Innate lymphoid cells in the intestines
and
The role of dietary compounds
Sigrid Wahlen
Master Infection and Immunity
Utrecht University
Master thesis
Supervisor: PhD student G. Goverse (VUmc)
Examiners: Prof. Reina Mebius (VUmc) and Assoc. Prof. Marianne Boes (UMCU)
Oct. 2013 – Jan. 2014
Figures on cover from left to right: Colour enhanced tissue section of Citrobacter Rodentium infected
murine colon (1); Intestine of healthy Rag1-/- mouse with stained epithelial cells (red), nuclei (blue)
and innate lymphoid cells (green) (2); Tissue of murine intestine with red fluorescent filamentous
actin (Alexa Fluor 568), green nuclei (SYTOX green) and N-acetylglucosamine and N-acetylneuraminic
residues stained blue (Alexa Fluor 350) (3).
Abstract
The incidence of intestinal related diseases such as inflammatory bowel disease annually
increases and progress in research is impeded by the complexity of the intestinal
environment characterized by interactions of the host, commensal and diet compartment.
The host immune system in the gut recently gained a new cell type namely the innate
lymphoid cells (ILC) which are classified in three families and exhibit functions ranging from
lymphoid organogenesis and tissue remodelling to cytotoxicity and immunity against
pathogens. Since the diet has been shown to have a large impact on gut immunity, the
association with ILCs is currently being investigated. Exogenous AhR ligands and vitamins
were found to ensure a tolerogenic environment in the intestines suggesting a beneficial
role during periods of excessive inflammation. However, studies which focus on vitamin A,
obtained contradictory findings indicating that the effect cannot be studied in the absence of
other factors such as the endogenous cytokines TGFβ or IL-15. Both AhR ligands and vitamins
determine the outcome of developmental or functional mechanisms concerning ILCs. In this
review we report the different ILC families and an update of findings regarding dietary
influences on ILCs in the intestines.
Contents
Introduction .................................................................................................................................... 1
The three families of innate lymphoid cells
Group 1 ILC ........................................................................................................................ 4
Group 2 ILC ........................................................................................................................ 6
Group 3 ILC ........................................................................................................................ 8
The role of dietary components in ILC development, survival and function
Aryl Hydorcarbon Receptor ligands ................................................................................... 12
Vitamins ............................................................................................................................. 16
Vitamin A .............................................................................................................. 16
Vitamin D .............................................................................................................. 21
Concluding remarks ........................................................................................................................ 23
Acknowledgments .......................................................................................................................... 25
References ...................................................................................................................................... 26
Appendix : Summary for general public ......................................................................................... 34
Introduction
Intestinal related diseases such as inflammatory bowel disease are widespread and affect
millions of patients every year. The symptoms range from diarrhoea, constipation, rectal
bleeding to severe bowel movement and abdominal pains which highly affect the quality of
life (4). Understanding the processes that occur in the intestines is crucial to design
treatments and eliminate disease. However, in recent years it has become clear that the
gastro-intestinal tract has an extremely complex environment characterized by the
interdependence of three key components including the nutrient, commensal and host
compartment. The latter compartment is mainly comprised of the local immune system and
an epithelial barrier separating the host from external factors in the lumen (5,6).
The intestinal immune system is an extensive network of immune cells and lymphoid tissues
including Peyer’s patches, cryptopatches and isolated lymphoid follicles (7). Although the
immune cells present in these tissues or diffused in the lamina propria are protected by a
dense mucus layer, foreign particles such as commensal or diet derived products are
constantly in close proximity which under normal circumstances do not occur elsewhere in
the body where they can have harmful effects. Therefore, to prevent unnecessary
inflammatory reactions, most gut immune cells differentiate to become unresponsive
towards compounds commonly found in the gut. However, these cells still need to be able to
mount an efficient immune response against invading pathogens, which highlights the
importance of tightly regulating gut immunity (5,8).
The delicate balance in the intestines is maintained by various immune cells and although LTi
cells were identified, one group of related lymphoid cells has been overlooked for a long
period of time. Evidence shows that the innate lymphoid cells (ILC) play a crucial role in
lymphoid tissue organogenesis, maintenance of epithelial integrity, tissue remodelling and
repair. Despite of their lymphoid morphology, they are also essential components in early
stages of innate immunity against microbes (7,9,10). Furthermore, ILCs lack myeloid and
dendritic cell markers and do not possess recombinant activating (Rag) gene dependent
rearranged antigen receptors, which distinguishes them from T- and B-cells (11,12). During
1
their development, ILCs use common cytokine receptor-γ chain signalling and the DNA
inhibitor Id2 suggesting a common lymphoid progenitor (CLP) either localized in the fetal
liver or adult bone marrow (10). Id2 is part of a helix-loop-helix protein family and is able to
heterodimerize with E proteins which blocks their DNA binding capacity thereby preventing
the transcription of genes essential for B-cell and in part for T-cell development,
subsequently stimulating the development of ILCs (Figure 1). The ratio of E and Id proteins
serves as a switch that controls the fate of precursor cells towards either adaptive or innate
lymphoid cells (12–14).
Figure 1. Overview of the three ILC families. Every ILC family descends from a CLP cell which requires Id2, the
common γ chain receptor (γcR) and the activity of specific transcription factors. The table describes signature
molecules, location in the body, effector functions and the equivalent T helper subsets. CD, Crohn’s disease;
FALC, fat-associated lymphoid cluster; MLN, mesenteric lymph node; RA, retinoic acid; AhR, aryl hydrocarbon
receptor.
2
Furthermore, three ILC subtypes have been identified and in order to avoid confusion, a
consensus has been made recently on the nomenclature which will be utilized in this review
including group 1, group 2 and group 3 ILC (9). Although the different ILC types seem to
mirror the T helper cell subsets, they are not only classified on the basis of specifically
secreted cytokines but also according to their function and phenotype due to the variability
in cytokine profile, which is depicted in figure 1.
The field of ILCs has gained much interest the past few years by using mice models which
mimic human diseases. Since research is only beginning to understand the role of ILCs in the
human intestinal environment, this review will focus on gut ILCs in mice.
As mentioned earlier, the host compartment is not the only factor that has an impact on
processes in the intestinal tract. Recent findings suggest commensal bacteria and ingested
food components influence the ILC populations in the gut, however the identification of
these particles is impeded by the enormous diversity of nutrients and by indirect sources
such as bacteria capable of transforming compounds into a form that is can be digested by
the host. The goal of this review is to provide a background on the three ILC families and an
overview of the discoveries that are being made on the effect of diet derived compounds
like AhR ligands and vitamins on the development, maintenance and function of intestinal
ILCs.
3
The three families of innate lymphoid cells
Group 1 ILC
Natural killer cells are the prototypical group 1 ILC and are characterized by the production
of IFN-γ and the murine surface markers CD27, CD11b, CD127, KLRG-1, the natural
cytotoxicity receptor (NCR) NKp46 and in some mouse strains NK1.1 (9,11). In addition, ILC1
cells are considered to be members of the group 1 ILC and produce IFN-γ similar to NK cells.
However, because ILC1 cells are able to originate from group 3 ILCs, it is hypothesized that
they do represent a distinct cell type (9).
The development of NK cells is induced in the bone marrow by IL-15 and Flt3 ligand and
requires the activity of transcription factors as T-bet, which is also specific for Th1 cells
(14,15). Moreover, research has shown that under the influence of IL-7, IL-15 and GATA-3,
NK cells could be derived from the thymus in addition to bone marrow (14,16). In contrast to
the development of NK cells, ILC1 cells can directly descend from CLPs or arise from a group
3 ILC. Independent studies demonstrated that IL-12 could transform NCR+ ILC3 into IFN-γ
producing ILCs with increased T-bet and decreased RORγt expression suggesting an inducible
plasticity of ILC3 cells with T-bet and RORγt as key regulators (9,17–19). Of note, this
plasticity is also present in Th1 and Th17 which further emphasizes the analogy of T helper
cells and ILCs (9).
Virus infected or cancerous cells are eliminated by NK cells through mechanisms involving
granule exocytosis or death receptor interactions. In the granules of NK cells, perforins and
granzymes reside that upon release form pores in the target cells and activate the
intracellular apoptotic machinery respectively. Furthermore, the interaction of death ligands
such as FasL and TNF-α to their receptors on affected cells will also induce programmed cell
death as depicted in figure 2 (7,10,14). Although it is known that ILC1s are weakly cytotoxic
and produce IFN-γ, the exact functions are unclear and need to be investigated further (10).
Group 1 ILCs might initially be stimulated to prevent disease, evidence suggests an
involvement in pathology due to an increase of ILC1s in patients suffering from Crohn’s
disease and in humanized mouse models with dextran sodium sulphate induced colitis (20).
4
These findings were also obtained in Rag-/- mice in which colitis was aggravated by CD40
monoclonal antibodies indicating that the recruitment and activation of ILC1 could
contribute to the disease (21).
Figure 2. Effector functions of NK cells. NK cells are able to eliminate target cells such as virus infected cells or
tumour cells via two mechanisms. Granzymes and perforins located in NK cell granules are released upon
recognition of target cells after which these molecules form pores in the plasma membrane and activate the
apoptotic machinery. FasL or TNFα are also used by NK cells to induce programmed cell death in affected cells.
5
Group 2 ILC
Several research groups have identified another ILC family residing in mesenteric fatassociated lymphoid clusters (FALC), mesenteric lymph nodes (MLN), spleen, liver, lungs and
intestines, which proliferated and produced the T helper type 2 cytokines IL-5 and IL-13
during helminth infection (10,22). Group 2 ILCs were isolated via unique surface markers
including chemokine receptors CXCR6, CXCR4 and CCR9, after which different subtypes of
the ILC2 family were identified: the natural helper cells, nuocytes and innate helper 2 cells. It
is not yet clear if these cells all represent either distinct cell types or only one cell at various
stages of activation (9,10).
Although the developmental relationships of group 2 ILCs remain to be clarified, evidence
has been gathered showing that development depends on the transcription factors RORα,
GATA-3 and Notch. Interestingly, studies have shown that Notch is only required in nuocytes
and that IL-7 signalling is involved in the development of natural helper cells and nuocytes
(23,24). Despite the implications that are being made, more studies are necessary to
determine the importance of different factors to the development of the individual ILC2
subtypes. Moreover, the inability of these cells to further differentiate upon cytokines
suggests terminal differentiation.
In addition to tissue repair and homeostasis by ILC2-derived amphiregulin after influenza
infections, the ILC2s were shown to be essential in intestinal immunity against helminths and
bacteria (7,10,11,13,14). These pathogens stimulate intestinal epithelial cells to secrete IL-25
and IL-33 which recruit and activate ILC2s to expand in the lamina propria, MLN and FALC
and to produce type 2 cytokines. Eosinophils, basophils, mast cells and IgE producing plasma
cells migrate towards the source of these cytokines in order to aid in expelling the parasites.
Goblet cells present in the epithelial layer are also triggered to increase the secretion of
mucus ensuring the inability of helminths to strongly attach to the luminal wall.
Simultaneously, alarmins derived from epithelial cells are released which induces a chain of
immunological reactions resulting in the activation and subsequent migration of Th2 cells
towards the infection site where a cross talk between the innate and adaptive lymphoid cells
enables the maintenance of both cell types and a boost for the local immune response
(7,13,14) (Figure 3).
6
Because Th2 cells and their cytokines have been implicated in allergic disorders, research has
now focused on involvement of ILC2s (7,9,11,14). The stimulating cytokines IL-25 and IL-33
and effector cytokines IL-5 and IL-13 are all effective in causing respiratory inflammation in
mice (25–28). Recent data shows that inflammation is reduced in ovalbumin induced asthma
models by inhibiting IL-25 or IL-33 signalling (27). Furthermore, RAG-/- mice treated with IL13, presented characteristic symptoms of asthma such as pulmonary eosinophil recruitment,
increased secretion of mucus and airway hyperresponsiveness (29).
Figure 3. ILC2 cells protect against helminths by inducing a type 2 immune response. Helminths induces
epithelial derived IL-25 and IL-33 thereby activating ILC2 cells to produce IL-5 and IL-13 which stimulate
eosinophils, basophils, IgE producing plasma cells and Mast cells to raise immunity against helminths. Goblet
cells are also induced to secrete high amounts of mucus which inhibits attachment of the parasite to the
intestinal wall. Th2 cells are recruited to the site of infection and provide the immune response with an extra
boost. Cross talk with the Th2 cells also maintains the local ILC2 population.
7
Group 3 ILC
While transcription factors such as Notch, Aryl Hydrocarbon Receptor (AhR) and Tox
contribute to the differentiation of group 3 ILC members, RORγt is the key regulator, which is
demonstrated by data gained with RORγt-/- mice lacking ILC3 cells. Moreover, these ILCs
were defined by the secretion of IL-22 and IL-17. In addition, all developing group 3 ILCs
were shown to require IL-7 signalling, however signature activating molecules have to be
investigated further. Three RORγt+ subsets were established including the lymphoid tissue
inducer cells, NCR+ ILC3 and NCR- ILC3 cells which seem to mirror the Th17 or Th22 cells
(7,12,13,30).
LTi. The formation and remodelling of lymphoid tissues during embryonic development and
adulthood is known to be regulated by lymphoid tissue inducer (LTi) cells, characterized by
the expression of IL-17, IL-22 and lymphotoxin-α1β2 and the following cell surface markers:
NCR-RORγt+CD4+/-CD45+LT+ CD127+CD117+c-kit+, which are specific for both fetal and adult
LTi cells (7,13,31). However, in order to identify other surface molecules that define these
two LTi cells more studies are required. Moreover, CLPs of fetal and adult LTi cells originate
from the liver or bone marrow respectively suggesting that pre- and postnatal lymphoid
organogenesis is initiated by two different mechanisms (14).
Before birth, the formation of the entire lymphoid system is orchestrated by interactions in
the mesenchyme of predestined sites between lymphotoxin-α1β2 on LTi cells and
lymphotoxin-β receptor of stromal cells, which leads to receptor ligation, stromal cell
activation and increased expression of chemokines such as CXCL13, CCL21 and CCL19 and
VCAM-1, ICAM-1 and MAdCAM-1 adhesion molecules. Consequently, additional LTi cells are
recruited to the developing tissue which induces the maturation of T- and B-cell specific
areas (Figure 4A). After birth, cryptopatches and isolated lymphoid follicles (ILF) are
developed by adult LTi cells in the gut. The formation of cryptopatches containing ILCs and
dendritic cells, relies on AhR signalling and is independent on commensal bacteria, whereas
after weaning, when the intestines are colonized by bacteria, the ILFs are formed by
stimulating LTi cells with CCL20 and β-defensin, derived from epithelial cells which are in
turn activated by microbial products. The interaction of LTi cells and stromal cells causes
recruitment of B-cells and subsequent IgA-production which maintains the luminal
8
restriction of commensal bacteria (13,30,32) (Figure 4B). In addition to lymphoid
organogenesis, due to the production of both IL-17 and IL-22, adult LTi cells were proposed
to play a role within mucosal immunity (7,33).
A
B
Figure 4. Group 3 ILCs orchestrate the development of lymphoid tissues before and after birth. (A) Before
birth, lymphoid tissues such as lymph nodes and Peyer’s Patches are formed by the interaction of LTi cells and
stromal cells, which leads to the recruitment of additional LTi cells and the division in T- and B-cell specific
zones. (B) Cryptopatches (CP) develop after birth and are transformed into ILFs by activating LTi cells with βdefensins and CCL20, derived from epithelial cells after commensal stimulation. The interaction of LTi and
stromal cells leads to the recruitment of B-cells which differentiate towards IgA-producing plasma cells
resulting in the containment of bacteria.
9
NCR+ ILC3. When the NCR+ ILC3 cells were isolated by independent research groups, they
were first believed to belong to group 1 ILCs and were only later shown to differ due to
increased expression of RORγt and to the absence of cytotoxic granules (13,34). In addition,
proper NK cell development requires IL-15 and Fα3 ligand while ILC3 development and
survival is sustained with IL-7r, Notch and RORγt signalling in lamina propria localized CLPs
(14). IL-22 is the main cytokine produced by NCR+ ILC3 cells and is known to function in antiinflammatory processes (7).
After maturation, NCR+ ILC3 cells are mainly situated near intestinal tissues and start to
secrete IL-22 which helps to prepare for bacterial colonization and maintains mucosal
immunity against microbes. Several studies reached this conclusion by demonstrating that
RORγt+ ILCs are the main source for IL-22 during Citrobacter Rodentium infections and that
mice deficient in IL-22 had increased susceptibility to the bacterium in addition to an
augmented mortality rate (7,35–37). During intestinal homeostasis, epithelial cells produce
IL-25 which indirectly via dendritic cells inhibits the NCR+ ILC3 cells to mount an antimicrobial
response. When a pathogen succeeds to penetrate the barrier, this inhibition is overruled
and dendritic cell-derived IL-23 in concert with the interaction of lymphotoxin-α1β2 and its
receptor induces IL-22 secretion, thereby stimulating epithelial cells to increase the secretion
of antimicrobial proteins such as RegIIIβ and RegIIIγ (7,10,22,38) (Figure 5).
Similar to T helper cells, plasticity in ILCs has been observed in murine NCR+ ILC3 cells being
able to differentiate to an ILC1 phenotype with increased T-bet activity and production of
IFNγ. The cytokine profile that is present in the extracellular space is hypothesized to be
crucial for determining the fate of these cells. In humans, isolated tonsil NCR+ ILC3 cells
produced IL-13 when cultured in an IL-2 and TLR2 ligand rich medium indicating that these
cells could have a larger plasticity than previously thought (17,19,39).
NCR+ ILC3 are associated with colitis, Crohn’s disease and even with cancer. Accumulation of
RORγt+ ILCs induced by IL-23 and subsequent elevated levels of IL-17 and IL-22 were found
in patients suffering from inflammatory bowel disease and dysregulation of IL-17 and IL-22 is
therefore proposed to trigger autoimmunity (14,40). Moreover, cancerous cells secrete
CCL21 to recruit and utilize the local innate immune system in order to survive and
proliferate. by stimulating tumour growth through the formation of an extracellular network
10
that mediates the recruitment and differentiation of immunosuppressive cells such as T
regulatory cells (41). In contrast, a positive impact of ILC3 was observed in studies using B16
melanoma cells, which revealed an antitumor effect of IL-12 induced IFNγ (42).
Figure 5. Group 3 ILCs help maintain epithelial integrity by mounting immunity against bacteria. Under
homeostatic conditions, the epithelial cells in the intestine produce IL-25 which inhibits the dendritic cells to
produce IL-23 and to interact with ILCs. When bacteria approach the barrier or when an infection is taking
place, the inhibition of IL-25 is not sufficient causing dendritic cells to produce IL-23 which stimulates NCR+ ILC3
cells to secrete IL-22. Consequently, epithelial cells release antimicrobial peptides to eliminate the pathogens.
NCR- ILC3. The last member of the group 3 ILCs was found in fetal and inflamed adult
intestines of mice and resembles the LTi cells in phenotype due to the absence of NKp46 and
dependence of IL-7 and RORγt signalling. The principal cytokine that is produced by these
cells is IL-17 which can either act pro-inflammatory by mediating neutrophil recruitment or
anti-inflammatory resulting in remodelling and tissue repair (13,14). The opposing outcomes
of IL-17 signalling could arise due to other cytokines working in concert with IL-17.
Furthermore, evidence was provided for a pathologic role of NCR- ILC3 in Helicobacter
hepaticus induced colitis, since depletion of these cells improved the condition of the mice
significantly (40). However, the processes concerning this ILC3 are not intensively studied
and more investigations need to be performed on this subject.
11
The role of dietary components in ILC development,
survival and function
ILCs are essential in maintaining gut homeostasis, which is difficult to achieve considering
the magnitude of insults occurring at the epithelial barrier. Intestinal ILCs continuously come
into contact with nutrients that are shown to have an impact on gut immunity. For instance,
exposure to a high fat diet for an extended period of time inevitably induces changes in the
local immune system (43). In the next section, we will discuss some examples of diet derived
substances that have either clear or poorly investigated influences on development,
maintenance or function of ILCs.
Aryl hydrocarbon receptor ligands
AhR is a member of the Per-ARNT-Sim (PAS) superfamily and functions as a nuclear receptor.
The structure is comprised of a basic helix-loop-helix domain, a PAS domain and two regions
involved in cellular localization namely nuclear export and nuclear localization signal which
ensure that AhR is constantly shuttled between the nucleus and cytosol (44–46). The PAS
domain is promiscuous towards binding partners that are either endogenous or exogenous
(45). Examples of endogenous ligands are heme metabolites, indigoids, arachidonic acid
metabolites and tryptophan metabolites such as indole 3-carbinol (I3C), 6-formylindolo[3,2b] carbazole and 6,12-diformylindolo[3,2-b] carbazole. Exogenous ligands are derived from
the environment and constitute toxins like polycyclic aromatic hydrocarbons, halogenated
aromatic hydrocarbons and polychlorinated biphenyls in addition to dietary compounds
including cruciferous vegetables (broccoli, cabbage, brussels sprouts and cauliflower) rich in
I3C and fruits and vegetables high in natural flavonoids (44,47,48).
AhR predominantly resides in the cytoplasm of NCR+ ILC3 and LTi cells where it interacts with
chaperone proteins such as Hsp90, AIP and p23 which all render AhR inactive (44,49–51).
Upon ligand binding, AhR undergoes a conformational change leading to the removal of
these chaperone proteins thereby exposing the nuclear localization signal. After
translocation to the nucleus, AhR forms a transcriptional complex with AhR nuclear
12
translocator (Arnt) and subsequently binds to AhR response elements on target genes such
as cytochromeP450 metabolizing enzymes and the Il22 gene (45,46,49,50,52). In addition, it
has been shown that AhR can have an impact on signalling independent of DNA binding
through the interaction with NFκB subunits and the release of the non-receptor tyrosine
kinase Src after activation (45,53,54). Furthermore, excessive or limited activity of AhR is
known to have detrimental effects and a tight regulation is thus essential for the shutdown
of AhR activity by either removing the activating signal, targeting AhR for proteasomal
degradation, AhR repressors or by exposing the nuclear export signal (45).
Recent studies demonstrated the impact of AhR on ILC3 cell development and function in
the intestinal environment. Although Kiss et al. showed that AhR was not important for
embryonic ILC3 cell development, a crucial role in maintenance and function during
adulthood was demonstrated. No significant difference in cell count could be observed in
new borns which were AhR deficient compared to wild type mice, while the ILC3 cells of 8week old AhR-/- mice were shown to be decreased indicating that the development was not
disturbed early in life (49,50). Moreover, it was proven that the maintenance was not
impeded due to an inadequate generation of intestinal ILC3 but because of an increased
susceptibility to apoptosis (51) (Figure 6).
Evidence was provided by Kiss et al. for the relevance of diet derived AhR ligands in the
maintenance of intestinal ILC3 cells. When wild type mice were given a phytochemical-free
diet, a reduction in the number of ILC3 cells was observed compared to identical mice fed a
standard diet and replenishing these mice with the phytochemical I3C was able to reverse
the outcome (49). Interestingly, another study was unable to show a decrease in intestinal
NCR+ ILC3 cells of mice fed a synthetic diet lacking vegetable products, which could be
explained by a discrepancy in the age of the mice (50). A significant effect could be observed
in 4-week-old mice while there was no difference in mice 6 to 8 weeks of age. Therefore, Kiss
et al. tested the same synthetic diet on older mice (6 weeks) with completely formed
intestinal lymphoid tissues and no significant loss of ILC3 cells in the gut could be detected
whereas I3C repletion was able to induce an expansion of the cell compartment. The authors
concluded that AhR activated by dietary ligands did not play an important role in ILC3 cell
survival after postnatal lymphoid organogenesis. Other possible explanations for the
conflicting results are variability in experimental settings, facilities or suppliers of materials.
13
It has also been proposed that AhR might not induce direct effects, but that it just alters or
integrates the cytokine signals originating from distinct sources (55).
In addition to cellular maintenance, AhR contributes to the cytokine production of ILC3 cells.
Research has proven the presence of an AhR-response element in the promoter region of
Il22 gene and IL-22 expression was markedly reduced in mice lacking the AhR gene (50).
However, AhR alone was not sufficient to stimulate IL-22 production and RORγt, which binds
ROR-responsive elements near the Il22 gene, was discovered to be required as well for
transcriptional initiation (51). In line with these observations, AhR was shown to be a
protective factor in C. Rodentium infections through the induction of IL-22 synthesis in the
lamina propria of the small intestine (50–52). From these findings it was concluded that AhR
is essential to induce IL-22 production in intestinal ILC3 cells which is explained in figure 6.
Due to the involvement of LTi cells in lymphoid tissue development, the effect of AhR activity
in intestinal lymphoid organogenesis has been examined. The intestines of AhR-/- mice had a
marked reduction in cryptopatches and ILFs in the lamina propria. However, no difference
was detected in Peyer’s Patches and MLN which suggested that AhR activity only affected
lymphoid tissues formed after birth and did not have any impact on the embryonically
imprinted lymphoid tissues (49–51). These observations are in line with experiments
performed with 4-week-old mice fed a phytochemical-free diet, which showed a decrease in
cryptopatches and ILFs. In contrast, identical experiments performed with mice having fully
formed intestinal lymphoid tissues revealed no significant change which indicated that the
active period of AhR ligands ranges from the postnatal period to the formation of ILFs (49)
(Figure 6).
Collectively, it has been elucidated that dietary AhR ligands do not interfere with group 3 ILC
development in the intestinal environment but do control the function and maintenance of
these ILCs postnatally possibly via anti-apoptotic mechanisms which has an impact on the
formation of cryptopatches and isolated lymphoid follicles.
14
Figure 6. AhR ligands influence group 3 ILCs and ILF formation. Diet derived AhR ligands were shown to induce
RORγt+ ILC expansion which ensures cellular maintenance. Since AhR binds to the promoter region of the Il-22
gene, it is able to stimulate the production of IL-22. AhR is also essential for ILF formation via RORγt+ ILC
activation.
15
Vitamins
A positive correlation between vitamins and health has long been established. They are
commonly known to function as antioxidants which eliminate harmful free radicals such as
reactive oxygen species, thereby preventing irreversible DNA damage (8). However, recent
studies have discovered an alternative mechanism of action of vitamins which involves the
intestinal immune system. Vitamins A and D were shown to have either a direct or indirect
effect on molecular processes in cells of both the innate and adaptive immune system,
which continue to be an interesting subject for research concerning ILCs in the gut
(8,43,56,57).
Vitamin A. Vitamin A is a fat soluble molecule present in the diet as retinyl esters or
precursor carotenoids (5,58). In the liver these substances are hydrolysed to retinol, which is
transported by retinol binding protein (RBP) to peripheral cells expressing the enzymes
alcohol dehydrogenase (ADH) and retinal dehydrogenase (RALDH). ADH catalyses the
reversible conversion of retinol into retinal after which the latter is irreversibly transformed
by RALDH into 9-cis retinoic acid or all-trans-retinoic acid (RA), the active metabolite of
vitamin A (5,8,43,58). In the intestinal environment, the expression of different RALDH
isoforms (RALDH1, 2, 3, 4) can be induced by retinoic acid and microbial stimuli in a vast
array of both hematopoietic and non-hematopoietic cells as CD103+ dendritic cells, epithelial
cells, CX3CR1+ macrophages and stromal cells of gut associated lymphoid tissues (GALT) (59).
Due to the presence of RA producing enzymes in gut immune cells, vitamin A can have an
important local immunomodulatory role in addition to established functions in forming bone
and epithelial linings, eyesight, organogenesis during embryonic development and
reproduction (59,60).
RA functions by inducing transcription of target genes through binding to the nuclear
retinoic acid receptor (RAR) α, β or γ and in some exceptional conditions to peroxisome
proliferator activated-receptor (PPAR) β. Heterodimerization with retinoic X receptor (RXR)
enables binding to retinoic acid response elements (RARE or RXRE) in the promoter region of
target genes (58,59) thereby competing with other nuclear receptors such as VDR which has
16
been suggested to prevent expression of their respective target genes (61,62). However,
evidence for this competition still needs to be gathered. RA is eventually inactivated by
CYP26A1, creating oxidative metabolites in the process (59).
Despite the extensive research that has been performed on the influence of vitamin A on the
immunological response, the exact outcome of RA signalling is not fully understood. Some
studies have shown that by removing RA excessive inflammation occurred, suggesting a
tolerogenic effect. However a pro-inflammatory phenotype of RA was also demonstrated
that poses vitamin A as instigator in autoimmune disorders such as inflammatory bowel
disease (IBD) (63–65). Despite these contradictions, some implications have been made in
the field of adaptive and innate lymphoid cells.
B lymphocytes are considered to be target cells of RA either directly or indirectly via
dendritic cells and are known to secrete various types of immunoglobulin of which IgA
dimers are most important in mucosal tissues, where they neutralize the pathogens close to
the epithelial barrier. A number of studies have proven that RA combined with IL-6 and IL-5
is necessary for local differentiation of IgA antibody secreting cells via dendritic or stromal
cell function (60,61,66–68).
In addition, RA was found to be in charge of controlling T-cell fate via RARα and RARγ (65).
The existence of RARE in the promoter region of the Foxp3 gene suggests that RA can
regulate T regulatory cell differentiation, which seems to additionally require TGFβ (69,70).
Moreover, the Th2 phenotype is favoured by RA through the stimulation of GATA3, Maf,
Stat6 and il-4 genes and by inhibiting the transcription of T-bet (71–74). A tolerogenic
environment is thus created in the gut by RA which is critical for oral tolerance. In contrast,
RA is still able to induce pro-inflammatory CD4+ T-cells via RARα, providing evidence that RA
is not the only factor that needs to be considered in regulating gut immunity (65).
Experiments were conducted to clarify the effect of RA on function and differentiation of
Th17 cells revealing contradictory results. Some in vitro studies demonstrated that Th17 cells
were inhibited by RA (75,76), while others highlighted the importance of RA for Th17 cell
differentiation (77,78). However it was concluded that the differentiation was dependent on
RA concentration in combination with currently unknown cytokines present in the
17
environment (79). For example, previously published work showed that RA was proinflammatory in concert with IL-15 and tolerogenic in the presence of TGF-β (59,80).
Research on the recently emerged ILCs and the effect of vitamin A is still in its infancy,
however it has been suggested these cells could play a large role in intestinal homeostasis. A
study performed by Mielke et al. was the first to show that RA induced IL-22 expression in
NCR3+ ILC3 in a ‘steady state’ after isolation from the lamina propria of the murine gut.
Furthermore, RA induced production of IL-22 continued during dextran sodium sulphate
(DSS) induced colitis and RA treatment reduced disease symptoms, which suggested that RA
has a positive impact on IL-22 expression in NCR3+ ILC3 in vivo and is thereby able to
promote tissue repair, cellular growth and the production of antimicrobial peptides by
intestinal epithelial cells (81).
Martin et al. investigated the secretion of IL-22 binding protein (IL-22BP), a soluble secreted
receptor known to form a high affinity bond with IL-22 thereby inhibiting its function. First
evidence for the role of RA in IL-22BP production was obtained from experiments performed
with splenic and intestinal dendritic cells from healthy rats which showed increased IL-22BP
expression in the intestinal derived cells compared to those found in the spleen. Local
diffusible factors in the intestines such as nutrients were described as cause for the marked
difference in IL-22BP expression and various diet derived molecules were tested on human
monocyte derived dendritic cells. Only the RA receptor agonist AM580 was able to increase
the expression of human IL-22BP whereas PGE2, a known antagonist of RALDH2, completely
abolished expression which strongly indicated that RA is also involved in the regulation of IL22BP production. Additional experiments with recombinant RA gathered proof to support
the hypothesis that IL-22BP is produced in homeostatic conditions, when the dendritic cells
are in an immature state. Upon activation and subsequent maturation, the dendritic cells
were demonstrated to lose their ability to produce IL-22BP (82).
These two studies showed that RA can induce both IL-22 and IL-22BP secretion, however
similar to the mechanism of RA in the adaptive immune system, the outcome is
hypothesized to be co-dependent on other environmental triggers. As shown in figure 7, RA
is considered anti-inflammatory in the presence of tolerogenic stimuli representing a
‘steady-state’ or a state in need for the restoration of balance in case of excessive
18
inflammation. Dendritic cells remain in an immature state and continue to secrete IL-22BP
which inactivates IL-22 and thereby functions as a negative feedback loop. However, when
an infection induces the activation and maturation of dendritic cells, the expression of IL22BP is downregulated and IL-22 is able to exert its effect on epithelial cells (82). In addition,
the rate of synthesis and thus the concentration of RA could also be altered upon
recognition of threads such as pathogens thereby causing a different overall effect (58).
However, there are a few shortcomings in the study of Martin et al. which should be further
investigated. Firstly, the cells that were used to demonstrate the effect of RA were derived
from humans and most findings were done on murine material. It is therefore not possible
to claim with certainty that what holds in humans can also be applied in mice. Nevertheless,
important implications can still be made. Secondly, although they have shown some findings
on the expression of IL-22BP, it would be interesting to investigate the effect on IL-22
expression and function. One could reason that if IL-22BP expression is increased, the effect
of IL-22 will reduce, however the mechanism may not be as simple.
A view is now emerging on the relationship between ILCs and RA as RA was shown to induce
IL-22 production. However, as previously stated RA is not the only factor contributing to the
intestinal immune response. Cytokines, commensal products and other dietary factors such
as AhR ligands could play an equivalent role.
19
Figure 7. Vitamin A derivative RA induces mucosal immunity via ILCs in the intestines. Dendritic cells express
RALDH enabling them to form RA from retinal. Under homeostatic conditions, RA can cause via an intracrine
mechanism the expression of IL-22BP in dendritic cells which binds and inhibits IL-22 produced by NCR+ ILC3
cells. However, during an infection dendritic cells in the lamina propria mature and cease to produce IL-22BP.
Consequently, IL-22 is free to exert its functions on epithelial cells.
20
Vitamin D. Dietary products such as fatty fish and endogenous synthesis by UVB radiation in
the skin are the two sources of vitamin D supplying the liver for further metabolism.
Moreover, 7-dehydrocholesterol is converted into vitamin D3 by UV radiation while vitamin
D3 or D2 can readily be absorbed by the small intestine. Through hepatic hydroxylation
vitamin D is metabolized into 25-hydroxyvitamin D (25OHD), which is the most abundant
form of vitamin D in the circulation. Until recently it was thought that cells of the proximal
tubule in the kidneys were the sole location in the body where the final conversion of
25OHD to the active vitamin D metabolite 1,25-dihydroxyitamin D (1,25(OH)2D) was
executed by the enzyme CYP27B1 (56,57,83). However, research has provided evidence for
the expression of CYP27B1 in other cell types such as dendritic cells, T- and B-lymphocytes,
macrophages and monocytes indicating that vitamin D has the ability to influence immunity
not only in an endocrine or paracrine, but even in an intracrine fashion, which means that
the local concentration of 1,25(OH)2D can reach high levels (83–86).
The active metabolite of vitamin D regulates transcription by interacting with the nuclear
vitamin D receptor (VDR). Identical to the mechanism of RA, the interaction of 1,25(OH)2D
with VDR results in heterodimerization with RXR, where after the complex binds to specific
sequences referred to as vitamin D responsive elements (VDRE), located in regulatory
domains of target genes. Transcription of vitamin D target genes is then either initiated or
repressed upon VDR-RXR complex docking.
Due to the expression of VDR by a wide range of immune cells, an extensive effect of vitamin
D can be observed on both innate and adaptive cells. Vitamin D activates innate immunity by
inducing the production of antimicrobial peptides including cathelicidin and β defensin 2 by
macrophages and monocytes (87,88). It was also demonstrated that other stimuli such as IL15 and TLR1/2 ligands increased the expression of CYP27B1 and VDR which in turn
augmented the production of antimicrobial peptides triggered by vitamin D highlighting the
importance of vitamin D in clearing pathogens (89,90).
Moreover, dendritic cells are subject to the effects of 1,25(OH)2D and are driven towards a
more tolerogenic phenotype which is accomplished by reducing the expression of MHC-II,
CD40, CD80 and CD86, resulting in impaired dendritic cell maturation and antigen
presentation. While the secretion of IL-2, IL-12, IL-6 and IL-23 by these antigen presenting
21
cells is additionally inhibited upon vitamin D signalling, the secretion of IL-10 is increased
which contributes to the 1,25(OH)2D induced tolerogenicity (57,62,91). Consequently, a shift
in the T-cell population towards more immunosuppressive T-cell types such as regulatory Tcells and Th2 cells will follow which can also be achieved directly via 1,25(OH)2D
transcriptional regulation within T-cells (92). Like T-cells, B-lymphocytes express the VDR and
thus have the ability to respond to 1,25(OH)2D leading to a reduced proliferative and
differential capacity and decreased production of IgG and IgM (43,83). Recent studies have
demonstrated that the effect can either be direct via VDR signalling in B-cells or indirect via
vitamin D induced Th2 cells (93). Furthermore, it is hypothesized that the adaptive immune
system is able to sense extracellular levels of vitamin D and respond appropriately by
favouring a more tolerogenic phenotype, thereby dampening the inflammatory response
which has been implicated as a protective property against autoimmune disorders (94).
To this day, few studies have been performed on the influence of vitamin D on ILCs in the
gut, while some findings on the remaining local immune cells suggest that this could be an
interesting research topic. Vitamin D could have a negative effect on the function of the IL22 producing ILCs, NCR+ ILC3 and LTi. Because studies have shown that vitamin D is able to
inhibit dendritic cell maturation, we hypothesize an indirect, inhibitory activity of vitamin D
on the IL-22 producing ILCs in figure 8 (57,95).
Figure 8. Vitamin D is
hypothesized to inhibit IL-22
production by LTi cells.
Although it remains to be
clarified, we hypothesize that
vitamin D could prevent IL-22
secretion by LTi cells via
inhibiting dendritic cells and
the production of IL-23.
22
Concluding remarks
The complex network of host, commensal and nutritional components makes the intestinal
environment challenging to study and without exact knowledge concerning the mechanisms
occurring in the intestines, related diseases are difficult to treat or cure. In addition, ILCs
were shown to be required for immunological processes including tissue repair, lymphoid
organogenesis and protection against helminths and bacteria. After their recent discovery
questions were raised regarding the involvement of these cells in intestinal homeostasis and
diseases. Nonetheless, progress has been made in the field of gut immunity and the effect of
diet derived compounds such as AhR ligands and vitamins.
AhR is believed to be involved in group 3 ILC maintenance, IL-22 production and generation
of postnatal lymphoid tissues suggesting intact embryonic development. Interestingly, the
relevance of AhR ligands derived from the diet has only been implicated in cellular
maintenance and lymphoid tissue formation with a loss of function after the cryptopatches
and ILFs are completely formed. Furthermore, it would be interesting to investigate whether
dietary AhR ligands would play a role in IL-22 production.
By regulating the expression of T-cell specific transcription factors such as GATA3 and RORγt,
vitamin A derivative RA was able to induce a shift towards more tolerogenic phenotypes.
Since identical transcription factors are used by ILCs, it remains to be elucidated whether RA
would have the same impact on ILC development. Moreover, RA was found to stimulate
both NCR+ ILC3 cells and dendritic cells to produce IL-22 and IL-22BP respectively depending
on the immunological state of the intestines.
While T- and B-cells are known to change phenotype upon activation by vitamin D, few
findings have been gathered concerning the impact on ILCs. The presence of VDR and
CYP27B1 in ILCs should be determined because they can provide a local source of
1,25(OH)2D, which would establish a direct mechanism of action. However, here we
hypothesize an indirect mechanism by inhibiting dendritic cells.
Aside from exogenous AhR ligands and vitamins, the dietary compartment is comprised of a
large number of compounds including carbohydrates and lipids which could have an effect
on the local ILCs. For example, oxysterols are hypothesized to be ligands of RORγt, meaning
23
that group 3 ILC development could be stimulated by these particles (96). In addition, the
microbiota provide an indirect source of diet derived substances by transforming these
molecules into a form that can be absorbed by the host. The vast amount of dietary
compounds and the emerging characteristics of ILCs together suggest that research is
currently just scratching the surface and that we should continue to investigate the ILCs and
the role of AhR ligands, vitamins and other factors in shaping ILCs in the intestine.
24
Acknowledgments
I would like to take the opportunity to thank everyone who guided me during the process of writing
the master thesis.
I want to thank my daily supervisor Gera Goverse in particular for reviewing my work multiple times
and helping me with my academic writing skills.
I would also like to address my gratitude to Assoc. Prof. M. Boes (UMCU) and Prof. R. Mebius (VUmc)
for reading and grading my work as the first and second reviewer.
25
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Appendix: Summary for general public
Many people suffer from intestinal related disorders such as Crohn’s disease and this number
increases every year. Although there are therapies available, research needs to gain more
information about the processes occurring in the intestine. However, the vast amount of cells and
molecules present in the gut makes it difficult to discover new information which could aid in finding
new and improved therapies. Furthermore, the intestine can be divided into three compartments
namely the bacterial, diet and host compartment. The local immune system is part of the host
compartment and plays a crucial role in maintaining balance. Recently emerged immune cells called
innate lymphoid cells are known to be involved in the generation of lymphoid tissues and the battle
against pathogens. Moreover, the diet compartment comprises many molecules that originate from
ingested food and was shown to have an impact on immune cells. Studies have shown that vitamins
and molecules in cruciferous vegetables called the AhR ligands have an anti-inflammatory effect on
intestinal ILCs. In contrast, others have shown the complete opposite concerning vitamin A which
suggested that variation in concentration or other molecules such as cytokines could alter the
outcome of these dietary components. In this report, we give an overview of the different ILC
families and discuss recent findings on the influence of AhR ligands and vitamins on ILCs in the
intestines.
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