Th17 Immunity in the Colon Is Controlled by Two Novel Subsets of Colon-Specific Mononuclear Phagocytes

Abstract

Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14⁺CD24⁺ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14⁺CD24⁺ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.

Keywords: Th17 immunity; colon; dendritic cells; intestine; macrophages; small intestine.

Figure 1

Identification of a novel population of CD14⁺CD24⁺CD88^– mononuclear phagocytes that reside in colon and are exclusive to this intestinal organ. Representative plot gated on live MHC-II⁺ APCs from small intestine lamina propria (SI-LP) (A) or colon lamina propria (C-LP) (B). (C, D) CD11b/CD11c⁺ APCs from SI-LP (C) gated in (A) or C-LP (D), gated in (B) evaluated for CD14 and CD24 expression. (E, F) CD14⁺ populations from SI-LP (E) gated in (C) or C-LP (F) gated in (D) evaluated for CD24 and CD64 expression. (G) The CD64^negative population from C-LP [gated in (F)] evaluated for Ly6C expression. (H) The Ly6C^– population gated in (G) evaluated for CD11b and CD11c expression. (I) Mean fluorescence intensity (MFI) of surface CD88 expressed by SI-LP macrophages and each of the three cDC subsets (See Figure S1 for macrophage and cDC gating strategy). Each dot represents one mouse. (J) Representative plot gated on total CD11b⁺CD24^– cells from SI-LP depicting populations representing the developmental transition of monocytes into intestinal macrophages (termed the monocyte-waterfall): (I) Ly6C⁺MHC-II^– monocytes; (II) Ly6C⁺MHCII⁺ transitioning monocytes; (III) Ly6C^–MHCII⁺ mature macrophages. (K, L) Gated populations shown in (J) and total cDCs were evaluated for CD88 (K) and CD64 expression (L): Dashed line, cDCs; (I) Green, monocytes; (II) Red, transitioning monocytes; (III) Gray, mature macrophages. (M) SI-LP APCs gated as in (A) were evaluated for CD14 and CD24. (N) CD14⁺CD24⁺ populations gated in (M) were evaluated for Ly6C. (O) Ly6C^– populations gated in (N) were evaluated for CD64 and CD88. (P) Contour plot overlay of SI-LP CD14⁺CD24⁺Ly6C^– populations (red, gated in (N)) and SI-LP cDCs (grey). (Q–T) C-LP APCs were gated and analyzed as in (M–P). (U–V) Representative plots of total live MHC-II⁺CD11b/CD11c⁺CD14⁺Ly6C^– APCs from SI-LP (U) or C-LP (V) evaluated for CD88 and CD24 expression (See Figure S3 for gating strategy). (W, X) C-LP mononuclear phagocytes of the indicated subset were co-cultured 1:1 with naïve OT-II CD4 T cells for 4 days. (W) T cells from each co-culture were restimulated and evaluated for the percentage of IL-17⁺ OT-II T cells. (X) The abundance of IL-17 protein in co-culture supernatant prior to stimulation was analyzed in parallel. Data in (W, X) is combined from three independent experiments each containing sorted APCs from at least 5 mice. Each dot represents one experiment or replicates within an experiment. Error bar represents mean ± SEM. Data in (A–V) is representative of >40 individual mice. ***p < 0.001, ****p < 0.0001, n.s., not significant (one-way ANOVA with Tukey’s post hoc test).

Figure 2

Colon-specific CD14⁺CD24⁺CD88^– APCs are composed of a CD26⁺CX3CR1^int/neg cDC subset and an atypical CD26^–CX3CR1^– macrophage subset. (A) Representative histogram of CX3CR1-GFP expression by C-LP macrophages and CD14⁺CD24⁺CD88^– APCs (gated as in Figure 1V ). Dashed line represents GFP expression by the respective populations from wild-type, GFP negative control mice. (B) Representative plot of CD26 expression on C-LP CD14⁺CD24⁺ CD88^– APCs. (C) Comparative CX3CR1-GFP expression by the indicated C-LP population. (D) Representative light microscopy image of sorted cells from the indicated C-LP mononuclear phagocyte subset. Data is representative of two independent experiments. (E, F) Absolute cell number of the indicated subset of C-LP mononuclear phagocyte (E), and macrophages (F) in Flt3l ^+/+ and Flt3l ^–/– mice. Data was combined from two independent experiments. (G–I) Hematopoietic chimera reconstituted with Zbtb46-DTR bone marrow were injected with diphtheria toxin (DT) to deplete Zbtb46-expressing cells. Control chimera injected with PBS alone were analyzed in tandem. Representative flow plot gated as in Figure 1V indicating the relative abundance of CD14⁺CD24⁺CD88^– APCs (G), the relative abundance of CD26⁺ and CD26^– subset among these APCs (gated in (G, H), and the absolute number (I) of the indicated mononuclear phagocyte subset in PBS-treated or DT-treated mice. Data is representative of two independent experiments. (J) The ratio of Ccr2 ^-/-:wild-type (WT) cells within each of the indicated subset of C-LP mononuclear phagocyte from hematopoietic chimera reconstituted with a 1:1 ratio of WT and Ccr2 ^-/- bone marrow. Data is representative of two independent experiments. (K) The geometric (geo) mean of acidified Zymosan pHrodo™ bioparticles within the indicated C-LP mononuclear phagocyte population after 1h incubation at 37°C. Fluorescence from cultures incubated at 4°C was subtracted. (L) The geometric mean of CFSE within the indicated C-LP mononuclear phagocyte population after 1h incubation with irradiated, CFSE labeled apoptotic thymocytes. CFSE fluorescence from cultures incubated at 4°C was subtracted. Data in (K, L) is combined from three independent experiments. Each dot represents pooled cells from 3-5 mice. (M) Percentage of IL-17⁺ OT-II CD4 T cells, and abundance of IL-17 protein (N) in co-cultures with CD26⁺ or CD26^– subsets within CD14⁺CD24⁺CD88^– APCs, analyzed as in Figures 1W, X . Data in (M, N) is combined from four independent experiments each containing sorted APCs from at least 5 mice. Each dot represents one experiment or replicates within an experiment. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant. (Statistical analysis for (J–I) was performed using one-way ANOVA with Tukey’s post hoc test. All other analyses were performed using unpaired Student’s t test).

Figure 3

CD26^–CD14⁺CD24⁺CD88^–macrophages are an atypical macrophage subset that expresses very little IL-10, and high levels of IL-6. Expression of Il10(A), Il6 (B), Il12b (C), and Il23a (D) in the indicated subset of C-LP mononuclear phagocyte. Each data point represents expression in sorted cells pooled from at least 5 mice. (E) Unsupervised, hierarchical cluster analysis of Euclidian distance between the indicated C-LP mononuclear phagocyte subset based on the relative expression of the indicated gene. Data within each gene set is relative to Hprt and normalized to the average expression among all cell types tested. Error bars represent mean ± SEM. **p < 0.01, ****p < 0.0001, n.s., not significant (one-way ANOVA with Tukey’s post hoc test).

Figure 4

Colon-specific CD14⁺CD24⁺CD88^– DCs and macrophages are absent from Irf4-cko mice yet persist in hCD207-DTA mice. (A) Representative histogram of intracellular IRF4 protein in the indicated subset of C-LP mononuclear phagocyte. Iso, isotype control antibody staining. Data is representative of three separate experiments. (B–F) C-LP mononuclear phagocytes were analyzed from co-housed littermate Irf4-wt and Irf4-cko mice. Representative plot gated on total DCs as in Figure S1 (B) and the absolute cell number (C) of the indicated cDC subset. Representative plot gated as in Figure 1V of the abundance of CD14⁺CD24⁺CD88^– APCs (D) and the absolute cell number (E) of CD26⁺CD14⁺CD24⁺ CD88^– DCs and CD26^–CD14⁺CD24⁺ CD88^– macrophages. (F) Absolute cell number of conventional macrophages from Irf4-wt or Irf4-cko mice. Data in (B–F) was combined from three independent experiments. (G–K) C-LP mononuclear phagocytes from co-housed littermate hCD207-DTA⁺ or hCD207-DTA^– mice were analyzed as in (B–F). Data are representative of three independent experiments. Error bars represent mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant (unpaired Student’s t test).

Figure 5

Colon-specific CD14⁺CD24⁺CD88^– APCs are required for Th17 immunity in the colon. (A–E) C-LP or SI-LP CD4 T cells were analyzed from co-housed littermate Irf4-wt and Irf4-cko mice. (A) Representative plot of C-LP CD4 T cells from mice of the indicated genotype analyzed for IFNγ and IL-17 production. The percent (B) and absolute cell number (C) of C-LP Th17 cells from all mice tested. The percent (D) and absolute cell number (E) of SI-LP Th17 cells in Irf4-wt and Irf4-cko mice analyzed in (B, C). Data in (B–E) was combined from three independent experiments. (F–J) C-LP and SI-LP CD4 T cell populations were analyzed from co-housed littermate hCD207-DTA^– and hCD207-DTA⁺ mice as in (A–E). Representative plot of C-LP CD4 T cells (F), the percent (G) and absolute cell number (H) of C-LP Th17 cells, and the percent (I) and absolute cell number (J) of SI-LP Th17 cells. Data are representative of three independent experiments. Error bars represent mean ± SEM. *p < 0.05, **p < 0.01, n.s., not significant (unpaired Student’s t test).