Research ArticleINFLAMMATION

IL-23–producing IL-10Rα–deficient gut macrophages elicit an IL-22–driven proinflammatory epithelial cell response

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Science Immunology  14 Jun 2019:
Vol. 4, Issue 36, eaau6571
DOI: 10.1126/sciimmunol.aau6571

A Colitis Circuit

Cytokines are known to play a critical role in maintaining gut homeostasis, but their specific cellular sources are less well understood. Here, Bernshtein et al. used a murine model of inflammatory bowel disease (IBD) in which macrophages specifically lack expression of interleukin-10 receptor (IL-10R), and mice developed symptoms of spontaneous colitis similar to that observed in children with IL-10R mutations. They identified macrophage-derived IL-23 as the cytokine critical to induce the pathology. IL-23 triggered accumulation and IL-22 production by TH17 cells that, in turn, promoted production of chemokines by colonic epithelial cells and destructive neutrophil recruitment. Together, these results reveal the mechanism by which intestinal IL-10R–deficient macrophages drive IBD pathogenesis.


Cytokines maintain intestinal health, but precise intercellular communication networks remain poorly understood. Macrophages are immune sentinels of the intestinal tissue and are critical for gut homeostasis. Here, we show that in a murine inflammatory bowel disease (IBD) model based on macrophage-restricted interleukin-10 (IL-10) receptor deficiency (Cx3cr1Cre:Il10rafl/fl mice), proinflammatory mutant gut macrophages cause severe spontaneous colitis resembling the condition observed in children carrying IL-10R mutations. We establish macrophage-derived IL-23 as the driving factor of this pathology. Specifically, we report that Cx3cr1Cre:Il10rafl/fl:Il23afl/fl mice harboring macrophages deficient for both IL-10R and IL-23 are protected from colitis. By analyzing the epithelial response to proinflammatory macrophages, we provide evidence that T cells of colitic animals produce IL-22, which induces epithelial chemokine expression and detrimental neutrophil recruitment. Collectively, we define macrophage-specific contributions to the induction and pathogenesis of colitis, as manifested in mice harboring IL-10R deficiencies and human IBDs.


Inflammatory bowel diseases (IBDs) such as Crohn’s disease (CD) and ulcerative colitis (UC) are two chronic and relapsing pathologies of the small and large intestine that affect millions of individuals worldwide (1). Extensive genome-wide association studies (GWAS) revealed 200 IBD-associated genetic loci, and recent high-resolution fine mapping identified statistically convincing causal variants (2). Studies in mice have provided insight into the cellular composition of the intestinal mucosa, including mononuclear phagocytes (3), adaptive and innate lymphoid cells (ILCs) (4), intercellular communication circuits maintaining gut homeostasis (5), and the impact of microbiota (6). Animal IBD models have better defined genetic factors causing, contributing to, or preventing intestinal inflammation (7). One such module consists of the cytokine interleukin-10 (IL-10), associated signaling components, such as Stat3 (8, 9), and IL-10 receptor (IL-10R) (9, 10).

Most of the intestinal macrophages reside in the connective tissue or lamina propria, underlying the monolayer of epithelial cells (ECs). As nonmigratory cells, they do not translocate to draining lymph nodes (LNs) and display limited or no potential to “prime” naive T cells (10). In adult mice, most lamina propria macrophages are continuously replenished by Ly6Chi blood monocytes (11). Monocyte-derived cells replace an embryonically derived population in the gut shortly after birth (1214). As opposed to many other tissue macrophages that self-renew in their entirety throughout adult life (15), adult intestinal macrophages comprise subsets with distinct half-lives (10). Constant replacement of short-lived gut macrophages necessitates ongoing adaption of monocyte precursors to the dynamic local gut environment, including exposure to microbial stimuli (16). IL-10, provided by regulatory T cells (17), is a critical factor ensuring colon homeostasis and preventing the emergence of proinflammatory monocyte-derived cells. Specifically, colonic macrophages unable to sense IL-10 due to Il-10ra deficiency exhibit unrestrained responses in patients (12, 13). Moreover, mice that also harboring IL-10Ra–deficient macrophages develop severe early onset colitis (14) and, hence, provide a valuable model for mechanistic studies of the human disorder caused by the Il-10ra loss-of-function mutation.

IL-10R-deficient macrophages display a proinflammatory expression signature, including up-regulation of IL-23 (14), a cytokine composed of two subunits: p19, which is unique to IL-23, and p40, which is shared with IL-12 (18). IL-23 was shown to contribute to colitis development in several mouse models. After Helicobacter hepaticus infection of lymphopenic Rag2−/− mice, antibody-mediated neutralization of p19 or p40, but not p35 (the second IL-12 subunit), ameliorated disease (19). Likewise, Il23a−/−:Rag1−/− and Il12b−/−:Rag1−/−, but not Il12a−/−:Rag1−/−, mice are protected from T cell transfer–induced colitis (19). Moreover, after a combination of H. hepaticus infection and IL-10R blockade, wild-type (WT) and Il12a−/− mice succumb to disease, whereas Il12b−/− mice are spared (20). The underlying mechanism by which IL-23 drives inflammation may involve interferon-γ–secreting T helper 1 (TH1) cells and reduced IL-17 levels, although differentiation of TH17 cells was unaltered in the absence of IL-23 (19). In support of IL-23 acting on T cells, adoptive transfers of IL-23R–deficient T cells into immunodeficient mice caused less severe colitis than WT T cell engraftment (21). Several cell types were suggested as the source of IL-23, including monocytes, Cx3cr1+ macrophages, and CD103+CD11b+ dendritic cells, although their respective contributions might differ in the small and large intestines (2225).

T cell responses to IL-23 include the production of IL-22, a cytokine acting on ECs, which, in the context of colitis, is widely considered anti-inflammatory. Thus, IL-22 therapy by gene transfer or IL-22 production by neutrophils was shown to ameliorate colitis (26). Likewise, IL-22–deficient mice displayed increased gut inflammation, both in the dextran sodium sulfate (DSS) and the T cell transfer colitis model (27). In contrast, in dermal inflammatory diseases such as psoriasis, IL-22 was shown to play a detrimental role (28). Proinflammatory activities of IL-22 in the gut were reported for an innate-driven colitis model based on the injection of anti-CD40 antibody, which stimulated myeloid cells (29) and caused dysbiosis favoring pathogen colonization potentially due to IL-22–induced antimicrobial peptide (AMP) production (29). Collectively, IL-22 is widely considered a beneficial agent in gut inflammation and may be involved in wound healing and bacterial protection (3032). Accordingly, IL-22 therapy has been suggested as a possible treatment for patients with IBD (28), although the potential exists for deleterious outcomes associated with chronic IL-22 stimulation of gut ECs.

Here, we investigated macrophage-driven colitis, as manifested in children and mice harboring IL-10R deficiencies (12, 13, 33). Specifically, we define causative cell responses and reveal the critical role of the IL-23/IL-22 pathway in this IBD pathology. We report that proinflammatory macrophages produce IL-23, which, in turn, recruits TH17 cells and induces secretion of IL-22. Colonic ECs respond to IL-22 exposure by expression of AMPs and neutrophil chemoattractants that appear to be critical for colitis development.


IL-10Ra–deficient macrophages confer spontaneous colitis involving the IL-23/IL-22 axis

In mice and humans, intestinal macrophages require IL-10 sensing to maintain gut homeostasis (1214). Accordingly, Cx3cr1cre:Il10rafl/fl mice harboring IL-10Ra–deficient macrophages develop spontaneous colitis manifested in IBD hallmarks, including tissue remodeling and immune cell infiltrates in the colon, but not the ileum (14).

Intestinal macrophages extracted from colonic lamina propria of 6- to 7-week-old precolitic Cx3cr1cre:Il10rafl/fl mice displayed a proinflammatory gene expression signature (Fig. 1, A and B, and fig S1). Mutant macrophages exhibit elevated expression of cytokine and chemokine genes, including Il23a, Il12b, and Ccl5; genes encoding activation markers such as trem1; and inflammatory molecules like nos2 and saa3. In total, 310 genes were significantly down-regulated, and 355 genes were significantly up-regulated in mutant cells, as compared with controls (fold change, >2; adjusted P < 0.05).

Fig. 1 IL-10R-deficient macrophages secrete IL-23, inducing IL-22 secretion by ILC3 and TH17 cells.

(A) Volcano plot of statistical significance (−log10 P value) against log2 ratio of macrophages sorted from the colonic lamina propria of 6- to 7-week-old Cx3cr1creIl10rafl/fl and Cx3cr1gfp/+ mice, based on RNA-seq data. Significantly up- or down-regulated genes (fold change, >2; adj. P < 0.05) are in black, and relevant proinflammatory up-regulated genes are highlighted in red. Data are representative of two independent experiments, n ≥ 3 for each group. (B) RNA-seq–normalized read numbers for single genes of interest are plotted separately; each dot represents one mouse. (C) qRT-PCR analysis of Il23p19 expression by sorted colonic macrophages from 6- to 7-week-old mice (left) or of Il22 expression (right) in colonic whole-tissue extracts of 6- to 7-week-old indicated mice, including antibiotics (Abx) treatment. Data were collected from two independent experiments, n ≥ 3 in each group. (D) qRT-PCR analysis of Il17 expression in colonic whole-tissue extracts of indicated mice. Data were collected from two independent experiments, n ≥ 3 in each group. (E) Whole-mount staining of colonic tissue of Cx3cr1cre:Il10rafl/fl:Rorgtgfp mice and Cre-negative littermates (age, 3 to 4 months), analyzed by confocal microscope, GFP in green, and CD3 in red. Indent in the bottom is in the white rectangle. Scale bars, 50 μm (top) and 20 μm (bottom). (F) Representative picture of flow cytometry analysis and sorting strategy of Cx3cr1cre:Il10rafl/fl:Rorgtgfp mice and Cre-negative littermates 3 to 4 months old. (G and H) qRT-PCR analysis of RNA extracted from sorted colonic TH17 cells (F) or ILC3s (G) for indicated genes. Data were collected from two independent experiments, n = 3 in each group. *P ≤ 0.05.

IL-23R was identified by GWAS as an IBD susceptibility gene (34), and IL-23–deficient mice are resistant to colitis induction in different IBD mouse models (19, 20, 35). IL-10ra–deficient macrophages expressed high levels of Il23a, as revealed by RNA sequencing (RNA-seq) (Fig. 1B) and confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) conducted on sorted colonic macrophages (Fig. 1C). Antibiotic treatment upon weaning abolished the proinflammatory signature of IL-10Ra–deficient colonic macrophages (fig. S1B) and IL-23 up-regulation (Fig. 1C), establishing microbiota-derived signals as macrophage activation stimuli in this model. IL-23 is known to induce IL-22 expression by TH17 cells and type 3 innate lymphoid cells (ILC3s) (36, 37). Accordingly, Il22 mRNA levels were elevated in the tissue of mice harboring IL-10Ra–deficient macrophages in a microbiota-dependent manner (Fig. 1C). In addition, and in line with possible involvement of TH17 cells in this colitis model, IL-17 transcript levels were significantly up-regulated in whole-tissue extracts of colitic mice (Fig. 1D). Differential contributions of TH17 cells and ILC3s to colitis development remain controversial (4). To study their role in the present colitis model, we crossed Cx3cr1cre:Il10rafl/fl animals to Rorgtgfp reporter mice (38), allowing identification and discrimination of ILC3 and TH17 cells by flow cytometry and histology (Fig. 1, E and F). The lamina propria of colitic Cx3cr1cre:Il10rafl/fl:Rorgtgfp animals was abundantly populated with ILC3s and TH17 cells (Fig. 1, E and F, and fig. S1C), both of which expressed elevated Il22 mRNA levels as compared with cells isolated from Il10rafl/fl:Rorgtgfp control littermates (Fig. 1, G and H). ILC3s extracted from colitic mice also expressed elevated levels of Cxcr6, a chemokine receptor critical for the localization and IL-22 production in the small intestine (Fig. 1H) (39).

Collectively, Cx3cr1cre:Il10rafl/fl mice display a microbiota-dependent activation of the IL-23/IL-22 axis. Proinflammatory intestinal macrophages that lack IL-10R produce IL-23 and promote the accumulation and IL-22 production of both TH17 cells and ILC3s.

ECs respond to proinflammatory macrophages by induction of IL-22–dependent genes

Cx3cr1cre:Il10rafl/fl bone marrow (BM) transplanted into lethally irradiated WT recipients induced spontaneous colitis, evident within 6 to 7 weeks after engraftment. Specifically, mice that received the colitogenic BM, but not recipients of Cx3cr1gfp/+ control BM, displayed attenuated weight gain and high colonoscopy scores 7 weeks after transplant (Fig. 2A). The main IL-22 sensors in the colon are ECs (4). To determine the epithelial response to IL-10Ra–deficient macrophages, we implemented a reporter-based system, avoiding contaminations by immune cells, which reside in the epithelial layer. Specifically, we took advantage of Villincre:R26-tdTomato mice (40), whose colonic and ileal ECs, but not CD45+ cells, express a tomato reporter protein detectable by histology and flow cytometry (Fig. 2, B and C). Of note, colonic ECs extracted from irradiated mice showed few differentially expressed genes as compared with ECs isolated from nonirradiated animals (fig. S2, A and C) and hence do not display persistent transcriptomic changes in agreement with their reported efficiency to cope with irradiation damage (41).

Fig. 2 The response of ECs to proinflammatory macrophages.

(A) Weight and colonoscopic analysis of [Cx3cr1cre:Il10rafl/fl > WT] or [Cx3cr1gfp/+ > WT] BM chimeras. Colonoscopy was performed 7 weeks after transplant. Data are representative of three independent experiments, n ≥ 3 for each group. *P < 0.05. (B) Representative picture of microscopic analysis of [Cx3cr1gfp > Villincre:R26-tdTomato] BM chimera. (C) Representative picture of flow cytometry analysis and sorting strategy of ECs from [Cx3cr1gfp > Villincre:R26-tdTomato] BM chimera. (D) Description of BM chimera experiment and timeline of EC harvest. (E) Volcano plots of statistical significance (log10 P value) against log2 ratio of ECs sorted from the colon of [Cx3cr1cre:Il10rafl/lf > Villincre:R26-tdTomato] BM chimeras and Villincre:R26-tdTomato mice, based on RNA-seq data. Significantly up- or down-regulated genes (fold change, >2; adj. P < 0.05) are in black and Il-22–induced genes are highlighted in red. Data were collected from one experiment, n ≥ 3 for each group. (F) Heatmap of RNA-seq data of colonic and ileal ECs extracted from the same mouse. Normalized reads number were log-transformed. Presented are genes of interest, significantly up-regulated in colonic but not in ileal ECs in response to proinflammatory macrophages at both disease stages [day 14 (d14) and d60 after transplant)]. (G) qRT-PCR analysis of Reg3b, Reg3g, Cxcl1, and Cxcl5 expression in whole-tissue extracts of colons of 6- to 7-week-old mice. Data were collected from two independent experiments, n ≥ 3 in each group.

Colitogenic Cx3cr1cre:Il10rafl/fl BM was engrafted into irradiated Villincre:R26-tdTomato recipients, and Tomato+ colonic and ileal ECs were isolated at two time points, i.e., before overt disease onset [14 days posttransplant (dpt)] and upon signs of colonic inflammation (60 dpt; Fig. 2D). RNA-seq analysis revealed significant transcriptomic changes of colonic ECs at 14 dpt, which increased at 60 dpt, with 316 and 834 differentially expressed genes, respectively (Fig. 2E). In contrast to the colonic ECs, ileal ECs showed minimal changes at both time points, although they had been isolated from the same animal (fig. S2B). Differentially expressed genes of colonic ECs at early and late time points comprised a prominent signature of IL-22–induced genes (4), including AMPs, CXC chemokines, and members of the calcium binding S100 protein family (S100a8 and S100a9) that encode calprotectin (Fig. 2F). These chemokines and S100 were absent from ileal EC transcriptomes (Fig. 2F). The two subunits comprising the IL-22 receptor, Il10rb and Il22ra, were expressed by both ileal and colonic EC under steady-state and inflammatory conditions (fig. S2D), indicating the ability of ECs to sense IL-22 in both ileum and colon under steady-state conditions. Expression of the IL-10–specific receptor subunit il10ra was below background levels. Robust AMP and chemokine up-regulation was not only restricted to colitic BM chimeras but also observed in Cx3cr1cre:Il10rafl/fl mice, which displayed the up-regulation of Reg3b, Reg3g, Cxcl1, and Cxcl5, already at 6 to 7 weeks of age when signs of colitis were mild, as compared with co-housed Il10rafl/fl littermate controls (Fig. 2G and fig. S2F). In conclusion, before overt signs of intestinal inflammation, colonic EC respond to proinflammatory macrophages, including the expression of AMPs and chemokines that are established to be IL-22–induced.

Mutant macrophage production of IL-23 drives colitis in Cx3cr1cre:Il10rafl/fl mice

The centraI role of IL-23 has been established in IBD models (19, 20, 35), but the critical cellular sources of this cytokine remain controversial and might differ between anatomic location and pathology (2225). Moreover, in our system, colonic IL-10R–deficient macrophages produce other proinflammatory factors, including Ccl5, Nos2, Saa3, and Il12b, in addition to IL-23 (Fig. 1, A and B); macrophage-derived IL-23 is unlikely to be the sole driver of pathology. To nevertheless probe for such a scenario in this colitis model, we generated Cx3cr1cre:Il10rafl/fl:Il23afl/fl mice (42). Notably, intestinal macrophages that failed to sense IL-10 and were unable to produce IL-23, also lost the complete proinflammatory signature, including expression of the aforementioned proinflammatory factors (Fig. 3, A to C). Thus, Il10ra/Il23 double-deficient cells displayed only seven differentially expressed genes compared with Cx3cr1gfp control macrophages (Fig. 3C); in contrast, 464 genes were found differentially expressed between macrophages isolated from Cx3cr1cre:Il10rafl/fl compared with the same Cx3cr1gfp macrophages (Fig. 3B).

Fig. 3 IL-23 deficiency prevents the colitogenic activity of IL-10R mutant macrophages.

(A) Heatmap of RNA-seq data of colonic macrophages sorted from 6- to 7-week-old mice. Normalized read numbers were log-transformed. (B and C) Volcano plots of statistical significance (log10 P value) against log2 ratio of macrophages sorted from the colonic lamina propria of (B) Cx3cr1cre:Il10rafl/fl and Cx3cr1gfp/+ mice or (C) Cx3cr1cre:Il10rafl/fl:Il23afl/fl and Cx3cr1gfp/+ mice, based on RNA-seq data. Significantly up- or down-regulated genes (fold change, >2; adj. P < 0.05) are in black, and relevant proinflammatory up-regulated genes are highlighted in red. In (B), data are representative of two independent experiments (B) or from one experiment (C), in each experiment, n ≥ 3. (D) Colonoscopic (left) and histopathological (right) analysis of 3- to 4-month-old mice. Data were collected from two independent experiments, n ≥ 3 in each group. (E) Representative images of histopathological analysis of indicated mouse strains. (F) qRT-PCR analysis of Il23a expression by sorted colonic macrophages of indicated mouse strains (left) or Il22, Nos2, and Reg3b expression in colonic whole-tissue RNA extracts of indicated mouse strains. Data were collected from two independent experiments, n ≥ 3 in each group. All mice were age-matched; each dot represents one mouse. n.s., non significant, P > 0.05; *P ≤ 0.05.

Mere genetic neutralization of macrophage-derived IL-23 rescued Cx3cr1cre:Il10rafl/fl:Il23afl/fl mice from colitis, as evident from colonoscopy and histopathology analysis of co-housed age-matched littermates (Fig. 3, D and E). In the absence of macrophage-derived IL-23, tissue Il22 expression returned to homeostatic levels, as did expression of genes coding for AMPs and Nos2 (Fig. 3F). Data obtained from corresponding BM chimeras corroborated the above observation (fig. S3). Collectively, these data establish that the critical IL-23 source driving colitis in Cx3cr1cre:Il10rafl/fl mice are macrophages and are the same cells that lack IL-10 signaling. Moreover, our results emphasize the importance of IL-23 for disease initiation and establish that, directly or indirectly, this factor induces a secondary proinflammatory gene signature in the macrophages.

IL-22 is required for IL-23–driven colitis in Cx3cr1cre:Il10rafl/fl mice

IL-23 induces intestinal ILC3s, TH17 cells, and neutrophils to produce IL-22 (43), which is widely considered to be beneficial (26, 27) but can be harmful under certain circumstances (29). Given the epithelial IL-22 response to IL-10Ra–deficient macrophages (Fig. 2F), we tested the impact of IL-22 in our colitis model. Specifically, we crossed Cx3cr1cre:Il10rafl/fl mice to Il22−/− animals (37). Cx3cr1cre:Il10rafl/fl:Il-22−/− mice did not develop signs of colitis, as determined by colonoscopy and histopathological evaluation (Fig. 4, A and B). Levels of Il22 and neutrophil-recruiting chemokines Cxcl1 and Cxcl5, as well as Reg3b, Reg3g, and Nos2 in colonic tissue of Cx3cr1cre:Il10rafl/fl:Il22−/− mice were similar to those of Il10rafl/fl:Il22−/− littermate controls (Fig. 4C). Collectively, this establishes IL-22 in the Cx3cr1cre:Il10rafl/fl model as a proinflammatory factor that is essential for colitis induction. Of note, although the Cx3cr1cre:Il10rafl/fl:Il22−/− mice did not exhibit intestinal pathology, their colonic macrophages displayed elevated levels of Il23a, as compared with Il10rafl/fl:Il22−/− littermates (Fig. 4D). In contrast and supporting the notion that their induction is a secondary effect potentially requiring the EC response, Ccl5 transcripts were not elevated in macrophages (Fig. 4D). In addition, levels of il17a transcripts were increased in colonic tissues of Cx3cr1cre:Il10rafl/fl:Il22−/− mice, suggesting that in our model, IL-17 alone is insufficient to induce AMP and chemoattractant expression by EC (Fig. 4, C and D). Last, flow cytometry analysis using intracellular staining of colonic lamina propria T cells of Cx3cr1cre:Il10rafl/fl:Il22−/− mice showed increased numbers of IL-17–producing T cells relative to littermate controls (Fig. 4E). Collectively, these data suggest that, in our model, IL-22 is a required factor for colitis initiation, and its induction is the critical colitogenic response to IL-23 produced by IL-10R–deficient proinflammatory macrophages.

Fig. 4 IL-22 is critical for colitis driven by IL-10R-deficient macrophages.

(A) Colonoscopic (left) and histopathological (right) analysis of 3- to 4-month-old mice. (B) Representative images of histopathological analysis of indicated mouse strains. Scale bar, 100 µm. (C) qRT-PCR analysis of Il22, Cxcl1, Cxcl5, Reg3b, Reg3g, and Nos2 expression in colonic whole-tissue extracts of indicated mouse strains. (D) qRT-PCR analysis of Il23a and Ccl5 by sorted colonic macrophages of indicated mouse strains (left), and whole-tissue Il17a levels of indicated mouse strains. (E) Flow cytometry analysis and quantification of lamina propria T cells extracted from the colons of indicated mouse strains. Data in (A), (C), (D), and (E) were collected from two independent experiments, n ≥ 3 in each group. All mice were age-matched; each dot represents one mouse.

Unexpectedly, WT mice that received Cx3cr1cre:Il10rafl/fl:Il22−/− BM grafts succumbed to colitis like [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras (Fig. 5A). qRT-PCR analysis of colonic tissue of colitic BM chimeras showed elevated levels not only of transcripts for the neutrophil recruiting chemokines Cxcl1 and Cxcl5 but also Il22 mRNA (Fig. 5B), suggesting derivation of the latter from host cells that survived irradiation. Irradiated Il22−/− recipients that received the otherwise colitogenic Cx3cr1cre:Il10rafl/fl:Il22−/− BM were devoid of Il22 and protected from colitis, as compared with WT recipients (Fig. 5C). While corroborating the notion of IL-22 as a proinflammatory factor, this suggests radio-resistant cells as a critical IL-22 source in the Cx3cr1cre:Il10rafl/fl colitis model. To probe for radio-resistant Rorgt-expressing host cells, we generated BM chimeras using Rorgtgfp mice as recipients. Green fluorescent protein–positive (GFP+) TH17 cells, but not GFP+ T cell receptor–negative ILC3, were abundantly found in the lamina propria of both [Cx3cr1cre:Il10rafl/fl > Rorgtgfp] and [Il10rafl/fl > Rorgtgfp] chimeras 3 weeks after BM transplantation (Fig. 5D). TH17 cell numbers were significantly higher in Cx3cr1cre:Il10rafl/fl BM recipients (Fig. 5D), and sorted TH17 cells from these colitic chimeras exhibited elevated Il22 transcription (Fig. 5E), indicating IL-23 encounter and activation. Last, [Cx3cr1cre:Il10rafl/fl > Il22−/−] BM chimeras exhibited reduced colitis progression (Fig. 5F) and low Il22 and Cxcl5 whole-tissue mRNA levels. This result excludes the possibility of IL-22 production by graft-derived cells, supporting the notion of a radio-resistant cell as the exclusive IL-22 source. Collectively, these data suggest that the critical colitogenic cellular source of IL-22 in the Cx3cr1cre:Il10rafl/fl colitis model is radio-resistant cells, which include TH17 cells.

Fig. 5 Definition of the cellular source of IL-22.

(A) Weight and colonoscopic analysis of [Cx3cr1cre:Il10rafl/fl > WT], [Cx3cr1gfp/+ > WT], or [Cx3cr1cre:Il10rafl/fl:Il-22−/− > WT] BM chimeras. Colonoscopy was performed 6 weeks after transplant. n.s., non significant, P > 0.05; *P ≤ 0.05. Data were collected from two independent experiments, n ≥ 3 in each. (B) qRT-PCR analysis of Il22, Cxcl1, and Cxcl5 expression in whole-tissue extracts of colons of indicated BM chimeric mice. Data were collected from two independent experiments, n = 3 to 5 in each group. (C) qRT-PCR analysis of Il22 expression in whole-tissue extracts of colons of indicated BM chimeric mice. Data are from one of two representative experiments. Weight (left) and colonoscopic (right) analysis of [Cx3cr1cre:Il10rafl/fl:Il22−/− > WT] and [Cx3cr1cre:Il10rafl/fl:Il22−/− > Il22−/−] BM chimeras. Colonoscopy was performed 7 weeks after transplant. Weight data are from one of two representative experiments, n = 3 to 4 in each group; colonoscopic data are collected from two experiments. n = 3 to 4 in each group. (D) Flow cytometry analysis of the lamina propria of [Cx3cr1cre:Il10rafl/fl > Rorgtgfp] and [Il10rafl/fl > Rorgtgfp] BM chimeras. Tcrb, T cell receptor beta chain. Data are representative of three experiments, n = 3 to 5 in each group. (E) qRT-PCR analysis of Il22 expression by sorted TH17 cells from indicated BM chimeras. Data were collected from three independent experiments, n = 3 to 5 in each group. (F) Weight and colonoscopic analysis of [Cx3cr1cre:Il10rafl/fl > WT] or [Cx3cr1cre:Il10rafl/fl > Il-22−/−] BM chimeras. Colonoscopy was performed 6 weeks after transplant. Data were collected from two independent experiments, n ≥ 3 in each group. (G) qRT-PCR analysis of Il22 and Cxcl5 expression in whole-tissue extracts of colons of indicated BM chimeric mice. Data were collected from two independent experiments, n = 3 to 5 in each group.

CD4+ cells are critical for IL-22 production and neutrophil accumulation in Cx3cr1cre:Il10rafl/fl colitis model

Next, we aimed to further define mechanistic aspects of the colitogenic activity of IL-22 observed in our mouse model. Transcripts encoding the neutrophil-recruiting chemokines Cxcl1 and Cxcl5 were consistently up-regulated in the colonic tissue of Cx3cr1cre:Il10rafl/fl mice (Fig. 2, G and F) in an IL-22–dependent manner (Fig. 4C). Cxcl1 and Cxcl5 were induced in colonic epithelium but not expressed by ileal ECs (Fig. 2F). In contrast, although induced in the inflamed colon, AMPs were constitutively expressed in ileal epithelium. This suggested that neutrophil attraction, rather than AMP induction, might be critical for the IL-22–driven pathology. In line with this notion, flow cytometry analysis revealed prominent neutrophil infiltrates in the colon of Cx3cr1cre:Il10rafl/fl mice, which were absent in the protected Cx3cr1cre:Il10rafl/fl:Il23afl/fl and Cx3cr1cre:Il10rafl/fl:Il22−/− strains (fig. S4 and Fig. 6, A and B). Neutrophils are known to cause collateral tissue damage (44), and their tissue infiltration and activities are tightly controlled (45). In line with their destructive potential, recruited neutrophils in the tissues of Cx3cr1cre:Il10rafl/fl mice displayed elevated reactive oxygen species production as compared with blood neutrophils of the same animals (Fig. 6C).

Fig. 6 Neutrophil recruitment to the colonic lamina propria depends on CD4+ T cells.

(A) Representative plots of flow cytometry analysis of the colonic lamina propria of indicated mouse strains. (B) Quantification of flow cytometry analysis according to gating strategy indicated in (A). n.s., non significant, P > 0.05; *P ≤ 0.05. (C) Representative plots of flow cytometry analysis of the colonic lamina propria of Cx3cr1cre:Il10rafl/fl mice (left). Quantification of flow cytometry analysis (right). (D) Schematic of T cell depletion protocol. (E) Quantification of flow cytometry analysis of mesenteric LNs indicating efficient ablation of CD4+ T cells but not CD8+ T cells. (F) Representative plots of flow cytometry analysis of the colonic lamina propria of [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras. (G) Representative immunofluorescence images of colon sections of [Il10rafl/fl > WT], [Cx3cr1cre:Il10rafl/fl > WT], and [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras treated with the anti-CD4 regimen. Scale bars, 50 μm. (H) qRT-PCR analysis of Il22, Cxcl1, and Cxcl5 expression in whole-tissue extracts of colons of indicated BM chimeric mice. Data are collected from two independent experiments, n ≥ 3 in each group.

To formally establish the link between IL-22–expressing CD4+ T cells and the neutrophil accumulation, we tested the impact of T cell ablation on neutrophil recruitment (Fig. 6D). Using an antibody regimen, CD4+ T cells were efficiently depleted from mesenteric LNs, whereas CD8+ T cells remained unaffected (Fig. 6E). [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras depleted of CD4+ T cells showed significant reduction of neutrophil recruitment (Fig. 6F). Histological analysis confirmed neutrophil infiltrates in [Cx3cr1cre:Il10rafl/fl > WT] mice and the associated loss of colon tissue architecture, which was, as determined by E-cadherin and collagen staining, prevented by the CD4+ T cell depletion (Fig. 6G). Moreover, reduced neutrophil infiltration correlated with lower Il22, Cxcl1, and Cxcl5 mRNA expression in BM chimeras depleted of CD4+ T cells (Fig. 6H). As we found CD4+ ILC3s, another prominent IL-22 source associated with Citrobacter rodentium infections (46), to be radiosensitive, these data support the notion that the cellular sources of procolitic IL-22 in our model are likely TH17 cells (Fig. 1E). The depletion experiment places CD4+ T cells and IL-22 upstream of neutrophil recruitment in the cascade of colitis progression. Collectively, our observations point to IL-22 as a proinflammatory factor that is likely secreted by TH17 cells and induces EC expression of Cxcl1 and Cxcl5, whereby promoting neutrophil recruitment to the intestinal lamina propria.

Ablation of neutrophil infiltrates ameliorates colitis

As neutrophil recruitment appeared to be a hallmark of macrophage-induced colitis, we next tested the contribution of these cells to tissue pathology. Neutrophil extracellular traps (NETs) were shown to promote inflammation in atherosclerosis and mediate tissue damage created by neutrophils in pulmonary infection (47, 48). However, histological analysis of colitic BM chimeras revealed the absence of NET formation in colonic tissue (fig. S6A). To probe for involvement of another prominent neutrophil activity, [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras and controls were treated with neutrophil elastase inhibitor (NEI; fig. S6, B and C). Although we observed a reduction in levels of the neutrophil activation marker CD63 after NEI administration (fig. S6C), the treatment had no effect on neutrophil accumulation and tissue Nos2 levels (fig. S6, D and E). Last, we ablated neutrophils using the anti-Ly6G antibody (Fig. 7A). Flow cytometry analysis confirmed the depletion of neutrophils from the colon of the antibody-treated [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras (Fig. 7B), as did histological analysis (Fig. 7C). The latter revealed significantly reduced tissue damage in the colons of neutrophil-depleted animals, as indicated by the absence of villi hypertrophy and loss of cell polarity that was observed in colon areas with high neutrophil infiltrates of the immunoglobulin G (IgG)–treated control mice (Fig. 7C). Whole-tissue RNA analysis by qRT-PCR revealed decreased levels of Nos2 expression in neutrophil-depleted BM chimeras (Fig. 7D). Of note, macrophages and EC also produced Nos2 under inflammatory conditions according to our RNA-seq data (Figs. 1, A and B, and 2F); the fact that neutrophil depletion was sufficient to reduce tissue Nos2 levels suggests that either these cells are the main source of this enzyme or macrophage Nos2 expression is downstream of neutrophil recruitment. To test for such a potential effect of the neutrophil ablation on macrophage transcriptomes, we performed RNA-seq analysis on these cells isolated from anti-Ly6G and control IgG–treated [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras (Figs. 1, A and B, and 2F). Neutrophil ablation did not significantly impair macrophage expression of proinflammatory factors, such as Nos2, Ccl5, and Saa3 (Fig. 7E and fig. S7A), but either the absence of neutrophils or the ensuing improved tissue context did affect the global macrophage transcriptomes (fig. S7B). Collectively, these data establish that the neutrophil recruitment induced by epithelial response to IL-22 has a major contribution to tissue damage in IL-10R–deficient macrophage–driven colitis (fig. S8).

Fig. 7 Neutrophil depletion ameliorates colitis in Cx3cr1cre:Il10rafl/fl BM chimeras.

(A) Schematic of neutrophil depletion protocol. (B) Representative plots of flow cytometry analysis of the colonic lamina propria of [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras treated with IgG control or anti-Ly6G antibody (left). Quantification of flow cytometry analysis (right). *P ≤ 0.05. (C) Representative immunofluorescence images of the colonic tissue of [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras treated with IgG control or anti-Ly6G antibody. Scale bars, 50 µm. (D) qRT-PCR analysis of Nos2 expression in whole-tissue extracts of colons of indicated BM chimeric mice. (E and F) Volcano plot of statistical significance (log10 P value) against log2 ratio of macrophages isolated from colonic tissue of [Cx3cr1cre:Il10rafl/fl > WT] BM chimeras treated with IgG control (F) or anti-Ly6G antibody (E) and [Il10rafl/fl > WT] BM chimeras, based on RNA-seq data. Significantly up- or down-regulated genes (fold change, >2; adj. P < 0.05) are in black, and relevant proinflammatory up-regulated genes are highlighted in red. Data in (A) to (D) are collected from two independent experiments, n ≥ 3 in each group.


Here, we focused on understanding the critical steps in the induction and progression of macrophage-driven colitis, as manifested in children and mice harboring IL-10R deficiencies (12, 13). Specifically, macrophages of pediatric patients harboring IL-10Ra mutations and Cx3cr1cre:Il10rafl/fl mice that fail to sense IL-10 develop proinflammatory gene expression signatures that lead to gut pathology.

Using a conditional double mutagenesis strategy, we established macrophage-derived IL-23 as the critical driver of the disease. Macrophages from Cx3cr1cre:Il10rafl/fl:Il23afl/fl mice that lack the ability to produce IL-23, in addition to the IL-10R deficiency, no longer develop a proinflammatory gene signature. Our results corroborate and strengthen the key role of IL-23 in colonic inflammation that has been suggested earlier for the other experimental models (19, 25, 35). Moreover, we establish a critical hierarchy of proinflammatory factors, with macrophage-derived IL-23 at the apex of the cascade. Of note, IL-23 expression in macrophages is tightly controlled, including dedicated posttranscriptional circuits (49, 50) that have been linked to IL-10 (51). Collectively, this highlights IL-23 as a prime target for colitis therapy, as also supported by the clinical success of Ustekinumab, a monoclonal antibody that specifically blocks the IL-23 subunit p40 (52, 53).

We found macrophage-derived IL-23 to induce colonic TH17 cells and ILC3 to secrete IL-22, as also suggested in other colitis models (25). However, unlike earlier studies that involved DSS or T cell transfers into lymphopenic mice (27), IL-22–deficient Cx3cr1cre:Il10rafl/fl animals were protected from disease, establishing that, akin to its role in psoriasis (36), IL-22 production is deleterious in our IBD model. Rather, colitis driven by IL-10R–deficient macrophages in animals that harbor normal T cell compartments, and hence probably also IL-10R–deficient pediatric patients, more closely resembles the pathology induced by anti-CD40 treatment (29). The latter model lacks conventional T cells and therefore implies ILC3s as the IL-22 source, corroborating an earlier study (54). In contrast, in our model that retains both conventional and unconventional T cells, we assigned IL-22 production to TH17 cells. Shouval and colleagues (55) reported enhanced TH17 responses in patients with IL-10R deficiency and infantile-onset IBD, the close human counterpart of our model (12, 13). The pathogenicity of TH17 cells was also highlighted in a recent study that reported a direct link between H. hepaticus colonization and the generation of these cells (56). H. hepaticus infection of IL-10–deficient mice induced inflammatory TH17 cells in the colon, but not in the ileum, in line with the notion that IBD pathology is restricted to the large intestine in both IL-10–deficient and Cx3cr1cre:Il10rafl/fl mice. Collectively, our study and the one by Littman and colleagues (56) highlight the importance to investigate key drivers of IBD pathology also in models that retain conventional T cells, in addition to lymphopenic or immunodeficient mice.

A proinflammatory role of IL-22 has been identified in skin pathologies such as acanthosis and psoriasis (36) and was suggested to be related to keratinocyte hyperplasia. In the context of the gut, Salmonella enterica ser. Typhimurium was shown to benefit from IL-22 induction because EC-derived AMPs provided a competitive colonization advantage for the pathogen (57). As to the mechanism of how IL-22 promotes inflammation, the data point to a critical role of EC-derived neutrophil chemoattractants in our model. Specifically, TH17-derived IL-22 induces ECs to express the chemokines Cxcl1 and Cxcl5 alongside AMPs (58). The observed tight link between neutrophil infiltrates and gut pathology in the Cx3cr1cre:Il10rafl/fl mouse colitis model suggests that these factors are a major components in its pathological cascade, as recently proposed for the anti-CD40 colitis model (29).

The Cx3cr1cre:Il10rafl/fl mouse provides a colitis model mimicking infantile-onset IBD (12, 13). A major difference between the murine model and human disorder is that, in the patients, all cells harbor the IL-10R deficiency, whereas in the mouse model, the deletion is restricted. Other cells that are impaired in IL-10 sensing, such as T regulatory cells, could hence contribute to pathology. We show that macrophage-derived IL-23 drives the pathology and establishes critical roles of the T cells, ECs, and neutrophils. Future studies will be needed to address the molecular nature and source of the stimuli that establish the full proinflammatory signature of the IL-10R–deficient macrophages, beyond IL-23 expression. Our data reveal distinct inducer and effector modules in macrophage-driven colitis, as manifested in children and mice harboring IL-10R deficiencies. Neutralization of both modules in patients that globally lack the critical signal transducer Stat3, due to loss-of-function mutations affecting the open reading frame, does not result in colitis but in other morbidities (59). Our studies predict that variants that affect Stat3 expression in myeloid cell only might confer IBD risk. Several of the STAT3 single nucleotide polymorphisms (SNPs) reported to be associated with IBDs are located within regulatory elements (11). Future studies using targeted CRISPR-Cas9 mutagenesis of these regulatory elements will be required to test our assumption. However, collectively our findings support the validity of conditional mutagenesis in mouse models to define critical cell type–specific activities that promote IBD pathology.

Together, this study provides mechanistic insight into the cascade of intercellular communication events triggered by IL-10R–deficient macrophages in the colon. IL-23 produced by the mutant cells emerged as a critical trigger of TH17 cells that secrete IL-22 and thereby initiate an epithelial cell response that results in deleterious recruitment of neutrophils. Our findings might have direct implications for pediatric patients harboring IL-10R deficiencies (60) and add to our general understanding of cell type–specific contributions to IBD pathology.



This study included the following animals: Cx3cr1Cre mice (JAX stock no. 025524, B6J.B6N(Cg)-Cx3cr1tm1.1(cre)Jung/J) (42), Il10rafl/fl mice (38), Il23afl/fl mice (61), Rorgtgfp reporter mice (40), R26-tdTomato mice [B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J] (37), VillinCre mice (62), and Il22−/− mice (63). Only male mice were used. BM chimeras were generated by engraftment of recipient mice that were irradiated the day before with a single dose of 950 cGy using a XRAD 320 machine (Precision X-Ray Inc.). All animals were on C57BL/6 background and bred at the Weizmann Animal Facility. Sentinels in our facility were typed H. hepaticus positive. Whenever possible, age-matched co-housed males were used for experiments. Animals were maintained under specific pathogen-free conditions and handled according to protocols approved by the Weizmann Institute Animal Care Committee as per international guidelines.

Cell isolation, flow cytometry analysis, and sorting of intestinal macrophages and intestinal ECs

For the isolation of colonic ECs, extra-intestinal fat tissue and blood vessels were carefully removed, and colons were then flushed of their luminal content with cold phosphate-buffered saline (PBS), opened longitudinally, and cut into 0.5-cm pieces. Colon pieces were incubated in RPMI 1640 medium supplemented with 2 mM EDTA, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% sodium pyruvate for 40 min at 37°C shaking at 250 rpm.

For the isolation of colonic lamina propria cells, EC and mucus were removed by 40-min incubation with Hanks’ balanced salt solution (without Ca2+ and Mg2+) containing 5% FBS, 2 mM EDTA, and dithiothreitol [0.15 mg/ml (1 mM); Sigma-Aldrich] at 37°C shaking at 250 rpm. Colon pieces were then digested in PBS+/+ containing 5% FBS, collagenase VIII (1 mg/ml; Sigma-Aldrich), and deoxyribonuclease I (0.1 mg/ml; Roche) for 40 min at 37°C shaking at 250 rpm. The digested cell suspension was then washed with PBS and passed sequentially through 100- and 40-mm cell strainers. The following antibodies were used to stain the surface markers of the cells: CD45 (30-F11), Ly-6C (HK1.4), CD11b (M1/70), CD11c (HL3), Ly6G (1A8), CD64 (X54-5/7.1) from BioLegend, Bio-gems, or eBioscience, and CD103 (M290) from BD Biosciences. Cells were analyzed with the LSRFortessa flow cytometer (BD Biosciences) or sorted using a FACSAria machine (BD Biosciences). Flow cytometry analysis was performed with the FlowJo software.

Real-time PCR

Total RNA was extracted from sorted cells with the RNeasy Micro Kit (Qiagen) and from murine colons with the PerfectPure RNA Tissue Kit (5 Prime). RNA was reverse-transcribed with a mixture of random primers and oligo(dT) with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR was performed with a SYBR Green PCR Master Mix kit (Applied Biosystems). Quantification of the PCR signals of each sample was performed by comparing the cycle threshold (Ct) values, in duplicate, of the gene of interest with the Ct values of the TATA-binding protein (TBP) housekeeping gene (for whole-tissue RNA) or β-actin (for sorted cells). See Table 1 for primers used for qPCR.

Table 1 Primers used in qPCR.

View this table:

RNA-seq and analysis

For EC mRNA sequencing core facilities total RNA was extracted from 0.1 to 1 × 106 sorted cells with the RNeasy Micro Kit (Qiagen). One-hundred nanograms of total RNA was processed using the TruSeq Stranded Total RNA HTSample Prep Kit (with Ribo-Zero Gold) of Illumina (RS-122-2303). Libraries were evaluated by Qubit and TapeStation. Sequencing libraries were constructed with barcodes to allow multiplexing of 24 samples ran on three lanes. A total of 20 million single-end 50–base pair reads were sequenced per sample on a Illumina HiSeq 2500 high output mode instrument. Macrophage mRNA was sequenced using MARSeq, as previously described (62). Briefly, 104 to 105 cells from each population were sorted into 50 μl of lysis/binding buffer (Life Technologies). mRNA was captured with 15 μl of DynaBeads oligo(dT) (Life Technologies), washed, and eluted at 85°C with 6 μl of 10 mM tris-Cl (pH 7.5). A total of 5 million reads were sequenced per library. In both cases, gene expression levels were calculated using the HOMER software package ( rna mm9 -d <tagDir> -count exons -condenseGenes - strand + -raw) (63). Normalization and differential expression analysis were perfomed using the DESeq2 R package.

Histopathological analysis

Analysis was performed as previously described with minor adjustments (14). Briefly, colon was fixed in 2% paraformaldehyde solution overnight at 4°C, then embedded in paraffin, and sectioned and stained with hematoxylin and eosin. Tissues were examined in a blinded manner by a pathologist. Three segments of the colon (proximal colon, medial colon, and distal colon/rectum) were given a score between 0 and 4, and the average of these scores provided a total colonic disease score.


For T cell and ILC3 staining, tissues were fixed in 2% paraformaldehyde at 4°C and stained for CD3 (SP7, Abcam) and GFP (polyclonal IgG ab6658, Abcam) overnight at 4°C. Alexa Fluor 488–conjugated donkey antigoat and Cy3-conjugated donkey antirabbit (Jackson ImmunoResearch) secondary antibodies were added for 1 hour. Nuclei were stained with Hoechst. Images were acquired using a Nikon Eclipse Ti inverted spinning disc confocal microscope with 20× air objectives, with Andor iQ software, and analyzed with Volocity 6.1.1 software (Improvision Ltd.).

For neutrophil and tissue damage staining after dewaxing and rehydrating, the formalin-fixed, paraffin-embedded gut sections were incubated in Target Retrieval solution (pH 9.0) (S236884-2, Agilent Technologies LDA UK Limited) at 97°C for 45 min. Sections were incubated with primary antibodies mouse anti–E-Cadherin antibody (1:500 dilution; 610181, BD Biosciences) and rabbit polyclonal to collagen IV (1:500 dilution; ab6586, Abcam PLC), followed by Alexa Fluor 488–conjugated donkey antimouse [1:200 dilution; A21202, Thermo Fisher Scientific (Life Technologies)], Alexa Fluor 568–conjugated donkey antirabbit, Alexa Fluor 647–conjugated anti-Ly6G, and DAPI (4′,6-diamidino-2-phenylindole). Then, tissues were mounted with ProLong Gold Antifade Mountant [P36934, Thermo Fisher Scientific (Life Technologies)] and examined with confocal microscopy. Image analysis was performed using ImageJ.

Antibiotic treatment

For antibiotic treatment, mice were given a combination of vancomycin (0.5 g/l), ampicillin (1 g/l), kanamycin (1 g/l), and metronidazole (1 g/l) in their drinking water upon weaning and continuously until they were euthanized. All antibiotics were obtained from Sigma-Aldrich.

In vivo cell depletion and NEI treatment

For CD4 depletion, mice were injected intravenously with 100 μg of the anti-CD4 antibody (clone GK1.5, BioXcell) or isotype control (clone LTF2, BioXcell). For neutrophil depletion, mice were injected intraperitoneally with 120 μg of anti-Ly6g antibody (clone 1A8, BioXcell) or rat serum IgG control (Sigma-Aldrich). NEI was purchased from Axon Medchem and injected intraperitoneally at 2.5 μg/g every 3 days.

Quantification and statistical analysis

In all experiments, data are presented as means if not stated otherwise; each dot represents one mouse. Statistical tests were selected on the basis of appropriate assumptions with respect to data distribution and variance characteristics. Student’s t test (two-tailed) was used for the statistical analysis of differences between two groups. One-way analysis of variance (ANOVA) followed by Bonferroni correction was used for the statistical analysis of differences between three or more groups. Statistical significance was defined as P < 0.05. Sample sizes were chosen according to the standard guidelines. The number of animals is indicated as “n”. Of note, sizes of the tested animal groups were also dictated by availability of the transgenic strains and litter sizes, allowing littermate controls. Pre-established exclusion criteria are based on Institutional Animal Care and Use Committee guidelines. Animals of the same age, sex, and genetic background were randomly assigned to treatment groups. The investigator was not blind to the mouse group allocation. Tested samples were blindly assayed.


Fig. S1. Gating strategies and transcriptome analysis of animals exposed to antibiotics.

Fig. S2. Transcriptome analysis of ECs of Villincre:R26-tdTomato mice.

Fig. S3. Lack of neutrophil infiltrates in the lamina propria of Cx3cr1cre:Il10rafl/fl:Il23afl/fl mice.

Fig. S4. Macrophage-derived Il23a is critical for colitis induction in the BM chimera model.

Fig. S5. Chimerism of blood monocytes and T cells.

Fig. S6. Analysis of NEi-treated BM chimeras.

Fig. S7. Analysis of transcriptomes of macrophages retrieved from BM chimeras treated with control IgG or neutrophil-depleting anti-Ly6G antibody.

Fig. S8. Summary schematic illustrating the finding of this study.

Data file S1.

Data file S2.


Acknowledgments: We would like to thank all members of the Jung Laboratory, as well as E. Zigmond and S. Yona for helpful discussion, A. Brauner for bioinformatic analysis, and G. Bar-Eli and I. Shteinberg for technical help. We also thank the staff of the Weizmann Animal facility, FACS facility, and INCPM for expert advice. We thank W. Mueller for IL-10Rfl/fl mice and M. Hegen (Pfizer) and F. Powrie for providing Il-23afl/fl mice. Funding: Work in the Jung laboratory was supported by the European Research Council (AdvERC grant 340345), an ERA-NET INFECT grant, and a research grant from Roland N. Karlen Foundation. Author contributions: B.B. and S.J. conceived the project and designed the experiments; B.B., M.G.-V., M.K., C.A.T., and C.C. performed the experiments; B.B. and E.D. performed the statistical analysis; L.C.-M. performed the RNA-seq, and E.D. analyzed the data. A.H. and E.E. advised on experiments; P.T. provided a critical tool; M.I., Q.W., and V.P. provided neutrophil data and expertise; B.B. and S.J. wrote the paper; and S.J. supervised the project. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The accession codes for the RNA-seq datasets reported in this paper can be found at GEO: GSE129215 (macrophage data) and GSE129212 (epithelial cell data). Additional data that support the findings of this study are available as source data in the Supplementary Materials. Other data are available from the corresponding author upon request. The mouse strains used in this study are available from the Jackson mouse repository or the authors upon request.

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