ARTICLE
Received 2 Oct 2014 | Accepted 2 Feb 2015 | Published 12 Mar 2015
Tegest Aychek1, Alexander Mildner1, Simon Yona1, Ki-Wook Kim1, Nardy Lampl1, Shlomit Reich-Zeliger1, Louis Boon2, Nir Yogev3, Ari Waisman3, Daniel J. Cua4 & Steffen Jung1
Gut homeostasis and mucosal immune defense rely on the differential contributions of dendritic cells (DC) and macrophages. Here we show that colonic CX3CR1 mononuclear phagocytes are critical inducers of the innate response to Citrobacter rodentium infection.
Specically, the absence of IL-23 expression in macrophages or CD11b DC results in the impairment of IL-22 production and in acute lethality. Highlighting immunopathology as a death cause, infected animals are rescued by the neutralization of IL-12 or IFNg. Moreover, mice are also protected when the CD103 CD11b DC compartment is rendered decient for IL-12 production. We show that IL-12 production by colonic CD103 CD11b DC is repressed by IL-23. Collectively, in addition to its role in inducing IL-22 production, macrophage-derived or CD103 CD11b DC-derived IL-23 is required to negatively control the otherwise deleterious production of IL-12 by CD103 CD11b DC. Impairment of this critical mononuclear phagocyte crosstalk results in the generation of IFNg-producing former TH17 cells and fatal immunopathology.
DOI: 10.1038/ncomms7525 OPEN
IL-23-mediated mononuclear phagocyte crosstalk protects mice from Citrobacter rodentium-induced colon immunopathology
1 Department of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel. 2 Bioceros, Yalelaan 46, 3584 CM Utrecht, The Netherlands. 3 Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg University, 55131 Mainz, Germany. 4 Merck Research Laboratories, 901 South California Avenue, Palo Alto, California 94304-1104, USA. Correspondence and requests for materials should be addressed to S.J. (email: mailto:[email protected]
Web End [email protected] ).
NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 1
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525
The intestinal lamina propria hosts a complex make-up of mononuclear phagocytes, including dendritic cells (DC) and macrophages that collectively contribute
to the maintenance of gut homeostasis and responses to pathogen challenges1. Murine intestinal DC subpopulations are currently dened by the expression of integrins CD11c (aX),
CD11b (aM) and CD103 (aEb7), as well as CD24 (ref. 2). CD103 CD11b DC depend, like classical splenic XCR1
CD8a DC, on the transcription factors Irf8, Id2, and Batf3 for their developmenton3. These cells migrate to the mesenteric lymph nodes to prime and polarize nave T cells and seem as their splenic counterpart specialized in cross-presentation4. In contrast, CD103 CD11b DC in the ileum were shown to be required for the efcient generation of Th17 cells58. In the colon,
CD103 CD11b DC are rare, but might be functionally replaced by a population lacking CD103 expression9. The ontogeny of CD103 and CD103 CD11b DC remains less well dened, as these cells are currently difcult to discern from monocyte-derived cells10. The most abundant intestinal mononuclear phagocytes are CX3CR1hi macrophages that develop from Ly6C blood monocytes and are conditioned in the healthy gut to become non-inammatory cells11,12. In steady state, these cells are non-migratory and are critically involved in the maintenance of gut homeostasis13. Specic activities of intestinal macrophages and the DC subsets, and in particular, potential crosstalk between these cells remain incompletely understood.
Attaching and effacing (A/E) enteropathogens adhere to intestinal epithelial cells of the infected host, efface their microvilli and reorganize their cytoskeleton. They control host cells by the injection of effector molecules via a type III secretion system, whose components are encoded on a conserved pathogenicity island14. In immunocompetent mice, the A/E pathogen Citrobacter rodentium, which serves as model organism for human enteropathogenic Escherichia coli and enterohaemorrhagic E. coli15, induces a transient, distal colitis followed by a systemic CD4 T-cell-dependent IgG response resulting in bacterial clearance16. Mice decient in the cytokine interleukin-23 (IL-23), a heterodimer of p19 and p40 polypeptide chains encoded by the il23a and il12b genes, respectively17, fail to control C. rodentium and die from the infection within a week18. IL-23 requirement has been linked to IL-22 secretion by RORgt-dependent innate lymphoid cell (ILC; now named group3 ILC, or ILC3;ref. 19). Thus, C. rodentium-challenged il23a-decient animals can be rescued by exogenous IL-22; moreover, also IL-22-decient mice die from C. rodentium infection, albeit with a delay20. IL-22 is believed to boost epithelial regeneration and induces epithelial cells to express antimicrobial peptides (AMPs), including defensins and lectins of the RegIII family20,21.
Here we investigated the cellular source of early IL-23 that protects Citrobacter-challenged mice. In line with recent reports, we establish that IL-23 production by colonic CX3CR1 mononuclear phagocytes, including CD103 CD11b DC and macrophagesbut not CD103 CD11b DCwas required for the induction of IL-22 and AMPs. Animals harbouring an IL-23 deciency acutely died from the Citrobacter challenge. Interestingly though, mice could be rescued by an additional IL-12 deciency in the CD103 CD11b DC compartment, as well as antibody-mediated neutralization of IL-12 and interferon-g (IFNg). Collectively, we establish that IL-23, in addition to its role as inducer of IL-22 and AMPs, suppresses IL-12 production by colonic CD103 CD11b DC. Impairment of this mono-nuclear phagocyte crosstalk results in lethal immunopathology that is associated with the generation of IFNg-producing TH1 cells from former TH17 cells.
ResultsHaematopoietic IL-23 is essential to survive C. rodentium infections. Host defense against C. rodentium involves the orchestrated expression of cytokines and AMPs20. Kinetics of the response can, however, considerably vary depending on the local microbiota composition22. We therefore analysed the colonic tissue isolated from Citrobacter-challenged C57BL/6 (wild-type) WT mice housed in our animal facility for the expression of IL-12 superfamily cytokines, for example, IL-12 and IL-23, composed of p35/p40 and p19/p40, respectively. We found the expression of il23a (encoding IL23p19) to be transiently upregulated on day 3 post infection (PI), alongside il12b (encoding IL12p40), while il12a (encoding IL12p35) was induced only by day 6 PI (Supplementary Fig. 1a, Supplementary Table 1). As previously reported20, the induction of IL-23 was followed by the production of IL-22 and the AMP RegIIIb, as well as members of the S100A family and IL-17A (Supplementary Fig. 1b,c). To probe the requirement of a haematopoietic IL-23 source, we generated chimeras by engrafting lethally irradiated WT mice with either il12b- or il23a-decient bone marrow (BM) cells. (il23a / 4WT) chimeras died from the C. rodentium challenge by day 12 PI; surprisingly though, (il12b / 4WT)
BM chimeras mice survived (Fig. 1a). Both the chimeras displayed an impaired induction of IL-22 and RegIIIb transcripts, compared with controls (Fig. 1b). The absence of these factors alone could hence not explain the differential lethality. To determine whether BM chimeras harbouring the il23a mutation succumb to the infection due to increased pathogen burden, we took advantage of the bioluminescence of the C. rodentium strain we used for the challenge23. The reporter signal intensities emitted from chimeras lacking IL-23 or IL-12/23 production were comparable (Fig. 1c), arguing against bacterial overload as the death cause. Challenged (il23a / 4WT) and (il12b / 4WT) chimeras displayed reduced mucosal hyper-plasia and goblet cell prevalence, as compared with (WT4WT) controls. However, these alterations were comparable in the two mutant groups (Fig. 1c). To gain further insight into the potential mechanism underlying the differential lethality of the chimeras, we measured cytokine levels in the supernatant of colon explant cultures by enzyme-linked immunosorbent assay (ELISA). Interestingly, samples of infected (il23a / 4WT) but not (WT4WT) chimeras displayed a signicant increase in IL-12, which was as expected absent from the (il12b / 4WT)
chimeras (Fig. 1d). On the other hand, IFNg levels were increased in both (il23a / 4WT) and (WT4WT) cultures, but low in (il12b / 4WT) cultures. Thus collectively, increased
IL-12 production in the colon correlates with lethality, as does IFNg production, in absence of IL-22.
Of note, the fact that (il12b / 4WT) chimeras survived theC. rodentium challenge contrasts the reported lethality observed for il12b / mice18. Indeed, also in our animal facility these mice died from infection, as did (il12b / 4il12b / ) BM chimeras, while animals with p40-procient donor or recipient genotype survived (Supplementary Fig. 2). This discrepancy might be related to a radio-resistant long-lived p40-expressing population or could result from the inherent difference between mice that lack p40 throughout development versus chimeras rendered p40 decient only in adulthood. While this nding warrants further exploration, it does not affect our study as all subsequent experiments were performed in BM chimeras and, wherever possible, included internal controls.
Collectively, our data conrm the earlier notion that haematopoietic cells are a critical source of IL-23. Surprisingly though, the resistance of (il12b / 4WT) BM chimeras to the C. rodentium challenge indicated that the IL-22 or
AMP deciency alone are not responsible for acute death.
2 NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525 ARTICLE
100
**
**
75
150
105
Survival (%)
IL-22 expression
(fold increase)
RegIIIexpression
(fold increase)
50
Donor BM:
100
104
25
il23a/ il12b/
WT
il23a/
50
103
0
0
0
102
5 10 15 20
WT
Days after infection
il23a/
il12b/
WT
il23a/
il12b/
109
10
Luminescence
0.2
il12b/
0.8
PAS
WT il12b/
il23a/
NS
**
Luminescence
(RLU)
WT
108
0.6
107
107
WT il23a/
il12b/
0.4
40
200
100
**
Colonic
hyperplasia (mm)% Of area of
goblet cells
30
20
0 WT il23a/ il12b/
0 WT il23a/ il12b/
200 m 200 m 200 m
400
300
15
4
** ** ***
IL-12p70 ng ml1
3
10
IFNg ng ml1
2
5
1
0 0
WT il23a/ il12b/ WT il23a/ il12b/
Figure 1 | Haematopoetic IL-23 is essential to survive C. rodentium infections. (a) Survival curves of C. rodentium-challenged (il12b / 4WT), (il23a / 4 WT) and (WT4WT) BM chimeras (n 6 per group, representative of three individual experiments). (b) Real-time PCR analysis of IL-22 and
RegIIIb expression in colonic tissues collected on day 7 PI. All groups, as in a (n 46 mice per group, representative of three individual experiments). Data
were analysed by analysis of variance (ANOVA) followed by Tukeys post hoc test, **Po0.01. (c) Tissue distribution of the luminescent C. rodentium strain at day 7. IVIS results are displayed as pseudo-colour images of peak bioluminescence, with variations in colour representing light intensity at a given location; right panel quantication of the luminescent C. rodentium signal. Lower panel, periodic acid-Schiff staining indicating goblet cell deletion, and decreased mucosal hyperplasia in (il12b / 4WT) and (il23a / 4WT) chimeras. (n 35 mice per group from three individual experiments). Data
were analysed by ANOVA followed by Tukeys post hoc test, **Po0.01. (d) ELISA for IL-12 and INFg performed on supernatants of colon explant cultures ofC. rodentium-challenged (il12b / 4WT), (il23a / 4WT) and (WT4WT) BM chimeras on day 7 PI (n 35 mice per group from three individual
experiments). Data were analysed by ANOVA followed by Tukeys post hoc test, **Po0.01, ***Po0.001.
Rather our data suggest that the lethality might be due to IL-12 overproduction.
CX3CR1 cell-derived IL-23 is critical to survive C. rodentium challenge. To further dene the cell type that produces IL-23 in the infected colon, we employed a conditional cell ablation strategy targeting intestinal mononuclear phagocytes. CD11c-DTR mice24 allow for diphtheria toxin (DTx)-induced conditional ablation of the three major colonic mononuclear phagocytes, including CD103 CD11b DC, CD103
CD11b DC and CX3CR1hi macrophages25.
To investigate the impact of IL-23 production by DC or macrophages on the anti-Citrobacter response, we took advantage of a mixed BM chimera approach26 (Fig. 2a). Specically, we generated three groups of chimeras by reconstituting WT mice with equal mixtures of DTR-transgenic BM and mutant BM. Upon DTx treatment, such mixed chimeras are left with mutant mononuclear phagocytes only,
while other cellular compartments remain composed of WT and mutant cells. Flow cytometric analysis of DTx-treated mixed BM chimeras conrmed the specic ablation of DTR-transgenic (CD45.1 ) cells from the colon (Fig. 2b). DTx-treated (il23a / /CD11c-DTR4WT) chimeras, retaining only IL-23-decient CD11c cells, including all intestinal DC and macrophages, died by day 8 PI (Fig. 2c). This establishes DC or macrophages as critical IL-23 sources in the colon. Interestingly, infected DTx-treated (il12b / /CD11c-DTR4WT) chimeras survived and the combined absence of IL-23 and IL-12 thus again conferred resistance to the challenge. Mice lacking either IL-23- or IL-12/23-producing CD11c cells displayed a signicant reduction in IL-22 transcripts in colonic tissue, when compared with WT chimeras (Fig. 2d). None of the BM chimeras exhibited a signicantly increased bacterial burden (Supplementary Fig. 3a).
Taking advantage of CX3CR1Cre mice27, we next developed an ablation strategy that spares CD103 CD11b DC. Flow cytometric analysis of the ileum and colon of CX3CR1Cre:
NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 3
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525
a b
105
103
105
103
0 w/o DTX
DTR
CD45.1/1
KO
CD45.2/2
103 0
103
0
50%
50%
0
0
105
105
0 DTX
KO DC/MF
CD11c
CD45.1
DTR DC/MF
0
CD11b
CD45.2
c
d
1,000
**
100
75 50 25
0
Survival (%)
IL-22 expression
(fold increase)
CD11c-DTR/il23a/
CD11c-DTR/il12b/
600
200
CD11c-DTR/WT
0 5 10 15 20
0 103 105 0 103 105
0 0 103 105 0 103 105
il23a/
0 WT il12b/
Days after infection
e f
100
600 *
* *** ***
10,000
Survival (%)
IL-22 expression
(fold increase)
RegIIIexpression
(fold increase)
8,000
50
CX3CR1- DTR/WT
400
6,000
CX3CR1- DTR/il23a/
CX3CR1- DTR/il12b/
200
4,000
2,000
0
0
0
5
10
15 20
0 WT il23a/ il12b/ WT il23a/ il12b/
Days after infection
Figure 2 | IL-23 production by colonic macrophages and CD103 CD11b DC is required to survive the C. rodentium challenge. (a) Schematic of mixed BM chimera approach. (b) Flow cytometric analysis of colon of DTx-injected mouse on day 5 after DTX treatment (lower panel) and control (upper panel)
showing the depletion of CD11c CD45.1 DTR-transgenic cells. Data represent one of the three independent experiments (n 3 mice per group).
(c) Survival curves of DTx-treated (il23a / /CD11c-DTR4WT), (il12b / /CD11c-DTR4WT) and (WT/CD11c-DTR4WT) chimeras following Citrobacter challenge (n 6 per group from three individual experiments). (d) Real-time PCR analysis of IL-22 expression in colonic tissues collected from
DTx-treated (il23a / /CD11c-DTR4WT), (il12b / /CD11c-DTR4WT) and (WT/CD11c-DTR4WT) chimeras on day 7 PI (n 46 per group from
three individual experiments). Data were analysed by analysis of variance (ANOVA) followed by Tukeys post hoc test, **Po0.01. (e) Survival curvesof DTx-treated (il23a / /CX3CR1-DTR4WT), (il12b / / CX3CR1-DTR4WT) and (WT/CX3CR1-DTR4WT) chimeras following Citrobacter challenge (n 7 per group from three individual experiments). (f) Real-time PCR analysis for IL-22 and RegIIIb expression of colonic tissues collected from
DTx-treated (il23a / / CX3CR1-DTR4WT), (il12b / / CX3CR1-DTR4WT) and (WT/CX3CR1-DTR4WT) chimeras on day 8 PI (n 5 per group from
three individual experiments). Data were analysed by ANOVA followed by Tukeys post hoc test *Po0.05, ***Po0.001. w/o, without.
R26-RFP reporter mice revealed that CD64 CD11b macrophages were, as expected, homogeneously labelled (Supplementary Fig. 4a,b; Supplementary Fig. 5a,b); also colonic CD103 CD11b DC showed frequent rearrangements. Of note, ileal CD103 CD11b DC were found to be efciently labelled in CX3CR1Cre:R26-RFP reporter mice as well (Supplementary Fig. 5a,b); however, these cells are rare in the colon (Supplementary Fig 4a,b). In contrast, few ileal and colonic CD103 CD11b DC showed signs of Cre recombinase activity. After crossing CX3CR1Cre mice to iDTR animals28, CX3CR1-expressing cells become sensitive to DTx. CX3CR1Cre:iDTR (CX3CR1-DTR) mice did not tolerate prolonged ablation, but (CX3CR1-DTR4WT) BM chimeras showed no adverse side effects of repetitive and prolonged DTx treatment. Flow cytometric analysis of both the colon and the ileum of DTx-injected CX3CR1-DTR chimeras revealed that
consistent with the above reporter gene expression prole intestinal macrophages were efciently ablated (Supplementary Fig. 4ce; Supplementary Fig. 5ce). In addition, colonic CD103 CD11b cells and ileal CD103 CD11b compartments were signicantly affected. In contrast, intestinal CD103
CD11b DC were largely resistant to the ablation. In summary, the CX3CR1Cre system spares (BatF3-dependent) CD103
CD11b DC, but targets both CX3CR1hi macrophages and
CD103 and CD103 CD11b DC; the latter might be due to their expression of the chemokine receptor or their derivation from CX3CR1-expressing cells27. For simplicity we will refer from here on to cells that are in CX3CR1-DTR mice sensitive to DTx as CX3CR1 cells. In analogy to the above CD11c-DTR experiment, we generated mixed chimeras with CX3CR1-DTR BM and either mutant or WT BM, and challenged the animals with C. rodentium. Mice that after DTx treatment specically lacked il23a in their CX3CR1 cell compartment died by day 10
12 after infection (Fig. 2e). Furthermore, also mice that lacked il12b in CX3CR1 cells, impairing both their IL-12 and IL-23 production, died. Both the groups of animals displayed a considerable reduction in IL-22 and RegIIIb transcripts, when compared with WT chimeras (Fig. 2f). These data establish that the production of the colonic IL-23, which induces IL-22 and AMPs in response to the C. rodentium challenge is restricted to CD103 CD11b DC or macrophages, but not CD103
CD11b DC. Our data thus support previous reports of IL-23 expression by these cells or their equivalents8,2931. Importantly, the fact that DTx-treated (il12b / /CX3CR1-DTR4WT)
chimeras die from the Citrobacter challenge, but (il12b / / CD11c-DTR4WT) chimeras survive, indicates that the rescue results from the introduction of the IL-12 deciency into the CD103 CD11b DC compartment. These data support our
4 NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525 ARTICLE
earlier observation that exacerbated IL-12 production by CD103 CD11b DC is associated with the lethality in our system (Fig. 1d).
IL-23 regulates IL-12 production by CD103 CD11b DC. To directly dene the cells that express IL-12 and IL-23 before and after C. rodentium challenge in vivo, we performed an reverse transcription PCR expression analysis on uorescence-activated cell sorting (FACS)-sorted cells (Supplementary Fig. 6a,b). Colonic CD11c CD64 CD24 macrophages, but neither
CD103 CD11b DC nor CD103 CD11b DC responded to the infection with il23a induction (Fig. 3a). CD103 CD11b
DC, on the other hand, downregulated il12a mRNA expression. Interestingly, CD103 CD11b DC, but not CD103 CD11b
DC seem to express the IL-23 receptor, as they displayed signals for both il23r and il12rb1 (Fig. 3a, Supplementary Fig. 6c) thus sensitizing these cells to IL-23. To examine whether the presence or absence of IL-23 expression from CX3CR1 cells affects IL-12 production by CD103 CD11b DC, we sorted these cells from infected DTx-treated (il23a / /CX3CR1-DTR 4WT) chimeras and compared them with cells isolated from infected DTx-treated control chimeras. In the absence of CX3CR1 cell-derived IL-23,
CD103 CD11b DC displayed signicantly enhanced il12a and il12b mRNA expression, whereas il12a mRNA expression by macrophages was unaffected (Fig. 3b).
These data suggest that CD103 CD11b DC are subject to IL-23-dependent paracrine negative control. Specically, colonic
CX3CR1 cells, including macrophages and possibly CD103 CD11b DC produce IL-23 that suppresses IL-12 expression by
CD103 CD11b DC. To directly investigate this putative negative control circuit, we generated mixed chimeras with WT and il-23rgfp/gfp BM32. The resulting mice harbour in their lamina propria both IL23R-decient (CD45.2) and -procient (CD45.1) CD103 CD11b DC (Fig. 4a). We then challenged these mice with C. rodentium. Conrming the genotype of the
BM grafts IL23R expression was absent from CD45.2 CD103 CD11b DC as compared with the CD45.1 WT DC equivalents (Fig. 4b). More importantly though and proving that CD103 CD11b DC are in the infected mice under negative control by IL-23, IL23R-decient DC expressed signicantly more il12a mRNA than WT DC isolated from the same animals (Fig. 4b). In further support of the critical role of IL-23 in controlling IL-12 production, supernatants of cultured colon explants of Citrobacter-infected (il23r gfp/gfp4WT) mice showed signicantly higher IL-12 levels than that of infected controls (Fig. 4c). Collectively, this establishes that intestinal CD103 CD11b DC are target of a critical IL-23-mediated negative control circuit that prevents overt and potentially deleterious IL-12 production by these cells.
IL-23 is required to prevent IFNc-driven immunopathology.C. rodentium-infected animals harbouring IL-23-decient CX3CR1 cells acutely succumb to infection. The above data suggest that IL-23 controls otherwise deleterious IL-12 production by CD103 CD11b DC. IL-12 is known to promote the
20
15
10
5
0
0
*
il23a /TBP
800
*
il12a /TBP IL-23R /TBP
600
400
200
10
8
6
4
2
0
C. rodentium + + + +
CD64+ CD24+
Macrophage
DC
CD64+
CD103+ CD11b
CD103 CD11b+
800
600
*
il12a expression
(fold increase)
IL-23R expression
(fold increase)
600
400
400
200
200
0 CX3CR1-DTR/WT
CX3CR1-DTR/ il23a/
0 CX3CR1-DTR/WT
0 CX3CR1-DTR/ WT
CX3CR1-DTR/ il23a/
30
40
20
30
20
10
10
Figure 3 | Analysis of intestinal mononuclear phagocytes for basal and PI cytokine production. (a) Reverse transcription PCR (RTPCR) analysis of FACS-sorted lamina propria DC and macrophages isolated from uninfected or infected WT mice 3 days PI. Data are representative of 36 independent experiments with DC and macrophages pooled from 710 mice (for sorting strategy see Supplementary Fig. 6). (b) RTPCR analysis of CD103
CD11b CD11c DC and CD103 CD11b CD11c cells, including macrophages and CD103 CD11b DC were sorted from DTx-treated mixed (WT/CX3CR1-DTR4WT) and (il23a / /CX3CR1-DTR4WT) BM chimeras. Data are presented as fold increase compared with the DC from uninfected mice. Data were obtained from three independent experiments with cells pooled from 911 chimeras. Data were analysed by two-tailed t-test *Po0.05. TBP, TATA binding protein.
0 CX3CR1-DTR/WT
CX3CR1-DTR/ il23a/
CX3CR1-DTR/ il23a/
8
8
il12b expression
(fold increase)
6
6
4
4
2
2
0 CX3CR1-DTR/ WT
CX3CR1-DTR/ il23a/
0 CX3CR1-DTR/ WT
CX3CR1-DTR/ il23a/
Macrophages
CD11b DC
NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 5
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525
105
WTCD45.1/1 50% 50%
IL-23R KO
CD45.2/2
CD103
105
0
103
CD45.2
103
0
IL-23R KO DC
WT DC
0 103
105
0
103
105
CD11b
300
20
30
*
il-12a expression
(fold increase)
200
100
4 3 2 1 0
il-12b expression
(fold increase)
IL-23R expression
(fold increase)
80
60
40
**
*
15
20
10
IL-12p70 ng ml1
20
10
5
0 0
CD45.1
IL-23R/
CD11b
DC
Figure 4 | IL-23 negatively controls IL-12 production by colonic CD103 CD11b DC in vivo. (a) Schematic of mixed chimera approach and ow cytometric analysis of colonic mononuclear phagocyte compartment of Citrobacter-infected (il-23rgfp/gfp (CD45.2)/WT (CD45.1)4WT (CD45.2)) mice indicating chimerism of CD103 CD11b DC population. Cells are gated according to scatter, doublets and CD11c expression. Data represent. oneof the three independent experiments (n 3 mice per group). Data were analysed by two-tailed t-test *Po0.05. (b) reverse transcription PCR analysis of
il12a, il12b and il23R transcripts measured in CD103 CD11b CD11c DC sorted from (IL-23Rgfp/gfp(CD45.2)/WT (CD45.1)4WT (CD45.2)) chimeras. (n 3 mice per group from three individual experiments). (c) ELISA for IL-12 performed on colon explant culture supernatants ofC. rodentium-challenged
(IL-23Rgfp/gfp4WT) and (WT4WT) chimeras on day 7 PI (n 34 mice per group from three individual experiments). Data were analysed by two-tailed
t-test *Po0.05, **Po0.01.
WT CD11b DC
IL-23R/
CD11b
DC
WT CD11b DC
IL-23R/
CD11b
DC
WT CD11b DC
0 IL-23R/
WT
polarization of nave T cells into TH1 cells and induce IFNg secretion by CD4 and CD8 T cells, as well as NK cells that can cause detrimental immunopathology33. To investigate whether the uncontrolled IL-12 production by colonic CD103
CD11b DC triggers such a cascade in our system, we neutralized IFNg in Citrobacter-infected mice that harbour IL-23-decient CX3CR1 cells using an antibody regimen. Indeed, this treatment efciently rescued infected DTx-treated (CD11c-
DTR/il23a / 4WT) and (CX3CR1-DTR/il23a / 4WT) chimeras from pathogen-induced mortality (Fig. 5a,b).
Moreover, also the neutralization of IL12p40, that is, the cytokine which is likely involved in the induction of IFNg33, improved the survival of the respective Citrobacter-infected animals (Fig. 5a,b).
The kinetics of the observed fatality suggested ILC or a preexisting T-cell population as IFNgsource. Fate-mapping studies recently established that TH17 cells can differentiate into pathogenic TH1 cells, both in the brain and gut3436. To evaluate the involvement of this TH17/TH1 cell axis in the Citrobacter-infected mice lacking either IL-23 or IL-12/-23, we analysed colonic tissue of whole and mixed BM chimeras generated with il23a / , il12b / , CD11c-DTR and WT BM cells. FACS analysis of T cells isolated from the colon of the various mice, as well as reverse transcription PCR analysis for IL-17A and IFNg expression indicated a shift towards TH1 cells in
IL-23-decient BM chimeras ((il23a / 4WT); Fig. 5c). Similar results were obtained when the IL-23 deciency was restricted to CD103 CD11b DC and macrophages (DTx-treated (il23a / /CD11c-DTR4WT) chimeras; Fig. 5d). To probe if the observed accumulating TH1 cells derive from TH17 cells, we analysed their CD121a (IL1R1) expression, which has been reported to identify exTH17 cells (ref. 35). As shown in Fig. 5e, CD121a was prominently displayed on a fraction of IFNg TH1 cells isolated from mice lacking CX3CR1 cell-derived IL-23. Of note, IFNg transcript levels and TH1 frequencies were also
elevated in WT chimeras; however, these cells did not display CD121a expression and were resistant to the infection. Collectively, these data suggest that TH17-derived TH1 cells, rather than conventional TH1 cells, are the source of the deleterious IFNg that causes the severe immunopathology in the absence of the IL-23-mediated mononuclear phagocyte crosstalk.
DiscussionThe aim of this study was to dene the differential contributions of the intestinal DC and macrophages in the immediate early innate host defense against C. rodentium challenge. Specically, we identied colonic CX3CR1 mononuclear phagocytes, including CD103 CD11b DC and macrophages, as early producers of the critical IL-23 required for the induction of IL-22 and AMPs20. Absence of IL-23, however, not only impaired the innate response, but also revealed the existence of a critical crosstalk between colonic CX3CR1 cells and CD103 CD11b
DC. Specically, we found that IL-23 suppressed IL-12 production by CD103 CD11b DC thereby preventing fatal immunopathology.
The host response to the A/E mouse pathogen C. rodentium can be segregated into early innate reactions and late adaptive immunity37. The former depend critically on IL-23, that is required to induce IL-22 production by ILC3 (refs 20,38). Notch2-dependent small intestinal CD11b CD103 cells, the likely equivalent of colonic CD103 CD11b DC, were shown to produce IL-23 upon Citrobacter infection30. Moreover, these
IRF4-dependent cells were reported as the IL-23 source that maintains the steady-state TH17 compartment in this tissue68. Recent studies further established intestinal macrophages as the IL-23 source31. We corroborate these ndings by showing that mice that harbour an IL-23 deciency in colonic CX3CR1 mononuclear phagocytes, comprising CD103 CD11b DC and macrophages, fail to induce IL-22 and AMPs.
6 NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525 ARTICLE
CD11c-DTR mixed BM chimeras
CX3CR1-DTR mixed BM chimeras
Days after infection
100
100
80
80
Survival (%)
60
P=0.0003
il23a/ +isotype
il23a/ +anti il12b
il23a/ +anti IFN
WT +PBS
10 7.8 2.9
6.4 9.7 3.9
il23a/ +PBS
Survival (%)
60
P=0.004
il23a/ +anti il12b
WT +PBS
il23a/ +anti IFN
40
40
il23a/ +PBS
20
20
0
0 5 10 15 20
0
0 5 10 15 20
Days after infection
Whole BM chimera CD11c-DTR mixed BM chimera
0
IL-17 A expression
(fold increase over day 0)
IFNexpression (fold increase over day 0)
IL-17 A expression
(fold increase over day 0)
0
25
*
20
*
10
IFNexpression
(fold increase over
day 0)
8
20
7.5
6
15
4
10
5
10
5
2
2.5
0 WT WT il23a/ il12b/ WT il12b/
0 WT il23a/ il12b/ WT il23a/
il23a/
0 WT il23a/ il12b/ il23a/ il12b/
WT il23a/ il12b/
10 3.2
WT il23a/ il12b/
8
*
5
*
Ratio (IFNg/IL-17)
Ratio (IFNg/IL-17)
10
6
10
2.2
10
2.5
10
3.7
4
3
10
10
10
10
10
10
IL-17
IL-17
4
10
10
10
10
10
10
2
10
10
10
2
10
10
10
0
4.8
0
7.9
0
0
0
3.8
1
0
0
10
10
10
10
0 10 10 10 10 0 10 10 10 10
0 10 10 10 0 10 10 10 0 10 10 10
0
IFN
IFN
il12b/
Gated on CD3+CD4+ IFNg+ cells
60
*
*
% CD121+
(out of CD4+ CD3+
IFN+)
wt il23a/ il12b/
0 wt il23a/
CD121 il12b/
50
100
100
40
0.945
100
8.48 3.77
Number
of cells
30
50
50
50
20
0
10
0 0 104 105
103
0 0 104 105
103 0 104 105
103
Figure 5 | Citrobacter-infected mice that lack IL-23-producing macrophages and CD103 CD11b DC succumb to IL-12- and IFNc-mediated immunopathology. (a) Survival curves of DTx-treated mixed (il23a / /CD11c-DTR4WT) BM chimeras infected by Citrobacter gavage and treated with
IFNg- and IL12p40-specic neutralizing antibodies by intravenous.injection or PBS and isotype control antibodies. (n 5 per group, representative of three
individual experiments). Statistical analysis was performed using the log-rank test. (b) Survival curves of DTx-treated mixed (il23a / /CX3CR1-DTR4WT) BM chimeras infected by Citrobacter gavage and treated with IFNg- and IL12p40-specic neutralizing antibodies by intravenous injection or PBS; (n 56 per group from three individual experiments). Statistical analysis was performed using the log-rank test. (c) (Top) real-time PCR analysis of IFNg
and IL-17A of colonic tissues collected on day 7 PI and (bottom) ow cytometric analysis of TH17 and TH1 isolated from the lamina propria of il23a / , il12b / and WT BM chimeras. Cells were pregated on CD3 CD4 cells. (n 36 mice per group from two individual experiments). Data were analysed
by analysis of variance (ANOVA) followed by Tukeys post hoc test *Po0.05. (d) (Top) real-time PCR analysis of IFNg and IL-17A of colonic tissues collected on day 7 PI and (bottom). Flow cytometric analysis of TH17 and TH1 isolated from the lamina propria of indicated chimeras generated with haematopoietic mixture of mutant BM with CD11c-DTR BM. Cells were pregated on CD3 CD4 cells. (n 45 mice per group from two individual
experiments). Data were analysed by ANOVA followed by Tukeys post hoc test *Po0.05. (e) Flow cytometric analysis of TH1 cells isolated from DTx-treated (il23a / /CD11c-DTR4WT), (il12b / /CD11c-DTR4WT) and (WT/CD11c-DTR4WT) BM chimeras (n 4 per group from four individual
experiments). Data were analysed by ANOVA followed by Tukeys post hoc test *Po0.05.
Surprisingly, Citrobacter-infected animals bearing only an IL-23 (p19) deciency in all the DCs and macrophages died from the bacterial challenge, whereas animals harbouring a combined IL-12/ 23 (p40) deciency were resistant, although both the mice display impaired IL-22 induction. Furthermore, animals with an IL-12/23 mutation restricted to CX3CR1 cells succumbed to the infection.
Collectively, these data suggested a critical deleterious role of CD103 CD11b DC-derived IL-12 and ensuing immunopathology as cause of the acute death of the animals. Indeed, antibody-mediated neutralization of IL-12 or IFNg rescued Citrobacter-challenged mice harbouring the IL-23 deciency.
Aside from orchestrating the early innate host response to Citrobacter infection, that is, IL-22 and AMP induction, IL-23 is thus also critically involved in a crosstalk of CX3CR1 cells and
neighbouring CD103 CD11b DC. Our conclusion is supported by the fact that (1) CD103 CD11b DC express
IL-23 receptor and seem within the colonic DC compartment to be uniquely sensitive to IL-23; (2) in absence of IL-23, IL12p35 and IL12p40 mRNA expression is signicantly increased in CD103 CD11b DC of Citrobacter-challenged mice, as was IL-12 secretion by colon explants. Most importantly, (3) IL-23 receptor-decient CD103 CD11b DC displayed prominent
IL12p35 mRNA expression over WT DC in the same Citrobacter-challenged mice. The fact that IL-23 can interfere with the IL-12 axis has been previously reported in refs 39,40. Sieve et al.40 showed that IL-23 could inhibit the IL-12-induced effector function of IFNg induction by T cells. However the authors argued that this effect was due to a direct antagonistic IL-23 effect
NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 7
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525
on the IL-12 receptor on T cells, rather than an IL23R-mediated effect on IL-12 production by myeloid cells, as shown in this study. Becker et al.39 showed that in an in vitro system IL-23 specically downregulated IL-12 secretion in LPS-exposed BM culture-derived DC. Focusing on IL23p19-decient DC, these authors concluded that they observed an autocrine regulatory loop, which they considered responsible for the aggravated colitis observed in IL23p19 / mice39. Our present data support the notion of such a less appreciated, but clinically probably highly relevant immunoregulatory role of IL-23. Moreover importantly, we extend the earlier reports by revealing a novel paracrine regulatory circuit between the specic intestinal mononuclear phagocyte subsets. Of note, this immunoregulatory role is distinct from reported effects of IL-23 on IL-12 receptor signalling in T cells, which limits their IFNg production40. The molecular details underlying the suppressive effect of IL-23 on CD103 CD11b
DC might be related to activation of signal transducer and activator of transcription 3 (STAT3) and thus similar to IL-10 suppression of IL-12 production41. However, the exact mechanism remains to be dened.
IL-12 is known to polarize nave T cells towards a TH1 fate42. However, given the short time frame of our experiments, the immunopathology that we observed was unlikely to result from exacerbated adaptive immunity. TH17 cells have been shown to retain plasticity and can under certain pathological conditions, such as experimental autoimmune encephalomyelitis34,35, but also following Helicobacter challenge in the intestine36 convert into TH1 cells. Given the presence of TH17 cells in the steady-state colon, we considered these rapidly generated exTH17 TH1 cells an attractive explanation for the IFNg pathology we observe.
Indeed, we found TH1 cells in our setting of immunopathology to express CD121a (IL1R1) indicative of their TH17 past35. However, we cannot include the involvement of other IL-12 responsive IFNg producers, such as ILC-1 (ref. 43), or additional direct IL-23 effects on ILC (ref. 44).
Intestinal homeostasis and gut resistance to pathogen challenge rely on a balance of pro- and anti-inammatory processes. This homeostastic inammation is established in response to the constant microbial product exposure of this unique organ45. Our ndings highlight the interplay of these processes by corroborating the role of myeloid cell-derived IL-23 as an inducer of IL-22, which is known to promote mucosal healing and stabilize barrier integrity. Importantly though, our data indicate that IL-23 curbs the simultaneously potential damaging immunopathology caused by IL-12 production. Of note, our data do not argue against the importance of IL-22 in the protection against Citrobacter challenge, which is well documented18,20. However, we show that under certain conditions, such as the absence of IL-12 production by CD103 CD11b DC,
Citrobacter-challenged mice can handle an impaired IL-22 response, probably since epithelial injury is less severe.
Genome-wide association studies have revealed a link between allelic variants of the IL-23 receptor, its signalling and the risk to develop Crohns disease or ulcerative colitis, although the underlying mechanistic details remain to be elucidated46,47. Given the appreciated role of IL-23 in the control of ILC3 and TH17 biology, efforts to understand the reason for genetic IBD predispositions are currently largely focused on the effect of this cytokine on innate and adaptive lymphocytes. Though not mutually exclusive, our results suggest that the IL-23 receptor variants could also affect a critical regulatory circuit between myeloid cells thatwhen disruptedmight result in deleterious hyperactivity of intestinal DC. Our results highlight the intricacy of the regulatory circuits that exist in the intestinal lamina propria to maintain immune protection and prevent collateral damage. Moreover, these ndings should be of clinical importance
highlighting a previously under-appreciated immunoregulatory role of IL-23 on mononuclear phagocytes.
Methods
Mice. This study involved the use of the following mice, all on C57BL/6 background: B6.129S1-Il12btm/Jm/J mice (p40 / )48 and B6.IL23p19tm (p19 / )
mice49; CX3CR1Cremice27 (deposited at the European Mouse Mutant Archive,EM:06349; JAX Stock No. 25524 B6.C-Cx3cr1otm1.1(cre)Jung4/J);
Rosa26-DTR (iDTR)28; CD11c-DTR mice24;IL-23Rgfp mice32. To analyse recombinase activity of Cx3cr1cre mice the animals were crossed with Rosa-26-rfp (ref. 50).For BM chimera generation, C57BL/6 mice were lethally irradiated with 950 rad. The next day, 5 106 BM cells isolated from the femora and tibiae of donor
mice were intravenously injected into the irradiated recipient mice, which were kept at rest for 68 weeks before the experiment. Animals were maintained under specic pathogen-free conditions and handled according to the protocols approved by the Weizmann Institute Animal Care Committee as per international guidelines.
Citrobacter infection and assessment of bacterial load. Mice were gavaged with a spontaneous naladixic acid-resistant derivative of the strain C. rodentiumICC168 (Citrobacter freundii biotype 4280), that is kanamycin-resistant and biolumines-cent, the strain ICC180 (ref. 23), a kind gift of Gad Fraenkel (Centre for Molecular Bacteriology and Infection, Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom). Mice were gavaged with 0.2 ml of PBS containing 109 c.f.u. Bacteria were grown at 37 C in Luria Bertani broth or on Luria Bertani agar with kanamycin (50 mg ml 1) After 3 h the bacterial density was assessed by measuring its absorbance at an optical density of 600 nm and conrmed by plating of serial dilutions. For the assessment of bacterial load, mice were anaesthetized and imaged using IVIS (Xenogen, Almeda, CA). Greyscale reference images taken under low illumination were collected and overlaid with images capturing the emission of photons from the bioluminescent C. rodentium using LIVING IMAGE software (Xenogen). For the analysis of the colon, mice were euthanized and imaged as above.
Cell preparations. For isolation of lamina propria DC and macrophages, intestinal epithelial cells were removed by incubation with the HBSS containing DTT and 5% of fetal calf serum and shaking in a 37 C incubator (300 r.p.m.) for 40 min. Intestinal pieces were digested in 5 ml HBSS containing 10% FBS, 1.2 mg ml 1 of collagenase VIII (Sigma) and shook (300 r.p.m.) at 37 C for 40 min. Digested colon tissue was passed through a 80-mm mesh and cells were collected by centrifugation at 1,400 r.p.m. for 10 min at 4 C. For isolation of intestinal T cells, lamina propria cells were after the Collagenase digest fractionated on a Percoll density gradient (40 and 80%). To allow for the intracellular cytokine staining of TH17 cells and TH1 cells, IL-17A and IFNg production was triggered and accumulated by PMA and ionomycin (Sigma) treatment for 2 h and treatment with Brefeldin A and Monensin (eBioscience) for 2 h at 37 C 5% CO2. Cells were permeabilized and xed for the intracellular staining by the BD perm/xation kit.
Antibody-mediated cytokine neutralization. For the neutralization experiments, indicated mice were intraperitoneally injected with anti-IFNg-specic mAb (Clone, XMG1.2) or anti-IL12p40-specic mAb (C17.8) on day 0, 2, 4, 6 and 8 after C. rodentium infection at a dose of 1 mg per mouse. The control groups received isotype control IgG2a mAb.
Cell ablation. For cell depletion in (CX3CR1-DTR4WT) BM chimeras, 25 ng g 1 body weight diphtheria toxin (DTx; Sigma-Aldrich) was injected IP every 48 h for 5 days and the mice were euthanized 1820 h after DTx injection. For prolonged cell ablation in the functional assays, the dose was reduced to 12.5 ng g 1 body weight after the rst 5 days (day 1 was one day before infection). (CD11c-DTR4WT)
BM chimeras were injected 12.5 ng g 1 in the rst 5 days and 6.25 ng g 1 body weight until the end of the assay.
cDNA synthesis and real-time PCR. Total RNA was extracted from the colon using the Gentle-MACS (MiltenyiBiotec) and RNA tissue kit (5prime) and from the sorted cells with the RNeasy Micro Kit (Qiagen). cDNA synthesis was performed with 0.52 mg of total RNA with High Capacity RNA-to-cDNA Kit (Applied Biosystems). Real-time PCR was performed with SYBR Green master mix (Applied Biosystems). Expression of each target gene was normalized to that of TATA binding protein using the comparative (DDCT) method.
Histology. Tissues were xed in 4% paraformaldehyde overnight at 4 C, embedded in parafn, sectioned and stained with periodic acid-Schiff. Slides were evaluated using an Olympus BX51 microscope, and image acquisition was conducted with the Olympus DP70 camera and DP-Manager software. The percent area of goblet cells and colonic hyperplasia were calculated using a technique established previously51.
8 NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525 ARTICLE
Flow cytometric analysis. The following uorochrome-labelled monoclonal antibodies and staining reagents were used according to manufacturers protocols: lamina propria cells were stained with anti-CD11c-APC, anti-CD64-APC (eBioscience), anti-CD11b-PerCP Cy5.5 (Biolegend), anti-CD103-PE/Horizon 421(BD Pharmingen), anti-MHC II-PE-CY7, anti-CD24- PE-CY7 (eBioscience), anti-CD45.2-FITC (eBioscience), anti-CD45.1-Pacic blue (Biolegend) and 40,6-diamidino-2-phenylindole. TH17 and TH1 cells were stained with anti-IL-17A-PE, anti-CD3-biotin, SA-PerCP, anti-CD4-APC, anti-CD8-FITC, anti-CD45-Pacic Blue anti-IFNg-PE-CY7 and anti-CD121A-PE. The cells were analysed with LSR
Fortessa ow cytometer (BD) or sorted with a FACSAria machine (BD). Flow cytometry analysis was done with the FlowJo software.
Analysis of colon explant cultures and ELISA. The colons of mice were ushed with RPMI and open along a longitudinal axis. Thereafter, 3-mm2 punch biopsies were obtained from the distal colon and incubated for 24 h in RPMI supplemented with 10% fetal calf serum and antibiotics (one punch biopsy per 100 ml medium). Supernatants were collected and kept frozen until assessed. Quantitative evaluation of IL-12 and IFNg levels in supernatant were done using an ELISA modication, with spectrally distinguishable beads (Spherotech) as solid phase. Primary antibodies were covalently linked to the beads, with spectrally different beads linked to antibodies against a unique analyte. The various coated beads were incubated simultaneously with the supernatant and secondary biotinylated antibodies for 2 h, washed and stained with streptavidin-PE. Bead uorescence level was used to separate the different analytes and then compared with a standard curve for quantication. The beads were analysed with LSRFortessa ow cytometer (BD).
Statistical analysis. Data were analysed by analysis of variance followed by Tukeys post hoc, two-tailed t-test, and long-rank analysis on the KaplanMeier plots using GraphPad Prism 4 (San Diego, CA). Data are presented as mean-s.e.m. values of Po0.05 were considered statistically signicant. *Po0.05, **Po0.01, ***Po0.001.
References
1. Cerovic, V., Bain, C. C., Mowat, A. M. & Milling, S. W. F. Intestinal macrophages and dendritic cells: whats the difference? Trends. Immunol. 35, 270277 (2014).
2. Farache, J., Zigmond, E., Shakhar, G. & Jung, S. Contributions of dendritic cells and macrophages to intestinal homeostasis and immune defense. Immunol. Cell. Biol. 91, 232239 (2013).
3. Mildner, A. & Jung, S. Development and function of dendritic cell subsets. Immunity 40, 642656 (2014).
4. Cerovic, V. et al. Lymph-borne CD8a( ) dendritic cells are uniquely able to
cross-prime CD8( ) T cells with antigen acquired from intestinal epithelial
cells. Mucosal Immunol. 4, 104113 (2014).5. Lewis, K. L. et al. Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine. Immunity 35, 780791 (2011).
6. Schlitzer, A. et al. IRF4 transcription factor-dependent CD11b dendritic cells
in human and mouse control mucosal IL-17 cytokine responses. Immunity 38, 970983 (2013).7. Persson, E. K. et al. IRF4 transcription-factor-dependentCD103( )CD11b( ) dendritic cells drive mucosal T helper 17 cell
differentiation. Immunity 38, 958969 (2013).8. Welty, N. E. et al. Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism. J. Exp. Med. 210, 20112024 (2013).
9. Cerovic, V. et al. Intestinal CD103 dendritic cells migrate in lymph and
prime effector T cells. Mucosal Immunol. 6, 104113 (2012).10. Schreiber, H. A. et al. Intestinal monocytes and macrophages are required for T cell polarization in response to Citrobacter rodentium. J. Exp. Med. 210, 20252039 (2013).
11. Rivollier, A., He, J., Kole, A., Valatas, V. & Kelsall, B. L. Inammation switches the differentiation program of Ly6Chi monocytes from antiinammatory macrophages to inammatory dendritic cells in the colon. J. Exp. Med. 209, 139155 (2012).
12. Zigmond, E. et al. Ly6Chi monocytes in the inamed colon give rise to proinammatory effector cells and migratory antigen-presenting cells. Immunity 37, 10761090 (2012).
13. Mortha, A. et al. Microbiota-dependent crosstalk between macrophages and ILC3 promotes intestinal homeostasis. Science 343, 12492881249288 (2014).
14. Lai, Y., Rosenshine, I., Leong, J. M. & Frankel, G. Intimate host attachment: enteropathogenic and enterohaemorrhagic Escherichia coli. Cell. Microbiol. 15, 17961808 (2013).
15. Mundy, R., Macdonald, T. T., Dougan, G., Frankel, G. & Wiles, S. Citrobacter rodentium of mice and man. Cell. Microbiol. 7, 16971706 (2005).
16. Maaser, C. et al. Clearance of citrobacter rodentium requires B Cells butnot secretory immunoglobulin A (IgA) or IgM antibodies. Infect. Immun. 72, 33153324 (2004).
17. Oppmann, B. et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13, 715725 (2000).
18. Mangan, P. R. et al. Transforming growth factor-b induces development of the TH17 lineage. Nature 441, 231234 (2006).
19. Sonnenberg, G. F., Fouser, L. A. & Artis, D. Border patrol: regulation of immunity, inammation and tissue homeostasis at barrier surfaces by IL-22. Nat. Immunol. 12, 383390 (2011).
20. Zheng, Y. et al. Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Nat. Med. 14, 282289 (2008).
21. Basu, R. et al. Th22 cells are an important source of IL-22 forhost protection against enteropathogenic bacteria. Immunity 37, 10611075 (2012).
22. Ivanov, I. I. et al. Induction of intestinal Th17 cells by segmented lamentous bacteria. Cell 139, 485498 (2009).
23. Wiles, S., Pickard, K. M., Peng, K., Macdonald, T. T. & Frankel, G. In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium. Infect. Immun. 74, 53915396 (2006).
24. Jung, S. et al. In vivo depletion of CD11c dendritic cells abrogates priming of
CD8 T cells by exogenous cell-associated antigens. Immunity 17, 211220
(2002).25. Varol, C. et al. Intestinal lamina propria dendritic cell subsets have different origin and functions. Immunity 31, 502512 (2009).
26. Sapoznikov, A. et al. Organ-dependent in vivo priming of naive CD4 ,
but not CD8 , T cells by plasmacytoid dendritic cells. J. Exp. Med. 204,
19231933 (2007).27. Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 7991 (2012).
28. Buch, T. et al. A Cre-inducible diphtheria toxin receptor mediatescell lineage ablation after toxin administration. Nat. Methods 2, 419426 (2005).
29. Kinnebrew, M. A. et al. Bacterial agellin stimulates Toll-like receptor 5dependent defense against vancomycin-resistant Enterococcus infection.J. Infect. Dis. 201, 534543 (2010).30. Satpathy, A. T. et al. Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching- and-effacing bacterial pathogens. Nat. Immunol. 14, 937948 (2013).
31. Longman, R. S. et al. CX3CR1 mononuclear phagocytes support colitis-
associated innate lymphoid cell production of IL-22. J. Exp. Med. 9, 143 (2014).32. Awasthi, A. et al. Cutting Edge: IL-23 receptor GFP reporter mice reveal distinct populations of IL-17-producing cells. J. Immunol. 182, 59045908 (2009).
33. Vignali, D. A. A. & Kuchroo, V. K. IL-12 family cytokines: immunological playmakers. Nat. Immunol. 13, 722728 (2012).
34. Kurschus, F. C. et al. Genetic proof for the transient nature of the Th17 phenotype. Eur. J. Immunol. 40, 33363346 (2010).
35. Hirota, K. et al. Fate mapping of IL-17-producing T cells in inammatory responses. Nat. Immunol. 12, 255263 (2011).
36. Morrison, P. J. et al. Th17-cell plasticity in Helicobacter hepaticusinduced intestinal inammation. Mucosal Immunol. 6, 11431156 (2013).
37. Ouyang, W., Kolls, J. K. & Zheng, Y. The biological functions of T helper 17 cell effector cytokines in inammation. Immunity 28, 454467 (2008).
38. Maynard, C. L. et al. Regulatory T cells expressing interleukin 10 develop from Foxp3 and Foxp3 precursor cells in the absence of interleukin 10.
Nat. Immunol. 8, 931941 (2007).39. Becker, C. et al. Cutting edge: IL-23 cross-regulates IL-12 production inT cell-dependent experimental colitis. J. Immunol. 177, 27602764 (2006).
40. Sieve, A. N., Meeks, K. D., Lee, S. & Berg, R. E. A novel immunoregulatory function for IL-23: Inhibition of IL-12-dependent IFN-g production. Eur. J.
Immunol. 40, 22362247 (2010).41. Kortylewski, M. et al. Regulation of the IL-23 and IL-12 balanceby Stat3 signaling in the tumor microenvironment. Cancer Cell 15, 114123 (2009).
42. Ogarra, A. Cytokines induce the development review of functionally heterogeneousT helper cell subsets. Immunity 8, 275283 (1998).
43. Fuchs, A. et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-g-producing cells. Immunity 38, 769781 (2013).
44. Buonocore, S. et al. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature 464, 13711375 (2010).
45. Aychek, T. & Jung, S. The axis of tolerance. Science 343, 14391440 (2014).
46. Duerr, R. H. et al. A genome-wide association study identies IL23R as an inammatory bowel disease gene. Science 314, 14611463 (2006).
47. Cho, J. H. The genetics and immunopathogenesis of inammatory bowel disease. Nat. Rev. Immunol. 8, 458466 (2008).
48. Magram, J. et al. IL-12-decient mice are defective in IFN gamma production and type 1 cytokine responses. Immunity 4, 471481 (1996).
NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications 9
& 2015 Macmillan Publishers Limited. All rights reserved.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7525
49. Cua, D. J. et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inammation of the brain. Nature 421, 744748 (2003).50. Luche, H., Weber, O., Nageswara Rao, T., Blum, C. & Fehling, H. J. Faithful activation of an extra-bright red uorescent protein in knock-in Cre-reporter mice ideally suited for lineage tracing studies. Eur. J. Immunol. 37, 4353 (2007).
51. Aychek, T., Vandoorne, K., Brenner, O., Jung, S. & Neeman, M. Quantitative analysis of intravenously administered contrast media reveals changes in vascular barrier functions in a murine colitis model. Magn. Reson. Med. 66, 235243 (2011).
Acknowledgements
We would like to thank Caterina Curato and members of the Jung laboratory for help and discussion and the staff members of the Weizmann sorting facility for technical support. We thank Thomas Korn for provision of bone marrow of the IL-23 receptor-decient mice. This work was supported by the Crohns and Colitis Foundation of America (CCFA), the United StatesIsrael Binational Science Foundation (BSF) and the Pasteur Weizmann Foundation.
Additional information
Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications
Web End =http://www.nature.com/ http://www.nature.com/naturecommunications
Web End =naturecommunications
Competing nancial interests: There are no competing nancial interests.
Reprints and permission information is available at http://npg.nature.com/reprintsandpermissions/
Web End =http://npg.nature.com/ http://npg.nature.com/reprintsandpermissions/
Web End =reprintsandpermissions/
How to cite this article: Aychek, T. et al. IL-23-mediated mononuclear phagocyte crosstalk protects mice from Citrobacter rodentium-induced colon immunopathology. Nat. Commun. 6:6525 doi: 10.1038/ncomms7525 (2015).
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Web End =http://creativecommons.org/licenses/by/4.0/
10 NATURE COMMUNICATIONS | 6:6525 | DOI: 10.1038/ncomms7525 | http://www.nature.com/naturecommunications
Web End =www.nature.com/naturecommunications
& 2015 Macmillan Publishers Limited. All rights reserved.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
Copyright Nature Publishing Group Mar 2015
Abstract
Gut homeostasis and mucosal immune defense rely on the differential contributions of dendritic cells (DC) and macrophages. Here we show that colonic CX3 CR1+ mononuclear phagocytes are critical inducers of the innate response to Citrobacter rodentium infection. Specifically, the absence of IL-23 expression in macrophages or CD11b+ DC results in the impairment of IL-22 production and in acute lethality. Highlighting immunopathology as a death cause, infected animals are rescued by the neutralization of IL-12 or IFNγ. Moreover, mice are also protected when the CD103+ CD11b- DC compartment is rendered deficient for IL-12 production. We show that IL-12 production by colonic CD103+ CD11b- DC is repressed by IL-23. Collectively, in addition to its role in inducing IL-22 production, macrophage-derived or CD103- CD11b+ DC-derived IL-23 is required to negatively control the otherwise deleterious production of IL-12 by CD103+ CD11b- DC. Impairment of this critical mononuclear phagocyte crosstalk results in the generation of IFNγ-producing former TH17 cells and fatal immunopathology.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer