Curriculum Vitae
Name: Ankit Bhatta
Email Address: [email protected]
Degree and Date to be Conferred: M.S., 2013
Collegiate Institutions Attended:
University of Maryland, Baltimore, Graduate School, Baltimore, MD
Dates Attended: Fall 2012-Fall 2013
Degrees: Masters of Science
Concentration: Molecular Medicine
Degree conferred: 2013
Fergusson College, Pune University, Pune, Maharashtra, India
Dates Attended: 2007-2010
Degrees: Bachelors of Science
Concentration: Microbiology
Degree conferred: 2010
ABSTRACT
Title of Thesis: Understanding the expression of IL15 and IL15Rα Splice variants in
Celiac Disease
Ankit Bhatta, Master of Science, 2013
Thesis Directed by: Dean L Mann, M.D., Professor Primary Appointment- Pathology,
Secondary Appointments- Microbiology and Immunology
Celiac disease (CD) is an autoimmune disease of the small intestine. Different factors
such as dietary components, genetics, immune response, and loss of intestinal
permeability work in concert to contribute to the pathogenesis of CD. In CD the cytokine
IL15, pleotropic cytokine reported to participate in many facets of the immune system,
and its receptor alpha (IL15Rα) have been reported to be upregulated. IL15 is regulated at
multiple distinct levels of transcription, translation, intracellular trafficking and also by
its receptor. Disturbance in any of these regulatory mechanisms may lead to a
dysregulated IL15 production. Understanding the complex machinery of the
IL15/IL15Rα system will thus help us to better understand its role in the pathogenesis of
CD. Quantitative real time PCR (QRTPCR) was used to investigate if the upregulated
IL15 and IL15 receptor alpha reported in CD is a consequence of differential exon
expression. Peripheral blood mononuclear cells (PBMCs) from patient and control
samples were used to carryout QRT PCR on IL15 and IL15Rα mRNA to investigate their
exon expression profile. We found a clear and significant difference in expression of
IL15 mRNA and saw differential expression of specific exons in IL15Rα mRNA in
PBMC from the control and patient groups. We also report that the major immune cell
subset responsible for the cytokine and its receptor alpha mRNA are the monocytes.
Overall this is a good pilot study with some interesting findings, which with further
investigations can help give a better insight to disease pathogenesis. We believe that
future studies on these reports may assist in establishing IL15 and its receptor isoforms as
a novel biomarkers for CD.
Understanding the expression of IL15 and
IL15Rα Splice variants in Celiac Disease
by
Ankit Bhatta
Thesis submitted to the faculty of the Graduate School
of the University of Maryland, Baltimore in partial fulfillment
of the requirements for the degree of
Master of Science
2013
Acknowledgement
I would like to acknowledge everyone who helped me in the completion of my Masters
Research. Foremost, I would like to express my sincere gratitude to my advisor, Dr. Dean
Mann, for giving me an opportunity to work under his guidance. I am also very grateful
to Dr. Kristina Harris for her guidance, support and patience in training me during this
endeavor. I would also like to thank Dr. Joseph Briggs for his scholarly input,
encouragement, and support in developing my research skills.
I would like to thank my thesis committee members Dr. Frank Margolis, Dr. Kamal
Moudgil and Dr. Sergi P Atamas for being generous to be a part of the thesis committee,
and for their invaluable expertise.
I also would like to thank my family for their constant unconditional support. I am
extremely thankful to my colleagues and friends for their technical support and for their
feedback when writing the thesis.
iii
Table of Contents
I.
Introduction ................................................................................................................. 1
II. Methods ..................................................................................................................... 23
III. Results ....................................................................................................................... 30
IV. Discussion .................................................................................................................. 39
V. Summary and Future Direction ................................................................................. 44
VI. References ................................................................................................................. 47
iv
List of Tables
Table 1: Dietary grains and the type of prolamines they contain ....................................... 3
Table 2: Study group design ............................................................................................. 25
Table 3: Primer sequence .................................................................................................. 28
v
List of Figures
Figure 1: Microscopic image (Left) and H&E stained section (Right) of small-intestinal
mucosal biopsy.................................................................................................................... 5
Figure 2: The various cellular responses elicited by the infiltration of epitopes of dietary
glutens that leads to the response observed in pathogenesis of CD .................................... 8
Figure 3: Human IL15 gene and its exon intron representation ....................................... 12
Figure 4: Human IL15Rα gene and its exon intron representation................................... 15
Figure 5: Various mechanisms of IL15 delivery. ............................................................. 17
Figure 6: Graphical representation of total IL15 mRNA quantification using QRT PCR 31
Figure 7: Analysis of IL15Ra exon expression in PBMC of different groups using real
time PCR. .......................................................................................................................... 33
Figure 8: Graphical representation of total IL15 and IL15 SSP mRNA quantification in
cell subsets using QRT PCR ............................................................................................. 35
Figure 9: Graphical representation of IL15Ra mRNA quantification in cell subsets using
QRT PCR .......................................................................................................................... 37
Figure 10: Graphical representation of IL15Ra exons for monocytes .............................. 38
vi
I.
Introduction
Overview of celiac disease
Under physiological conditions the gut associated lymphoid tissue induces tolerance to
antigens [1]. An exception to this is seen in celiac disease (CD), where certain
susceptible individuals lose tolerance induced by dietary antigens. CD is a chronic
inflammatory enteropathy induced by dietary grains in certain genetically predisposed
individuals. Patients suffering from this disease show diverse clinical manifestations,
which typically include, but are not restricted to, discomfort in the digestive tract,
diarrhea, anemia, fatigue, and weight loss. CD is predominantly seen in Western
countries, where it is estimated to affect approximately 1% of the population [2, 3]. CD is
also evident in patients suffering from various autoimmune diseases such as rheumatoid
arthritis, Addison’s disease, Sjogren’s syndrome, Graves’ disease, etc. [4-6]. Different
factors such as dietary components, genetics, immune responses, and loss of intestinal
permeability work in concert to contribute to the pathogenesis of CD [7]. The only
treatment available is a dietary regimen free from gluten.
Genetic Factors
Genetic factors play a major role in the predisposition to CD. The involvement of a
genetic component in the disease manifestation has been corroborated by the high
predisposition of siblings and family members to the disease. A major genetic
predisposition has been shown to be associated with the human leukocyte antigen (HLA)
molecules [8] . The HLA, major histocompatibility complex (MHC) in humans, are a
group of cell surface proteins, which helps present antigenic peptides to the T helper cells
1
(TH cells). TH cells are a subgroup of lymphocytes which play an important role in
initiating the adaptive immune response. The HLA- DP,DM,DOA,DOB,DQ and DR
corresponds to the MHC II antigen presenting molecules, heterodimeric complex
composed of a β-chain and the α-chain, which presents extracellular antigens to T cells.
The β-chain is encoded by DQB1*0201 or *0202, whereas the α-chain is encoded by
DQA1*0501.
The primary HLA heterodimeric molecules associated with CD and expressed on the
surface of antigen presenting cells are the HLA-DQA1*0501, DQB1*0201 (DQ2)
molecules and the DQA1*0301, DQB*0302 (DQ8) molecules [9]. The HLA molecule
most strongly associated with CD is primarily HLA-DQ2, which is expressed in more
than 90% of patients. Together HLA-DQ2 and DQ8 have been found to occur in
approximately 95% of most patients [10]. The HLA-DQ8 that is found in the minority of
celiac patients is made up of a heterodimer formed by a β-chain encoded by the
DQB*0302 allele and an α-chain that is encoded by the DQA1*0301 allele. Less
frequently, combination of alleles DR7-DQ2 is observed in CD, where in the
DQA1*0201/DQB1*0201 encodes the DQ allele [11-13]. Some CD patients also have
DQ8 on a DR4 haplotype encoded by DQA1*0301/DQB1*0302 [13, 14].
Approximately 30% of the populations have the disease associated allele – DQ2-DQ8;
however, only 1% develop the disease [15-17]. This is because although the DQ2-DQ8
allele increases the risk of CD, it usually predisposes individuals to gluten sensitivity.
One of the reasons that this may happen is because the isoforms of the HLA formed in
some individuals are not able to present all the antigens of gluten completely; therefore,
these individuals have a smaller antigenic response. Some isoforms of the HLA on the
2
other hand, can present a large repertoire of antigenic peptides from gluten leading to
higher antigenicity [18-20]. A higher gliadin specific T cell response has been shown to
be elicited by APC with two copies of DQ2 compared to those with only one copy [20].
Individuals with homozygous DQA1*05/DQB1*0201 genes in cis or trans have a higher
disease manifestation compared to individuals carrying heterozygous DQ2/X or DQ8/X
[21]. Thus, though the HLA allele is a predisposing factor, it is not the only driving factor
in CD. Forty percent of the heritability of CD can be explained by the HLA alleles; the
remaining 60% is thought to be driven by non-HLA factors. The disease is not driven by
one factor alone; multiple conditions need to be working in concert for the disease to
manifest itself.
Etiologic agent
Dietary grains such as wheat, rye and barley rich in prolamines (Table 1), that are storage
proteins rich in amino acids: proline and glutamine, have been shown to trigger the
immune response on an enteric challenge in susceptible individuals [22].
Table 1: Dietary grains and the type of prolamines they contain
Dietary grains
Type of prolamines
Wheat
gliadins and glutenins
Rye
secalins
Barley
hordeins
Patients with a genetic predisposition are intolerant to cereals with high prolamine
content. A typical feature of CD is a gluten driven autoantibody response against tissue
3
transglutaminase tTG2 [23].The enzyme tTG2 is a member of the transglutaminase
family, which catalyzes Ca2+ dependent post translational modifications of glutamine
residues. In the intestinal lamina propria tTG2 deaamidates gliadin, due to its high
glutamine residue, producing peptides with high affinity for HLA-DQ2 and HLA-DQ8,
which can be efficiently presented to T lymphocytes [14, 24-27]. The increase in tissue
transglutaminase-2 has been attributed to the leakage of gliadin in the intestinal
epithelium. Some of the biological effects induced by gliadin include: the reorganization
of actin cytoskeletal components in the epithelial cells of intestinal origin [28], an
increase in mucosal permeability [29] , an increase in nitric oxide and other cytokines in
peritoneal macrophages[30], and maturation of bone marrow derived dendritic cells
[31].The increase in permeability allows gliadin to easily translocate to the intestinal
lamina propria, thus making the dietary antigen accessible to immune and non-immune
cells.
Pathology
The major histological changes associated with CD include lesions in the small intestine,
villous atrophy (Figure 1), increased intra epithelial lymphocytes (IEL) and crypt
hyperplasia [32]. There is also an increase of other cellular infiltrates such as plasma
cells, eosinophils, mononuclear cells and others. The truncation of villi leads to nutrient
malabsorption and thus failure to gain weight. Long term CD leads to vitamin deficiency,
osteoporosis, dermatitis herpetiformis, arthritis, other extra-intestinal complications and
occasionally death secondary to neoplastic complications.
4
Figure 1: Microscopic image (Left) and H&E stained section (Right) of smallintestinal mucosal biopsy.
Top panel represents normal biopsy with numerous villi. Bottom panel shows microscopic and
stained intestinal section representing villous atrophy in intestinal biopsy from individual with
CD.
Panels reprinted from Gastroenterology [33] with permission from the American
Gastroenterological Association.
Diagnosis of CD can be made by the presence of specific anti-transglutaminase
antibodies, IgA anti-endomysial antibodies and IgG gliadin autoantibodies. However,
5
testing for the IgA anti-endomysial antibodies are not completely reliable, since small
populations of CD patients are IgA deficient and thus can give a false negative result. The
only available definitive treatment is to follow a gluten free diet regimen, which leads to
both clinical and mucosal recovery in most CD patients. However, some patients progress
to refractory disease, which increases the risk of gastrointestinal malignancy [34].
Immune Response
The immune system is composed of a complex series of interactions among various cells.
The immunological events responsible for the aberrant response that leads to CD have
not been fully determined. Gluten has been shown to disrupt the immune homeostasis in
the gut of patients with CD. In addition to normal circulating immune cells, the intestinal
immune system has been shown to have mucosal immune cells, which comprise of cells
such as lamina propria mononuclear cells (LPMC) and IEL. The mucosal immune cells
and the peripheral circulating immune cells orchestrate the complex immune reaction
existing in the gut.
LPMC are a part of the mucosal immune system comprising of different types of gut
resident immune cells that provide a barrier and defensive function in the intestine. Early
during CD, epithelia of patients are infiltrated by IEL [35], and these cells are activated
independent of the T cell receptor (TCR). The majority of IEL present in healthy
individuals are TCRαβ IEL. The remaining IEL are made up of CD7+CD3- IEL
(including NK) 13%, TCRγδ IEL 10% and CD8αα IEL 10% [36]. This distribution is
permanently skewed in CD, with an increase in TCRγδ IEL and the loss of CD7+CD3IEL. TCRαβ IEL is also increased in celiac patients and this increase has been shown to
6
be gluten driven [37-39]. Thus, IEL serves as an important histological marker in
diagnosis.
CD is generally considered to be an adaptive immune response of T cells directed
towards gliadin. Tissue transglutaminase leads to deamidation of gluten and thus converts
neutrally charged glutamine to negatively charged glutamic acid. This assists in the
formation of peptides that are tailored to fit in the HLA DQ2 and DQ8 peptide binding
grooves of the antigen presenting cells (APC) with a higher affinity, which leads to
presenting the antigen to TH cells in the lamina propria of the small intestine. It has been
shown that the gliadin induced lymphocytes are predominantly of the TH1 phenotype and
produce interferon gamma (IFN-γ) along with other cytokines. The transcription factor Tbet, which drives the differentiation to TH1 phenotype, is upregulated in CD [40]. Flow
cytometric analysis of peripheral blood mononuclear cells (PBMC) from untreated celiac
patients were shown to have an increased pSTAT1 expression in the monocyte subset
and a T-bet increase in the CD4+ T cell, CD8+ T cell and CD19+ B cell subset of PBMC
[41]. IFN-γ also leads to the secretion of CXCL10, which acts as a chemo attractant for
monocytes/macrophages, DC, T cells, and NK cells.
Gliadin stimulated CD4+ TH cells have been shown to prime B cells and induce their
differentiation into plasma cells that produce the specific anti-gliadin and anti-tissue
transglutaminase antibodies. The immune response leads to the infiltration of
lymphocytes in the lamina propria of the gut and also induces production of pro
inflammatory cytokines known to cause remodeling of tissue. PBMC from children with
untreated CD have been shown to produce type 1 cytokines after gluten challenge [42].
7
Thus from what has been described so far, gliadin or any prolamine has the ability to
induce many cellular responses that contribute to the pathogenesis of CD (Figure 2).
Figure 2: The various cellular responses elicited by the infiltration of epitopes of
dietary glutens that leads to the response observed in pathogenesis of CD
Adapted from Clinical and Experimental Immunology doi:10.1111/j.1365-2249.2005.02783.x
IL-15R; interleukin-15 receptor, MIC-A; MHC class I polypeptide-related sequence A, COX-2;
Cyclooxygenase 2, HLA; human leukocyte antigen, IEL; intraepithelial lymphocyte, LPMC;
lamina propria mononuclear cell, DC; dendritic cell, TCR; T cell receptor, tTG; tissue
transglutaminase, IFN; interferon, FasL; FAS ligand, Ig; Immunoglobulin.
IFN-γ and the other cytokines produced during this immune response lead to the
alteration of key mucosal functions, activation and release of matrix metalloproteinase
8
(MMP) [43] and other enzymes which causes damage to the mucosal epithelium.
Experiments with anti IFN-γ have been shown to prevent gluten induced mucosal damage
[44].
It has been reported that IL15 induces most of the epithelial changes in response to
gliadin challenge. Dietary antigen gluten has been shown to up regulate IL15 in the
epithelium and lamina propria of the gut of celiac patients. The cytokine has been shown
to drive the expansion and survival of innate immune IEL, which in turn leads to an
autoimmune damage of the gut. Though the downstream effects of IL15 have been
described, the precise role of the ligand and receptor interaction has yet to be elucidated
in CD. Understanding the role of IL15 in CD has proven to be a challenge. Not only does
the cytokine have many effector functions, little is understood about the receptor and
ligand interaction. The pleotropic function of the cytokine IL15 and its role in the
development of CD are discussed below.
Interleukin 15
The cytokine Interleukin 15 (IL15) is a pleotropic pro-inflammatory cytokine, discovered
in 1994 by two different laboratories [45, 46] and was able to induce IL2 independent T
cell growth. It belongs to the four alpha helix bundle family, which includes many other
cytokines, some of which are IL2 , IL6, GMCSF [47].
Though being similar to IL2 in function, IL15 has a different spectrum of target cells on
which they are expressed, when compared to IL2 receptors which are mostly present on
T-cells [48]. Activated lymphocytes produce IL2, whereas IL15 are produced by the
antigen presenting cells and stromal cells of numerous tissues. The cellular distribution of
9
IL15 mRNA differs from IL2, since IL15 mRNA has shown to be distributed in a broad
spectrum of immune and non-immune cells. The IL15-IL15 receptors are not only
important for regulation of immune and stromal functions, but also in the development of
immune subsets. IL15-IL15Rα deficient mice have been shown to have a significant
reduction in the number of CD8 T cells, NK cells, NKT cells and IEL [49, 50]. IL2 has
been shown to cause activation induced cell death of αβT cells [51], whereas IL15 on the
other hand, has been shown to suppresses apoptosis of cytokine deprived activated Tcells [52] and lead to the activation and maintenance of memory T cells [53].
The proinflammatory response of IL15 has been reported in murine and cell line
experiments, where the cytokine lead to an induction of nitric oxide synthase (iNOS)
which contributes to immune defense [54, 55]. Upregulation of IL15 has also been
reported to be induced by bacterial and viral pathogens [56, 57]. IL15 has been shown to
induce an innate anti-tumor response independent of NK and CD8 T cells [58]. The
cytokine IL15 has also been described as a skeletal muscle anabolic agent [59] and to
support the proliferation of intestinal cells [60].
The human IL15 locus maps to chromosome 4 region q25-35 [61]. The IL15 gene
(Figure 3) encodes 9 exons and 8 introns [62], which includes the alternative exon 4a that
was later described [63-65]. Though being widely distributed, the expression of IL15 is
regulated at the level of transcription, translation and intra-cellular trafficking [64, 6668]. This tight post transcriptional regulation was reported in monocytes exposed to LPS,
where only IL15 mRNA was increased without a significant increase in circulating IL15
[67]. The dominant regulatory mechanism of IL15 has been shown to be post
translational and at the level of intracellular trafficking [69].
10
Two IL15 isoforms exist that arise due to alternative splicing of IL15 pre-mRNA (Figure
3), differing only in the length of their signal peptide [63, 65, 69], which differ in their
intra-cellular localization [70]. The IL15 isoform signal peptides are encoded by more
than one exon. The long signal peptide (LSP) form contains a sequence of 48 amino
acids, which is encoded by exons 3 through 5 of the IL15 gene [46]. The short signal
peptide (SSP) form contains a 21 amino acids signal peptide which shares 11 amino acid
sequence with LSP encoded by exon 5 and is also encoded by exon 4 -5 [65, 68, 71]. This
happens due to the presence of exon 4a which inserts premature termination codons and
an alternative downstream translation site with a poor Kozak consensus sequence.
11
Figure 3: Human IL15 gene and its exon intron representation
Adapted from BioEssays28:362–377,2006
the splicing of which leads to the formation of two isoforms differing in length of signal peptide
that dictates the trafficking of these two isoforms.
12
It has been shown that the different isoforms of IL15 have different tissue distributions.
The expression of IL15 SSP mRNA has been described in the testis, heart, and thymus.
The IL15 LSP mRNA has been shown to be present in the skeletal muscle, placenta,
heart, lung, liver, thymus, and kidney [64, 65, 69]. The presence of these signal peptide
variants dictates the trafficking of these two isoforms. Isoforms with LSP are shuttled
through the ER, whereas SSP are found localized in the nucleus and cytoplasm [72, 73].
The differential trafficking points to the mechanism that regulates the secretion of IL15.
The translation of IL15 mRNA is affected by the presence of multiple AUG codons. The
IL15 LSP mRNA contains multiple AUGs upstream of the actual translation start site that
have been shown to dramatically reduce translational efficiency. The translation of IL15
SSP mRNA is not restricted by such codons. Another complex mechanism regulating
IL15 presentation is by existing as a heteromer with its receptor; which is possible since
IL15 has been shown to carry two binding sites for IL15 Rα [74]. The presence of two
isoforms of IL15 suggests that each isoform may have a distinct biological role.
Interleukin 15 Receptor
The IL15 receptor is a heterotrimeric receptor, which is made up of a unique α chain [48]
and also utilizes the IL2β and γ chain [75] that belongs to the hematopoietin receptor
superfamily [76-78]. The IL15 receptor alpha (IL15Rα) contains a sushi domain required
for cytokine binding. The hinge, stalk, trans-membrane region and the cytoplasmic tail
are all structurally similar to those present on the IL2 receptor [48, 79]. The IL2 receptor
β chain is utilized exclusively by IL2 and IL15 receptors. Whereas, the common γ chain
is utilized by many other cytokines, such as IL4, IL7, and IL9 [80-83]. IL15 binds with
13
high affinity to its receptor α chain, even in the absence of the IL2β and the common γ
chain. However, high affinity binding for IL2 is possible only when all the components of
its receptor are present [79].
The human IL15Rα locus maps to chromosome 10 in regions p14-15, the IL2Rα locus is
also present in this same region [79, 84]. The IL2Rβ and common γ chain are located on
chromosome 22 and the X chromosome [85] respectively. The IL15Rα gene (Figure 4)
was reported to be encoded by seven exons which could lead to the formation of eight
different isoforms by alternative use of these exons [86]. However, recently a new exon
present between exon 1 and 2, exon 2A, has been described [87], which was shown to
prevent the cleavage of cell surface expressed IL15/IL15Rα heterodimeric complex. IL15
has been reported to form a heteromeric complex with IL15Rα intracellularly and
extracellularly, which has been reported to assist in trafficking, presentation and stability
of the cytokine and for the purpose of this dissertation will be called as the IL15-IL15Rα
system interchangeably. Exon 2A was shown to be deleted from one of the C-terminal
isoforms of IL15Rα that lead to the presence of secretable IL15/IL15Rα heterodimeric
complex. With the recent characterization of the exon 2A, there are now four different
kinds of splicing events (Figure 4) shown in humans: loss of exon 2, loss of exon 2A, loss
of exon 3 and alterative usage between exon 7 and exon 7`[86]. Exon 2 encodes the sushi
domain and also a nuclear localization signal (NLS), the isoforms lacking exon 2 have
been found in non-nuclear compartments. Another difference between the IL15 receptor
and the IL2 receptor is the presence of a single sushi domain in case of IL15 receptor in
contrast to two sushi domains in IL2 receptor [79]. The sushi domain also known as the
glycoprotein-1 motif contains four cysteines forming two disulfide bonds in 1-3, 2-4
14
patterns. It is present in some complement and adhesion molecules. The sushi domain is
essential for ligand binding by the IL15 receptor [88]. Exon 3 encodes the linker region,
which leads to improved binding ability of the receptor to the ligand [89]. Deletion of this
domain has been shown to cause an inability of the receptor to bind IL15 [86]. The
cytoplasmic domain of the receptor is encoded by exon 7, and is not required for binding
or signaling, but required for cell surface expression of the receptor. The IL2 receptor β
and the common γ subunits are integral for cell signaling, and utilize the JAK/STAT
pathway of signal transduction. The IL2 β functions through the JAK1 phosphorylating
STAT3, whereas STAT5 is phosphorylated by JAK2 in the case of common γ chain [90,
91].
Figure 4: Human IL15Rα gene and its exon intron representation.
Adapted from BioEssays28:362–377, 2006
Various isoforms of Human IL15Rα due to alternative splicing viz. Δ2; exon two missing, Δ3;
exon three missing, Δ4; exon four missing, Δ2,3; exons two and three missing, Δ3,4; exons three
and four missing, Δ3,4,5; exons three, four and five missing.
15
The presence of three distinct subunits of IL15 receptor is a limiting factor, since all three
receptors subunits at many times, are not expressed on the same cell. For this reason the
IL15 and its receptor system exhibit a diverse pattern of ligand and receptor interaction as
listed below –

One such mechanism is trans- presentation / juxtacrine signaling (Figure 5A).
Here the cell surface bound IL15/IL15 Rα heteromeric complex can present the
cytokine to neighboring cells expressing the weak affinity IL15Rβ/γ [92]. These
surface bound IL15-IL15Rα receptor complexes, get recycled by endocytosis and
presented again on the surface [92].

Circulating IL15 has also been shown to exist in complex with soluble IL15Ra
complex (Figure 5C), and such complexes have been shown to have an improved
half-life [93].

In addition to its soluble form, IL15 also exists as a membrane bound nonsecretable form [94]. Such membrane bound IL15 (Figure 5E) has been reported
to exist independent of the receptor in monocytes, fibroblasts and keratinocytes
[11, 95].

Reverse signaling has been shown to occur on membrane bound IL15, in which
signaling has been shown to be induced by an anti-IL15 antibodies or soluble
form of IL15Rα. Reverse signaling leads to activation and phosphorylation of the
MAPK (mitogen associated protein kinase) family members and the FAK (focal
adhesion Kinase) members that are responsible for signal transduction [96].
16
Figure 5: Various mechanisms of IL15 delivery.
Adapted from ELSEVIER Immunology Letters127 (2010) 85–92
IL15 gets associated with IL15Rα in the Endoplasmic Reticulum (ER) and is transported to the
surface where it can encounter IL15Rβγ in either [A] trans or (on a different cell) [B] cis (on the
same cell). [C] Soluble IL15Rα associated with IL15 can act as an agonist to stimulate
neighboring cell and even cells at a distant location. [D] Unbound soluble IL15Rα can also act as
an antagonist by sequestering free IL15 thus antagonizing IL15 activity. [E] IL15 can also be
present on cell surface of various cell types, and it does so independent of IL15Rα.
The receptor subunits are not only important for transduction, but they also serve other
functions for the IL15-IL15Ra system. It has been shown that similar to LSP the
secretion of IL15 SSP is chaperoned by the IL15Ra and this complex leads to higher
cytokine concentration in circulation and improved half-life of the cytokine [97]. Given
the pleotropic nature of IL15, such complex mechanisms act as checkpoints in the IL15
17
and IL15Rα system that helps regulate IL15 response to prevent immuno-pathological
reactions.
Interleukin 15 in celiac disease:
The immune system plays a major role in surveillance against pathogens and thus is
important for host defense. The pleotropic cytokine IL15 has been shown to play a role in
both innate and adaptive immunity, which leads to a broad spectrum of immunological
outcomes. It has also been reported to be involved in the maintenance of memory T cells
(CD4 and CD8) [98]. IL15 further influences immune outcome by its involvement in
protecting lymphocytes and dendritic cells against apoptosis, by acting as an inducer of
anti-apoptotic genes of the Bcl-2 family of proteins [52, 99]. Various inflammatory and
autoimmune diseases such as multiple sclerosis [100], diabetes [101], inflammatory
bowel disease [102], have been shown to be a consequence of upregulated IL15 [103].
Uncontrolled expression of IL15 in transgenic mice was shown to cause autoimmune
intestinal damage, similar to that seen in CD, and antibody mediated blockade of IL15
was reported to reverse such intestinal damage [104]. Gliadin has been shown to activate
the innate immune response, and also lead to upregulation IL15 in the epithelium and
lamina propria of the gut of celiac patients [105]. The expansion of IEL is one such
hallmark of innate immune cells seen in CD. IL15 has been shown to be a potent inducer
of IELs [106]. The presence of IL15 in gut has also been reported to induce an increase in
intraepithelial TCRγδ+ and CD94+ cells [107]. It has been shown in in-vivo experiments
analyzing celiac patients and healthy controls that the dysregulated IL15 leads to TCR
independent cytotoxicity targeted towards enterocytes. This has been attributed to the
18
oligo-clonal expansion of CD8 T cells that have been reprogrammed into lymphokine
activated killer cells or NK like cells by IL15 [108]. Lamina propria and intestinal
epithelium from active celiac patients have been reported to have an overexpression of
IL15 on their surface [109]. Enterocytes have been shown to produce and respond to
IL15 that acts as a potent stimulator of IEL [60, 106].
MICA (MHC Class –I chain related gene) is a cell surface protein related to MHC Class
–I family, but they do not associate with the β-2-microglobulin protein seen in MHC-I
complex. Active celiac patients and patients challenged with gliadin or its peptide p31-49
have shown to have an overexpression of stress molecule MICA on the surface of
epithelial cells, this effect has been shown to be relayed by IL15. These molecules are
induced by stress and recognized by NK cells, NKT cells and most subtypes of T cells.
IELs from active celiac patients having NKG2D and CD94 on their surface, which
recognize MIC present on the mucosa, are selectively increased [110]. The cytokine also
induces the expression of NKG2D on IELs that enables the IELs to attack MICA
expressing enterocytes [108, 111]. An antibody against IL15 was shown to abrogate this
gluten mediated MICA expression [112].
Gluten challenge in CD patients has been shown to cause an accumulation of monocyte
derived cells in their intestines [113, 114]. Furthermore, IL15 exposure to monocytes has
been shown to drive TH1 and TH17 response to gliadin in healthy individuals, who are
normally well tolerated to wheat gliadin [115]. In addition to the T cell mediated
response mediated by IL15 in CD, Yokoyama et al. have demonstrated a B cell mediated
pathological response in transgenic mice expressing human IL15 in enterocytes. There is
extensive plasmacytosis in the lamina propria and the presence of auto-antibodies against
19
tissue transglutaminase2 in these transgenic mice, pointing to the possibility that IL15
drives both T cell and B cell response in CD [116].
It was shown that in active celiac patients, IL15 interfered with T regulatory cells (Tregs)
and disrupted their function [117] and that IL15Rα was overexpressed on Tregs from
patients. The sustained proinflammatory response seen in CD has been attributed to an
impairment in the TGFβ signaling, which acts as an immune modulator. IL15 was shown
to inhibit the TGFβ signaling in a Smad dependent manner in T cells and in the intestinal
mucosa of celiac patients [118]. Thus, it could be possible that the pathologic immune
response seen in CD is due to impairment of the TGFβ signaling, leading to a disruption
in immune homeostasis and as a result the overt proinflammatory response. IL15 has also
been shown to contribute to the proinflammatory response by conferring resistance
against the immune modulatory action of Tregs on effector T cells [119].
Mention et al. [109] have shown in enterocytes isolated from biopsies that thought there
is a significant increase in surface expression of IL15 in active celiac patients compared
to controls, there was no difference in IL15mRNA between the groups [109]. The
mechanism that regulates the surface expression of IL15 has yet to be elucidated in CD.
One of the reasons for IL15 upregulation in active celiac patients could be due to the
changes in post transcriptional regulation, or as a consequence of IL15Rα mediated
stability of the cytokine. Trans-presentation by IL15Rα has been suggested for the
membrane bound expression of IL15 [92], but this has yet to be fully confirmed .
Bernardo et.al have shown that duodenal biopsies of active celiac patients compared to
controls have higher IL15Rα mRNA expression. Celiac patients even on a gluten free
diet, thus, respond more efficiently to IL15 compared to non-celiac individuals [120].
20
The difference in pathology seen between healthy patients and active celiac patients may
thus be a consequence of differential responsiveness to the pleotropic cytokine IL15.
Genetic and/or environmental factors that control IL15 and its receptor alpha expression
may be the reason behind the hypersensitivity of IL15 in the intestine and likely to
participate in the pathogenesis of CD. The overall goal of our research was to shed light
on the Cytokine and its receptor in the context of CD. The tight regulation and posttranscriptional modification of IL15 and its receptor α chain by alternative splicing has
not yet been explored in CD. The pleotropic nature of IL15 ranging from breakage of
self-tolerance, inhibition of apoptosis in [121], to its inflammatory role in other
autoimmune diseases in other models has intrigued us to investigate further and
characterize its role in CD. It has been reported that IL15 induces most of the epithelial
changes in response to gliadin challenge. Investigators have also previously shown that
innate immune activation was inhibited by anti-IL15 antibodies [122]. Therefore
identifying the mechanism of upregulation of IL15 in CD may contribute to deciphering
targets that are the cause of pathogenesis and hence important for therapeutic
intervention.
Hypothesis:
IL15 expression and secretion is a tightly regulated system and is known to be controlled
by the presence of different isoforms of IL15 and IL15Rα. This cytokine has also been
described to be involved in CD, where it is markedly upregulated on gluten challenge.
The upregulated cytokine has been shown to influence immune and stromal components
21
of the gut. We hypothesize that the gliadin induced activation of IL15-IL15Rα system in
CD is due to the aberrant isoform expression that leads the pro-inflammatory response.
Objectives:
Aim 1: To analyze the differential exon expression of IL15 and IL15Rα in celiac
patients
We wanted to investigate if there are differential splice variants of the cytokine and its
receptor in peripheral blood mononuclear cells from various celiac patients. The presence
of a particular exon in an isoforms may lead to different effector functions of the IL15
cytokine system. Thus we wished to investigate if the dominance of certain exon
expression profile in celiac patients compared to healthy controls could be the
predisposing factor, and if differential exon expression is affected by gluten consumption.
Aim 2: To analyze differential exon expression in immune cell subsets so as to
answer if the isoform expression is a particular cell subset effect or a total PBMC
effect.
It has been shown that IL15 is overexpressed in CD and has been reported to drive the
adaptive and innate immune response. We wish to characterize which immune cell
subset, if any, contribute to this overexpression. Since IL15Rα has been reported to affect
the secretion and stability of the cytokine, we wished to investigate if there are
differential exon expressions in IL15 and IL15Rα in various immune cell subsets.
22
II.
Methods
Study Subjects
Though HLADQ2/8 is strongly associated with the disease, it fails to explain why many
individuals with the allele never develop CD on gluten exposure. Therefore, we wanted
to investigate if there is a gliadin mediated alteration of the IL15 isoforms in the various
HLA allele individuals. We stratified the patients into four groups (Table 2) on the basis
of HLA typing (DQ2/8 + and DQ2/8-), health status (Healthy control and celiac), gluten
consumption and DQ2/8+ allele (healthy patients on gluten diet, treated with gluten free
diet and active on gluten diet), in an attempt to understand the relationship of these
etiologic factors in the manifestation of disease.
Patients used in the study were Caucasians with an average age of 39 (21-69) and
included both male and female individuals. Patients were separated into various groups
using HLA typing, tissue transglutaminase IgA antibodies (Phadia), Endomysial
antibody, and confirmed by biopsy in the case of active celiac patients. For tissue
transglutaminase IgA antibodies the threshold for antibody titers used were – less than 7
(negative); greater than 10 (positive). All celiac patients were confirmed by biopsies and
tested positive.
Healthy controls comprised of two groups that were separated on the basis of HLA
DQ2/8. The DQ2/8+ healthy group comprised of 7 individuals with an average age of 40
ranges being from 28 to 60. For the purpose of this experiment these individuals will be
called Healthy DQ2/8+. The non DQ2/8 healthy group comprised of 12 individuals with
23
an average age of 34 ranging from 21 to 59. For the purpose of this experiment these
individuals were called healthy non DQ2/8 or DQ2/8 negative.
HLA DQ2/8+ patients on a gluten free diet for less than 16 weeks and those who tested
positive for Endomysial antibody and tissue transglutaminase IgA were grouped together,
and for the purpose of this experiment these individuals were called active CD patients.
There were 8 patients in this group with age ranging from 21 to 52 and an average age of
34.
HLA DQ2/8+ patients on a gluten free diet for more than 6 months and those who tested
negative for Endomysial antibody and tissue transglutaminase IgA were grouped together
and for the purpose of this experiment these individuals were called treated CD patients
or CD on gluten free diet. The age of the 7 patients in this group ranged from 33 to 69
with the average age being 48.
The lack of access to gut tissue samples limited our experiments only to PBMC from
patients.
24
Table 2: Study group design
CONTROLS
PATIENTS
Group name
Non-DQ2/8
DQ2/8
Active Patients
CD on GFD
HLA
DQ2/8 -ve
DQ2/8 +ve
DQ2/8 +ve
DQ2/8 +ve
Average Age (years)
34
40
34
48
Age range (years)
21-59
28-60
21-52
33-69
No. of subjects
12
7
8
7
GFD time
None
None
<16 weeks
>6 months
Endomysial Ab
Negative
Negative
Positive
Negative
tTG IgA
Negative
Negative
Positive
Negative
Cells
Peripheral blood mononuclear cells (PBMC) were isolated from celiac patients and
healthy donors whole blood by density gradient centrifugation in Lymphocyte Separation
Medium (ICN Biomedicals Inc.). PBMC were viably cryopreserved in RPMI-1640 media
(Invitrogen Corp.) containing 20% human AB serum (hAB) (Gemini Bioproducts) and
10% Dimethylsulfoxide (Sigma) using an automated cell freezer (Gordinier Electronics),
and stored in the vapor phase of liquid nitrogen until used. All individuals gave informed
consent for peripheral blood drawn for this study. The study protocol was approved by
the Institutional Review Board at the University Of Maryland School Of Medicine. Upon
25
thawing PBMC, cell viability was determined by trypan blue exclusion. All cells were
cultured in RPMI-1640, 5% heat inactivated FBS.
Cell Separation
Positive selection of monocytes was carried out using MACS Separator Human CD14
Microbeads and MS columns (Miltenyi Biotec), cells not separated by the column were
labeled CD14- monocyte depleted leukocytes, herein called lymphocytes. All separations
were carried out according to manufacturer’s instructions. And the purity of separation
was validated by flow cytometry.
RNA Extraction
Total RNA was extracted from cell populations using TRizol (Life Technologies) reagent
according to the manufacturer’s recommendations, with the following modification.
Following chloroform treatment, the aqueous phase was transferred to a new tube and
mixed with equal volume of 70% ethanol. Total RNA was then purified using a
QIAGEN RNeasy Mini Kit according to the manufacturer’s instructions. The total RNA
concentration was assessed by measuring absorbance at 260nm using a
spectrophotometer (NANO DROP spectrophotometer ND-1000, Thermo Scientific,
USA). The 260/280 ratio was also determined as a measure of purity and all ratios were
between 1.8 and 2.0. Working stocks of total RNA were prepared at a final concentration
of 10ng/ul in nuclease free H2O. All samples were stored at -80°C until used.
26
cDNA Preparation
Reverse transcription was carried out using 100ng of total RNA using a BIO RAD iScript
cDNA Synthesis Kit according to the manufacturers recommendation. cDNA synthesis
reactions were done using a BIO RAD iQ5 Thermocycler and run using the following
parameters – 25°C for 5 minutes; 42°C for 30 minutes; 85°C for 5 minutes; 4°C hold
After completion of the reverse transcription, total volume of cDNA mixture was brought
up to 100ul using nuclease free H2O so as to have a final cDNA concentration of 1ng/ul.
Samples were stored at -20°C to -80°C until further use.
Real Time PCR
Quantitative real time PCR was carried out on 2ul of cDNA samples using BIO RAD iQ
SYBER Supermix according to manufacturer’s instructions. cDNAs were analyzed in
triplicates. Primer concentration was 200nM each for sense and anti-sense primers.
Primer sequence and annealing temperature are given below (Table 3). BIO RAD iQ5
optical system software Ver2.1 was used to analyze raw data in order to determine
threshold cycle (Ct) values. Expression of 18S RNA was used as an internal reference
transcript for normalization between samples, which was validated to be invariantly
expressed in all samples. The final quantification of cDNA was done using the ∆∆Ct
method [123] by normalizing to 18S RNA followed by comparison of relative
expression to Control .
27
As there are at least eight different isoforms of IL15Rα, primers were designed to span
across two exons. Primer name and sequences are listed below (Table 3). IL15 was
quantified using primers for Total IL15 and IL15 SSP.
Table 3: Primer sequence
Transcript
(RefSeq)
18S
Sequence
Name
18S for
18S
18S rev
Exon 1 sense
IL15Ra
1d for
IL15Ra
2b rev
IL15Ra
1-2A
IL15Ra
2b rev
IL15Ra
2 for
IL15Ra
3 rev
IL15Ra
3 for
IL15Ra
4 rev
IL15Ra
4 for
IL15Ra
5 rev
Exon 2 anti-sense
Exon 1-2A sense
NM_001256765
Exon 2 anti-sense
Exon 2 sense
Exon 3 anti-sense
Exon 3 sense
Exon 4 anti-sense
Exon 4 sense
Exon 5 anti-sense
Oligo Sequence (5’3’)
5’ – acc cgt tga acc
cca ttc gtg a –3’
5’ – gcc tca cta aac
cat cca atc gg –3’
5’– gac gcg ggg cat
cac gtg cc –3’
5’– gtt gtc cag tgg
gcg aca ttc –3’
5’- gac gcg gga tgc
aag aga cag g – 3’
5’– gtt gtc cag tgg
gcg aca ttc –3’
5’– gaa tgt cgc cca
ctg gac aac –3’
5’– gga gag gct ctc
tgg ctg tgg –3’
5’– cca cag cca gag
agc ctc tcc –3’
5’– ctc atg act gct
tat ctc tgt gg –3’
5’– cca cag aga taa
gca gtc atg ag –3’
5’– gtg gcc ctg tgg
ata cac ac –3’
28
Exon
Pair
18S
Annealing
Temperature (°C)
56.5
1d-2b
60
1-2A2
2-3
58
3-4
58
4-5
58
Table 3: Primer sequence Continued
Exon 6 sense
Exon 7 anti-sense
IL15 Total
(NM_172175.2,
NM_000585.4)
IL15 Total
(NM_172175.2,
NM_000585.4)
IL15 SSP
(NM_172175.2)
IL15 SSP
(NM_172175.2)
IL15Ra
6 for
IL15Ra
7 rev
IL15
total for
5’– tgg cta tct cca
cgt cca ctg t –3’
5’– cat ggc ttc cat
ttc aac gct gg –3’
5’– aac aga agc caa
ctg ggt gaa tg –3’
IL15
total rev
5’– ctc caa gag aaa
gca ctt cat tgc –3’
IL15
SSP for
IL15
SSP rev
5’– agt ttg ccc aaa
gca cct aac cta –3’
5’– cct gca ctg aaa
cag ctg cac aaa –3’
6-7
56.5
Total
60
SSP*
60
* Short Signal Peptide (SSP)
Statistics
Statistical analysis were conducted using GraphPad (Prism software) and for all statistical
graphical representation. Data were presented as mean value ± Standard deviation.
Wilcoxon signed-rank test was used for paired samples. When multiple groups were
involved, one way ANOVA with Holm-Sidak adjustment for multiple comparisons was
used. Significance was considered at P < 0.05.
29
III.
Results
Results supporting Aim 1 -To analyze the differential exon expression of IL15 and
IL15Rα in celiac patients
IL15 SSP mRNA is significantly higher in Active celiac patients.
To analyze whether celiac patients showed specific splicing of IL15 Gene leading to
preferential isoform expression, using quantitative real time PCR, we investigated the
sample cDNA library with primer pairs targeted for SSP and the total IL15 cDNA.
Healthy, Treated, Active and non DQ2/8+ samples were used. There was no difference in
total IL15 between the groups under investigation (Figure 6A).
However, as analyzed by one way ANOVA, a significant difference was seen in healthy
vs. active, where there was ~3 fold increase in IL15 SSP. And ~ 2 fold increase in IL15
SSP in healthy non DQ2/8 vs. active (Figure 6B). No significant difference was found
between treated CD and controls.
30
Figure 6: Graphical representation of total IL15 mRNA quantification using QRT
PCR
(A) The various groups showed no difference in the amount of total IL15 present.
(B) IL15 SSP mRNA levels was shown to be different when comparing DQ2/8+ and nonDQ2/8+ healthy patients with active celiac patients. This difference was statistically significant.
Data indicates mean value with error bars representing s.d.
Active patients are HLA DQ2/8+ individuals, who were on gluten free diet for less than
16weeks. Whereas treated patients belong to the same HLA, but have been on a gluten
free diet for more than 6 months. The result suggests that the differential SSP mRNA
expression is induced in susceptible HLA DQ2/8+ individuals on gluten challenge. And
this increase in IL15 SSP mRNA decreases on gluten withdrawal as seen in treated
31
patients (CD on GFD). However, healthy DQ2/8+ patients though being on a normal diet
do not show this SSP increase.
Differences in IL15Ra exon expression
The IL15Ra gene encodes seven exons and can lead to the formation of eight different
isoforms of the receptor.
To determine if the disease state causes preferential splice variants of IL15Ra gene, we
investigated the sample cDNA library from DQ2/8 + Healthy, CD on gluten free diet,
Active and non DQ2/8 Healthy by quantitative real time PCR. We found that nonHLADQ2/8 healthy samples had a higher expression of IL15Rα exons 1-2, 2-3 and 3-4
when compared to healthy HLA DQ2/8+ samples (Figure 7). Active celiac patients
showed higher expression of IL15Rα exons 3-4 compared to CD on gluten free diet and
DQ2/8+ healthy individuals. There were no differences in IL15Ra exons 4-5 or exons 6-7
between any of the groups.
When we looked at non DQ2/ vs. DQ2/8 we saw that for the first 3 exon profiles, the non
DQ2/8 patients had higher expression of the exon compared to the DQ2/8. This suggests
that though there is environmental trigger (gliadin) related effect within the DQ2/8
population of patients, there is also a constitutive genetic difference between the two
DQ2/8 populations for IL15Rα.
32
Figure 7: Analysis of IL15Ra exon expression in PBMC of different groups using
real time PCR.
Figure represents log fold difference between various groups relative to non DQ2/8 healthy
patients. Data indicates mean value with error bars representing +/- s.d.
33
In the previous experiments we saw a gluten dependent change in expression of the exons
for IL15 and its receptor between HLA DQ2/8+ active and CD groups on GFD. This
change in IL15 and its receptor exons was not seen in healthy individuals of the same
HLA DQ2/8+ who were on a regular diet. We next wanted to investigate what immune
cell subsets are involved in the differential exon expression. We used HLA DQ2/8+
samples, 3 samples each from the healthy DQ2/8 on a regular diet and CD on a gluten
free diet, to make our experimental group. Two populations of cells were derived from
PBMC as described in methods, which were selected monocytes (CD14+) and leukocytes
depleted of monocytes (CD14-)/lymphocytes. We used quantitative real time PCR to
analyze the sample cDNA library using primer pairs targeted for the IL15 and IL15α
exons.
34
Results supporting Aim 2 - To analyze differential exon expression in immune cell
subsets
Monocytes are the predominant producers of total and SSP IL15 mRNA in PBMC
from celiac patients
s
te
cy
o
ph
m
Ly
s
te
cy
o
ph
m
Ly
s
te
cy
o
on
M
s
te
cy
o
on
M
Figure 8: Graphical representation of total IL15 and IL15 SSP mRNA
quantification in cell subsets using QRT PCR.
Higher amount of total IL15 and SSP IL15 mRNA in monocytes compared to paired
lymphocytes isolated from the same individuals (n=6). Data indicates mean value with error bars
representing s.d.
QRT PCR was used to analyze IL15 mRNA and IL15Rα mRNA using the cDNA library,
in the lymphocyte and monocyte populations. Monocytes showed significantly higher
35
levels of total IL15 mRNA compared to lymphocytes (Figure 8). Monocytes are the
predominant producers of IL15 mRNA. Similar results were seen for IL15 SSP mRNA.
There was no intrinsic difference in exon expression for IL15 mRNA, under the disease
free state, in healthy and treated patients. Data was analyzed using paired non-parametric
two-tailed t-test.
Monocytes compared to lymphocytes have higher IL15Rα mRNA in PBMC from
celiac patients
We next wanted to quantitate IL15Rα exon mRNA level between monocytes and
lymphocytes from DQ2/8+ samples. Figure 9 represents a log (2) fold difference between
paired lymphocytes and monocytes. The Wilcoxon sign ranked test of the two matched
samples (non-parametric) was used. All exon pairs of IL15Rα, excluding exon 1-2, were
shown to be upregulated in the monocyte population compared to lymphocytes (Figure
9).
Recently a new exon, exon 2A present between exon 1 and 2 has been described in
human monocytes and DC [87]. We were also able to validate the exon expression, by
quantitative real time PCR, in monocytes from the DQ2/8+ individuals.
36
Figure 9: Graphical representation of IL15Ra mRNA quantification in cell subsets
using QRT PCR.
Monocytes were shown to have higher mRNA for all the exon pairs, excluding exon 1-2,
compared to lymphocytes isolated from same individuals (n=6). Data indicates mean value with
error bars representing s.d. *p<0.05
37
Figure 10 represents, only the log (2) fold difference of monocyte relative to matched
lymphocytes isolated from the same individuals, for all exon pairs of IL15Rα.
Figure 10: Graphical representation of IL15Ra exons for monocytes
Graphical representation of IL15Ra exons for monocytes relative to matched lymphocytes
isolated from the same individuals (n=6). Data indicates mean value with error bars representing
s.d.
38
IV.
Discussion
The low detectable levels of IL15 have been thought to be attributed to multiple
regulations at the level of transcription, translation, intracellular trafficking and secretion
[56, 66, 69]. While the increased expressions of IL15 and IL15Rα have been investigated
in CD, the role of the various isoforms of the cytokine and its receptor system in the
pathogenesis of the disease is poorly understood. We proposed that preferential isoform
expression due to an alternative splicing event of the IL15 and IL15Rα transcript could
be the reason for the increased expression of the cytokine system observed. We analyzed
gene expression at the RNA level of IL15 and its receptor IL15Rα PBMCs from celiac
patients compared to healthy controls.
The mature form of both SSP and LSP are the same. Of the two isoforms of IL15, the
LSP IL15 is either secreted or present as a cell surface bound cytokine. SSP on the other
hand is not secreted, but is localized diffusely in the cytoplasm and also in the nucleus,
and is thought to play a role in its transcriptional regulation [65]. The low level of
secretion of the cytokine makes it difficult to detect IL15 in the serum and supernatants
of cultured cells [124]. IL15 has been reported to also exist as a cell surface bound form,
which allows the cytokine to be presented in trans to neighboring cells (Figure 5A).
However, the mechanism of its expression on the cell surface has been under debate.
Dubois et al [92] report that IL15 is present on the cell surface in association with
IL15Rα. However, others have shown that IL15 exists as a membrane anchored integral
membrane protein, which exists independent of IL15 Rα [95]. Membrane bound IL15
39
can cause receptor clustering at immunological synapse, and is thought to make such
membrane anchored IL15 more potent than soluble IL15. Others have shown that LPMC
from active CD patients secrete IL15 even in the absence of any stimuli. The release of
the cytokine was significantly amplified in active CD than in treated CD and controls,
suggesting that active patients have a lower threshold for cytokine activation and release
[125]. Even with all these results, it is difficult to understand what regulates the
dichotomy between the two isoforms. The function of the SSP isoform has yet to be
characterized, some have proposed that the SSP isoforms are involved in regulatory
functions [65, 70]. Furthermore, the pleotropic functions of the cytokine suggest that
though the mature forms of the isoforms are same, they may have distinct functions as
immature isoforms.
IL15Rα has been shown to form intracellular complexes when co-expressed with IL15,
which leads to increased stability of the cytokine [126]. The heteromeric complex of
SSP/IL15Rα was shown to increase the stability and secretion of the cytoplasm restricted
peptide. Such receptor mediated rescue was more efficient for IL15 LSP/IL15Rα
complex, where IL15Rα co-expression leads to the higher secretion of the peptide with
greater half-life (t1/2), when compared to SSP [97]. Bergamaschi et al. [97] have shown
that IL15 SSP acts as a regulator by competing with IL15 LSP for IL15Rα, which leads
to lower levels of secreted IL15.
40
We report that PBMC from active celiac patients have higher levels of IL15 SSP mRNA
compared to healthy controls and treated patients on GFD. These results depict a
preferential expression of IL15 SSP mRNA driven by gluten consumption, in active
patients but not in CD on GFD. However, there was no significant difference in the level
of total IL15 between the various groups. Since IL15 SSP primers could not be designed
for QRT PCR, we can only make conjectures about the questions regarding the level of
LSP mRNA. IL15 SSP and IL15 LSP together make up the total pool of IL15 mRNA
present. When translated, the levels of SSP and LSP are relative to the total IL15 present.
Since the total IL15 levels remain unchanged, but the SSP levels increase in active CD,
this would mean that LSP levels are reduced in active CD. Thus in celiac patients, dietary
glutens may regulate the levels of both SSP and LSP mRNAs. If glutens only increased
SSP mRNA and the LSP mRNA remained constant, then total IL15 mRNA would also
have gone up. However, since total IL15 mRNA remained the same and SSP mRNA
increased, we suggested that gluten may also induce the reduction of IL15 LSP mRNA.
Because of the unique functions of IL15Rα in signaling and regulation of the cytokine,
the receptor has been a target for investigation in CD. Bernardo et al. have investigated
IL15Rα mRNA in duodenal biopsies, using primers that detected non secretable isoform
containing exon 3. They report that there was a higher expression of IL15Rα mRNA in
active CD compared to treated individuals and controls [120]. This IL15Rα was found to
be mostly associated with the nuclear membrane [120], which was similar to the results
reported previously [86]. Bernardo et al. have reported that IL15Rα expression in cells
from duodenal biopsies was modulated by IL15 stimulation. In another study IL15Rα
41
was also shown to be higher in intra epithelial lymphocytes (IEL) from active CD, which
favor a TH1 profile of cytokine production when treated with IL15 [125].
Our experiments on PBMCs did not show an overexpression of IL15Rα in the patient
groups compared to healthy controls. We report here from our experiments that PBMC
from active CD patients have higher expression of IL15Rα exons 3-4 compared to CD
on gluten free diet and DQ2/8+ healthy patients. Exon 3 has been shown to contribute to
increased binding of the cytokine [89]. In our experiments we saw no difference in
IL15Ra Exons 6-7 between the groups, which encodes the domain for non-secretable
membrane bound receptor. However, the IL15Rα exon expression profile between the
non DQ2/8 healthy group and the active CD group was similar. These results indicate the
need for more investigation to understand, how the exon expression profile of isoforms
may lead to homeostasis in one case and disease in the other.
A gluten challenge to CD patients has been shown to cause an accumulation of monocyte
derived cells in the intestine of patients [113, 114]. Exposure of IL15 monocytes have
been shown to drive TH1 and TH17 responses to gliadin in healthy individuals, who are
normally tolerant to wheat gliadin [115]. We report that in the PBMC, most of the
IL15/IL15Rα mRNA is derived from the monocytes. This is evident from the QRT-PCR
experiments which showed significantly higher levels of total IL15 and SSP IL15 in
monocytes. Most exons of the IL15Rα transcript were also expressed at higher levels in
monocytes compared to lymphocytes, when analyzed by QRT-PCR. Our studies are
consistent with the experiments on monocytes addressing the recently characterized exon
2A that is responsible for cell surface associated IL15Rα [87]. We were able to show that
IL15 Rα exons 1-2A-2 are significantly higher in mRNA from enriched monocytes
42
compared to lymphocytes from HLA DQ2/8+ samples. Thus in monocytes, such IL15 Rα
would mostly be membrane associated due to the exon 2A encoded domain. This
assumption fits well with the results presented by Musso et al. [95], who reported that
monocytes contain membrane bound, biologically active IL15/IL15Rα complexes.
The cells used in these experiments were from healthy DQ2/8+ and treated patients.
Thus, the quantification of exon 2A needs to be done on monocytes from active celiac
patients to analyze, what gluten consumption does to the exon expression profile of
DQ2/8+ patients.
43
V.
Summary and Future Direction
The data presented here suggests that gluten drives higher expression of IL15 SSP
mRNA in active celiac patients. Differential expression of exons 3-4 was also shown to
be preferentially induced by gluten in active celiac patients, and monocytes were shown
to be the primary source of IL15 SSP and total mRNA. Though the experiments
performed here do not permit us to draw definitive conclusions; they do present some
interesting observations. These results lead us to believe that the differential expression
of exon mRNA was observed due to gluten related effects on patients with the HLA DQ
2/8 genetic background. However, despite being on a normal diet, healthy DQ2/8+
patients did not show this differential expression in mRNA. This suggests that healthy
DQ2/8+ individuals are either protected from the antigenic activation by gluten, or they
are intrinsically different from celiac patients in their ability to recognize gluten as an
antigen. Furthermore, healthy non DQ2/8 individuals express a similar expression
compared to the active CD group for IL15Rα. But the healthy non DQ2/8 group, does not
show a differential expression of IL15 SSP mRNA. This could mean that IL15 SSP may
be an essential driving factor for CD.
This poses an intriguing question as to why IL15 SSP mRNA expression is gluten
dependent for celiac patients and not for healthy patients of the two genetic backgrounds.
Our results on DQ2/8+ (healthy and treated CD on GFD) monocytes and lymphocytes
show that monocytes are the population of cells predominantly responsible for the
differential exon expression. Additionally we describe a functional assay to investigate
44
the multiple exon expression profile for IL15 and IL15Rα, and thus can be used for future
studies on gut samples.
Our data only answers the relative expression of an exon cassette between groups. Since
we do not know the absolute quantification, we cannot make deductions about the
relative differences between the various exon cassettes within a group that would be
required to make assumptions of the type of isoform predominating. Despite our findings
of the differential expression of exons for IL15 and IL15Rα from mRNA in PBMC, we
do not know about the stability of the transcripts, their translational success or efficiency.
Based on these results, we need to determine if the response seen in active CD patients
can be recapitulated in treated patients on GFD by stimulation with gluten. Additionally,
in most cases the HLA DQ2/8 allele possessing population are gluten intolerant and do
not have CD. Therefore future investigations need to be carried out on non DQ2/8 active
celiac patients, to see if they too can recapitulate the same response as seen in DQ2/8+
active celiac patients. This is because though the HLA DQ2/8 allele is associated with
CD, it is only a predisposing factor, since 30% of the population have the disease
associated allele – DQ2-DQ8; however, only 1% develop the disease [15-17].
If so, future studies will need to be carried out to determine the functional role of IL15
SSP, and why SSP mRNA is overexpressed in patients and not in healthy controls. The
higher expression of IL15Rα exons in healthy non DQ2/8 compared to healthy DQ2/8+
needs to be further investigated to understand the role IL15Rα plays.
Even though our results are confounded by these factors, they do present with some new
and interesting observations, and they emphasize why there is a pressing need for further
45
investigation in this system, to validate and draw conclusions from these observations.
Since IL15 is regulated at multiple distinct levels of transcription, translation,
intracellular trafficking and also by its receptor, disturbance in any of these regulatory
mechanisms may thus lead to an increased IL15 production. Understanding the complex
machinery of the IL15/IL15Rα system will help us to better understand its role in the
pathogenesis of CD, and assist in the development of possible therapeutic strategies.
46
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