Monocyte and M1 Macrophage-induced Barrier Defect Contributes to Chronic Intestinal Inflammation in IBD

Abstract

Background: Macrophages are key players in inflammatory bowel diseases (IBD). This study aimed to determine site-specific effects of defined macrophage subtypes on the integrity of the intestinal epithelial barrier.

Methods: Macrophage subtypes in situ in intestinal specimens of patients with IBD were visualized by immunohistochemistry. In vitro polarization of human peripheral CD14 cells yielded M1 or M2 macrophages. The influence of primary monocytes or macrophage subtypes on epithelial barrier integrity was analyzed by transepithelial resistance measurements, Western blot analysis, confocal laser scanning microscopy, and cytometric bead array in a coculture model of primary human macrophages and layers of intestinal epithelial cell lines.

Results: The lamina propria of the inflamed intestine in patients with IBD, predominantly in Crohn's disease, is massively infiltrated by CD68 cells also positive for inducible nitric oxide synthase and tumor necrosis factor (TNF) α. The presence of M1 macrophage shifted the balance in the local macrophage compartment towards a proinflammatory state. In the coculture model, monocytes and M1 macrophages reduced transepithelial resistance as a marker for epithelial barrier integrity. The mechanisms for paracellular leakage included intracellular relocalization of tight junction proteins like claudin-2 and epithelial cell apoptosis. Determined by specific cytokine blockade, M1 macrophages exerted their deleterious effect mainly through TNF-α, whereas monocyte-mediated damage was driven by the inflammasome effector cytokines, interleukin-1β and interleukin-18.

Conclusions: Lamina propria monocytes and M1 macrophages invading intestinal tissues directly contribute to disrupting the epithelial barrier through deregulation of tight junction proteins and induction of epithelial cell apoptosis, thus driving intestinal inflammation in IBD.

FIGURE 1

Monocytes and macrophage subpopulations in the LP of patients with IBD and normal intestinal tissue. Colon tissues samples from patients with CD, ulcerative colitis (UC), or normal tissue were stained by immunohistochemistry (A) for CD68 as a pan-macrophage marker, (B) the M2-macrophage markers CD163 and stabilin-1, and (C) for the M1 macrophage markers iNOS and TNF-α. Panels at the left hand side show representative images for immunohistochemical staining. Original magnification: 400-fold; bars represent 50 µm. Positive cells appear in red. Panels at the right hand side summarize the respective positive cells counted in 10 high power fields. Median and range of n = 4 to 11. D, Ratio of iNOS⁺ to CD163⁺ cells as counted in 10 high power fields; *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 2

Effect of monocytes and macrophage subpopulations on the epithelial barrier. A, Monocytes (Mo), M1 or M2 macrophages were cocultured with the epithelial cell line T84 and LPS (100 ng/mL) was added to the lower chamber. Transepithelial resistance of the epithelial cell layer was measured over time after initiation of the coculture. Mean ± SEM of n = 8. **P < 0.01, ***P < 0.001 compared with cocultures with monocytes; ###P < 0.001 compared with cocultures with M1 macrophages; †P < 0.05, ††P < 0.01. B, Monocytes (Mo), M1 or M2 macrophages were cocultured with the intestinal epithelial cell lines T84, Caco-2, or HT-29/B6. If applicable, LPS (100 ng/mL) was added to the lower chamber. Transepithelial resistance of the epithelial cell layer was measured after 54 hours of coculture and is expressed as percent of initial resistance. Mean ± SEM of n = 8 to 22. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 3

Effect of monocytic cells on tight junction proteins. After coculture with monocytes or macrophage subpopulations or without monocytic cells for control for 54 hours without LPS, claudin-1, claudin-2, JAM-A, and E-cadherin were detected in T84 cells by Western blot analysis. β-actin served as loading control. Upper panels show representative blots; lower panels show the corresponding densitometric analysis. Mean ± SEM of n = 4; **P < 0.01.

FIGURE 4

Effect of monocytic cells on the integrity of the epithelial layer. T84 cells cocultured with monocytes (Mo), M1 or M2 macrophages or without immune cells (control, ctrl) were immunostained for ZO-1 (red) and JAM-A (green). Nuclei were stained with DAPI (blue). A, Panels show collapsed z-stack projections (n = 4). In areas of interest, stainings are shown in single color mode. Bars represent 10 μm. B, Distribution of ZO-1 within the cell layer is shown in XZ sections of immunostained T84 cell layers. Upper panels show representative images. Lower panels show the corresponding quantification of ZO-1, expressed as percent of maximum intensity in thirteen horizontal sections of the epithelial cell layer (from apical to basal).

FIGURE 5

Induction of apoptosis in epithelial cells. After coculture with monocytes or macrophage subpopulation or without monocytic cells for control for 54 hours without LPS, cleaved caspase-3 and -8 were detected in T84 cells by Western blot analysis with β-actin as loading control. Staurosporine (Stp) served as positive control for apoptosis. Upper panels show representative blots; lower panels show the corresponding densitometric analysis. Mean ± SEM of n = 4; **P < 0.01.

FIGURE 6

Effect of cytokine inhibition on monocyte-/macrophage-mediated disruption of epithelial barrier. A, CD14⁺ cells cultured for 1 day (Mo; monocytes) or polarized to M1 and M2 macrophages for 6 days were subsequently stimulated with LPS (100 ng/mL) for additional 24 hours. Cytokine production was determined in supernatants by cytometric bead array. Median and range of n = 8 to 10; *P < 0.05; **P < 0.01; ***P < 0.001. B, Monocytes (Mo), M1 or M2 macrophages were cocultured with Caco-2 cells. LPS (100 ng/mL) and anti-TNF-α (10 µg/mL), a soluble IL-1R (15 µg/mL) or anti-IL-18 antibody (1 µg/mL) were present in the lower chamber. Transepithelial resistance of the epithelial cell layer after 54 hours of coculture is expressed as percent of initial resistance. Mean ± SEM of n = 5 to 12; *P < 0.05; **P < 0.01; ***P < 0.001.