Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon

Abstract

Dendritic cells (DCs) and macrophages (MPs) are important for immunological homeostasis in the colon. We found that F4/80(hi)CX3CR1(hi) (CD11b(+)CD103(-)) cells account for 80% of mouse colonic lamina propria MHC-II(hi) cells. Both CD11c(+) and CD11c(-) cells within this population were identified as MPs based on multiple criteria, including an MP transcriptome revealed by microarray analysis. These MPs constitutively released high levels of IL-10 at least partially in response to the microbiota via an MyD88-independent mechanism. In contrast, cells expressing low to intermediate levels of F4/80 and CX3CR1 were identified as DCs based on phenotypic and functional analysis and comprise three separate CD11c(hi) cell populations: CD103(+)CX3CR1(-)CD11b(-) DCs, CD103(+)CX3CR1(-)CD11b(+) DCs, and CD103(-)CX3CR1(int)CD11b(+) DCs. In noninflammatory conditions, Ly6C(hi) monocytes (MOs) differentiated primarily into CD11c(+) but not CD11c(-) MPs. In contrast, during colitis, Ly6C(hi) MOs massively invaded the colon and differentiated into proinflammatory CD103(-)CX3CR1(int)CD11b(+) DCs, which produced high levels of IL-12, IL-23, iNOS, and TNF. These findings demonstrate the dual capacity of Ly6C(hi) blood MOs to differentiate into either regulatory MPs or inflammatory DCs in the colon and that the balance of these immunologically antagonistic cell types is dictated by microenvironmental conditions.

Figure 1.

Definition and characterization of four DC/MP subsets in the steady-state colon. (A) Representative FACS analysis showing the gating strategy to define colonic MP and DC subsets. After pregating on CD45⁺MHC-II^hilin⁻ cells, cLP cells were subdivided into four different populations (MP subsets 1 and 2 and DC subsets 3 and 4) based on their CD11c and F4/80 expression. (B) CX3CR1/CD103 and CD11b/CD103 expression profiles on the four MP and DC populations defined in A. Plots are representative of >20 experiments with 3–10 pooled mouse colons. (C) Quantification of the four cLP MP/DC populations (defined in A), expressed as percentages of total MHC-II^hilin⁻ cells. Data are mean ± SEM of three independent experiments with at least three pooled mouse colons. (D) Electron micrographs of cLP subsets 1–4 FACS sorted from 10 pooled mouse colons. (E) Surface phenotype of subsets 1–4. Specific markers (black lines) and isotype controls (gray-filled areas) are shown. Plots are representative of at least three independent experiments with 3–10 pooled colons each. (F) Microarray analyses of the genes differentially expressed in subsets 1–4. (H) Microarray analyses of the genes differentially expressed in subset 1 versus 2. Each replicate represents data obtained for one subset FACS sorted from 10 pooled colons.

Figure 2.

Functional characterization and localization of cLP MP/DC subsets and T cell priming capacities of subsets 1–4. (A and B) Phagocytic capacities of subsets 1–4. cLP cells from C57BL/6 mice were incubated in the presence of fluorescent microbeads for 45 min and analyzed for their bead content. (A) Percentage of each subset containing at least one bead. (B) Percentage of each subset containing one bead, two beads, three beads, four beads, or more after pregating on total bead⁺ cells. Results are mean ± SD and are representative of two independent experiments with three mice each. (C) Allo-stimulatory capacities of C57BL/6 cLP subsets 1–4 and control splenic DCs and MPs co-cultured with CD4⁺ T cells from the spleen of BALB/c at the indicated ratios. (D and E) Proliferation of OT-II (D) and OT-I (E) cells after co-culture with graded doses of OVA protein–pulsed cLP subsets 1–4 and control splenic (SPL) DCs and MPs. Proliferation was determined by thymidine incorporation assay after 4 d (C and D) or 3 d (E) of culture. Results are expressed as mean ± SD of cpm triplicates and are representative of at least two independent experiments using pooled cells from 10 mice per experiment. Unpaired Student’s t tests were performed to compare subset 2 versus 3 (blue statistics) and subset 2 versus 4 (green). The comparison of subset 2 versus 1 never reached statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F) In situ expression of CD11c (red) and F4/80 (green; top and middle) or CD11c (red) and CD11b (green; bottom) on C57BL/6 mouse colon sections. Hoechst counterstain is blue. Three mice were analyzed in three separate experiments. Bars, 50 µm.

Figure 3.

Subsets 1 and 2 spontaneously release the antiinflammatory cytokine IL-10 and respond to LPS stimulation with an antiinflammatory signature. (A) Real-time RT-PCR analysis of IL-10 mRNA expression by freshly isolated FACS-sorted cLP subsets from C57BL/6 mice. Results are mean real amounts ± SD, normalized to GAPDH (1/2^ΔCt), of at least three independent experiments with 10 pooled mice in each. (B) Spontaneous release of IL-10 measured by ELISA after overnight culture of freshly isolated FACS-sorted colonic subset 1 or 2, compared with splenic (SPL) DCs, splenic MPs, or MOs. Results are mean ± SD of three experiments with 5–10 pooled mice in each. (C and D) Representative plots (C) and quantification (D) of IL-10–GFP reporter expression by cLP subsets 1–4 freshly isolated from Vert-X mice. FACS plots present the IL-10–GFP/CD11b staining profile after pregating on each individual cLP subset. Quadrant gates were set using isotype controls. For IL-10–GFP negative controls, cells were prepared from WT mice. Results are mean ± SD of three mice analyzed separately and are representative of two experiments. (E) Real-time RT-PCR analysis of cytokine mRNA expression in FACS-sorted cLP DC/MP from C57BL/6 mice. Results are represented as real amounts, normalized to GAPDH (1/2^ΔCt). Data are mean ± SEM of at least three independent sorting experiments performed in duplicate. ND, not detected. (F) Quantification of the cytokines secreted by cLP subsets 1 and 2 and control splenic MPs and DCs after 24 h of culture in media alone (open bars) or in the presence of 1 µg/ml LPS (closed bars), using the SearchLight multiplex cytokine immunoassays. Results are mean ± SD of three independent experiments with 10 pooled colons and 3 pooled spleens each. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 4.

IL-10 production by cLP subsets 1 and 2 is partially dependent on the presence of commensal bacteria and regulates the production of proinflammatory cytokines by cLP DC/MP subsets. (A) Absolute numbers of cLP subsets 1–4 in WT and GF mice. (B) Real-time RT-PCR analysis of IL-10 mRNA expression by cLP subsets 1 and 2 FACS sorted from GF, MyD88^−/−, or WT mice. Results are mean ± SD of real amounts normalized to GAPDH (1/2^ΔCt) and are representative of at least two experiments with five mice each. (C) Real-time RT-PCR analysis of IL-12p35, IL-23p19, and IL-6 mRNA expression in cLP subsets 1–4, FACS-sorted WT (open bars), or IL-10^−/− mice (closed bars) at 6 wk of age. Results are mean ± SD of real amounts normalized to GAPDH (1/2^ΔCt) and are representative of two experiments with 10 mice each. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results are mean ± SD of three experiments with three to five pooled colons per group.

Figure 5.

MOs generate mostly the immunoregulatory cLP subset 2 after adoptive transfer into CD11cDTR → C57BL/6 bone marrow chimera. (A) FACS strategy for the sorting of CD115⁺lin⁻ (CD3, CD4, CD8α, NK1.1, B220, CD11c, CD117 [c-kit]) Ly6C^hiCD11b⁺ bone marrow of MOs. (B) Postsorting flow cytometry plots. Data are representative of at least four experiments with 10 pooled bone marrows. (C) FACS analysis of the sorted MO surface phenotype. Plots are representative of three independent experiments with 10 pooled bone marrows. (D) Schematic diagram depicting the strategy and time points used for the depletion of CD11c⁺ cells in CD11cDTR → C57BL/6 bone marrow chimeric mice, the adoptive transfer of purified CD45.1⁺ MOs, and the analysis of MO progeny in the colon. (E and F) FACS analysis of colonic cells from recipient CD11cDTR → C57BL/6 chimera at day 8 and 15 after i.v. graft of 2–3 × 10⁶ purified CD45.1⁺ bone marrow MOs. A nongrafted chimera is shown as a negative control (w/o graft). Data are representative of at least two separate experiments with three MO-grafted mice and controls. (G) Real-time RT-PCR analysis of cytokine mRNA expression in CD45.1⁺ (MO derived) colonic cells FACS sorted from CD11cDTR → C57BL/6 chimera 7 d after MO graft. Results are mean real amounts (1/2^ΔCt) ± SD, have been normalized to GAPDH, and are representative of two experiments with five to six MO-transferred mice each.

Figure 6.

MOs generate an inflammatory CD103⁻ DC subset 3 after adoptive transfer into RAG^−/− colitic mice. (A) Strategy used for the induction of colitis in Rag^−/− mice, the adoptive transfer of purified MOs, and the analysis of MO progeny in the colon. (B) Quantification of the total number of colonic CD45⁺ and MHC-II^hi cells expressed as fold change in CD45RB^hi (colitic) versus CD45RB^hi+lo (control) animals. Results are mean ± SD of three independent experiments with at least five mice per group. (C and D) Representative FACS plots (C) and quantification (D) of subsets 1, 2, 3 CD103⁻, 3 CD103⁺, and 4 in the colon of colitic versus control mice 7 wk after transfer of CD45RB^hi or CD45RB^hi+lo CD4⁺ T cells, respectively. Results are mean ± SD of three independent experiments with at least five mice per group. *, P < 0.05; ***, P < 0.001. (E) CD11b/CD103 expression on subset 3 in colitic versus control mice. (F and G) Analysis of the colon of colitic mice 3 d after engraftment or not with 2–3 × 10⁶ purified bone marrow MOs. The data presented are representative of at least two separate experiments with three MO-transferred mice each. (H) Real-time RT-PCR analysis of cytokine mRNA expression in CD45.1⁺ (MO derived) colonic cells FACS sorted from recipient colitic mice 3 d after adoptive transfer of 3 × 10⁶ bone marrow MOs (closed bars). The mRNA profile of MO-derived cells sorted from noncolitic CD11cDTR → C57BL/6 chimeric mice (data duplicated from Fig. 5 G) has been plotted here for comparison purposes (open bars). Results are mean ± SD of two experiments with five to seven pooled colons/group.

Figure 7.

CD103⁻ subset 3 exhibits potent T cell priming capacities and induces the differentiation of IFN-γ–producing T cells in vitro. (A) Proliferation of OT-II cells after co-culture with graded doses of OVA protein–pulsed cLP subsets 1, 2, 3 CD103⁻, 3 CD103⁺, and 4 and control splenic (SPL) DCs and MPs. Proliferation was determined by thymidine incorporation assay after 4 d of culture. Data are mean ± SD and are representative of two experiments performed in triplicates. Subset 3 CD103⁻ was compared with subset 1 or 2 and splenic MPs using an unpaired Student’s t test, and the less significant p-values were plotted (black statistics). Subset 3 CD103⁻ was also compared with subset 3 CD103⁺ (green statistics), subset 4 (violet statistics), and splenic DCs (orange statistics). foxp3, IFN-γ, and IL-17A intracellular FACS staining in OT-II cells co-cultured for 5 d with FACS-sorted OVA-loaded colonic subsets 1, 2, and 3 (CD103⁻ or CD103⁺) from WT mice. (B) Representative plots. (C) Quantification of the absolute numbers of live foxp3⁺, IFN-γ⁺, and IL-17A⁺ OT-II cells/well in the co-cultures. (D) Quantification of IFN-γ and IL-17A production by OT-II cells after 4.5 d of culture. (C and D) Data are mean ± SD of two separate experiments performed in triplicates. *, P < 0.05; **, P < 0.01; ***, P < 0.001.