The role of indoleamine 2,3-dioxygenase in virus-induced asthma exacerbations

Virus-induced exacerbations and the concomitant enhanced inflammatory responses
present the major clinical manifestation of asthma but is poorly understood
from a mechanistic point of view. This hampers the development of adequate
prophylaxis and treatment of asthma exacerbations. Recent ex vivo studies have
implicated a defective interferon (IFN)-beta and IFN-lambda production by
airway epithelial cells and BALF cells in response to rhinovirus (RV) infection
resulting in deficient apoptosis of virus-infected cells in asthma. There is as
yet no in vivo data corroborating these findings, nor is known by which
mechamism apoptosis is induced. Indoleamine 2,3-dioxygenase (IDO) is a key
anti-viral protein. In vitro and in vivo studies have revealed that IDO
expression limits viral replication and inflammatory responses by promoting
apoptosis of virus-infected cells and inflammatory cells (T cells and
granulocytes), respectively. Interestingly, IDO expression is induced by Th1
cytokines (IFN-gamma) and reduced by Th2 cytokines (IL-4). As Th2 cytokines
are relatively over-represented in airways of patients with asthma, we
hypothesize that virus-induced IDO expression is reduced relative to that in
healthy individuals and therefore viral infection and the inflammatory response
are resolved to a lesser degree in asthma.

With this project we aim to clarify the differences in airway pathophysiology
between asthmatics and healthy controls that underlie the prolonged survival of
virus and the exaggerated inflammation in virus-induced exacerbations of
asthma. We will study apoptosis (acting as antiviral mechanism and as a
mechanism resolving inflammation) in an experimental rhinovirus infection model
in asthmatics and healthy controls in vivo.

Specific questions:
1) Is IDO expression and/or IDO activity impaired during rhinovirus infection
in the airways of asthma patients versus healthy subjects and does it correlate
with viral replication, T cell responses, eosinophil and neutrophil numbers and
inflammatory mediator production?
2) Is rhinovirus infection in asthma patients associated with reduced apoptosis
of airway epithelial cells, and of inflammatory cells, compared to that in
healthy subjects?
3) Is IDO expression/activity is associated with clinical outcome after
rhinovirus infection (asthma symptoms, spirometry, hyperresponsiveness)?

The answers to these specific question may help us to identify novel targets
for therapeutic intervention.

Healthy individuals and intermittant to mild persistent allergic asthma
patients will be experimentally infected with rhinovirus type 16 (RV16). The
procedure of RV16 exposure will be performed at day 0. Two days prior to
infection (day -2) the volunteers will undergo a bronchoscopy to obtain
bronchoalveolar lavage fluid (BALF) and four epithelial brushes. The collected
materials will be used to determine baseline levels of the primary study
parameters. The bronchoscopy, including BALF and the epithelial brush, will be
repeated on day 6 after RV16 exposure (RV16). In addition, lungfunctiontests
(FEV1 and PC20histamine) will be performed on day 4 after RV16 exposure
(baseline FEV1 and PC20histamine have been determined at the screeningsvisit).
Six weeks after RV16 exposure, blood will be drawn to confirm infection on
basis of neutralizing antibodies.

Prior to this study we will perform a pilot study (5 patients in each group)
using low-dose RV16, which was successfully used in a study-cohort of COPD
patients. Based on the results of this pilot study, we will decide whether we
use this low-dose RV16 or the normal RV16 dose that has successfully been used
in previous studies with allergic asthma patients. Based on inflammatory
mediator production in previous studies, we calculated that 14 individuals in
each group is sufficient for our study. Anticipating on a drop-out rate of 15%,
we need to include 16 individuals per group. We therefore need to recruit 5
(pilot study) + 16 individuals = 21 individuals per group.

After anamnesis and a physical examination, individuals will be subjected to a
lungfunction test, 10 ml of blood will be drawn and a skin-prick test will be
performed, which are considered to be a mild burden. Bronchoscopy, used to
obtain BAL fluid and epithelial brushes, is an invasive technique that, despite
the use of the anaesthetic lidocain, inflicts an unpleasant feeling and thus a
considerable burden to the individual. A bronchoscopy may give rise to a dry
cough and some distress to the nose, where the bronchoscope is inserted. During
brushing a superficial bleeding may develop which normally stops rapidly. The
bronchoscopy will take about 15 minutes to complete.

Experimental RV16 infections will cause mild commom-cold symptoms in both
healthy individuals and stable allergic asthma patients. RV16 infection will
evoke a transient exacerbation of asthma symptoms. The RV16 infection protocol
is a standard procedure to challenge healthy individuals, intermittent and mild
persistent (allergic) asthmatics and COPD patients. The rationale for using
RV16 is that this rhinovirus strain causes mild common-cold symptoms as
compared to other rhinovirus strains. In addition, rhinoviruses are endemic,
causing common colds in the general population. No adverse events of using
RV16 inoculation in healthy indivuals and patients (asthma and COPD) have been
reported. Exposure to RV16 will cause considerable burden that will last for
several days (up to a week).