AbstractBackgroundA20 and TAX1BP1 interact to negatively regulate NF-κB-driven inflammation. A20 expression is altered in F508del/F508del patients. Here we explore the effect of CFTR and CFTR genotype on A20 and TAX1BP1 expression. The relationship with lung function is also assessed.MethodsPrimary nasal epithelial cells (NECs) from CF patients (F508del/F508del, n=7, R117H/F508del, n=6) and controls (age-matched, n=8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene expression was determined by qPCR.ResultsSilencing of CFTR reduced basal A20 expression. Following LPS stimulation A20 and TAX1BP1 expression was induced in control NECs and reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del had lower expression than R117H/F508del. A20, but not TAX1BP1 expression, was proportional to FEV1 in all CF patients (r=0.968, p<0.001).ConclusionsA20 expression is reduced in CF and is proportional to FEV1. Pending confirmation in a larger study, A20 may prove a novel predictor of CF inflammation/disease severity

Elborn, J. Stuart

Ennis, Madeleine

Kelly, Catriona

Schock, Bettina C.

Williams, Mark T.

Elsevier - Publisher Connector 

Expression of the inflammatory regulator A20 correlates with lung function in patients with cystic fibrosis 

Background



A20 and TAX1BP1 interact to negatively regulate NF-κB-driven inflammation. A20 expression is altered in F508del/F508del patients. Here we explore the effect of CFTR and CFTR genotype on A20 and TAX1BP1 expression. The relationship with lung function is also assessed.



Methods



Primary nasal epithelial cells (NECs) from CF patients (F508del/F508del, n = 7, R117H/F508del, n = 6) and controls (age-matched, n = 8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene expression was determined by qPCR.



Results



Silencing of CFTR reduced basal A20 expression. Following LPS stimulation A20 and TAX1BP1 expression was induced in control NECs and reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del had lower expression than R117H/F508del. A20, but not TAX1BP1 expression, was proportional to FEV1 in all CF patients (r = 0.968, p &#60; 0.001).



Conclusions



A20 expression is reduced in CF and is proportional to FEV1. Pending confirmation in a larger study, A20 may prove a novel predictor of CF inflammation/disease severity

Schock, Bettina

Enlighten

Expression of the inflammatory regulator A20 correlates with lung function in patients with cystic fibrosis

Catriona Kelly

Mark T. Williams

J. Stuart Elborn

Madeleine Ennis

Bettina C. Schock

Crossref

Journal of Cystic Fibrosis

Background: A20 and TAX1BP1 interact to negatively regulate NF-¿B-driven inflammation. A20 expression is altered in F508del/F508del patients. Here we explore the effect of CFTR and CFTR genotype on A20 and TAX1BP1 expression. The relationship with lung function is also assessed. Methods: Primary nasal epithelial cells (NECs) from CF patients (F508del/F508del, n= 7, R117H/F508del, n= 6) and controls (age-matched, n= 8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene expression was determined by qPCR. Results: Silencing of CFTR reduced basal A20 expression. Following LPS stimulation A20 and TAX1BP1 expression was induced in control NECs and reduced in CF NECs, broadly reflecting the CF genotype: F508del/F508del had lower expression than R117H/F508del. A20, but not TAX1BP1 expression, was proportional to FEV1 in all CF patients (r= 0.968, pb0.001). Conclusions: A20 expression is reduced in CF and is proportional to FEV1. Pending confirmation in a larger study, A20 may prove a novel predictor of CF inflammation/disease severity

ResearchOnline@GCU

Enlighten: Publications

Journal of Cystic Fibrosis 12 (2013) 411–415
www.elsevier.com/locate/jcfShort Communication
Expression of the inﬂammatory regulator A20 correlates with lung function
in patients with cystic ﬁbrosis
Catriona Kelly a, b, Mark T. Williams a, J. Stuart Elborn a, Madeleine Ennis a, Bettina C. Schock a,⁎
a Centre for Infection and Immunity, Queen's University of Belfast, Belfast BT97AE, UK
b Institute for Science and Technology in Medicine, Keele University, Keele ST55BG, UK
Received 24 August 2012; received in revised form 18 October 2012; accepted 22 October 2012
Available online 17 November 2012Abstract
Background: A20 and TAX1BP1 interact to negatively regulate NF-κB-driven inﬂammation. A20 expression is altered in F508del/F508del
patients. Here we explore the effect of CFTR and CFTR genotype on A20 and TAX1BP1 expression. The relationship with lung function is also
assessed.
Methods: Primary nasal epithelial cells (NECs) from CF patients (F508del/F508del, n=7, R117H/F508del, n=6) and controls (age-matched,
n=8), and 16HBE14o- cells were investigated. A20 and TAX1BP1 gene expression was determined by qPCR.
Results: Silencing of CFTR reduced basal A20 expression. Following LPS stimulation A20 and TAX1BP1 expression was induced in control
NECs and reduced in CF NECs, broadly reﬂecting the CF genotype: F508del/F508del had lower expression than R117H/F508del. A20, but not
TAX1BP1 expression, was proportional to FEV1 in all CF patients (r=0.968, pb0.001).
Conclusions: A20 expression is reduced in CF and is proportional to FEV1. Pending conﬁrmation in a larger study, A20 may prove a novel
predictor of CF inﬂammation/disease severity.
© 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.Keywords: A20 protein; FEV1; NF kappa B; Airway epithelial cells; Cystic Fibrosis; Chronic inﬂammation1. Introduction
Cystic Fibrosis (CF) lung disease is heterogenous in nature
and the severity of disease in patients with the same genotype
can vary significantly. Activation of TLR4 by Gram-negative
bacteria such as Pseudomonas aeruginosa (P. aeruginosa)
leads to pro-longed NF-κB (p65) signalling in CF primary
epithelial cells and CF cell lines [1,2]. A20 (TNFAIP3, tumor
necrosis factor, alpha-induced protein 3) is an endogenous
negative regulator of the NF-κB pathway and is rapidly and
transiently induced in response to bacterial and viral stimuli.
A20 terminates NF-κB driven inflammation by inhibiting the⁎ Corresponding author at: Centre for Infection and Immunity, Queen's
University of Belfast, Health Sciences Building, 97 Lisburn Road, Belfast BT9
7AE, UK. Tel.: +44 28 9097 5876; fax: +44 28 90 972671.
E-mail address: b.schock@qub.ac.uk (B.C. Schock).
1569-1993/$ -see front matter © 2012 European Cystic Fibrosis Society. Pub
http://dx.doi.org/10.1016/j.jcf.2012.10.009lishedpolyubiquitination and activation of the central adaptor protein
TNF receptor-associated factor (TRAF) 6 [3]. Desensitized
pathogen recognition is thought to be secondary to hyperactive
TRAF6 signalling in chronic lung diseases including CF [4].
Despite varied pathogeneses, A20 is a critical regulator of
inflammatory processes in rheumatoid arthritis, systemic lupus
erythematosus, type 1 diabetes, multiple sclerosis, psoriasis and
inflammatory bowel disease [5]. Furthermore, A20 is a sus-
ceptibility gene for the development of Crohn's [6] and irritable
bowel disease [7]. In the lung, P. aeruginosa challenge rapidly
induces A20 in mice [8], while A20 is essential for termination
of TLR2/4 mediated IL-8 release from primary airway epithelial
cells [9]. We recently showed that A20 is reduced in CF airway
epithelial cells after stimulation with LPS. The action of A20 is
dependent on an adapter protein called TAX1BP1, which is also
significantly reduced in LPS-treated CF cells [1,10]. Further-
more, this work suggested that CF airway epithelial cells lackby Elsevier B.V. All rights reserved.
412 C. Kelly et al. / Journal of Cystic Fibrosis 12 (2013) 411–415essential interactions between A20, TAX1BP1 and TRAF6,
which may partly explain the associated chronic activation of
NF-κB [1].
Here we further investigate the relationship between A20,
TAX1BP1 and CFTR as well as any association with lung func-
tion (FEV1) in patients homozygous for F508del and heterozy-
gous for R117H/F508del.
2. Materials and methods
Primary nasal epithelial cells (NEC) were obtained from CF
patients (F508del/F508del {n=7}, R117H/F508del {n=6}) and
age matched controls {n=8} as previously described [11] and
were fully differentiated at air–liquid interface (ALI). NECs were
treated with LPS (P. aeruginosa, Sigma, 50 μg/ml) for 24 h. TheFig. 1. The effect of CFTR and genotype on A20 and TAX1BP1 mRNA expression
16HBE14o- cells treated with siRNA against CFTR. Data are presented as mean±
different CF genotypes (R117H/F508del and F508del/F508del) on basal A20 (C) an
was expressed relative to Jurkat cells, which acted as an internal calibrator for all e
TAX1BP1 (F) was also determined by qPCR. Expression was calculated after 24
indicated in the figure. *pb0.05, ***pb0.001 between groups as indicated in the fiResearch Ethics Committee of Northern Ireland approved the
study and all participants provided informed consent (07/NIR02/
23). In CF patients lung function (FEV1) was also determined at
time of cell sampling.
The 16HBE14o- bronchial epithelial cell line (D. Gruenert
UCSF, USA) was grown in submersion and transfected at
80% confluence with commercially available CFTR siRNA
(GenomeWide siRNA, Qiagen) and RNAiFect™ Transfection
Reagent (Qiagen). Experiments included mock (transfection
reagent only) and scrambled (Allstars Neg siRNA, Qiagen)
controls. Transfection efficiency was confirmed by qPCR as
72%±5.34%.
Total RNA was extracted using an RNeasy kit (Qiagen) and
quantified on a Nanodrop (Thermo Scientific). Equal amounts of
RNA were reverse transcribed into cDNA (Sensiscript Reverse. The basal expression of A20 (A) and TAX1BP1 (B) was assessed by qPCR in
SEM with n=3. ***pb0.001 compared with scrambled siRNA. The effect of
d TAX1BP1 (D) mRNA expression was determined by qPCR. Basal expression
xperiments. Furthermore, the mRNA expression of LPS-induced A20 (E) and
h simulation with LPS and data are presented as mean±SEM with n=6–8 as
gure.
413C. Kelly et al. / Journal of Cystic Fibrosis 12 (2013) 411–415Transcription Kit, Qiagen). A20, TAX1BP1 and p65 expression
was assessed by qPCR [1]. Relative expression to β-actin was
calculated using the ΔΔCt method. To overcome inter-patient
variability in basal gene expression levels, mRNA expression
24 h after LPS stimulation was compared with mRNA expres-
sion at 0 h (standardized to 1) for each individual sample. cDNA
obtained from Jurkat cells acted as an internal calibrator for all
experiments and was used to determine differences in basal gene
expression.
All data are presented as the means±SEM. Differences be-
tween groups were analysed using non-parametric tests and
were considered to be significant if pb0.05.
3. Results
CFTR silencing in 16HBE14o- cells caused a 43% reduction
(pb0.001) in basal A20 expression, but did not alter basal
TAX1BP1 levels (Fig. 1A and B). A reduction in basal A20 and
TAX1BP1 expression was also confirmed in CFNECs comparedFig. 2. Relationship between A20, TAX1BP1, p65 and lung function in different CF
F508del) on LPS-induced p65 (A) expression was determined by qPCR. Expression wa
with n=6–8 as indicated in the figure. *pb0.05, **pb0.01 between groups as ind
(C) expression was assessed by Spearman rank test. Significant correlations are indicate
(E) expression was assessed by Spearman rank test. Significant correlations are indicatto control NECs (Fig. 1C and D). Following LPS stimulation,
A20 was induced in control NECs and reduced in CF NECs.
F508del homozygotes showed a more pronounced reduction in
A20 expression than R117H/F508del heterozygotes (pb0.05,
Fig. 1E). However, a reduction in TAX1BP1 expression was only
observed in F508del homozygotes (pb0.05, Fig. 1F).
To provide a basic indication of how reduced A20 expression
may relate to NF-κB and the inflammatory response we assessed
the expression of p65 (NF-κB subunit) by qPCR Following LPS
stimulation, p65 expression was higher in F508del homozygotes
than R117H/F508del heterozygotes (pb0.05, Fig. 2A). Basal A20
expression was inversely proportional to p65 levels (Fig. 2B). A
relationship between p65 and TAX1BP1 was not observed, but a
clear separation of the two genotypes was observed (Fig. 2C).
We also assessed the relationship between basal A20 and
TAX1BP1mRNA expression and the FEV1 of CF cohort (Fig. 2D
and E). We only included CF patients who had also had FEV1
measured on the same day as their nasal brushing sample was
taken in this analysis. The median FEV1 for the R117H/F508delgenotypes. The effect of different CF genotypes (R117H/F508del and F508del/
s calculated after 24 h simulation with LPS and data are presented as mean±SEM
icated in the figure. The relationship between p65 and A20 (B) or TAX1BP1
d in the figure (r value). The relationship between FEV1 and A20 (D) or TAX1BP1
ed in the figure (r value).
Table 1
Patient demographics.
Patient demographics including FEV1 are shown in Table 1.
Group 1: Non-CF,
healthy individual
Group 2: R117H/F508del
CF patients
Group 3: F5808del /F508del
CF patients
% Female 55 40 60
Median Age 27 (20, 29.5) 29 (28, 35) 25 (15, 35)
Median BMI 25.3 (22.4, 26.8) 24.5 (21.6, 27.5) 22.3 (20.7, 26.3)
FEV1 (% predicted) – 82.5 (70, 95.3) 50 (36, 62)
sputum for+ve P.aeruginosa – – 100%
Smokers – – –
414 C. Kelly et al. / Journal of Cystic Fibrosis 12 (2013) 411–415heterozygous and F508del homozygous groups were 82.5 and
50.05% predicted (Table 1), respectively, and FEV1 was sig-
nificantly correlated with A20 expression (Fig. 2D; Spearman
rank test r=0.968, pb0.001, n=9), but not TAX1BP1 expression
(Fig. 2E).4. Discussion
A20 is an important regulator of inflammatory signalling,
which inhibits p65 translocation to the nucleus. Conversely, si-
lencing of A20 increases p65 activation and potentiates the
inflammatory response [12]. The ability of A20 to inhibit NF-κB
activation is dependent on interaction with the TRAF6 binding
protein TAX1BP1 [13,14]. In complex, A20 and TAX1BP1 act
directly to prevent TRAF6 polyubiquitination and terminate
TLR-driven inflammatory signalling [14]. In the absence of
TAX1BP1, A20 cannot bind or act on target proteins [13], lead-
ing to persistent NF-κB activation and chronic inflammation.
Using LPS stimulated CFBE41o cells we have previously
shown that A20 does not interact with TAX1BP1 or TRAF6.
This, together with persistently upregulated nuclear p65 protein
expression, suggests that A20 action is dysfunctional in CF
epithelial cells [1].
Here, we extend our previous findings to different CF geno-
types and investigate the association of reduced A20/TAX1BP1
expressionwith levels of inflammation and lung function (FEV1).
Basally and following LPS stimulation, A20 expression was
reduced in CF cells in a manner that largely reflected genotype;
the F508del/F508del group had a lower expression than the
R117H/F508del group. Furthermore, basal A20 expression in CF
patients (both genotypes) was inversely proportional to p65
levels. LPS induces A20, which in turn inhibits NF-κB activity.
LPS selectively increases the transcriptional activity of p65, but
not p50, in intestinal epithelial cells [15]. In line with this,
overexpression of p65 increases NF-κB transcriptional activity
[16]. Furthermore, A20 is itself regulated by NF-κB and p65 has
been shown to bind to endogenous A20 [17]. Our previous work
has shown increased and sustained activation of p65 in CF airway
epithelial cells [1]. We therefore measured p65 mRNA expres-
sion to provide an indication of how reduced A20 expression
may relate to NF-κB and the inflammatory response. Our results
suggest that A20 expression may be directly related to CFTR
expression/function and underlines the important role for A20 in
the regulation of NF-κB activation in CF.Finally, and in line with the role of A20 in regulating in-
flammatory responses, our data show a significant correlation
between FEV1 and basal A20 expression. Notably, those with
the poorest lung function were those with the most pronounced
reduction in A20 expression irrespective of genotype. Data from
CFTR silenced 16HBE14o- cells support a possible direct link
between A20 and CFTR. Our work suggests that CFTR expres-
sion can modify the transcription of A20; however, the mech-
anism of this modification is not yet known.We are examining a
number of potential intermediaries including the A20 binding
inhibitors of NF-κB (ABIN) proteins. Although TAX1BP1 ex-
pression was not altered by CFTR silencing, the genotype specific
expression profile would suggest that TAX1BP1 is regulated by
an intermediary (potentially TRAF6 or other protein(s) within the
TRAF6 feedback loop), which will also be investigated in future.
In conclusion, LPS-stimulated expression of A20 mRNA is
reduced in CF in a manner that broadly reflects genotype. More
specifically, basal A20 expression is altered in a manner pro-
portional to FEV1. Pending confirmation in a larger scale study,
A20 may prove a novel predictor of inflammation and disease
severity in the CF population and future studies will investigate
if pharmacological induction of A20 can modulate NF-κB
driven inflammation in CF airways.References
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Expression of the inflammatory regulator A20 correlates with lung function in patients with cystic fibrosis

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