The Human IL-23 Receptor rs11209026 A
Allele Promotes the Expression of a Soluble
IL-23R−Encoding mRNA Species
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of June 17, 2017.
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J Immunol 2015; 194:1062-1068; Prepublished online 31
December 2014;
doi: 10.4049/jimmunol.1401850
http://www.jimmunol.org/content/194/3/1062
http://www.jimmunol.org/content/suppl/2014/12/31/jimmunol.140185
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Supplementary
Material
Raymond Y. Yu, Jonathan Brazaitis and Grant Gallagher
The Journal of Immunology
The Human IL-23 Receptor rs11209026 A Allele Promotes
the Expression of a Soluble IL-23R–Encoding mRNA Species
Raymond Y. Yu, Jonathan Brazaitis, and Grant Gallagher
G
enome-wide association studies andsingle nucleotide
polymorphism (SNP) analysis are powerful ways to
identify disease susceptibility variants and have provided
insight to a number of complex autoimmune diseases in humans
(1–7). Although such SNPs can have functional and phenotypic
consequences (8, 9), the molecular basis of these discoveries often
remains unclear. Pinpointing such changes in function is one of
the challenges in unraveling the genetic basis of complex disease
predisposition and severity. The SNP rs11209026 (G1142A), located in exon 9 of IL23R (Supplemental Fig. 1), has been associated with strong protection against Crohn’s disease (allele A);
that is, allele A is underrepresented in the disease population (10).
This observation has subsequently been replicated in multiple
studies encompassing a range of autoimmune disorders (11–15).
Given the widely observed protective nature of rs11209026 allele
A, it is important to determine whether this allele itself has a protective function or merely a protective association. Interestingly, the
A allele causes an amino acid change close to the end of the coding
sequence of exon 9; the more common (allele G) arginine at residue
381 becomes a glutamine (R381Q). To test whether this generated
Genetic Immunology Laboratory, HUMIGEN, The Institute for Genetic Immunology, Genesis Biotechnology Group, Hamilton, NJ 08690
Received for publication July 22, 2014. Accepted for publication November 19,
2014.
R.Y.Y. designed and executed experiments, analyzed data, and wrote and finalized the
manuscript and revision; J.B. carried out experiments; G.G. designed experiments,
analyzed data, and wrote and finalized the manuscript and revision.
Address correspondence and reprint requests to Prof. Grant Gallagher, HUMIGEN
LLC, Institute for Genetic Immunology, Genetic Immunology Laboratory, 2439
Kuser Road, Hamilton, NJ 08690. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: AON, antisense oligonucleotide; BAC, bacterial
artificial chromosome; ESE, exonic splicing enhancer; qRT-PCR, quantitative RTPCR; siRNA, small interfering RNA; SNP, single nucleotide polymorphism; SR,
serine/arginine-rich.
Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401850
a functional difference, de Paus et al. (16) transfected T cells with
expression plasmids encoding the IL-23R in its R or Q form.
However, this study demonstrated that the two receptor forms
functioned similarly, ruling out different functions of the two forms
of the receptor protein itself (16). Then, Di Meglio et al. (17) examined healthy individuals and compared IL-23 responsiveness in
allele A carriers compared with allele G carriers. They demonstrated reduced IL-17A and STAT3 phosphorylation in the presence of allele A and suggested that the protective effects of the IL23R R381Q were mediated through selective attenuation of IL-23–
induced Th17 effector function (17). This study was accompanied
by an independent parallel study from Sarin et al. (18) showing that
healthy carriers of allele A showed decreased IL-23–dependent IL17 and IL-22 production. Using a similar population of healthy
donors, Pidasheva et al. (19) concluded that IL23R-Q381 (allele A)
is a loss-of-function allele and furthermore showed that carriers of
this allele had decreased surface IL-23R expression while retaining
similar levels of IL23RmRNA (19). These studies on healthy
donors have been complemented by studies in human disease.
Oosting et al. (20) showed that A allele resulted in lower levels of
IL-17 production in a Borrelia burgdorferi stimulation model,
whereas Hazlett et al. (21) suggested that in rheumatoid arthritis
patients, the presence of this allele required higher IL-23 concentrations to produce similar amounts of IL-17A concentrations. Finally and most recently, Di Meglio et al. (22) studied memory
T cells from healthy individuals and psoriasis patients to show that
allele A reduced responsiveness to IL-23, citing an allele–dosage
effect and commenting that individuals in their study who were
AA homozygous were almost unresponsive to IL-23. (Interestingly,
recent data in inflammatory bowel disease patients suggests that this
AA genotype is very rare; in fact, only 0.12% of inflammatory
bowel disease cases were homozygous for the A allele, less than
one-third of those observed in healthy, ethnically matched individuals of European descent; R. Duerr, personal communication.)
Thus, multiple studies from independent laboratories, in healthy
individuals and human patients, have shown that although the
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The human IL23R gene single nucleotide polymorphism rs11209026 A allele confers protection against inflammatory diseases.
However, although this difference has been associated with reductions in IL-23–induced IL-17A production and STAT3 phosphorylation, the molecular mechanism underlying these changes remains undefined. Th17 cell maturation depends on IL-23
signaling. Multiple splice forms of the human IL23R transcript exist, and one, D9, encodes a soluble form of the receptor. In
this study, we asked whether this protective allele was associated with mRNA splicing. Using mini-gene constructs and competitive
oligonucleotide binding, we showed that the A allele alters IL-23R a-chain mRNA splicing and favors exon 9 skipping by reducing
the binding of the splicing enhancer SF2. This enhances expression of the D9 mRNA and consequently diminishes IL-23 signaling.
Thus, the presence of the A allele increases expression of the soluble form of IL23R mRNA (which then functions as a decoy receptor)
and lowers the ability to develop a Th17 phenotype upon IL-23 stimulation. We further showed that antisense oligonucleotides
targeting the SF2 binding site could efficiently induce exon 9 skipping in the presence of the G allele, and thereby replicate the
effect of the A allele. Antisense oligonucleotide treatment caused dose-responsive induction of the IL23RD9 mRNA and interfered
with in vitro differentiation of human Th17 cells, reducing their expression of the signature Th17 cytokines IL-17A and IL-17F.
This may represent a novel approach to therapy of Th17-mediated diseases by elevating soluble IL-23R while simultaneously
reducing the remaining cell surface receptor density. The Journal of Immunology, 2015, 194: 1062–1068.
The Journal of Immunology
Materials and Methods
Construction of minigene plasmids
The RP11-684P13 bacterial artificial chromosome (BAC) clone carrying
138 kb human chromosome 1 was purchased from Invitrogen (Carlsbad,
CA). This fragment of human chromosome 1 contains 33 kb DNA sequence
upstream of the IL23R transcription start, all the exonic and intronic
sequences, and 12 kb DNA sequence downstream of the transcription stop.
The common (G) allele minigene plasmid (as illustrated in Fig. 1A) was
constructed by the PCR using the following primer pairs: exon 8/intron 8
fragment: 8F, 59-AAGCTAGCTCCCCAGGTCACATCAAAAG-39, and 8R,
59-AAGGTACCAAAATTAGCTGGGCGTGATG-39; intron 8/exon 9/intron
10 fragment: 9F, 59-AAGGTACCCCTGTGTCAGACAAGCCAAA-39, and
9R, 59-AAGGATCCAAGGCAACCCTGGAGTCTTT-39; intron 10/exon 10
fragment: 9F, 59-AAGGATCCTCTGTTGCCCAGAGTGAGTG-39, and 9R,
59-AACTCGAGTTCACAACATTGCTGTTTTTCA-39.
The PCR fragments were subcloned into the mammalian expression
vector, pCDNA3.1. The minigene plasmid was sequence verified. PCR-
mediated site-directed mutagenesis was used to change the common allele sequence G into the protective allele sequence A.
Construction of SF2 and SRP40 expression plasmids
Expression plasmids of SF2 and SRP40 were constructed by amplifying SF2
and SRP40 coding sequences using the following primers: SF2 F, 59ACAAGCTTGCCACCATGTCGGGAGGTGGTGTGATT-39, and SF2 R, 59ATCTCGAGTTATGTACGAGAGCGAGATCT-39; and SRP40 F, 59-ACAAGCTTGCCACCATGAGTGGCTGTCGGGTATTC-39, and SRP40 R, 59ATCTCGAGTTAATTGCCACTGTCAACTGA-39.
The PCR fragments were subcloned into the mammalian expression
vector, pcDNA3.1. The expression plasmids were sequence verified.
AON-mediated exon skipping
The RNA AON, 59-accuacccaguucggaauGauc-39, was synthesized by Integrated DNA Technologies. The phosphodiester bond was modified, replacing
one of the nonbridging oxygens by sulfur. The RNA AON containing the
sulfur-substituted oligonucleotides has a phosphorothioate linkage and
29-O-methyl RNA bases. The AON was transfected into the 293T cells
(American Type Culture Collection) and primary human cells (such as
CD4+ T cells and PBMCs) using Lipofectamine 2000 (Invitrogen) and the
human T cell transfection kit (Amaxa).
In vitro Th17 cell differentiation
CD4+ T cells were negatively enriched using human CD4+ T cells enrichment kit (StemCell Technologies). A total of 1 3 106 cells/ml CD4+
naive T cells were differentiated into Th17 cells under Th17 culture condition (CD3/28 beads, 10 ng/ml IL-1b, 10 ng/ml IL-6, 10 ng/ml IL-23, and
1 ng/ml TGF-b) for 5 d. All cytokines were purchased from Humanzyme.
The CD3/28 beads were used according to the manufacturer’s instructions
(Miltenyi Biotec). The differentiated cells were subjected to RNA extraction and real-time PCR (quantitative RT-PCR [qRT-PCR]) to analyze gene
expression.
Real-time PCR
Naive T cells were differentiated under Th17 conditions for 5 d. Differentiated cells were collected and RNA was extracted using TRIzol (Invitrogen).
RNA was reverse transcribed into cDNAwith the AffinityScript QPCR cDNA
Synthesis Kit (Stratagene), according to the manufacturer’s instructions. The
real-time PCR was performed using Brilliant II SYBR Green QPCR Master
Mix (Stratagene). The following primers were used in the study: IL17A, F,
59-CTGGGAAGACCTCATTGGTGTCAC-39, and R, 59-CGGTTATGGATGTTCAGGTTGACC-39; IL17F, F, 59-CCTCCCCCTGGAATTACACTGTC-39, and R, 59-CAGGGTCTCTTGCTGGATGGG-39; GAPDH, F,
59-GAGTCAACGGATTTGGTCGT-39, and R, 59-GACAAGCTTCCCGTTCTCAG-39.
SF2 RNA interference
293T cells were transfected with 20 pmoles antisense SF2 RNA (Dharmacon).
Transfected cells were collected, washed in PBS, and lysed in ProteoJET
mammalian cell lysis reagent (Fermentas) with protease and phosphatase
inhibitors (Sigma-Aldrich), 48 h posttransfection. Lysates were cleared by
centrifugation, and supernatants were prepared for SDS-PAGE by addition of
sample loading buffer (Bio-Rad). Lysates were subjected to 4–12% gradient
PAGE (Bio-Rad) and transferred to Immun-Blot polyvinylidene difluoride
membrane (Bio-Rad), as per the manufacturer’s recommendations. Membranes were blocked in 5% milk/TBST at room temperature for 1 h. Membranes were first probed with Abs against SF2 (Santa Cruz Biotechnology)
and then stripped and reprobed for actin (Santa Cruz Biotechnology).
Fragment analysis
Total RNA was isolated from PBMCs with the Absolutely RNA miniprep kit
(Stratagene) following the manufacturer’s instructions. Purified RNA was
reverse transcribed into cDNA using AffinityScript cDNA synthesis kit
(Stratagene). PCR was carried out using Expand Long Template Enzyme
mix (Roche Applied Science) with forward (59-AATGCTGGGAAGCTCACCTACATA-39) and reverse (59-D3-GCTTGTGTTCTGGGATGAAGATTTC-39) primers, which was fluorescent labeled with D3 dye. The amplified
product was then analyzed in the Beckman CEQ8000 using their Fragment
Analysis Program. 1 ml (5%) of PCR product was denatured in 39 ml sample
loading solution buffer (Beckman) containing DNA standard size markers.
Two DNA standard size markers, DNA size standard marker kit 600 (0.5 ml/
reaction; Beckman) and a custom-made D1 labeled 600–1200 size marker
(1 ml/reaction; Bioventures) were used in to cover the DNA size range from
60 to 1200 nt.
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function of the R and G form proteins may be similar if transfected,
the natural presence of the alleles encoding these proteins leads
inevitably to reduced IL-23 responsiveness in individuals carrying
the A allele, originally shown to be protective by Duerr et al. (10).
Nonetheless, the mechanism has remained undefined until now.
The rs11209026 SNP is located within exon 9 at the 39 end
(encoding either R or Q at residue 381). Exon sequences not only
encode information for amino acids but also contain cis-acting
elements that influence the use of flanking splice sites and so can
regulate mRNA processing (23–25). Often, such cis-acting elements function as binding sites for serine/arginine-rich (SR) proteins, a family of essential splicing factors. Exon-bound SR
proteins promote splicing of adjacent introns and are also involved
in the regulation of alternative splicing (26). Coding-region SNPs
within these cis-acting elements have been reported to affect the
patterns or efficiency of mRNA splicing, which in turn can cause
phenotypic variation (27–31).
We previously demonstrated that the IL23R gene transcript
undergoes extensive alternative splicing (32, 33), including the
generation of one form (IL23RD9; Genbank AM990318) that encodes a soluble version of the entire external domain of the receptor chain. More recently, we showed that this IL23RD9 mRNA
encodes a D9 protein that constitutes a soluble IL-23R protein.
This soluble protein binds human IL-23 in solution and dosedependently inhibits STAT3 phosphorylation and functional maturation of human Th17 cells in vitro (34). In the present study, we
hypothesized that the coding SNP rs11209026 may affect exon 9
splicing of IL23R during mRNA maturation.
In this paper, we describe the role of the protective A allele in
modulating mRNA splicing of exon-9 of the human IL23R chain by
modifying the binding site for the SF2 splice enhancer protein. In
so doing, this allele enhances the expression of our previouslyreported soluble version of the IL-23R-chain protein (34). These
data therefore explain the molecular mechanism underlying the
protective nature of this IL-23R polymorphism, in a manner that is
consistent with the studies described above (10, 16–22).
Finally, we extend these studies to demonstrate a candidate therapeutic approach, targeting the SF2 binding site with antisense oligonucleotides to effect a powerful conversion of the full-length
mRNA (which encodes the cell surface receptor) to the IL23RD9
mRNA form (which encodes the soluble, IL-23–adsorbing version
of the receptor). Therefore, this new approach is effective in two
fronts; because the overall level of IL23R mRNA transcript is not
changed, antisense oligonucleotide (AON) treatment diminishes
IL-23 signaling by both increasing the absolute levels of the soluble receptor while simultaneously reducing, on a mole-for-mole
basis, the number of cell surface receptors available for receiving
IL-23 and transmitting its signal.
1063
1064
HUMAN IL-23R SNP PROMOTES ALTERNATIVE SPLICING
Isolation and culture of human PBMCs and immune cells
PBMCs were isolated from heparinized whole venous blood of healthy donors
by density gradient centrifugation using Ficoll-Paque (Sigma-Aldrich) according to the manufacturer’s instructions. Blood was purchased as anonymous buffy coats from New Jersey blood transfusion service with no donor
identifying details. Isolated PBMCs were maintained in RPMI 1640 medium
(Invitrogen-Life Technologies, Carlsbad, CA) supplemented with 10% heatinactivated FBS (Invitrogen-Life Technologies) and 1 mM glutamine
(Invitrogen-Life Technologies).
In vitro oligonucleotide binding assay
Biotinlyated RNA oligonucleotides containing either common allele G or
protective allele A were used: common allele (G): biotin, 59-ATCATTCC-GAACTGGGTAGGT-39; and protective allele (A), biotin, 59-ATCATTCC-AAACTGGGTAGGT-39.
The RNA oligonucleotides were incubated with either whole-cell lysates
from the 293T cells or SF2 recombinant protein for 1 h at room temperature.
The biotinlyated RNA molecules were then precipitated by streptavidin
agarose (Pierce). The precipitates were extensively washed with PBS (pH 7.4).
The amount of SF2 bound to the RNA oligonucleotides was assayed by
immunoblot using anti-SF2 Ab (Santa Cruz Biotechnology).
As previously described (34), a sandwich ELISA was developed to detect
D-9 using 5 mg/ml mouse anti-hIL23R (R&D Systems) as capture Ab and
1.6 mg/ml goat biotinlyated anti-hIL23R (R&D Systems) as the detection
Ab. The capture Ab was first coated on the microtiter plate in 50 mM
bicarbonate buffer (pH 9.6) at 4˚C overnight. The plate was then blocked
with 10% FBS/TBST at room temperature for 2 h. Samples were added to
the well and incubated at 4˚C overnight. Detection Ab in TBST was added
to the wells and incubated at room temperature for 2 h. The plate was
extensively washed with TBST during each change. The immunocomplex
was detected by addition of Streptavidin-HRP (R&D Systems) and tetramethylbenzidine substrate (eBioscience). The plate was read at OD 450 nm.
Statistical analysis
Student t test was used throughout. Unless otherwise indicated, p values
are shown as being , 0.05.
Results
Minigene analysis demonstrates alternative splicing with the A
allele
We previously demonstrated that the IL23Ra gene undergoes extensive alternative splicing (32, 33), including the generation of
one form (IL23RD9; Genbank AM990318) that encodes a soluble
version of the entire external domain of the receptor chain (34). In
this article, we hypothesized that the coding SNP rs11209026
(which is located at the 39-end of exon 9) may affect exon 9 splicing
of IL23R during mRNA maturation (Fig. 1A, Supplemental Fig. 1).
To test this hypothesis, a mini-gene was constructed, which contained the genomic sequence of IL-23Ra from the start of exon 8
to the end of exon 10 but with deletion of internal intronic regions
to minimize the size of the plasmid. The intronic regions retained
included essential elements for RNA splicing, such as the 59-donor
splice site, the A branch point and the 39-acceptor splice site. Two
minigene plasmids were made, one containing the common allele
G on exon 9 and one carrying the protective allele A, to examine
the effect of this SNP on exon 9 splicing (Fig. 1A).
Minigene constructs were positioned under the control of a
constitutive promoter and transfected into 293T cells. As shown in
Fig. 1B and 1C, transfection of the mini-gene carrying the common
allele mainly expressed mature RNA containing exons 8, 9, and 10;
weak expression of a transcript lacking exon 9 was also detected.
This result was consistent with our observation that ∼10% of total
IL23R mRNA in human PBMCs lacked exon 9 (34). Intriguingly,
the minigene containing the protective allele A showed enhanced
expression of exon 8/10 transcript, corresponding to this IL23RD9
mRNA species (Fig. 1B, 1C). Although the protective allele showed
no effect on the total expression level of minigene (transcripts of
exon 8/9/10 and exon 8/10) quantitatively measured by qRT-PCR,
the mini-gene containing the variant allele A showed ∼2.5-fold increased exon 8/10 (p , 0.05), compared with the minigene carrying
the common allele G (Fig. 1C). Therefore, this observation indicated
that the DNA sequence at the end of exon 9 contains a regulatory
element for RNA splicing and that the protective allele A favors
exon 9 skipping.
Protective allele A disrupts an SF2 binding site
RNA splicing is a regulated process that involves intronic and exonic
cis-elements important for correct splice site identification. These
elements can act either by stimulating (enhancers) or repressing
(silencers) splicing. Exonic splicing enhancers (ESEs) in particular
appear to be very prevalent and may be present in most, if not all
exons (35). We predicted that the end of exon 9 contained an essential element for regulation of exon 9 splicing. ESE Finder 3.0
(http://rulai.cshl.edu/tools/ESE) was used to predict putative splice
enhancer binding sites. Binding sites for SR protein family members SF2 and SRp40 were predicted in the common allele G and
protective allele A respectively. In order to examine the effect of
these two proteins on exon 9 splicing, mini-gene constructs containing either the common allele G or the protective allele A were
coexpressed with either SF2 or SRp40 in 293T cells.
In both minigenes, forced overexpression of SF2 strongly enhanced exon 9 splicing, which resulted in a marked reduction in
exon 8/10 expression (p , 0.05). However, similar expression of
SRp40 showed no significant effect (p = ns), suggesting that SF2
was the more important factor in regulating exon 9 splicing
(Fig. 2A). To examine this phenomenon in a less synthetic context,
BAC RP11-684P13 (140 kb, containing the complete IL23R gene,
including 33 kb upstream of transcription start and 11 kb
downstream of transcription stop, resulting in the expression of
IL23R under its authentic promoter) was cotransfected with SF2
into 293T cells; again, SF2 overexpression profoundly diminished
exon 8/10 expression (data not shown). To confirm these obser-
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ELISA
FIGURE 1. The effect of SNP rs11209026 on IL23R splicing. (A) Schematic representation of the minigene plasmids. The minigene plasmids contain exons 8, 9, and 10, and the 59- and 39-regions of their flanking introns.
Two minigene plasmids were generated, one containing the common allele
G and one containing the protective allele A. (B and C) Either G or A allele
minigene plasmids were transfected into 293T cells. RNA was prepared
48 h posttransfection and reverse transcribed to cDNA. The expression
levels of two mRNA transcripts, exon 8/9/10 and exon 8/10, were detected
by RT-PCR (B) and real-time qRT-PCR. (C) *p , 0.05. The results demonstrate that the A minigene spontaneously expressed higher levels of exon
8/10 transcript than did the G minigene.
The Journal of Immunology
vations, SF2 protein levels were knocked down with small interfering RNA (siRNA) in 293T cells; this resulted in elevation of the
levels of exon 8/10 (i.e., encoding IL23RD9 (34)) transcript from
the allele G minigene (p , 0.05; Fig. 2B).
Interestingly, overexpression of SF2 was also able to diminish
the expression of the exon 8/10 mRNA form from the allele A
minigene (p , 0.05). Again, SF2 functioned as the splice enhancer, whereas SRp40 played no apparent role in exon 9 splicing
or skipping (Fig. 2A). Overexpression of SF2 therefore appears
able to repair the splicing defect caused by allele A.
1065
Antisense RNA oligonucleotide targeting mimics allele A
FIGURE 2. The IL23R protective allele A disrupts an SF2 binding site.
Threshold analysis using the ESE finder program disclosed a predicted SF2
binding site in the presence of the G allele, which was replaced with a predicted SRp40 site when the protective A allele was present. (A) 293T cells
were cotransfected with either the common or protective allele minigenes,
each in the presence of one of the following: the control plasmid pcDNA, an
SF2 expression construct, or an SRp40 expression construct. Expression
levels of exon 8/9/10 and exon 8/10 transcript were determined by qRTPCR. Overexpression of SF2 diminished exon 8/10 levels from either
minigene (*p , 0.05), whereas SRp40 overexpression had no effect. (B)
293T cells were cotransfected with the common or allele minigene and
either control siRNA or SF2-specific siRNA. The effectiveness of SF2
knockdown was determined by Western blotting (inset). Knockdown of
SF2 enhanced the level of exon 8/10 transcript, mimicking the presence of
the protective allele (*p , 0.05). (C) An in vitro binding assay for SF2
was performed on 293T cell lysates. (C, upper panel) Biotinylated oligonucleotides representing the common or protective alleles were titrated
(50–200ng) into the lysate and precipitated with streptavidin–agarose.
The degree of SF2 binding was visualized by Western blotting. The protective allele oligonucleotide consistently attracted less SF2 than the
common allele. (C, lower panel) Unlabeled common or protective allele
oligonucleotides were used to compete SF2 binding to biotinylated common
allele oligonucleotide. The protective allele failed to compete.
The Crohn’s disease protective SNP rs11209026 allele A caused
exon 9 skipping and resulted in increased expression of the
IL-23RaD9 variant that encodes a soluble receptor. To examine
further the function of this locus and to begin to explore its use as
a therapeutic target in individuals lacking the protective allele,
AONs were designed to block the SF2 binding site on exon 9 and
the 59-splice donor site on intron 9 to induce exon 9 skipping.
The allele G minigene was cotransfected with AONs into
293T cells. We found that AONs induced exon 9 skipping in a dosedependent manner (Fig. 3A). AONs were also able to increase
exon-9 skipping by 4-fold (p , 0.05) when cotransfected with the
BAC clone containing the IL23R gene used previously (Fig. 3B).
Exon 9 encodes the transmembrane region of IL-23R. Skipping of
exon 9 resulted in a frame shift with premature stop codon at exon
10 (Supplemental Fig. 1). Thus, the Δ9 protein lacks the sequence
that corresponds to the transmembrane and intracellular domains
but does contain the entire extracellular domain (32). Because this
Δ9 protein retains the natural signal sequence, we hypothesized
that Δ9 protein would be secreted from the cell and so would
represent a soluble IL-23Ra-chain. Soluble IL-23Ra was quantitated in the culture medium obtained from the BAC-transfection
experiments, using a sandwich ELISA that specifically detects the
soluble form of the IL-23Ra (34). The soluble IL-23Ra level was
increased from 0.5 to 0.8 ng/ml (p , 0.05) upon treatment with
AONs (Fig. 3C). This confirmed that targeting the SF2 binding
site on exon 9 mimicked the effect of the protective A allele,
resulting in elevated IL23RΔ9 mRNA expression with corresponding elevated secretion of the Δ9 protein.
AON targeting diminishes Th17 cell development
This observation was extended by examining the effect of AONs
on the natural expression of IL23R mRNA in human PBMCs.
Once again, AONs dose-dependently increased (p , 0.05) the
expression of Δ9 mRNA (Fig. 4A, 4B). To examine the specificity
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We had observed that the minigene carrying the variant allele
favored exon 9 skipping (Fig. 1B, 1C). In addition, binding sites
for SF2 and SRp40 were predicted in the sequences containing
common allele G and protective allele A, respectively (data not
shown). Because SF2 functioned as an activator whereas SRp40
played no obvious role during exon 9 splicing with either allele,
and overexpression of SF2 corrects the defect in exon 9 splicing
generated by the presence of the variant allele A (Fig. 2A), we
hypothesized that the mechanism of enhanced exon 9 skipping in
the protective allele’s minigene could be a reduced binding ability
of SF2 on exon 9 of the pre-mRNA (Supplemental Fig. 2).
To examine the SF2 binding affinity on the common allele and
variant allele sequences, an oligonucleotide pull down experiment
was performed (Fig. 2C). Equal amounts of biotin-labeled RNA
oligonucleotides containing either the common allele G or protective allele A were incubated with 293T whole-cell lysate. The
RNA/protein complex was then precipitated using streptavidin–
agarose. SF2 was successfully precipitated in this assay (but not
SRp40; data not shown). The protective allele’s oligonucleotide
bound SF2 more weakly than did that of the common allele, in
a dose-responsive manner (Fig. 2C, upper panel). A competition
experiment was performed to confirm this observation. The unlabeled G allele oligonucleotide competed efficiently with the
biotin-labeled G oligonucleotide, resulting in reduced SF2 pulldown; unlabeled A oligonucleotide failed to compete (Fig. 2C,
lower panel). Therefore, the results indicated that variant allele A
in exon 9 of the IL23R pre-mRNA binds SF2 less efficiently,
which results in enhanced exon 9 skipping.
1066
of AONs targeting, we conducted a semiquantitative fragment
analysis assay to evaluate their ability to enhance the Δ9 species
relative to other known and detectable splice forms. As shown in
Fig. 4C, treatment of human PBMCs with AONs resulted in an
elevation only of Δ9; full-length mRNA or Δ5, Δ8 or Δ8,9 were
unchanged or moderately diminished. Thus, AONs specifically
induced the expression of Δ9 without enhancing the expression of
other variants.
Th17 cells play an important role in Crohn’s disease and other
autoimmune disorders; IL-23R signaling is essential for terminal
maturation of Th17 cells (36–38). Therefore, we used our novel
AON approach to convert the cell surface IL-23R into the soluble
IL23RΔ9 form, using in vitro–differentiated human CD4+ T cells
to determine whether this could modulate their ability to develop
a Th17 phenotype and specifically, what the effect of AON on IL23–induced expression of IL-17A/F would be. CD4+ T cells were
purified and driven toward a Th17 phenotype cells under the influence of CD3/28 and a Th17 differentiation cytokine mixture
comprising IL-1, TGF-b, and IL-6 (Fig. 5A), as described previ-
FIGURE 4. AON treatment elevates D9 expression in human lymphocytes. Human PBMCs were isolated from fresh buffy coats over Ficoll–
Hypaque and transfected with control or IL23R-specific AONs (50 or
100 pmol). (A and B) RNA was prepared after 24 h and reverse transcribed.
AON treatment resulted in a dose-responsive increase in D9 mRNA by RTPCR and qRT-PCR. (B) *p , 0.05, +p = 0.051. (C). The global expression
profile of IL23R mRNA splice variants was quantified by fragment analysis.
Treatment with AON specifically elevated the D9 mRNA and had no effect
or diminished the other detectable IL23R mRNA species.
ously (34). Development of the Th17 phenotype was determined
by measuring IL17A and IL17F mRNA levels. Treatment with
AON increased the relative D9 mRNA by ∼4-fold (p , 0.05;
Fig. 5B). Exposure of developing cells to IL-23 induced IL17A
and IL17F mRNA by 2.5- and 4-fold, respectively (p , 0.05;
Fig. 5C). Of great interest was the observation that treatment of
these developing cells with AON reduced the IL17 mRNA induction by IL-23 by .50% for IL17F and almost to background
for IL17A. This result was mirrored in the AON-mediated reduction of secreted IL-17A or IL-17F proteins (Fig. 3C and data
not shown). Taken together, these data demonstrate that AON
treatment can upregulate the proportion of the soluble IL23RΔ9
form and reduce the development of functional human Th17 cells.
Discussion
The SNP rs11209026 is found in the 39-region of exon 9 of the
human IL23R gene. Allele A of this SNP is significantly underrepresented in patients suffering from Crohn’s disease and ulcerative colitis (10) as well as a number of chronic autoimmune
inflammatory disorders (11–15, 39), suggesting that it mediates a
protective effect.
To date, eight literature reports from seven independent groups
have addressed various aspects of the functional nature of this
SNP and the protective effect of its A allele. Although the (R381Q)
change in amino acids does not appear to be functional in and of
itself, there is unanimous agreement that there exists an A-related
protective phenotype of reduced IL-23 responsiveness in human
T cells; in at least one case, a clear allele-dosage effect has been
reported (10, 16–22). Despite these important associative and
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FIGURE 3. RNA AONs induce exon 9 skipping. (A) 293T cells were
transfected with the common allele minigene plasmid and cotransfected
with either control AON or IL23R exon 9–specific AONs. RNA was prepared after 24 h, and the levels of 8/9/10 or 8/10 transcripts were visualized
by RT-PCR. Increasing levels of specific AONs elevated the proportion of
8/10 transcript dose-responsively. (B) The experiment was repeated using
the IL23R gene in its full genomic context by transfecting 293T cells with
the BAC RP11-684P13 clone. A 4-fold elevation of the corresponding D9
transcript was induced by specific AON cotransfection, whereas the total
level of IL23R mRNA remained constant (*p , 0.05). (C) In these same
experiments, supernatants were surveyed for the presence of soluble IL23R protein (i.e., the translation product of the AM990318, D9 mRNA).
Levels of D9 protein were elevated following specific AON treatment
(*p , 0.05).
HUMAN IL-23R SNP PROMOTES ALTERNATIVE SPLICING
The Journal of Immunology
functional studies, the mechanism through which the A allele
affects the IL-23R and modulates IL-23 signaling remained unresolved until now.
Being mindful of the location of the polymorphism at the 39-end
of exon 9, and having previously demonstrated that a rich variety
of IL-23Ra splice variants exists (32, 33), we hypothesized that
instead of altering the function of the cell surface receptor, the
protective allele modulated splicing of the IL23R primary transcript.
Our minigene approach defined clear differences in the inclusion
of exon 9 in the transcript according to the presence or absence of
the protective A allele. They also showed that this effect was leaky,
in as much as both G and A allelic transcripts contained a proportion of the D9 message (greater in A). Thus, G/G individuals
will always have some D9 protein present (Fig. 1, Supplemental
Fig. 1) and similarly, homozygosity for A would never lead to
a complete absence of IL-23R protein from the cell surface, although it may be too sparse to be functionally effective. Interestingly, Di Meglio et al. (22) showed that A/A individuals were
almost unresponsive to IL-23. It may well be the case that under
the experimental conditions used in that report, the single stimulating dose of IL-23 (100 ng/ml) is insufficient to trigger the lower
density of functionally intact IL-23Rs on the surface of the
memory T cells in the face of the enhanced levels of soluble IL23R that would be present in these cultures; resolving this apparent conflict will require further experimentation.
Changes in mRNA splicing suggested a variation in the role or
efficiency of splice enhancer proteins in the processing of this
mRNA, and we identified SF2 as the key protein in this study.
Identical results were obtained with the minigene and BAC
approaches and confirmed in SF2 overexpression and knockdown
experiments. The results suggested that the protective allele A
binds SF2 less efficiently and competitive oligonucleotide pulldown experiments demonstrated that the protective allele consistently bound SF2 more weakly than did that of the the common
allele (Fig. 2). Interestingly, forced overexpression of SF2 appeared able to repair the splicing defect caused by allele A.
In Crohn’s disease and other disorders, the protective allele is
less frequent in the patient population. To mimic its protective
effects, we used AONs, designed to block the SF2 binding site on
exon 9 and the 59-splice donor site on intron 9 and so induce exon
9 skipping. We found that AONs induced exon 9 skipping in
a dose-dependent manner when directed against the allele G
minigene. AONs were also able to induce exon 9 skipping when
cotransfected with the BAC clone containing the IL23R gene
(Fig. 3). Exon 9 encodes the transmembrane region of IL-23R.
Skipping of exon 9 resulted in a frame shift generating a premature stop codon in exon 10 (Supplemental Fig. 1). Thus, the Δ9
protein lacks the sequence that corresponds to the transmembrane
and intracellular domains but does contain the entire extracellular
domain (34). Because the Δ9 protein has the natural signal sequence, we hypothesized that AON-induced Δ9 protein would be
secreted from the cell and function as a soluble IL-23R chain. This
was confirmed by ELISA (Fig. 3). Thus, targeting the SF2 binding
site on exon 9 mimicked the effect of the protective A allele,
resulting in elevated IL23RΔ9 mRNA expression with corresponding elevated secretion of the Δ9 protein. We extended this
observation by examining the effect of AONs on the natural expression of IL23R in human PBMCs. AON specifically induced
the expression of Δ9 without enhancing the expression of other
variants (Fig. 4). To verify that AON treatment of human PBMCs
would effect a modulation of Th17 cell function, we subjected
developing human Th17 cells to AON treatment (Fig. 5) and demonstrated that such treatment could reduce the maturation of functional human Th17 cells.
In conclusion, we show in this study that the protective
rs11209026 allele A disrupts a binding site for the splice enhancer
protein SF2, resulting in lower activity of this protein in allele A
carriers. This permits elevated expression of one particular splice
form, IL23RΔ9. Because exon 9 encodes the transmembrane domain of IL23R and its deletion joins exon 10 out of frame,
a truncated form of the protein, Δ9, is produced. This is unable to
anchor in the membrane and is consequently secreted from the
cell. As we have previously shown, this soluble external domain of
the IL-23R has the ability to interfere with IL-23 signaling (34).
Thus, the protective A allele functions to elevate the expression
of a soluble form of the human IL-23R chain, accounting for the
previous observations of Pidasheva et al. (19). These novel findings, although wholly consistent with the independent observations of others, provide a framework for hypothesis-driven testing
in healthy individuals of known genotype. Our data predict that
individuals bearing the A allele would have less IL-23R on their
cells’ surface [and this has already been independently observed
(19)]; in addition, they ought to secrete increased levels of soluble
IL-23R compared with G/G individuals, present a greater proportion of their mature IL-23R mRNA as lacking exon 9 and be
relatively refractory to the action of IL-23. Thus, our hypothesis
can readily be tested. Were this to be confirmed, recombineering
of the BAC RP11-684P13 (which contains the entire genomic
region of IL-23R and has the G allele) would allow the effect
of the A allele to be directly compared with G and allow the
mechanism of action to be explored in more detail.
Our findings therefore provide a molecular mechanistic explanation of the protective effect of SNP rs11209026 allele A. A strategy
to induce IL23RΔ9 mRNA and protein in the absence of the protective A allele was developed and demonstrated to diminish the
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FIGURE 5. AON treatment diminished the maturation of human Th17
cells. (A) Human CD4+ T cells were prepared from PBMCs by negative
enrichment and differentiated into Th17 cells as shown. AON (control or
IL23R specific) were transfected into these primary human CD4+ cells on
day 1; IL-23 was added on day 2, and cells were harvested on day 4. (B)
IL-23–treated, differentiated primary Th17 cells did not change their
overall level of IL23R mRNA when transfected with specific AONs but did
upregulate the proportion of D9 mRNA, demonstrating that AON treatment
can induce D9 expression in human Th17 cells (*p , 0.05). (C) mRNA
levels for IL17A and IL17F were quantitated by qRT-PCR in the in vitro–
differentiated, AON-treated Th17 cells. Neither control nor specific AONs
modified IL17 mRNA levels, and control AON did not prevent efficient
induction of IL17 mRNA by IL-23 in the absence of IL-23 treatment.
However, IL23R-specific AONs, previously shown to elevate D9 mRNA
and protein, inhibited the induction of IL17A and IL17F mRNA in human
in vitro differentiated Th17 cells (*p , 0.05).
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HUMAN IL-23R SNP PROMOTES ALTERNATIVE SPLICING
induction of functionally mature human Th17 cells in vitro. This
provides a new potential therapeutic avenue in Crohn’s disease,
using AON-mediated exon 9 skipping to minimize the further
maturation of Th17 cells.
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Acknowledgments
We thank HUMIGEN and Genesis Biotechnology Group colleagues for
helpful discussions.
Disclosures
The authors are full-time employees of HUMIGEN LLC, a for-profit
company. The authors are named inventors on granted and pending
U.S. patents describing the work presented here.
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