T regulatory cells from people with asthma show a Th2-like
phenotype
Kirstin Jansen1, Oliver F. Wirz1,
Willem van de Veen1,2, Ge Tan1,3,
Milena Sokolowska1, Simon D.
Message4, Tatiana Kebadze4, Nicholas
Glanville4, Patrick Mallia4, Cezmi
A. Akdis1,2, Sebastian L. Johnston4,
Kari Nadeau5 and Mübeccel Akdis1*
1 Swiss Institute of Allergy and Asthma Research (SIAF), University of
Zurich, Davos, Switzerland.
2 Christine Kühne – Center for Allergy Research and Education
(CK-CARE), Davos, Switzerland.
3 Functional Genomics Center Zürich, ETH Zürich/University of Zürich,
Zürich, Switzerland.
4 National Heart and Lung Institute, Imperial College London, United
Kingdom.
5 Sean N. Parker Center for Allergy and Asthma Research, Department of
Medicine, Stanford University, Palo Alto, CA, USA.
* Corresponding author:
Mübeccel Akdis, MD, PhD.
Swiss Institute of Allergy and Asthma Research (SIAF)
Herman-Burchard-Strasse 9
CH-7265 Davos-Wolfgang, Switzerland
E-mail: akdism@siaf.uzh.ch
Tel.: +41 81 410 08 48
Declaration of fundingM. Akdis has received research support from the Swiss National Science
Foundation No. 320030-159870/310030-179428 and PREDICTA (No: 260895) and
the Sean N Parker Center for Allergy and Asthma Research at Stanford
University. C.A. Akdis is employed by the Swiss Institute of Allergy and
Asthma Research, University of Zurich; the Swiss National Science
Foundation No. 310030-156823, and the Christine Kühne – Center for
Allergy Research and Education (CK-CARE). M. Sokolowska received
research grant from the Swiss National Science Foundation No.
310030_189334/1 and from the GSK. The experimental infection study was
supported by a Medical Research Council Clinical Research Fellowship (to
S.D.M.), a British Medical Association H.C. Roscoe Fellowship (to
S.D.M.), British Lung Foundation/Severin Wunderman Family Foundation
Lung Research Program Grant P00/2, Asthma UK Grants 02/027 and 05/067,
Welcome Trust Grants 063717 and 083567/Z/07/Z for the Centre for
Respiratory Infection, Imperial College, and the National Institute for
Health Research (NIHR) Biomedical Research Center funding scheme. S. L.
Johnston is the Asthma UK Clinical Professor (grant CH11SJ), is an NIHR
Emeritus Senior Investigator and was supported by MRC Centre Grant
G1000758, Asthma UK Centre Grant AUK-BC-2015-01 and European Research
Council Advanced Grant 788575. K.C. Nadeau is supported by NIH grant U19
AI104209 (Asthma and Allergic Diseases Cooperative Research Center), U01
AI140498 and R01 AI140134 and the Naddisy Foundation.
To the editor,
Asthma is the most common chronic inflammatory disease of the lung,
characterised by wheezing, shortness of breath and variable airflow
obstruction. It is a heterogeneous disease that can be classified into
different endotypes of which T2-high - allergic asthma is one of the
most common forms, especially in children. Allergic asthma is
characterised by increased IgE and type-2 cytokines, including IL-5,
IL-4 and IL-131.Thus far, it is not completely
understood why these type-2 responses are poorly controlled in asthma. T
regulatory cells (Treg cells) are key mediators in controlling type 2
responses. However, under certain conditions, Treg cells can display a
pathogenic and proinflammatory phenotype and contribute to disease
pathogenesis2. Treg cells of food allergic children
showed a T helper 2 (Th2)-like phenotype. Whether this Th2-like
phenotype of Treg cells is also present in asthmatic individuals is
unknown.
Therefore, in this exploratory study, we compared the gene-expression
profile of Tregs from people with stable allergic-asthma to non-allergic
controls without asthma. We isolated PBMCs from 5 people with asthma and
4 controls (Table S1) and sorted Treg cells with flow cytometry
(CD3+CD4+D25hiCD127low).
Then, we isolated RNA from the sorted Treg cells and performed RNA-seq
(See Supplemental information for detailed methods). In total, 369 genes
were differentially expressed between Treg cells from asthmatic
individuals and controls (P<0.01) (Supplemental Figure 1). We
clustered the genes into different groups: Treg cell markers, cytokine
receptors, virus related, transcription factors, cytokines and others
(Figure 1A). Interestingly, we found that the expression of FOXP3was reduced in Treg cells from asthmatic individuals (Figure 1B). This
is in line with a previous study that observed a lower expression ofFOXP3 in Treg cells from individuals with
asthma3. Interestingly FOXP3 expression
inversely correlated with the IgE levels found in the serum (Figure 2A),
supporting the finding that Treg cells can suppress IgE
production4.
In addition, we found a significant upregulation of IL13 mRNA
expression and a trend to increased expression of IL4 andIL5 mRNAs in Tregs in asthma, indicating a Th2-like phenotype as
was reported in Tregs from children with food
allergies2. Furthermore, we found an upregulation of
the prostaglandin D2 receptor (PTGDR2 ) or CRTH2, in line with a
previous study that reported an increased amount of
CRTH2+ Tregs in asthma5.
Interestingly, several cytokine receptors were differentially expressed
between Tregs from asthmatic individuals compared to controls. The IL-4
receptor alpha transcript IL4RA was significantly reduced in
asthma. The expression of IL4RA also strongly correlated with the
levels of IgE in the serum (Figure 2A). Previously, it was shown in mice
that IL-4 receptor signalling is essential in controlling Th2 responses
and airway inflammation6. Our data suggest a similar
role of IL4RA in humans. Likewise, we observed a downregulation
of TNF receptor superfamily member 25 (TNFRSF25 ), which was shown
to contribute to preventing allergic lung
inflammation7 and downregulation of OX40
(TNFRSF4 ).
Additionally, we observed a difference in virus/type-I
interferon(IFN)-related genes in asthma, which was also observed in
single-cell transcriptomic data of allergen-specific Tregs from
individuals with asthma8. Curiously, the expression of
the type 1 IFN receptors IFNAR1/2 were lower expressed in asthma,
which could indicate a deficiency against respiratory viruses and
chronicity.
Lastly, we performed an enrichment analysis to see up or downregulation
of pathway maps, process networks and go processes with MetaCore (Table
1). The pathway maps and process networks included upregulation of
pathways related to immune functions already described. However, the
affected GO processes were mostly related to epigenetic mechanisms
including nucleosome organisation, nucleosome assembly and chromatin
organisation. With the tool STRING, we performed a pathway analysis that
showed a cluster of histone genes (Figure 2B). So far, there is no data
reporting the function of histone genes in Tregs or related to asthma,
but perhaps this finding could be related to changes in epigenetics. It
was reported that in asthma Tregs have increased CpG methylation of theFOPX3 locus compared to individuals without
asathma3.
In conclusion, Tregs from individuals with asthma show reduced
expression of several molecules related to Treg suppressive
functionality, while having increased expression of Th2-like
characteristics that could lead to their reduced control of allergic
airway inflammation. Further studies are needed to confirm these
findings in a larger population and investigate their contribution to
disease pathology.
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Figure 1: Tregs from asthmatic individuals show a distinct
phenotype compared to controls. (A) Genes that are significantly changed in Tregs cells from asthmatic
individuals compared to controls (log 2 ratio)– clustered in the
groups: Treg markers, cytokine receptors, virus related, transcription
factors, cytokines and others. (B) Fragments per kilo base per million
mapped reads (FPKM) values of genes of interest (FOXP3, IL13, IL5, IL4,
IL4R, PTGDR2, TNFRSF25, TNFRSF4, IFNAR1, IFNAR2) of all donors. N = 4
(healthy), 5 (asthma). *** p<0.001 , ** p<0.01, *
p<0.05
Figure 2: Phenotype of Tregs might be associated to Treg
function . (A) Correlation between expression of FOXP3 (left) and IL4RA
(right) with IgE serum levels. (B) Satellite plot showing a cluster of
known interactions related to nucleosome assembly. Genes higher
expressed in asthmatic individuals are shown in red, and lower
expression in blue.