Responsivity and reproducibility of messenger and micro RNA airway inflammatory markers - a pilot study

Progressive airway inflammation and remodelling represent the main underlying
features in the pathogenesis of chronic obstructive pulmonary disease (COPD)
(Wessler & Kirkpatrick, 2008). Changes of pulmonary function represent the
mainstream outcome measure regarding the assessment of response to treatment in
COPD in daily clinical practice, but it reflects only poorly the underlying
pathology as well as the burden to the patient. Therefore, there is an
increased interest to identify sensitive airway biomarkers in order to evaluate
the potential and efficacy of anti-inflammatory and *remodelling therapeutic
interventions (Singh, Edwards, Tal-Singer, & Rennard, 2010; Kistemaker, Oenema,
Meurs, & Gosens, 2012).

Since the respiratory tract extends from the nose to the lungs, there are
several procedures to analyse inflammatory processes of the airway mucosa,
which might contribute to identify such airway biomarkers.

Because the inflammatory process among others leads to an accumulation of
inflammatory mucous in the lumen, the analysis of sputum represents a
non-invasive method, which allows the objective assessment of response to
treatment and disease activity in the lower airways (Hogg et al., 2004). It has
been repeatedly shown that COPD patients exhibit increased numbers of
inflammatory cells, as well as increased concentrations of various inflammatory
markers in induced sputum (O*Donnell et al., 2004; Rutgers et al., 2000; Singh
et al., 2010). However, the reproducibility and responsivity of cytokine
measurements in sputum exhibit a number of limitations. Several factors
contribute to this, including the inability to cough up spontaneous sputum in
some patients, variable dilution due to inducing sputum by nebulized saline
solutions, variable concentration effects of treatment such as
anticholinergics, and the dissolution of disulfide bridges in many cytokine
proteins upon DTT pretreatment needed to dissolve the sticky sputum. These
problems have provided a major contribution to two studies failing to
demonstrate consistent associations between reduction of COPD exacerbations and
cytokine concentrations in induced sputum of patients who were treated with
anticholinergics (Powrie et al., 2007; Perng et al., 2009). In both studies
methodological problems in the determination of protein levels and in variable
dilution were a major drawback.

It has been shown that airway inflammatory responses can be found at different
airway epithelial sites, including the nasal mucosa. It has been shown that
cytokine responses in the nasal epithelium might be used as suitable surrogate
for epithelial cells of the lower airways in patients with airway inflammatory
diseases (Comer, Elborn, & Ennis, 2012; Huang et al., 2016; Zhang et al.,
2010). However, scientific knowledge is still limited regarding the
relationship of cytokine gene expression between nasal and lower airway
epithelium and requires further studies for validation.

In the light of prospective COPD interventional studies it is necessary to
identify first stable airway biomarkers that overcome microbiological
limitations of measuring cytokine concentrations in order to study possible
anti-inflammatory and *remodelling effects of therapeutic interventions, later
on. A promising approach is the analysis of m (messenger) RNA as an
inflammatory marker. To our knowledge, there is only little data regarding the
analysis of cytokine mRNA expression induced sputum as well as nasal epithelium
in COPD patients. Even less information is available regarding mi (micro) RNA.

We aim to investigate cytokine mRNA and miRNA levels of IL-6, IL-8, IL-17,
TNF-alpha, MCP-1, MIP-1 beta, ECP and TGF-beta expression regarding their
reproducibility and responsivity in induced sputum and nasal mucosa in COPD
patients in order to assess their potential as an objective outcome measure.

Primary objectives:
- Determination of the reproducibility and responsivity of mRNA levels of IL-6,
IL-8, TNF-alpha, MCP-1, MIP-1 beta and TGF-beta as inflammatory markers in
induced sputum

- Determination of the reproducibility and responsivity of mRNA and miRNA
levels of IL-6, IL-8, IL-17, TNF-alpha, MCP-1, MIP-1 beta and TGF-beta as
inflammatory markers in nasal epithelium.



Secondary objectives:
Analyzes of the measurement characteristics of inflammation cell profiles, LTB4
and protein levels of IL-6, IL-8, TNF-alpha, MCP-1, MIP-1 beta, ECP and
TGF-beta in induced sputum.

This is a prospective pilot study that will be conducted across seven weeks.
Subjects with an initial COPD exacerbation will be recruited and followed for
seven weeks. At three moments (3-4 days after the start of acute COPD
exacerbation, after 42 days and after 44-51 days) sputum as well as nasal
mucosa samples will be collected by sputum induction, as well as nasal brushes,
respectively.

Burden and risk associated with participation:
Sputum induction includes the inhalation of nebulised sterile hypertonic saline
solution and can lead to transient bronchoconstriction. This can lead to
coughing and shortness of breath. Participants will be previously informed
about the potential side effect and pretreated with inhaled salbutamol and
monitored during the process of sputum induction.

Nasal swab collections have the potential to irritate the intranasal cavity and
lead to acute epistaxis; however the risks associated with discomforts from
such events are minimal.