Download Th17 development

Links Between IL-7Rα
Ligands and Th17 Cells
in Immune Disorders
Masters’ Thesis by
Tamar Tak, Bsc
January 2009
Supervisors:
Dr. Joel A.G. van Roon
Angela Bikker, Msc
Department of Rheumatology & Clinical Immunology, UMCU
Contents
Abstract ................................................................................................................................. 3
Introduction ........................................................................................................................... 4
The Th1/Th2 paradigm ...................................................................................................... 4
New kids on the block ........................................................................................................ 4
Th17 development................................................................................................................. 5
Murine Th17 development ................................................................................................. 5
Human Th17 development ................................................................................................ 6
Th17 effector functions in healthy individuals ........................................................................ 8
Th17cells target specific pathogens ................................................................................... 8
IL-17: a cytokine important for the immune system ............................................................ 8
IL-17 receptor .................................................................................................................... 9
IL-22 in defence and epithelial barrier regulation ............................................................... 9
Multiple roles for IL-21 ......................................................................................................10
Th17 and immunopathology .................................................................................................11
Systemic Lupus Erymathosus (SLE).................................................................................11
Experimental autoimmune encephalomyelitis (EAE) and MS............................................11
Dermal inflammation, psoriasis and acanthosis ................................................................12
Primary Sjögrens Syndrome .............................................................................................12
RA and its experimental models .......................................................................................12
Evidence for protection by Th17 cells or cytokines ...........................................................14
IL-7Rα ligand functioning in developing and mature T cells ..................................................15
IL-7Rα associates with the γc or TSLPR-chain .................................................................15
IL-7Rα, γc and TSLPR signalling ......................................................................................15
IL-7Rα is crucial in lymphocyte development ....................................................................16
IL-7Rα ligands in peripheral T-cell homeostasis ...............................................................17
IL-7 concentrations are regulated by consumption............................................................18
IL-7 and TSLP influence antimicrobial defence by influencing Th subtype commitment ....18
The link between IL-7Rα ligands and Th17 cells in immunopathology ..................................20
IL-7Rα ligand Th subtype commitment in immunopathology .............................................20
IL-7 promotes expression of other cytokines resulting in positive feedback loops .............21
Concluding remarks .............................................................................................................23
References ...........................................................................................................................24
2
Abstract
The interleukin (IL)-7 receptor α-chain (IL-7Rα) is expressed on effector T cells, including T
helper (Th) 17 cells. Its ligands IL-7 and thymic stromal lymphopoietin (TSLP) are suggested
to play a role in regulating naive T cell differentiation into the different Th subsets. Of these
subsets, Th17 cells are most recently discovered and associated with several immune
disorders. This thesis discusses the IL-7Rα ligands and Th17 cells in health and disease and
the relation between the two cytokines in immune disorders. IL-7 is proposed to promote Th1
and Th17 generation. In turn, cytokines produced by these cells prevent Th2 generation.
TSLP drives Th2 development and inhibits differentiation into Th1 or Th17 cells. The balance
between these different Th subtypes may be disturbed in immune disorders and could
therefore be modulated by IL-7Rα ligands. As well, IL-7 induces secretion of proinflammatory
cytokines TNF-α, IL-17 and IL-21. These cytokines have the potential to induce a positive
feedback loop, resulting in more cytokine secretion and Th17 generation. Hence, they might
contribute to immune disorders.
3
Introduction
Every day, the body is threatened by many pathogens which differ greatly in appearance,
route of entry etc. In order to protect from this wide range of threats, the immune system has
many different branches cooperating in one complex system. A first important distinction that
can be made is between the innate and adaptive immune system. The adaptive part
recognizes pathogens by certain patterns that are conserved between species of pathogens
and are crucial for its function. Exactly what patterns an individual’s immune system will
recognize are largely genetically determined and are stable during its lifetime. The adaptive
or specific immunity is the other side of the coin: it does not recognize patterns, but highly
specific epitopes. Also, it is not (entirely) genetically determined and can learn to cope with
new pathogens.
The Th1/Th2 paradigm
Adaptive immunity in turn has traditionally been split in T helper (Th) 1 and Th2 responses,
two different subtypes of CD4+ T helper cells1. Th1 mediates defence against intracellular
pathogens like viruses and parasites, while Th2 is associated with protection against
extracellular pathogens1. Differentiation to Th1 eventually leads to cytotoxic T cell (CTL)
activity and Th2 differentiation leads to a humoral immune response involving antibody
producing B cells, mast cells and eosinophils.
Both Th cell types arise from a common precursor, the naïve Th cell. The decision to mature
into one or the other type depends on cytokine signals present in the environment inducing
gene expression. Presence of interleukin (IL) -12 skews differentiation into Th1 and IL-4 on
the other hand results in Th2 cells. Cytokines secreted by one type of cell inhibits
differentiation of precursors in the other cell subset2. Interferon-gamma (IFN-γ) for instance is
produced by the Th1 subset and down-regulates proliferation of Th2 cells. In turn, Th2
cytokines IL-4 and IL-10 can block IL-12 production, thereby reducing differentiation into
Th12. This cross-regulation results in a balance between the two subsets with their
differential effector mechanisms. Distortion of this balance has been implicated to lie at the
basis of diseases like allergy asthma and autoimmunity3.
New kids on the block
Although the existence of T-cell subtypes gave more insight in the mechanisms underlying
immunity, it could not explain the body’s unresponsiveness to non-pathogenic antigens like
intestinal bacteria. These phenomena turned out to be mediated by so-called regulatory T
cells (Tregs), cells suppressing immune responses4. Tregs are important in the immune
system, but lie beyond the scope of this paper and will not be discussed thoroughly.
Besides Tregs, another new Th subtype has been identified, called Th17 for its IL-17
secretory capabilities5. After its discovery, it was soon implied to be involved in many immune
disorders and will therefore be subject of this thesis.
The discovery of these new T cell subtypes clearly indicate that the immune system is more
complex than proposed in the Th1/Th2 paradigm. Similar as in the Th1/Th2 paradigm, it is
proposed that different Th subtypes are involved in different immune diseases. Therefore,
understanding of how this balance is regulated is important to understand the mechanisms
underlying these diseases.
In this regulation of the differentiation into Th subtypes the Interleukin (IL)-7 Receptor αchain (IL-7Rα) and its ligands IL-7 and thymic stromal lymphopoietin (TSLP) are involved.
The IL-7Rα is expressed on effector T cells, but not on Tregs (van Roon, unpublished data)
and its different ligands can drive differentiation in different Th subtypes. Therefore, IL-7Rα
and its ligands will be discussed as well and integrated with our current knowledge of Th17
cells in the context of immunopathology.
4
Th17 development
In 1989 Mossman and Cofman6 first proposed the Th1/Th2 paradigm, in which Th1 cells
assist in eradicating viruses and other intracellular pathogens, and Th2 cells are important in
humoral immunity and control of parasite infections7. However, this paradigm could not fully
explain pathological exacerbations of the immune system. Attempts to understand this led to
the discovery of a new Th subset, called Th17 after its first discovered cytokine
Murine Th17 development
As stated above, Th1 and Th2 cells depend on their effector cytokines for differentiation from
naive T cells8. These same cytokines (interferon (INF)-γ and IL-4 respectively) block Th17
development. In mice, Th17 cells are shown to depend on TGF-β and IL-6 for differentiation
9
. IL-6 is a proinflammatory cytokine produced in large amounts by innate immune cells upon
activation of pattern recognition receptors8. In contrast, transforming growth factor (TGF)-β is
regarded as an anti-inflammatory cytokine since loss of the cytokine leads to deadly immune
overactivation10 and was already known to be required for differentiation of naive T cells into
Tregs. Therefore, IL-6 prevents generation of Tregs in favour of Th17 cells9. Upon stimulation
with these cytokines, naive T cells upregulate retinoic acid-related orphan receptor (ROR)γt
and, as more recently discovered, RORα in a signal transducer and activation of transcription
(STAT) 3-dependent way. RORγt in turn, acts as a master regulator driving differentiation to
the Th17 phenotype (Fig 1)11-13.
Also involved in Th17 generation is IL-21, this cytokine produced by natural killer (NK) and
Th17 cells is described to drive differentiation of naive T cells into Th17, although not as
strong as IL-614. IL-21-/- mice Th17 cells are still generated, indicating that the cytokine is not
essential for Th17 development14. However, Th17 cell numbers in these mice are
significantly reduced, indicating that the cytokine does play a role in development8. During
differentiation it is produced in large amounts by newly formed Th17 cells and acts as a
positive feedback loop driving other naive cells to differentiate into Th17 as well (fig 1. 8)8, 14.
After differentiation of the Th17 cell, another cytokine, IL-23, comes into play. Its presence is
required by IL-6 and TGF-β stimulated cells (like Th17 cells) in order to be able to induce
tissue inflammation and it downregulates expression of IL-1015. This led to the suggestion
that IL-23 stabilizes the
phenotype of newly formed
Th17 cells8. IL-10 is one of
the cytokines produced by
Tregs, but not by Th17
cells16,
which
might
indicate that the cells used
for this experiment were
not fully developed into
Th17 cells, despite IL-6
stimulation. This close
affiliation of Tregs and
Th17 cells starts early in
differentiation; both cell
types require TGF-β, but
presence of IL-6 or IL-21
determines
whether
a
Fig 1. Stimulation of naive T cells with a combination of TGF-β and IL-6
naive
cell
will
be
or IL-21 induces expression of RORγt and differentiation into Th17 cells.
proinflammatory (Th17) or
IL-21 promotes proliferation of newly differentiated Th17 cells and is
immunosuppressive
produced by these same cells, forming a positive feedback loop. IL-23, in
turn, stabilizes the Th17 phenotype and the cell starts producing all of its
(Treg). Even after being
characterizing cytokines8.
fully differentiated, murine
5
Tregs can be reprogrammed into Th17 cells by DCs activated with an antibody crosslinking
B7-DC (B7-DC Xab)17 or by adding a mixture of Th17 inducing cytokines (IL-1, IL-21 and
most potently: IL-6)11. IL-6 was also shown to downregulate FOXP3 expression11. This
important Treg transcription factor in turn, can bind to RORγt, preventing Th17 differentiation.
Taken together, these findings implicate that (in mice) generation of Tregs and Th17 cells is
related and depends on presence of IL-6. As inducers of both cell types inhibit differentiation
into the other subset, this could result in a balance between Tregs and Th17 cells, similar as
described for Th1/Th2 helper cells.
Human Th17 development
The origin and differentiation signals of human Th17 cells are very different from those in
murine cells. First TGF-β, of undeniable importance for murine Th17 development, shows
contradicting effects. Several studies show Th17 development independent of TGF-β, but
this might be due to bovine cytokine present in the culture medium used in these
experiments18. Others indicate TGF-β to be indispensable for Th17 generation19-21. One of
these19 shows that TGF-β upregulates RORγt, but simultaneously inhibits its ability to
upregulate IL-17 expression. A second paper shows TGF-β, IL-6, IL-21 and IL-1β all to be
required, but to have differential effects, dependent on the relative concentrations of the
cytokines20. More important, lack of TGF-β induced a Th1-like profile in this experiment. The
third article21 states that both IL-21 and TGF-β are indispensable for differentiation and
induce RORγt expression.
To complicate this model even further, a recent article shows Th17 cells to originate
exclusively from a certain subset of CD4+ naive Tcells expressing CD161, a transmembrane
protein with an up-to-day unknown function22. These cells did not depend on TGF-β for
RORγt expression, but expressed it constitutively, in contrast to the CD161- cells which did
not express it at all and were restricted to Th1 or Th2 commitment. In the CD161+ cells
expression of both RORγt and Tbet (a transcription factor critical for Th1 development) was
enhanced upon stimulation with IL-23 and IL-1β, but not by IL-6. Addition of TGF-β did
enhance RORγt expression and decreased Tbet, but was not indispensable. Furthermore,
the proportion of IFN-γ producing (Th1-like) cells decreased. In turn, IL-4 or IL-12 diminished
RORγt expression in favour of Tbet (Th1) or GATA3 (Th2) respectively18. Taken together,
this could implicate that although TGF-β is not a critical factor for Th17 development. Instead,
it decreases skewing towards a Th1 phenotype in favour of Th17 (Fig 2). Another aspect in
Th17 development supporting this theory is the existence of Th cells expressing both IFN-γ
and IL-17, so-called Th17/Th1 cells23. It suggests even more flexibility between the two
subsets, with variations in TGF-β concentrations determining whether Th1, Th17 or
Th17/Th1 cells arise.
In summary, the exact factors required for human Th17 development remain unclear, but
several factors, being IL-1β, IL-6, IL-21, IL-23, and TGF-β, stimulate differentiation and could
be required. As well, since Th17 only arise from CD161+ cells, processes leading to
expression of the protein are likely to promote Th17 generation as well. Which of these
factors is most important in Th17 differentiation and how skewing towards this subtype
exactly works, remains to be shown conclusively.
6
IL-1β/IL-6/
IL-21/IL-23
Fig 2. In absence of TGF-β,
stimulation with a combination
of IL-1β, IL-6, IL-21 and IL-23
induces both Th1 and Th17
development, but Th17 cells
arise exclusively from CD161+
progenitors. In presence of
TGF-β, Th1 differentiation is
inhibited,
favouring
Th17
development. Adapted from18.
IL-1β/IL-6/
IL-21/IL-23
7
Th17 effector functions in healthy individuals
Th17cells target specific pathogens
Just like Th1 and Th2 cells, Th17 cells are believed to be required for clearance of a certain
type of pathogens. Several different classes of pathogens are shown to initiate a Th17
response; Gram-positive, Gram negative and acid-fast bacteria , fungi and even the parasite
toxoplasmosis gondii24-29. A major role of Th17 appears to be attracting other immune cells,
being neutrophils, monocytes, CD4+ memory T cells and B-cells30. Knockouts of the IL-17
cytokine or receptor, as well as mice treated with blocking antibodies, show a defective
granulocyte response and enhanced susceptibility to infection with K. pneumoniae24, 31. In
these mice lung epithelial tissue shows decreased production of CXC chemoattractants,
stemm cell factor (SCF) and Granulocyte-Colony Stimulating Factor (G-CSF) in response to
infection32(Fig 3). Besides attracting neutrophils, IL-17 has been shown to indirectly attract
Th1 effector cells to M. tuberculosis granulomas by regulation of CXCR3 ligands MIG, IP-10
and I-TAC27. In this experiment, however, IL-17 was not required for the primary control of M.
tuberculosis, since no increased susceptibility for infection was seen in knockout mice27.
However, other Th17 cytokines might be relevant defence against M. tuberculosis, but were
not tested in this experiment. For another intracellular pathogen, Listeria monocytogenes, IL17 knockouts showed no increased susceptibility either32. This indicates that IL-17 is more
important for defence against extracellular pathogens rather than intracellular. Since Th17
cells appear to be suited best for targeting extracellular pathogens, cooperation with Th2
cells appears likely. As mentioned above, however, IL-4 inhibits Th17 development and in
turn, TGF-β prevents Th2 differentiation.
Besides the clear role of IL-17 in host defence, another Th17 derived cytokine, IL-22, is of
importance as well. This cytokine is indispensible in fighting off extracellular pathogens32, 33
and will be discussed more thorough below.
As mentioned above, fungal infections are shown to induce a Th17 response28, but the role
of Th17 in fungal infections remains a matter of debate. IL-17 has been shown to
downregulate Th1 responses towards fungal infection34 in vivo and to inhibit antifungal
activity in vitro16. As well, two separate experiments show a lack of IL-1735 or IL-2136 to
improve immunopathology caused by fungal infection. Taken together, these findings
indicate a role in pathology instead of host defence upon fungal infection. However,
neutrophils are indispensable in defence against fungi and IL-17 attracts these cells, a
positive role in fighting off fungi cannot be excluded.
For an adequate antiviral response, Th1 cells are known to be indispensable. Since IL-17 is
known to inhibit maturation of other Th subsets, IL-17 induced inhibition of Th1 development
could be beneficial for a virus. Indeed, an IL-17 homologue is described in the genome of a
herpesvirus and is considered relevant for the virus’ persistence in its host37, 38. As well, a
vaccinia virus complemented with the IL-17 gene showed increased replication in vivo, but
not in vitro, indicating Th17 responses to be beneficial for evading the immune system
instead of increased viral replication per se39.
Summarizing, Th17 responses probably hamper antiviral immunity and play an unknown role
in fighting off fungal infections. On the other hand, Th17 responses beneficial and
indispensable in immunity against extracellular bacteria and, in some diseases, cooperate
with the Th1 subset in clearing intracellular infections.
IL-17: a cytokine important for the immune system
IL-17A is the first member of the IL-17 family discovered and has four other family members,
designated IL-17B-F16. Though, since IL-17B-D are not produced by Th17 cells and the origin
of IL-17E is still unknown, this thesis will focus on the other two family members. IL-17A and
IL-17F are found as IL-17A homodimers or IL-17A-F heterodimers. Specifically blocking IL17A has more effect than blocking IL-17F40. Therefore, we will speak of the combination of
homo- and heterodimers as IL-17 in this article.
8
As described above, IL-17 signalling induces production of CXC chemokines, G-CSF and
SCF. The chemokine production can synergize with TNF-α signalling, giving a higher
production than induced the sum of both cytokines16.
Besides indirectly attracting immune cells, IL-17 can also contribute to granulopoiesis. IL17RA knockouts only show a slight decrease in neutrophils counts, but a significantly
impaired recovery of neutrophil counts after sublethal irradiation41. Systemic overexpression
of IL-17, in turn, leads to massive haematopoiesis caused by the induction of G-CSF and
SCF production42.
IL-17 receptor
The first discovered receptor for IL-17 is IL-17RA. It is has a 293 amino acid (aa)
extracellular domain, a short transmembrane (23aa) domain and a long intracellular tail
consisting of 525aa38. In mice, IL-17RA mRNA is expressed in the lungs, kidneys, spleen and
liver as well as in isolated fibroblasts, mesothelial, epithelial and various myeloid cells38. Mice
lacking the receptor are unable to bind IL-17 to T or B lymphocytes24. In humans, receptor
mRNA is found in epithelial cells, fibroblasts, T and B lymphocytes, marrow stromal cells and
myelomonocytic cells16, 43. The protein itself is reported on human T lymphocytes and
vascular epithelial cells16. Moreover, IL-17 induced G-CSF and CXCL1 can be blocked
completely using neutralizing antibodies for IL-17RA16. However, IL-17RA binds its ligand
with relatively low affinity; only one-tenth of what is to be expected based on the effects it
induces16, 38. This led to the hypothesis that another receptor would have to be involved for
proper IL-17 signalling. One candidate is IL-17RC, another transmembrane receptor. The
two (human) receptors are shown to co-immunoprecipitate when expressed in a murine (IL17-/-) cell line and to bind and respond to IL-17 only when co-expressed in the same cell44. As
well, cells from IL-17RC deficient mice fail to generate a response to both IL17 and IL-17F45.
Taken together, this could indicate that IL-17RA and IL-17RC heterodimers are required for
IL-17 signalling, but experiments were performed on mice or on transfected murine cells, not
on human primary cells. Besides, IL-17RC expression is reported in human cartilage,
prostate, liver, heart, kidney and muscle cells46, which differs from IL-17RA expression. And
of course, if a requirement for IL-17RA/C heterodimers exists, the two receptors will have to
be co-expressed in all cells shown to respond to IL-17 stimulation. Summarizing, IL-17RA
and C cooperation might explain why IL-17RA alone has such a low affinity for its ligand, but
biological relevance in human cells remains to be determined.
IL-22 in defence and epithelial barrier regulation
IL-22 is a cytokine produced by various immune cells. Anti-CD3 stimulated T cells, NK cells
and Th1 cells are all described to produce the cytokine, but Th17 cells are by far the
dominant IL-22 producers16. Its production is stimulated by IL-6 and IL-2345, both essential for
Th17 development. Therefore, it will come to no surprise that IL-17 and IL-22 are normally
co-expressed in Th17 cells16. In vivo studies demonstrated that IL-22 is indispensable to fight
various extracellular infections, as are Th17 cells in general32, 45. The IL-22 receptor is
composed of two chains, the ubiquitously expressed IL-10R2 and the IL-22R chain, which is
not expressed on immune cells16. Instead, it is expressed on and elicits responses from
many types of epithelial cells16. One of these responses is inducing a proinflammatory
response47-49, such as the production of acute-phase proteins, chemokines and other
cytokines. A second class of molecules produced upon IL-22 stimulation is a broad variety of
antimicrobial peptides like β-defensins and S100-family proteins47-49. A third mode of action
does not involve production of cytokines, but a mechanical effect instead. Together with IL17, IL-22 can disrupt tight junctions in epithelial barriers50, possibly to allow other cells to
enter immune-privileged compartments, for example by allowing them to cross the bloodbrain barrier. And although not mentioned by the authors, it could explain why Th1 cells can
only enter the lungs with Th17 involvement upon M. tuberculosis infection (however, this
effect is only described as an IL-17 dependent effect).
9
Multiple roles for IL-21
As mentioned above, IL-21 can favour development of Th17cells over Tregs and its
autocrine loop amplifies Th17 responses and its own expression14. Its receptor shows
similarities with the IL-2R, including being associated with the γc-chain51. It is expressed on
macrophages, dendritic, epithelial, B, T and NK cells and has a function broader than merely
regulating differentiation of the Th17 subtype 16. First its influence on the other T cell
subtypes; IL-21 can promote both cellular and humoral responses which are traditionally
considered Th1 (IFN-γ) and Th2 (IL-4) mediated. Cellular immunity is augmented in several
ways. First, it stimulates IFN-γ production by both Th1 and NK cells 52, 53. In addition, it
stimulates in vitro NK cell maturation 54 and once maturated these cells show enhanced
proliferation and cytotoxicity upon IL-21 stimulation, while survival is decreased55. However,
IL-21-/- mice show normal NK cell numbers 56, indicating that IL-21is not required for NK
development. However, biological functions of IL-21 for human NK cells in vivo will have to
be examined more thoroughly.
Other effector cells of cellular immunity are CD8+ T cells, which are augmented by IL-21.
Both naive and memory CD8+ T cells are enhanced in proliferation and activation by the
cytokine57. On the other hand, IL-21 is also shown to inhibit CD8+ proliferation58. Normally,
DCs present antigens to CD8+ cells. When the CD8+ cells’ TCR binds an antigen presented
on an MHC molecule, the DC activates it. After short stimulation of DCs with IL-21, they
completely lose the capability to induce CD8+ proliferation. Even further, mice injected with
these IL-21 stimulated DCs fail to develop T cell mediated contact hypersensitivity (CHS)
(while control mice do develop this immunopathology)58. This suggests a protective role for
Th17 cells in CHS, but this will be discussed more thoroughly below.
Another protective role for IL-21 lies in the development of allergy. Allergy is mediated by
Th2 cytokines, such as IL-4 which induce IgE production by B-cells. IL-21 is shown to play a
critical role in B-cell functioning as well, being the most potent T cell derived cytokine
promoting B-cell proliferation59. It can induce transcription factors like Bcl-6 and Blimp-1,
regulating B-cell maturation and terminal differentiation60. IL-21-/- mice show no defects in Bcell development, though56, they have lowered levels of antigen-specific IgG1 in their blood
and substantially higher amounts of the allergy inducing IgE56. Consequently, administration
of exogenous IL-21 at the time of immunization leads to decreased production of antigenspecific IgE by inhibiting transcription of a factor required for IgE production (germ line Cε) 61.
Human Th17 (and Th17/Th1) cells are also shown to exhibit this, as they can induce
production of IgM, IgG, and IgA, but not IgE23. However, whether human Th17 cells can
suppress IgE and whether this effect is IL-21 dependent has not been reported to date. Even
though, these reports demonstrate that Th17 cells can modulate Th2 responses and that IL21 has potential protective role against allergy.
Th17
Fig 3. Th17 derived IL-17 stimulates
epithelial expression of CXC ligands, GCSF and SCF, thus indirectly attracting
innate immune cells. IL-22 promotes
expression of anti-bacterial peptides and
controls epithelial barrier function and
repair, thus contributing to bacterial
defense as well. Adapted from.
, SCF
10
Th17 and immunopathology
Since Th17 cells and cytokines appear to have a pivotal role in regulating immune
responses, it will come to no surprise that these cells play a major role in immunopathology
as well. In several autoimmune diseases previously thought to be Th1 mediated, Th17 cells
appear to be more pathogenic. IL-17, IL-22 and IL- 23 are all shown to be upregulated in
several autoimmune diseases which will be discussed below. As well, Th17 cells are found to
infiltrate diseased tissue in patients with rheumatoid arthritis (RA), multiple sclerosis (MS),
psoriasis, Crohn disease and primary Sjögren’s Syndrome (pSS) 62, 63. In short, activated
Th17 cells attract neutrophils to inflamed tissue. Overactivation of these cells leads to tissue
damage as seen in many diseases34, but most work performed in the field involves the
cytokine patterns regulating the immune response. The following section will describe the
role of Th17 cells in several autoimmune diseases and experimental disease models and will
focus on these cytokine patterns.
Systemic Lupus Erymathosus (SLE)
SLE is an autoimmune disease with a broad range of clinical manifestations in skin, kidneys,
lungs, brain, heart, blood vessels and cells, serosal surfaces and joints59. In several disease
models, IL-21 is shown to be a contributing factor. First, BXSB-Yaa mice, a strain
spontaneously developing SLE-like symptoms, express high levels of IL-2160. Neutralization
of IL-21 early in disease, increased disease severity, whereas late neutralization increased
survival64. Implications of these findings are unclear, but could point to different effects of IL21 stimulation on T and B-cells, inducing increased Th17 activation and B-cell apoptosis
respectively59. Second, the sanroque mouse, having a mutation leading to excessive IL-21
expression, develops SLE-like symptoms as well. A third mouse model, MRL-lpr, does not
have increased levels of IL-21 upon stimulation with anti-CD3, but a combination of anti-CD3
and anti-CD28 resulted in a 10-fold higher IL-21 expression compared to control65. As well,
neutralization of the cytokine led to a reduction of skin lesions, anti-dsDNA antibodies,
absolute T cell numbers in the spleen, proteinuria and lymphadenopathy. Since a reduced
concentration of autoreactive antibodies was found neutralizing IL-21 must affect both B and
T cells65.
In human SLE, peripheral B cells show a reduced expression of IL-21R, but this might be
caused by expansion of altered B cell subsets59. Furthermore, IL-21 concentrations were
elevated in SLE-patients, but so were several other Th1 and Th17 cytokines and no
correlation between IL-21 levels and disease severity or anti-dsDNA could be shown59. On
the other hand, IL-21 polymorphisms are shown to associate with SLE66. In conclusion, the
role of IL-21 in SLE remains unclear, but these contradicting reports could be due to IL-21
having different effects in early and late disease, as seen in the BXSB-Yaa mice.
Experimental autoimmune encephalomyelitis (EAE) and MS
EAE is an experimental animal model for human MS. This disease is characterized by
autoreactive Th cells causing inflammatory lesions in the central nervous system (CNS)59.
Like other autoimmune diseases, EAE was previously thought to be Th1 mediated30.
However, mice deficient in IL-12Rβ-chain and thus unable to elicit Th1 responses showed
increased inflammation in the CNS and a more rapid progression to paralysis upon
immunization with an encephalitogenic peptide (MOG)67. In this model, increased levels of IL23mRNA expression were detected compared to control mice with normal IL-12Rβ
expression. Antigen stimulated splenocytes of IL-23-/- mice had increased IL-17 and TNF-α
production and less IFN-γ67. In another study with these knockout mice mice, EAE could not
be induced and IL-6, IL17 and TNF-α expression was completely absent upon restimulation
with the MOG peptide in vitro5. IL-23 blocking antibodies elicit a similar phenotype as the
knockout mice5. Blocking IL-23 before immunization prevents induction of EAE and
administration of the antibody after immunization ameliorates the disease68. Anti-IL-17 did
decrease disease severity, but not as profound as anti-IL-23, probably due to effects of TNF11
α and IFN-γ produced by Th17 cells68, whereas in IL-23-/- and IL-21-/- mice no Th17 cells
develop at all.
Human Th17 cells were shown to kill CNS derived foetal neuronal cells in vitro50. They were
shown to be more capable of crossing a layer of blood-brain-barrier epithelial cells and allow
influx of other CD4+ T cells, serum albumin and neutrophils50. However, in the EAE model no
difference in infiltration of immune cells was seen in IL-23 deficient mice, questioning the
contribution of decreased blood-brain-barrier functioning in the EAE model system5. The role
of IL-17 and IL-22 on human brain epithelial cells, astrocytes and microglial cells could be of
importance as well, but remain to be characterized30.
Taken together, Th17 cells appear to be the main contributors to pathology in MS and are a
promising target for drug development.
Dermal inflammation, psoriasis and acanthosis
Even before Th17 cells were recognized to be a separate Th subset, IL-23 was implicated to
be involved in psoriasis69. In this chronic inflammatory skin disease leukocyte infiltration of
dermis and epidermis, dilation and growth of blood vessels and hyperplasia of the skin
(acanthosis) is seen. In these skin lesions, IL-23 mRNA was shown to be increased more
than 10 and 20-fold respectively for its two subunits69. (The p40 subunit is shared with IL-12,
which’s levels were not elevated, explaining the discrepancy between the two subunits.)
Subsequently, injection of IL-23 or IL-6 in mice ear can induce dermal inflammation and
acanthosis33. The infiltrating CD4+ T cells showed a Th17 phenotype, expressing IL-17 and
IL-22. Mice lacking IL-22 showed dramatically decreased pathology and in a mouse model
for psoriasis, IL-22 blocking antibodies ameliorated the disease70. In humans similar findings
are described. Firstly, IL-22 expression is shown to be increased in psoriatic skin47.
Secondly, IL-22 induces psoriatic symptoms in cultured human epidermis49 and thirdly, IL-22
levels in plasma correlate with disease severity71. Taken together there is strong evidence
that Th17 produced IL-23 induces IL-22 expression, which in turn leads to dermal
inflammation and psoriasis.
Therefore, blocking antibodies for these two cytokines are promising as a new treatment for
psoriasis and indeed, a phase II clinical trial with antibodies for the IL-12/IL-23 p40 subunit
are shown to dramatically decrease disease severity72.
Primary Sjögrens Syndrome
Sjögrens Syndrome is an autoimmune disease affecting exocrine glands, resulting in
destruction of salivary and lachrymal glands. The disorder is called pSS when encountered
alone and secondary Sjögrens Syndrome when it is combined with another autoimmune
disorder. PSS is described to be Th1 dependent, but expression of IL-17 and IL-23 is
reported to be upregulated in cells infiltrating the glands (mainly CD4+ T cells) and glandular
epithelial cells from pSS patients63, 73. As well, IL-17 producing cells are described to infiltrate
the glands of pSS. Taken together, this suggests that Th17 are involved in pSS as well, but
very little is known about the contribution of Th17 cells in pSS and will therefore have to be
investigated further 63.
RA and its experimental models
The disorder in which Th17 involvement is studied most intensively, is RA. Chronic
inflammation of synovial tissue associated with bone and cartilage damage hallmark this
disease. As in most diseases described above, RA was initially thought to be Th1 mediated,
as well as its prototypical mouse model, collagen-induced arthritis (CIA)30. These studies
showed IFN-γ to be expressed in serum and synovial fluid of RA patients and administration
of IFN-γ to worsen CIA severity 74, 75. In cotrast, IFN-γ and IFN-γ receptor deficient mice are
shown to develop more severe CIA76, 77 and IFN-γ deficiency makes mouse strains that are
normally resistant to CIA susceptible78. These disrepancies clearly demonstrate that IFN-γ
can either be protective protective or attributing to in CIA 75. Indeed, when using IFN-γ
blocking antibodies, the moment of administration determines whether the antibodies are
protective or not30. As well, IL-12-/- mice are protected from CIA79. Since these mice are
12
driven towards a Th1 profile but protected from CIA, it further supports the hypothesis that
Th1 cell are not the subtype responsible for pathology in this experimental model.
For IL-17, its role in RA is demonstrated as well. IL-17-/- mice develop less arthritis and
blocking antibodies reduce joint inflammation, bone erosion and cartilage destruction in
CIA80, 81. As well, two other mouse models for RA in which the disease develops
spontaneously require IL-17 for disease development, namely the IL-1Ra deficient and SKG
mice82, 83. In humans, IL-17 and TNF-α mRNA expression in the synovium are shown to
correlate with joint damage progression, while IFN-γ mRNA expression was predictive for
protection from joint damage progression84, which clearly illustrates the importance of IL-17
in the pathogenesis of RA. As described above, IL-17 induces expression of CXC-ligands
and G-CSF, leading to attraction of neutrophils, CD4+ T cells, monocytes and T cells, all of
which populate the inflamed joint30.
Two other cytokines overexpressed in RA are TNF-α and IL-1β. Both cytokines promote
Th17 generation 8 and can synergize with IL-17 in inducing inflammation and joint damage 30.
The other way around, IL-17 can induce expression of these cytokines30. Moreover, TNF
blocking agents are widely used for treating RA patients with significant efficacy. However,
by blocking IL-1β or IL-17 as well, efficacy could be improved and treat patients
nonresponsive to TNF blockers.
IL-23 is already implicated in other diseases and might play a role in RA as well. It is
upregulated in serum and synovial fluid of RA patients 85 and STAT4, one of its downstream
signalling molecules is identified as a susceptibility gene for RA 86. Further supporting the
role of IL-23, mice deficient of the cytokine or STAT4 develop less severe arthritis 79, 87.
Also upregulated in serum and synovial fluid are IL-21 and IL-2288, 89. For IL-22, its effects on
the joints of RA patients is still unknown30, High expression levels of the IL-21 receptor are
found on synovial cells90 and blocking IL-21 with an IL-21R fusion protein decreased disease
severity in the CIA model91, implicating a role for this cytokine as well.
The last interleukin implicated in Th17-disfunction, especially in RA, is IL-15. Just like the
other cytokines, it is overexpressed in RA patients’ synovial fluid and blood and it can induce
IL-17 secretion from PBMCs92. Synovial fibroblasts from RA patients produce IL-15, which
induces expression of IL-17 and TNF-α from T cells. In turn, these cytokines stimulate
fibroblasts to produce IL-6 and IL-15, creating a positive feedback loop93. Injecting CIA mice
with an IL-15 receptor antagonist downregulated expression of Th17 cytokines and
ameliorated disease94 and IL-15-/- showed slightly decreased disease incidence and severity
as well95. The other way around, IL-15 overexpression in mice induced a higher incidence
and severity of CIA95.
The previous sections illustrate that high Th17 cytokine concentrations are found in synovium
of RA patients. These cytokines are proposed to be activating T cells without involvement of
antigen stimulation as large numbers of T cells are found in the synovium which functionally
resemble T cells solely activated by cytokines in vitro.
Beside these high cytokine levels, RA is characterized by bone destruction in the joints.
Since blocking Th17 cytokines has effect on disease severity, Th17 might be involved in
bone destruction as well. Indeed, IL-17 was shown to promote proteoglycan and collagen II
release (indicative for matrix destruction) for bovine cartilage explants96. In an in vitro study
with mice metatarsils, IL-17 alone was unable to induce cartilage destruction. TNF-α
however, did induce damage, which was even more profound in combination with IL-1797. In
this second study, explants were depleted from osteoclasts, professional bone resorbing
cells, indicating that IL-17 and TNF-α might induce proliferation of osteoclasts or induce other
mechanisms of bone destruction. In human bone and synovium samples from RA patients,
IL-17 induced collagen destruction and prevented new collagen synthesis98, indicating that
IL-17 plays a role in human joint destruction as well. A first way to induce bone destruction is
by upregulating or activating collagen and proteoglycan degrading proteins. Addition of
exogenous IL-17 was shown to increase expression of one of these proteins, matrix
metalloproteinase-1 (MMP-1), by 5-fold in isolated synoviocytes isolated from RA patients99.
However, the same experiment with cultured whole explants from the synovium did not show
any effect of IL-17 on MMP-1 production99. A second way to induce bone destruction is by
13
generation of osteoclasts. Th1 and Th2 cells were shown to be unable to induce
osteoclastogenesis in two different experimental systems100. In presence of Th17 cells,
though, osteoclasts were efficiently formed. Th17 cells from IL-17-/- mice showed a reduced
ability to generate osteoclasts. Addition of IL-17 or IL-23 could only induce
osteoclastogenesis in coculture with osteoblasts, cells supporting osteoclastogenesis100. This
induction by osteoblasts was shown to be dependent on, receptor activator of nuclear factor
kappa B ligand (RANKL) which is well known to induce ostoclastogenesis100, 101.
Taken together, there is strong evidence supporting the pathological role of Th17 cells in RA
and its animal models. However, in another animal model, proteoglycan-induced arthritis is
shown to depend on IFN-γ, suggesting that Th1 might be more dominant in RA in some
patients, while in others the Th17 subtype plays has a more prominent role102.
Evidence for protection by Th17 cells or cytokines
Though IL-22 is shown to be required for chronic inflammation in psoriasis, it can also be
protective in chronic liver inflammation. It can protect hepatocytes from serum starvationinduced apoptosis in vitro in a STAT3 dependent manner and administration of IL-22 prior to
injecting proinflammatory molecules reduces the amount of damage in the liver in vivo 103. On
the other hand, liver specific STAT-3 deficient or IL-22-/- mice have a reduced ability to
recover from liver injury in vivo 104 and IL-22 blocking antibodies exacerbate injuries during
acute liver inflammation103. After the discovery of the Th17 subset, IL-17 was tested for
protective effects in liver inflammation, but although IL-17RA and IL-17RC are expressed in
these cells, the cytokine induced neither protective nor proinflammatory effects 105. Taken
together these experiments show a protective role for a Th17 derived cytokine in contrast to
many other diseases in which the T cell subtype contributes to pathological symptoms. This
decreases the cytokines potential as a drug target, since increased IL-22 levels might induce
autoimmune diseases and decreased levels could be dangerous in combination with liver
damage.
As stated above, IL-21 is also implicated to have suppressive capabilities58. In a murine T cell
mediated contact hypersensitivity (CHS) model, IL-21 abrogated the ability of DCs to
stimulate CD8+ T cells in vitro, thus preventing activation and proliferation. In this model,
mice are injected with FITC stimulated DCs. After administration of FITC on the skin two
weeks later, these mice develop a CHS response. However, stimulating these DCs with IL21 prior to injection abrogated their ability to induce CHS in vivo as well. Subsequent
injection of non-IL21 stimulated, FITC incubated DCs in the same mice could not induce
CHS anymore, demonstrating that IL-21 incubated DCs might also induce antigen-specific
tolerance.
14
IL-7Rα ligand functioning in developing and mature T cells
Th17 cells and other effector T cells show surface expression of IL-7Rα. The receptor is
essential in survival and development of cells differentiating to T cells as well as in mature T
cells106. Also, the receptor and its ligands play a role in Th subtype differentiation and are
implicated in immunopathology. Therefore, the following section will discuss the roles of IL7Rα and its ligands in healthy individuals and immune disorders.
IL-7 is a cytokine essential in lymphocyte development and survival, and expressed in many
tissues by epithelial, stromal and dendritic cells107. Most IL-7 producing cells are not immune
cells, while the targets of the cytokine are106, 108. The cytokine is a 177 amino acid
glycosilated peptide which has a 81% homology between human and mice109. IL-7 is tissuederived, since it is primarily produced in epithelia, but expression is also found in the liver
and dendritic cells (DCs)109. Its production does not seem to be tightly regulated under
normal circumstances, though overexpression of the cytokine is implied to play a role in
autoimmunity, as will be discussed below. In a non-inflamed environment, TGF-β is shown to
influence IL-7; it decreases mRNA levels and protein secretion from human stromal cells and
inhibits IL-7 induced proliferation of human B-cell precursors110. In turn, IL-7 can downregulate TGF-β, pointing to a balance between production of these cytokines and a possible
immunoregulatory role109, 111. The lack of regulation at the level of expression and secretion
suggests that IL-7 signalling is regulated at another level, probably by regulating expression
of its receptor, which will be discussed below.
Similar to IL-7, TSLP is not produced by most immune cells. Mast cells are the only
hematopoietic cells expressing the cytokine112. Non-heamatopoietic cells expressing TSLP
are lung fibroblasts, smooth muscle cells, skin keratinocytes and bronchial epithelial cells 113.
Little is known about the regulation of TSLP expression, but mast cells are shown to
upregulate expression upon stimulation by IgE crosslinking113.
IL-7Rα associates with the γc or TSLPR-chain
IL-7 signals through the IL-7 Receptor (IL-7R), a heterodimeric protein consisting of a 440aa
IL-7Rα-chain and a common cytokinereceptor γ-chain (γc). This common chain is shared by
receptors for IL-2, IL-4, IL-9, IL-15 and IL-21, which compete for the limited amount of
available γc109, 114. As well, IL-7Rα can associate with another γc-like chain, called the TSLP
receptor (TSLPR)-chain. Heterodimerization of IL-7Rα and TSLPR results in a new receptor
specifically binding thymic stromal lymphopoietin (TSLP)115.Therefore, IL-7 and IL-7Rα
knockout mice show different phenotypes, as TSLP signalling is abrogated in the receptor
knockout as well116. For IL-7 to signal properly, both IL-7Rα and γc are required, but since γc
is ubiquitously expressed in lymphocytes, surface expression of IL-7Rα on a lymphocyte
indicates that the cell is able to respond to IL-7109. This expression has been seen on
immature B and T cells and most mature T cells109, 117, but also on DCs and monocytes118.
The TSLPR chain shows an overlap in expression, with protein or mRNA reported on DCs,
pre-activated T cells and mast cells 118-120. Moreover, NKT cells are implicated to respond to
TSLP as well 121.
IL-7Rα, γc and TSLPR signalling
As stated above, binding of IL-7 to its receptor induces several reactions by the cell, such as
proliferation, cell cycling or survival. Upon ligand binding, the two IL-7R chains
heterodimerize (Fig. 4)122. Both chains activate an associated Janus kinase (JAK), being
JAK-1 and JAK-3 for IL-7Rα and γc, respectively123. Upon activation, these JAKs
phosphorylate the IL-7Rα chain, creating docking sites for signal transducers and activators
of transcription (STAT)-1, STAT-3 and especially STAT-5124, 125. In turn, the STAT molecules
are phosphorylated by the JAKS and dimerize126. The STAT dimers translocate to the
nucleus where they modulate the expression of several genes involved in cell survival and
proliferation, like the previously mentioned Bcl-2127. As well, STAT-5 is described to function
15
in the cytoplasm by binding scaffold protein Gab2 and activate the PI3K/Akt and Ras/MAPK
pathways which are both involved in cell proliferation128. However, these experiments were
performed in cell lines and the Ras/MAPK pathway does not appear to be activated in
peripheral T cells 129, whereas the contribution of the PI3K/Akt pathway is still a matter of
debate126.
When stimulating cells of several lymphocyte derived cell lines with TSLP, STAT5 activation
is seen as well130. However, where IL-7 requires recruitment and phosphorylation of JAK
kinases, none of four known JAK kinases is phosphorylated upon binding of TSLP to its
receptor131. On the other hand, the TSLPR chain contains a box-1 domain critical for JAKbinding and this domain is shown to be required not only for IL-7 induced STAT5 activation,
but also by TSLP induced activation130. Also, IL-7 and TSLP can both induce STAT5
activation, but exert different roles in the immune system, indicating that other signals have
to be present, in order to discriminate between signals from the two cytokines. Taken
together, these findings show that more research has to be done in order to find out how
exactly TSLP and IL-7 induce responses by their target cells.
Fig 4. Upon binding of IL-7 or TSLP,
the two chains of their respective
receptor sdimerize. For the IL-7R,
the associated JAK-1 and JAK-3
phosporylate the IL-7Rα chain, so
STAT5 can bind and subsequently
phosphorylate
STAT5.
The
phosphorylated STAT5 dimerizes,
translocates to the nucleus and
induces transcription of genes
involved in survival and proliferation.
As well, STAT-5 can activate the
PI3K/Akt pathway, inducing survival
and proliferation as well. During
TSLPR
signalling,
no
JAK
phosphorylation is seen and no
PI3K activation has been described.
However, STAT5 is activated,
translocates to the nucleaus and
induces transcription
IL-7Rα is crucial in lymphocyte development
The nonredundant role for IL-7 in lymphopoesis is most clearly demonstrated by mice
deficient in either IL-7 or IL-7Rα. In these mice, B and T-cell development is severely
impaired and γδT cells are completely absent 116. TSLP-/- mice show no changes in
lymphocyte development, but a combination of γc and TSLP impairment is more severe than
in mice only deficient in γc 116. As well, IL-7-deficient mice show less severe impairment than
IL-7Rα -/- mice, suggesting that TSLP can substitute for IL-7 to some extent116. Even further,
overexpression of TSLP in IL-7-/- mice can rescue B and T cell development 132, providing
more evidence that TSLP can substitute for IL-7.
In humans, no TSLP or IL-7 specific deficiencies have been described, but patients with an
IL-7Rα mutation are. This impairment of IL-7Rα leads to T-B+NK+ SCID confirming that IL-7
plays a role in human T-cell development as well133. However, the presence of B-cells in
16
these patients shows that IL-7 is not absolutely required for B-cell lymphopoiesis, but a role
for the cytokine in promoting proliferation under normal circumstances is likely.
As stated above, a cells ability to respond to IL-7 is based on IL-7Rα expression and can
therefore be easily assessed. During lymphopoiesis, IL-7Rα expression is first seen on the
common lymphoid progenitor (CLP) in the bone marrow, which can differentiate into T cells,
but preferentially to B-cells107. However, other early lymphocyte progenitors are not
expressing IL-7Rα, yet able to efficiently generate T cells107. Altogether, some early
progenitors express IL-7Rα, but this is in no way a universal feature, indicating that IL-7 is
not absolutely required in the earliest stages of T cell development.
After the first stages of T-cell development, IL-7 is crucial in differentiation through four socalled double-negative (DN) stages, wherein cells express neither CD4 nor CD8 and reside
in the thymus. In these stages IL-7 serves as a potent growth factor, implicating a role in
proliferation. As well, inhibition of apoptosis was shown to be important as well, since T-cell
development in IL-7Rα knockouts can partially be rescued by overexpression of Bcl-2 or
deletion of the Bax gene (leading to impaired apoptosis)134, 135. Other experiments show that
IL-7 is mainly required to overcome inhibitory signals, since certain chromosomal deletions
allow mice to generate T cells in absence of IL-7136.
It is in these stages that VDJ rearrangement of the TCR γ-chain takes place in γδT cells.
During this process, IL-7 signalling leads to histone acetylation, making the VDJ locus
accessible for rearrangement137.
During the next step of development, the intermediate single positive (SP) stage, IL-7Rα
expression is lost. Moreover, forced expression of IL-7Rα in this stage leads to decreased
thymic output. This effect may occur due to a higher consumption of IL-7, but forced
expression has also shown to inhibit transcription factors required for progression to the next
stage107, 138. During this next (double positive) stage, IL-7Rα expression is still repressed.
Even further, forced expression of the receptor is shown to be unable to signal into the cell,
as an inhibitor (suppressor of cytokine signalling, SOCS-1) is highly expressed at this stage
139
. After its double positive stage, the cell regains its IL-7Rα expression while progressing
into its final, SP, stage and leaves the thymus as a naive CD4+ Th cell or a CD8+ cell 107.
Whether the cell differentiates into a CD8+ or a CD4+ cell is also influenced by IL-7Rα, as
the receptor is shown to be involved in commitment to the CD8+ lineage 140. TSLP on the
other hand, shows preference for CD4+ T cell expansion, indicating a possible difference
between stimulation with TSLP of IL-7.
IL-7Rα ligands in peripheral T-cell homeostasis
After leaving the thymus, T cells are still influenced by IL-7. It plays a major role in regulating
the amount of T cells in the periphery, as can be seen most strikingly in lymphodepleted
hosts. After lymphocytes are depleted, there is no increase in lymphocyte production in the
thymus. Instead, the little cells produced or still present in the periphery proliferate at high
rate to increase the lymphocyte pool107. This process is called homeostatic peripheral
expansion (HPE). During HPE IL-7 levels rise and the T-cell pool grows rapidly. This growth
has been shown to be dependent on both IL-7 and TCR signalling141; without IL-7, only cells
bearing TCRs with high affinity for antigen can proliferate141. The role of IL-7 is shown to be
independent of the method by which lymphopenia is induced107 and is seen in neonatal mice
as well142. The role of TSLP in HPE is shown in sublethally irradiated mice only and effects
are not as profound as for IL-7116. However, TSLPR knockouts show weakened recovery of
lymphocyte numbers, indicating that this cytokine is required for HPE as well.
Besides having effects in lymphodepleted hosts, IL-7 is important under physiologic
conditions. Cells that have just left the thymus, recent thymic emigrants (RTEs), express high
levels of IL-7Rα and both their survival and proliferation are promoted by IL-7 in vitro143. In
vivo, peripheral naive T cells depend on IL-7 for survival and proliferation as well143, 144. Also,
memory T cells require IL-7 for survival and the cytokine has been implicated to be involved
in commitment of CD8+ cells to the memory phenotype, but this is still a matter of debate143.
17
IL-7 concentrations are regulated by consumption
As mentioned above, inactive T cells (naive and memory) require IL-7 to maintain their
numbers. When IL-7 levels rise during HPE or after injection of exogenous IL-7, T cells start
proliferating at a high rate until a new equilibrium is reached145. The other way around, after
administration of IL-7 blocking antibodies, the T-cell pool cannot be maintained146, indicating
a role for IL-7 in regulating the size of the inactive T-cell pool. Upon encountering of antigen
or activation by other cytokines, T cells downregulate IL-7Rα expression146. These activated
cells proliferate, but since they do not express IL-7Rα anymore, they do not consume the
cytokine and the pool of inactive cells is maintained107, 143, 146. After antigen is cleared and
proinflammatory cytokines are no longer present to provide anti-apoptotic signals, they go
into apoptosis unless they express IL-7Rα and form memory T cells143.
IL-7 and TSLP influence antimicrobial defence by influencing Th subtype commitment
As stated above, the immune system requires different Th subsets to clear different
pathogens. For efficient clearing of a certain pathogen, the right Th cells have to be
activated. In this regulation of Th lineage decision lays another important role for the IL-7Rα
ligands. Firstly, IL-7 is shown to induce production of Th1 cytokines in mononuclear cells
isolated from synovial fluid of RA patients147. As well, activated T cells preincubated with IL-7
showed an increased production of TNF-α and IFN-γ147. Besides this direct effect of IL-7 on
T-cells, the cytokine was also shown to indirectly induce IFN-γ secretion in an IL-12
dependent manner147. As well, IL-7 is also shown to induce IL-17 production from peripheral
blood monocytes isolated from pSS patients (Bikker, A et al. In press) and in monocytes
isolated from RA patients148.
In contrast, TSLP skews the immune system to a Th2 response. In mice, TSLP was shown
to directly induce a Th2 phenotype (IL-4 production) in CD4+ T cells149. In humans, however,
TSLP fails to directly induce Th2 proliferation150. This inability to respond to TSLP could be
contributed to the observation that TSLPR is not expressed on naive T cells, but only on
preactivated cells151. When stimulating these activated cells with TSLP, they show and
increased expression of IL-2Rα, thus increasing the sensitivity IL-2 and augmenting
proliferation120. Although, TSLP is unable to directly drive naive cells towards a Th2 profile, it
can in presence of DCs. DCs stimulated with the cytokine express Th2 cytokines and OX40
ligand150, activate naive T cells and induce differentiation to Th2 cells120. This induction is
shown to depend on the expression of OX40L
In summary, IL-7Rα ligands can induce differentiation of naive T cells and modulate the
immune system by promoting either Th2 or Th1/Th17 responses (Fig 5). Choosing the right
type of response is critical in pathogen defence and can have implications for the
development of immunopathology. The following section will discuss the potential roles of IL7Rα ligands in disease.
18
Fig 5. IL-7Rα ligands differentially induce naive T cell differentiation. TSLP stimulated DCs drive T cells to a Th2
phenotype by expression of OX40 ligand and secreting Th2 cytokines. IL-7 can induce IFN-γ, TNF-α and IL-17 secretion
in a direct and an indirect manner by inducing IL-12 production from DCs. Secretion of these cytokines points towards
differentiation into a Th1, Th17 or possibly to a Th17/Th1 phenotype.
19
The link between IL-7Rα ligands and Th17 cells in
immunopathology
IL-7Rα ligand Th subtype commitment in immunopathology
Besides their normal roles in the immune system, IL-7Rα and its ligands are implicated in
immune disorders. The following section will discuss these proteins in pathology and
integrate their role with that of the Th17 subtype.
A first indication of IL-7Rα being involved in autoimmunity is overexpression of IL-7 in several
diseases. Increased expression is seen in inflamed tissue in juvenile idiopathic arthritis152,
psoriasis153, ulcerative colitis154, polychondritis (not significant) 155, primary Sjögren’s
Syndrome148, sponylarthritis156 and RA157. As well, CD4+ cells in the synovium of RA patients
are shown to be hyperresponsive to IL-7157. In vitro, the cytokine upregulates expression of
several costimulatory factors, such as CD40, CD69, CD80, CD86 and LFA-1157, 158,
implicating it could play a role in activating T cells. IL-7 is shown to induce proliferation of
autoreactive T cells and these autoreactive cells are in a more activated state in MS159. Also,
the cytokine can induce expression of Th1 cytokines in joints of RA patients147 and IL-17 from
PBMCs of pSS patients148. IL-7 promoting IL-17 and Th1 cytokine production suggests that it
can induce generation of Th1, Th17 and Th17/Th1 cells. Therefore, it might contribute to
pathology in disorders which are associated with these Th subtypes by driving naive T cell
differentiation towards these potentially pathological Th subtypes.
For TSLP, overexpression is seen in typical Th2 mediated disorders. In normal skin,
histologic staining does not show any TSLP at all. In patients with atopic dermatitis, the
cytokine is seen in normal skin neither, but highly expressed in acute and chronic atopic
dermatitis lesions113. As well, high levels of TSLP are associated with airway inflammatory
disease in both human and mice112, 150. Moreover, TSLPR-/- mice are unable to develop
inflammatory allergic responses in the lung 160 and overexpression of TSLP induces both
spontaneous airway inflammation and atopic dermatits161, 162.
In contrast, TSLP is suggested to be protective for inflammatory bowel disease163, which is
considered Th1 or Th17 driven62, 163 70% of patients with Crohn disease had no TSLP mRNA
expression in d gut epithelial tissue, suggesting that dysregulation of TSLP expression might
contribute to pathology or that pathology dysregulates TSLP expression163,
Taken together, IL-7Rα ligands are clearly involved in Th differentiation and might contribute
to several diseases by skewing differentiation towards a specific Th subtype (Fig 6). On the
other hand, these cytokines could promote differentiation of the subtype which is not
associated with the disease. In turn, these cells would prevent generation of the diseaseassocated subtype, thereby ameliorate or prevent disease (Fig 6).
20
Fig 6. IL-7Rα ligands
drive
proliferation
towards different Th
subsets, which are
associated
with
different diseases. Th
cells
prevent
generation of other Th
subtypes, which could
prevent or ameliorate
diseases
associated
with these other Th
subtypes. Also, Th
specific
cytokines
might
directly
ameliorate or prevent
disease.
IL-7 promotes expression of other cytokines resulting in positive feedback loops
Besides its potential role in Th subtype commitment, IL-7 is shown to induce several other
cytokines involved in immune disorders. Firstly, IL-7 is shown to induce TNF-α production164,
a cytokine clearly shown to be an important mediator in RA158. In turn, TNF-α increases IL-7
production by synovial fibroblasts and bone marrow stromal cells, creating a positive
feedback loop165, 166(Fig 7). Since generation of Th17 cells is shown to be promoted by TNFα and these cells coexpress the cytokine together with IL-17, this suggests that IL-7 might
contribute to Th17 functioning. A similar positive feedback loop exists for IL-15 and TNF-α, to
which IL-7 might contribute by promoting TNF-α expression8, 30, 93.
In autoimmune diseases, Tregs are considered to be protective, preventing disease onset or
progression. In vitro, IL-7 and IL-15 are shown to directly impair suppressive capabilities of
Tregs 152, 167. As well, IL-7 can interfere with Treg generation, since it can downregulate TGFβ production 168. Disabling Tregs to suppress activated immune cells or to develop at all
could be a factor contributing to autoimmune diseases, but not only Tregs depend on TGF-β,
Th17 cells require it as well. Even further, TGF-β inhibits IL-7 expression168. Taken together,
this would imply that compartments with high IL-7 concentrations prevent Th17 development
and that compartments in which Th17 cells do develop IL-7 expression is suppressed.
However, both IL-7 and Th-17 cytokines are upregulated in inflamed tissue of several
immune diseases and seem to cooperate in inducing inflammation.
Another Th17 cytokine influenced by IL-7 is IL-21. IL-7 and IL-15 both induce expression of
the Th17 cytokine169. These three cytokines are all shown to synergize in the induction of
CD8+ T cell activation57, 109, providing more evidence for a relation between IL-7 and Th17
cells. However, IL-7 and IL-21 both require the γc chain for signalling. Therefore, both
receptors might compete with each other for γc association and signalling143, but whether this
takes place in vivo or influences a cells response to the cytokines is unknown.
Another convergence of Th17 and IL-7 functioning is in the production of MMPs, proteinases
contributing to bone destruction in RA170. MMP-1 production by isolated synoviocytes is
increased by IL-1799. In turn, IL-7 induced MMP-13 production by human chondrocytes and
proteoglycan release from cartilage explants170.
A last process in immunopathology promoted by both Th17 cytokines and IL-7 is the
formation of osteoclasts in RA. As stated above, IL-17 and IL-23 can both induce RANKL
expression on osteoblast, which stimulate precursor cells to differentiate in bone resorbing
21
osteoclasts100. In vitro, IL-7 stimulates osteoclast formation by promoting release of soluble
RANKL. This effect could either be direct stimulation100, or indirect via upreglation of TNF-α
and LFA-1, which in turn upregulate RANKL on T cells164, 171. However, since neutralizing
RANKL leads to a decrease of only 60% of osteoclastogenesis, there has to be a RANKLindependent way of inducing T-cell mediated osteoclastogenesis166. This pathway is
suggested to be mediated by TNF-α and IL-1166. As well, in vivo administration of IL-7
induces bone loss172, but not in T-cell deficient mice, showing that the bone loss is T cell
mediated164. This and the suggestion that, in part, IL-7 induced TNF-α production is
responsible for the increased RANKL expression, suggests that Th17 cells are involved. IL-7
could initiate the IL-7/TNF-α feedback loop, activating Th17 cells and even further increasing
RANKL expression, which in turn promotes osteoclastogenesis.
Fig 7. Differentiation of naive T cells into Th17 cells is promoted by several cytokines. Together, these cytokines
form a complicated network in which they stimulate or inhibit each other, with some forming positive feedback loops
which might lead to autoimmune diseases.
22
Concluding remarks
Taken together, there is strong evidence of Th17, as well as IL-7 involvement in autoimmune
diseases. All cytokines involved show several overlapping positive feedback loops (Fig 7),
inducing more and more production of proinflammatory cytokines. These feedback loops are
likely to lay at the basis of all autoimmune diseases. However, differences in patients and
diseases might result in different feedback loops, cytokines and Th subsets being involved in
each individual case. Therapies aiming to stop these feedback loops by inhibiting one or
multiple cytokines seem promising, though. Anti-TNF-α has already been used with great
success, but a large group of patients does not respond to treatment. This nonresponsiveness could be due to different mechanisms being involved in non-responders
compared to good responders.
Even though, our knowledge of immune disorders increases rapidly. Blockade or
administration of other cytokines might be beneficial in treating immune disorders, providing
hope that in the near future progression of many of these diseases can be stopped, helping
large numbers of patients untreatable today.
23
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