Intestinal proinflammatory macrophages induce a phenotypic switch in interstitial cells of Cajal

Abstract

Interstitial cells of Cajal (ICCs) are pacemaker cells in the intestine, and their function can be compromised by loss of C-KIT expression. Macrophage activation has been identified in intestine affected by Hirschsprung disease-associated enterocolitis (HAEC). In this study, we examined proinflammatory macrophage activation and explored the mechanisms by which it downregulates C-KIT expression in ICCs in colon affected by HAEC. We found that macrophage activation and TNF-α production were dramatically increased in the proximal dilated colon of HAEC patients and 3-week-old Ednrb-/- mice. Moreover, ICCs lost their C-KIT+ phenotype in the dilated colon, resulting in damaged pacemaker function and intestinal dysmotility. However, macrophage depletion or TNF-α neutralization led to recovery of ICC phenotype and restored their pacemaker function. In isolated ICCs, TNF-α-mediated phosphorylation of p65 induced overexpression of microRNA-221 (miR-221), resulting in suppression of C-KIT expression and pacemaker currents. We also identified a TNF-α/NF-κB/miR-221 pathway that downregulated C-KIT expression in ICCs in the colon affected by HAEC. These findings suggest the important roles of proinflammatory macrophage activation in a phenotypic switch of ICCs, representing a promising therapeutic target for HAEC.

Keywords: Gastroenterology; Inflammatory bowel disease; Macrophages.

Figure 1. Enterocolitis score increases and macrophages infiltrate in the dilated colon of HAEC patients.

(A) H&E staining of the proximal resection margin, dilated segment, transition zone, and narrowed colon of HSCR and HAEC patients. Scale bars: 100 μm. (B) An enterocolitis grading system was used to evaluate inflammation. (C) CD68 and iNOS immunofluorescence double staining to identify M1 macrophages. Red, CD68; green, iNOS; blue, DAPI. Scale bars: 100 μm. Data shown are representative of n = 75 samples of HSCR patients and n = 21 samples of HAEC patients. One-way ANOVA: ***P < 0.001.

Figure 2. Proinflammatory cytokines increase, whereas C-KIT⁺ ICCs decrease, in the dilated colon of HAEC patients.

(A) Immunohistochemistry staining of ICCs of HSCR and HAEC patients for C-KIT. Scale bars: 100 μm. Data are representative of n = 75 samples of HSCR patients and n = 21 samples of HAEC patients. Arrows indicate C-KIT⁺ ICCs. (B) Western blotting of colon of HSCR and HAEC patients. (C) Semiquantitative analysis of protein expression levels, with each protein being normalized to β-actin. One-way ANOVA: ***P < 0.001.

Figure 3. Enterocolitis score increases in the proximal colon of 3-week-old Ednrb^–/– mice.

(A) H&E staining of proximal, middle, and distal colon sections from 3-week-old Ednrb^+/+ and Ednrb^–/– mice. Scale bars: 100 μm. (B) An enterocolitis grading system was used to evaluate inflammation. Data shown represent results from 6 independent experiments. One-way ANOVA: ***P < 0.001.

Figure 4. Proinflammatory cytokines increase but C-KIT⁺ ICCs decrease in the proximal colon of 3-week-old Ednrb^–/– mice.

(A) Immunohistochemistry staining of ICCs from 3-week-old Ednrb^+/+ and Ednrb^–/– mice for C-KIT. Scale bars: 100 μm. (B) Western blotting of colon from 3-week-old Ednrb^+/+ and Ednrb^–/– mice. (C) Semiquantitative analysis of protein expression levels, with each protein being normalized to β-actin. Data shown represent results from 3 independent experiments. One-way ANOVA: ***P < 0.001.

Figure 5. Proinflammatory M1 macrophages increase in the proximal colon of 3-week-old Ednrb^–/– mice.

(A) Representative analysis of CD45⁺F4/80⁺, CD11b⁺CD11c^–, iNOS⁺, and TNF-α⁺ cells in the proximal colon from 3-week-old Ednrb^+/+ and Ednrb^–/– mice. (B) Percentage of CD45⁺F4/80⁺ cells among total viable cells in colonic tissue. (C) Percentage of CD11b⁺CD11c^– cells among CD45⁺F4/80⁺ cells. (D) Percentage of iNOS⁺ cells and TNF-α⁺ cells among CD11b⁺CD11c^– cells. Data shown represent results from 3 independent experiments. One-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Clod treatment alleviates colitis in 3-week-old Ednrb^–/– mice.

(A) H&E staining of proximal, middle, and distal colon from 3-week-old Ednrb^+/+ and Ednrb^–/– mice treated with Clod. Scale bars: 100 μm. (B) An enterocolitis grading system was used to evaluate inflammation. Data shown represent results from 6 independent experiments. One-way ANOVA: *P < 0.05; ***P < 0.001.

Figure 7. Proinflammatory M1 macrophages decrease in the proximal colon of 3-week-old Ednrb^–/– mice after Clod treatment.

(A) Representative analysis of CD45⁺F4/80⁺, CD11b⁺CD11c^–, iNOS⁺, and TNF-α⁺ cells in the proximal colon from 3-week-old Ednrb^+/+ and Ednrb^–/– mice treated with Clod. (B) Percentage of CD45⁺F4/80⁺ cells among total viable cells in colonic tissue. (C) Percentage of CD11b⁺CD11c^– cells among CD45⁺F4/80⁺ cells. (D) Percentage of iNOS⁺ cells and TNF-α⁺ cells among CD11b⁺CD11c^– cells. Data shown represent results from 3 independent experiments. One-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8. Clod treatment suppresses inflammatory cytokine production and promotes recovery of ICC phenotype in 3-week-old Ednrb^–/– mice.

(A) Immunohistochemistry staining for C-KIT in ICCs from 3-week-old Ednrb^+/+ and Ednrb^–/– mice treated with Clod. Scale bars: 100 μm. (B) Western blot analysis of colon from 3-week-old Ednrb^+/+ and Ednrb^–/– mice treated with Clod. (C) Semiquantitative analysis of protein expression levels, with each protein being normalized to β-actin. Data shown represent results from 3 independent experiments. One-way ANOVA: **P < 0.01; ***P < 0.001.

Figure 9. Clod treatment restores murine colon slow waves.

(A) Colonic slow waves from 1-, 2-, and 3-week-old Ednrb^+/+ and Ednrb^–/– mice, and 3-week-old Ednrb^+/+ and Ednrb^–/– mice treated with Clod. s/div, seconds/division. (B and C) The amplitudes (B) and frequencies (C) of colonic slow waves of 1-, 2-, and 3-week-old Ednrb^+/+ and Ednrb^–/– mice are shown. Data shown represent results from 3 independent experiments. One-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 10. TNF-α induces downregulation of C-KIT and inhibits the pacemaker currents of isolated ICCs.

(A) C-KIT and CD34 immunofluorescence double staining was used to identify the ICC phenotype with or without TNF-α treatment. Green, CD34; red, C-KIT; blue, DAPI. Scale bars: 100 μm. (B) Pacemaker currents of ICCs were recorded using the whole-cell mode of the patch clamp technique. (C) Amplitudes of pacemaker currents with or without TNF-α treatment. Data shown represent results from 3 independent experiments. Student’s t test: ***P < 0.001.

Figure 11. The TNF-α/NF-κB/miR-221 signaling pathway is involved in C-KIT expression in isolated ICCs.

(A) TargetScan software was used to predict the possible miRNAs that could target the 3′-UTR of c-Kit mRNA in humans and mice. (B) Western blotting of isolated ICCs with or without TNF-α and PDTC treatment. (C) Semiquantitative analysis of protein expression levels with each protein normalized to β-actin. (D) MiR-221 expression levels with or without TNF-α and PDTC treatment. (E and F) Western blot analysis of C-KIT and CD34 in isolated ICCs with or without TNF-α and miR-221 inhibitor treatment. Semiquantitative analysis of protein expression levels with each protein normalized to β-actin. Data shown represent results from 3 independent experiments. (G) The signaling pathway involving TNF-α, miR-221, and C-KIT in ICCs. One-way ANOVA: *P < 0.05; ***P < 0.001.