MCB Accepted Manuscript Posted Online 27 April 2015 Mol. Cell. Biol. doi:10.1128/MCB.01498-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Noncanonical effects of IRF9 in intestinal inflammation: more than type I and type III
2
interferons
3 4
Isabella Rauch*†/, Felix Rosebrock*†, Eva Hainzl#, Susanne Heider§, Andrea Majoros*,
5
Sebastian Wienerroither*, Birgit Strobl#, Silvia Stockinger#, Lukas Kenner§, Mathias Müller#
6
and Thomas Decker*
7 8
*Department of Microbiology and Immunobiology, Max F. Perutz Laboratories, University of
9
Vienna, Austria
10
§Ludwig Boltzmann Institute for Cancer Research, Medical University of Vienna, Vienna,
11
Austria
12
#Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna,
13
Austria
14 15
†
16
/
17
Pathogenesis, University of California, Berkeley, California, USA
these authors contributed equally
current affiliation: Department of Molecular & Cell Biology, Division of Immunology &
18 19 20
Running title: IFNλ and IRF9 in colitis
21 22 23
address correspondence to
24
Thomas Decker
25
Phone 00431427754605
26
Fax 0043142779546
27
[email protected]
28 29 30 31 32 33 34 1
35
Abstract
36
The ISGF3 transcription factor with its Stat1, Stat2 and IRF9 subunits is employed for
37
transcriptional responses downstream of receptors for type I interferons (IFN-I) that include
38
IFNα and IFNβ and type III interferons (IFN-III), also called IFNλ. Here we show in a
39
murine model of dextran sodium sulfate (DSS)-induced colitis that IRF9 deficiency protects
40
animals whereas the combined loss of IFN-I and IFN-III receptors worsens their condition.
41
We explain the different phenotypes by demonstrating a function of IRF9 in a noncanonical
42
transcriptional complex with Stat1, apart from IFN-I and IFN-III signaling. Together
43
Stat1/IRF9 produce a proinflammatory activity that overrides the benefits of the IFN-III
44
response on intestinal epithelial cells. Our results further suggest that the CXCL10 chemokine
45
gene is an important mediator of this proinflammatory activity. We thus establish IFNλ as
46
potentially anti-colitogenic cytokine and propose an important role for IRF9 as component of
47
noncanonical Stat complexes in the development of colitis.
48
2
49
Introduction
50
Interferons (IFN) are subdivided into three distinct types, type I IFN (IFN-I, mainly IFNα/β),
51
type II IFN (IFN-II or IFNγ) and type III IFN (IFN-III or IFNλ/IL28/IL29). Collectively IFN
52
are potent regulators of immune responses to pathogens (1, 2). In addition they contribute to
53
autoimmunity-related or other types of sterile inflammation (3-5). Biological responses to
54
IFN require transcription of a large number of IFN-induced genes (ISG), regulated by signal
55
transducers and activators of transcription (Stat) and interferon regulatory factors (IRF). In
56
their canonical signaling pathways the type I and type III IFN receptors stimulate the
57
assembly of the IFN-stimulated gene factor -3 (ISGF3) complex that contains tyrosine-
58
phosphorylated Stat 1 and 2 in association with IRF9, whereas the IFNγ receptor employs
59
Janus tyrosine kinases (Jaks) to produce the gamma-interferon-stimulated factor (GAF), a
60
homodimer of tyrosine-phosphorylated Stat1 (6). In addition, non-canonical complexes of
61
Stats1/2 and IRF9 can be formed in response to IFN-I or IFNγ signaling and contribute to
62
gene-selectivity of the transcriptional response (7-12). Stat1 homodimers bind to gamma
63
interferon activated sequences (GAS) in target promoters whereas ISGF3 binds to IFN
64
stimulated response elements (ISRE), which can be found in a large number of antimicrobial
65
and antiviral genes. Noncanonical complexes containing IRF9 would similarly be expected to
66
associate with the ISRE, consistent with the DNA-binding specificity of this subunit.
67
In keeping with the common deployment of ISGF3, immunological activities of IFN-I and
68
IFN-III appear to be very similar. However, the IFN-I receptor (IFNAR) is expressed on
69
virtually all nucleated cells, whereas IFN-III receptor expression in mice is restricted to
70
epithelial tissues (13). It is not entirely clear yet, whether IFN-I and III lead to completely
71
identical signaling outputs, as their receptors may differ in their ability to activate Stats other
72
than 1 and 2 or the MAPK pathway (14, 15).
73
Inflammatory bowel disease (IBD) is a health problem affecting a rising number of
74
individuals especially in the western world. A multi-step process initiates this chronic disease,
75
with a disturbance of the epithelial layer as an early event (16). Host factors as well as gut
76
microbiota have been implicated in the development and maintenance of IBD, with specific
77
innate and adaptive immune signaling pathways as well as specific bacterial species such as
78
members of the Enterobacteriaceae shown to be associated with the disease (17, 18). IFN-I
79
both reduce or enhance colitis in animal models, depending on the intensity of inflammation
80
and whether the acute or resolution phases are examined (5, 19). Clinical studies testing IFN-I
81
in the treatment of IBD support this view as they led to conflicting results concerning their
82
therapeutic potential (5, 20). Loss of IFNγ signaling exacerbated disease in some reported 3
83
animal studies (21, 22) while it was protective in others (23, 24). Blockade of IFNγ caused a
84
fairly subtle improvement of symptoms in human IBD patients (25, 26). IFN-III have not
85
been studied in the context of colitis. However, such studies appear of high priority in the
86
light of recent reports showing they control rotavirus infections by targeting the intestinal
87
epithelium (27). This study further demonstrated that polarized intestinal epithelial cells (IEC)
88
respond to IFN-III from the basolateral and apical but to IFN-I only from the apical side. The
89
authors suggested IFN-III might be more relevant for immune signaling in IEC than IFN-I.
90
Earlier in vitro studies showed that IFN-III decrease proliferation and induce antiviral proteins
91
in human IEC lines (28). Also, infection of IEC lines with various Gram-positive bacteria
92
induced production of IFN-III (29).
93
The aim of our study was to determine the effect of simultaneous elimination of IFN-I and
94
IFN-III responses by inducing colitis with the chemical dextran sodium sulfate (DSS) in mice
95
lacking the ISGF3 subunit IRF9. We report that mice were strongly protected from colitis
96
when deficient for IRF9. Surprisingly, the opposite effect was observed after combined
97
deletion of IFN-I and IFN-III receptors. Our results suggest that the procolitogenic activity of
98
IRF9 results from its participation in a noncanonical complex with Stat1 independently of
99
IFN-I and IFN-III receptor signaling. In addition, they support the notion that IFN-III prevent
100
damage of the gut mucosa after DSS treatment and might provide a novel therapeutic option.
101 102
4
103
Material and Methods
104 105
Mice, animal experiments. Animal experiments were approved by the University of
106
Veterinary Medicine Vienna institutional ethics committee and carried out in accordance with
107
protocols approved by the Austrian law (BMWF-66.006/002-II/10b/2010).
108
Mice lacking functional type III IFN receptors (IL28rα-/-) were provided by Bristol-Myers-
109
Squibb Company, New Jersey, USA. B6.A2G-Mx1 wild-type mice carrying intact Mx1
110
alleles (Mx1), B6.A2G- Mx1-IL28rα-/- mice lacking functional type III IFN receptors (Mx1-
111
IL28rα-/-), and B6.A2G-Mx1-IL28rα-/-Ifnar1-/- double-knockout mice (Mx1-IL28rα-/-Ifnar1-/-;
112
30) lacking functional receptors for both type I and type III IFN were provided by Peter
113
Stäheli (Freiburg, Germany). The mice were backcrossed to C57BL/6 mice as previously
114
described for Balb/c (31). C57BL/6N mice, Ifnar1-/-, Irf9-/- (32), Stat1-/- (33) and Stat2-/- mice
115
(34) backcrossed for more than 10 generations on a C57BL/6N background were housed in
116
the same specific pathogen free (SPF) facility under identical conditions according to
117
recommendations of the Federation of European Laboratory Animal Science Association,
118
additionally monitored for being norovirus negative. Colitis experiments where performed in
119
individually ventilated cage isolators.
120
To induce colitis, mice were provided with 2% dextran sodium sulfate (DSS, molecular
121
weight: 36–50 kDa, MP Biomedicals) in autoclaved drinking water ad libitum for 7 days,
122
after which they received DSS-free autoclaved drinking water for 2 days. Mice were weighed
123
daily and sacrificed on day 0 (untreated), 5 or 10 of the colitis protocol. Upon sacrifice the
124
intestine was removed and flushed with phosphate-buffered saline (pH 7.2). The colon was
125
halved lengthwise and one half was snap frozen while the other half was fixed in 2%
126
paraformaldehyde, prepared as a swiss role, and embedded in paraffin. Sections of whole
127
intestine were stained with H&E using a standard protocol and blind-scored by a pathologist
128
using an established method that evaluates inflammation severity, crypt damage,
129
inflammation extent and percent tissue involvement (35).
130
Immunohistochemistry. Tissue sections were rehydrated and boiled 20 min in a citric buffer
131
(pH 6) to unmask antigens. For proliferation assessment slides where incubated with rabbit
132
polyclonal anti-Ki67 antibody (NovoCastra) diluted 1:1000 and to evaluate apoptosis with
133
rabbit polyclonal anti-cleaved Caspase-3 antibody (Cell Signaling) diluted 1:200. Epitope
134
binding was revealed with peroxidase-conjugated secondary antibodies. Sections were
135
counterstained with hematoxylin and imaged on an epifluorescence microscope (Zeiss
136
AxioImager). IHC images were photographed and quantified with the HistoQUEST software 5
137
(TissueGnostics GmbH). At least 3 pictures per colon were taken and the percentage of
138
positive stained cells per number of nucleated cells was calculated.
139
RNA isolation, cDNA synthesis and qPCR. For RNA preparation colon tissue was
140
homogenized / BMDM were lysed in 700 µl RA1 buffer of the NucleoSpin II RNA isolation
141
kit (Macherey and Nagel) and processed according to the manufacturer’s protocol. cDNA was
142
prepared as described (36). PCRs were performed with 60°C annealing temperature on an
143
Eppendorf cycler. Relative expression of every sample was calculated to the housekeeping
144
gene OAZ1. Primer sequences are summarized in supplemental table 1.
145
Chromatin immunoprecipitation (ChIP) and ChIP-Seq. ChIP was performed as described
146
(37). 2µl (anti-Stat1, anti-Stat2, Santa Cruz) or 4ml (anti-IRF9, Santa Cruz) antiserum was
147
used per 50µg Chromatin. ChIP data were normalized to and expressed as a percentage of
148
input. Primers used for qPCR of the Cxcl10 enhancer regions (E1 and E2) and IFN response
149
region of Mx1 are listed in table S1. Next generation sequencing was carried out by the
150
Vienna Biocenter Campus Support Facilities (CSF). 5-10 ng of DNA precipitate was used for
151
the generation of sequencing libraries using the KAPA library preparation kit for Illumina
152
systems. Libraries were quantified with a Bioanalyzer dsDNA 1000 assay kit (Agilent) and a
153
QPCR NGS library quantification kit (KAPA). Cluster generation and sequencing was
154
performed with a HiSeq 2000 system with a read length of 100 nucleotides according to the
155
manufacturer's guidelines (Illumina).
156
In the re-ChIP experiments (12), the immune complexes were eluted by adding 10 mM
157
dithiothreitol (DTT) and incubating for 40min at room temperature. The samples were diluted
158
40-fold and reimmunoprecipitated.
159
Cell isolation and treatment. For IEC isolation, colons where washed in cold PBS
160
containing 50µg/ml gentamycin, cut into pieces and incubated for 10 min in 15mM
161
EDTA/PBS at 37°C shaking. After 1 g sedimentation to remove cell sheets, supernatants
162
where collected, spun down, washed in PBS and resuspended in buffer RA1 for RNA
163
isolation. For western blot, cells where resuspended in RPMI 1640 with or without 5ng/ml
164
IFNγ (eBioscience) for 15min and processed as described (36).
165
For lamina propria mononuclear cell (LPMC) isolation, colons where incubated in RPMI
166
1640 containing gentamycin and 5mM EDTA to fully remove IEC 3 times 15 min at 37°C
167
shaking. Remaining tissue pieces where washed in RPMI and incubated for 90 min in RPMI
168
containing 15mM HEPES and 0.2 mg/ml type VIII collagenase (Sigma). The digest was
169
filtered through a 70µm cell strainer, spun down, washed in PBS and resuspended in buffer
170
RA1. These fractions contained >50% LPMC. 6
171
Bone marrow-derived macrophages (BMDMs) were differentiated from bone marrow isolated
172
from femurs and tibias of 6-8 week old mice. Cells were cultured in DMEM (Sigma-Aldrich)
173
supplemented with 10 % of FCS (Sigma-Aldrich), 10 % of L929-cell conditioned medium as
174
a source of CSF-1 and 100 units/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich) as
175
previously described (36). The culture contained >99 % of F4/80+ cells. BMDMs were
176
treated with 5 ng/ml of IFNγ (Affymetrix - eBioscience).
177
CXCL10 determination. Frozen tissue was homogenized in PBS containing proteinase
178
inhibitors (Phenylmethylsulfonylfluorid, Roche Complete Protease Inhibitor Cocktail), 1mM
179
vanadate and 1mM DTT. The homogenate was freeze-thawed twice. Total protein in the
180
supernatants was determined by BCA assay (Pierce) and CXCL10 levels where determined
181
using FlowCytomix kit (eBioscience) on 25µl supernatant according to the manufacturer’s
182
instructions. The CXCL10 amount was normalized to total protein.
183
Western blot. Organs or isolated IEC where homogenized in Frackelton buffer containing
184
protease and phosphatase inhibitors and centrifuged at 12000 g. The supernatant was used for
185
western blotting as described (36). Total Stat1 was detected using mAb to the Stat1 N
186
terminus (BD Biosciences) at a dilution of 1:1000. Tyrosine 701 phosphorylated Stat1 was
187
detected using a 1:1000 diluted antibody (Cell Signaling). As a loading control, the blot was
188
probed with Abs to the Erk1 and Erk2 kinases (panErk) (BD Transduction Laboratories) at a
189
dilution of 1:2000.
190
Statistics. Statistical analysis was done using the Mann Whitney U test, considering p
1
Noncanonical effects of IRF9 in intestinal inflammation: more than type I and type III
2
interferons
3 4
Isabella Rauch*†/, Felix Rosebrock*†, Eva Hainzl#, Susanne Heider§, Andrea Majoros*,
5
Sebastian Wienerroither*, Birgit Strobl#, Silvia Stockinger#, Lukas Kenner§, Mathias Müller#
6
and Thomas Decker*
7 8
*Department of Microbiology and Immunobiology, Max F. Perutz Laboratories, University of
9
Vienna, Austria
10
§Ludwig Boltzmann Institute for Cancer Research, Medical University of Vienna, Vienna,
11
Austria
12
#Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna,
13
Austria
14 15
†
16
/
17
Pathogenesis, University of California, Berkeley, California, USA
these authors contributed equally
current affiliation: Department of Molecular & Cell Biology, Division of Immunology &
18 19 20
Running title: IFNλ and IRF9 in colitis
21 22 23
address correspondence to
24
Thomas Decker
25
Phone 00431427754605
26
Fax 0043142779546
27
[email protected]
28 29 30 31 32 33 34 1
35
Abstract
36
The ISGF3 transcription factor with its Stat1, Stat2 and IRF9 subunits is employed for
37
transcriptional responses downstream of receptors for type I interferons (IFN-I) that include
38
IFNα and IFNβ and type III interferons (IFN-III), also called IFNλ. Here we show in a
39
murine model of dextran sodium sulfate (DSS)-induced colitis that IRF9 deficiency protects
40
animals whereas the combined loss of IFN-I and IFN-III receptors worsens their condition.
41
We explain the different phenotypes by demonstrating a function of IRF9 in a noncanonical
42
transcriptional complex with Stat1, apart from IFN-I and IFN-III signaling. Together
43
Stat1/IRF9 produce a proinflammatory activity that overrides the benefits of the IFN-III
44
response on intestinal epithelial cells. Our results further suggest that the CXCL10 chemokine
45
gene is an important mediator of this proinflammatory activity. We thus establish IFNλ as
46
potentially anti-colitogenic cytokine and propose an important role for IRF9 as component of
47
noncanonical Stat complexes in the development of colitis.
48
2
49
Introduction
50
Interferons (IFN) are subdivided into three distinct types, type I IFN (IFN-I, mainly IFNα/β),
51
type II IFN (IFN-II or IFNγ) and type III IFN (IFN-III or IFNλ/IL28/IL29). Collectively IFN
52
are potent regulators of immune responses to pathogens (1, 2). In addition they contribute to
53
autoimmunity-related or other types of sterile inflammation (3-5). Biological responses to
54
IFN require transcription of a large number of IFN-induced genes (ISG), regulated by signal
55
transducers and activators of transcription (Stat) and interferon regulatory factors (IRF). In
56
their canonical signaling pathways the type I and type III IFN receptors stimulate the
57
assembly of the IFN-stimulated gene factor -3 (ISGF3) complex that contains tyrosine-
58
phosphorylated Stat 1 and 2 in association with IRF9, whereas the IFNγ receptor employs
59
Janus tyrosine kinases (Jaks) to produce the gamma-interferon-stimulated factor (GAF), a
60
homodimer of tyrosine-phosphorylated Stat1 (6). In addition, non-canonical complexes of
61
Stats1/2 and IRF9 can be formed in response to IFN-I or IFNγ signaling and contribute to
62
gene-selectivity of the transcriptional response (7-12). Stat1 homodimers bind to gamma
63
interferon activated sequences (GAS) in target promoters whereas ISGF3 binds to IFN
64
stimulated response elements (ISRE), which can be found in a large number of antimicrobial
65
and antiviral genes. Noncanonical complexes containing IRF9 would similarly be expected to
66
associate with the ISRE, consistent with the DNA-binding specificity of this subunit.
67
In keeping with the common deployment of ISGF3, immunological activities of IFN-I and
68
IFN-III appear to be very similar. However, the IFN-I receptor (IFNAR) is expressed on
69
virtually all nucleated cells, whereas IFN-III receptor expression in mice is restricted to
70
epithelial tissues (13). It is not entirely clear yet, whether IFN-I and III lead to completely
71
identical signaling outputs, as their receptors may differ in their ability to activate Stats other
72
than 1 and 2 or the MAPK pathway (14, 15).
73
Inflammatory bowel disease (IBD) is a health problem affecting a rising number of
74
individuals especially in the western world. A multi-step process initiates this chronic disease,
75
with a disturbance of the epithelial layer as an early event (16). Host factors as well as gut
76
microbiota have been implicated in the development and maintenance of IBD, with specific
77
innate and adaptive immune signaling pathways as well as specific bacterial species such as
78
members of the Enterobacteriaceae shown to be associated with the disease (17, 18). IFN-I
79
both reduce or enhance colitis in animal models, depending on the intensity of inflammation
80
and whether the acute or resolution phases are examined (5, 19). Clinical studies testing IFN-I
81
in the treatment of IBD support this view as they led to conflicting results concerning their
82
therapeutic potential (5, 20). Loss of IFNγ signaling exacerbated disease in some reported 3
83
animal studies (21, 22) while it was protective in others (23, 24). Blockade of IFNγ caused a
84
fairly subtle improvement of symptoms in human IBD patients (25, 26). IFN-III have not
85
been studied in the context of colitis. However, such studies appear of high priority in the
86
light of recent reports showing they control rotavirus infections by targeting the intestinal
87
epithelium (27). This study further demonstrated that polarized intestinal epithelial cells (IEC)
88
respond to IFN-III from the basolateral and apical but to IFN-I only from the apical side. The
89
authors suggested IFN-III might be more relevant for immune signaling in IEC than IFN-I.
90
Earlier in vitro studies showed that IFN-III decrease proliferation and induce antiviral proteins
91
in human IEC lines (28). Also, infection of IEC lines with various Gram-positive bacteria
92
induced production of IFN-III (29).
93
The aim of our study was to determine the effect of simultaneous elimination of IFN-I and
94
IFN-III responses by inducing colitis with the chemical dextran sodium sulfate (DSS) in mice
95
lacking the ISGF3 subunit IRF9. We report that mice were strongly protected from colitis
96
when deficient for IRF9. Surprisingly, the opposite effect was observed after combined
97
deletion of IFN-I and IFN-III receptors. Our results suggest that the procolitogenic activity of
98
IRF9 results from its participation in a noncanonical complex with Stat1 independently of
99
IFN-I and IFN-III receptor signaling. In addition, they support the notion that IFN-III prevent
100
damage of the gut mucosa after DSS treatment and might provide a novel therapeutic option.
101 102
4
103
Material and Methods
104 105
Mice, animal experiments. Animal experiments were approved by the University of
106
Veterinary Medicine Vienna institutional ethics committee and carried out in accordance with
107
protocols approved by the Austrian law (BMWF-66.006/002-II/10b/2010).
108
Mice lacking functional type III IFN receptors (IL28rα-/-) were provided by Bristol-Myers-
109
Squibb Company, New Jersey, USA. B6.A2G-Mx1 wild-type mice carrying intact Mx1
110
alleles (Mx1), B6.A2G- Mx1-IL28rα-/- mice lacking functional type III IFN receptors (Mx1-
111
IL28rα-/-), and B6.A2G-Mx1-IL28rα-/-Ifnar1-/- double-knockout mice (Mx1-IL28rα-/-Ifnar1-/-;
112
30) lacking functional receptors for both type I and type III IFN were provided by Peter
113
Stäheli (Freiburg, Germany). The mice were backcrossed to C57BL/6 mice as previously
114
described for Balb/c (31). C57BL/6N mice, Ifnar1-/-, Irf9-/- (32), Stat1-/- (33) and Stat2-/- mice
115
(34) backcrossed for more than 10 generations on a C57BL/6N background were housed in
116
the same specific pathogen free (SPF) facility under identical conditions according to
117
recommendations of the Federation of European Laboratory Animal Science Association,
118
additionally monitored for being norovirus negative. Colitis experiments where performed in
119
individually ventilated cage isolators.
120
To induce colitis, mice were provided with 2% dextran sodium sulfate (DSS, molecular
121
weight: 36–50 kDa, MP Biomedicals) in autoclaved drinking water ad libitum for 7 days,
122
after which they received DSS-free autoclaved drinking water for 2 days. Mice were weighed
123
daily and sacrificed on day 0 (untreated), 5 or 10 of the colitis protocol. Upon sacrifice the
124
intestine was removed and flushed with phosphate-buffered saline (pH 7.2). The colon was
125
halved lengthwise and one half was snap frozen while the other half was fixed in 2%
126
paraformaldehyde, prepared as a swiss role, and embedded in paraffin. Sections of whole
127
intestine were stained with H&E using a standard protocol and blind-scored by a pathologist
128
using an established method that evaluates inflammation severity, crypt damage,
129
inflammation extent and percent tissue involvement (35).
130
Immunohistochemistry. Tissue sections were rehydrated and boiled 20 min in a citric buffer
131
(pH 6) to unmask antigens. For proliferation assessment slides where incubated with rabbit
132
polyclonal anti-Ki67 antibody (NovoCastra) diluted 1:1000 and to evaluate apoptosis with
133
rabbit polyclonal anti-cleaved Caspase-3 antibody (Cell Signaling) diluted 1:200. Epitope
134
binding was revealed with peroxidase-conjugated secondary antibodies. Sections were
135
counterstained with hematoxylin and imaged on an epifluorescence microscope (Zeiss
136
AxioImager). IHC images were photographed and quantified with the HistoQUEST software 5
137
(TissueGnostics GmbH). At least 3 pictures per colon were taken and the percentage of
138
positive stained cells per number of nucleated cells was calculated.
139
RNA isolation, cDNA synthesis and qPCR. For RNA preparation colon tissue was
140
homogenized / BMDM were lysed in 700 µl RA1 buffer of the NucleoSpin II RNA isolation
141
kit (Macherey and Nagel) and processed according to the manufacturer’s protocol. cDNA was
142
prepared as described (36). PCRs were performed with 60°C annealing temperature on an
143
Eppendorf cycler. Relative expression of every sample was calculated to the housekeeping
144
gene OAZ1. Primer sequences are summarized in supplemental table 1.
145
Chromatin immunoprecipitation (ChIP) and ChIP-Seq. ChIP was performed as described
146
(37). 2µl (anti-Stat1, anti-Stat2, Santa Cruz) or 4ml (anti-IRF9, Santa Cruz) antiserum was
147
used per 50µg Chromatin. ChIP data were normalized to and expressed as a percentage of
148
input. Primers used for qPCR of the Cxcl10 enhancer regions (E1 and E2) and IFN response
149
region of Mx1 are listed in table S1. Next generation sequencing was carried out by the
150
Vienna Biocenter Campus Support Facilities (CSF). 5-10 ng of DNA precipitate was used for
151
the generation of sequencing libraries using the KAPA library preparation kit for Illumina
152
systems. Libraries were quantified with a Bioanalyzer dsDNA 1000 assay kit (Agilent) and a
153
QPCR NGS library quantification kit (KAPA). Cluster generation and sequencing was
154
performed with a HiSeq 2000 system with a read length of 100 nucleotides according to the
155
manufacturer's guidelines (Illumina).
156
In the re-ChIP experiments (12), the immune complexes were eluted by adding 10 mM
157
dithiothreitol (DTT) and incubating for 40min at room temperature. The samples were diluted
158
40-fold and reimmunoprecipitated.
159
Cell isolation and treatment. For IEC isolation, colons where washed in cold PBS
160
containing 50µg/ml gentamycin, cut into pieces and incubated for 10 min in 15mM
161
EDTA/PBS at 37°C shaking. After 1 g sedimentation to remove cell sheets, supernatants
162
where collected, spun down, washed in PBS and resuspended in buffer RA1 for RNA
163
isolation. For western blot, cells where resuspended in RPMI 1640 with or without 5ng/ml
164
IFNγ (eBioscience) for 15min and processed as described (36).
165
For lamina propria mononuclear cell (LPMC) isolation, colons where incubated in RPMI
166
1640 containing gentamycin and 5mM EDTA to fully remove IEC 3 times 15 min at 37°C
167
shaking. Remaining tissue pieces where washed in RPMI and incubated for 90 min in RPMI
168
containing 15mM HEPES and 0.2 mg/ml type VIII collagenase (Sigma). The digest was
169
filtered through a 70µm cell strainer, spun down, washed in PBS and resuspended in buffer
170
RA1. These fractions contained >50% LPMC. 6
171
Bone marrow-derived macrophages (BMDMs) were differentiated from bone marrow isolated
172
from femurs and tibias of 6-8 week old mice. Cells were cultured in DMEM (Sigma-Aldrich)
173
supplemented with 10 % of FCS (Sigma-Aldrich), 10 % of L929-cell conditioned medium as
174
a source of CSF-1 and 100 units/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich) as
175
previously described (36). The culture contained >99 % of F4/80+ cells. BMDMs were
176
treated with 5 ng/ml of IFNγ (Affymetrix - eBioscience).
177
CXCL10 determination. Frozen tissue was homogenized in PBS containing proteinase
178
inhibitors (Phenylmethylsulfonylfluorid, Roche Complete Protease Inhibitor Cocktail), 1mM
179
vanadate and 1mM DTT. The homogenate was freeze-thawed twice. Total protein in the
180
supernatants was determined by BCA assay (Pierce) and CXCL10 levels where determined
181
using FlowCytomix kit (eBioscience) on 25µl supernatant according to the manufacturer’s
182
instructions. The CXCL10 amount was normalized to total protein.
183
Western blot. Organs or isolated IEC where homogenized in Frackelton buffer containing
184
protease and phosphatase inhibitors and centrifuged at 12000 g. The supernatant was used for
185
western blotting as described (36). Total Stat1 was detected using mAb to the Stat1 N
186
terminus (BD Biosciences) at a dilution of 1:1000. Tyrosine 701 phosphorylated Stat1 was
187
detected using a 1:1000 diluted antibody (Cell Signaling). As a loading control, the blot was
188
probed with Abs to the Erk1 and Erk2 kinases (panErk) (BD Transduction Laboratories) at a
189
dilution of 1:2000.
190
Statistics. Statistical analysis was done using the Mann Whitney U test, considering p
