UNRAVELLING long-term chronic morbidity (Wenzel et al.,

Thesis submitted for the Degree of Doctor of Philosophy
In Pharmacology (Pharmacy)
Registration No.: 0942 of 2012 – 2013

Asthma is a disease which has reached epidemic proportions affecting 334 million people of
all ages, ethnic groups and countries worldwide and it is the 14th most important disorder in the
World in terms of the extent and duration of disability (Network et al., 2014). It is estimated that
around 250,000 people die prematurely each year because of asthma (Bousquet et al., 2007).
However, due to geographical diversity, there is a considerable heterogeneity of asthma in terms
of gene-environment interactions, pathophysiological mechanisms, environmental exposures,
comorbidities, age, underlying disease severity, responsiveness of disease to therapy, and burden
of disease including asthma exacerbations and death as well as long-term chronic morbidity
(Wenzel et al., 2006). So, though all asthmatics manifest airflow limitation on spirometry, and
similar symptoms but they have a great deal of heterogeneity with respect to severity of airflow
limitation, degree of reversibility, therapeutic response, asthma triggers and long term outcomes
(Szefler et al., 2002, Martin et al., 2007). Cluster based multi-variate approach had been used to
analyze this heterogeneity at clinical, cellular and molecular levels which revealed that earlyonset
atopic asthma is the most common phenotype (Haldar et al., 2008). It is estimated that the
population-based proportion of asthma cases attributable to atopy is between 50%-60% (Pearse
et al., 1999; Arbes et al., 2007).
It is now accepted that environmental triggers can activate airway epithelial cells to initiate
allergic airway responses and asthma in susceptible children because they have pre-existing
atopy and specific genetic risk factors. Environmental triggers that can activate epithelial cells
include oxidants (cigarette smoke, vehicle exhaust), aeroallergens and microbial infections,
especially viruses (Sarinho et al., 2009; El-shariff et al., 2006). The aeroallergens of relevance to
asthma include pollen grains, house dust mite proteins, and proteins from furred animals which
are otherwise considered innocuous and should induce immune tolerance when inhaled.
The breach in immune tolerance that takes place in asthmatic airways is incompletely
understood. To understand the mechanism for allergic airway inflammation we need a
framework based on animal model and an aeroallergen such as pollen.
Approximately 50% of the asthmatic patients are sensitized to specific proteins present in the
pollen (Brozek, et al., 2010; Xiao et al., 2013; Gaur et al., 2007; Giolekas, et al., 2004). Local
and regional flora determines the type and concentration of the pollen grains which gets
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dispersed in the ambient air (Bush, et al., 1989; Mandal, et al., 2008; Spieksma, et al., 1989;
Banik, et al., 1990). This results in geographical variation in the prevalence of sensitization to
various pollen grain and the fluctuations in the incidence of respiratory allergy symptoms in the
sensitized individuals which parallels with the flowering season of the offending plant
(Bousquet, et al., 2008; Delgado, et al., 2013).
Alstonia scholaris an evergreen tropical tree of the Apocynaceae family (commonly known as
Indian devil tree or Chhatim) native to the Indian subcontinent, Southeast Asia and Australasia
(Hussain et al., 2014). This tree flowers in the month of September to November (Mondal, et al.,
1998). Respiratory allergy to Alstonia scholaris pollen is common in tropics and it is known to
cause asthma exacerbations, allergic rhinitis and atopic dermatitis during its flowering season. In
Eastern India it is known to initiate about 15-20% sensitization in respiratory allergic patients
(Banik, et al., 1992).
Study of allergic asthma requires a suitable animal model which will simulate the disease in
humans. Several animals like guinea pigs, mice, rats, sheep and dogs have been used as popular
models for asthma (Zosky, et al., 2007; Karol, et al., 1994). Over the past several years,
experimental induction of asthma using immune-based models of allergic asthma has increased
(Lloyd, et al., 2001). These models provide a better understanding of how and by what
mechanism, aeroallergens like pollen grains may contribute to the frequency and severity of
asthma attacks and to the exacerbation of allergic responses.
Of various animals used, rat in comparison to other animals demonstrates many features of
airway allergy and allergic asthma that are similar to those exhibited by humans (Pauluhn, et al.,
2001; Kucharewicz, et al., 2008; Tulic, et al., 2002).
During the past 3 decades much has been learned about the pathogenesis of allergen-induced
airway inflammation. Human allergic asthma involves an initial exposure to an allergen that
results in T-helper 2 (Th2) cell dependent stimulation of the immune response that mediates the
production of increased levels of IgE and cytokines like Interleukin 4, 5 and 13 (Kim, et al.,
2010; Oettgen, et al., 2001). IL-4 is the most potent Th2 polarizing factor (Ansel, 2006). There is
evidence in human asthma for an excess of CD4+Th2 lymphocytes in the airways (Robinson, et
al., 1992). The majority of secreted IgE is bound by high affinity IgE receptor on mast cells, and
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IgE-bound Fc?RI cross-linking by a specific antigen mediates the release of inflammatory
mediators by mast cells leading to the inflammatory response (Holgate, et al., 2008).
These inappropriate Type 2 immune responses in the lower airway are the central abnormality in
asthma. But the mechanisms of persistent type 2 immune responses in asthma are not well
understood. One of the mechanism could be a defect in the production of the interferons (IFN)
by the airway epithelial cells, as data from mouse models and epidemiologic studies suggest that
a deficiency of IFN signaling in airways leads to the development of asthma (Holtzman, et al.,
2012; Gavala, et al., 2011). But it is not yet established that a defect in IFN-dependent pathway
is a mechanism of asthma initiation or exacerbation.
Also recent data suggest that mechanisms involved in the regulation of the survival and apoptosis
of inflammatory cells may play a central role in the persistent inflammatory process
characterizing allergy and asthma. Apoptosis serves to control the “excess” of inflammatory
cells, limiting tissue damage, and ease the inflammation (Haslett, 1999). Several diseases, like
nasal polyps and rheumatoid arthritis, suggest that the chronicity of the disease is associated with
failure or delay of apoptosis in inflammatory cells and that these cells survive in inflammatory
regions (Galli, 2008; Simon, 2003). However, most studies focused so far on apoptosis of
eosinophils (Vignola, et al., 2000). T lymphocytes, especially Th2 cells, have a central role in the
regulation of the immune system.
In asthma, stimuli that promote repair mechanisms may act as a switch that induce cellular
proliferation and inhibit apoptosis. Prolonged survival of inflammatory cells may contribute to
the respiratory symptoms (Vignola, et al., 2000; Ramos-Barbón, et al., 2005). Selective
resistance to Fas-dependent apoptosis reflects altered Ag-driven, accessory cell-dependent
signalling and that ineffective activation of Fas signal transduction may contribute to Tcelldependent
immune inflammation in asthma (Jayaraman, et al., 1999). In normal airways,
vascular smooth muscle cells and bronchial epithelium express Fas, which undergoes apoptosis
upon Fas cross-linking with FasL, which is a general mechanism for bronchial homeostasis
(Ramos-Barbón, et al., 2005). The ratio of anti-apoptotic proteins vs. pro-apoptotic proteins is
important in determining the resistance of a cell to apoptosis (Susin, et al., 2000). The severity of
asthma is also inversely correlated with the apoptosis of the eosinophils in the airways (Simon,
2003). The excess lymphocytic burden after an inflammatory response is controlled by apoptosis.
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This mechanism seems to be impaired in asthmatic subjects. The role of T lymphocyte apoptosis
in allergic diseases has not been defined.
Another putative mechanism for the breach in immune tolerance and persistence of inflammatory
response in the airways is compromised T regulatory cell (Treg) function (Karagiannidis, et al.,
2004; Ling, et al., 2004; Karlsson, et al., 2004). A breakthrough in our understanding of Tregs
occurred with the discovery of the intracellular transcription factor forkhead box P3 (Foxp3)
which is a master control gene in the development and function of Tregs that act as a repressor of
transcription and regulator of T cell activation (Lim, et al., 2006, Fontenot, et al., 2003; Schubert,
et al., 2001). Tregs may induce peripheral tolerance through direct interactions with antigen
presenting cells and other lymphocytes through its various cell surface molecules (Ozdemir, et
al., 2009; Oderup, et al., 2006; Liao, et al., 2010; Takeda, et al., 2004; Diaglio, et al., 2007).
Another possible mechanism is that Tregs characteristically secrete IL-10 and TGF? and these
cytokines have multiple activities relevant to tolerance, including synthesis of non-inflammatory
IgG4 and IgA isotypes and regulatory effects on T cells and dendritic cells (Yadav, et al., 2013).
Given that Tregs play a vital role in the control of airway inflammation, there is an urgent need
to understand both the cellular and molecular mechanisms contributing to Treg homeostasis in
allergic asthma. Unlike effector T cells, Tregs are considered to be anergic. However, Tregs
undergo expansion and vigorous proliferation, particularly in a lymphopenic host, which
suggests that these cells are active under such circumstances (Annacker, et al., 2001). Though it
is known that Treg mediated suppression is reduced in asthma and treatment improves Treg
suppression of effector cells (Wing, et al., 2006), but it is not known whether the presence of low
numbers of Treg cells at the site of inflammation could be due to increased rates of apoptosis of
these cells, or to downregulation of their characteristic CD25 expression (Gavin, et al., 2002;
Banz, et al., 2002). So, though the central role of Treg cells in controlling immune responses is
well recognized in human diseases but there is still paucity of specific evidences for Treg cell
dysfunction in allergic asthma and that too for tropical aeroallergens like the pollen of Alstonia
Control of a disease requires the availability of effective treatment modalities. Though the
available pharmacologic therapies for allergic asthma are effective in reducing and preventing
symptoms development, but none of them alter the progression of this disease. Allergen specific
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immunotherapy (SIT) is the only treatment that has been shown to improve allergic dysfunction
and to re-direct the immune system away from the allergic response (Bousquet, et al., 1994). But
the specific immunologic mechanism(s) by which immunotherapy achieves its effects is still not
completely understood. Various theories for the immunological mechanisms underlying this
treatment have been proposed like, shifting of Th2 dominated allergen specific immune
responses towards a Th1 response, induction of allergen specific T-cell tolerance and increased
or restored Tregs function (Akdis, et al., 2007; Larche, et al., 2006; Wing, et al., 2006; Zhang, et
al., 2014). The mechanism which is important in various different clinical conditions needs to be
studied. Furthermore, though epicutaneous SIT is a clinically effective treatment, it has been
shown that IgE-mediated allergen uptake by mucosal APCs may lead to better
immunomodulation (Allam, et al., 2010; Dahl, et al., 2008). So, the best route for specific
allergen immunotherapy with an aeroallergen also needs to be investigated.
Considering the above scenario, the present pre-clinical study was conceived to develop a novel
sensitization and challenge model of allergic asthma in rats with a clinically important aeroallergen
of tropical region i.e. Alstonia scholaris, and to unravel the molecular mechanisms of
allergic airway inflammation along with the study of effector T cell and also regulatory T cell
apoptosis. This study further envisages to use this model in studying the immunologic
mechanism behind effective allergen specific immunotherapy (SIT) in allergic asthma and to
evaluate the difference (if any) in the immunological response between two routes of
immunotherapy vis-a-vis intranasal and intraperitoneal.
The present study thus for the first time offers a multi-targeted approach towards various
pathways of apoptosis of splenic T lymphocytes by specific allergen immunotherapy and also
attenuation of airway allergy by the generation of CD4+CD25+Foxp3+T cells and other subsets
of Treg cells like Tr1 cells, Th3 cells. It also emphasizes Treg sensitivity to apoptosis by
intranasal immunotherapy in the same animal model. The present investigation will thus help to
chart out newer molecular targets for treating allergic asthma or rhinitis.

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