Gut Health and Asthma

The gut and lungs are anatomically distinct, but potential anatomic communications and complex pathways involving their respective microbiota have reinforced the existence of a gut–lung axis (GLA). Compared to the better-studied gut microbiota, the lung microbiota, only considered in recent years, represents a more discreet part of the whole microbiota associated to human hosts. Gut health is not the only area to think about.

While the majority of studies focused on the bacterial component of the microbiota in healthy and pathological conditions, recent works highlighted the contribution of fungal and viral kingdoms at both digestive and respiratory levels. Moreover, growing evidence indicates the key role of inter-kingdom crosstalks in maintaining host homeostasis and in disease evolution.

In fact, the recently emerged GLA concept involves host–microbe as well as microbe–microbe interactions, based both on localized and long-reaching effects. GLA can shape immune responses and interfere with the course of respiratory diseases. In this review, we aim to analyze how the lung and gut microbiota influence each other and may impact on respiratory diseases.

Due to the limited knowledge on the human virobiota, we focused on gut and lung bacteriobiota and mycobiota, with a specific attention on inter-kingdom microbial crosstalk. These are able to shape local or long-reached host responses within the GLA.


Recent advances in microbiota explorations have led to an improved knowledge of the communities of commensal microorganisms within the human body. Human skin and mucosal surfaces are associated with rich and complex ecosystems (microbiota) composed of bacteria (bacteriobiota), fungi (mycobiota), viruses (virobiota), phages, archaea, protists, and helminths (Cho and Blaser, 2012).

The role of the gut bacteriobiota in local health homeostasis and diseases is being increasingly investigated, but its long-distance impacts still need to be clarified (Chiu et al., 2017). Among the relevant inter-organ connections, the gut–lung axis (GLA) remains less studied than the gut–brain axis.

So far, microbiota studies mainly focused on the bacterial component, neglecting other microbial kingdoms. However, the understanding of mycobiota involvement in human health and inter-organ connections should not be overlooked (Nguyen et al., 2015; Enaud et al., 2018).

Viruses are also known to be key players in numerous respiratory diseases and to interact with the human immune system, but technical issues still limit the amount of data regarding virobiota (Mitchell and Glanville, 2018). Therefore, we will focus on bacterial and fungal components of the microbiota and their close interactions that are able to shape local or long-reached host responses within the GLA.

While GLA mycobiota also influences chronic gut diseases such as IBD, we will not address this key role in the present review: we aimed at analyzing how lung and gut bacteriobiota and mycobiota influence each other, how they interact with the human immune system, and their role in respiratory diseases.

Gut Health

Microbial Interactions Within the Gut–Lung Axis

The gut microbiota has been the most extensively investigated in gut health. The majority of genes (99%) amplified in human stools are from bacteria, which are as numerous as human cells and comprise 150 distinct bacterial species, belonging mainly to Firmicutes and Bacteroidetes phyla. Proteobacteria, Actinobacteria, Cyanobacteria, and Fusobacteria are also represented in healthy people (Sekirov et al., 2010; Human Microbiome Project Consortium, 2012).

More recently, fungi have been recognized as an integral part of our commensal flora, and their role in health and diseases is increasingly considered (Huffnagle and Noverr, 2013; Huseyin et al., 2017). Fungi are about 100 times larger than bacteria, so even if fungal sequences are 100 to 1,000 times less frequent than bacterial sequences, fungi must not be neglected in the gastrointestinal ecosystem.

Mycobiota Diversity

In contrast with the bacteriobiota, the diversity of the gut mycobiota in healthy subjects is limited to few genera, with a high prevalence of Saccharomyces cerevisiae, Malassezia restricta, and Candida albicans (Nash et al., 2017).

Note from the WAF editorial board. We wish to acknowledge and thank Raphaël Enaud, Renaud Preve, Eleonora Ciarlo, Fabien Beaufils, Gregoire Wieërs, Benoit Guery and Laurence Delhaes for their support of Asthma education and research. For more information about Asthma or Gut Health, visit the World Asthma Foundation.

Although often dichotomized due to technical and analysis sequencing issues, critical interactions exist between bacteriobiota and mycobiota (Peleg et al., 2010). The most appropriate approach to decipher the role of gut microbiota is therefore considering the gut as an ecosystem in which inter-kingdom interactions occur and have major implications as suggested by the significant correlations between the gut bacteriobiota and mycobiota profiles among healthy subjects (Hoffmann et al., 2013).


Yeasts, e.g., Saccharomyces boulardii and C. albicans, or fungus wall components, e.g., ?-glucans, are able to inhibit the growth of some intestinal pathogens (Zhou et al., 2013; Markey et al., 2018). S. boulardii also produces proteases or phosphatases that inactivate the toxins produced by intestinal bacteria such as Clostridium difficile and Escherichia coli (Castagliuolo et al., 1999; Buts et al., 2006).

In addition, at physiological state and during gut microbiota disturbances (e.g., after a course of antibiotics), fungal species may take over the bacterial functions of immune modulation, preventing mucosal tissue damages (Jiang et al., 2017). Vice versa, bacteria can also modulate fungi: fatty acids locally produced by bacteria impact on the phenotype of C. albicans (Noverr and Huffnagle, 2004; Tso et al., 2018).


Beside the widely studied gut microbiota, microbiotas of other sites, including the lungs, are essential for host homeostasis and disease. The lung microbiota is now recognized as a cornerstone in the physiopathology of numerous respiratory diseases (Soret et al., 2019; Vandenborght et al., 2019).

Inter-Kingdom Crosstalk Within the Lung Microbiota

The lung microbiota represents a significantly lower biomass than the gut microbiota: about 10 to 100 bacteria per 1,000 human cells (Sze et al., 2012). Its composition depends on the microbial colonization from the oropharynx and upper respiratory tract through salivary micro-inhalations, on the host elimination abilities (especially coughing and mucociliary clearance), on interactions with the host immune system, and on local conditions for microbial proliferation, such as pH or oxygen concentration (Gleeson et al., 1997; Wilson and Hamilos, 2014).

The predominant bacterial phyla in lungs are the same as in gut, mainly Firmicutes and Bacteroidetes followed by Proteobacteria and Actinobacteria (Charlson et al., 2011). In healthy subjects, the main identified fungi are usually environmental: Ascomycota (Aspergillus, Cladosporium, Eremothecium, and Vanderwaltozyma) and Microsporidia (Systenostrema) (Nguyen et al., 2015; Vandenborght et al., 2019).

In contrast to the intestinal or oral microbiota, data highlighting the interactions between bacteria and fungi in the human respiratory tract are more scattered (Delhaes et al., 2012; Soret et al., 2019). However, data from both in vitro and in vivo studies suggest relevant inter-kingdom crosstalk (Delhaes et al., 2012; Xu and Dongari-Bagtzoglou, 2015; Lof et al., 2017; Soret et al., 2019).

Several Pathways

This dialogue may involve several pathways as physical interaction, quorum-sensing molecules, production of antimicrobial agents, immune response modulation, and nutrient exchange (Peleg et al., 2010). Synergistic interactions have been documented between Candida and Streptococcus, such as stimulation of Streptococcus growth by Candida, increasing biofilm formation, or enhancement of the Candida pathogenicity by Streptococcus (Diaz et al., 2012; Xu et al., 2014).

In vitro studies exhibited an increased growth of Aspergillus fumigatus in presence of Pseudomonas aeruginosa, due to the mold’s ability in to assimilate P. aeruginosa-derived volatile sulfur compounds (Briard et al., 2019; Scott et al., 2019). However, the lung microbiota modulation is not limited to local inter-kingdom crosstalk and also depends on inter-compartment crosstalk between the gut and lungs.
Microbial Inter-compartment Crosstalk

From birth throughout the entire life span, a close correlation between the composition of the gut and lung microbiota exists, suggesting a host-wide network (Grier et al., 2018). For instance, modification of newborns’ diet influences the composition of their lung microbiota, and fecal transplantation in rats induces changes in the lung microbiota (Madan et al., 2012; Liu et al., 2017).

Gut-Lung Interaction

The host’s health condition can impact this gut–lung interaction too. In cystic fibrosis (CF) newborns, gut colonizations with Roseburia, Dorea, Coprococcus, Blautia, or Escherichia presaged their respiratory appearance, and their gut and lung abundances are highly correlated over time (Madan et al., 2012). Similarly, the lung microbiota is enriched with gut bacteria, such as Bacteroides spp., after sepsis (Dickson et al., 2016).

Conversely, lung microbiota may affect the gut microbiota composition. In a pre-clinical model, influenza infection triggers an increased proportion of Enterobacteriaceae and decreased abundances of Lactobacilli and Lactococci in the gut (Looft and Allen, 2012). Consistently, lipopolysaccharide (LPS) instillation in the lungs of mice is associated with gut microbiota disturbances (Sze et al., 2014).

Although gastroesophageal content inhalations and sputum swallowing partially explain this inter-organ connection, GLA also involves indirect communications such as host immune modulation.

Gut–Lung Axis Interactions With Human Immune System

Gut microbiota effects on the local immune system have been extensively reviewed (Elson and Alexander, 2015). Briefly, the gut microbiota closely interacts with the mucosal immune system using both pro-inflammatory and regulatory signals (Skelly et al., 2019). It also influences neutrophil responses, modulating their ability to extravasate from blood (Karmarkar and Rock, 2013).

Receptor Signaling

Toll-like receptor (TLR) signaling is essential for microbiota-driven myelopoiesis and exerts a neonatal selection shaping the gut microbiota with long-term consequences (Balmer et al., 2014; Fulde et al., 2018). Moreover, the gut microbiota communicates with and influences immune cells expressing TLR or GPR41/43 by means of microbial associated molecular patterns (MAMPs) or short-chain fatty acids (SCFAs) (Le Poul et al., 2003).

Data focused on the gut mycobiota’s impact on the immune system are sparser. Commensal fungi seem to reinforce bacterial protective benefits on both local and systemic immunity, with a specific role for mannans, a highly conserved fungal wall component. Moreover, fungi are able to produce SCFAs (Baltierra-Trejo et al., 2015; Xiros et al., 2019). Therefore, gut mycobiota perturbations could be as deleterious as bacteriobiota ones (Wheeler et al., 2016; Jiang et al., 2017).

Lung Microbiota and Local Immunity

A crucial role of lung microbiota in the maturation and homeostasis of lung immunity has emerged over the last few years (Dickson et al., 2018). Colonization of the respiratory tract provides essential signals for maturing local immune cells with long-term consequences (Gollwitzer et al., 2014).

Pre-clinical studies confirm the causality between airway microbial colonization and the regulation and maturation of the airways’ immune cells. Germ-free mice exhibit increased local Th2-associated cytokine and IgE production, promoting allergic airway inflammation (Herbst et al., 2011).

Consistently, lung exposure to commensal bacteria reduces Th2-associated cytokine production after an allergen challenge and induces regulatory cells early in life (Russell et al., 2012; Gollwitzer et al., 2014). The establishment of resident memory B cells in lungs also requires encountering lung microbiota local antigens, especially regarding immunity against viruses such as influenza (Allie et al., 2019).

Interactions between lung microbiota and immunity are also a two-way process; a major inflammation in the lungs can morbidly transform the lung microbiota composition (Molyneaux et al., 2013).

Gut Health, Long-Reaching Immune Modulation Within Gut–Lung Axis

Beyond the local immune regulation by the site-specific microbiota, the long-reaching immune impact of gut microbiota is now being recognized, especially on the pulmonary immune system (Chiu et al., 2017).

The mesenteric lymphatic system is an essential pathway between the lungs and the intestine, through which intact bacteria, their fragments, or metabolites (e.g., SCFAs) may translocate across the intestinal barrier, reach the systemic circulation, and modulate the lung immune response (Trompette et al., 2014; Bingula et al., 2017; McAleer and Kolls, 2018).

SCFAs, mainly produced by the bacterial dietary fibers’ fermentation especially in case of a high-fiber diet (HFD), act in the lungs as signaling molecules on resident antigen-presenting cells to attenuate the inflammatory and allergic responses (Anand and Mande, 2018; Cait et al., 2018).

SCFA receptor–deficient mice show increased inflammatory responses in experimental models of asthma (Trompette et al., 2014). Fungi, including A. fumigatus, can also produce SCFAs or create a biofilm enhancing the bacterial production of SCFAs, but on the other hand, bacterial SCFAs can dampen fungal growth (Hynes et al., 2008; Baltierra-Trejo et al., 2015; Xiros et al., 2019). The impact of fungal production of SCFAs on the host has not been assessed so far.

Other Elements

Other important players of this long-reaching immune effect are gut segmented filamentous bacteria (SFBs), a commensal bacteria colonizing the ileum of most animals, including humans, and involved in the modulation of the immune system’s development (Yin et al., 2013). SFBs regulate CD4+ T-cell polarization into the Th17 pathway, which is implicated in the response to pulmonary fungal infections and lung autoimmune manifestations (McAleer et al., 2016; Bradley et al., 2017).

Recently, innate lymphoid cells, involved in tissue repair, have been shown to be recruited from the gut to the lungs in response to inflammatory signals upon IL-25 (Huang et al., 2018). Finally, intestinal TLR activation, required for the NF-?B–dependent pathways of innate immunity and inflammation, is associated with an increased influenza-related lung response in mice (Ichinohe et al., 2011).


Other mechanisms may be involved in modulating the long-reaching immune response related to gut microbiota, exemplified by the increased number of mononuclear leukocytes and an increased phagocytic and lytic activity after treatment with Bifidobacterium lactis HN019 probiotics (Gill et al., 2001). Diet, especially fiber intake, which increases the systemic level of SCFAs, or probiotics influence the pulmonary immune response and thus impact the progression of respiratory disorders (King et al., 2007; Varraso et al., 2015; Anand and Mande, 2018).

The GLA immune dialogue remains a two-way process. For instance, Salmonella nasal inoculation promotes a Salmonella-specific gut immunization which depends on lung dendritic cells (Ruane et al., 2013). Respiratory influenza infection also modulates the composition of the gut microbiota as stated above. These intestinal microbial disruptions seem to be unrelated to an intestinal tropism of influenza virus but mediated by Th17 cells (Wang et al., 2014).

In summary, GLA results from complex interactions between the different microbial components of both the gut and lung microbiotas combined with local and long-reaching immune effects. All these interactions strongly suggest a major role for the GLA in respiratory diseases, as recently documented in a mice model (Skalski et al., 2018).
Gut–Lung Axis in Respiratory Diseases

Acute Infectious Diseases

Regarding influenza infection and the impact of gut and lung microbiota, our knowledge is still fragmentary; human data are not yet available. However, antibiotic treatment causes significantly reduced immune responses against influenza virus in mice (Ichinohe et al., 2011). Conversely, influenza-infected HFD-fed mice exhibit increased survival rates compared to infected controls thanks to an enhanced generation of Ly6c-patrolling monocytes. These monocytes increase the numbers of macrophages that have a limited capacity to produce CXCL1 locally, reducing neutrophil recruitment to the airways and thus tissue damage. In parallel, diet-derived SCFAs boost CD8+ T-cell effector function in HFD-fed mice (Trompette et al., 2018).

Both lung and gut microbiota are essential against bacterial pneumonia. The lung microbiota is able to protect against respiratory infections with Streptococcus pneumoniae and Klebsiella pneumoniae by priming the pulmonary production of granulocyte-macrophage colony-stimulating factor (GM-CSF) via IL-17 and Nod2 stimulation (Brown et al., 2017).

Gut Health and Lung Bacterial Infections

The gut microbiota also plays a crucial role in response to lung bacterial infections. Studies on germ-free mice showed an increased morbidity and mortality during K. pneumoniae, S. pneumoniae, or P. aeruginosa acute lung infection (Fagundes et al., 2012; Fox et al., 2012; Brown et al., 2017). The use of broad-spectrum antibiotic treatments, to disrupt mouse gut microbiota, results in worse outcome in lung infection mouse models (Schuijt et al., 2016; Robak et al., 2018).

Mechanistically, alveolar macrophages from mice deprived of gut microbiota through antibiotic treatment are less responsive to stimulation and show reduced phagocytic capacity (Schuijt et al., 2016). Interestingly, priming of antibiotic-treated animals with TLR agonists restores resistance to pulmonary infections (Fagundes et al., 2012). SFBs appear to be an important gut microbiota component for lung defense against bacterial infection thanks to their capacity to induce the production of the Th17 cytokine, IL-22, and to increase neutrophil counts in the lungs during Staphylococcus aureus pneumonia (Gauguet et al., 2015).

Modulating chronic infectious diseases will similarly depend on gut and lung microbiotas. For instance, Mycobacterium tuberculosis infection severity is correlated with gut microbiota (Namasivayam et al., 2018).

Chronic Respiratory Diseases

Multiple studies have addressed the impact of gut and lung microbiota on chronic respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and CF (Table 1).

Table 1. Gut–lung axis in human chronic respiratory diseases. Gut Health.

Decreased lung microbiota diversity and Proteobacteria expansion are associated with both COPD severity and exacerbations (Garcia-Nuñez et al., 2014; Wang et al., 2016, 2018; Mayhew et al., 2018). The fact that patients with genetic mannose binding lectin deficiency exhibit a more diverse pulmonary microbiota and a lower risk of exacerbation suggests not only association but also causality (Dicker et al., 2018).

Besides the lung flora, the gut microbiota is involved in exacerbations, as suggested by the increased gastrointestinal permeability in patients admitted for COPD exacerbations (Sprooten et al., 2018). Whatever the permeability’s origin (hypoxemia or pro-inflammatory status), the level of circulating gut microbiota–dependent trimethylamine-N-oxide has been associated with mortality in COPD patients (Ottiger et al., 2018). This association being explained by comorbidities and age, its impact per se is not guaranteed. Further studies are warranted to investigate the role of GLA in COPD and to assess causality.

Early Life Perturbation

Early-life perturbations in fungal and bacterial gut colonization, such as low gut microbial diversity, e.g., after neonatal antibiotic use, are critical to induce childhood asthma development (Abrahamsson et al., 2014; Metsälä et al., 2015; Arrieta et al., 2018).

This microbial disruption is associated with modifications of fecal SCFA levels (Arrieta et al., 2018). Causality has been assessed in murine models. Inoculation of the bacteria absent in the microbiota of asthmatic patients decreases airways inflammation (Arrieta et al., 2015).


Furthermore, Bacteroides fragilis seems to play a major role in immune homeostasis, balancing the host systemic Th1/Th2 ratio and therefore conferring protection against allergen-induced airway disorders (Mazmanian et al., 2005; Panzer and Lynch, 2015; Arrieta et al., 2018). Nevertheless, it is still not fully deciphered, as some studies conversely found that an early colonization with Bacteroides, including B. fragilis, could be an early indicator of asthma later in life (Vael et al., 2008).

Regarding fungi, gut fungal overgrowth (after antibiotic administration or a gut colonization protocol with Candida or Wallemia mellicola) increases the occurrence of asthma via IL-13 without any fungal expansion in the lungs (Noverr et al., 2005; Wheeler et al., 2016; Skalski et al., 2018). The prostaglandin E2 produced in the gut by Candida can reach the lungs and promotes lung M2 macrophage polarization and allergic airway inflammation (Kim et al., 2014).

Mouse & Human Gut Health

In mice, a gut overrepresentation of W. mellicola associated with several intestinal microbiome disturbances appears to have long-reaching effects on the pulmonary immune response and severity of asthma, by involving the Th2 pathways, especially IL-13 and to a lesser degree IL-17, goblet cell differentiation, fibroblasts activation, and IgE production by B cells (Skalski et al., 2018).

These results indicate that the GLA, mainly through the gut microbiota, is likely to play a major role in asthma.

Cystic Fibrosis and Gut Health

In CF patients, gut and lung microbiota are distinct from those of healthy subjects, and disease progression is associated with microbiota alterations. (Madan et al., 2012; Stokell et al., 2015; Nielsen et al., 2016). Moreover, the bacterial abundances at both sites are highly correlated and have similar trends over time (Madan et al., 2012). This is especially true regarding Streptococcus, which is found in higher proportion in CF stools, gastric contents, and sputa. (Al-Momani et al., 2016; Nielsen et al., 2016).

Moreover, CF patients with a documented intestinal inflammation exhibit a higher Streptococcus abundance in the gut (Enaud et al., 2019). That suggests the GLA’s involvement in intestinal inflammation. Of note, gut but not lung microbiota alteration is associated with early-life exacerbations. Some gut microbiota perturbations, such as a decrease of Parabacteroides, are predictive of airway colonization with P. aeruginosa (Hoen et al., 2015).

Furthermore, oral administration of probiotics to CF patients leads to a decreased number of exacerbations (Anderson et al., 2016). While the mycobiota has been recently studied in CF (Nguyen et al., 2015; Soret et al., 2019), no data on the role of the fungal component of the GLA are currently available in CF. This deserves to be more widely studied.

Improving Health in the Gut

The role of inter-compartment and inter-kingdom interactions within the GLA in those pulmonary diseases now has to be further confirmed and causality assessed. Diet, probiotics, or more specific modulations could be, in the near future, novel essential tools in therapeutic management of these respiratory diseases.


The gut–lung axis or GLA has emerged as a specific axis with intensive dialogues between the gut and lungs, involving each compartment in a two-way manner, with both microbial and immune interactions (Figure 1). Each kingdom and compartment plays a crucial role in this dialogue, and consequently in host health and diseases. The roles of fungal and viral kingdoms within the GLA still remain to be further investigated. Their manipulation, as for the bacterial component, could pave the way for new approaches in the management of several respiratory diseases such as acute infections, COPD, asthma, and/or CF.

WAF: Gut health is an important area of research for the foundation.

Gut Health and asthma, an interview with Rodney Dietert, PhD.

See also Dr. Dietert’s interview about the Gut and Lung connection.

Non-Eosinophilic Asthma (NEA)

Although non-eosinophilic asthma (NEA) is not the best known and most prevalent asthma phenotype, its importance cannot be underestimated. NEA is characterized by airway inflammation with the absence of eosinophils, subsequent to activation of non-predominant type 2 immunologic pathways. This phenotype, which possibly includes several not well-defined subphenotypes, is defined by an eosinophil count <2% in sputum. NEA has been associated with environmental and/or host factors, such as smoking cigarettes, pollution, work-related agents, infections, and obesity. These risk factors, alone or in conjunction, can activate specific cellular and molecular pathways leading to non-type 2 inflammation.

Note from the WAF: We wish to acknowledge and thank Darío Antolín-Amérigo, Javier Domínguez-Ortega,3,4 and Santiago Quirce Department of Allergy, Hospital General de Villalba, Madrid, Spain, for their contribution to Asthma education and research.

The most relevant clinical trait of NEA is its poor response to standard asthma treatments, especially to inhaled corticosteroids, leading to a higher severity of disease and to difficult-to-control asthma. Indeed, NEA constitutes about 50% of severe asthma cases. Since most current and forthcoming biologic therapies specifically target type 2 asthma phenotypes, such as uncontrolled severe eosinophilic or allergic asthma, there is a dramatic lack of effective treatments for uncontrolled non-type 2 asthma. Research efforts are now focusing on elucidating the phenotypes underlying the non-type 2 asthma, and several studies are being conducted with new drugs and biologics aiming to develop effective strategies for this type of asthma, and various immunologic pathways are being scrutinized to optimize efficacy and to abolish possible adverse effects.

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.


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].


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.


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 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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Defeating Asthma Series Announced for World Asthma Day, May 5, 2020


World Asthma Foundation is supporting care of Asthma and asthmatics around the world through a new series focused on Defeating Asthma with the aim of shining a spotlight on getting to a cure

The World Asthma Foundation (WAF) exists for education and advocacy for people with asthma who suffer medically with health issues that make them highly vulnerable to the COVID-19 virus and other diseases.

We’ve hunkered down close to home here at the WAF. While doing so, we’re poring over volumes of available Asthma research data to share our understanding of the root causes of Asthma with emphasis on Severe Asthma.

Our ultimate goal is to understand the root cause of Severe Asthma (already considered a pandemic by many) while we aim for a cure. By banding together with other Asthmatics, including those that care about Asthmatics and clinicians that treat, we can defeat Asthma and we can do so now.

Why this Matters:

  • Asthma is not one disease but many and the causes underlying its development and manifestations are many including environmental issues
  • Asthma has reached pandemic levels around the globe
    Asthma is a chronic lung disease that affects over 300 million worldwide
  • The projected rate will reach 400 million by 2025
  • Environmental exposures have been proven to play a significant role in the development of asthma and as triggers
  • Asthma is believed to be determined by a complicated set of one’s own genetics and environmental exposures including a multitude of toxic chemicals and the overuse of antibiotics
  • In the U.S., African Americans are almost three times more likely to die from asthma-related causes than the white population
  • Australia reported the highest rate of doctor diagnosed, clinical/treated asthma, and wheezing
  • Defining asthma remains an ongoing challenge and innovative methods are needed to identify, diagnose, and accurately classify asthma at an early stage to most effectively implement optimal management and reduce the health burden attributable to asthma
  • According to the U.S. Centers for Disease Control, The total annual cost of asthma in the United States, including medical care, absenteeism and mortality, was $81.9 Billion a year.

We can move the needle by taking action now to make the difference for those that suffer from Asthma.” – Alan Gray, Director WAF Australia

What you can expect from the WAF Severe Asthma Series

Follow along with the series (click here) as we cover a variety of topics of interest to Asthmatics. 

  • What are the various types of Severe Asthma
  • What drives Severe Asthma
  • Impact of the environment on Severe Asthma
  • For additional on Asthma and the Microbiome click here 
  • What are the treatment options for Severe Asthma
  • Real world case studies with in-depth analysis
  • University research
  • Live expert podcast and interviews
  • Healthy lifestyle resources
  • Asthma advocacy guide and communication strategies for talking with your medical team

WAF will bring fresh perspectives from experts in the field that affects Asthmatics.

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