Unique CD14 intestinal macrophages contribute to the pathogenesis of Crohn disease via IL-23/IFN-gamma axis

Abstract

Intestinal macrophages play a central role in regulation of immune responses against commensal bacteria. In general, intestinal macrophages lack the expression of innate-immune receptor CD14 and do not produce proinflammatory cytokines against commensal bacteria. In this study, we identified what we believe to be a unique macrophage subset in human intestine. This subset expressed both macrophage (CD14, CD33, CD68) and DC markers (CD205, CD209) and produced larger amounts of proinflammatory cytokines, such as IL-23, TNF-alpha, and IL-6, than typical intestinal resident macrophages (CD14-CD33+ macrophages). In patients with Crohn disease (CD), the number of these CD14+ macrophages were significantly increased compared with normal control subjects. In addition to increased numbers of cells, these cells also produced larger amounts of IL-23 and TNF-alpha compared with those in normal controls or patients with ulcerative colitis. In addition, the CD14+ macrophages contributed to IFN-gamma production rather than IL-17 production by lamina propria mononuclear cells (LPMCs) dependent on IL-23 and TNF-alpha. Furthermore, the IFN-gamma produced by LPMCs triggered further abnormal macrophage differentiation with an IL-23-hyperproducing phenotype. Collectively, these data suggest that this IL-23/IFN-gamma-positive feedback loop induced by abnormal intestinal macrophages contributes to the pathogenesis of chronic intestinal inflammation in patients with CD.

Figure 1. CD14-expressing cells were increased in the intestinal mucosa of patients with IBD.

(A) LP macrophages of normal intestinal tissue specimens and of patients with active IBD were analyzed by FACS for CD14 and CD33 cell-surface expression. Numbers indicate the percentage of CD14⁺ cells present in human tissue. (B) Percentage of CD14⁺ intestinal macrophages among CD33⁺ cells from normal control subjects, noninflamed mucosa of patients with UC (UCn), inflamed mucosa of patients with UC (UCi), noninflamed mucosa of patients with CD (CDn), and inflamed mucosa of patients with CD (CDi). *P < 0.05, ***P < 0.001. (C) Fluorescence microscopy of human intestine from CD patients stained with anti-CD14 (green), anti-CD68 (red), and DAPI (blue). CD14⁺CD68⁺ macrophages were present in the intestinal LP. CD14^–CD68⁺ macrophages were also observed. (D) Sorted CD14⁺CD33⁺ intestinal macrophages were analyzed for the expression of CD14 and CD33, and these cells were reseeded and analyzed for CD68. Numbers indicate the percentage of CD14⁺ cells per sorted cells. Scale bar: 20 μm.

Figure 2. CD14⁺CD33⁺ cells in the human intestinal LP revealed unique phenotypes and produced larger amounts of proinflammatory cytokines than CD14^–CD33⁺ intestinal macrophages.

(A) Flow cytometry for the surface phenotypes of intestinal CD14⁺CD33⁺ and CD14^–CD33⁺ cells. The shaded histogram shows the profiles of the indicated Ab staining and the open histogram shows staining with isotype controls. The data shown are representative of 5 independent experiments on normal control subjects or noninflamed mucosa of CD patients. (B) Proinflammatory cytokine production by E. coli or E. faecalis heat-killed antigen-stimulated CD14⁺CD33⁺ or CD14^–CD33⁺ intestinal macrophages from the inflamed mucosa of CD patients. Control stimulation used is cell culture medium alone. N.D., not detected. Data represent mean ± SEM from at least 3 independent experiments.

Figure 3. CD14⁺ intestinal macrophages from patients with CD produced abundant levels of IL-23 and TNF-α in response to commensal bacteria antigen stimulation.

(A) Quantitative RT-PCR of basal mRNA expression levels in isolated CD14⁺ macrophages from normal and IBD patients. (B) Cytokine production by CD14⁺ intestinal macrophages stimulated by heat-killed E. coli or E. faecalis (1 × 10⁸ CFU/ml) for 24 hours. (C) Quantitative RT-PCR of IL-12–related cytokines by CD14⁺ intestinal macrophages stimulated by heat-killed E. coli or E. faecalis (1 × 10⁸ CFU/ml) for 24 hours. All CD14⁺ macrophages used in this experiment are from inflamed mucosa of IBD patients and noninflamed mucosa of normal control subjects. Data are expressed as mean ± SEM of individual patients or controls (normal, n = 9; UC, n = 9; CD, n = 13). Statistical analysis was performed using Kruskal-Wallis 1-way ANOVA and the Tukey-Kramer test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal control subjects; ^#P < 0.05, ^##P < 0.01 versus UC.

Figure 4. Commensal bacteria induced IFN-γ but not IL-17 by LPMCs via IL-23 produced by CD14⁺ intestinal macrophages.

(A) Th1 and Th17 cytokine production by LPMCs (1 × 10⁶ cells/ml) treated with heat-killed E. faecalis antigen for 24 hours. Data represent mean ± SEM (normal control, n = 5; UC, n = 8; CD, n = 8). LPMCs of IBD patients were isolated from inflamed mucosa both in UC and CD. Statistical analysis was performed using Kruskal-Wallis 1-way ANOVA and the Tukey-Kramer test for multiple comparisons. *P < 0.05, **P < 0.01 versus normal control; ^#P < 0.05, ^###P < 0.001 versus UC; ^ζP < 0.05, ^ζζP < 0.01, ^ζζζP < 0.001 versus unstimulated controls. (B) Th1- and Th17-related cytokine mRNA expression after commensal bacteria stimulation (12 hours) by LPMCs from inflamed mucosa of patients with CD. Data represent mean ± SEM of at least 6 individuals. p35, IL12p35; p40, IL12/IL23p40; p19, IL23p19; p28, IL27p28. Statistical analysis was performed using Wilcoxon test. *P < 0.05, **P < 0.01. (C) Basal and commensal-induced cytokine production by LPMCs or CD14⁺ cells depleted LPMCs from inflamed mucosa of patients with CD. Data represent mean ± SEM from 6 independent experiments.

Figure 5. Intestinal macrophage-derived IL-23 induced IFN-γ production by LPMCs, and LP CD4⁺ T cells synergize with TNF-α in patients with CD.

(A) IL-23–induced proinflammatory cytokine production by LPMCs from normal control subject or inflamed mucosa of patients with IBD. Data represent mean ± SEM (normal control, n = 5; UC, n = 8; CD, n = 8). *P < 0.05 versus normal control; ^##P < 0.01 versus UC; ^ζP < 0.05, ^ζζP < 0.01 versus unstimulated controls. (B) Synergistic effect of TNF-α and IL-6 on the IL-23–induced IFN-γ production by LPMCs and LP CD4⁺ T cells from inflamed mucosa of patients with CD. Data represent mean ± SEM from 4 individuals. (C) Analysis of the suppressive effect of anti-p40 or anti–TNF-α Abs on the commensal bacteria-induced IFN-γ production by LPMCs from inflamed mucosa of CD patients. α-p40 Ab, α–IL-12/IL-23p40 Ab. Data represent mean ± SEM from at least 4 individuals. Statistical analysis was performed using Kruskal-Wallis 1-way ANOVA and the Tukey-Kramer test for multiple comparisons.

Figure 6. The intestinal inflammatory microenvironment affects macrophage differentiation and induces an IL-23–producing phenotype.

(A) Morphological findings of in vitro–differentiated macrophages from peripheral CD14⁺ monocytes of normal controls with or without LPMC-CM. Scale bar: 20 μm. (B) Flow cytometry for the surface phenotypes of LPMC-CM–induced in vitro–differentiated macrophages. The shaded histogram shows profiles of indicated Ab staining and the open histogram shows staining with isotype controls. The data shown are representative of 5 independent experiments. (C) Cytokine production by LPMC-CM–induced in vitro–differentiated macrophages stimulated with heat-killed E. coli for 24 hours. Data represent mean ± SEM from 6 independent experiments. (D) Cytokine production by macrophages differentiated from normal and CD monocytes with or without UC- and CD-CM. Data represent mean ± SEM from 5 independent experiments. All data used at least 3 different CM from individual patients and at least 3 different monocytes from individual patients and controls. Statistical analysis was performed using Kruskal-Wallis 1-way ANOVA and the Tukey-Kramer test for multiple comparisons. **P < 0.01, ***P < 0.001 versus M-CSF induced macrophages; ^#P < 0.01, ^##P < 0.01 comparison between normal control monocytes and monocytes from CD patients.

Figure 7. IFN-γ in CD-CM promotes IL-23–hyperproducing proinflammatory macrophage differentiation.

(A) Quantification of IFN-γ and IL-6 in LPMC-CM. Data are shown as mean ± SEM from 3 individual normal controls and 4 individual patients with IBD used for macrophage differentiation experiments. (B) Flow cytometry for the surface phenotypes of IFN-γ–induced in vitro–differentiated macrophages. The shaded histogram shows the profiles of the indicated Ab staining and the open histogram shows staining with isotype controls. (C) Production of IL-12/IL-23p40 and IL-23 by bacteria-stimulated macrophages differentiated with or without IFN-γ. Data represent mean ± SEM from 3 independent experiments. (D) Effect of IFN-γ signal blocking using anti–IFN-γ Ab (α-IFNγ Ab) (1 μg/ml) combination with anti–IFN-γ receptor 1 Ab (α-IFNγR Ab) (10 μg/ml) or same amount of those isotype controls (mouse IgG; mouse IgG_2A for α-IFNγ Ab, and mouse IgG₁ for α-IFNγR Ab) from CD-CM on macrophage differentiation. Statistical analysis was performed using paired t test. Data represent mean ± SEM from 5 independent experiments. *P < 0.05 compared with controls.