PATH3205 Topic C: Asthma Research Laboratory
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Introduction
Asthma can be described as a chronic airway inflammatory disorder, with episodes of
acute inflammation, often referred to as ‘exacerbations’ (Global Initiative for Asthma,
2015). An exact method of diagnosing asthma, and its underlying aetiology however,
remains elusive. Of immediate concern are these asthma exacerbations, which in terms
of costs is the most significant contributor to direct health-care expenditure (Dougherty
& Fahy, 2009).
Asthma incidence and exacerbation has been generally agreed to be higher in
metropolitan areas (Gem, 2010), however other recent studies have failed to find
significantly higher asthma risk and incidence in metropolitan areas (Keet et al. 2015).
Thus we need to determine exactly what it is, in the environment that exacerbates
asthma. A few studies (Tecer, Alagha, Karaca, Tuncel & Eldes, 2008; Lin, Chen, Burnett,
Villeneuve & Krewski, 2002) have explored the effects of particulate matter of various
sizes, including those <10µm aerodynamic diameter (PM10) as is used in this
experiment, and found a positive correlation between concentration of ambient
particulate matter and incidence of asthma exacerbations, measured by number of
hospitalisations.
This experiment parallels that outlined in the method of “Ambient particulate matter
induces an exacerbation of airway inflammation in experimental asthma: role of
interleukin-33” by Shadie, Herbert & Kumar, 2014. We hypothesised and found that
ambient particulate matter exacerbates airway inflammation in asthma. More
specifically, that ambient (PM10) particulate matter induces acute exacerbation of
airway inflammation in the BALB/c asthma mouse model. Furthermore, we suspected
and found evidence to support that Interleukin-33 (IL-33) is a key player in PM10
induced asthma exacerbation development, as was suggested in previous studies
(Oboki, Nakae, Matsumoto & Saito, 2011), in the decreased level of inflammation of antiIL-33 antibody treated, chronically challenged mice in comparison to the non-treated
mice.
The aim of this experiment is to determine whether Sydney’s ambient (PM10)
particulate pollutants can drive acute inflammation of airways in asthma, as measured
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PATH3205 Topic C: Asthma Research Laboratory
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by the level of TNF-alpha expressed and histologically assessed inflammation. That is,
the number of eosinophils, a hallmark of asthmatic airway inflammation (Oboki et al.,
2011). The administration of anti-IL-33 antibodies was also performed here, to
determine the effect on level of inflammation induced, as asthma exacerbation.
Materials and Methods
The BALB/c mouse model for asthma was used, which was a previously described,
effective model of asthma (Temelkovski, Hogan, Shepherd, Foster & Kumar, 1998). The
weanling mice were systemically sensitized to ovalbumin, as was done in Herbert et al.,
2012. The features of chronic asthma were induced by challenging the mice with
aerosolized OVA over 4 weeks.
Four experimental groups (eight mice each), were utilized: naïve mice, chronically
challenged, chronically challenged + Sydney PM10, and chronically challenged + Sydney
PM10 +IL-33 neutralising antibody. The Sydney PM10 particulate matter was
intranasally administered. Inflammation was assessed histologically and in terms of
TNF-alpha levels in bronchoalveolar lavage (BAL) fluid, measured using ELISA.
Results
Histologically, it was found that the presence of eosinophils were not elevated in
chronically challenged only mice. In those who were chronically challenged, and treated
with Sydney PM10, number of eosinophils were significantly elevated, consistent with
asthma exacerbation. In the group of mice, chronically challenged, treated with Sydney
PM10 and also treated with anti-IL-33 antibodies, eosinophil infiltration was not as high
as in those without anti-IL-33 antibodies.
Quantitatively, we found that TNF-alpha levels in the chronic challenged mice were
slightly elevated (~122pg/ml) compared to the naïve mice (~108pg/ml). The chronic
challenged mice treated with Sydney PM10 exhibited much higher levels (~468pg/ml),
and those also treated with anti-IL-33 antibodies exhibited slightly lower levels at
(~275pg/ml) (see Figure 1).
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PATH3205 Topic C: Asthma Research Laboratory
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Concentration of TNF-alpha (pg/ml)
TNF-alpha in bronchoalveolar lavage fluid
from mice
Naïve mice
600
500
Chronic challenged mice
400
300
Chronic challenged plus
SydPM10 mice
200
Chronic challenged plus
SydPM10 + anti-IL-33 antibody
mice
100
0
Mice Groups
Figure 1 – Concentration of TNF-alpha in bronchoalveolar lavage fluid for
experimental groups, as measured using ELISA.
From the data used to construct Figure 1, the difference between the naïve and chronic
challenged plus Sydney PM10 mice is not significant (p>0.05). The difference between
the naïve and chronic challenged plus Sydney PM10 mice ± anti-IL-33 antibody are both
significant (P<0.0001). Similarly, the difference between the chronic challenged mice
and the chronic challenged plus Sydney PM10 mice ± anti-IL-33 antibody are both
significant (P<0.0001). Finally the difference between the chronic challenged plus
Sydney PM10 mice and the chronic challenged plus Sydney PM10 + anti-IL-33 antibody
is also significant (P<0.0001). (Significance calculated using unpaired t-test)
Thus results of this experiment demonstrate a significant difference in inflammation
levels between the naïve/chronic challenged mice and the chronic challenged plus
Sydney PM10 mice ± anti-IL-33 antibody mice, as well as between the chronic
challenged plus Sydney PM10 mice, and the chronic challenged mice plus Sydney PM10
+ anti-IL-33 antibody mice.
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Discussion
In this report, we determined that Sydney’s ambient (PM10) particulate matter can
drive acute inflammation of airways in asthma, as measured by the level of TNF-alpha
expressed and histologically assessed inflammation. The difference in TNF-alpha levels
in BAL, between the chronically challenged mice and the chronically challenged mice
treated with Sydney PM10 were found to be statistically significant, as consistent with
the asthma exacerbation.
To better quantify the effect of the treatments investigated in this experiment, more
control groups would be desirable. In particular, groups of naïve mice given intranasal
Sydney PM10 (50 µg/ml) ± anti-IL-33 antibody treated, naïve anti-IL-33 treated and
chronically challenged, anti-IL-33 treated mice. These would allow factoring in of the
treatment’s baseline effects, thus pinpointing with a greater degree of certainty the
source of TNF-alpha level differences.
The administration of anti-IL-33 antibodies performed here, had a suppressive effect on
the level of inflammation induced (decreased concentration of TNF-alpha), as can be
seen in Figure 1. This suggests that IL-33 indeed plays a key role in inducing acute
asthma exacerbation as its deficiency resulted in decreased levels of TNF-alpha
expression. As such, the supposition that IL-33 neutralisation may be a useful treatment
option for asthma exacerbation, is supported by this experiment. Other recent studies
such as those by Oboki, et al., 2011, Lee et al., 2014, and Gallelli, Busceti, Vatrella,
Maselli & Pelaia, 2013, likewise has delineated that IL-33 levels are elevated in asthma
exacerbation, and indicating its potential for a future asthma exacerbation treatment
target.
Further study needs to be done to verify the potential of anti-IL-33 antibodies as
treatment. The exploration of drug interactions, systemic effects in humans, and
effectiveness in the real world needs to be investigated.
In conclusion, in the controlled setting of a mouse asthma model, we have shown
ambient particulate matter can induce inflammation of the airway, similar to asthma
exacerbation, with IL-33 playing a key role as a mediator of inflammation.
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Reference:
Dougherty, R., & Fahy, J. (2009). Acute exacerbations of asthma: epidemiology, biology
and the exacerbation-prone phenotype. Clinical & Experimental Allergy, 39(2),
193-202. doi:10.1111/j.1365-2222.2008.03157.x
Gallelli, L., Busceti, M., Vatrella, A., Maselli, R., & Pelaia, G. (2013). Update on
Anticytokine Treatment for Asthma. Biomedical Research International, 2013, 110. doi:10.1155/2013/104315
Global Initiative for Asthma. (2015). Pocket Guide for Asthma Management and
Prevention (for adults and children older than 5 years) (p. 5). Global Initiative for
Asthma. Retrieved from
http://www.ginasthma.org/local/uploads/files/GINA_Pocket_2015.pdf
Keet, C., McCormack, M., Pollack, C., Peng, R., McGowan, E., & Matsui, E. (2015).
Neighborhood poverty, urban residence, race/ethnicity, and asthma:
Rethinking the inner-city asthma epidemic. Journal Of Allergy And Clinical
Immunology, 135(3), 655-662. doi:10.1016/j.jaci.2014.11.022
Lee, H., Rhee, C., Kang, J., Byun, J., Choi, J., & Kim, S. et al. (2014). Blockade of IL-33/ST2
ameliorates airway inflammation in a murine model of allergic asthma.
Experimental Lung Research, 40(2), 66-76.
doi:10.3109/01902148.2013.870261
Lin, M., Chen, Y., Burnett, R., Villeneuve, P., & Krewski, D. (2002). The Influence of
Ambient Coarse Particulate Matter on Asthma Hospitalization in Children:
Case-Crossover and Time-Series Analyses. Environmental Health
Perspectives, 110(6), 575-581. doi:10.1289/ehp.02110575
Oboki, K., Nakae, S., Matsumoto, K., & Saito, H. (2011). IL-33 and Airway
Inflammation. Allergy, Asthma and Immunology Research, 3(2), 81.
doi:10.4168/aair.2011.3.2.81
Shadie, A., Herbert, C., & Kumar, R. (2014). Ambient particulate matter induces an
exacerbation of airway inflammation in experimental asthma: role of
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interleukin-33. Clinical and Experimental Immunology, 177(2), 491-499.
doi:10.1111/cei.12348
Tecer, L., Alagha, O., Karaca, F., Tuncel, G., & Eldes, N. (2008). Particulate Matter (PM 2.5
, PM 10-2.5 , and PM 10) and Children's Hospital Admissions for Asthma and
Respiratory Diseases: A Bidirectional Case-Crossover Study. Journal Of
Toxicology And Environmental Health, Part A, 71(8), 512-520.
doi:10.1080/15287390801907459
Temelkovski, J., Hogan, S., Shepherd, D., Foster, P., & Kumar, R. (1998). An improved
murine model of asthma: selective airway inflammation, epithelial lesions and
increased methacholine responsiveness following chronic exposure to
aerosolised allergen. Thorax, 53(10), 849-856. doi:10.1136/thx.53.10.849
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Reflection
Aside from the obvious learning of new knowledge, overall, I was glad we were given
the opportunity to both learn from and speak to a variety of researchers from different
areas of medical science. Given that I intend on doing honours next year, I feel that this
course has provided me with valuable insight into the common laboratory techniques
employed, a number of different research areas and potential supervisors, as well as
encouraging the development of independent and critical thinking skills.
Regarding the asthma topic, I felt that the research laboratory was particularly valuable.
Prior to doing it, I was not specifically interested in asthma despite its prevalence in our
society. Afterwards, having seen and better understood the difficulties faced in research
of this disease, asthma seems a more captivating disease, full of variables and potential.
I especially appreciated the way this lab was conducted, with a more group-oriented,
conversational brainstorm approach to the question: “Can exposure to particulate
pollutants drive exacerbations of asthma?” with some guidance from the tutors. It was
interesting the way different groups of students all thought of different methods of
investigating the same research question. I felt the lab in this way, more effectively
nurtured our critical thinking skills and encouraged us to think on our own.
All in all, I did not particularly feel that this course developed my teamwork skills as
much as it facilitated its maintenance. However, with the group presentation, I know it
necessitated my further developing my presentation skills with the seemingly
mammoth task of a 10minute presentation. Prior to this, speeches extended to an
average of a mere 5 minutes. I felt the more scientific style and question/answer format,
also shaped us to be better prepared for the style of honours and beyond.
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