Impaired resolution of inflammation in the Endoglin heterozygous mouse model of chronic colitis

Abstract

Endoglin is a coreceptor of the TGF-β superfamily predominantly expressed on the vascular endothelium and selective subsets of immune cells. We previously demonstrated that Endoglin heterozygous (Eng (+/-)) mice subjected to dextran sulfate sodium (DSS) developed persistent gut inflammation and pathological angiogenesis. We now report that colitic Eng (+/-) mice have low colonic levels of active TGF-β1, which was associated with reduced expression of thrombospondin-1, an angiostatic factor known to activate TGF-β1. We also demonstrate dysregulated expression of BMPER and follistatin, which are extracellular regulators of the TGF-β superfamily that modulate angiogenesis and inflammation. Heightened colonic levels of the neutrophil chemoattractant and proangiogenic factor, CXCL1, were also observed in DSS-treated Eng (+/-) mice. Interestingly, despite increased macrophage and neutrophil infiltration, a gut-specific reduction in expression of the key phagocytic respiratory burst enzymes, NADPH oxidase 2 (Nox-2) and myeloperoxidase, was seen in Eng (+/-) mice undergoing persistent inflammation. Taken together, these findings suggest that endoglin is required for TGF-β superfamily mediated resolution of inflammation and fully functional myeloid cells.

Figure 1

Persistent colonic inflammation in DSS-treated Eng ^+/− mice is associated with changes in a major gut bacterial group. (a) Eng ^+/+ and Eng ^+/− mice were given a 3% DSS solution orally for 5 days (red), followed by return to normal drinking water (blue). The peak of inflammation occurred at days 7–9 for both genotypes; however, by days 18–23, Eng ^+/− mice showed signs of persistent inflammation, while Eng ^+/+ mice were undergoing resolution of inflammation. (b) Representative hematoxylin and eosin stained distal colonic sections from colitic mice illustrate massive infiltration of leukocytes in the lamina propria (black arrows) in both Eng ^+/+ and Eng ^+/− mice during the acute phase (day 9). Eng ^+/− mice show persistent leukocyte infiltration and incomplete crypt regeneration at day 19, whereas Eng ^+/+ mice show minimal signs of inflammation. All images are at the same magnification (Bar = 100 μm). (c) Relative abundance of various gut bacterial groups, Bacteroides, Clostridium coccoides (Cluster XIVa), and Clostridium leptum (Cluster IV), against total eubacteria in Eng ^+/+ and Eng ^+/− mice at days 18–23 was determined by real-time polymerase chain reaction (PCR) using group-specific 16S rRNA primers. Results represent mean ± SEM (N = 4 mice for Eng ^+/+group and 6 for Eng ^+/− group). *P < 0.05 versus corresponding Eng ^+/+ group.

Figure 2

Reduced active TGF-β1 levels and altered expression of factors that regulate angiogenesis and resolution in Eng ^+/− mice undergoing persistent inflammation. (a) Endogenously active (without acid treatment) and total (with acid treatment) TGF-β1 levels were measured by ELISA in colonic tissue of Eng ^+/+ and Eng ^+/− mice at days 0 and 18–23 (N = 6 mice for day 0 and 9-10 mice for days 18–23). ∗P < 0.05 versus corresponding day 0, ^† P < 0.05 versus corresponding Eng ^+/+ group. (b) The mRNA expression profile of several angiogenic and TGF-β pathway-related genes was assessed in colons of Eng ^+/+ and Eng ^+/− mice during the resolution phase. The results are expressed as a fold-change (Eng ^+/− over Eng ^+/+ mice) and only significantly (P ≤ 0.05) altered genes with a 2-fold or greater change are shown (N = 4 mice for Eng ^+/+ group and 5 for the Eng ^+/− group; BMP endothelial cell precursor-derived regulator (Bmper), follistatin (Fst), and thrombospondin-1 (Tsp-1)). (c) Representative immunoblot and densitometric analysis of TSP-1 expression, normalized to β-actin, in colons of Eng ^+/+ and Eng ^+/− mice at days 18–23 of colitis. TSP-1 levels were nondetectable at days 0 and 7–9 (N = 6 mice for Eng ^+/+ group and 5 for the Eng ^+/− group). ^‡ P = 0.072 versus Eng ^+/+ mice. Results represent mean ± SEM.

Figure 3

Higher number of infiltrating myeloid cells in colonic lamina propria of colitic Eng ^+/− mice. (a) Representative flow cytometry contour plots of colonic lamina propria cells, isolated at day 0 and day 22 of a DSS-induced colitis experiment, show the distribution of CD11b⁺F4/80⁺ and CD11b⁺F4/80⁻ cells. All plots were first gated for CD45⁺CD11b⁺ cells (not shown) and the percentage of CD45⁺ leukocytes is indicated. (b) The distribution and (c) total number of CD11b⁺F4/80⁺ cells in Eng ^+/+ and Eng ^+/− mice at days 0, 7–9, and 18–23. (d) Representative flow cytometry contour plots of colonic lamina propria cells isolated from both groups of mice at days 8 and 18 of the colitis course. Cells were initially gated for CD11b⁺F4/80⁻ cells and analyzed for neutrophils (Ly6C⁺/⁻Ly6G⁺) and monocytes (Ly6C⁺Ly6G⁻). Histograms in (e) and (f) show the number of neutrophils and monocytes per mouse, respectively. Results represent mean ± SEM (N = 3 experiments for day 0, 5 for days 7–9, and 4 for days 18–23, with 3-4 mice/experiment). *P < 0.05 versus corresponding day 0.

Figure 4

Reduced colonic MPO and Nox-2 expression in colitic Eng ^+/− mice are not associated with an intrinsic bone marrow defect. (a) Representative Western blots show colonic expression levels of MPO and Nox-2 in basal and colitic mice, normalized to β-actin (days 0 and 18–23 images are derived from a single immunoblot). Histograms illustrate densitometric analysis of (b) MPO and (c) Nox-2 expression (for MPO: N = 6–8 mice for day 0 and 4–9 mice for days 7–9 and 18–23; for Nox-2: N = 4-5 mice for day 0, 3–7 for days 7–9 and 18–23). (d) Colonic MPO enzymatic activity was assessed in Eng ^+/+ and Eng ^+/− mice at days 0, 9–12, and 19 of colitis (N = 6–8 mice for days 0, 9–12, and 19). (e) The H₂O₂ levels in colons of mice at days 0 and 18–23 were measured using an Amplex Red assay. Apocynin (APO) was utilized as an NADPH oxidase inhibitor (N = 6–8 mice for day 0 and 4–7 for days 18–23). (f) Total bone marrow cells were isolated and MPO and Nox-2 expression was assessed using Western blot analysis. Representative images show bone marrow expression levels of MPO and Nox-2 in basal and colitic Eng ^+/+ and Eng ^+/− mice, with β-actin as the normalizing factor (days 0 and 18–23 images are derived from a single immunoblot). Histograms illustrate densitometric analysis of (g) MPO and (h) Nox-2 expression at all-time points tested (for MPO and Nox-2: N = 3 mice for day 0 and 3–5 mice for days 7–9 and 18–23). *P < 0.05 versus corresponding day 0, ^† P < 0.05 versus corresponding Eng ^+/+ group, and ^# P < 0.05 versus corresponding untreated group. Results represent mean ± SEM.

Figure 5

Eng ^+/− mice show normal bone marrow-derived neutrophil function. (a) Superoxide (O₂ ⁻) production, measured by a dihydroethidium (DHE) based assay, was tested in isolated bone marrow-derived neutrophils from days 0, 12, and 25, with or without PMA stimulation. The results are plotted as fold change relative to the unstimulated Eng ^+/+ control for each time point. *P < 0.05 versus corresponding unstimulated group. (b) Neutrophil migration capacity (displacement and velocity) was determined using fMLP as a chemotactic agent in both Eng ^+/+ and Eng ^+/− mice. Results represent mean ± SEM (for DHE assay: N = 4–8 samples for days 0, 12, and 25; for chemotaxis assay: N = 3 experiments for each genotype).