Results
PD-L1 is induced in blood cells of pre-school asthmatic
children with a virus-induced asthma phenotype and associated with the
presence of rhinovirus in their
airways
We recently described that acute in vitro infection of peripheral
blood mononuclear cells from preschool children with and without asthma
with rhinovirus, a single stranded RNA picornavirus, is associated with
the upregulation of Interferon regulated genes like STAT1, STAT2 and
Interferon Regulatory Factor (IRF) 1 (Bergauer et al., 2017b; a).
Moreover, paradoxically, IFNγ upregulates also PD-L1, a factor involved
in silencing/exhausting of activated T cells by ligating PD1 on the
surface of T cells (Mandai et al., 2016). Consistently, we found that
acute rhinovirus infection ex vivo induced PD-L1 and CTLA4 in the
PBMCs of asthmatic children (Bielor et al., 2017). We thus wanted to
follow up these in vitro observations in the two cohorts of our
study and analyzed 21 control children and 24 children with asthma(Fig. 1a) . The clinical data of these cohorts of children were
recently reported (Bergauer et al., 2017b; Bielor et al., 2017;
Hentschke et al., 2017) and are summarized in Table 1 andTable 2 . By looking at the PD-L1 mRNA expression in blood, we
found that PD-L1 mRNA expression was induced in children with a
virus-induced asthma phenotype (in accordance to PRACTALL guidelines
2008 (Bacharier et al., 2008)) compared to healthy control children(Fig. 1b) . Children with this asthma phenotype shows symptom
free periods, whereas the most common precipitating factor are colds by
respiratory viruses, like humane rhinovirus (Bacharier et al., 2008).
Furthermore, by trend, we observed an induction of PD-L1 mRNA in the
blood cells of asthmatic children as compared to control children(Fig. 1c) . We next analyzed PD-L1 expression after allergen and
rhinovirus challenge. Considering the presence of rhinovirus (+RV) in
the airways, we found that, by trend, asthmatic children with rhinovirus
in the airways, have an increased PD-L1 mRNA expression in total blood
cells (Fig. S1a). This is also associated with increased
expression of the Low density lipoprotein receptor (LDLR) (Fig.
S1b), which is one of the main receptors used by the viruses,
especially for RV1b, to entering the cells.
PD-L1 is upregulated in blood cells of asthmatic children
with increased
bronchoconstriction
We then asked if the lung function, especially the FEV1% as well as the
PEF% (peak expiratory flow, predicted), of the cohorts would correlate
with increased PD-L1 expression in blood. The FEV1 (Forced expiratory
volume in 1 second) / FVC (Forced vital capacity) ratio (FEV1%), is a
calculated ratio used in the diagnosis of obstructive and restrictive
lung disease. It represents the proportion of a person’s vital capacity
that they are able to expire in the first second of forced expiration
(FEV1) to the full, forced vital capacity (FVC). The result of this
ratio is expressed as FEV1% (Swanney et al., 2008). Lower values of
FEV1% represent airway obstruction. In our cohort of children with
asthma, but not in control children, we found a PD-L1 induction in
children with higher bronchoconstriction (Fig. 1d) and an
inverse correlation between PD-L1 and FEV1% (Fig. 1e) ,
indicating that worse asthma is associated with induction of PD-L1 mRNA
in blood cells of children with asthma. We then further investigated the
role of another lung function parameter, the PEF% (peak expiratory
flow) value (Fig. 1f, g) . The PEF% is defined as the largest
expiratory flow, which is achieved with a maximum forced effort after
maximum inspiration and is used as a control parameter during asthma
therapy. Similarly to the FEV1% we found a significant PD-L1 induction
in children with worse asthma (Fig 1f) as well as an inverse
correlation between PD-L1 expression and the PEF% (Fig 1g) . We
also found that increased PD-L1 mRNA expression correlated with reduced
FEV1% and PEF% (Fig. 1h) (I do not see Fig 1h) ,
indicating that asthmatic preschool children with rhinovirus
colonization in the airways have worse respiratory function associated
with PD-L1 induction in their peripheral blood mononuclear cells
(PBMCs). By contrast, healthy control children with and without
rhinovirus in the airways as well as in asthmatic children without
rhinovirus colonization in the airways no correlation between FEV1% or
PEF% and PD-L1 was observed (Fig S1c, d) .
IFNβ correlated with better lung function in asthmatic
children
We next reasoned that in the case of asthma induced by
infections, especially rhinovirus
infections, IFN-type I and specifically IFNβ might be of importance
(Staples et al., 2015). Thus, we next analyzed the IFNβ level in cell culture supernatants
of untreated PBMCs from healthy and asthmatic children with and without
rhinovirus in the airways (Fig. 2a) as well as after a re-stimulation
with RV1b in vitro (Fig. S2a, b) and correlated them with their
FEV1% and PEF% (Fig. 2a,b; Fig. S2c-e and S3). Here we found that,
only asthmatic children and especially asthmatic children with RV in
their upper airways show a direct correlation between the IFNβ level and
the FEV1% and PEF%, respectively, indicating that a subpopulation of
children could respond to rhinovirus infection with IFNβ
production.
PD-L1 levels correlated with IFNβ-production in healthy but
not in asthmatic
children
Since it is known that Interferon induces PD-L1 (Friedrich et al., 2018)
we correlated the IFNβ expression in the supernatants of untreated and
with RV1b re-stimulated PBMCs and the PD-L1 expression in total blood
cells and found a direct correlation in control children, but not in
asthmatic children (Fig. 3, S4a, b) . These data indicate that
IFNβ is associated with PD-L1 in control children and that asthmatic
children have a disturbed IFNβ mediated PD-L1 induction.
PD-L1 is upregulated in blood cells of asthmatic children
with high C-reactive protein (CRP) serum levels and correlated with RV
in the
airways
We next reasoned that not only rhinovirus but also other infection or
inflammatory agents could cause PD-L1 induction in asthmatic children.
We thus next looked at C-reactive protein (CRP) level in serum of our
cohorts of children. CRP binds to the phosphocholine expressed on the
surface of dead or dying cells and some bacteria and leading to the
activation of the complement system and promotion of phagocytosis by
macrophages (Bray et al., 2016). Higher levels are found in
inflammation, viral infections (10–40 mg/L), active bacterial infection
(40–200 mg/L), severe bacterial infections and burns (>200
mg/L) (Chew, 2012). We considered high CRP levels as an indicator of
ongoing infection and inflammation and found that children with asthma
and a CRP value over 5 mg/l had a significantly higher PD-L1 mRNA
expression in total blood cells as compared to the control children(Fig. 4a) . Moreover, in both healthy and asthmatic children,
CRP was found to be associated with high PD-L1 levels in the serum(Fig. 4b; S4c) . Finally, in the presence of rhinovirus in the
airways, CRP correlated with PD-L1 expression in healthy children(Fig. 4c) . Taken together, these data suggest the presence of
induced PD-L1+ cells in the blood of asthmatics with
worse asthma and ongoing inflammation and infection.