Phenotyping of asthma by multidimensional analysis: Focus on peripheral blood characteristics of severe asthma patients

In this thesis the activation state of blood eosinophils and neutrophils was discovered
to contribute significantly to the diagnostic accuracy of a quick and low-invasive test for
eosinophilia asthma. In patients with eosinophilic airway inflammation, blood eosinophils
are more refractory to stimulation with fMLF.

A multi-dimensional reduction approach based on Non-linear PCA reduced dimensions
of cellular and clinical parameters, making use of the multivariate advantage. Subsequent
Discriminant Analysis of PCA scores of the Utrecht asthma cohort lead to a prediction model
that was validated by the independently measured Oxford cohort of asthma patients. In
contrast to blood, sputum eosinophils and neutrophils were found to be overall highly
activated, independent of the asthma phenotype being eosinophilic or non-eosinophilic.
Hence, sputum eosinophil and neutrophil activation do not have diagnostic value for
asthma phenotypes.

In the subsequent two chapters, the MFC analysis method FLOOD was described. FLOOD
identifies and describes subpopulations of cells that are changed or mobilized as a result
of a stimulus. FLOOD showed its potential in the human LPS challenge study by clearly
visualizing two subpopulations in an unsupervised fashion that were already known to be
present but were not yet described in so much detail regarding specific receptor profile.
FLOOD also identified exercise-associated neutrophils in elite rowers after intense anaerobic
exercise. The exercise-associated neutrophils show similarities to banded cells appearing
after LPS challenge. Additional functional assays (e.g. bacterial killing capacity) will be
required to verify whether these neutrophils are in fact similar.

DAMACY, a second multivariate MFC analysis method, proved helpful in diagnosing asthma in
general and eosinophilic asthma specifically. Moreover, it identified an important unknown
subset of CD3+CD8+CRTH2+ cells, known to be Tc2 cells. In the last chapter of this thesis Tc2
cells are found to be present in higher numbers in peripheral blood and airways of patients
with severe eosinophilic asthma. In vitro assays show an activation response of Tc2 cells
by PGD2 and LTE4, both lipid mediators that were measured in higher concentrations in the
airways of patients with severe eosinophilic asthma. Therefore Tc2s might play an important
role in severe eosinophilic asthma and could be a promising target of therapy.

In summary, this thesis shows that a combination of detailed cellular measurements and
improved multi-dimensional and unbiased analysis methods of MFC data lead to the
development of promising diagnostic steps to further phenotype asthma with the ultimate
goal to improve asthma care.