Becky Kelly et al., 2003).
Mechanisms regulating generation of force by airway smooth muscle actin–myosin coupling

Force generation by airway smooth muscle is mediated by interactions between actin and myosin that depend on phosphorylation of the myosin light chain by the serine–threonine kinase, myosin light chain kinase (Fig. 3). This process is negatively regulated by myosin phosphatase. Increases in intracellular calcium concentration in smooth muscle cells induce contraction by two parallel pathways. When bound to calcium, the serine–threonine kinase calmodulin directly phosphorylates, and thereby activates, myosin light chain kinase. Increased calcium also increases GTP loading of the GTPase, RhoA, which increases the activity of its downstream effector kinases Rho-associated coiled-coil–containing protein kinases 1 and 2 (ROCK 1 and 2). ROCKs directly phosphorylate myosin light chain phosphatase, an effect that inactivates the phosphatase, further enhancing myosin phosphorylation. RhoA can also be activated independently of increases in intracellular calcium.

Core signaling pathways responsible for airway smooth muscle contraction. Airway smooth muscle contractile force is generated by cyclic cross-bridging of actin and smooth muscle myosin, which depends on myosin phosphorylation. Myosin phosphorylation is regulated by cyclic increases in cytosolic calcium (Ca2+) that activate calmodulin (CaM) to phosphorylate myosin light chain kinase (MLCK), which directly phosphorylates myosin. In parallel, the small GTPase, RhoA, is activated by both calcium-dependent and -independent pathways. Rho directly activates Rho-associated coiled-coil protein kinase (ROCK) which, in turn, phosphorylates and thereby inactivates myosin light chain phosphatase (MLCP), which normally dephosphorylates myosin. The most important physiological pathway for increasing cytosolic calcium in airway smooth muscle involves activation of G?q by G protein–coupled receptors that respond to extracellular contractile agonists, such as methacholine (Mch), serotonin (5-HT), and histamine. G?q activates phospholipase C ? (PLC?), which generates IP3 to bind to IP3 receptors on the sarcoplasmic reticulum and release sequestered Ca2+.

There are multiple upstream paths to increased i[Ca] in airway smooth muscle. Acetylcholine, released from post-ganglionic parasympathetic efferent nerves that innervate the muscle, activates G protein–coupled M2 muscarinic receptors, which are coupled to G?q. GTP-loaded G?q activates its downstream effector, PLC?, which phosphorylates PIP2 to generate IP3. IP3, in turn, binds to IP3 receptors on the sarcoplasmic reticulum to trigger translocation of calcium into the cytosol. Other contractile agonists, including histamine, bradykinin, and serotonin (5-HT; the specific agonists and receptors vary across mammalian species) bind to different G protein–coupled receptors to trigger the same pathway. Agonist-induced airway smooth muscle contraction is usually associated with cyclic oscillations in i[Ca], thought to be induced by local changes in cytosolic calcium triggering reuptake of calcium by the sarcoplasmic reticulum, and the magnitude of contractile force induced is most closely associated with the frequency of these calcium oscillations rather than their amplitude (Bergner and Sanderson, 2002).

Increases in cytosolic calcium concentration can also be induced by an influx of calcium from the extracellular space, generally due to the opening of voltage-gated calcium channels in the plasma membrane. These channels can be opened experimentally by increasing the extracellular concentration of potassium ions, which also induces airway smooth muscle contraction. Increased extracellular potassium concentrations also increase release of acetylcholine from post-ganglionic efferent nerves, so proper interpretation of the effects of KCl requires simultaneous addition of a muscarinic antagonist such as atropine.
Regulation of airway smooth muscle force generation by integrin-containing adhesion complexes

For smooth muscle cell contraction to be translated into the force required for airway narrowing, the contracting smooth muscle cell must be firmly tethered to the underlying ECM. Linkage to the ECM is accomplished through the organization of multi-protein complexes nucleated by integrins. The short cytoplasmic domains of integrins can organize surprisingly large multi-protein machines that modulate multiple signaling pathways and link integrins (and thus their ECM ligands) to the actin–myosin cytoskeleton (Yamada and Geiger, 1997; Zaidel-Bar et al., 2007). Many of the contractile agonists that stimulate myosin phosphorylation and actin–myosin interaction simultaneously enhance the formation of integrin signaling complexes, induce actin polymerization at sites of adhesion, and strengthen coupling between the actin–myosin cytoskeleton and the ECM (Mehta and Gunst, 1999; Tang et al., 1999, 2003; Gunst and Fredberg, 2003; Gunst et al., 2003; Opazo Saez et al., 2004). These events appear to also be quite important for generation of maximal contractile force because interventions that inhibit the formation or activity of adhesion complexes can inhibit the strength of contraction without affecting myosin phosphorylation (Mehta and Gunst, 1999; Tang et al., 2003; Opazo Saez et al., 2004).
Lessons from abnormal behavior of airway smooth muscle in animal models
Mice lacking ?9?1 integrin in airway smooth muscle.

Although there are large differences between the organization of airways in mice and humans, in vivo abnormalities in airway narrowing seen in mouse models do provide some insight into pathways that potentially contribute to abnormal airway smooth muscle contraction in asthma. For the purposes of this review, we will cite three illustrative examples. The integrin ?9?1 is highly expressed in airway smooth muscle (Palmer et al., 1993). Conditional knockout of the integrin ?9 subunit (uniquely found in the ?9?1 integrin) results in a spontaneous increase in in vivo airway responsiveness (as measured by increases in pulmonary resistance in response to intravenous acetylcholine), and to increased contractile responses to cholinergic agonists of both airways in lung slices and tracheal rings studied in an organ bath (Chen et al., 2012). Interestingly, although tracheal rings from these mice also have increased contractile responses to other G protein–coupled receptor agonists (e.g., serotonin), they have normal contractile responses to depolarization with KCl. These findings suggest that loss of ?9?1 increases airway responsiveness at some step upstream of calcium release from the sarcoplasmic reticulum (Fig. 4 A). In this case, increased airway responsiveness appears to be due to loss of co-localization of the polyamine-catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT), which binds directly to the ?9 cytoplasmic domain (Chen et al., 2004), and the lipid kinase, PIP5K1?, which binds directly to talin, an integrin ?1 subunit binding partner. Spermine and spermidine are critical cofactors for PIP5K1?, so its juxtaposition with SSAT effectively reduces enzymatic activity. PIP5K1? converts PI4P to PIP2 and is responsible for most of the PIP2 produced in airway smooth muscle cells (Chen et al., 1998). PIP2 is the substrate for IP3 generation by PLC?, so when ?9?1 is present and ligated, contractile agonists that activate receptors coupled to G?q induce less IP3 generation (Chen et al., 2012) and thus less Ca2+ release through IP3 receptors in the sarcoplasmic reticulum. The importance of this pathway was confirmed by the observations that the frequency of Ca2+ oscillations induced by cholinergic agonists was reduced in lung slices from mice lacking ?9?1, and that all of the abnormalities in smooth muscle from these animals could be rescued by addition of a cell-permeable form of PIP2 (Chen et al., 2012).

Pathways that negatively regulate airway smooth muscle contraction. (A) The integrin ?9?1 negatively regulates airway smooth muscle contraction by colocalizing the polyamine-catabolizing enzyme, spermine spermidine acetyltransferase (SSAT), which directly binds to the ?9 subunit with the lipid kinase, PIP5K1?, the major source of PIP2 in airway smooth muscle, which binds to talin, a direct interactor with the ?1 subunit. PIP5K1? depends on spermine and spermidine for maximal activity, so the local breakdown of spermine and spermidine reduces PIP5K1? activity, thereby decreasing PIP2 concentrations and the amount of IP3 that is generated by activation of contractile G protein–coupled receptors (such as those activated by acetylcholine or serotonin [5-HT]). (B) The secreted scaffold protein, milk fat globule-EGF factor 8 (MFGE8), inhibits the smooth muscle hypercontractility induced by IL-13, IL-17, and tumor necrosis factor ? (TNF) by inhibiting the induction and activation of the small GTPase, RhoA. Active RhoA contributes to smooth muscle contraction by directly activating Rho-associated coiled-coil protein kinase (ROCK) which, in turn, phosphorylates and thereby inactivates myosin light chain phosphatase (MLCP), which normally dephosphorylates myosin.
Effects of T cell cytokines on airway smooth muscle contractility.

Several studies conducted over the past 15 years have suggested that cytokines released from T cells can contribute to airway hyperresponsiveness in allergic asthma (Locksley, 2010). The Th2 cytokine IL-13 has been most extensively studied, and can induce both mucous metaplasia and airway hyperresponsiveness when administered directly into the airways of mice (Grünig et al., 1998; Wills-Karp et al., 1998). In vitro, incubation of tracheal rings or lung slices increases narrowing of airways in lung slices and increases force generation by mouse tracheal rings, at least in part by inducing a dramatic increase in expression of the small GTPase, RhoA (Chiba et al., 2009), which is a critical effector of airway smooth muscle contraction (Fig. 4 B). Chronic allergen challenge or direct administration of IL-13 into the airways of mice also increased RhoA expression, in association with induction of airway hyperresponsiveness. A recent study suggested that IL-17 can also increase airway smooth muscle contractility and airway narrowing by induction of RhoA in airway smooth muscle cells (Kudo et al., 2012). In that study, mice lacking the ?v?8 integrin specifically on antigen-presenting dendritic cells were protected from allergen-induced airway hyperresponsiveness. These mice had the same degree of general airway inflammation and mucous metaplasia in response to allergen as wild-type control mice, but had a very specific defect in the generation of antigen-specific Th17 cells, an important source of IL-17 in lungs (Kudo et al., 2012). In vitro, IL-17 was shown to directly increase the contractility of mouse tracheal rings and to increase the levels of RhoA protein and its downstream effector, ROCK2, and to increase phosphorylation of the direct ROCK target, myosin phosphatase. Phosphorylation of myosin phosphatase inhibits its function, and IL-17 was also shown to consequently increase phosphorylation of myosin light chain kinase. Importantly, all of these biochemical effects were dramatically induced in vivo in airway smooth muscle of control mice in response to allergen sensitization and challenge, but all were markedly reduced in mice lacking ?v?8 on dendritic cells. Furthermore, tracheal rings removed from these knockout mice after allergen challenge had decreased in vitro contractility compared with rings from allergen challenged control mice, but this difference in contractility was eliminated by exogenous addition of IL-17. These findings strongly suggest that both IL-13 and IL-17 can contribute to airway hyperresponsiveness by directly inducing RhoA expression in airway smooth muscle (Fig. 4 B). Tumor necrosis factor ?, also implicated in asthma pathogenesis, has been shown to increase airway smooth muscle contractility by a similar mechanism (Goto et al., 2009).
Enhanced cytokine-mediated airway smooth muscle contraction in MFGE8-deficient mice.

Milk fat globule EGF factor 8 (MFGE8) is a secreted protein composed of two EGF repeats and two discoidin domains. MFGE8 was originally described to facilitate uptake of apoptotic cells by phagocytes (Hanayama et al., 2004). Mice lacking MFGE8 have normal baseline lung morphology and function, but have exaggerated airway responsiveness after allergen sensitization and challenge (Kudo et al., 2013). However, this abnormality did not appear to be related to any effects on reuptake of apoptotic cells. Immunostaining demonstrated that secreted MFGE8 was concentrated adjacent to airway smooth muscle. Tracheal rings removed from MFGE8 knockout mice had normal contractile responses at baseline, but had markedly enhanced contractile responses after overnight incubation with IL-13, and this increase in contractility could be rescued by addition of recombinant MFGE8 to the muscle bath. Importantly, rescue required the presence of at least one of the discoidin domains and of the integrin-binding RGD motif of the second EGF repeat. In mouse tracheal rings and cultured airway smooth muscle, loss of MFGE8 greatly enhanced the IL-13–induced increase in RhoA protein. These findings suggest that ligation of one or more RGD-binding integrins on airway smooth muscle by extracellular MFGE8 normally serves as a brake on cytokine-mediated RhoA induction and thereby limits maximal cytokine-induced airway hyperresponsiveness (Fig. 4 B). The specific integrin(s) involved in this response, the molecular mechanisms linking integrin ligation to inhibition of RhoA, and the role and binding partner(s) of the MFGE8 discoidin domains that are required for RhoA inhibition all remain to be determined.

Conclusions

Rapid progress has been made toward identifying epithelial and smooth muscle cell molecules and pathways that can produce many of the abnormalities found in individuals with asthma. Because these discoveries were made in diverse experimental systems, we still face major challenges in understanding how these molecules and pathways interact in vivo and in identifying the pathways that are most relevant in people with asthma. Asthma is a heterogeneous disease, and recent progress toward identifying subtypes with distinct pathophysiologic mechanisms promises to focus attention on certain pathways in epithelial and smooth muscle cells (Lötvall et al., 2011). It will be especially important to understand mechanisms underlying severe asthma. Approximately 5–10% of individuals with asthma have severe disease, with symptoms that persist despite standard therapy with bronchodilators and inhaled corticosteroids (Brightling et al., 2012). These individuals have high rates of asthma exacerbations leading to hospitalization and are at relatively high risk for fatal asthma attacks. Continued attention to the study of the cell biology of asthma will be crucial for generating new ideas for asthma prevention and treatment based on normalizing epithelial and smooth muscle function.

Aspirin Exacerbated Respiratory Disease

What is aspirin-exacerbated respiratory disease (AERD)

Aspirin-exacerbated respiratory disease (AERD) is a clinical tetrad of nasal polyps, chronic hypertrophic eosinophilic sinusitis, asthma and sensitivity to any medication that inhibits cyclooxygenase-1 (COX-1) enzymes, namely aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) Ingestion of aspirin, and most NSAIDs, results in a spectrum of upper and/or lower respiratory reactions, to include rhinitis, conjunctivitis, laryngospasm and bronchospasm.1,2 AERD affects 0.3-0.9% of the general population, but the prevalence rises to 10-20% of asthmatics and up to 30-40% in those asthmatics with nasal polyposis.3-7 The average age of onset is 34 years in a US study and is thought to be acquired between teenage to middle adulthood years with no ethnic predilection and rare familial associations.3-7 AERD is more commonly reported in females (57% vs. 43%).

Note from the WAF editorial board:  The WAF would like to acknowledge and thank  Rachel U. Lee1 and Donald D. Stevenson, Division of Allergy, Asthma & Immunology, Naval Medical Center Portsmouth, Portsmouth, VA, USA. and the Division of Allergy, Asthma & Immunology, Scripps Clinic, San Diego, CA, USA for their continued support to Asthma education,.

Genetic Basis of Asthma

Asthma is the most common chronic childhood disease in developed nations and its prevalence has increased in the world over the last 25 years. It is a complex disease with both genetic and environmental risk factors. Asthma is caused by multiple interacting genes, some having a protective effect and others contributing to the disease pathogenesis, with each gene having its own tendency to be influenced by the environment. This article reviews the current state of the genetics of asthma in six categories, viz. epidemiology, management, aetiology, family and twin studies, segregation and linkage studies, and candidate genes and single nucleotide polymorphisms (SNPs).

Asthma is one of the most serious allergic diseases and the most common chronic childhood disease in developed nations1. It has been characterized by increased responsiveness of the tracheobronchial tree to a multiplicity of stimuli2–4, increased infiltration of various inflammatory cells especially eosinophils into the airway, epithelial damage, airway smooth-muscle hypertrophy5, constriction, variable airway obstruction usually associated with inflammation in the conducting airways of the lungs6 and mucous hypersecretion in the bronchiolar walls of the lung7. Asthma is critically dependent on a series of cell adhesion molecule-mediated interactions between vascular endothelium and leukocytes7, leading to symptoms8 and elevation in total serum IgE9. It is manifested physiologically by widespread narrowing of the air passages and clinically by paroxysms of dyspnoea, cough, wheezing and tightness, provoked by one or more triggers such as physical exertion and airway irritants (cold, dry air, smoke, etc.)4,10. It is an episodic disease, with acute exacerbations interspersed with symptom-free periods. Typically, most attacks are short-lived, lasting minutes to hours, and clinically the patient seems to recover completely after an attack. However, there can be a phase in which the patients experience some degree of airway obstruction daily. This phase can be mild, with or without superimposed severe episodes, or can be much more serious, with severe obstruction persisting for days or weeks; the latter condition is known as “acute severe asthma”. In unusual circumstances, acute episodes can cause death4. Asthma exacerbations are characteristically worse at night and can progress to severe airflow obstruction, shortness of breath, and respiratory distress and insufficiency. Rarely, severe sequel such as hypoxic seizures, respiratory failure, and death can occur.

Here we review the latest information on the genetic basis of asthma which is one of the most intriguing diseases affecting people of all ages, gender, race and ethnicities. Familial and segregation studies have an important role in asthma aetiology and several candidate genes on all the human chromosomes play their roles in initiation and/or inhibition of different pathways of asthma disease.

Note from the WAF editorial board: We wish to acknowledge and thank Mahdi Bijanzadeh, Padukudru A. Mahesh,* and Nallur B. Ramachandra
at the Indian Journal of Medical Research for their dedication to Asthma education and research.

Conclusion and future prospects

Asthma is one of the most serious and intriguing allergic diseases. Asthma aggregates within families and is a complex multifactorial disease with the involvement of environment and genetic components. Our preliminary pedigree analysis revealed that autosomal recessive pattern of inheritance was prominent in asthma; parental consanguinity100 and serum intracellular cell adhesion molecule-1 (ICAM-1)101 was significantly associated with asthma, whereas the ABO blood system102, IL-4 and ADAM33 specific gene variants81, and serum E-selectin101 were not associated with asthma. More than 100 loci have been reported to be associated with asthma and there are also indications that mutation in a major gene can cause asthma. Due to an increasing number of current studies being done in genetics of asthma, there is an increasing list of inducer and inhibitor candidate genes for asthma. There are more than 100 candidate genes in every chromosome which are identified to have an association with asthma and the strength of association of these SNPs with asthma varies in different parts of the world. More studies are needed to determine the exact function of these genes, gene-gene interactions and the gene-environment interactions which are undoubtedly complex and remain elusive for the time being even with whole genome-wide association studies.

Further studies on asthma with the genomics data and tools, to map, identify the specific gene/s, and phenotype specific SNPs will help to unravel the pathways involved in asthma aetiology and employ pharmacogenomics to design better drugs for an individualized treatment plan. Thus with a fruitful interaction among researchers involved in pathophysiology, epidemiology, clinical research and genetics of asthma, this century holds promise for a better understanding of the pathology, diagnosis, prevention, treatment and management of asthma.

Learn About Biomarkers in Asthma and Why they Matter

Asthma is a heterogenous disease characterized by multiple phenotypes driven by different mechanisms. The implementation of precision medicine in the management of asthma requires the identification of phenotype-specific markers measurable in biological fluids. To become useful, these biomarkers need to be quantifiable by reliable systems, reproducible in the clinical setting, easy to obtain and cost-effective.

Using biomarkers to predict asthma outcomes and therapeutic response to targeted therapies has a great clinical significance, particularly in severe asthma. In the last years, significant research has been realized in the identification of valid biomarkers for asthma. This review focuses on the existent and emerging biomarkers with clinical higher applicability in the management of asthma.

Note from the WAF: The WAF editorial board wishes to acknowledge and thank Angelica Tiotiu Pulmonology Department, University Hospital, 9, Rue du Morvan, 54511 Nancy, Vandœuvre-lès-Nancy France National Heart and Lung Institute, Airway Disease Section, Imperial College London, London, UK for their contribution to Asthma education.

Asthma is a heterogeneous disease diagnosed by the presence of intermittent symptoms of wheeze, cough and chest tightness, typically related to a reversible airflow obstruction, usually resolves spontaneously or with asthma treatment [1, 2]. Over the years, clinicians have defined several phenotypes based on the presentation and age of onset of symptoms, the severity of the disease, and the presence of other conditions such as allergy and eosinophilia with different long-terms outcomes and response to therapy with corticosteroids Despite the recognition of these phenotypes of asthma, the approach to the management of asthma recommended by the international Global Initiative for Asthma (GINA) guidelines continues to be based on the severity of the condition, with drugs added on the basis of asthma control

In the era of the personalized medicine, in order to deliver this approach for asthma, it is important to be able to phenotype the condition in an unbiased way and to define biomarkers able to predict the course of the disease and the response to therapy [2, 3]. A biomarker is a measurable indicator that can evaluate a normal or pathological biological processes or pharmacologic response to a therapeutic intervention [2]. A valid biomarker would have several key characteristics: to distinguish between disease and health with high positive and negative predictive values, to provide information about disease prognosis and clinical outcomes, to change with disease progression and “normalize” with successful treatment, to be reliable and reproducible in the clinical setting with little or no day-to-day variation, to be easy to collect in the “real-world” setting, to be quantifiable in an analytical system with well-defined performance, and to be cost-effective

Despite the sustained research efforts during the last years focused on the identification of biomarkers applicable in clinical practice for the management of asthma, only a few biomarkers indicative of T2-high asthma have been described (e.g. IgE, eosinophils in blood and/or sputum, Fractional Exhaled Nitric Oxide [FeNO], periostin), and their utility in diagnosis, prognosis and therapy is still controversial

This review will summarize the recent knowledge about the biomarkers (proteins and related substances) identified of asthma with special focus on those with higher clinical applicability.

Blood cells and serum biomarkers

Using the blood for requiring biomarkers is micro-invasive (the procedure can be painful and difficult in some patients) and easy to realize in the clinical setting, requires minimal patient effort, could be collected across the age spectrum, and it is cost-effective

Blood eosinophil count is not useful for the diagnosis of asthma (GINA), but it can serve as prognostic biomarker and to predict several therapeutic responses in asthmatic patients with type 2 inflammation.

A recent study realized on a large cohort in UK, showed that patients with blood eosinophil counts greater than 400 cells/?L experienced significantly more severe exacerbations (adjusted rate ratio RR 1·42) and acute respiratory events (RR 1·28) than those with counts of 400 cells/ ?L or less and had significantly lower odds of achieving overall asthma control (odds ratio OR 0·74) [7]. Another study found that blood eosinophilia (>?400 cells/ ?L) is a risk factor for airflow obstruction in asthmatic patients (even in those without symptoms) and predicts an enhanced longitudinal decline in lung function, independently of smoking status [8].

Similarly, in a pediatric cohort [9], blood eosinophilia (? 300 cells/?L) is associated with asthma severity (p?=?0.036), high atopy (p?=?0.001), more exacerbations (p?=?0.022), FEV1/FVC (p?=?0.004), and bronchial hyperresponsiveness (p?=?0.002).

Blood eosinophils counts can predict responsiveness to corticosteroid therapy. In atopic asthmatic children with blood eosinophilia (? 300 cells/?L), daily inhaled corticosteroids use is associated with more asthma control days and lower exacerbations rate [10]. Previous data showed that blood eosinophils count could be useful to monitor the response to oral corticosteroids because the adjustment of dose to maintain blood eosinophilia ?61% [68]. Sputum neutrophilia is associated with asthma severity and poor response to corticosteroids [64, 69]. Macrolide treatment could be a possible therapeutic intervention for these patients. Clarithromycin administration (500?mg twice daily) in patients with refractory asthma reduced the airway neutrophil counts and improved the quality of life in patients undergoing active treatment. A subgroup analysis in patients with sputum neutrophilia of >?61% showed that they had greater improvements in quality of life scores compared with those without sputum neutrophilia [70]. A more recent trial (AMAZES) [71] confirmed the benefice of the macrolide treatment with a reduction in exacerbation rate and an improvement of quality of life in patients with refractory asthma who took azithromycin 500?mg three times per week for 48?weeks. Prior data suggested that activation of CXCR2 resulted in increased airway neutrophilia, thus contributing to the pathogenesis of non-eosinophilic asthma, but a recent trial with a CXCR2 antagonist in severe neutrophilic asthma (sputum neutrophils >?40%) not showed a significant improvement in asthma outcomes despite the reduction of sputum neutrophilia [72].

Recent data found that changes in sputum eosinophil count over time reflect fluctuations in clinical asthma control [73]. The high level of Group 2 ILC in the sputum is corelated with severe asthma whose airway eosinophilia is greater than 3%, despite normal blood eosinophil numbers (Discussion

In asthma, and particularly in the severe asthma, many biomarkers have been investigated but only few of them, so far, can be easily used in clinical practice [121]. The Table 1 summarizes the advantages, the limits and the utility in the clinical setting of major biomarkers.
Table 1

Summary of major biomarkers’ characteristics

Biomarker Advantages Limits Utility

Blood eosinophils -Minimal invasive
-Minimal patient effort
-Easy to measure and collect in the clinical setting
-Correlates with sputum eosinophilia -Painful and difficult in some patients
-Varying cut-offs used to determine predictive characteristics
-Can be elevated due to other causes, such as parasitic infection -Defines the inflammatory phenotype
-Predicts exacerbations, poor asthma control and greater airway obstruction
-Predicts therapeutic responses to corticosteroids and biotherapies
Serum IgE -Easy to measure
-Identifies patients who may be candidates for Anti-IgE therapy -Not predictive of response to Anti-IgE
-Outperformed by other markers of T2 inflammation and allergen specific IgE -Associated with asthma severity and airway remodelling
Serum periostin -Marker of Il-13 activity and T2 airway inflammation -Not currently realised in the clinical setting
-Can be elevated in growing children -Predicts a greater airway obstruction and decline of lung function
-Predicts therapeutic responses to biotherapies
Sputum eosinophils -Non invasive
-Reflects the upper airways -Difficult to collect
-Not all patients can provide adequate samples
-Not universally available
-Requires specialized training, equipment, laboratory -Defines the inflammatory phenotype
-Predicts responses to corticosteroids and biotherapies
FeNO -Non invasive
-Minimal patient effort
-Easy to collect in the clinical setting -Multiple confounders
-Requires specialized equipment -Identifies airways inflammation
-Predicts exacerbations and airways hyperreactivity
-Predicts responses to corticosteroids and several biotherapies

An ideal biomarker should be suitable to identify the disease as well the specific endotype/phenotype, useful in the monitoring of the disease and to determine the prognosis, easily to obtain with minimum discomfort or risk to the patient [3, 4, 121].

According to the presence of assessable biomarkers of T2 mediated airway inflammation, the cluster-analysis identified several asthma phenotypes. The T2-high phenotype includes the classical allergic one (mild blood eosinophilia, high levels of FeNO, high level of serum total IgE) and the late-onset, nonallergic but highly eosinophilic one, frequently associated to chronic rhinosinusitis with nasal polyps (high FeNO but serum total IgE normal or elevated but probably with a lower etiopathogenetical importance) [1, 121]. The eosinophilic phenotype is associated with an intense production of IL-5 and IL-13. The T2-low phenotypes are more diversified and less well defined, with predominant neutrophilic airway inflammation, higher frequency of recurrent airway infections, higher prevalence of obesity and cigarette smoking. The mechanisms implicated in these phenotypes are the TNF? and IL-17 inflammatory pathways [69].

Unfortunately, at the moment, an ideal biomarker doesn’t exist and the overlap between the biomarkers is a reality. Using panels of biomarkers could improve probably the identification of asthma endotypes in the era of the precision medicine.

Other desired characteristics of a biomarker are the easiness and non-invasiveness of assessment. The development of point-of-care testing and non-invasive devices (one validated recently for the blood eosinophil count, others in study for the assessment of serum IgE and periostin) could accelerate the path leading to a precision medicine approach and clinical management of severe asthma [121].

Biomarkers, in addition to their role in defining phenotypes and endotypes may also have a predictive value for the response to biologic treatments. Serum total IgE is used in practice to verify that a patient with severe allergic asthma could be a candidate for omalizumab therapy and blood eosinophils count (usually ?300 cells/?L) to prescribe biological agents such as anti-IL5 antibody in the eosinophilic refractory severe asthma. If in the last 10?years, only omalizumab was available, followed by mepolizumab, we will move in the next few years to a situation in which we will have to choose one monoclonal antibody among many (benralizumab, an IL-5 receptor antagonist; dupilumab, an IL-4 receptor alpha antagonist; tezepelumab, an anti-thymic stromal lymphopoietin antibody). This implies the need of more selective biomarkers (or panels of them) in order to identify the right biologic therapy for each single patient, in a more personalized and precise medicine approach to the disease treatment [2, 121].

Conclusions

The implementation of the precision medicine in the management of asthma in clinical practice requires the detection of valid biomarkers. A variety of biomarkers have been used clinically to predict the response to steroid therapy, and in the clinical trial setting to identify patients that will respond to biologic therapies, but currently available biomarkers are limited in number and precision. At the moment, for a patient with a severe allergic asthma (high level of serum total IgE, high FeNO, normal or mild blood eosinophilia) uncontrolled despite a Step 4 or 5 treatment of GINA guideline, omalizumab seems to be the most adapted therapeutic option. If failure, another biologic therapy such as mepolizumab or reslizumab could be prescribe if blood eosinophilia (? 300 cells/?L, respectively ?400 cells/?L). In the refractory eosinophilic asthma without atopic background (high blood eosinophilia, high FeNO, normal IgE), an anti-IL5 antibody seems to be the most appropriate. Macrolides could be an interesting therapeutic option for the patients with severe uncontrolled asthma with T2-low inflammatory pattern, as well the bronchial termoplasty in patients with airways remodeling.

Further research and validation of emerging biomarkers are needed to define the molecular phenotype of asthma, particularly in the non-T2 pathways, to predict outcomes and therapeutic response to more specific targeted therapies. The use of omics data from multiple platforms (transcriptomics, proteomics, or metabolomics) appears as a promising tool to obtain endotypes. Viewing the heterogeneity of asthma, to predict therapeutic response, the development of composite biomarkers from blood, urine and exhaled breath seams to be a more appropriate solution in practice.

Asthma’s Inner World – a patients journey of discovery

By Alan Gray

World Asthma Foundation (WAF) is supporting care of Asthma and asthmatics around the world through a new Severe Asthma Series focused on “Defeating Asthma” with the aim of shining a spotlight on a deeper understanding and getting to a cure.
I’m Alan Gray, the Director of the World Asthma Foundation (WAF) located in Adelaide, Australia. Today, I’m talking to Bill Cullifer, in Northern California, he’s the founder of the World Asthma Foundation (WAF) and a Severe Asthmatic. I’m hoping to spend some socially distanced time with Bill to get his perspective on why he chose to establish the WAF in 2003 and what he finds important about Severe Asthma. We’ll also cover what he’d like me to accomplish heading up the Severe Asthma project as the Director in Australia.

Backstory

Bill retired in 2013 from his Web professional career as a result of battling severe respiratory issues. Complicated by anaphylaxis to Aspirin and allergy to Aspergillus, a common and ubiquitous Fungi in the air we breathe every day. Bill has debilitating Severe Asthma. Severe Asthmatics are at high risk for COVID19, so reaching out to Bill today is timely since he’s isolated like many other Asthmatics. As a colleague and friend, Bill has asked me to lend my web publishing experience to share his 17-year personal journey of discovery with Asthmatics everywhere. I’m pleased to be a supporter of the Asthma community and to lend a hand.

Question and Answer session with Bill Cullifer, Severe Asthmatic and Founder WAF

Alan: Good morning Bill and thanks for making yourself available.

Bill: Good morning Alan and thanks for the kind words and the gracious support. Nice to hear from you today.

Alan: Bill, we’ve known each other for over 20 years dating back to your Web professional efforts to educate and certify Web workers around the globe. I appreciate you reaching out to me to support the Severe WAF and the Severe Asthma Series. To that end, I have a few questions for you.

Bill: Ok, great thanks Alan and thanks for your support.

Alan: Why does Severe Asthma matter to you?

Bill: Great question. Severe Asthma is a global health crisis that affects over 300 million people worldwide. Asthma has already reached Pandemic levels by definition standards published by the World Health Organization (WHO). For those that suffer, Severe Asthma can be very debilitating and can cause premature death. I know first hand because Severe Asthma has dogged me personally for the last 17 years. While inhalers can be effective treatment for some, many Severe Asthmatics require daily systemic steroids, expensive treatment options and physical therapy.

Asthma rates are just getting worse. The projected rate for Asthma tops 400 million worldwide in the middle part of this decade. This is unacceptable really. Despite significant advances in our understanding, Severe Asthma continues to wreak havoc on individuals and our global economy. Given the toll on individuals, the burden on society and the huge financial cost, we need an “all hands on deck” to turn this around. Asthma education and advocacy are an integral piece for solving this puzzling disease in my opinion.

Alan: What can we expect from the WAF Severe Asthma series?

Bill: For a number of Severe Asthmatics, getting to a definitive diagnosis, can take years. In fairness, Severe Asthma is a complex disease, it’s confusing and frustrating for clinicians alike as well.

The Severe Asthma Series is about my own personal journey of discovery. A research journey that’s still unfolding actually. With encouragement from family and friends to share my story with others, I’ve turned over my 17 binders of notes, assembled my documents and medical records. I hope others can benefit from my story.

Alan: Any key takeaways?

Bill: For starters, Asthma is way more complicated than experts first realized actually. Also, Asthma is not a single disease but rather a syndrome. That’s major progress because it’s not only descriptive, it’s the truth. I’ve struggled to understand this for decades. We can’t defeat what we don’t understand and I think that unlocking the mystery is part of the Asthma solution I’d say.

Alan: How are you now and how are you holding up with the global COVID19 pandemic?

Bill: Severe Asthmatics are at high risk for COVID19 according to health experts around the globe. Like many in the over 60 crowd with underlying health issues, I’m hunkering down. I’m trusting my own instincts and following health guidelines by avoiding outside contact by staying indoors and hopefully out of harm’s way. Severe Asthma and COVID19 are both as much mystifying as they are isolating. I empathize with Asthmatics everywhere. It’s really a tough and uncertain time. Playing it smart, I think we’ll get through this.

Alan: Why did you establish the World Asthma Foundation (WAF) and what do you hope to accomplish with the Severe Asthma Series?

Bill: Alan, It’s human nature to want to learn more when you or someone close to you is diagnosed with a potential life threatening illness. To help me improve my personal understanding and diagnosis, I created a simple website at http://worlsasthmafoundation.org in 2003 and registered the WAF on the web. More of a newsfeed really than a website, The goal was to harness and publish daily Asthma news from around the world and to automate the delivery to my email every 24 hours. Community forums were not as robust as they are today. Automation saved me time from manually searching for the daily news. I learn something new about Asthma every day. Way more informational than I ever gleaned from reading the pamphlets at the doctors office. Today, the WAF has evolved to include a lot more than just the news. Over 8k subscribers last I checked. A lot has changed since 2003. Advances in research and technology, along with a number of very passionate researchers is on the rise and its a good thing to be reporting on. Ideally, and if you’re willing, I’m hoping to leverage your web publishing background to provide timely and relevant Asthma information that will benefit those that suffer. Asthma education matters and my hunch is that my findings can go a long way in moving the needle to our collective understanding of Severe Asthma.

Alan: What would you like Asthmatics to know about this series?

Bill: Severe Asthmatics like myself have daily struggles trying to breathe and living to see another day. I’m hopeful that my journey of discovery of the past 17 years will improve the level of understanding for the Asthma community. Asthma for example, is driven by both genetics and environmental factors, We’ve known that for sometime now. But what does that mean exactly? It’s been my mission to unpack this mystery. The genes we inherit are important but what impact does the environment have on our dna? Activation of the immune system has plagued researchers for years and it would also be nice to unpack this mystery as well. To be clear, I’m not a physician, and this should not serve as medical advice. I’m just a regular guy with Severe Asthma that’s trying to figure Severe Asthma out like everyone else. Science is about unlocking the truth and the truth is, together Asthmatics can ultimately prevail in getting the answers to a multitude of questions. Leading to a cure would be fantastic.

Alan: What would you like me to do to help Bill?

Bill: Alan, you’re an experienced web publisher. I’d like you to publish my findings and journey of discovery – a patient perspective to support those that suffer and those that support them. Interview the experts too and support the community with their expertise too. You’re good at this and it will help a lot. I’d be greatly appreciative and I know others will as well.

Alan: Thanks Bill. I appreciate your support as well. Asthma is a worthy cause. Take care of yourself and stay safe!

Bill: Thanks and you as well.

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COVID-19 Clues -Shortness of breath is a critical differentiator from other common illnesses

Interesting News Asthmatics

Harvard Medical School is reporting a couple of days ago on their news its news website, that “What can we learn from people with coronavirus who seek care at outpatient clinics?

NOTE from WAF: We salute Harvard Medical School for the sharing of this research and all of those on the front line. If your an Asthmatic and you experience any of these symptoms including severe shortness of breathe call someone right away. 

Since the early days of the COVID-19 pandemic, scientific literature and news reports have dedicated much attention to two groups of patients—those who develop critical disease and require intensive care and those who have silent or minimally symptomatic infections. This article is part of Harvard Medical School’s continuing coverage of medicine, biomedical research, medical education and policy related to the SARS-CoV-2 pandemic and the disease COVID-19.

Such accounts have mostly overlooked another large and important category of patients—those with symptoms concerning enough to seek care, yet not serious enough to need hospital treatment.

Now, a new analysis by researchers at Harvard Medical School and Harvard-affiliated Cambridge Health Alliance offers insights into this in-between category based on data collected from people presenting at an outpatient COVID-19 clinic in Greater Boston.

The team’s observations, published April 20 in the journal Mayo Clinic Proceedings, are based on data from more than 1,000 patients who visited the clinic for respiratory illness since COVID-19 was declared a pandemic in March.

The findings offer a compilation of clues that can help clinicians distinguish between patients with COVID-19 infections and those with other conditions that may mimic COVID-19 symptoms.

Such clues are critical because early triage and rapid decision-making remain essential even now that testing is becoming more widely available than it was in the early days of the pandemic, the research team said. Testing remains far from universal, and even when available, tests still may have a turnaround time of one to three days. Additionally, some rapid point-of-care tests that have emerged on the market have not been entirely reliable and have caused false-negative readings.

“Early recognition and proper triage are especially important given that in the first days of infection, people infected with SARS-CoV-2 may experience symptoms indistinguishable from a variety of other acute viral and bacterial infections,” said study lead author Pieter Cohen, an associate professor of medicine at Harvard Medical School and a physician at Cambridge Health Alliance. “Even when point-of-care diagnostic tests are available, given the potential for false-negative results, understanding the early natural history of COVID-19 and good old-fashioned clinical skills will remain indispensable for proper care.”

A nuanced understanding of the typical presentation of COVID-19 in the outpatient setting can also help clinicians determine how often to check back with patients, the researchers added. For example, those who have started developing shortness of breath demand very close monitoring and frequent follow-up to check how the shortness of breath is evolving and whether a patient may be deteriorating and may need to go to the hospital.

According to the report, COVID-19 typically presents with symptoms suggestive of viral infection, often with low-grade fever, cough and fatigue, and, less commonly, with gastrointestinal trouble. Shortness of breath usually emerges a few days after initial symptoms, becomes most pronounced upon exertion and may involve sharp drops in blood oxygen levels.

Chief among the team’s findings:

* Fever is not a reliable indicator. If present, it could manifest only with mild elevations in temperature.
* COVID-19 may begin with various permutations of cough without fever, sore throat, diarrhea, abdominal pain, headache, body aches, back pain and fatigue
* It can also present with severe body aches and exhaustion.
* A reliable early hint is loss of the sense of smell in the first days of disease onset.
* In serious COVID-19, shortness of breath is a critical differentiator from other common illnesses.
* Almost no one, however, develops shortness of breath, a cardinal sign of the illness, in the first day or two of disease onset.
* Shortness of breath can appear four or more days after onset of other symptoms.
* The first days after shortness of breath begins are a critical period that requires close and frequent monitoring of patients by telemedicine visits or in-person exams.
* The most critical variable to monitor is how the shortness of breath changes over time. Oxygen saturation levels can also be a valuable clue. Blood oxygen levels can drop precipitously with exertion, even in previously healthy people.
* A small number of people may never develop shortness of breath, but may have other symptoms that could signal low oxygen levels, including dizziness or falling.
* Anxiety—common among worried patients with viral symptoms suggestive of COVID-19—can also induce shortness of breath.

Distinguishing between anxiety-induced shortness of breath and COVID-19-related shortness of breath is critical. There are several ways to tell the two apart.

Key differentiators include:

Time of onset: Anxiety-induced shortness of breath occurs rapidly, seemingly out of the blue, while COVID-19 shortness of breath tends to develop gradually over a few days.
Patient description of sensation: Patients whose shortness of breath is caused by anxiety often describe the sensation occurring during rest or while trying to fall asleep but does not become more pronounced with daily activities. They often describe a sensation of inability to get enough air into their lungs. By contrast, shortness of breath induced by COVID-19-related drops in oxygen gets worse with physical exertion, including performing simple daily activities like walking, climbing stairs or cleaning.
Anxiety-related shortness of breath does not cause drops in blood oxygen levels

During a clinical exam, a commonly used device, the pulse oximeter, can be valuable in distinguishing between the two. The device measures blood oxygen levels and heart rate in a matter of seconds when clipped onto one’s finger.

Several types of pneumonia—a general term denoting infection in the lungs—can present with striking similarity to COVID-19. For example, COVID-19 respiratory symptoms appear to closely mimic symptoms caused by a condition known as pneumocystis pneumonia, a pulmonary infection predominantly affecting the alveoli, the tiny air sacs lining the surface of the lungs. Both COVID-19 patients and patients with pneumocystis pneumonia experience precipitous drops in oxygen levels with exertion and shortness of breath. However, in the case of pneumocystis pneumonia, the shortness of breath typically develops insidiously over weeks, not within days, as is the case with COVID-19. Here, a careful patient history detailing evolution of symptoms would be critical, the authors said.

Likewise, during the initial days of infection, both the flu and COVID-19 may have identical presentations, but thereafter the course of the two infections diverges. People with uncomplicated flu rarely develop significant shortness of breath. When they do experience trouble breathing, the shortness of breath is mild and remains stable. On the rare occasion of when flu causes a viral pneumonia, patients deteriorate rapidly, within the first two to three days. By contrast, patients with COVID-19 don’t begin to develop shortness of breath until several days after they first become ill.

Study co-investigators include Lara Hall, Janice Johns and Alison Rapaport.

Fragranced consumer products: effects on asthmatics

WAF Salutes Anne Steinemann, Department of Infrastructure Engineering, Melbourne School of Engineering, The University of Melbourne, Melbourne, VIC 3010 Australia

Fragranced consumer products, such as cleaning supplies, air fresheners, and personal care products, can emit a range of air pollutants and trigger adverse health effects. This study investigates the prevalence and types of effects of fragranced products on asthmatics in the American population. Using a nationally representative sample (n?=?1137), data were collected with an on-line survey of adults in the USA, of which 26.8% responded as being medically diagnosed with asthma or an asthma-like condition.

Results indicate that 64.3% of asthmatics report one or more types of adverse health effects from fragranced products, including respiratory problems (43.3%), migraine headaches (28.2%), and asthma attacks (27.9%). Overall, asthmatics were more likely to experience adverse health effects from fragranced products than non-asthmatics (prevalence odds ratio [POR] 5.76; 95% confidence interval [CI] 4.34–7.64). In particular, 41.0% of asthmatics report health problems from air fresheners or deodorizers, 28.9% from scented laundry products coming from a dryer vent, 42.3% from being in a room cleaned with scented products, and 46.2% from being near someone wearing a fragranced product. Of these effects, 62.8% would be considered disabling under the definition of the Americans with Disabilities Act. Yet 99.3% of asthmatics are exposed to fragranced products at least once a week. Also, 36.7% cannot use a public restroom if it has an air freshener or deodorizer, and 39.7% would enter a business but then leave as quickly as possible due to air fresheners or some fragranced product. Further, 35.4% of asthmatics have lost workdays or a job, in the past year, due to fragranced product exposure in the workplace. More than twice as many asthmatics would prefer that workplaces, health care facilities and health care professionals, hotels, and airplanes were fragrance-free rather than fragranced. Results from this study point to relatively simple and cost-effective ways to reduce exposure to air pollutants and health risks for asthmatics by reducing their exposure to fragranced products.

The online version of this article (10.1007/s11869-017-0536-2) contains supplementary material, which is available to authorized users.
Keywords: Asthma, Fragranced consumer products, Indoor air quality, Fragrance, Health effects, Volatile organic compounds, Semi-volatile organic compounds

Introduction

Fragranced consumer products pervade society and emit numerous volatile organic compounds, such as limonene, alpha-pinene, beta-pinene, acetaldehyde, and formaldehyde (Steinemann 2015; Nazaroff and Weschler 2004), and semi-volatile organic compounds, such as musks and phthalates (Weschler 2009; Just et al. 2010). However, ingredients in fragranced products are exempt from full disclosure on product labels or safety data sheets (Steinemann 2015), limiting awareness of potential emissions and exposures. Fragranced products have been associated with a range of adverse health effects including work-related asthma (Weinberg et al. 2017), asthmatic exacerbations (Kumar et al. 1995; Millqvist and Löwhagen 1996), respiratory difficulties (Caress and Steinemann 2009), mucosal symptoms (Elberling et al. 2005), migraine headaches (Kelman 2004), and contact dermatitis (Rastogi et al. 2007; Johansen 2003), as well as neurological, cardiovascular, cognitive, musculoskeletal, and immune system problems (Steinemann 2016).

This article investigates specifically the effects of exposure to fragranced products on asthmatics in the US population. In addition to health impacts, it also investigates societal access, preferences for fragrance-free environments, awareness of fragranced product emissions, and implications for air quality and health. It compares results from the sub-population of asthmatics with non-asthmatics, as well as with the general US population, as reported in Steinemann (2016). The study provides important data on the extent and severity of the problem, pointing to opportunities to reduce the adverse health, economic, and societal effects by reducing exposure to fragranced products.

Methods

A nationally representative on-line survey was conducted of the US population, representative of age, gender, and region (n?=?1137, confidence limit?=?95%, confidence interval?=?3%). The survey drew upon a large web-based US panel (over 5,000,000 people) held by Survey Sampling International, using randomized participant recruitment (SSI 2016). The survey instrument was developed and tested over a two-year period before full implementation in June 2016. The survey response rate was 95% (responses to panel recruitment 1201; screen-outs 13; drop-outs 46; completes 1137), and all responses were anonymous. The research study received ethics approval from the University of Melbourne. Details on the survey methodology are provided as a supplemental document.

This article extends and deepens the general population study of Steinemann (2016) by analyzing specifically the effects on asthmatics and compared to non-asthmatics and the general population. Of the general population surveyed, 26.8% responded as being medically diagnosed with either asthma (15.2%, n?=?173) or an asthma-like condition (12.5%, n?=?142) or both (26.8%, n?=?305). For the purposes of the article, the sub-population of “asthmatics” will be those medically diagnosed with asthma, an asthma-like condition, or both; the sub-population of “non-asthmatics” will be those in the general population other than asthmatics.

Survey questions investigated use and exposure to fragranced products, both from one’s own use and from others’ use, exposure contexts and products, health effects related to exposures, impacts of fragrance exposure in the workplace and in society, awareness of fragranced product ingredients and labeling, preferences for fragrance-free environments and policies, and demographic information.

Specific exposure contexts included air fresheners or deodorizers used in public restrooms and other environments, scented laundry products coming from a dryer vent, being in a room after it was cleaned with scented cleaning products, being near someone wearing a fragranced product, entering a business with the scent of fragranced products, fragranced soap used in public restrooms, and ability to access environments that used fragranced products.

Fragranced products were categorized as follows: (a) air fresheners and deodorizers (e.g., sprays, solids, oils, disks); (b) personal care products (e.g., soaps, hand sanitizer, lotions, deodorant, sunscreen, shampoos); (c) cleaning supplies (e.g., all-purpose cleaners, disinfectants, dishwashing soap); (d) laundry products (e.g., detergents, fabric softeners, dryer sheets); (e) household products (e.g., scented candles, restroom paper, trash bags, baby products); (f) fragrance (e.g., perfume, cologne, after-shave); and (g) other.

Health effects were categorized as follows: (a) migraine headaches; (b) asthma attacks; (c) neurological problems (e.g., dizziness, seizures, head pain, fainting, loss of coordination); (d) respiratory problems (e.g., difficulty breathing, coughing, shortness of breath); (e) skin problems (e.g., rashes, hives, red skin, tingling skin, dermatitis); (f) cognitive problems (e.g., difficulties thinking, concentrating, or remembering); (g) mucosal symptoms (e.g., watery or red eyes, nasal congestion, sneezing); (h) immune system problems (e.g., swollen lymph glands, fever, fatigue); (i) gastrointestinal problems (e.g., nausea, bloating, cramping, diarrhea); (j) cardiovascular problems (e.g., fast or irregular heartbeat, jitteriness, chest discomfort); (k) musculoskeletal problems (e.g., muscle or joint pain, cramps, weakness); and (j) other. Categories were derived from prior studies of fragranced products and health effects (Caress and Steinemann 2009; Miller and Prihoda 1999) and pre-tested before full survey implementation.

Results

Main findings are presented in this section, and full results for asthmatics, non-asthmatics, and the general population are provided as supplemental documentation. Demographic information is provided in Table ?Table11.

Table 1

Demographic information
Asthmatics Non-asthmatics General population
N N N
% of column total N
% of general population row N
% of column total
% of column total % of general population row
Total 305 305 832 832 1137
100.0% 26.8% 100.0% 73.2% 100.0%
Male/female
?All males 136 136 389 389 525
44.6% 25.9% 46.8% 74.1% 46.2%
?All females 169 169 443 443 612
55.4% 27.6% 53.2% 72.4% 53.8%
Gender–age
?Male 18–24 16 16 31 31 47
5.2% 34.0% 3.7% 66.0% 4.1%
?Male 25–34 36 36 94 94 130
11.8% 27.7% 11.3% 72.3% 11.4%
?Male 35–44 42 42 94 94 136
13.8% 30.9% 11.3% 69.1% 12.0%
?Male 45–54 30 30 78 78 108
9.8% 27.8% 9.4% 72.2% 9.5%
?Male 55–65 12 12 92 92 104
3.9% 11.5% 11.1% 88.5% 9.1%
?Female 18–24 26 26 52 52 78
8.5% 33.3% 6.3% 66.7% 6.9%
?Female 25–34 40 40 95 95 135
13.1% 29.6% 11.4% 70.4% 11.9%
?Female 35–44 43 43 112 112 155
14.1% 27.7% 13.5% 72.3% 13.6%
?Female 45–54 41 41 103 103 144
13.4% 28.5% 12.4% 71.5% 12.7%
?Female 55–65 19 19 81 81 100
6.2% 19.0% 9.7% 81.0% 8.8%
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Fragranced product exposure

Among asthmatics, 99.0% are exposed to fragranced products at least once a week, from their own use (71.1% air fresheners and deodorizers; 85.9% personal care products; 78.4% cleaning supplies; 81.3% laundry products; 76.7% household products; 67.5% fragrance; 3.6% other). Further, 94.8% are exposed to fragranced products at least once a week, from others’ use. Combined, 99.3% of asthmatics are exposed to fragranced products through their own use, others’ use, or both. Among non-asthmatics, 98.1% are exposed to fragranced products at least once a week from their own use, 91.1% from others’ use, and 98.9% from either or both. Thus, asthmatics are more likely to be exposed to fragranced products, from their own use and others’ use and both, than non-asthmatics (POR, 1.66; 95% CI, 0.36–7.71).
Adverse health effects

Among asthmatics, 64.3% reported one or more types of adverse health effects from exposure to one or more types of fragranced products (43.3% respiratory problems; 27.2% mucosal symptoms; 28.2% migraine headaches; 19.0% skin problems; 27.9% asthma attacks; 15.1% neurological problems; 14.1% cognitive problems; 12.1% gastrointestinal problems; 9.8% cardiovascular problems; 11.1% immune system problems; 9.5% musculoskeletal problems; and 1.3% other). Among non-asthmatics, 23.8% reported one or more types of adverse health effects from exposure to one or more types of fragranced products (see Table ?Table2).2). Thus, among all types of health effects (excepting asthma attacks), asthmatics are more likely to be affected than non-asthmatics (POR 5.76; 95% CI, 4.34–7.64).
Table 2

Frequency and types of adverse health effects reported from exposure to fragranced consumer products
Asthmatics Non-asthmatics General population
305 832 1137
26.8% 73.2% 100.0%
Migraine headaches 86 93 179
28.2% 11.2% 15.7%
Asthma attacks 85 6 91
27.9% 0.7% 8.0%
Neurological problems 46 36 82
15.1% 4.3% 7.2%
Respiratory problems 132 79 211
43.3% 9.5% 18.6%
Skin problems 58 63 121
19.0% 7.6% 10.6%
Cognitive problems 43 23 66
14.1% 2.8% 5.8%
Mucosal symptoms 83 101 184
27.2% 12.1% 16.2%
Immune system problems 34 11 45
11.1% 1.3% 4.0%
Gastrointestinal problems 37 26 63
12.1% 3.1% 5.5%
Cardiovascular problems 30 20 50
9.8% 2.4% 4.4%
Musculoskeletal problems 29 14 43
9.5% 1.7% 3.8%
Other 4 15 19
1.3% 1.8% 1.7%
Total 196 198 394
(One or more health problems) 64.3% 23.8% 34.7%
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Of the 64.3% of asthmatics reporting adverse health effects from fragranced products, proportionately more males report adverse effects than females, relative to non-asthmatics (asthmatic 52.0% female, 48.0% male; non-asthmatic 60.1% female, 39.9% male) (POR 1.39; 95% CI, 0.93–2.97) (see Table ?Table3).3). Among all age groups, proportionately more asthmatics in age group 25–34 report adverse effects relative to non-asthmatics (asthmatic 69.7%; non-asthmatic 23.3%) (POR 7.59; 95% CI, 4.19–13.76). Among all gender and age groups, proportionately more males age 25–34 report adverse effects relative to non-asthmatics (asthmatic 83.3%; non-asthmatic 18.1%) (POR 22.65; 95% CI, 8.15–62.92).
Table 3

Demographic information for individuals reporting adverse effects from exposure to fragranced products
Asthmatics Non-asthmatics General population
N
% of column total N
% of asthmatics row, Table ?Table11 N
% of column total N
% of non-asthmatics row, Table ?Table11 N
% of column total N
% of general population row, Table 1
Total 196 196 198 198 394 394
100.0% 64.3% 100.0% 23.8% 100.0% 34.7%
Male/female
?All males 94 94 79 79 173 173
48.0% 69.1% 39.9% 20.3% 43.9% 33.0%
?All females 102 102 119 119 221 221
52.0% 60.4% 60.1% 26.9% 56.1% 36.1%
Gender–age
?Male 18–24 8 8 6 6 14 14
4.1% 50.0% 3.0% 19.4% 3.6% 29.8%
?Male 25–34 30 30 17 17 47 47
15.3% 83.3% 8.6% 18.1% 11.9% 36.2%
?Male 35–44 31 31 24 24 55 55
15.8% 73.8% 12.1% 25.5% 14.0% 40.4%
?Male 45–54 17 17 15 15 32 32
8.7% 56.7% 7.6% 19.2% 8.1% 29.6%
?Male 55–65 8 8 17 17 25 25
4.1% 66.7% 8.6% 18.5% 6.3% 24.0%
?Female 18–24 12 12 8 8 20 20
6.1% 46.2% 4.0% 15.4% 5.1% 25.6%
?Female 25–34 23 23 27 27 50 50
11.7% 57.5% 13.6% 28.4% 12.7% 37.0%
?Female 35–44 28 28 33 33 61 61
14.3% 65.1% 16.7% 29.5% 15.5% 39.4%
?Female 45–54 27 27 26 26 53 53
13.8% 65.9% 13.1% 25.2% 13.5% 36.8%
?Female 55–65 12 12 25 25 37 37
6.1% 63.2% 12.6% 30.9% 9.4% 37.0%
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Specific exposure contexts

Air fresheners and deodorizers were associated with health problems for 41.0% of asthmatics (54.4% respiratory problems, 39.2% asthma attacks, 29.6% mucosal symptoms, 36.8% migraine headaches, 15.2% neurological problems, 26.4% skin problems, and others), and for 12.9% of non-asthmatics (see Table ?Table4).4). Thus, asthmatics were more likely to experience adverse effects from air fresheners than non-asthmatics (POR 4.71; 95% CI, 3.47–6.39).
Table 4

Frequency and types of health problems experienced by asthmatics, non-asthmatics, and the general population from exposure to four types of fragranced consumer products
Air fresheners or deodorizers Scented laundry products Scented cleaning products Fragranced person
Asth Non-asth Gen Pop Asth Non-asth Gen Pop Asth Non-asth Gen Pop Asth Non-asth Gen Pop
Health problem 125 107 232 88 54 142 129 95 224 141 127 268
41.0% 12.9% 20.4% 28.9% 6.5% 12.5% 42.3% 11.4% 19.7% 46.2% 15.3% 23.6%
Migraines 46 36 82 24 13 37 42 33 75 45 51 96
36.8% 33.6% 35.3% 27.3% 24.1% 26.1% 32.6% 34.7% 33.5% 31.9% 40.2% 35.8%
Asthma attacks 49 4 53 27 1 28 42 4 46 41 3 44
39.2% 3.7% 22.8% 30.7% 1.9% 19.7% 32.6% 4.2% 20.5% 29.1% 2.4% 16.4%
Neurological 19 17 36 16 8 24 28 19 47 27 14 41
15.2% 15.9% 15.5% 18.2% 14.8% 16.9% 21.7% 20.0% 21.0% 19.1% 11.0% 15.3%
Respiratory 68 40 108 34 12 46 67 42 109 77 41 118
54.4% 37.4% 46.6% 38.6% 22.2% 32.4% 51.9% 44.2% 48.7% 54.6% 32.3% 44.0%
Skin 33 32 65 22 19 41 25 20 45 24 15 39
26.4% 29.9% 28.0% 25.0% 35.2% 28.9% 19.4% 21.1% 20.1% 17.0% 11.8% 14.6%
Cognitive 15 16 31 9 6 15 21 10 31 21 9 30
12.0% 15.0% 13.4% 10.2% 11.1% 10.6% 16.3% 10.5% 13.8% 14.9% 7.1% 11.2%
Mucosal 37 49 86 27 21 48 35 48 83 40 58 98
29.6% 45.8% 37.1% 30.7% 38.9% 33.8% 27.1% 50.5% 37.1% 28.4% 45.7% 36.6%
Immune system 16 5 21 16 3 19 18 5 23 17 2 19
12.8% 4.7% 9.1% 18.2% 5.6% 13.4% 14.0% 5.3% 10.3% 12.1% 1.6% 7.1%
Gastrointestinal 18 13 31 20 9 29 17 15 32 21 10 31
14.4% 12.1% 13.4% 22.7% 16.7% 20.4% 13.2% 15.8% 14.3% 14.9% 7.9% 11.6%
Cardiovascular 18 12 30 11 4 15 16 10 26 15 5 20
14.4% 11.2% 12.9% 12.5% 7.4% 10.6% 12.4% 10.5% 11.6% 10.6% 3.9% 7.5%
Musculoskeletal 19 8 27 21 2 23 13 10 23 15 2 17
15.2% 7.5% 11.6% 23.9% 3.7% 16.2% 10.1% 10.5% 10.3% 10.6% 1.6% 6.3%
Other 2 6 8 1 3 4 2 2 4 2 5 7
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Scented laundry products coming from a dryer vent were associated with health problems for 28.9% of asthmatics (38.6% respiratory problems, 30.7% asthma attacks, 30.7% mucosal symptoms, 27.3% migraine headaches, 18.2% neurological problems, 25.0% skin problems, and others), and for 6.5% of non-asthmatics (see Table ?Table4).4). Thus, asthmatics were more likely to experience adverse effects from scented laundry products coming from a dryer vent than non-asthmatics (POR 5.84; 95% CI, 4.03–8.46).

Being in a room after it has been cleaned with scented products was associated with health problems for 42.3% of asthmatics (51.9% respiratory problems, 32.6% asthma attacks, 27.1% mucosal symptoms, 32.6% migraine headaches, 21.7% neurological problems, 19.4% skin problems, and others), and for 11.4% of non-asthmatics (see Table ?Table4).4). Thus, asthmatics were more likely to experience adverse effects from being in a room after it has been cleaned with scented products than non-asthmatics (POR 5.69; 95% CI, 4.16–7.77).

Being near someone wearing a fragranced product was associated with health problems for 46.2% of asthmatics (54.6% respiratory problems, 29.1% asthma attacks, 28.4% mucosal symptoms, 31.9% migraine headaches, 19.1% neurological problems, 17.0% skin problems, and others), and 15.3% of non-asthmatics (see Table ?Table4).4). Thus, asthmatics were more likely to experience adverse effects from being near someone wearing a fragranced product than non-asthmatics (POR 4.77; 95% CI, 3.56–6.40).

Exposure to fragranced products can trigger disabling health effects, according to criteria from the Americans with Disabilities Act (ADA 1990): “Do any of these health problems substantially limit one or more major life activities, such as seeing, hearing, eating, sleeping, walking, standing, lifting, bending, speaking, breathing, learning, reading, concentrating, thinking, communicating, or working, for you personally?” Among asthmatics reporting health problems, 62.8% reported that the severity of the health effect from fragranced product exposure was potentially disabling. Thus, asthmatics were more likely to report disabling health effects from fragranced products than non-asthmatics (POR 7.13; 95% CI, 5.11–9.95).
Ingredient disclosure and product claims

Among asthmatics, 41.3% were not aware that a “fragrance” in a product is typically a chemical mixture of several dozen to several hundred chemicals, 57.4% were not aware that fragrance chemicals do not need to be fully disclosed on the product label or material safety data sheet, and 58.0% were not aware that fragranced products typically emit hazardous air pollutants such as formaldehyde. Further, 64.3% of asthmatics, and 75.7% of non-asthmatics, were not aware that even so-called natural, green, and organic fragranced products typically emit hazardous air pollutants (28.9% of asthmatics and 15.7% of non-asthmatics were aware). However, 60.3% of asthmatics, and 60.1% of non-asthmatics, would not still use a fragranced product if they knew it emitted hazardous air pollutants.
Societal and workplace effects

Fragranced products can also present barriers for asthmatics in public places and the workplace. Among asthmatics, 36.7% are prevented from using the restrooms in a public place, because of the presence of an air freshener, deodorizer, or scented product. Also, 28.9% are prevented from washing their hands with soap in a public place, if the soap is fragranced. Further, 43.9% are prevented from going to some place because they would be exposed to a fragranced product that would make them sick. Notably, 39.7% report that if they enter a business, and smell air fresheners or some fragranced product, they want to leave as quickly as possible.

Significantly, 35.4% of asthmatics, and 7.7% of non-asthmatics, have become sick, lost workdays, or lost a job, in the past 12 months, due to fragranced products in their work environment. Thus, asthmatics were more likely to have lost workdays or lost a job due to illness from fragranced products in their work environment than non-asthmatics (POR 6.58; 95% CI, 4.65–9.30).

Fragrance-free policies receive a strong majority of support. Among asthmatics, 66.2% would be supportive of a fragrance-free policy in the workplace (compared to 16.1% that would not). Thus, more than four times as many asthmatics would prefer a fragrance-free workplace than fragranced. Also, 72.1% of asthmatics would prefer that health care facilities and health care professionals be fragrance-free (compared to 14.8% that would not). Thus, nearly five times as many asthmatics would prefer fragrance-free health care facilities and professionals than fragranced.

Among non-asthmatics, 48.3% would support a fragrance-free workplace (compared with 21.0% that would not), and among the general population, 53.1% would support a fragrance-free workplace (compared with 19.7% that would not). Thus, regardless of population, fragrance-free workplaces receive more than twice as many in support as not.

Asthmatics also strongly prefer fragrance-free airplanes and hotels. If given a choice between flying on an airplane that pumped scented air throughout the passenger cabin, or did not pump scented air throughout the passenger cabin, 63.6% of asthmatics would choose an airplane without scented air (compared to 24.9% with scented air). Similarly, if given a choice between staying in a hotel with fragranced air, or without fragranced air, 63.0% would choose a hotel without fragranced air (compared to 28.5% with fragranced air).

Among non-asthmatics, 57.6 and 52.9% would prefer fragrance-free airplanes and hotels, respectively (compared with 23.1 and 27.5% that would not) and among the general population, 59.2 and 55.6% would prefer fragrance-free airplanes and hotels, respectively (compared with 23.6 and 27.8% that would not). Thus, overall, more than twice as many asthmatics, as well as the general population, would prefer that airplanes and hotels were fragrance-free rather than fragranced.

Discussion

Asthma is a serious and increasing health condition, affecting an estimated 25 million Americans, and costing an estimated $56 billion annually in medical expenses, missed school and work days, and premature deaths (CDCP 2017a). Nearly 12 million Americans had an asthma attack in 2015, many of which could have been prevented (CDCP 2017b).

Results from this study show that asthmatics are profoundly, adversely, and disproportionately affected by exposure to fragranced consumer products. While non-asthmatics are also affected, asthmatics are more likely to experience adverse health effects from exposure (POR 5.76; 95% CI 4.34–7.64).

Of particular concern are involuntary exposures to fragranced products, such as in health care facilities and workplaces. Asthmatics are prevented from accessing public toilets, businesses, and workplaces due to adverse health effects from fragranced products. Further, 35.4% have lost workdays or a job, in the past year, due to fragranced product exposure in the workplace. More than twice as many asthmatics would prefer that workplaces, health care facilities, health care professionals, airplanes, and hotels were fragrance-free than fragranced.

Limitations of the study include the following: (a) data were based on self-reports, although a well-established method for survey research; (b) all possible products and health effects were not included, although the low percentages for responses in the “other” category indicates the survey captured the primary products and effects; (c) product emissions and exposures were not measured directly; (d) the cross-sectional design of the study, while useful for determining prevalence, provides data that represent just one point in time, limiting the analysis of risk factors, temporal relationships between exposures and effects, and trends in prevalence, and (e) only adults (ages 18–65) were included in the survey, which overlooks the effects of fragranced products on children (such as in day care facilities and schools) and on seniors (such as in retirement communities and assisted living facilities).

Results of this study provide strong evidence that fragranced consumer products can harm health for both asthmatics and non-asthmatics, with asthmatics more affected. Understanding why these products are associated with a range of health problems is a critical topic that requires further research. Fragranced products emit a range of volatile and semi-volatile organic compounds, some of which are associated with adverse health effects, but virtually none of which need to be disclosed (Steinemann 2009, 2015), thus limiting scientific inquiry and public awareness of potential exposures to problematic compounds. A broader mechanistic framework is needed to understand which ingredients, or combinations of ingredients, could be associated with the adverse health outcomes reported in this study. In the meantime, a prudent and practical approach, and one that would provide direct and immediate benefits, would be to limit exposure to fragranced consumer products.

Perfumes, Magazines and Severe Asthma

Perfumes Strips and Scents in Magazines “Negatively Affect Asthmatics and adverse respiratory reactions to perfumes says study. In honor of #AsthmaAwarenessWeek and #WorldAsthmaDay can we stop doing this?

Note from the World Asthma Foundation. This study dates back to 1994. How much education is needed to change behavior? Can we PLEASE stop this practice already? It’s 2020 and we all know this to be true already right? Just saying People @people magazine.

Background

Perfume- and cologne-scented advertisement strips are widely used. There are, however, very few data on the adverse effects of perfume inhalation in asthmatic subjects.

OBJECTIVES:

This study was undertaken to determine whether perfume inhalation from magazine scent strips could exacerbate asthma.

METHODS:

Twenty-nine asthmatic adults and 13 normal subjects were included in the study. Histories were obtained and physical examinations performed. Asthma severity was determined by clinical criteria of the U.S.National Heart, Lung, and Blood Institute (NHLBI). Skin prick tests with common inhalant allergens and with the perfume under investigation were also performed. Four bronchial inhalation challenges were performed on each subject using commercial perfume scented strips, filter paper impregnated with perfume identical to that of the commercial strips, 70% isopropyl alcohol, and normal saline, respectively. Symptoms and signs were recorded before and after challenges. Pulmonary function studies were performed before and at 10, 20, and 30 minutes after challenges.
RESULTS:

Inhalational challenges using perfume produced significant declines in FEV1 in asthmatic patients when compared with control subjects. No significant change in FEV1 was noted after saline (placebo) challenge in asthmatic patients. The percent decline in FEV1 was significantly greater after challenge in severely asthmatic patients as compared with those with mild asthma. Chest tightness and wheezing occurred in 20.7% of asthmatic patients after perfume challenges. Asthmatic exacerbations after perfume challenge occurred in 36%, 17%, and 8% of patients with severe, moderate, and mild asthma, respectively. Patients with atopic asthma had greater decreases in FEV1 after perfume challenge when compared with patients with nonallergic asthma.

CONCLUSIONS:

Perfume-scented strips in magazines can cause exacerbations of symptoms and airway obstruction in asthmatic patients. Severe and atopic asthma increases risk of adverse respiratory reactions to perfumes.

U.S National Institutes of Health stands with Asthma patients, families, advocates, researchers, and health care professionals

Today on @WorldAsthmaDay, the U.S National Institutes of Health stands with patients, families, advocates, researchers, and health care professionals to raise awareness about this common chronic respiratory disease, the people it affects, and the biomedical research that improves its prevention and treatment.

Asthma is a chronic lung disease that causes periods of wheezing, chest tightness, shortness of breath, and coughing. It is a major contributing factor to missed time from school and work, with severe attacks requiring emergency room visits and hospitalizations. Sometimes these asthma attacks can be fatal.

This year, we recognize that the coronavirus disease 2019 (COVID-19) pandemic is creating concern and uncertainty for many people around the globe, including those with asthma. The disease can affect the nose, throat, and lungs, cause an asthma attack, and possibly lead to pneumonia and acute respiratory disease. According to the Centers for Disease Control and Prevention(link is external), people with asthma should continue their current asthma medications and discuss any concerns with their healthcare provider. Researchers at NIH and elsewhere are working to learn more about COVID-19 and to develop specific treatments and vaccines.

Three NIH institutes support and conduct studies on asthma — the National Institute of Environmental Health Sciences (NIEHS); the National Heart, Lung, and Blood Institute (NHLBI); and the National Institute of Allergy and Infectious Diseases (NIAID). Institute scientists and grantees made several important advances in understanding, treating, and managing asthma in 2019. These findings and other highlights are featured in five topic areas below:

Relationship between asthma and COVID-19
Populations at risk of developing asthma
Potential new treatments
Genes involved in asthma
Asthma management

Relationship between asthma and COVID-19

NIAID is initiating a home-based study to assess the incidence of infection with SARS-CoV-2, the virus that causes COVID-19, in children and their caregivers and siblings. A key objective of this observational study will be to determine if infection rates or immune responses to SARS-CoV-2 infection differ in children who have asthma or other allergic conditions compared to those who have not been diagnosed with or treated for these conditions.

NIAID also is starting an observational study in patients hospitalized for COVID-19 to understand if specific characteristics of the immune response influence or reflect the severity of infection. This study may help determine whether underlying diseases, such as asthma, influence the body’s response to SARS-CoV-2 infection.
Populations at risk of developing asthma

Children

NIH scientists are making progress in understanding the underlying factors that contribute to the development of asthma in U.S. children. This year, an international collaboration led by NIEHS scientists reported that the presence of newly discovered novel epigenetic markers — or chemical tags that attach to DNA — may indicate a newborn’s risk of developing asthma. The data were generated by the Pregnancy and Childhood Epigenetics Consortium and may help researchers find asthma biomarkers, or molecular indicators of asthma, and identify at birth which children will eventually develop the condition.

At NHLBI, researchers discovered a link(link is external) between childhood asthma flare ups and changes in the lung microbiome, the communities of bacteria and other microorganisms that are normally present in the lung and usually do not cause symptoms. The scientists determined that children with mild to moderate asthma who experienced early signs of an upcoming asthma flare up tended to have higher levels of certain types of disease-causing bacteria in their lungs. The study could lead to a precision medicine approach for treating mild to moderate childhood asthma by altering the number and types of bacteria in a child’s airways.

Ongoing NIAID-funded clinical studies focus on interventions to prevent asthma development in children at high risk of developing the condition. One team of researchers studied a large group of children who were hospitalized as infants with bronchiolitis, a common early-life lung infection usually caused by a virus. The scientists found that recurrent wheezing by age 3 is at least three times more likely to occur in children whose bronchiolitis was associated with a rhinovirus C infection and who also had early signs of allergy to foods or inhaled allergens.

African Americans and people of African ancestry

Another group that bears a disproportionate burden of asthma is African Americans. In an NHLBI-funded study that is the largest genome-wide association study of asthma in African ancestry populations to date, researchers identified two novel regions on a specific chromosome that may be linked to asthma risk. The scientists theorize that a better understanding of the genetic risk factors for asthma in African ancestry populations will lead to development of better therapeutic interventions.
Potential new treatments

Using a mouse model of asthma, NIEHS researchers reported a possible treatment for neutrophilic asthma, a particularly severe form that responds poorly to the standard asthma therapy of corticosteroids. The orally available drug VTP-938 made it easier for the mice to breathe after they were exposed to house dust extracts. The results suggest that VTP-938 may be an innovative treatment for humans with this steroid-resistant form of asthma.
Genes involved in asthma

An NIAID-funded study sought to understand why some, but not all, colds lead to asthma attacks among children with asthma. The scientists obtained nasal washings from 106 children with severe asthma who experienced cold symptoms. Members of the research team compared samples from those who required corticosteroids after a cold-induced asthma attack and those who did not have an asthma attack following a cold. The research team found that colds that led to an asthma attack caused changes in the production of six families of genes that are associated with maintaining the function of the outermost layer of tissue lining the respiratory tract and with the responses of immune cells in close contact with this layer.

Variations in two genes — the aryl hydrocarbon receptor nuclear translocator (ARNT) and the protein tyrosine phosphatase, nonreceptor type 22 (PTPN22) — are associated with immune-mediated diseases, such as asthma, in several ethnicities, according to NIEHS researchers and their collaborators. Because ARNT and PTPN22 are sensitive to environmental factors, this study is the first to demonstrate across ethnicities the combined role of these genes and environmental changes in the development of immune-related conditions like asthma.
Asthma management

NHLBI’s National Asthma Education Prevention Program is coordinating the 2020 focused updates to the 2007 Asthma Management Guidelines. These guidelines are designed to improve the care of people living with asthma as well as help primary care providers and specialists make decisions about asthma management. NHLBI released the updated focus areas of the guidelines for public comment, and the final recommendations for these areas are expected to be published later this year. They will address several priority topic areas listed below:

Using inhaled medications when needed
A new type of inhaled medication called long acting muscarinic antagonists
Treating allergies by exposure to low doses of allergens by mouth or with shots
Reducing indoor asthma triggers
A new procedure for asthma known as bronchial thermoplasty
A fractional exhaled nitric oxide test that may be helpful in diagnosing or managing asthma

Experts hope that these guidelines will help reduce the burden of asthma nationwide and improve the quality of life for those living with the condition.

Difficult Asthma and Fungus

 

If you or someone you know suffers from Severe Asthma, then it will be worth your time to learn about the world of Asthma and Fungi. While this paper is pretty technical and lengthy by design, it will provide you with a glimpse of how fungi and asthma are linked.

The World Asthma Foundation (WAF) salutes the Department of Paediatric Respiratory Medicine, Royal Brompton Hospital Harefield NHS Foundation and Paediatric Respiratory Medicine, National Heart and Lung Institute, Imperial College, Sydney Street for their contribution to medicine and those that suffer from Asthma. The WAF will cover this topic in more depth in future post.

Fungi have many potential roles in paediatric asthma, predominantly by being a source of allergens (severe asthma with fungal sensitization, SAFS), and also directly damaging the epithelial barrier and underlying tissue by releasing proteolytic enzymes (fungal bronchitis). The umbrella term ‘fungal asthma‘ is proposed for these manifestations. Allergic bronchopulmonary aspergillosis (ABPA) is not a feature of childhood asthma, for unclear reasons. Diagnostic criteria for SAFS are based on sensitivity to fungal allergen(s) demonstrated either by skin prick test or
specific IgE. In children, there are no exclusion criteria on total IgE levels or IgG precipitins because of the rarity of ABPA. Diagnostic criteria for fungal bronchitis are much less well established. Data in adults and children suggest SAFS is associated with worse asthma control and greater susceptibility to asthma attacks than non-sensitized patients. The data on whether antifungal therapy is beneficial are conflicting. The pathophysiology of SAFS is unclear, but the epithelial alarmin interleukin-33 is implicated. However, whether individual fungi have different pathobiologies is unclear. There are many unanswered questions needing further research, including how fungi interact with other allergens, bacteria, and viruses, and what optimal therapyshould be, including whether anti-neutrophilic strategies, such as macrolides, should be used.

Considerable further research is needed to unravel the complex roles of different fungi in severe asthma.

  1. Introduction
    The important role of fungi in worsening asthma has long been appreciated. In 2006, the term
    ‘severe asthma with fungal sensitization (SAFS)’ was first proposed in a review article that rightly
    acknowledged the historical evidence implicating fungi in the pathophysiology of asthma going
    back to the seventeenth century [1]. The role of fungi in asthma remains controversial to the present
    day, and these issues are reviewed below. What is certainly beyond dispute is that (a) although
    most acute attacks of asthma are precipitated by a viral infection, a sudden heavy aeroallergen load,
    such as grass pollen (“thunderstorm asthma”) [2] or soya bean (ships unloading in the docks of
    Barcelona) [3], can precipitate severe attacks, which might be eosinophilic rather than neutrophilic
    [4]; and, (b) fungal allergens can also cause acute attacks of asthma [5–7].
    The term SAFS focuses on allergic sensitization, but this is quite restrictive, because allergy is
    not the only mechanism whereby fungi can modulate asthma. Additional to allergic sensitization,
    which does not necessarily require airway fungal infection, is the release of tissue damaging
    proteases and other enzymes, which might disrupt the airway epithelial barrier and cause mucosal
    damage and airway remodeling [8]. For this to happen, a chronic fungal bronchitis needs to be
    J. Fungi 2019, 6, 55 2 of 17
    established. Sensitization and tissue damage both may co-exist. Here, I propose that the more
    general term ‘fungal asthma’ is used to encompass allergic sensitization (SAFS), fungal bronchitis,
    and combined sensitization/bronchitis (Figure 1). In adults, this would also include ABPA, not
    discussed here because of the rarity of this condition in children with asthma. All three entities may
    potentially benefit from anti-fungals, but fungal bronchitis without sensitization should not require
    the intensification of anti-Type 2 inflammatory medications. Of course, the pro-inflammatory
    effects of tissue damaging enzymes may merit treatment (as, for example, the anti-inflammatory
    strategies that may be used in cystic fibrosis (CF) [9,10] to counter the effects of infection driven,
    neutrophilic tissue destruction. The picture might also be dynamic; increasing inhaled steroids may
    cause topical immunosuppression (discussed in more detail below) and, thus, predispose to fungal
    bronchitis as a secondary phenomenon.
    Figure 1. Schematic of fungal involvement in asthma in children, in whom the diagnosis of allergic
    bronchopulmonary aspergillosis (ABPA) is rarely made.
    The justification for this sort of phenotyping is that it is clinically useful, because defining it
    leads to a change in management. Unfortunately, much of the paediatric guidance has had to be
    extrapolated from work in adults. The aim of this review is to assess the clinical utility of current
    concepts of fungal asthma (as defined above) in children, and suggest new approaches and where
    future work is needed. Although this review will focus as far as possible on children, it inevitably
    has to supplement this with adult experience and animal and cellular models where paediatric data
    are not available. Prior to writing this manuscript, a literature search was performed while using
    the search term limited to English Language papers,
    which was supplemented from the author’s personal archive of references.
  2. Definition of SAFS and Fungal Bronchitis
    2.1. SAFS in Adults
    SAFS was first defined in adults [11], and it has been suggested that it is a more severe
    phenotype than seen in unsensitized patients. For the purposes of the definition of SAFS, severe
    asthma is defined as treatment with 500 mcg Fluticasone/day or equivalent, or continuous oral
    corticosteroids, or four prednisolone bursts in the previous 12 months or six in the previous two
    years (as with so many definitions of severity, the figures are fairly arbitrary). The immunological
    J. Fungi 2019, 6, 55 3 of 17
    criteria for SAFS in adults also include a total immunoglobulin (Ig)-E < 1000, and negative IgG
    precipitins to Aspergillus fumigatus (AF) because allergic bronchopulmonary aspergillosis (ABPA) is
    a diagnostic consideration in adults, and in order to differentiate between ABPA and SAFS.
    Additionally, there needs to be evidence of sensitization (skin prick test wheal (SPT) ? 3 mm,
    specific IgE (sIgE) ? 0.4) to at least one of seven fungi, namely AF, Cladosporium herbarum, Penicillium
    chrysogenum (notatum), Candida albicans, Trichophyton mentagrophytes, Alternaria alternate, and Botrytis
    cinerea. The question as to whether sensitization is best determined by sIgE or SPTs was addressed
    in 121 patients with severe asthma (British Thoracic Society/SIGN steps 4 and 5) who underwent
    both tests to all the above fungi, except Trichophyton mentagrophytes [12]. Fungal sensitivity was very
    common, but concordance between skin prick tests and sIgE tests was poor (77% overall, but only
    14–56% for individual fungi). Hence, both of the tests need to be undertaken to rigorously diagnose
    SAFS.
    2.2. SAFS in Children
    There is no consensus definition of SAFS in children. Empirically, we define SAFS as severe,
    therapy resistant asthma [13] (STRA, with any pattern of symptoms), and we have used the same
    sensitization criteria as in adults, although in fact in a clinical setting we usually can only test for
    AF, Cladosporium and Alternaria alternate. For reasons that are unclear, ABPA is rarely, if ever, seen
    in children with asthma, despite being relatively common in children with CF [14], and so we do
    not adopt the IgE and IgG precipitin criteria of the adult definition. It is likely, but unproven, that
    there will also be discordance between sIgE and SPT results in children also [15], so both tests are
    needed.
    Table 1 contrasts the diagnosis of SAFS in adults and children.
    Table 1. Diagnostic criteria for severe asthma with fungal sensitization (SAFS) in adults and
    children.
    Fungal Sensitization
    (Positive Skin Prick Test
    and/or Specific IgE to One or
    More Fungus)
    Other Adult Criteria
    Other Paediatric
    Criteria
    Aspergillus fumigatus
    Cladosporium herbarum
    Penicillium chrysogenum
    (notatum)
    Candida albicans
    Trichophyton mentagrophytes
    Alternaria alternate
    Botrytis cinerea
    Treatment with 500 mcg Fluticasone
    Propionate/day, or Continuous oral
    corticosteroids, or 4 prednisolone bursts in
    12 months or 6 bursts in 24 months
    Severe, therapy
    resistant asthma
    (ERS/ATS Task Force
    criteria)
    IgE < 1000 IgE can be any level
    Negative IgG precipitins to Aspergillus
    fumigatus
    IgG precipitins to
    Aspergillus fumigatus
    can be positive or
    negative
    J. Fungi 2019, 6, 55 4 of 17
    2.3. Beyond SAFS: Fungal Detection in the Airway, Fungal Bronchitis and Asthma
    There is no requirement to detect fungi within the airway in order to diagnose SAFS, although
    fungal infection might be part of the syndrome. However low-grade fungal infection might drive
    asthma without inducing sensitization, for example, by the release of tissue damaging enzymes
    disrupting epithelial barrier function (below). In CF, AF bronchitis is associated with worse
    outcomes [16–18], giving biological plausibility to this mechanism in asthma. The isolation of
    fungus from airways of SAFS patients is unsurprisingly very common. AF sputum positivity by
    PCR was 70% in SAFS patients not taking anti-fungals [19], but the frequency was reduced in those
    prescribed these medications, and in a small subgroup in whom serial samples were obtained,
    itraconazole therapy resulted in sputum reverting from a positive to negative PCR. The
    sensitization to multiple molds is also common in asthma. In one study, 60% of patients were polysensitized,
    most frequently to Aspergillus fumigatus (32%) and A. Alternata (28%), Penicillium
    chrysogenum, Penicillium brevicompactum, Cladosporium cladosporioides, and Cladosporium
    sphaerospermum [20,21]
    There is also the issue of how intensively the presence of fungi should be sought. CF
    definitions are largely based on positive cultures, although whether repeated cultures or a single
    culture is needed for diagnosis is controversial. Much of the focus has been on AF, not least because
    it grows at 37 degrees (body temperature) and the spores are aerodynamically well suited to
    lodging in the lower respiratory tract, but as already stated, many other fungi may be important. In
    a study in which 69 adults underwent FOB and BAL, no fewer than 86% had fungi detectable by
    PCR on BAL, 46% of which were AF. Although a positive BAL was associated with increased BAL
    and plasma cytokines, there was no relation to asthma severity [22]. Molecular techniques may be
    even more sensitive. This study suggests that, the harder fungi are sought, the more they will be
    found. This group reported no increased asthma severity in SAFS adults; and importantly,
    potentially broadened the spectrum of fungi to which the patient may be sensitized.
    2.4. Fungal Asthma or Fungal Asthmas?
    It should be noted that the danger of umbrella definitions is that it could be taken to assume
    that all fungi have equal effects. The magnitude of the effects might be different, and will likely also
    be dependent on levels of exposure, but, more importantly, the pathophysiological pathways may
    be different. Clearly, if the approach is treatment with anti-fungals, this is irrelevant, but any
    molecular therapies may need to be fungus-specific (below).
  3. Paediatric and Adult Severe Asthma and the Atopies: Important Differences Relevant to
    Fungal Asthma
    The vast majority of children with severe asthma are markedly atopic [23], with multiple
    sensitizations to aeroallergens, such as house dust mite, grass and tree pollens, cockroach, and furry
    pets. By contrast, much severe adult asthma is neutrophilic, often in the obese and with other comorbidities,
    and with a female preponderance [24,25]. It is also increasingly being realized that
    atopy is not ‘all-or-none‘ and can be quantified [26]. Different atopies have differing significances
    [27–29]. Furthermore, complex interactions between allergens may be more important than
    individual results [30]. Sensitization to fungi is one part of the atopies; the question is, whether
    there is a discrete entity of SAFS in children, or whether fungal sensitization is one facet of asthma
    with polysensitzation to aeroallergens; to some extent, this remains unresolved. It might also be
    that the significance of fungal sensitization will be different in adults, and more likely to be a
    discrete entitity rather than mark of multiple sensitization, and this needs further exploration.
    However, the issue of anti-fungal treatment for paediatric SAFS is more one of ‘does it work?‘
    rather than ‘should it work?‘
    J. Fungi 2019, 6, 55 5 of 17
  4. Epidemiological Data: Associations between Fungi and Asthma Severity
    4.1. Cross-Sectional Studies
    Most of the big studies are in adults. The European Community Respiratory Health Survey
    [31] studied 1132 adults aged 20–44 years with current asthma. The frequency of mold sensitization
    (Alternaria alternata or Cladosporium herbarum, or both) increased significantly with increasing
    asthma severity across Europe, but there was no association between asthma severity and
    sensitization to pollens or cats. However, Dermatophagoides pteronyssinus sensitization was also
    positively associated with asthma severity. Thus, mold sensitization was highly associated with
    severe asthma in adults, but not uniquely so. In a systematic review and meta-analysis of 20 studies
    from 13 African countries [32] the mean asthma prevalence was 6%. The prevalence of fungal
    sensitization, mostly on skin prick testing, ranged from 3% to 52%, mean 28% with a pooled
    estimate of 23.3%. Aspergillus species were commonest. The prevalence of ABPA was estimated at
    1.6–21.2%. A similar study related fungal allergy to asthma severity, and there were no paediatric
    data.
    Another such study in severe asthma (GINA step 4 or 5 treatment) [33] enrolled 124 patients. A
    variety of markers were collected, including spirometry, exhaled nitric oxide, serum cytokines, and
    IgE. Fungal sensitization was assessed from IgE specific to fungal allergens (AF, Alternaria, Candida,
    Cladosporium, Penicillium, and Trichophyton species and the Schizophyllum commune). Thirty-six of
    124 patients (29%) were sensitized to at least one fungal allergen, most commonly Candida (16%),
    AF (11%), and Trichophyton (11%). Early-onset asthma (<16 years of age) was more common in
    patients with fungal sensitization (45% vs 25%; p = 0.02, see below). Interleukin-33 levels were also
    higher in patients with fungal sensitization, as discussed in more detail in the sections on
    pathophysiology. Asthma Control Test scores were worse in patients with multiple when compared
    with single fungal sensitizations and non-sensitized controls.
    4.2. SAFS and Control of Asthma
    Adult SAFS patients are more likely to have uncontrolled symptoms [34–39]. In a retrospective
    review of urban adult asthma patients, total serum IgE was highest in the 53 patients (17.3%) with
    fungal sensitization (median, 825 IU/mL vs. 42 non-atopic (n=137, 44%) vs. 203 other allergen
    sensitized (n=117, 38.1%), p < 0.001). The fungal sensitized patients were more likely to have been
    admitted to the intensive care unit (ICU) admission and been ventilated (13.2% vs. 3.7% non-atopic
    vs. 3.4% other sensitization p = 0.02; and 11.3%, 1.5%, and 0.9%, respectively, for ventilation, p <
    0.001). There are two possible interpretations of these data; firstly, polysensitized atopic asthmatics
    do worse, or that fungal sensitization is a discrete entity and an independent risk factor for bad
    outcomes.
    A study [40], which evaluated 206 adults with severe asthma (GINA step 4 or 5 treatment,
    mean age 45 ± 17 years, 99 [48%] male), of whom 78% had a positive SPT to one or more allergens.
    The most common allergen reported was house dust mites (Blomia tropicalis, Dermatophagoides
    pteronyssinus and Dermatophagoides farinii), but 11.7% were sensitized to Aspergillus species, and this
    was associated with uncontrolled asthma. In particular, Aspergillus sensitization was independently
    associated with the need for ?2 steroid bursts in the past year (odds ratio 3.05, 95% confidence
    interval 1.04–8.95). There was no association between asthma control and corticosteroid bursts with
    sensitization to any other allergen. Importantly, this study suggests that all fungi do not necessarily
    have equivalent effects.
    4.3. SAFS and Asthma Attacks: Children
    Paediatric data are much scantier, but the conclusions are very similar. A German group
    reviewed 207 children with a diagnosis of asthma of varying severity (25% had mild, 31%
    moderate, and 44% severe; 26% had a previous history of hospitalization for an asthma attack [35]).
    Alternaria was the leading mold causing sensitization, but this did not correlate with hospitalization
    J. Fungi 2019, 6, 55 6 of 17
    due to asthma attacks or other parameters of asthma severity. The prevalence of Alternaria
    sensitization increased with age and there was a significant association with the sensitization to
    other molds and aeroallergens, grass pollen, and cat epithelia. Alternaria sensitization in this study
    was thus not a risk factor for severe asthma and hospitalization. However, it should be noted that
    the risk might be a composite, both of sensitization, but also level of exposure; to take an absurd
    example, a sensitized patient who never subsequently encountered the allergen could not have an
    asthma attack triggered by that allergen.
    The Melbourne Air Pollen Children and Adolescent study [41] recruited 644 children and
    adolescents (aged 2–17 years) that were hospitalized for asthma and showed that exposure to
    Alternaria, less well known taxa, including Leptosphaeria, Coprinus, and Drechslera, and total spore
    counts were significantly associated with admissions for asthma independent of rhinovirus
    infection. Surges of spores of Alternaria, Leptosphaeria, Cladosporium, Sporormiella, Coprinus, and
    Drechslera were associated with significant effects delayed for up to three days, and Cladosporium
    sensitization was associated with significantly greater effects than the other fungi. Importantly, this
    study broadens the range of fungi that might need to be considered as part of fungal asthma,
    although the alternative explanations are that the effects were not mediated by allergic
    sensitization, or less likely, that that these spores were merely possibly markers of some
    unidentified root cause.
    4.4. SAFS and Lung Tissue Destruction
    In a cross-sectional study [42], 329 (76.3%) of adult asthmatics were sensitized to at least one
    fungus and this was related to the development of lung destruction, as assessed by postbronchodilator
    spirometry and computed tomographic (CT) scans. The sensitization to AF and/or
    Penicillium chrysogenum was associated with a lower first second forced expired volume (FEV1)
    when compared with those not sensitized, independent of atopic status, and an increased frequency
    of CT abnormalities, bronchiectasis, tree-in-bud, and collapse/consolidation. Cluster analysis
    identified three clusters: (i) hypereosinophilic hypothetically, true SAFS; (ii) high immunological
    biomarker load and high frequency of radiological abnormalities (hypothetically, fungal bronchitis
    dominant; and, (iii) low levels of fungal biomarkers (fungi not relevant). The authors concluded
    that AF sIgE was a risk factor for lung damage irrespective of ABPA.
    4.5. Fungi and Risk Assessment
    GINA and other guidelines have rightly stressed the importance of risk assessment as well as
    asthma control. There is no question that fungal sensitization is a marker of future risk of poor
    control and asthma attacks. Whether this is true for fungal bronchitis, as it is in CF, has yet to be
    explored.
  5. Clinical Features of SAFS in Children
    We have reported the largest, most detailed series of children with SAFS [43]. We studied 82
    children (median 11.7 years) with severe, therapy resistant asthma (STRA), who had undergone a
    protocolised series of investigations [44–46], including fibreoptic bronchoscopy (FOB),
    bronchoalveolar lavage (BAL), and endobronchial biopsy (EBx). Thirty eight were defined as SAFS,
    with a specific IgE or SPT to AF, Alternaria alternate or Cladosporium (in practice, we do not have
    access to testing for other fungi). We also found that children with SAFS had an earlier onset of
    symptoms (0.5 as compared with 1.5 years), a higher IgE (637 vs. 177) and were sensitized on
    testing sIgE to more non-fungal inhalant allergens, when compared with non-SAFS STRA. They
    were more likely to be prescribed maintenance oral corticosteroids (42% vs. 14%, p = 0.02).
    However, on BAL and EBx, the severity of airway inflammation and remodelling (absolute
    thickness of reticular basement membrane thickness and airway smooth muscle mass) did not
    differ between SAFS and control STRA, despite the greater use of anti-inflammatory medications.
    J. Fungi 2019, 6, 55 7 of 17
    Eight of 10 (80%) SAFS children responded to omalizumab, similar to STRA controls (11/18, 84%, p
    = NS). Mepolizumab was not licensed in children at the time of this study.
    Another paediatric study enrolled 64 children, of whom 25 (39%) had evidence of sensitization
    to at least one fungus [47]. Nineteen of 25 (76%) sensitized children had severe persistent asthma
    when compared to 13 of 39 (33%) non-sensitized (p = 0.0014). Nineteen of 32 (59%) severe persistent
    asthmatics had fungal sensitization, and these also had higher serum IgE and worse spirometry.
    Bronchial biopsy of sensitized children revealed that these children exhibited basement membrane
    thickening and eosinophil infiltration on bronchial biopsy.
    In a USA study of 126 children, Alternaria skin test reactivity was associated with severe,
    persistent asthma. Importantly, this was an independent risk factor to that of the total positive skin
    tests, suggesting there is an independent effect of this fungus unrelated to degree of atopy [48].
  6. Treatment of SAFS and Fungal Asthma
    The possible aspects of treatment are: (a) the reduction of allergic inflammation; (b) reduction
    of fungal burden; (c) reduction of tissue damage; and, (d) modulating the pro-inflammatory effects
    of tissue destruction. Most focus has been on the reduction of fungal burden, but without
    necessarily ensuring that there is a fungal infection.
    6.1. Adult Data
    Most of the data on antifungal therapy are in adults, and the results are conflicting. The FAST
    study [11] enrolled 58 adults into a double blind, randomized controlled trial of oral itraconazole or
    placebo for 32 weeks, with a follow up period of 16 weeks. The primary end point was the Asthma
    Quality of Life Questionnaire (AQLQ), with secondary endpoints being rhinitis score, total IgE and
    respiratory function. The study was positive, with improvements in AQLQ and rhinitis scores, an
    improved morning peak flow (20.8 l/min.) with itraconazole, and total IgE dropped (-510 iU
    itraconazole when compared with +30 placebo). Seven patients in the itraconazole group, and two
    placebo patients discontinued treatment. Interestingly, 60% had big improvements in QoL with
    itraconazole. The benefits of itraconazole declined rapidly in the washout period. By contrast,
    EVITA3 was a randomized, double blind, placebo controlled trial of Voriconazole in SAFS [49]. Of
    note, Voriconazole does not increase steroid bioavailability, unlike itraconazole [50,51]. The study
    duration was three months with a nine-month follow period. Fifty-six adults with SAFS were
    recruited. The inclusion criteria were at least two severe asthma attacks (defined as the prescription
    of oral corticosteroids) in the previous year, and a positive specific IgE or skin prick test to AF. The
    voriconazole levels were measured to optimize therapy. The primary endpoints were quality of life
    and asthma attacks. The study was negative. Neither trial mandated a positive airway fungal
    culture. In another report, 41 patients were studied retrospectively [52]. In those who received
    treatment (n = 32), this was with any of terbinafine, fluconazole, itraconazole, voriconazole, or
    posaconazole combined with standard treatment, by comparison with nine patients who had
    standard asthma therapy only. Those that were treated with anti-fungals showed improvement in
    Asthma Control Test, peak flow rate, and IgE. The response was better with longer treatment
    periods, and it was well tolerated, but relapse was common after the discontinuation of treatment.
    It should be noted that all of these data largely predate the widespread introduction of biological
    and, therefore, should be interpreted with caution in light of new therapies [53].
    In another study [54], 110 STRA GINA stage 4 adult asthmatics were randomly assigned to 200
    mg itraconazole twice a day or 10 mg prednisolone once daily for four months. There was no
    requirement for the demonstration of fungal sensitization or any other manifestation of fungal
    asthma. The study was not blinded. 71% of the itraconazole group improved and there were very
    few side-effects, whereas there was minimal change with prednisolone.
    In terms of acute asthma, there is a single case report of an 83 years old woman [55], with a 33-
    year history of asthma prescribed inhaled and oral corticosteroids. She presented with an acute
    attack of wheeze that did not respond to oral corticosteroids and antibiotics. She was found to
    culture AF in her sputum, a positive AF sIgE and IgG precipitins, and a positive galactomannan.
    J. Fungi 2019, 6, 55 8 of 17
    Voriconazole was added with a good response. Perhaps the most likely explanation is that this was
    treating acute AF bronchitis in an immunosuppressed adult. There is no general role for antifungals
    in acute asthma.
    6.2. Paediatric Data
    There are no randomized controlled trials of treatment in children. On general principles, we
    try to minimize fungal exposure, especially advocating for rehousing if there is visible mold in the
    house; we would check any nebulizers which might be being used for fungal contamination; and
    we would advise against children going into stables and barns [56], where mold abounds.
    However, although the reducing the burden of fungal allergen exposure seems sensible, the
    relationship between mold exposure, mold sensitization, and asthma severity is complex.
    Approximately 90% of homes in one case control study were contaminated with mold [20]. The
    sensitization to AF, but not to other molds, was associated with asthma severity. Whether or not the
    child was sensitized, AF and Penicillium spp in dust was associated with severe asthma; the latter
    was associated with worse lung function. The lessons of this study are that environmental AF
    exposure should be minimized, and that not all molds have the same effects. However, although
    exposure to mold may limit airway infection, it should be borne in mind that allergen reduction
    strategies have sometimes had unexpected effects. In some cases, high level exposure might induce
    tolerance, and reduction in levels lead to increased sensitivity; and there is marked variation
    between allergens in the relationship between environmental concentrations and likelihood of
    sensitization or tolerance [57].
    Additionally, we would also optimize standard asthma management and treatment [6–8],
    including treatment with omalizumab and mepolizumab if indicated, before going on to ‘beyond
    guidelines’ therapy with antifungals. Anecdotally, a child with refractory asthma, persistently
    abnormal spirometry, total E >20,000 IU/mL, and severe airway eosinophilia was sensitized to
    multiple fungi and responded dramatically to itraconazole [58]. Additionally, anecdotally,
    omalizumab might effectively treat the occasional case of SAFS [59], alone or combined with
    itraconazole which may be used to reduce IgE levels into the omalizumab range [60]. Also
    anecdotally, we have seen the occasional SAFS child who appeared to improve with antifungals.
    6.3. Conclusions: What is the Role of Antifungals in SAFS?
    It is suggested that the individual facets, or treatable traits of fungal asthma, are determined on
    an individual basis and a bespoke treatment plan developed. Clearly, the evidence for the use of
    antifungals is conflicting and of low quality. Part of the reason might be that SAFS as
    conventionally declined does not require the presence of fungal bronchitis. It is difficult to see how
    anti-fungal therapy would benefit SAFS if there were no fungal infection, and symptoms were
    solely due to sensitization to fungal spores. Logically, future trials of anti-fungal therapy in
    SAFS/fungal asthma should mandate the presence of fungal bronchitis.
    Our current approach is to address environmental exposures and optimize standard therapy in
    children with SAFS. If asthma control is optimal and there are no other markers of ongoing risk,
    such as a persistently raised exhaled nitric oxide or a past history of really severe attacks, with no
    present side-effects, then we would not use antifungals. However, if asthma control remains
    suboptimal, or significant risks persist, then we would consider adding an antifungal, such as
    itraconazole. It is important to note that there is a potential interaction between corticosteroids and
    azoles at the cytochrome p450 level [61], such that the combination of itraconazole and inhaled
    budesonide has led to iatrogenic Cushing Syndrome [50,51].
  7. Risk Factors for SAFS: Genetic Studies
    Although there is expanding literature on the genetic associations of ABPA, SAFS has been
    little investigated. There might indeed be genetic factors that are associated with SAFS, but, to my
    knowledge, there has been no large scale Genome Wide Association Study (GWAS) to confirm or
    J. Fungi 2019, 6, 55 9 of 17
    otherwise this suggestion. In a small, preliminary study [62], 325 haplotype-tagging single
    nucleotide polymorphisms (SNPs) in 22 previously suggested candidate genes were studied in
    SAFS (n = 47), atopic asthmatics (n = 152), and healthy control patients (n = 279). There were
    significant associations of Toll-like receptors (TLR) 3 and 9 (TLR3), C-type lectin domain family
    seven member A (dectin-1), IL-10, mannose-binding lectin (MBL2), CC-chemokine ligand 2 (CCL2)
    and CCL17, plasminogen, and the adenosine A2a receptor, different from those reported in asthma
    complicated by ABPA. Some of these hits are supported by cell and animal data (below). The main
    weakness of this study was the absence of a second validation cohort, without which the findings
    are, at best, preliminary. In an initial small study comprising 76 adults with chronic cavitatory
    pulmonary aspergillosis (n = 40), ABPA (n = 22), and SAFS (n = 14), no genetic associations of SAFS
    could be determined, unsurprisingly with such a small number of patients [63]. However, in one
    intriguing study, six SAFS children were heterozygous for a 24-base pair duplication in the CHIT1
    gene [64]. This duplication associates with an increased susceptibility to fungal infection and
    decreased circulating chitotriosidase levels [65]. Clearly there is a need for more work in this area.
  8. Pathophysiology of SAFS and Fungal Asthma
    8.1. Introduction
    As discussed, there are two pathological mechanisms, whereby fungi, especially AF, can cause
    disease in children with asthma [8]. These are as a source of allergen(s) to which the child is
    sensitized, leading to wheeze on exposure, and driving a Type 2 inflammatory response; and, the
    release of tissue damaging enzymes by fungi that have infected the airway (not dissimilar to, for
    example, house dust mite, which is allergenic and tissue damaging), and that might also generate
    an allergic response. It should be noted that other proteins could generate an allergic response
    without requiring airway infection, for example, in sensitization to furry pets.
    Any account of the potential role of exogenous infection of any cause must consider the
    possibility that this is iatrogenic, secondary to the use of corticosteroids. It is known that systemic
    corticosteroids are immunosuppressive, and also that mucosal immunity is essential for normal
    host defence [65]. It is biologically plausible that topical steroids would be immunosuppressive, and
    indeed their use is associated with increased prevalence of tuberculosis, [66] atypical Mycobacterial
    infection [67], and, in patients with COPD, pneumonia [68]. It is virtually impossible to dissect out
    the contribution of inhaled corticosteroids (ICS) to SAFS, because, by definition, all SAFS patients
    will be prescribed ICS. In one study [69], the fungal microbiome (mycobiome) was determined on
    bronchoscopic samples. The investigators reported that the mycobiome was highly varied with the
    biggest load in severe asthmatics. Healthy controls had low fungal loads; the most common fungus
    detected was the poorly characterized Malasezziales. AF was most the common in fungus in
    asthmatics and accounted for the increased fungal burden. Corticosteroid treatment was
    significantly associated with an increased fungal load. These interesting data cannot unravel
    whether inhaled corticosteroids caused SAFS, or SAFS led to the prescription of more inhaled
    corticosteroids.
    8.2. Cell and Animal Studies
    A number of different pathways have been implicated in SAFS, including the pattern
    recognition receptors (PRRs) TLR3, TLR9, and Dectin-1 and IL-7, Il-10, IL-22, CCL2, and CCL17
    [70,71]. IL-33 has been implicated in both adult [36] and paediatric SAFS [5]. IL-33 is an epithelial
    alarmin, together with IL-25 and TSLP. It is a member of the eleven member IL-1 family of
    cytokines. Of these, seven are proinflammatory (IL-1?, IL-1?, IL-18, IL-33, IL-36?, IL-36?, and IL-
    36?) and four probably immunomodulatory (IL-1 receptor antagonist [IL-1RA], IL-36Ra, IL-37, and
    IL-38). A recent manuscript [72] demonstrated that IL-1? and IL-1? are elevated in the BAL and
    sputum from adult SAFS patients. The same group used a murine model utilizing the AF challenge
    to show that IL-1R1 signaling promotes increased airway hyper-responsiveness and neutrophilic
    inflammation associated with type 1 (IFN-?, CXCL9, CXCL10) and type 17 (IL-17A, IL-22)
    J. Fungi 2019, 6, 55 10 of 17
    responses, each exacerbated in IL-1RA?/? mice. The administration of human recombinant IL-1RA
    (Kineret/anakinra) abrogated these responses, all suggesting that IL-1R1 signaling via type 1 and
    type 17 responses is an important and potentially treatable pathway of SAFS.
    A murine model further explored the links between Alternaria and asthma [73]. Wild-type and
    mice lacking the IL-33 receptor (ST2?/?) underwent inhalational challenge with inhaled house dust
    mite, cat dander, or Alternaria. Mice that were sensitized with house dust mite were subsequently
    challenged with Alternaria (with or without serine protease activity having been knocked down),
    and inflammation, remodeling, and lung function assessed 24 h after the challenge. Only Alternaria
    possessed intrinsic serine protease activity that led to the release of IL-33 into the airways via a
    mechanism that is dependent on the activation of protease activated receptor-2 and adenosine
    triphosphate signaling. This led to more pulmonary inflammation relative to that produced by the
    house dust mite challenge. IL-1? and matrix metalloproteinase (MMP) 9 release were also features
    of Alternaria challenge. Furthermore, Alternaria triggered a rapid, augmented inflammatory
    response, mucus hypersecretion, and airway obstruction. The effects of Alternaria were critically
    dependent on ST2 signaling. Hence, Alternaria-specific serine protease activity resulted in rapid IL-
    33 release, leading to TH2 inflammation and exacerbation of allergic airway disease.
    Alternaria proteases may have an important role. One study [74] used cells from normals or
    patients with severe asthma. They used both 16HBE cells and fresh bronchial epithelial cells
    cultured to air-liquid interface (ALI), and challenged them apically with extracts of Alternaria in
    order to further explore the role of Alternaria proteases. Alternaria extract protease activity was

Keywords: atopy; aspergillus bronchitis; fungal sensitization; itraconazole; severe asthma;
voriconazole