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. 2014 Jul 28;211(8):1571-83.
doi: 10.1084/jem.20140678. Epub 2014 Jul 14.

CX₃CR1⁺ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

Randy S Longman  1 , Gretchen E Diehl  2 , Daniel A Victorio  3 , Jun R Huh  2 , Carolina Galan  2 , Emily R Miraldi  4 , Arun Swaminath  5 , Richard Bonneau  6 , Ellen J Scherl  7 , Dan R Littman  8
Affiliations

CX₃CR1⁺ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

Randy S Longman et al. J Exp Med. .

Abstract

Interleukin (IL)-22-producing group 3 innate lymphoid cells (ILC3) promote mucosal healing and maintain barrier integrity, but how microbial signals are integrated to regulate mucosal protection offered by these cells remains unclear. Here, we show that in vivo depletion of CX₃CR1⁺ mononuclear phagocytes (MNPs) resulted in more severe colitis and death after infection with Citrobacter rodentium. This phenotype was rescued by exogenous IL-22, which was endogenously produced by ILC3 in close spatial proximity to CX₃CR1⁺ MNPs that were dependent on MyD88 signaling. CX₃CR1⁺MNPs from both mouse and human tissue produced more IL-23 and IL-1β than conventional CD103(+) dendritic cells (cDCs) and were more efficient than cDCs in supporting IL-22 production in ILC3 in vitro and in vivo. Further, colonic ILC3 from patients with mild to moderate ulcerative colitis or Crohn's disease had increased IL-22 production. IBD-associated SNP gene set analysis revealed enrichment for genes selectively expressed in human intestinal MNPs. The product of one of these, TL1A, potently enhanced IL-23- and IL-1β-induced production of IL-22 and GM-CSF by ILC3. Collectively, these results reveal a critical role for CX₃CR1⁺ mononuclear phagocytes in integrating microbial signals to regulate colonic ILC3 function in IBD.

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Figures

Figure 1.
Figure 1.
Intestinal CX3CR1+ cells protect mice from C. rodentium-induced colitis. (A and B) Depletion efficiency in the Cx3cr1DTR/GFP mice was assessed by flow cytometry of small intestinal lamina propria cells Cx3crDTR/GFP or littermate control mice after administration of DT to both groups daily for 2 d. (A) Surface staining for CD11b versus CX3CR1-GFP. (B) MHCII+ CD11c+ cells were assessed for expression of CX3CR1, CD103, and CD14. Results are representative of five independent experiments with a minimum of three animals per group. (C) Total number of MHCII+ CD11c+CD103+ (left) and MHCII+ CD11c+CX3CR1+ cells (right) per intestine as determined by flow cytometry analysis. n.s., P > 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent the SEM. Results are representative of five independent experiments with a minimum of 3 animals per group. (D) Weight of DT-treated littermate WT (control) mice or Cx3cr1DTR/+ mice following infection with C. rodentium (n = 7 mice/group). DT was administered at days −2, −1, and 0 and every other day after infection. Data are representative of two independent experiments. (E) Representative colonic histology from littermate control mice or Cx3cr1DTR/+ mice (analyzed in D) infected with C. rodentium at day 7 after infection. <, areas of lymphocyte infiltration; *, areas of epithelial erosion. Bar, 100 µm. (F) Survival curves of DT-treated Cx3cr1DTR/+ and littermate control mice infected with C. rodentium or uninfected (n = 7 mice/group). Data are representative of three independent experiments. Animals were treated with DT as in D. (G) Bacterial CFUs of spleens from littermate WT (control, n = 5) mice or Cx3cr1DTR/+ mice (n = 5) infected with C. rodentium and treated with DT as above. *, P ≤ 0.05. Two-tailed Student’s t test. Error bars represent the SEM. One of two representative experiments is shown.
Figure 2.
Figure 2.
CX3CR1+ cells support colonic ILC3 production of IL-22. (A) Survival curves of C. rodentium–infected Myd88f/f littermate controls (n = 10, open circle) as compared with CD11c-cre/Myd88f/f mice without (n = 13, filled circle) or with (n = 5, open triangle) exogenous hydrodynamic delivery of an IL-22–producing plasmid. Results are a composite of two independent experiments. (B) Survival curves of DT-treated Cx3cr1DTR/+ mice infected with C. rodentium after hydrodynamic delivery of a plasmid expressing IL-22 (n = 8) or control vector (n = 9). DT was administered at days −2, −1, and 0 and every other day after infection. Results are a composite of three independent experiments. (C–E) Percentage (C and D) and total number (E) of colonic Lin− CD90.2+ ILCs producing IL-22 from DT-treated Cx3cr1DTR/+ (n = 10) and littermate control mice (n = 9) 7 d after C. rodentium infection and from uninfected mice (NT; n = 3). Results are a composite of two independent experiments. DT was administered at days −2, −1, and 0 and every other day after infection. Intracellular IL-22 was assayed by flow cytometry after 4-h culture. A representative flow cytometry plot from each group is shown in C. **, P ≤ 0.01. One way ANOVA with Bonferroni’s correction. Error bars represent the SEM. (F) Total number of ILC3 per colon in Cx3cr1DTR/+ (n = 9) or control (n = 8) mice administered DT. Error bars represent the SEM. Results are one of three representative experiments. (G) Total number of IL-17+ or IL-22+ colonic CD4+ T cells from DT-treated Cx3cr1DTR/+ (n = 10) and littermate control mice (n = 9) 7 d after C. rodentium infection and from uninfected mice (n = 3). Intracellular IL-22 and IL-17 was assayed by flow cytometry after 4-h culture. *, P ≤ 0.05. One way ANOVA with Bonferroni’s correction. Error bars represent the SEM. Results are a composite of two independent experiments. (H) Confocal immunofluorescence of colonic samples from Cx3Cr1GFP/+ mice stained for CD3 and RORγt. Bar, 10 µm. White arrows indicate sites of MNP and ILC3 juxtaposition.
Figure 3.
Figure 3.
TLR-stimulated CX3CR1+ MNPs are stronger inducers of ILC3 production of IL-22 than CD103+ CD11b+ DCs and monocytes. (A–C) CD103 or CX3CR1+ MHCII+ CD11c+ CD11b+ cells were isolated from the lamina propria of CX3CR1GFP/+ mice (sort strategy shown in A and co-cultured with Lin− RORγt-GFP+ ILCs with or without the indicated bacterial TLR ligands or IL-23. IL-22 was assessed by intracellular staining of CD90.2+ ILCs after 18 h. A representative flow cytometry plot is shown in B. (C) Percent IL-22+ ILCs is shown from seven independent experiments. **, P ≤ 0.01; *, P ≤ 0.05. One way ANOVA with Bonferroni’s correction. Error bars represent SEM. (D–F) Ly6C+ MHCIIlo (monocytes) and Ly6C− MHCIIhi (MNPs) were isolated from CX3CR1+ CD11b+ lamina propria cells (sort strategy is shown in D and co-cultured with intestinal ILCs with LPS or IL-23 as indicated. Intracellular cytokine staining for IL-22 is shown after 18 h (E). Supernatants were harvested after 18 h and IL-22 production quantitated by ELISA (F). Results are representative of two independent experiments performed in triplicate. ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.
Figure 4.
Figure 4.
CX3CR1+ MNP-derived IL-23 and IL-1β activate ILC3 to produce IL-22. (A) Phenotype analysis of colonic LPMCs from Cx3cr1STOP-DTR/GFP mice with or without CD11c-cre after DT injection for 2 d. (top) Selective depletion of CX3CR1hi MNPs. (bottom) Expression of Ly6C and MHCII on CX3CR1hi and CX3CR1int populations. (B) Expression of IL-22 in Lin− CD90.2+ colonic ILCs from Cx3cr1STOP-DTR/+ (Stop-DTR) or CD11c-Cre x Cx3cr1STOP-DTR/+ (Cre-DTR) mice at 7 d after C. rodentium infection. DT was administered at days −2, −1, and 0 and every other day postinfection. One representative intracellular cytokine flow cytometry plot is shown on the left and a composite graph (n = 6/group) on the right. *, P ≤ 0.05, two-tailed Student’s t test. Error bars represent the SEM. Results are a composite of two independent experiments. (C) Supernatants from APC-ILC co-cultures (Fig. 3, B and C) were harvested after 18 h and assayed for IL-23 by ELISA. Results are averages of three independent experiments and the SEM is shown. (D) CX3CR1+ MNPs or CD103+ CD11b+ DCs were sorted and incubated with media or CpG for 18 h and supernatants were assayed for IL-1β by ELISA. Results are the mean of two independent experiments performed in duplicate and the SEM is shown. *, P ≤ 0.05; **, P ≤ 0.01. (E) Lin− CD90.2hi ILCs from WT or Il1r−/− mice were co-cultured with sorted intestinal MNPs from WT or Il23p19−/− mice, with or without CpG, as indicated. IL-22 production by the ILCs was assessed after 18 h by ELISA. Data are combined from three independent experiments performed in duplicate. *, P ≤ 0.05; ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.
Figure 5.
Figure 5.
Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from descending colon biopsies from patients with endoscopically mild to moderate Crohn’s’ disease (n = 8, gray) or ulcerative colitis (n = 6, black; Table S1), as well as age-matched non-IBD control patients undergoing routine screening colonoscopy (n = 8, white), were stimulated ex vivo with PMA/ionomycin and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3+ or CD3− fraction expressing IL-17 or IL-22 is indicated. *, P ≤ 0.05, two-tailed Student’s t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3− IL-22+ (black lines) and CD3− IL-22− (gray) LPMCs. (C) Expression of RORγt by c-Kit+CD56+ LPMCs. Lin− cells (CD14/CD19/CD3/CD11b/CD11c/TCRγδ−; Fig. S3 A) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORγt. Lin− CD56+ c-Kit+ ILC3 (black line) were compared with Lin− CD56+ c-Kit− NK cells (gray) for RORγt expression. (D) Surface staining of Lin− c-Kit+ ILCs for the indicated markers (black line) compared with isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with control media (dotted line). Cells shown were gated on Lin− CD56+ c-Kit+. The isotype control is in gray. (F) CD11c+ MHCII+ human colonic APCs were electronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed (prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry. (left) Result from one representative donor. (right) Percentage of IL-22+ ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients. **, P ≤ 0.01, two-tailed Student’s t test. Black bars represent the geometric mean.
Figure 6.
Figure 6.
Human ILC3 production of IL-22 is supported by IL-23 and IL-1β produced by TLR-stimulated CD14+ and CX3CR1+ MNPs. (A–C) HLA-DR+ CD11c+ cells from intestinal resection tissue were sorted into CD103+ DCs and CD14+ MNPs subpopulations and transcriptional profiles were assessed by RNA-seq. (A) Sorting strategy. (B) Each subset was examined for expression of the indicated cell surface markers. Isotype controls are shown in gray. One of three donors is shown. (C) Heatmap of relative expression of relevant MNP-related genes. Values represent the mean of two independent donors, and an asterisk denotes individual genes differentially expressed at an FDR = 0.01. (D and E) Induction of IL-22 in human ILCs in co-culture with CD14+ MNPs or CD103+ DCs in the presence of media alone, LPS, or flagellin, as indicated. c-Kit+ cells were examined for intracellular IL-22 production after 18-h culture. Data are representative of five independent experiments. (F) CD14+ MNPs or CD103+ DCs sorted from human intestine (as in A) were stimulated with the indicated TLR ligands for 18 h and qPCR or cytometric bead array analysis were used to quantitate IL-23p19 and IL-1β, respectively. Results are averaged from three independent donors, and technical replicates were performed in duplicate or triplicate, respectively. *, P ≤ 0.05. N.D., not detected. Two-tailed Student’s t test. Error bars represent the SEM. (G) Sorted human intestinal CD14+ MNPs and ILCs were left unstimulated or were co-cultured in the presence of LPS with or without neutralizing antibodies against IL-1β and IL-23. IL-22 ELISA was performed after 18h. Results are averaged from two independent donors performed in duplicate. *, P ≤ 0.05. Two-tailed Student’s t test. Error bars represent the SEM.
Figure 7.
Figure 7.
CX3CR1+ MNP-derived TL1A synergizes with IL-23 and IL-1β to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis was used to determine whether the indicated disease-related SNP were differentially expressed between CD14+ MNPs and CD103+ DCs. Significance was estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two independent donors. (B) B cells (CD3− CD19+), CX3CR1+ MNPs, CD103+ DCs, and Ly6C+ monocytes were sorted from the intestinal lamina propria of Cx3cr1GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by ΔCt normalized to GAPDH expression. Data are from two biological replicates performed with two technical replicates. *, P ≤ 0.05. Two-tailed Student’s t test. (C–E) Sorted intestinal ILCs from mice (C and E) or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1β, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments. (F–H) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured with media alone (-) or IL-23 and TL1A or co-cultured with CX3CR1+ MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin. *, P ≤ 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent SEM.
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