Environmental genotoxic factors pose a serious and constant peril to the genomic integrity
of cells at barrier surfaces with the environment. They can induce mutations that,
if they occur in epithelial stem cells, contribute to malignant transformation and
cancer development1–3. Genome integrity in epithelial stem cells is closely guarded
by an evolutionary conserved, cellular response pathway, the DNA damage response (DDR).
The DDR culminates in either transient cell cycle arrest and DNA repair or elimination
of damaged cells by apoptosis4,5. Here we show, that the cytokine interleukin (IL-)22
produced by group 3 innate lymphoid cells (ILC3) and γδ T cells is an important rheostat
of the DDR machinery in intestinal epithelial stem cells. Using a new mouse model
allowing for the sporadic inactivation of the IL-22 receptor in colon epithelial stem
cells, we demonstrate that IL-22 is required for an effective initiation of the DDR
following DNA damage. In consequence, stem cells deprived of IL-22 signals and exposed
to carcinogens escaped DDR-controlled apoptosis, contained more mutations, and were
more likely to give rise to colon cancer. We identified metabolites of glucosinolates,
a group of phytochemicals contained in cruciferous vegetables, to be an commonplace
source of genotoxic stress in intestinal epithelial cells. Glucosinolate metabolites
are ligands of the aryl hydrocarbon receptor (AhR)6 and AhR signaling in ILC3 and
γδ T cells controlled their production of IL-22. Mice fed with diets deprived of glucosinolates
produced only very low levels of IL-22 and, consequently, the DDR in epithelial cells
of mice on a glucosinolate-free diet was crippled. Collectively, we identify a homeostatic
network protecting stem cells against perils to their genome integrity by AhR-mediated
“sensing” of genotoxic components contained in diets. AhR signaling in turn ensures
on-demand production of IL-22 by innate lymphocytes directly regulating components
of the DDR in epithelial stem cells.
To model colitis-associated colon cancer (CAC), we challenged mice with the pro-carcinogen
azoxymethane (AOM), and then treated with dextran sodium sulfate (DSS) resulting in
intestinal inflammation fueling tumor growth. AOM is an alkylating agent that generates
meG) adducts in DNA that lead to O6
meG:T mispairings and eventually to mutations7. These mutations often affect the glycogen
synthase kinase (GSK)-3β phosphorylation site in exon 3 of the β-catenin gene similar
to the mutations8–10 found in the majority of human colon cancers. Mice genetically
and constitutively lacking IL-22 developed more tumors11 (Extended Data Fig. 1a,b),
that were mainly found in the distal part of the colon (Extended Data Fig. 1c). IL-22
is a cytokine selectively acting on non-hematopoietic cells including epithelial stem
cells12. While the results may suggest that IL-22 restrains tumor development, these
data were difficult to interpret since Il22
-/- mice have dysbiotic changes in microbial communities13 and show increased inflammation11
after DSS application (Extended Data Fig. 1d-f). Thus, the enhanced inflammation itself
could explain the higher number of tumors in Il22
-/- mice also developed more tumors when treated with AOM injections only (Fig. 1a).
To differentiate direct effects of IL-22 signaling on tumor transformation in colon
stem cells from confounding differences in inflammation, microbiota, and/or other
stem cell extrinsic factors inherent to the comparison of mutant and wildtype mice,
we conceived a mouse model allowing for the inducible and sporadic deletion of the
gene encoding IL-22 receptor a1 (Il22ra1) in intestinal epithelial stem cells (Fig.
1b, Extended Data Fig. 1g), the primary target of carcinogenesis14. To achieve this,
we first generated Il22ra1
fl/fl mice (Extended Data Fig. 2 a-g) and crossed these to Lgr5
CreERt2/+ mice allowing for tamoxifen-inducible Cre activity in crypt-based columnar
stem cells15. To report Cre activity and gene inactivation, Il22ra1
CreERt2/+ mice were further mated to Rosa26R-Confetti mice, containing four colors
that are stochastically activated after Cre-mediated removal of a transcriptional
roadblock16. After tamoxifen application, we targeted on average 15-30% of crypts
(Fig. 1c) and the fraction of crypts with one or more colors was not affected by Il22ra1
expression (Extended Data Fig. 2h,i). In contrast to the comparison of mutant and
wildtype mice (Extended Data Fig. 1d), we did not detect any differences in DSS-induced
weight loss between Il22ra1
CreERt2/+;R26R-Confetti and Il22ra1
CreERt2/+;R26R-Confetti mice (Extended Data Fig. 2j). Importantly, Confetti+ tumors
CreERt2/+;R26R-Confetti mice did not express IL-22Ra1 whereas Confetti- tumors in
the same mouse were IL-22Ra1-positive (Extended Data Fig. 3 a-e). This approach allowed
us to interrogate IL-22-dependent programs in stem cells within the same mouse by
creating a mosaic of IL-22Ra1+ and IL-22Ra1- crypts (Fig. 1b).
Considering the labelling efficiency (Fig. 1c, Extended Data Fig. 2h,i), we would
expect 15-30% of Confetti+ tumors which is what we observed in mice having one functional
allele of Il22ra1 (Fig. 1d). In striking contrast, the fraction of Confetti+ tumors
in mice with sporadic inactivation of the Il22ra1 gene was substantially increased,
demonstrating that absence of IL-22 signaling in colon epithelial cells pre-disposes
them for tumor development (Fig. 1d-f). In the colon, roughly half of the IL-22 producers
were CD4+ T cells, a third ILC3 and ca. 6% each, Foxp3+ CD4+ T cells and γδ T cells
(Extended Data Fig. 4a). ILC3 were the dominant source of IL-22 in the small intestine
(Extended Data Fig. 4b). Less sheltered mice showed a larger fraction (79-88%) of
CD4+ T cells among IL-22 producers (Extended Data Fig. 4c). Collectively, the data
demonstrate that IL-22 signaling in colon epithelial cells is a significant barrier
to tumor development.
Stem cells are the origin of cancer4,14. IL-22 is continuously produced at the steady-state17
but the IL-22-controlled transcriptional networks in colon stem cells are unknown.
Using RNA-seq, we found that during the steady-state 350 genes (with FC>2) were changed
in expression between Lgr5+ colon stem cells of Il22+/+
mice (Extended Data Fig. 5a,b). Gene Set Enrichment Analysis (GSEA) revealed, that
stem cells from Il22-/-
mice were depleted of transcripts associated with ‘DNA Repair’ (Fig. 2a) and ‘DNA
double-strand break processing’ (Extended Data Fig. 5c). Considering these biological
pathways, we performed RNA-seq of sorted colonic Lgr5+ stem cells from Il22+/+
mice 24 hours after inducing DNA double strand breaks when the cellular response to
DNA damage was maximal (Extended Data Fig. 5d). GSEA of the expressed genes showed
significant enrichment of gene signatures such as ‘Hallmark Apoptosis’ and ‘DNA damage
response effector genes’ in Lgr5+ stem cells from Il22
+/+ but not from Il22
-/- mice (Fig. 2b, Extended Data Fig. 5e). IL-22BP is an antagonist of IL-22 produced
to blunt excessive and potentially pathologic IL-22 production11. IL-22BP production
was reduced following DNA damage allowing for higher concentrations of bioactive IL-22
(Extended Data Fig. 5f). These data indicate that stem cells deprived of IL-22 signals
lack transcriptional signatures of the DDR and DNA repair pathways.
To pinpoint the IL-22-dependent processes within the DDR, we performed a systematic
analysis of critical checkpoints. Expression of MRN complex genes (Mre11, Rad50, Nbn),
which sense DNA damage, was independent of IL-22 (Extended Data Fig. 5g). The MRN
complex recruits and activates a group of kinases (i.e., ATM, ATR, DNA-PK) which initiate
the DDR cascade and are expressed in almost all living cells for an immediate response
to genotoxic stress18. We found a reduction of Atm expression in colon stem cells
-/- mice already in the absence of genotoxic stress (Fig. 2c,d, Extended Data Fig.
5h,6a). Atm is a STAT3 target gene19 and IL-22 induces STAT3 signaling in epithelial
cells20. STAT3 ChIP using primers for three STAT3 binding sites in the Atm gene regulatory
elements (Extended Data Fig. 6b) revealed reduced STAT3 occupancy at all three binding
sites in epithelial cells from Il22-/-
mice during the steady-state (Extended Data Fig. 6c). Exogenous in vivo application
of IL-22 significantly enhanced STAT3 binding (Extended Data Fig. 6c) and increased
ATM levels in colon epithelial stem cells (Fig. 2d). After DNA damage, ATM phosphorylates
histone variant H2AX (referred to as γH2AX) which flanks the break site21. While Il22
+/+ mice showed maximal γH2AX levels in colon crypts 8 hours after irradiation, there
was virtually no increase in Il22
-/- or Il22ra1
fl/fl;Vil1-Cre mice at any time point investigated (Fig. 2e,f and Extended Data Fig.
6d,e). H2AX protein levels did not differ between Il22
+/+ and Il22
-/- mice (Extended Data Fig. 6f). While we used irradiation here as the paradigm to
cause double strand DNA breaks, AOM treatment led to the same extent of IL-22-dependent
γH2AX formation (Extended Data Fig. 6g,h). Collectively, our data demonstrate that
during the steady-state IL-22 regulates the expression of ATM via STAT3 in colon epithelial
stem cells, supervising the initiation of the DDR following genotoxic stress.
Phosphorylation of p53 and SMC (structural maintenance of chromosome) proteins, both
direct targets of ATM4,5,18, was reduced in colon epithelial cells of irradiated Il22
-/- mice (Extended Data Fig. 7a). Based on our RNA-seq data, expression of various
p53 target genes (e.g., p21, Puma, hnRNPK, Susd6, Btg2 and Mdm2)22,23 was diminished
in colon stem cells of Il22
-/- mice (Fig. 2a,b, Extended Data Fig 5b). In particular, p21, a cell cycle arrest
gene, and Puma, a pro-apoptotic gene24 were not efficiently induced in Il22
-/- mice following irradiation (Fig. 3a,b, Extended Data Fig. 7b) or AOM treatment
(Extended Data Fig. 7c). Increased expression of these genes after DNA damage required
IL-22 signaling in epithelial cells as documented in mice with deletion of IL-22Ra1
or STAT3 in all intestinal epithelial cells (Fig. 3b, Extended Data Fig. 7d). Exogenous
application of IL-22 rescued Puma levels in colon stem cells depending on IL-22R expression
We hypothesized that colon stem cells experiencing DNA damage in the absence of IL-22
signals, do not undergo PUMA-mediated apoptosis or p21-controlled cell cycle arrest.
Genetic inactivation of IL-22Ra1 in epithelial cells or antibody-mediated neutralization
of IL-22 resulted in a lower fraction of apoptotic (i.e., cleaved (c)Caspase 3+) Lgr5+
stem cells in colon crypts following irradiation (Fig. 3d,e, Extended Data Fig. 7e-g,
and 8a-d). Using sporadic inactivation of IL-22Ra1 in colon epithelial stem cells,
we found a highly significant reduction of apoptosis in IL-22Ra1-deficient crypts
(i.e., Confetti+ crypts) compared to IL-22Ra1-proficient crypts (i.e., Confetti-)
within the same mouse (Fig. 3f,g). Inhibition of apoptosis with zVAD, a pan-caspase
inhibitor, in Il22
+/+ mice led to significantly increased numbers of tumors comparable to Il22
-/- mice (Fig. 3h). These data demonstrate that the reduced rate of apoptosis observed
after DNA damage in IL-22-deprived colon stem cells may explain the higher number
Cells that continue to cycle in the presence of DNA breaks are prone to mitotic defects
leading to the formation of micronuclei, a sign of genomic instability. Colonic epithelial
cells of irradiated Il22
-/- mice showed increased numbers of micronuclei when compared to wildtype controls
(Fig. 3i). Conversely, treatment of an intestinal epithelial cell line with IL-22
reduced the numbers of micronuclei after irradiation (Extended Data Fig. 8e). IL-22
has also been described as promoting proliferation of damaged stem cells12 and sustaining
proliferation of already transformed cells25. We found an increase in proliferation
after DNA damage in Il22
+/+ mice which was absent in Il22
-/- mice (Extended Data Fig. 8f). The DDR is a powerful cellular program that prevents
accumulation of mutations after genotoxic stress. We isolated colon epithelial cells
from mice eight weeks after AOM administration and quantified the frequency of exon
3 mutations in the Ctnnb1 gene which are strongly associated with various forms of
human cancer including colon cancer9,10. Colon epithelial cells from Il22
-/- mice accumulated a much higher frequency of Ctnnb1 mutations than cells from Il22
+/+ mice (Fig. 3j). Thus, IL-22, a cytokine produced by lymphocytes, acts as an important,
epithelial cell-extrinsic regulator of the DDR in colon stem cells preventing the
accumulation of potentially dangerous mutations.
Mutations accumulate in colon stem cells during life, partially due to environmental
genotoxic compounds3. Cruciferous vegetables of the Brassicaceae family contain large
amounts of a group of phytochemicals referred to as glucosinolates6, some of their
metabolites are potent genotoxins26. For example, the glucosinolate metabolite 1-methoxy-3-indolylmethyl
alcohol (1-MIM-OH) leads to the formation of DNA adducts and activates the DDR26 (Extended
Data Fig. 9a). We applied a single dose of 1-MIM-OH by gavage and monitored the expression
of PUMA and p21 (Fig. 4a, Extended Data Fig. 9b), which were induced in colon epithelial
stem cells in an IL-22-dependent manner. Indole 3-carbinol (I3C), a glucosinolate
metabolite without DNA damaging qualities, did not trigger the DDR (Fig. 4b). Regardless
of their DNA-damaging qualities, a single application of 1-MIM-OH or I3C significantly
increased the production of IL-22 by ILC3 and γδ T cells but not by CD4+ T cells (Fig.
4c,d and Extended Data Fig. 9c,d). IL-10 production by FoxP3+ T cells was not affected
either (Extended Data Fig. 9e). Glucosinolate-induced IL-22 expression enhanced expression
of Reg3g, an antimicrobial gene regulated by IL-22 (Extended Data Fig. 9f,g).
The increase of IL-22 production in response to 1-MIM-OH or I3C required AhR expression
in ILC3 and T cells (Extended Data Fig. 9h)27–29. Mice genetically lacking AhR in
ILC3 and T cells had a phenotype comparable to IL-22-deficient mice with reduced expression
of ATM (Fig. 4e), and, following genotoxic stress, with diminished phosphorylation
of H2AX (Fig. 2e,f, Extended Data Fig. 6d), absent induction of p21 and PUMA expression
(Fig. 4f) and reduced apoptosis of crypt-resident stem cells (Extended Data Fig. 8a,b).
Importantly, IL-22 application to Ahr
fl/fl;Rorc(γt)-CreTg mice restored PUMA expression (Fig. 3c).
We wondered whether glucosinolate-deprived diets would be disadvantageous to the host
to deal with consecutive genotoxic stress. We fed adult mice, either with an experimental
defined diet (EDD) that lacks phytochemicals (including glucosinolates; EDD-) or with
a defined diet supplemented with I3C, EDD+ (Extended Data Fig 9b). Normal mouse chow,
naturally containing bioactive concentrations of AhR ligands, served as control diet
(CD). Colon ILC3 and γδ T cells from mice on EDD- did not produce IL-22, whereas they
produced high levels of IL-22 when mice were fed EDD+ or CD (Fig. 4g and Extended
Data Fig. 10a,b). Epithelial cells from mice on EDD- showed reduced expression of
ATM (Fig. 4h), and, following DNA damage, a profound reduction in γH2AX (Fig. 4i,
Extended Data Fig. 10c), in induction of p21 and Puma expression (Fig. 4j) and in
apoptosis observed in colon crypts (Fig. 4k, Extended Data Fig. 10d). Importantly,
I3C addition to the diet restored all of these functions. Thus, AhR-mediated regulation
of IL-22 provided a substantial barrier against DNA damaging agents as part of our
Collectively, we uncovered a novel homeostatic control circuit by which IL-22-producing
and tissue-resident innate lymphocytes regulate the response of epithelial stem cells
to genotoxic dietary compounds at border surfaces with the environment, thereby effectively
limiting the number of mutations (Extended Data Fig. 10e). The AhR expressed by ILC3
and γδ T cells serves as a “sensor” of genotoxic phytochemicals for on-demand production
of IL-22 supervising the DDR machinery in epithelial stem cells. The AhR may turn
out to be an important node for cancer development as AhR signaling within epithelial
cells was recently shown to reduce colon cancer development30. Finally, we expect
such communication between innate lymphocytes and their tissue client cells to be
operative at other border surfaces which may be exploited as an adjunctive therapy
to avoid genotoxic sequelae associated with various forms of clinical therapy such
as chemotherapy, radiation therapy and bone marrow transplantation.
Methods (online only)
Conventional (specific pathogen-free) C57BL/6N mice were purchased from Janvier Laboratories.
EGFP-IRES-CreERt2/+ (here referred to as Lgr5
CreERt2/+ or Lgr5
Egfp/+ mice)15, Rosa26R-Confetti16 and Rorc(γt)-CreTg mice31 were purchased from Jackson
Laboratories and crossed in our animal facility. Il22
-/- mice were a kind gift of Jean-Christophe Renauld and Laure Dumoutier (Ludwig Institute
for Cancer Research, Brussels, Belgium)32. Mice with a floxed Stat3 allele33 were
kindly provided by Veronika Sexl (University of Veterinary Medicine Vienna) with permission
of Valeria Poli (Universita di Torino, Torino, Italy). Villin1-CreTg mice34 were kindly
provided by Marc Stemmler (Max-Planck-Institute of Immunobiology & Epigenetics, Freiburg,
Germany) with permission of Sylvie Robine (Institute Curie, Paris, France). Mice with
an IEC-specific deletion of Stat3 (Stat3
;Vil1-Cre) were generated by breeding Stat3
fl/fl mice with Villin1-CreTg mice. Il22ra1
;Vil1-Cre mice were generated by crossing of Il22ra1
fl/fl mice to Villin1-CreTg mice. Ahr-/-
mice35 and Ahr
fl/fl mice36 were kindly provided by Charlotte Esser (Leibniz Research Institute for
Environmental Medicine, Düsseldorf, Germany). Ahr
;Rorc(γt)-Cre mice were obtained by crossing Ahr
fl/fl mice with Rorc(γt)-CreTg mice27. To identify Lgr5+ stem cells, we backcrossed
+/+ and Il22
-/- mice to Lgr5
EGFP-IRES-CreERt2/+ mice (referred to as Il22
Egfp/+ mice or Il22
Egfp/+ mice). Mice were 8 to 10 weeks of age, unless indicated otherwise. Cohoused
littermates were used as controls whenever possible. Animals were housed in accordance
with the guidelines by the Federation for Laboratory Animal Science Associations and
the national animal welfare body Gesellschaft für Versuchstierkunde. Experiments were
in compliance with the German animal protection law (TierSchG) and were approved by
the local animal care committee (G15-1-034, Landesuntersuchungsamt Rheinland-Pfalz,
Generation of Il22ra1
fl/fl mice were generated using EUCOMM ES cells subclone JM8.N4 (Il22ra1
tm1a(EUCOMM)Wtsi)37. In brief, a floxed Il22ra1 allele was created by inserting an
FRT-flanked IRES-LacZ-Neo resistance cassette into intron 1 of the Il22ra1 gene. LoxP
sites were inserted to flank exon 2 (Extended Data Fig. 2a-g). After germ-line transmission,
the FRT cassette was removed by crossing to FLPe deleter mice38. For inactivation
of the Il22ra1 allele in intestinal stem cells, Il22ra1
CreERt2/+;Rosa26R-Confetti mice were fed for two weeks with diets containing 400 mg/kg
tamoxifen citrate (Envigo). Tamoxifen was stopped seven days before tumor induction.
Acute DNA damage was induced by i.p. injection of 25 mg/kg (body weight) azoxymethane
(AOM) or by exposure of cells or mice to a 137Caesium (Cs) source in an OB58/905-2
irradiator (Buchler, Germany). For acute DNA damage, mice were exposed to 8Gy of irradiation
if not indicated otherwise. For the analysis of IL-22 production, mice were injected
with 25mg/kg (body weight) AOM and lamina propria lymphocytes were analysed 24h later.
IL-22 neutralization and reconstitution
Monoclonal anti-IL-22 (Clone 8E11) blocking antibody was kindly provided by Genentech
(South San Francisco, USA). Anti-IL-22 (150μg/mouse) or control IgG were injected
36h, 12h before and 12h after inducing DNA damage. For reconstitution, mice were injected
with recombinant IL-22 (400ng/mouse, Peprotech) i.p. every 8h for three days prior
to the damage induction and at 8h and 16h afterwards. Epithelial cells were isolated
24h after damage. For the stimulation of ATM expression, mice were injected with recombinant
IL-22 (500 ng/mouse, Peprotech) i.p. 12h and 2h prior to epithelial isolation.
Mice of various genotypes were co-housed and injected with 10mg/kg body weight azoxymethane
(AOM, Sigma-Aldrich) i.p. on day 1. Starting from day 7, mice were given 2% Dextrane
Sulfate Sodium (DSS) in the drinking water for 7 days. The weight of the mice was
monitored weekly. The approved experimental procedures limited maximal colorectal
tumor burden to a 20% decrease in body weight. None of the experimental animals reached
this threshold. Tumor formation was assessed starting from day 80 after AOM treatment.
For AOM-only induced cancer, mice were injected three times in weekly intervals with
10mg/kg AOM i.p.. No DSS was given in the drinking water and tumor formation was assessed
4 months after the first AOM application. For the blockade of apoptosis, a pan-Caspase
inhibitor zVAD-FMK (Invivogen) was injected at 2mg/kg i.p. daily on day 0 - day 5,
with AOM being injected on day 1. Tumor formation was assessed on day 90.
Epithelial cell isolation and quantitative PCR
The colon, including the anorectal section, was opened longitudinally and cleaned
from feces and fat tissue. Epithelial cells were harvested by incubating the tissue
for 45 min in Hank’s Balanced Salt Solution (HBSS) supplemented with 5 mM EDTA and
10 mM HEPES at 37 °C. Cells were pelleted and resuspended in 500 μl Trizol (Life Technologies)
and macerated using MP FastPrep-24 (MP Biomedicals). RNA was extracted according to
the ImmGen protocol (https://www.immgen.org/Protocols/Total%20RNA%20Extraction%20with%20Trizol.pdf)
and cDNA was transcribed using High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems). Quantitative PCR was performed using qPCR SYBR Green Master Mix (Applied
Biosystems) on a 7900HT Fast Real-Time PCR System (Applied Biosystems). Primer sequences
are in Supplementary Information Table 1.
Lamina propria lymphocyte isolation
Lamina propria lymphocytes were isolated as described previously39. In brief, small
intestine or colon was removed and cleaned from feces and fat tissue. Peyer’s patches
were removed and epithelial tissue was dissociated by incubation in HBSS supplemented
with 5mM EDTA and 10mM HEPES at 37°C. Tissue was minced and subsequently digested
in HBSS containing Dispase (5U/ml; BD Biosciences), collagenase D (0.5mg/ml; Roche)
and DNaseA (0.5mg/ml; Sigma-Aldrich) and 3% FCS for 1h at 37°C. Lymphocytes were enriched
using an 80/40 Percoll gradient (Sigma-Aldrich). For cytokine staining, cells were
resuspended in RPMI medium with 10% FCS, 80μM 2-mercaptoethanol, 10mM HEPES, 1mM sodium
pyruvate, non-essential amino acids (Gibco), 100units/ml penicillin, 0.4mg/ml gentamicin
and 100mg/ml streptomycin and 8mg/ml glutamine and stimulated for 4h at 37°C in the
presence of brefeldin A (Sigma-Aldrich), IL-1β (10ng/mL, Peprotech) and IL-23 (20ng/mL,
Thermo Fisher) or for IL-10 production with PMA (25ng/mL, Sigma-Aldrich) and ionomycin
(500ng/mL, Sigma-Aldrich). Cells were fixed with FoxP3 fixation/permeabilization kit
(eBioscience) overnight and stained with fluorochrome coupled antibodies for 2h at
4°C. Cells were washed and analyzed on a FACS Canto II flow cytometer (BD Biosciences)40.
Data was analyzed using FlowJo Software V10.3 (Treestar). All antibodies used are
listed in Supplementary Information Table 2.
Colon was opened longitudinally and cleaned from feces and fat tissue. “Swiss rolls”
were prepared by winding the tissue on a wooden toothpick and tissue was fixed for
2h in 4% paraformaldehyde at 4°C. The tissue was washed and incubated overnight in
30% sucrose in phosphate buffer. Rolls were frozen in O.C.T. Tissue Tek (Sakura) and
stored at -80°C. For analysis, 6μm sections were cut using a CM1950 Cryotome (Leica).
Sections were dried, rehydrated and blocked for 1h with 10% bovine serum albumin (BSA).
Primary antibodies specific for cleaved Caspase-3 (Asp175; 5A1E; Cell Signaling Technology),
IL-22ra1 (496514, Thermo Fisher) and γH2AX (20E3; Cell Signaling Technology) in 0.1%
Triton X-100 in PBS were incubated overnight at 4°C. Secondary (α-rabbit-Alexa555,
α-rabbit-APC, α-rat-Alexa647; Life Technologies) and directly conjugated antibodies
(α-EpCam-APC; eBioscience) were incubated 1h at room temperature. Slides were mounted
using PermaFluor Aqueous Mounting Medium (Thermo Scientific) and imaged on an AF7000
or a TCS SP5 microscope (Leica). Image analysis was done using ImageJ analysis software41.
For quantification of cleaved Caspase-3 and γH2AX, whole colon sections were filtered
for EpCam-positive cells and within these, marker-positive cells were counted. Positive
cells were normalized to the total amount of epithelial cells (i.e., DAPI+ EpCam+
RNA-seq analysis of Lgr5+ colon stem cells
The colon, including the anorectal section, was removed, opened longitudinally and
cleaned from feces and fat tissue. Epithelial cells were harvested by incubating the
tissue for 45min in HBSS supplemented with 5mM EDTA and 10mM HEPES at 37°C. Supernatant
was collected, pelleted and afterwards digested for 30min at 37°C in HBSS containing
3% FCS, Dispase (5U/ml; BD Biosciences), collagenase D (0.5mg/ml; Roche) and DNaseA
(0.5mg/ml; Sigma-Aldrich). For flow cytometry analysis, cells were stained in PBS
containing 2% FCS. Colon stem cells were identified as DAPI- EpCam+ CD45- CD24int
Lgr5+ cells (Extended Data Fig. 5a), were highly purified using a FACS Aria III Cell
Sorter (BD Biosciences) and taken up in Trizol (Life Technologies). RNA was extracted,
and libraries were prepared with SMARTer Ultra Low Input RNA (Clontech), according
to the manufacturer’s instructions. Material was enriched for polyA sequences and
Next Generation Single-end sequencing was done on a HiSeq 2500 machine (Illumina)
with a maximal readlength of 50 bases. Bioinformatic processing was performed using
the Cufflinks pipeline42. Fragments Per Kilobase Million (FPKM) values were used for
transcriptional comparison. Computational processing was done using RStudio Software
(The R Foundation). Genes without any mappable reads were excluded from the analysis.
Gene Set Enrichment Analysis (GSEA) was done with Broad Institute GSEA Software43.
Genesets were taken from MSigDB. The “DNA Damage Response Effector Genes” geneset
was created from the literature4 (Extended Data Fig. 5e). Full lists of Gene Sets
with expression heatmaps can be found in Supplementary Information Table 3.
Western blot analysis
Epithelial cells were harvested as described above. Cells were lysed using NP-40 lysis
buffer (20 mM TRIS-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, Protease & Phosphatase
Inhibitor w/o EDTA; Thermo Fisher) for 1h on ice and additionally sonicated for 3
cycles using Bioruptor Plus (Diagenode). SDS-PAGE was performed with gels containing
5% or 10% polyacrylamide and blotted 16h with 40mA on a PVDF membrane (Carl Roth).
Detection antibodies p21 (F5; Santa Cruz), PUMA, p-p53 (all Abcam), Actin (AC15; Sigma-Aldrich),
p-SMC (D7S8Y), mouse ATM (D2E2), SMC (8E6), γH2AX (D17A3) (all Cell Signaling Technology)
were diluted in 5% BSA and incubated overnight at 4°C. Secondary antibodies were coupled
to HRP (α-mouse-HRP, α-rabbit-HRP; Jackson Immuno Research) and visualized using enhanced
chemiluminescence (PerkinElmer) and Hyperfilm (GE Healthcare). All antibodies used
are listed in Supplementary Information Table 2.
Mice were injected 1 μg recombinant IL-22 (Peprotech) or PBS i.p. 1h prior to epithelial
cell isolation. Epithelial cells were isolated as described above. Cells were resuspended
in PBS and fixed with 0.75% paraformaldehyde. Fixation was stopped with glycine and
cells were washed and lysed in 50mM Tris, 2.5mM EDTA, 0.1% NP-40 and 10 % Glycerol.
Nuclei were lysed in 50mM Tris, 5mM EDTA and 0.25% SDS and subsequently sonicated
in a Bioruptor Plus (Diagenode). Lysates were cleared by centrifugation and stored
at -80°C. Protein-A-coated magnetic beads (Diagenode) were blocked with yeast tRNA
(Life Technologies) and BSA (New England Biolab). Nuclear lysates were split in 10%
input and 90% immunoprecipitation solutions. The latter was incubated with α-STAT3
(C-20; Santa Cruz Biotechnology) overnight rotating at 4°C. Protein-A coded magnetic
beads were added and incubated for another 30min rotating at 4°C. Beads were purified
and washed repeatedly. Bound DNA was eluted in 10mM TRIS, 1 mM EDTA and 2% SDS at
37°C for 15min. De-crosslinking was performed overnight at 65°C. RNA was removed by
1h incubation with RNAse A (Peqlab) at 37°C, followed by 1h incubation with Proteinase
K (Peqlab) at 42°C. DNA was purified using PCR Purification Kit (Qiagen) according
to the manufacturer’s instructions. All antibodies used are listed in Supplementary
Information Table 2.
IEC6 cells44 were cultured for 48h in DMEM (Life Technologies) with 10% FCS, 80μM
2-mercaptoethanol, 10mM HEPES, 1mM sodium pyruvate, non-essential amino acids (Gibco),
100units/ml penicillin, 0.4mg/ml gentamicin and 100mg/ml streptomycin and 8mg/ml glutamine
and 0.1U/ml insulin (Sigma-Adrich), with or without 100ng/mL IL-22 (Peprotech). After
culturing cells in the presence of IL-22 for three days, cells were irradiated with
2Gy. Afterwards they were cultured for another 48h in the presence of cytochalasin
B (4.5μg/ml, Sigma-Aldrich) to stop cytokinesis. Nuclei of cells that had undergone
division, and the occurrence of micronuclei within these cells, were counted under
a fluorescent microscope. Between 500 and 1000 Nuclei per replicate were analysed.
For analysis of primary epithelium ex vivo, mice were irradiated with 2Gy and epithelial
cells were isolated by EDTA-based dissociation as described above. For micronucleus
assessment cells were settled on glass slides and fixed for 2min in ice-cold methanol.
DNA was stained with DAPI (Sigma-Aldrich) and images were acquired on an Axiovert
200M microscope (Zeiss).
Mice were co-housed and injected 10mg/kg body weight azoxymethane (Sigma-Aldrich)
i.p. on day 1, day 8 and day 15. Mice were sacrificed on day 75, when no macroscopic
tumor development could be observed. Epithelial cells from the colon were isolated
as described above and genomic DNA was extracted using QIAamp Fast DNA Tissue Kit
(Qiagen) according to the manufacturer’s instructions. Competitive Allele-Specific
TaqMan (cast) PCR (Thermo Fisher) was used to detect single basepair mutations in
the Ctnnb1 gene. AOM-induced mutations were derived from the literature45 and compared
to OMIM database to find conserved mutations and high frequency targets were chosen
(CTNNB1_5676_mu, b.C122I, p.T41I; CTNNB1_5671_mu, b.G101A, p.G34E; CTNNB1_5672_mu,
b.G94A p.D32N). For reference quantification of total Ctnnb1 gene expression, quantitative
RT-PCR was performed as described above. TaqMan PCR was carried out according to the
manufacturer’s instructions (Thermo Fisher).
Mice were generally fed ad libitum with standard grain-based rodent diet (R/M-H Extrudat,
sniff). This diet is also referred to as control diet (CD) during the experiments
with defined diets. To analyze the impact of dietary glucosinolates, mice were switched
to an experimental defined diet at the age of 6 weeks and fed with this diet for 6
weeks when the analyses were performed. The defined diet (EDD-) (EF/RM low chlorophyll;
sniff) is devoid of any plant-derived phytochemicals and is regularly tested to be
free of chlorophyll, flavonoids and glucosinolates. The defined diet was supplemented
with 2g/kg indole-3-carbinol (EDD+) (I3C, Sigma-Aldrich). All diets contained the
same amount of metabolizable energy and equivalent compositions of proteins, carbohydrates,
fat, trace elements, minerals and vitamins27.
Treatment with AhR ligands
1-methoxy-3-indolylmethyl alcohol (1-MIM-OH, synthesized as described elsewhere26)
and I3C were dissolved in corn oil (Sigma-Aldrich) and gavaged at a concentration
of 106mg/kg body weight. After the indicated timepoints, mice were sacrificed and
small intestine and colon were processed, as described above. Caecum was removed,
cleaned and snap frozen in liquid nitrogen. Tissue was later homogenized and genomic
DNA was extracted using QIAamp Fast DNA Tissue Kit (Qiagen) according to the manufacturer’s
DNA adduct analysis
Caecal tissue was harvested as described before and genomic DNA was extracted using
QIAamp Fast DNA Tissue Kit (Qiagen) according to the manufacturer’s instructions.
25μg of isolated DNA were spiked with stable isotopically labeled adduct standards
and hydrolyzed to 2’-deoxynucleosides (dN) according to our optimized protocol46.
After analyte enrichment by C18 solid phase extraction, the predominant DNA adducts
after 1-MIM-OH exposure, namely N
2-(1-MIM)-dG and N
6-(1-MIM)-dA, were analyzed by isotope-dilution liquid chromatography tandem-mass
spectrometry (LC-MS/MS) as described previously47 using an Agilent 1260 Infinity LC
system coupled to an Agilent 6490 triple quadrupole-mass spectrometer (Agilent Technologies)
interfaced with an electrospray ion source operating in the positive ion mode (ESI+).
Further, in the DNA hydrolyzate prior to sample purification, we analyzed the amounts
of dC and 5mdC (5-methyl-2’-deoxycytidine) by isotope-dilution LC-MS/MS as recently
published48. We calculated the amount of DNA that was actually used for the adduct
analysis knowing that dC (in fact the sum of dC and 5mdC) accounts for 21% of the
total dN in the mouse genome.
p-value of data sets was determined by unpaired two-tailed Student’s t-test with 95%
confidence interval. Normal distribution was assumed. All statistical tests were performed
with Graph Pad Prism V7 software. (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001; n.s.,
not significant). All plots are shown as mean ± SEM.
Extended Data Figure 1
Increased inflammation in Il22
-/- mice during AOM/DSS carcinogenesis
(a-f) Mice were given one dose of AOM i.p. followed by one week of 2% DSS in drinking
water. (a) Tumor development in Il22
+/+ and Il22
-/- mice. Scale bar=1cm. Tumor number (b) (Il22
+/+ n=9, Il22
-/- n=8, mean±SEM; p=0.022) and regional distribution of tumors (c) (Il22+/+
n=10, mean±SEM; pRectum=0.0007, pTotal=0.0078). (d) Body weight as percent of initial
+/+ n=7, Il22
-/- n=10, mean±SEM). (e) Expression of the indicated inflammatory and tumor-promoting
genes (n=4-6, mean±SEM) in colonic mucosa was assessed by qRT-PCR two days after DSS
treatment was stopped. (f) Expression of the indicated genes was analyzed by qRT-PCR
within tumors (n=3-9, mean±SEM).
(g) Schematic representation of the CAC treatment regimen in mice with sporadic inactivation
of the Il22ra1 gene (Il22ra1
CreERt2/+;R26R-Confetti and Il22ra1
Data are representative of two biologically independent experiments.
Extended Data Figure 2
Generation and validation of Il22ra1
(a) Schematic representation of the Il22ra1
tm1a(EUCOMM)Wtsi allele in targeted ES cells. The position of PCR primers and Southern
blot probes is indicated.
(b) Verification of construct integration into JM8.N4 ES cells by genomic PCR. DNA
from untargeted ES cells (Bruce4) served as a control.
(c) Southern blot after BamHI digestion confirming the correct integration of the
construct, which introduced a novel BamHI recognition site into the genomic DNA.
(d) Germline transmission was assessed by genomic PCR in the F1 generation.
(e) LacZ reporter activity was assessed in F1 mice.
(f) Gene expression of the IL-22Ra1 binding partners in colonic epithelial cells of
the indicated mouse strains was assessed by qRT-PCR (Il22
+/+ n=5, Il22
-/- n=4, Il22ra1
;Vil1-Cre n=4, Ahr
;Rorc(γt)-Cre n=3, mean±SEM).
(g) Representative immunofluorescence images of IL-22Ra1 (red) in colonic epithelial
cells. Scale bar=50 μm.
CreERt2/+;R26R-Confetti or Il22ra1
CreERt2/+;R26R-Confetti mice were fed with a tamoxifen-containing diet for two weeks.
(h) Labeling efficiency (%) of colon crypts at different time points after stopping
the tamoxifen treatment (Il22ra1
fl/+; day1 n=8:10; day7 n=7:8; day14 n=7:7 visual fields, mean±SEM). (i) Fraction
(%) of Confetti+ crypts (mean) with one, two or three colors.
(j) The indicated mouse strains were fed with tamoxifen-containing diets and subjected
to AOM/DSS treatment as indicated. Body weight as percent of initial weight (Il22ra1
fl/+ n=6, Il22ra1
fl/fl n=10, mean±SEM).
Data are representative of two (f-j) biologically independent experiments.
Extended Data Figure 3
IL-22Ra1 expression in tumors from Il22ra1
(a-d) Representative immunofluorescence images for IL-22Ra1 expression (violet) by
Confetti-negative (a,c) and Confetti-positive (b,d) colon tumors from Il22ra1
;R26R-Confetti (a,b) and Il22ra1
;R26R-Confetti (c,d) mice.
(e) Mean Fluorescence Intensity (MFI) of IL-22Ra1 fluorescende across tumor area of
the indicated mouse strains with and without prior Cre-activation (n=7; except Il22ra1
fl/fl Confetti-negative n=8, mean±SEM).
Extended Data Figure 4
Identification of IL-22-producing lymphocytes
(a-c) Mice of the indicated genotypes or strains were injected with AOM or PBS (Ctrl).
24h later, IL-22+ cells among the indicated lymphocyte subsets and absolute numbers
of IL-22+ cells were determined. (a) Colon C57BL/6 mice (n=6, mean±SEM, p=0.0154),
(b) small intestine C57BL/6 (n=6, mean±SEM). (c) Colon (n=2, mean±SEM).
Data are representative of two (a-c) biologically independent experiments.
Extended Data Figure 5
Initiation of the DDR is impaired in Il22
Egfp and Il22
Egfp mice (n=3 each) were irradiated with 8 Gy (“DNA damage”) or left untreated (“steady-state”).
Lgr5 (GFP)+ colon epithelial stem cells were highly purified and gene expression was
analyzed by RNA-seq. (a) Sorting strategy for colonic Lgr5+ stem cells. Stem cells
were highly purified (>98%) for RNA-seq analysis. (b) Modified Venn diagram. All expressed
genes with FC>2 were extracted and plotted. Bottom half represents genes differentially
expressed between Il22
+/+ and Il22
-/- mice at the steady-state. “Core expression” contains all genes with detectable
transcripts that were not differentially expressed. Upper half depicts genes differentially
expressed 24h after irradiation in comparison to untreated mice. Of these, 596 genes
differed in both genotypes, whereas 638 genes were only regulated in stem cells of
+/+ mice and 392 in Il22
-/- mice. (c) Gene set enrichment analysis (GSEA) was performed on all expressed transcripts
represented in Lgr5+ colon stem cells from Il22
+/+ (n=3) and Il22
-/- (n=3) mice at the steady-state. Nominal p-value (Kolmogorov-Smirnov) was calculated
for GO_DNA_Double_Strand_Break_Processing (p=0.01).
(d) Mice were were irradiated with 8 Gy. Expression (n=3, mean±SEM) of DDR effector
genes p21 and Puma in colonic epithelial cells was determined by qRT-PCR at the indicated
timepoints after irradiation.
(e) List of genes included in the GSEA gene set “DDR_Effector_Genes” (Fig. 2b)4.
(f) Mice were irradiated with 8 Gy. Il22bp (Il22ra2) gene expression in lamina propria
cells or colon epithelial cells was determined by qRT-PCR at the indicated timepoints
(g,h) Expression of the indicated genes in colon epithelial cells was determined by
qRT-PCR at steady-state. (g) MRN-complex genes (Il22
+/+ n=7, Il22
-/- n=6, mean±SEM) (h) DDR transducer genes (Il22
+/+ n=10, except Atmin n=7 and Parp1 n=5, Il22
-/- n=7, except Atmin n=4 and Parp1 n=5, mean±SEM).
Extended Data Figure 6
IL-22 regulates ATM expression and formation of γH2AX
(a) Immunoblot analysis of ATM from colon epithelial cells. Densitometric quantification
of signal intensity normalized to loading controls (n=3, mean±SEM; p=0.004).
(b) Schematic representation of STAT3 binding sites in the Atm gene locus.
(c) STAT3 ChIP was performed to determine specific pulldown at the transcription factor
binding sites (Start, Mid, End) in the promoter regions of the Atm gene and at a control
site within the Stat3 gene (Cold). Genomic DNA from colon epithelial cells of Il22
+/+ and Il22
-/- mice before and after in vivo stimulation with recombinant IL-22. Results represent
specific pull down relative to a DNA input sample (Start n=14, Mid n=7, End n=12,
Cold n=8, mean±SEM).
(d,e) Mice of the indicated genotypes were irradiated with 8 Gy. (d) Immunofluorescence
analysis of γH2AX (red) and of EpCam (blue) of the entire colon was performed 8h after
irradiation. Scale bar=500μm. (e) Quantification (Ctrl, 4h and 24h n=2, 8h n=3, mean±SEM)
of γH2AX+ nuclei per colon before and at the indicated timepoints after irradiation.
(f) Immunoblot analysis of H2AX protein and densitometric quantification of signal
intensity (normalized to loading controls) in primary colon epithelial cells of Il22+/+
mice (mean±SEM, n=2).
(g,h) Mice were irradiated with 8 Gy or injected with AOM i.p. Immunofluorescence
analysis of colon sections was performed 8h after damage. Quantification (g) (n=3,
mean±SEM) of γH2AX+ EpCam+cells and representative immunofluorescence images (h) after
γH2AX (red) and EpCam (blue) staining. Scale bar=25μm.
Extended Data Figure 7
Impairment of p53 activation and DDR effector genes in Il22
(a-g) Mice were irradiated with 8 Gy or injected with AOM. (a,b) Immunoblot analysis
of the indicated proteins from primary colon epithelial cells 24h after irradiation.
Densitometric quantification of signal intensity (mean±SEM, n=2). p-p53 and Actin,
p-SMC and SMC, PUMA and Actin, and p21 immunoblots were derived from one gel each.
Normalization was done to loading (p-p53, PUMA) or sample processing (p-SMC, SMC,
p21) controls. (c,d) Fold induction of p21 and Puma gene expression in colon epithelial
cells 24h after irradiation or AOM treatment was determined by qRT-PCR (AOM n=10,
8 Gy n=5, Stat3
fl/fl n=4, Stat3
;Vil1-Cre n=5, mean±SEM). (e) Apoptosis (red: cleaved (c)Caspase-3) in Lgr5+ (blue)
colon stem cells (arrows). Representative immunofluorescence images 8h after irradiation.
(f,g) Quantification of cleaved Caspase-3+ EpCam+ colonic epithelial cells 8h after
treatment with AOM or 8 Gy (f) (n=3, mean±SEM) and representative immunofluorescence
images (g) (cleaved Caspase-3, red and EpCam, blue). Scale bar=25μm
Data are representative of two (d-g) or three (a-c) biologically independent experiments
Extended Data Figure 8
Impairment of DDR-triggered apoptosis in Il22
(a-c) Mice of the indicated genotypes were irradiated with 8 Gy and analyzed 8h later.
(a) Immunofluorescence for cleaved Caspase-3 (red) and EpCam (blue). Scale bar=50μm.
Quantification of cCasp-3+ cells by histology (b) (Il22
+/+ n=3, Il22
-/- n=6, Il22ra1
;Vil1-Cre n=3, Ahr
;Rorc(γt)-Cre n=4, mean±SEM) or by flow cytometry (c) (n=3, mean±SEM, p=0.003).
(d) IL-22 neutralizing antibody or control IgG was injected i.p. 36h, 12h before and
12h after AOM application. Representative immunofluorescence images of the colon 24h
after AOM treatment stained for Lgr5 (blue) and cleaved Caspase-3 (red; arrows indicate
apoptotic stem cells). Quantification of cCasp-3+ crypts (n=3, mean±SEM, p=0.0039).
(e) IL-22-responsive IEC6 cells were cultured with or without recombinant IL-22. Cells
were irradiated with 2 Gy or left untreated and cultured in the presence of cytochalasin
B. DNA was visualized with DAPI 48h after irradiation. Number of cells containing
one or more micronuclei (n=4, mean±SEM). Representative images of untreated (Ctrl)
or irradiated (2 Gy) IEC6 cells. Arrow illustrates a micronucleus.
(f) Mice of the indicated genotypes were irradiated with 8 Gy. Quantification of BrdU+
cells in colonic crypts. (n=3, mean±SEM)
Data are representative of two biologically independent experiments
Extended Data Figure 9
Dietary AhR-ligands promote IL-22 production
(a) Wildtype mice were given 1-MIM-OH or carrier control (Ctrl) by gavage. Quantification
of DNA adducts at the indicated time points (Ctrl 4h n=2, Ctrl 8h n=4, Ctrl 24h n=2,
1-MIM-OH 4h n=4, 1-MIM-OH 8h n=8, 1-MIM-OH 24h n=6, mean±SEM).
(b) Schematic representation of the different treatment regimens.
(c-g) Mice of the indicated genotypes were exposed to 1-MIM-OH (c,f) or I3C (d,e,g)
by gavage and analyzed 8h later. (c,d) Representative flow cytometry analysis of IL-22
production by the indicated lymphocyte populations after gavage with 1-MIM-OH or I3C.
CD4+ T cells were defined as CD45+ lineage marker (Lin)+ CD4+ cells, ILC3 as CD45+
Lin- RORγt+ cells, and γδ T cells as CD45+ Lin+ γδTCR+ cells. Numbers indicate percent
of cells in gate. (e) Representative flow cytometry analysis and quantification of
IL-22 and IL-10 production by Lin+ CD4+FoxP3+ T regulatory cells 8h after gavage with
I3C (Ctrl n=4, I3C n=4, mean±SEM). (f,g) Expression of the IL-22 response gene Reg3g
in primary colon epithelial cells was determined by qRT-PCR 8h after gavage with 1-MIM-OH
+/+ n=5:4, Il22
-/- n=3:6, mean±SEM) or I3C (n=5, mean±SEM).
(h) Percentage of IL-22+ cells among the indicated lymphocyte subsets 8h after 1-MIM-OH
treatment of Ahr-/-
mice (Ctrl n=3, 1-MIM-OH n=4, mean±SEM).
(i) Numbers of IL-22+ ILC3 at different time points after 1-MIM-OH gavage (Ctrl n=2,
1-MIM-OH n=4, 1-MIM-OH 24h n=6, mean±SEM).
Data are representative of two biologically independent experiments.
Extended Data Figure 10
Diet-derived AhR-ligands control IL-22 production
(a-d) Mice were fed for 6 weeks with either normal, grain-based chow (CD), a phytochemical-free,
experimental defined diet (EDD-) or EDD supplemented with the AhR-ligand I3C (EDD+).
(a) Representative flow cytometry analysis of IL-22 production by the indicated lymphocyte
populations. Numbers indicate percent of cells in gate. (b) Expression of the IL-22
responsive gene Reg3g in primary colon epithelial cells was determined by qRT-PCR
(n=5, mean±SEM). (c,d) Representative colon immunofluorescence images 8h after irradiation
and stained for γH2AX (red) (c) or cCasp-3+ (red) (d) and EpCam (blue). Scale bar=50μm.
Data are representative of three biologically independent experiments.
(e) Model of IL-22-mediated protection of epithelial stem cells against genotoxic
stress. Epithelial stem cells at the intestinal barrier are continuously exposed to
a variety of insults. Among them are threats to genomic integrity represented by genotoxins,
contained in diets or produced by bacteria. Genome integrity in stem cells requires
a functioning DNA damage response pathway to limit damage to the genetic code in order
to prevent malignant transformation. Our data implicate a homeostatic regulatory loop
that assesses the degree of “genotoxic danger” by AhR-mediated sensing of nutrient-derived
genotoxins that serve as AhR ligands. AhR signaling in ILC3 and γδ T cells controls
IL-22 production. IL-22 signaling in intestinal epithelial stem cells in turn controls
components of the DDR. In particular ATM, a PI3-kinase-like molecule and an important
upstream transducer of the DDR, was tuned-up by IL-22 signaling in stem cells. Following
genotoxic stress, ATM controls downstream events of the DDR such as p53 activation
and expression of the DDR effector molecules p21 and PUMA that control DNA repair
and apoptosis, respectively. Disturbances of this pathway in mice genetically lacking
IL-22, the IL-22 receptor or the AhR led to an impaired DDR to various forms of genotoxic
stress. Consequently, intestinal epithelial stem cells from mice with impairment of
IL-22 signaling or production accumulated cancer-promoting mutations and showed accelerated
development of cancer.
Extended data figure 3
Extended data figure 4
Extended data figure 5
Extended data figure 6
Extended data figure 7
Extended data figure 8