The Role of Epithelial Cells in the Allergic Asthma Immune

Response
Emily Kim

2017SS Human Physiology

Capstone Project

College of Medicine
University of Cincinnati
 Abstract

The role of epithelial cells in the allergic asthma immune response

Emily Kim. College of Medicine, University of Cincinnati, 2017SS.

It is increasingly clear that pulmonary epithelial cells, which have the capacity to
sense both environmental allergens and allergen-driven immune responses, are
key mediators of allergic asthma, a non-communicable disease that affects over
300 million people worldwide and one in nine children. In particular, the symptoms
and pathophysiology of allergic asthma are precipitated by the action of Th2
cytokines such as IL-13 on airway epithelial cells. Consistent with an association
between IL-17A and more severe forms of asthma, we have shown that IL-17A
exacerbates IL-13-driven immunopathology. However, the effects of combined IL-
13 and IL-17A exposure on pulmonary epithelial cells remains incompletely
defined. Our goal is to elucidate the effects of IL-13, in the presence and absence
of IL-17A, on in vitro differentiated and polarized murine tracheal epithelial cells
(mTECs). Our data show that IL-17A regulates IL-13-mediated signaling and gene
expression in a concentration dependent manner. Furthermore, the cytokine-
mediated effects differ when the cytokines are applied on the apical or the basal
side of the differentiated and polarized mTECs. These results suggest that the
airway epithelium is a potential target for therapeutic endeavors in cases of IL-17A-
associated severe allergic asthma.

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Epithelial Cells in Asthma Immune Response Kim, Emily

Introduction

An allergy is classically defined as the tendency of ones immune system to overreact to molecules or

proteins to which most people do not react. This allergic reaction is generally characterized by T-cell proliferation

after initial exposure to the allergen that is biased towards the production of interleukin (IL)-4 by T helper 2 (Th2)

cells, which stimulates immunoglobulin (Ig) E synthesis. In the category of allergies, asthma can be defined as

a chronic inflammatory lung disease that is characterized by symptoms of wheezing, tightness in the chest,

coughing, and breathlessness. An asthmatic response may be induced by initial exposure to allergens such as

dander or pollen, pollutants such as cigarette smoke, a physical response such as exercise, or an infection.

Allergic asthma is defined by the presence of IgE antibodies specific for these common allergens, although both

allergic and non-allergic asthma see populations of Th2 cells secrete IL-4, IL-13, and tumor necrosis factor (TNF).

Specifically, through release of these cytokines, Th2 cells create several responses such as goblet cell

hyperplasia and bronchial hyperreactivity. Allergic asthma can be promoted through both genetic and

environmental factors. It is commonly known that exposure to cigarette smoke is a risk factor for Th2 sensitization

to common allergens such as Der p 1, a protein from Aspergillus spores that decreases transepithelial resistance.

House dust mite (HDM) allergens cleave tight junction-associated proteins such as ZO-1 and occludin (13).

Exposure to rhinoviruses can lead to disrupted tight junctions as well (8). Abnormal expression of genes

associated with the airway epithelium may contribute to an exacerbated inflammatory response (11). However,

other environmental factors can be protective against the onset of asthma, such as growing up on a farm (2).

One of the main issues with finding treatments for asthma is that because of these multiple factors, every person

could react a bit differently even with identical conditions and medication.

These contributing factors of the environment and genetics specifically interfere with the cytokinic

communication between the dendritic cells (DCs) and epithelial cells of the airway, and therefore these cytokines

could be used as potential therapeutic targets. The classic model of this communication starts with exposure to

the allergen. Then, epithelial cells attract immature DCs through the release of chemokines, which signal the

DCs to induce Th2-mediated responses via cytokines such as IL-25, IL-33, and granulocyte macrophage colony-
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Epithelial Cells in Asthma Immune Response Kim, Emily
stimulating factor (GM-CSF) (9). The DCs then travel up towards the lumen and sample the allergen by extending

their processes between the epithelial cells directly into the lumen. This extension provides a mechanism for

airway surveillance. The DCs engulf the allergen and present the antigen to nave T-cells via draining mediastinal

lymph nodes on class II major histocompatibility complexes (MHCII). This activates the nave T-cells towards

Th2-driven responses. DCs in asthmatics are incredibly sensitive as compared to non-asthmatics, especially to

proteolytic activity of common allergens such as Der p 1 (4).

Classically, the epithelial cell layer has been thought of as a passive barrier to protect against allergens

in a non-specific manner because of tight junctions in epithelia. However, in recent years, the epithelium is now

seen as a key player in the allergic asthma immune response by influencing the function of DCs and therefore

influencing Th2 function. This is done through pattern recognition receptors (PRRs) to quickly react to damage-

associated molecular patterns and through Toll-like receptors (TLRs), through which their ligation leads to signal

cascades to produce chemokines and cytokines that attract other cells to the airways. Epithelial cells also

participate in airway remodeling, which is a long-term change in the structure of the airway characterized by

thickening of the smooth muscle wall surrounding the airway, increase in extracellular matrix components under

the basement membrane that creates thickening, goblet cell hyperplasia, and reduced transepithelial resistance.

These symptoms lead to reduced surface area through which gas exchange can occur, leading to the phenotypic

symptoms described in the beginning of this paper. Through these factors, one can conclude that epithelial cells

have great implications in affecting the symptoms and severity of allergic asthma. Currently, inhaled

corticosteroids are widely used to treat allergic asthma by reducing the number of DCs in the airway, but this is

detrimental because it supports immunodeficiency in the pulmonary system. Therefore, cytokines that affect the

interplay between epithelial cells and DCs have potential to be better therapeutic targets, especially for severe

asthmatics.

Discussion

Allergic asthma affects over 300 million people worldwide; targeting IL-13 and IL-17A could be the key to

reducing mortality in severe steroid-refractory asthma. Eliminating the presence of IL-13 in the airway stops

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Epithelial Cells in Asthma Immune Response Kim, Emily
allergen-driven reactions, while reinstatement of the presence of IL-13-mediated signaling in the airway

epithelium will reestablish most symptoms of allergic asthma (5). Therefore, the presence of IL-13 is necessary

for allergic asthma responses to occur, making it a potential target for pharmaceuticals. IL-13 has been previously

shown to induce changes in bronchial smooth muscle cells, which results in bronchoconstriction or airway

hyperreactivity (AHR), a classic symptom of asthma that occurs when the airway constricts to nonspecific stimuli.

Additionally, IL-13 can activate expression of certain eotaxins, including CCL24 and CCL26, which have elevated

expression in asthmatics (10). Finally, IL-13 decreases the strength of the tight junctions in the epithelium and

prolongs allergic inflammatory responses (1). Meanwhile, IL-17 has been shown to induce expression of

granulocyte colony-stimulating factor (G-CSF), which recruits neutrophils to the site of inflammation as well as

promotes their survival and activation (7). These mechanisms are shown in a simplified form in Figure 3.

Mouse models that evoke mixed Th2 and Th17 responses, which produce IL-13 and IL-17A respectively,

are associated with more severe symptoms including airway inflammation and dysfunction than an isolated Th2-

mediated response (6). Mice exposed intratracheally to both IL-13 and IL-17A had increased AHR, goblet cell

hyperplasia, more dramatic airway inflammation, and enhanced IL-13 induced gene expression (3). The

presence of both Th2 and Th17 cells in the airway enhances airway inflammation by eosinophils. Increased

levels of eosinophilic inflammation are also produced by increased expression of IL-23 secreted by epithelial

cells, which increases the number of Th17 cells (12). Additionally, IL-17A creates steroid insensitivity in bronchial

epithelial cells, which may account for reduced steroid efficacy in severe asthmatics (14). Like IL-13, IL-17A

modulates bronchial hyperreactivity and airway remodeling, but it does not affect Th2 recruitment to the airways,

suggesting that IL-17A exacerbates IL-13-mediated responses, but IL-13 does not enhance IL-17A-mediated

responses (6). Previous data from Hall et al suggests that IL-17A accomplishes this exacerbation by increasing

STAT6 signaling by IL-13, which is the first molecular mechanistic explanation of how IL-17A exacerbates IL-13-

mediated responses (3).

Now, the role of epithelial cells in terms of IL-13-mediated responses and modulation by IL-17A is being

investigated via RNA isolation, complementary DNA synthesis, and analysis by real time PCR to show changes

in gene expression of epithelial cells grown in vitro. This was done because in the previous study described done

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Epithelial Cells in Asthma Immune Response Kim, Emily
by Hall, investigators were unsure whether increased levels of gene expression of IL-13-mediated genes in vivo

was due to increased numbers of cells recruited to the airspace or increased expression in the epithelial cells

themselves. Not many studies are being done on what effects the gene products of airway epithelial cells have

on other innate and adaptive immune cells, therefore this study investigates effects in the airway after the initial

exposure to the allergen, and where mature, polarized T cells are already present in the airway. The aim of this

study is to understand the role of these two cytokines together through the context of airway epithelial cells

because they are a major cell population targeted by this response and are also major mediators of the downward

immune response to inhaled allergens as described in the introduction. To do this, we have cultured mouse

tracheal epithelial cells isolated from the tracheas of 4-6-week-old C3H mice. We expose the differentiated and

polarized cell population, which includes ciliated cells, nonciliated cells, goblet cells, and Clara cells, either

apically or basally to IL-13 and IL-17A in varying concentrations, and then gene expression changes are

measured through RT-PCR. Referring to Figure 1, on the x-axis, the cells were exposed to either media alone,

or increasing concentrations of IL-13 up to 10ng/mL for 18-20 hours. On the y-axis, the ratio of IL-13 mediated

genes to a housekeeping gene is displayed. We tend to see a bell-shaped curve when all treatment groups are

examined. Similarly, when mTECs are exposed to IL-17A alone and when looking at IL-17A-mediated genes,

the cells again responded to the stimulation on both the apical and basal sides, exhibiting a bell-shaped curve.

As shown by Figure 2, when examining IL-13 mediated genes when cells were exposed to both IL-13 and IL-

17A, the mTECs can either show a concentration-dependent apical or basal response when exposed to a

constant concentration of IL-13 and increasing concentrations of IL-17A that is much more exacerbated than

exposure to IL-13 or IL-17A alone. On the x-axis, you can see the results of exposing the cells to no cytokines,

.5ng/mL of IL-13, and then .5ng/mL of IL-13 and increasing concentrations of IL-17A. On the y-axis, we have

compared levels of gene expression in an IL-13 mediated gene with a housekeeping gene to form a ratio of units

of gene expression. When the cells are exposed to media only, there is very little to no change in gene expression

when compared to a housekeeping gene. With exposure to .5 ng/mL of IL-13 alone for 18-20 hours, we can see

a small increase in levels of gene expression. However, when exposed to the same concentration of IL-13 and

increasing concentrations of IL-17A up to 10ng/mL, there is a positive concentration-dependent response on

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Epithelial Cells in Asthma Immune Response Kim, Emily
either the apical or basal side of the cell. This shows that mTECs have the ability to respond to both cytokines

as well as demonstrating supporting evidence for the exacerbation of IL-13-mediated responses by IL-17A.

Figure 1. Mouse Tracheal Epithelial Cells (mTECs) respond to IL-13 and IL-17A. Cultured and differentiated

mTECs were treated apically or basally with the indicated concentrations of (A) IL-13 or (B) IL-17A for ~20 hours.

Cells were collected from the membrane, total RNA was isolated and IL-13- or IL-17A-mediated gene expression

was analyzed by Real-Time PCR. The expression profiles of two repesentative genes are shown. Data shown

represent meanS.E.M. from one experiment with 3-4 replicates per treatment group. *p<0.05; **p<0.01;

***p<0.001.

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Epithelial Cells in Asthma Immune Response Kim, Emily

Figure 2. Mouse Tracheal Epithelial Cells (mTECs) increase IL-13-mediated gene expression with the

addition of IL-17A. Cultured and differentiated mTECs were treated apically or basally with 0.5ng/ml IL-13 and

0.25-10ng/ml IL-17A for ~20 hours. Cells were collected from the membrane; total RNA was isolated and IL-13-

mediated gene expression was analyzed by Real-Time PCR. The expression profiles of two repesentative genes

are shown. Data shown represent meanS.E.M. from one experiment with 3-4 replicates per treatment group.

*p<0.05; **p<0.01; ***p<0.001.

This differential response is quite intriguing because of the culturing conditions. Typically, treatment for

asthma only involves exposure of the apical side of the epithelial cells to corticosteroids, but now it is quite

evident that some responses facilitated by IL-17A are basolateral-specific. This could be due to a differential

number of receptors on either side of the cells due to the culturing method, where polarization of the cells was

promoted to form structures like cilia and goblet cells that secrete mucus on the apical side. This finding that the

basolateral sides of the airway epithelial cells have a great impact on asthma pathogenesis has great implications

for new targets for pharmaceuticals to treat steroid-refractory asthmatic patients. Although technically we have

not ruled out the possibility that cytokines could migrate through the membrane to the other side of the cells, if

this did happen, we would not expect to see such a differential response. In the future, experiments may be

completed to expose the mTECs to Th2 and Th17 cells instead of their respective cytokines. In addition, more

extensive analysis may be done through time courses and RNA-seq as well as by examining phosphorylation of

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Epithelial Cells in Asthma Immune Response Kim, Emily
STAT6 in the epithelial cells after exposure to cytokines through Western blots. Finally, the relationship between

IL-13, IL-17A, epithelial cells, and DCs may be examined.

Conclusion

In conclusion, asthma is a complex, chronic inflammatory lung disease that can be induced through

exposure to allergens, physical responses, infections, or genetic predispositions. In the humoral aspect of

immunity, it is defined by the presence of IgE antibodies, and cellularly, Th2 cells are known to secrete several

cytokines, including IL-13, which elicit physiological effects that lead to symptoms such as wheezing, tightness,

and breathlessness. The environmental and genetic factors alter the cytokinic communication between dendritic

cells and epithelial cells in the airway, and this balance in communication is essential to maintaining normal

airway function. Even though epithelial cells were not thought to play an active or immediate role in the allergic

asthma response in the past, it is evident that epithelial cells play a pertinent role in facilitating allergic asthmatic

reactions, including cases of steroid-refractory asthma characterized by the presence of both IL-13 and IL-17A

where typical treatments are not effective. Investigating the role of epithelial cells and the cytokines affecting

these cells may provide insight as to how to better treat allergic asthma, the most common non-communicable

disease in United States. A recent study has found that cytokinic exposure to mouse tracheal epithelial cells

leads to differential gene expression either apically or basally in the airway epithelium, an intriguing discovery

that could have implications on therapeutic targets for severe, steroid-refractory asthma that is currently not well

treated by common therapies.

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Epithelial Cells in Asthma Immune Response Kim, Emily
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Figure 3. The central role of epithelial cells in allergic asthma.

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