The Origins of Pediatric Respiratory diseases: laying groundwork for genetic, cellular and immunological analyses. (OPeRA)

Many respiratory diseases, such as asthma and cystic fibrosis, as well as low
lung function, which predisposes to COPD, arise in childhood. These diseases
are the outcome of the interaction of a genetically susceptible host with a
permissive environment. Most lung diseases can therefore be conceived as a
developmental disease, as these arise in a child with a developing lung and
immune system. Therefore, to identify the early mechanisms underlying disease
development, we will need to study children instead of adults, investigate
cells from the bronchial airways, and develop models that reflect bronchial
epithelial cell function. However, mechanistic, invasive studies in young
children with respiratory diseases are limited by ethical constraints.
In Erasmus MC - Sophia Children*s Hospital yearly 100-150 flexible
bronchoscopies are performed in children for various clinical indications. In
general during bronchoscopy a bronchoalveolar lavage (BAL) will be performed
and BAL fluid (BALF) will be cultured (bacteria, fungi, yeast) and analysed for
cells and lipid laden macrophages. Excessive BALF and/or leftover BALF after
these examinations is lost. In selected cases (in particular in asthma or
asthmatic symptoms) endobronchial biopsies are taken during bronchoscopy for
analysis of the reticular basement membrane thickness and inflammatory cells.
The bronchial epithelial cell is a key cell orchestrating the response of the
airways and the immune system, and bronchial epithelial cells express many
respiratory disease genes.
We hypothesize that curative, early childhood treatment for lung disease needs
to be based on detailed, mechanistic insights into the inception of disease in
the bronchial epithelium rather than established disease in adults. In contrast
to the bronchial epithelium, the nasal epithelium is accessible in young
children. However, it is not known how well the upper airway reflects the lower
airways in children with different disease conditions

The main objective of this proposal is to establish ex-vivo models of bronchial
epithelial cells from children. Use of (1) epithelial organoid cultures derived
from bronchial or nasal epithelial cells; and of (2) bronchial brushings; (3)
nasal brushings and (4) bronchoalveolar lavage fluid (BALF) allows us to study
epithelial cell function at the genetic, cellular and immunological level.
These models will be validated by comparison of gene expression from epithelial
cells of these three models to the gold standard: bronchial (single cell) gene
expression in biopsies of bronchial airway wall. Furthermore, this will enable
future mechanistic and intervention studies, as we will know which aspects of
the bronchial epithelium will be reflected in nasal cells, and which are
retained in epithelial organoid cell culture.
Another primary objective is to establish a biobank of bronchial biopsies,
bronchial and nasal epithelial cells and BALF of children with different
respiratory diseases.

This study will be an observational, cross-sectional cohort study that will
describe characteristics of developing pulmonary disease, including asthma,
severe wheeze and cystic fibrosis.
Children that will undergo a bronchoscopy based on clinical indications during
office hours will be asked to participate. With an estimated inclusion of 20
children per year, the study duration will be four years. The total inclusion
will be 80 children. The study will take place in Erasmus MC - Sophia
Children*s Hospital from 1 October 2022 to 30 October 2026.

We will obtain the following materials:
- 4 bronchial biopsies, processed as single cell suspension for RNA-seq.
- 3 bronchial brushes (2 for epithelial organoid culture, 1 for RNA isolation)
- 3 nasal brushes (2 for epithelial organoid culture, 1 for RNA isolation)
- Excess BALF will be stored
- 1 blood sample (total amount of 10ml) for peripheral blood mononuclear
cells.

Children will undergo:
- lung function assessments, if not performed < 12 weeks of bronchoscopy for
clinical reasons.

Baseline data on the clinical symptoms, environmental exposures, treatment, and
medical history will be retrieved from the patient files.

Nature and extent of the burden and risks associated with participation,
benefit and group relatedness: Ethical concerns are highly relevant when
performing invasive research in children. We propose to perform research in
(young) children, who already undergo bronchoscopy for clinical indications,
where a BAL is part of the routine investigations. We aim to store excessive
BALF for future research; this BALF is now lost after culturing and pathology
analysis. A bronchial biopsy, bronchial and nasal brushes and 1 blood sample
will be added as extra handlings for the study. This adds no extra risks to the
procedure. During bronchoscopy the major risk is that of anesthetics. It has
been well documented that risks related to a bronchoscopy itself are limited to
the incidental need for bronchodilators, minor bleeding that always stops
spontaneously, and fever. The main ethical aspect of our project therefore is
obtaining (additional) primary bronchial epithelial and nasal cells for
research purposes, which will prolong the planned bronchoscopy with
approximately 10 minutes. The benefits of this research will relate to the
validation of less or non-invasive methods to study airway epithelial cells
from children, which may reduce the need for future invasive studies and will
allow mechanistic studies into disease inception.