Information

Do allergens have structural similarities to pathogens?

Do allergens have structural similarities to pathogens?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

The conventional popular explanation of allergies is that the immune system confuses allergens with pathogens and reacts to them as such. Is there any merit to this explanation? If so, I would expect it to be possible to predict which proteins would be allergens by their structural similarity to pathogens. Is it? If not, how else could we explain which proteins are allergens and which aren't?


Biology 171

By the end of this section, you will be able to do the following:

  • Describe the cytoskeleton
  • Compare the roles of microfilaments, intermediate filaments, and microtubules
  • Compare and contrast cilia and flagella
  • Summarize the differences among the components of prokaryotic cells, animal cells, and plant cells

If you were to remove all the organelles from a cell, would the plasma membrane and the cytoplasm be the only components left? No. Within the cytoplasm, there would still be ions and organic molecules, plus a network of protein fibers that help maintain the cell’s shape, secure some organelles in specific positions, allow cytoplasm and vesicles to move within the cell, and enable cells within multicellular organisms to move. Collectively, scientists call this network of protein fibers the cytoskeleton . There are three types of fibers within the cytoskeleton: microfilaments, intermediate filaments, and microtubules ((Figure)). Here, we will examine each.


Microfilaments

Of the three types of protein fibers in the cytoskeleton, microfilaments are the narrowest. They function in cellular movement, have a diameter of about 7 nm, and are comprised of two globular protein intertwined strands, which we call actin ((Figure)). For this reason, we also call microfilaments actin filaments.


ATP powers actin to assemble its filamentous form, which serves as a track for the movement of a motor protein we call myosin. This enables actin to engage in cellular events requiring motion, such as cell division in eukaryotic cells and cytoplasmic streaming, which is the cell cytoplasm’s circular movement in plant cells. Actin and myosin are plentiful in muscle cells. When your actin and myosin filaments slide past each other, your muscles contract.

Microfilaments also provide some rigidity and shape to the cell. They can depolymerize (disassemble) and reform quickly, thus enabling a cell to change its shape and move. White blood cells (your body’s infection-fighting cells) make good use of this ability. They can move to an infection site and phagocytize the pathogen.

To see an example of a white blood cell in action, watch white blood cell chases bacteria a short time-lapse of the cell capturing two bacteria. It engulfs one and then moves on to the other.

Intermediate Filaments

Several strands of fibrous proteins that are wound together comprise intermediate filaments ((Figure)). Cytoskeleton elements get their name from the fact that their diameter, 8 to 10 nm, is between those of microfilaments and microtubules.


Intermediate filaments have no role in cell movement. Their function is purely structural. They bear tension, thus maintaining the cell’s shape, and anchor the nucleus and other organelles in place. (Figure) shows how intermediate filaments create a supportive scaffolding inside the cell.

The intermediate filaments are the most diverse group of cytoskeletal elements. Several fibrous protein types are in the intermediate filaments. You are probably most familiar with keratin, the fibrous protein that strengthens your hair, nails, and the skin’s epidermis.

Microtubules

As their name implies, microtubules are small hollow tubes. Polymerized dimers of α-tubulin and β-tubulin, two globular proteins, comprise the microtubule’s walls ((Figure)). With a diameter of about 25 nm, microtubules are cytoskeletons’ widest components. They help the cell resist compression, provide a track along which vesicles move through the cell, and pull replicated chromosomes to opposite ends of a dividing cell. Like microfilaments, microtubules can disassemble and reform quickly.


Microtubules are also the structural elements of flagella, cilia, and centrioles (the latter are the centrosome’s two perpendicular bodies). In animal cells, the centrosome is the microtubule-organizing center. In eukaryotic cells, flagella and cilia are quite different structurally from their counterparts in prokaryotes, as we discuss below.

Flagella and Cilia

The flagella (singular = flagellum) are long, hair-like structures that extend from the plasma membrane and enable an entire cell to move (for example, sperm, Euglena, and some prokaryotes). When present, the cell has just one flagellum or a few flagella. However, when cilia (singular = cilium) are present, many of them extend along the plasma membrane’s entire surface. They are short, hair-like structures that move entire cells (such as paramecia) or substances along the cell’s outer surface (for example, the cilia of cells lining the Fallopian tubes that move the ovum toward the uterus, or cilia lining the cells of the respiratory tract that trap particulate matter and move it toward your nostrils.)

Despite their differences in length and number, flagella and cilia share a common structural arrangement of microtubules called a “9 + 2 array.” This is an appropriate name because a single flagellum or cilium is made of a ring of nine microtubule doublets, surrounding a single microtubule doublet in the center ((Figure)).


You have now completed a broad survey of prokaryotic and eukaryotic cell components. For a summary of cellular components in prokaryotic and eukaryotic cells, see (Figure).

Components of Prokaryotic and Eukaryotic Cells
Cell Component Function Present in Prokaryotes? Present in Animal Cells? Present in Plant Cells?
Plasma membrane Separates cell from external environment controls passage of organic molecules, ions, water, oxygen, and wastes into and out of cell Yes Yes Yes
Cytoplasm Provides turgor pressure to plant cells as fluid inside the central vacuole site of many metabolic reactions medium in which organelles are found Yes Yes Yes
Nucleolus Darkened area within the nucleus where ribosomal subunits are synthesized. No Yes Yes
Nucleus Cell organelle that houses DNA and directs synthesis of ribosomes and proteins No Yes Yes
Ribosomes Protein synthesis Yes Yes Yes
Mitochondria ATP production/cellular respiration No Yes Yes
Peroxisomes Oxidize and thus break down fatty acids and amino acids, and detoxify poisons No Yes Yes
Vesicles and vacuoles Storage and transport digestive function in plant cells No Yes Yes
Centrosome Unspecified role in cell division in animal cells microtubule source in animal cells No Yes No
Lysosomes Digestion of macromolecules recycling of worn-out organelles No Yes Some
Cell wall Protection, structural support, and maintenance of cell shape Yes, primarily peptidoglycan No Yes, primarily cellulose
Chloroplasts Photosynthesis No No Yes
Endoplasmic reticulum Modifies proteins and synthesizes lipids No Yes Yes
Golgi apparatus Modifies, sorts, tags, packages, and distributes lipids and proteins No Yes Yes
Cytoskeleton Maintains cell’s shape, secures organelles in specific positions, allows cytoplasm and vesicles to move within cell, and enables unicellular organisms to move independently Yes Yes Yes
Flagella Cellular locomotion Some Some No, except for some plant sperm cells
Cilia Cellular locomotion, movement of particles along plasma membrane’s extracellular surface, and filtration Some Some No

Section Summary

The cytoskeleton has three different protein element types. From narrowest to widest, they are the microfilaments (actin filaments), intermediate filaments, and microtubules. Biologists often associate microfilaments with myosin. They provide rigidity and shape to the cell and facilitate cellular movements. Intermediate filaments bear tension and anchor the nucleus and other organelles in place. Microtubules help the cell resist compression, serve as tracks for motor proteins that move vesicles through the cell, and pull replicated chromosomes to opposite ends of a dividing cell. They are also the structural element of centrioles, flagella, and cilia.

Free Response

What are the similarities and differences between the structures of centrioles and flagella?

Centrioles and flagella are alike in that they are made up of microtubules. In centrioles, two rings of nine microtubule “triplets” are arranged at right angles to one another. This arrangement does not occur in flagella.

How do cilia and flagella differ?

Cilia and flagella are alike in that they are made up of microtubules. Cilia are short, hair-like structures that exist in large numbers and usually cover the entire surface of the plasma membrane. Flagella, in contrast, are long, hair-like structures when flagella are present, a cell has just one or two.

Describe how microfilaments and microtubules are involved in the phagocytosis and destruction of a pathogen by a macrophage.

A macrophage engulfs a pathogen by rearranging its actin microfilaments to bend the plasma membrane around the pathogen. Once the pathogen is sealed in an endosome inside the macrophage, the vesicle is walked along microtubules until it combines with a lysosome to digest the pathogen.

Compare and contrast the boundaries that plant, animal, and bacteria cells use to separate themselves from their surrounding environment.

All three cell types have a plasma membrane that borders the cytoplasm on its interior side. In animal cells, the exterior side of the plasma membrane is in contact with the extracellular environment. However, in plant and bacteria cells, a cell wall surrounds the outside of the plasma membrane. In plants, the cell wall is made of cellulose, while in bacteria the cell wall is made of peptidoglycan. Gram-negative bacteria also have an additional capsule made of lipopolysaccharides that surrounds their cell wall.

Glossary


Humans And Plants Share Common Regulatory Pathway

In findings that some might find reminiscent of science fiction, scientists at the Scripps Research Institute have shown for the first time that humans and plants share a common pathogen recognition pathway as part of their innate immune systems. The data could help shed fresh light on how pathogen recognition proteins function and the role they play in certain chronic inflammatory diseases.

The study provides new evidence that Nod1, a member of the Nod-like Receptor (NLR) protein family, is activated by the protein SGT1, which also activates Resistance (R) proteins in plants R proteins protect plants from various pathogens. The study also confirms structural similarities between the Nod1 protein, which plays a pivotal role in the innate immune system's recognition and response to bacterial infection and members of the R protein family.

"There has been a great deal of speculation that R proteins and Nod1 are related, but our study provides the first direct link between plants and humans," said Richard Ulevitch, the Scripps Research scientist whose laboratory conducted the study. "Plants have Nod-like receptors and similar immune responses to bacteria and other pathogens-the R proteins evolved to counteract these pathogenic effects. Our study provides a new perspective on the Nod1 pathway in mammalian cells as well as the value of drawing on plant studies of R protein pathways to better understand the pathogen recognition functions of these proteins."

The Nod proteins recognize invasive bacteria, specifically distinct substructures found in Gram-negative and Gram-positive organisms. Once activated, Nod1 produces a number of responses that include activation of intracellular signaling pathways, cytokine production and apoptosis or programmed cell death. Despite the fact that various models of Nod1 activation have been described, little has been known about other proteins that might affect the protein's activation. In contrast, a number of additional proteins have been linked to the activation pathways of the R protein in plants.

"The NLR family has clear links to human disease," Ulevitch said. "Out of the more than 20 proteins in the NLR family, several mutations are linked to diseases that involve chronic inflammation or autoimmune consequences. Up to now, there has been a limited understanding of the regulatory pathways of Nod1. By identifying SGT1 as a positive regulatory protein, our study offers new insights into the entire family."

SGT1 is a protein found in yeasts, plants, and mammals in both the nucleus and the cytosol. It functions in several biological processes through interaction with different multi-protein complexes. A large body of evidence also suggests that the protein plays a role in regulating pathogen resistance in plants. Various genetic studies have identified SGT1 as a crucial component for pathogen resistance in plants through regulation of expression and activities of some R proteins

Although there is a significant genetic crossover between plants and mammals, very little is known about this common human-plant regulatory pathway. Ulevitch speculated that certain protein regulatory structures might exist in both plants and humans simply because they do the same thing in much the same way.

"In reality," he said, "there are only so many ways to accomplish related biological responses."

The study also showed that a heat shock protein, HSP90, helped stabilize Nod1.

"Inhibiting HSP90 resulted in a significant reduction of Nod1 protein levels," Ulevitch said. "That clearly suggests that this protein plays a key role in stabilizing Nod1 and protecting it from degradation. In contrast, turning off SGT1 did not alter levels of Nod1."

In an earlier study, Ulevitch's laboratory reported that Nod1 also interacted with the COP9 complex, a multiprotein complex that is known to play a role in a number of development pathways in plants and that has a mammalian counterpart. This interaction, Ulevitch noted, provides a second link between Nod1 and plant R protein pathways.

"The association of Nod1 with SGT1 and the COP9 complex suggests that one possible role of SGT1 could be to target resistance-regulating proteins for degradation," he added. "In this hypothesis, the target protein would be a negative regulator of immune responses."

Future studies, Ulevitch said, will focus on the extensive literature that exists describing the R protein dependent immunity in plants to better understand human NLR pathways, especially those dependent on Nod1.

The study was supported by the National Institutes of Health and Novartis. The study was published in an advance online edition of the Proceedings of the National Academy of Sciences during the week of April 9, 2007.

Other authors of the study, "SGT1 Is Essential for NOD1 Activation," are Jean da Silva Correia, Yvonne Miranda, and Nikki Leonard.

Story Source:

Materials provided by Scripps Research Institute. Note: Content may be edited for style and length.


The cell biology of asthma

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.

Introduction

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.

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-13Rα1 and IL-4Rα. 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).

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 (p38δ-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-α, heparin-binding EGF, amphiregulin, β-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, 2002Koff 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 δ and PKC θ, leading to recruitment of the NADPH oxidase subunits p47 phox and p67 phox 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-α–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 γ-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 γ-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 γ-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 1β (IRE1β), a transmembrane ER stress sensor. IRE1β is found in mucus-producing cells in the intestine and the airways, but not in other cells. IRE1β regulates AGR2 transcription, and mice deficient in IRE1β had reduced AGR2 expression and impaired airway mucin production in an allergic asthma model (Martino et al., 2013). AGR2 and IRE1β 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.

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 Gαq. GTP-loaded Gαq activates its downstream effector, PLCβ, 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 α9β1 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 α9β1 is highly expressed in airway smooth muscle (Palmer et al., 1993). Conditional knockout of the integrin α9 subunit (uniquely found in the α9β1 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 α9β1 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 α9 cytoplasmic domain (Chen et al., 2004), and the lipid kinase, PIP5K1γ, which binds directly to talin, an integrin β1 subunit binding partner. Spermine and spermidine are critical cofactors for PIP5K1γ, so its juxtaposition with SSAT effectively reduces enzymatic activity. PIP5K1γ 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 PLCβ, so when α9β1 is present and ligated, contractile agonists that activate receptors coupled to Gαq induce less IP3 generation (Chen et al., 2012) and thus less Ca 2+ release through IP3 receptors in the sarcoplasmic reticulum. The importance of this pathway was confirmed by the observations that the frequency of Ca 2+ oscillations induced by cholinergic agonists was reduced in lung slices from mice lacking α9β1, 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).

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 αvβ8 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 αvβ8 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 α, also implicated in asthma pathogenesis, has been shown to increase airway smooth muscle contractility by a similar mechanism (Goto et al., 2009).

Enhanced cytokine-mediated airway smooth muscle contraction in MFGE8-deficient mice.

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

Conclusions

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


Results and discussion

Since our knowledge on peanut allergy has been limited to the crop species and its two genomic ancestors, and considering the findings that single amino acid substitutions could have substantial impact on allergenicity [14, 31, 32], it is pertinent to explore single amino acid substitutions in the wild species of Arachis and evaluate their impact on allergenicity. To do so, we sequenced the two immunodominant allergens Ara h 2 and Ara h 6 from 24 species representing the major lineages in Arachis and used multiple approaches to characterize molecular alterations and determine their impact on allerginicity. Three striking outcomes from this study have emerged. One, the two allergen homologues studied, Ara h 2 and Ara h 6, followed very contrasting evolutionary pathways. Specifically, Ara h 2 has undergone a higher proportion of amino acid substitutions compared with Ara h 6 and has accumulated by far a greater number of losses and gains of motifs ranging from 1–24 amino acids (S1 Fig). Two, and quite importantly, these molecular alterations were mostly concentrated in the immunodominant epitope-rich regions, specifically in Epitopes 6 and 7 (Fig 2, S1 Fig). Three, the mutational events (substitutions and insertions/deletions) particularly in Ara h 2 appear to follow phylogenetic trends from the base of the Arachis tree to the terminal branches. These findings raise the questions of the underlying factors that differently affected Ara h 2 and Ara h 6 modes of evolution and whether such molecular alterations are linked to potential accentuation of allergenicity. To address this curious biological situation, we will compare the two homologues at various molecular tiers and discuss the findings in a phylogenetic/evolutionary framework.

Gaps and epitopes are labeled. Numbers above the columns denote alignment positions.

Comparative molecular composition across the genus Arachis

One of the prominent findings in this study is that Ara h 2 and Ara h 6 homologs from wild Arachis species differ substantially in mutational patterns and ORF length. To shed light on the underlying factors that might account for these differences and assess the impact of this contrasting mode of evolution on these proteins, we evaluated molecular compositions at two tiers: nucleotide and amino acid levels. The nucleotide compositions for the entire ORFs of Ara h 2 and Ara h 6 homologs as well as the nucleotide frequencies per codon position are presented in Fig 3. Overall, the two homologues did not differ significantly in their nucleotide composition across the genus since the t-test rejected the null hypothesis. This underscores notable constraints on their mode of nucleotide substitutions to maintain similar compositions.

Average percentages of Adenine (A), Thymine (T), Guanine (G), Cytosine, and GC ratios. Calculations were done for overall DNA sequences and individual codon positions of Ara h 2 and Ara h 6 from the Arachis species. The notations 1, 2 and 3 denote 1 st , 2 nd , and 3 rd codon positions. Data are presented as mean ± SD. A two-way ANOVA with Sidak post-test was applied. The two homologues were not statistically different in nucleotide composition.

It has been demonstrated that higher GC content correlates positively with rates of substitutions [62, 63]. A recent study focusing on A. duranensis estimated an average GC content of 31.8% for the whole genome, which is in line with most legumes examined [9]. The overall GC ratio for both homologues was highly similar (Ara h 2 = 55.7± 0.8 Ara h 6 = 56.1± 0.5, (Fig 3). The average 56% GC content of Ara h 2 and Ara h 6, being substantially higher (75%) than that reported for the whole A. duranensis genome points to a suitable genetic landscape for accelerated rates of mutations in both. Nevertheless, the frequency of amino acid substitutions remained lower in Ara h 6. One notable difference is that Ara h 2 exhibited relatively higher GC ratios in its 1 st and 2 nd codon positions contrasted with Ara h 6 (Fig 3). This difference may account in part for the elevated amino acid substitutions in Ara h 2 since mutations in 1 st and 2 nd codon positions are translated into 96% and 100% nonsynonymous substitutions, respectively, compared with 31% for the 3 rd position [64].

The two homologues, although being highly allergenic, display disparity in their number and modes of amino acid substitutions (Fig 4, S1 Fig). Reciprocal amino acid sequence identities for Ara h 2 across the Arachis species ranged between 65.5% to 99.4% (mean 89.4%± 8.2) compared with 75.7%-99.3% for Ara h 6 (mean 93.2%± 5.3), underscoring the higher frequency of substitutions in the former. In the case of Ara h 2, the ORF length ranges from 156 to 181 amino acids (S1 Table). The amino acid alignment of Ara h 2 required the insertion of 17 gaps ranging from 1 to 29 amino acids, resulting in 203 alignment positions (S1 Fig). Importantly, amino acid positions 59–90 in epitope 5 and the immunodominant epitopes 6, 7 and the newly recognized 7b display residue substitutions and losses/gains that may potentially impact allergenicity (Fig 2). Thirteen gaps in the sector that includes epitopes 6, 7 and 7b are identified and labeled A-L (Fig 2). The mutation in position 64 presents a molecular marker for the genomic ancestry of the tetraploid crop since it is a glycine (G) in all three accessions of A. ipaensis and glutamic acid (E) in all A. hypogaea and A. duranensis accessions (Fig 2).

A two-way ANOVA with Sidak post-test was applied. All data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001.

The Ara h 6 ORF, in comparison, displayed a narrower range of amino acid residues across the genus, 145 to 147 (S1 Table). Only two sequence gaps were required for the alignment, corresponding to a methionine at position 22 and an arginine at position 69 (S1 Fig). Interestingly, this apparent in-frame initiation codon at position 22 evolved in species that emerged after the early diverging A. triseminata, A. paraguariensis and A. macedoi. This methionine residue could not be considered as an alternate initiation codon since it occurs downstream of the signal peptide region. Remaining amino acid differences were due to replacements, underscoring the contrasting patterns of evolution undergone by Ara h 2 and Ara h 6 across the genus. The infrequent loss/gain of amino acid residues in Ara h 6 across the genus Arachis compared with Ara h 2 is intriguing and could be attributed to structural/functional limitations on this allergen protein.

To assess potential heterogeneity in amino acid composition across the Arachis genus evolutionary history due to the noted mutational events, we analyzed amino acid compositions of the Ara h 2 and Ara h 6 homologs. The two were comparable in amino acid composition across the genus, and were low in tryptophan (W) and high in arginine (R) and glutamine (Q) (Fig 4 and S2 Table). These findings are in agreement with previous studies on peanuts [65]. Tryptophan was lacking in Ara h 2 of three species and the Ara h 6 of 14 species (S2 Table). It is observed that Ara h 2 accommodated higher variation across the genus Arachis in glutamine (Q), proline (P), and aspartate (D), whereas Ara h 6 displayed higher variation in glutamate (E), histidine (H) and arginine (R) (S2 Fig and S2 Table). Despite the contrasting patterns of variability, the two homologues are similar in composition in terms of the five amino acid physicochemical groups and they are rich in hydrophobic amino acids and low in aromatic amino acids (Fig 5). Therefore, mutational events in the two homologues appear to be constrained to maintain the physicochemical groups composition in order to conserve the functional and structural integrity of these proteins.

The means, standard deviations (noted as bars) and the statistical p values for significance of difference were calculated in Graphpad Prism. A two-way ANOVA with Sidak post-test was applied. The two homologues were not statistically different in sidechain compositions.

The hydrophobic nature of the protein is important for the functionality of the signal peptides of these allergens [30]. Ara h 2 and Ara h 6 are synthesized in the cytoplasm as precursor proteins with an additional N-terminal amino acid signal peptide required for transport to the vacuoles [19, 30, 66]. The signal peptides (amino acid residues 1–7 [29] or residues 1–21 [67] contain 2-residue gaps in Ara h 2 and display a number of substitutions in both Ara h 2 and Ara h 6 (Fig 6 and S1 Fig). Despite these mutations, the region remained highly constrained to maintain the hydrophobic physicochemical property required for its crucial function (S1 Fig). In summary, although the two allergen proteins have undergone contrasting patterns of amino acid substitutions and losses/gains over the evolutionary history of the genus, they maintained comparable amino acid composition and physicochemical groups.

Shown are a per-codon distribution of synonymous mutations (red), nonsynonymous mutations (gray) and indels (blue). IgE-binding epitopes are highlighted beneath the amino acid residues. 5A. Ara h 2 homologue. 5B. Ara h 6 homologue.

Signatures of natural selection in Ara h 2 and Ara h 6

The contrasting mutational patterns observed in Ara h 2 and Ara h 6 (Figs 4 and 6, S1 Fig) raise the question of whether these evolutionary modes are driven by natural selection, positive-diversifying or negative-purifying. Assessment of signatures of natural selection operating on coding genomic regions is a major challenge. Murrell et al. (2015) [45] noted that there is not yet an uncontroversial way to answer the question of whether a gene evolved under positive selection. Underlying factors that contribute to the difficulties in determining positive selection signature include intrinsic patterns of gene substitutions, biological assumptions made, and models used. To assess potential natural selection signatures acting on Ara h 2 and Ara h 6, given the prominent differences in mutational patterns, we selected and contrasted results of three independent methods (distance–based, BUSTED, and FUBAR) that adopt different biological assumptions and use metrics for site-specific or whole gene estimation of natural selection signatures.

One of the simplest methods is that of Nei and Gojobori (1986) [43] that focuses on the detection of natural selection using the rates of synonymous vs. nonsynonymous substitutions in protein coding genes. The algorithms compute variance and covariance of dS (synonymous) and dN (nonsynonymous) mutations and the proportions of synonymous (pS) and nonsynonymous (pN) differences. Using SNAP for these computations, the averages of all pairwise comparisons for the Ara h 2 data set showed dS = 0.0142, dN = 0.0475, dS/dN = 0.3319, and pS/pN = 0.3379. In contrast, the Ara h 6 data set revealed dS = 0.0831, dN = 0.0247, dS/dN = 5.8627, and pS/pN = 5.5914. The 0.332 dS/dN for Ara h 2 implies that it has experienced positive selection along the genus evolutionary history whereas the 5.863 dS/dN calculated for Ara h 6 indicates purifying selection. A dS/dN value below 1 implies positive/diversifying selection while above 1 signifies purifying selection (SNAP output is presented as dS/dN not dN/dS). BUSTED, a gene-wide and positive selection-focused method, reported evidence of gene-wide episodic diversifying selection in Ara h 2 across the branches of the phylogeny (p = 0.005 ≤ 0.05) but no evidence of episodic diversifying selection in Ara h 6 (p-value = 0.151 ≥ 0.05). Therefore, the BUSTED finding is in agreement with the Nei and Gojobori (1986) approach, with both showing that Ara h 2 is experiencing positive selection. Ara h 6, on the other hand, was shown to be under purifying selection using Nei and Gojobori (1986) approach (BUSTED tests for positive selection).

The site-specific, Bayesian-based FUBAR method detected in Ara h 2 diversifying selection and purifying selection at 5 sites each with 0.9 posterior probability. For Ara h 6, this method detected diversifying selection at 2 sites but purifying selection at 8 sites (posterior probability 0.9). The detected sites are highlighted in S3 Table. FUBAR (Murrell et al. 2013) [46] has more power for detecting weak positive selection (at low ω>1 values). Contrasting the gene-wide and the site-specific methods used here, it is believed that the gene wide methods are more effective in detecting natural selection signatures. Murrell et al. (2013) [46] noted that when positive selection is undetectable on any one codon site or branch in isolation, pooling evidence for positive selection across multiple sites and branches can render their cumulative effect more evident. Similar inferences were reached by others [68, 69]. This seems to be the case in the detection of positive selection by the gene-wide methods used in this study. In conclusion, the gene-wide approaches have shown that Ara h 2 is evolving under positive/diversifying selection. The Nei and Gojobori (1986) [43] and the FUBAR approaches (BUSTED tests for positive-selection) demonstrated that Ara h 6 is evolving under negative/purifying selection. FUBAR detected sites under positive selection in both Ara h 2 and Ara h 6 but more sites under purifying selection in Ara h 6 (2 vs. 8 sites, respectively). These findings raise the curious questions of whether positive selection in Ara h 2 played a role in the emergence of immunodominant motifs, whereas purifying selection in Ara h 6 maintained a status que for a gene that has emerged initially with strong allergenic features when Arachis diverged from its common ancestry.

To evaluate the potential consequences of the positive selection detected in Ara h 2, two concepts will need to be addressed. One relates to the detection of progressive increase in allergenicity across the evolutionary history of the genus and the other pertains to its potential adaptive advantages for allergenicity. Insight into the relationship between the progressive expansion of the region encompassing the immunodominant epitopes 6, 7 and 7b, including the progressive additions of DPYSPS motifs in Ara h 2 (Fig 2) and the enhancement of allergenicity is provided by the immunoblot experiments. These experiments (detailed later) revealed a progressive increase in allergenicity from the basal species A. triseminata to the terminal species in the phylogenetic tree, A. hypogaea. The changes correspond to expansions of the immunodominant motifs region (Fig 2), pointing to a possible association between the two phenomena. The second point to be addressed is the availability of evidence for adaptive advantages allergenicity may confer on peanut seeds. Direct experimental studies addressing this question in the wild species of Arachis are unavailable in the literature. However, some work exists that links the allergen proteins to defense against insects and pathogens. Ara h 2 is a trypsin inhibitor [7] and this function has been shown to control insect attacks, a feature that is being used in breeding programs for insect tolerance during seed storage [70]. The 2S albumin protein family, to which Ara h 2 and Ara h 6 belong, was demonstrated to be effective in inhibiting the growth of fungi and bacteria in radish seeds and four other members of the mustard family Brassicaceae [71]. Similarly, antifungal properties are known for a 2S albumin-homologous protein from passion fruit seeds [72]. Considering these findings in peanuts and other flowering plants, positive/diversifying selection pressure that promotes the augmentation of allergenicity may be advantageous to increase the chances of seed survival via protection from insects and fungi. The patterns of molecular evolution documented in Ara h 2 and the corresponding progressive increase in allergenicity resolved in the immunoblots encourages direct studies on wild species seeds to evaluate selective advantages for protein allergenicity.

Phylogenetic and evolutionary assessment of epitopes

The pattern of substitution (mutations and indels), particularly nonsynonymous substitutions, could have dual structural and functional implications in protein coding genes. In allergen genes, nonsynonymous substitutions in epitope-rich sections or duplications of allergenic epitopes may substantially impact allergenicity [14, 17, 30]. It has been shown in peanuts that a single residue substitution expresses up to 99% reduction in allergenicity by impacting the IgE-binding ability [14, 31, 32]. We will discuss within a phylogenetic platform the mutational events in the allergenic epitopes of the two homologues. Identification of the Ara h 2 and Ara h 6 epitopes across the genus is based on epitopes recognized in the crop and its putative ancestors by Staley et al. (1997) and Ratnaparkhe et al. (2014) [14, 29].

The Ara h 2 homologue

Ten epitopes have been recognized in Ara h 2 [14]. Epitopes 6 and 7 are unique to this homologue and contain the characteristic immunodominant hexapeptide DPYSPS motif (Fig 2 and S1 Fig) located between helices 2 and 3. The other eight epitopes are split among the upstream region (epitope 1–5) and the downstream regions (epitopes 8–10). Epitope 1–4 ( HASARQQWEL , QWELQGDR , DRRCQSQLER , LRPCEQHLMQ , respectively), and epitopes 8–10 ( LQGRQQ , KRELRN , QRCDLDVE , respectively) are completely to highly conserved across Arachis (S1 Fig). Epitope 5 ( KIQRDEDS ), in contrast, has undergone complex mutational events. Although its KIQR motif is unchanged throughout the genus, the remaining sector (DEDS) has accommodated a number of mutations and indels across the genus history, appearing as DQD-Q and DED-S (dashes denote gaps) in the basal clade of A. macedoi, A. lutescens, and A. triseminata, mutating to DQS—, EED-Q, DEDSS in species of subsequent lineages, and reverting back to the original sequence of DED-S in the crop and its related species of the terminal lineage (Fig 1 and S1 Fig). It is to be noted that the early diverging A. triseminata with the truncated DQS—motif displayed the lowest degree of allergenicity in our immunoblot experiment compared with the peanut (Fig 7, discussed below). These natural mutations have the potential of providing valuable guidelines for examining the impact of loss/gain and residue mutations on allergenicity.


Author information

These authors jointly supervised this work: Hugh H. Reid, Jamie Rossjohn.

Affiliations

Infection and Immunity Program and The Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute Monash University, Clayton, Victoria, Australia

Jan Petersen, Laura Ciacchi, Mai T. Tran, Khai Lee Loh, Nathan P. Croft, Anthony W. Purcell, Hugh H. Reid & Jamie Rossjohn

Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia

Jan Petersen, Laura Ciacchi, Hugh H. Reid & Jamie Rossjohn

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, the Netherlands

Yvonne Kooy-Winkelaar & Frits Koning

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

Melinda Y. Hardy & Jason A. Tye-Din

Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

Melinda Y. Hardy & Jason A. Tye-Din

Department of Microbiology & Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia

Zhenjun Chen & James McCluskey

ImmusanT, Cambridge, MA, USA

Department of Gastroenterology, The Royal Melbourne Hospital, Parkville, Victoria, Australia

Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

You can also search for this author in PubMed Google Scholar

Contributions

J.P., H.H.R., L.C., M.T.T., K.-L.L., Y.K.-W., N.P.C. and M.Y.H. contributed to data generation and analysis. Z.C. and J.M. provided key reagents. R.P.A., A.W.P., J.A.T-D. and F.K. contributed to data analysis and manuscript writing. H.H.R. and J.R. are joint senior and corresponding authors and, with J.P., conceived the study, analyzed data and co-wrote the manuscript.

Corresponding authors


Abstract

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling describes the structure–function of this protein family, which is spread widely among gram-positive bacteria and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.


For plant and animal immune systems the similarities go beyond sensing

Superposition of the HeLo domains of plant (yellow), human (blue), and mouse MLKL (pink). Credit: Takaki Maekawa

Although profoundly different in terms of physiology, habitat and nutritional needs, plants and animals are confronted with one shared existential problem: how to keep themselves safe in the face of constant exposure to harmful microorganisms. Mounting evidence suggests that plants and animals have independently evolved similar receptors that sense pathogen molecules and set in motion appropriate innate immune responses.

Now, in a study published in the journal Cell Host & Microbe, senior author Takaki Maekawa together with co-first authors Lisa K. Mahdi, Menghang Huang, Xiaoxiao Zhang and colleagues have discovered that plants have evolved a family of proteins that bear a striking resemblance to proteins called mixed lineage kinase domain-like proteins (MLKLs), which trigger cell death in vertebrates as part of the immune response. In uncovering and characterizing an important new family of plant immune proteins, the authors' study, which involved collaboration with fellow MPIPZ researchers Paul Schulze-Lefert, Jane Parker and Jijie Chai, provides intriguing new insights into how plants protect themselves from microbial invaders.

Regulated cell death often accompanies immunity against infection in plants, animals and fungi. One pervasive theory suggests that highly localized cell death responses serve to strictly limit the spread of infection. Although starting from independent origins, this shared response seems to also involve highly similar machinery: many proteins involved in cell death in different kingdoms of life contain a so-called HeLo domain, a bundle structure made up of four helices, which causes resistance and cell death by disturbing the integrity of cellular membranes or forming ion channels.

Based on the similarities between animal and plant immune systems and on the key role played by HeLo domains in cell death, Maekawa hypothesized that plants might also contain other proteins with HeLo domains. Making use of bioinformatic and structural analysis, he and his team discovered a new family of HeLo domain-containing proteins that are widely shared among different plant species, indicating that they are important for plant physiology.

Maekawa termed the proteins plant MLKLs, and for further studies he focused on MLKLs expressed in the model plant Arabidopsis thaliana. He and his team isolated MLKL proteins from A. thaliana and determined that plant MLKLs possess the same overall protein architecture as their vertebrate counterparts and also assemble into tetramer, likely auto-inhibited, structures when they're not active. Importantly, plant MLKLs also play a role in immunity, as plants in which genes encoding these proteins were mutated and thus non-functional were susceptible to pathogen infection.

Further investigation revealed additional similarities with vertebrate MLKLs: plant MLKLs are also trafficked to cellular membranes as part of their function, and activation of these proteins leads to cell death. Maekawa now aims to discover the molecular details underlying the function of plant MLKLs in immunity: "It will be exciting to uncover exactly how MLKLs are activated upon pathogen infection and how this activation is translated into effective plant protection."


Food allergen protein families and their structural characteristics and application in component-resolved diagnosis: new data from the EuroPrevall project

A large number of food allergens able to induce allergic symptoms in predisposed individuals, including severe, even life-threatening reactions, have been identified and characterized. However, proteins able to cause such IgE-mediated reactions can be assigned to only a limited number of protein families. Detailed knowledge about the characteristics of food allergens, their 3D structures, biological activity and stability, will help to improve diagnosis of food allergy, avoid unnecessary exclusion diets and assess the risk of cross-reactive allergies to other food sources. This review is dedicated to summarizing current knowledge about the most important food allergen protein families and to presenting data from the EuroPrevall allergen library, a proof-of-concept collection of highly purified, characterized and authenticated food allergens from animal and plant food sources to facilitate improved diagnosis of food allergies.

Relevant food allergen sources

This is a preview of subscription content, access via your institution.


METHODS

Animals

Female BALB/c mice, aged 8–12 weeks (n=12 per group), were purchased from Charles River Laboratories (Saint-Constant, Quebec, Canada) and housed in environmentally controlled specific-pathogen-free conditions for 1 week prior to study, and throughout the experiments. All procedures were reviewed and approved by the Animal Research Ethics Board at McMaster University.

Allergen sensitization and exposure

Mice were sensitized and exposed to OVA, HDM or a combination of the two allergens (HDM and OVA) according to established chronic allergen exposure protocols (Fig. 1) (Inman et al., 1999 Leigh et al., 2002 Leigh et al., 2004c Leigh et al., 2004a Leigh et al., 2004b Southam et al., 2007).

OVA exposure

All mice receiving OVA were sensitized to OVA through intraperitoneal (IP) injection on day 1 followed by both IP injection and intranasal (IN) installation on day 11. IP OVA injections involved precipitating 10% aluminum potassium sulfate with 0.05% OVA, adjusting the pH to 6.5, then centrifuging and resuspending the pellet in saline, followed by a 200 μl IP injection. This was followed by five periods of IN OVA exposure, each 2 weeks apart. During each 2-day period, OVA was administered on consecutive days at a concentration 100 μg/25 μl. Mice receiving OVA also received sham exposure to the HDM allergen (in the form of IN SAL), according to the HDM exposure protocol (Fig. 1).

HDM exposure

HDM extract from Dermatophagoides pteronyssinus (Greer Laboratories, Lenoir, NC) was resuspended with sterile phosphate buffered saline to reach a concentration of 15 μg/25 μl, which was subsequently aliquoted and frozen at −20°C. The HDM was thawed at 4°C overnight to be administered the next day. All mice receiving HDM were initially exposed to the allergen through a daily 25 μl (15 μg/25 μl) IN installation, for two periods of 5 days each (days 29–33 and 36–40). This was followed by an 8-week period of HDM exposure, during which mice were exposed to IN HDM three times per week, on every other day of the week (Mondays, Wednesdays and Fridays), starting on day 43. Mice receiving HDM also received sham exposure to the OVA allergen (in the form of IN SAL), according to the OVA exposure protocol (Fig. 1).

HDM-OVA exposure (combination)

Using the same methods of administration used for the single allergens alone, all mice receiving the combination of HDM and OVA were first sensitized to OVA and, following a 16-day rest period (days 12–28), were exposed to HDM on 5 days per week for a period of 2 weeks. In a pilot study conducted before the current study (data not shown), we attempted to sensitize mice to both allergens simultaneously. However, this resulted in poor health and death. Therefore, the 16-day rest period between sensitization to each allergen was necessary to ensure that the mice remained healthy during the sensitization phase. Following sensitization, mice were exposed to both OVA and HDM, combining both allergen exposure protocols (Fig. 1).

Sham exposure (saline control mice)

Control mice were sensitized and exposed to SAL (25 μl IN) by following the same chronic allergen protocols illustrated in Fig. 1 and by using the same methods of administration.

Outcome measurements

Outcome measurements were made at two time points: 24 hours following the final allergen exposure, to investigate the association between airway inflammation and AHR, and at 4 weeks following the final allergen exposure, to investigate the association between airway remodeling and AHR at a time when airway inflammation had completely subsided (Fig. 1). Outcome measurements were assessed in eight groups of mice, consisting of four different groups of mice at each of the two time points. Group 1 was control mice exposed to SAL, group 2 was mice exposed to OVA, group 3 was mice exposed to HDM and group 4 was mice exposed to the combination of allergens (HDM and OVA) (n=12 mice per group). The primary outcome measurement at both time points was an in vivo assessment of airway responsiveness (RRS to MCh). Additional outcome measurements at both time points included total and differential cell counts (eosinophils, neutrophils, lymphocytes and macrophages) in the BAL fluid, and airway morphometry using a computer-based image analysis system (Northern Eclipse, Version 7.0 Empix Imaging Inc., Mississauga, Ontario, Canada) to quantify the number of mast cells in a given area of lung tissue. Further outcome measurements made at 4 weeks following the final allergen exposure included splenocyte recall for the TH2 cytokine IL-4, to confirm allergen sensitization, and airway morphometry using Northern Eclipse to quantify sustained structural changes in the airway.

Airway responsiveness

Airway responsiveness was assessed by measuring the RRS response to increasing doses of intravenous MCh using the flexiVent small animal ventilator (SCIREQ, Montreal, Canada), as described previously (Hirota et al., 2006).

Splenocyte recall

Splenocyte recall was performed as described previously (Johnson et al., 2004). Briefly, spleens were harvested, and splenocytes were isolated and diluted to a concentration of 8×10 6 cells/ml in complete RPMI. Splenocytes were cultured in complete RPMI alone, or in complete RPMI supplemented with either OVA (40 μg/ml) or HDM (40 μg/ml) in a flat-bottom, 96-well plate (Becton Dickinson) in quadruplicate. After 5 days of culture, supernatants were harvested and quadruplicates were pooled and frozen at −80°C until cytokine measurements were ready to be made.

Cytokine analysis

The levels of IL-4 in the supernatant of the splenocyte culture were measured using an ELISA kit for IL-4 (Quantikine R&D Systems, Minneapolis, MN).

BAL fluid collection and analysis

BAL fluid was collected as described previously (Inman et al., 1999 Leigh et al., 2002 Leigh et al., 2004c Leigh et al., 2004a Leigh et al., 2004b Southam et al., 2007). Differential cell counts were performed on 400 cells. Cells were classified, based on morphological criteria, as eosinophils, neutrophils and lymphocytes.

Lung histology and morphometry

Lung histology and morphometry were performed as described previously (Ellis et al., 2003 Inman et al., 1999 Leigh et al., 2002 Leigh et al., 2004c Leigh et al., 2004a Leigh et al., 2004b Southam et al., 2007). Briefly, 3 μm-thick lung sections were cut and stained with: PSRed to quantify the presence of subepithelial collagen, PAS to demonstrate the presence of goblet cells, and Toluidine Blue to demonstrate the presence of mast cells. Additional sections were prepared for immunostaining using a monoclonal antibody against α-SMA (Clone 1A4, DAKO, Denmark) to quantify the amount of α-smooth muscle actin contractile proteins in the airway. Morphometric quantification of stained lung sections was performed using a customized digital image analysis system (Northern Eclipse). Quantification of mast cells, which has not been described previously, was performed by collecting an image of the entire stained tissue section under a 1×objective to calculate the area of lung tissue. An individual blinded to the study codes then counted all of the mast cells under a 40×objective and the result was expressed as MCs/mm 2 .

Statistical analysis

Values are expressed as mean±s.e.m. Student’s t-tests were used to compare levels of IL-4, airway reactivity (the slope of the RRS-MCh dose-response curve), Max RRS, total and differential cell counts, and indices of airway remodeling between saline control mice and mice receiving allergen. Comparisons between the groups of mice receiving single allergens and the group of mice receiving the allergen combination were made using ANOVA (Statistica version 10.0). Post-hoc multiple-comparison testing was performed by using Duncan’s test. All comparisons were two-tailed and P values less than 0.05 were considered to be statistically significant.