Intestinal inflammation marker calprotectin regulates epithelial intestinal zinc metabolism and proliferation in mouse jejunal organoids

Abstract

Calprotectin (CP), a heterodimer of S100A8 and S100A9, is expressed by neutrophils and a number of innate immune cells and is used widely as a marker of inflammation, particularly intestinal inflammation. CP is a ligand for toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE). In addition, CP can act as a microbial modulatory agent via a mechanism termed nutritional immunity, depending on metal binding, most notably Zn²⁺. The effects on the intestinal epithelium are largely unknown. In this study we aimed to characterize the effect of calprotectin on mouse jejunal organoids as a model epithelium, focusing on Zn²⁺ metabolism and cell proliferation. CP addition upregulated the expression of the Zn²⁺ absorptive transporter Slc39a4 and of methallothionein Mt1 in a Zn²⁺-sensitive manner, while downregulating the expression of the Zn²⁺ exporter Slc30a2 and of methallothionein 2 (Mt2). These effects were greatly attenuated with a CP variant lacking the metal binding capacity. Globally, these observations indicate adaptation to low Zn²⁺ levels. CP had antiproliferative effects and reduced the expression of proliferative and stemness genes in jejunal organoids, effects that were largely independent of Zn²⁺ chelation. In addition, CP induced apoptosis modestly and modulated antimicrobial gene expression. CP had no effect on epithelial differentiation. Overall, CP exerts modulatory effects in murine jejunal organoids that are in part related to Zn²⁺ sequestration and partially reproduced in vivo, supporting the validity of mouse jejunal organoids as a model for mouse epithelium.

Keywords: Calprotectin; Intestinal epithelium; Intestinal inflammation markers; Organoid; Proliferation; Zinc.

Fig. 1.

Regulation of Zn²⁺ metabolism in mouse jejunal organoids by calprotectin. Calprotectin (CP, 10 μM) was added to the cell culture medium and incubated for 24 hours. RT-qPCR results are shown (mean ± SEM). n=10+29 from 2 to 5 separate experiments. A t-test was performed: *p<0.05 vs. CONTROL.

Fig. 2.

Regulation of Zn²⁺ metabolism in mouse jejunal organoids by S1S2 calprotectin, a mutant lacking transition metals binding sites. S1S2 Calprotectin (S1S2 CP, 10 μM) was added to the cell culture medium and incubated for 24 hours. RT-qPCR results are shown (mean ± SEM, n=5). A t-test was performed: *p<0.05 vs. CONTROL.

Fig. 3.

Influence of Zn²⁺ in the effects of calprotectin in mouse jejunal organoids. Calprotectin (CP, 10 μM) and/or ZnSO₄ (50 μM) were added to the culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM (n=5). One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#p<0.05 vs. ZnSO₄.

Fig. 4.

Effect of calprotectin on the expression of genes involved in proliferative response. Calprotectin (CP, 10 μM) and/or ZnSO₄ (50 μM) were added to organoids culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM (n=5). One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#p<0.05 vs. ZnSO₄.

Fig. 5.

Effect of calprotectin on cell proliferation by microscopic analysis. Calprotectin or S1S2 calprotectin (10 μM) were added to organoids culture medium and incubated for 3 days. A. Data are expressed as mean ± SEM of calculated area (arbitrary units) with Image J software. Two-way ANOVA was performed: *p<0.05 vs. control D0; ⁺p<0.05 vs. C day 3; ^#p<0.05 vs. CP day 3. B. Representative images of organoids at day 0 and 3.

Fig. 6.

Calprotectin effect on EdU, TUNEL and LDH proliferation assay in mouse jejunum organoids. Two days cultured organoids were used, and CP or S1S2 CP (10 μM) were added for 4 hours before experiments were carried out. A. Data are expressed as mean ± SEM. One way ANOVA was performed: *p<0.05 vs. CONTROL. B. Representative images of TUNEL and EdU.

Fig. 7.

Effect of calprotectin on the expression of genes involved in intestinal epithelial cell differentiation. Calprotectin (CP, 10 μM) and/or ZnSO₄ (50 μM) were added to the culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM (n=5). One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#p<0.05 vs. ZnSO₄.

Fig. 8.

Regulation of Zn²⁺ metabolism in mouse jejunal organoid monolayers by calprotectin and a sterile bacterial homogenate. Calprotectin (CP, 10 μM) and/or SBH (5 mg/l) were added to the culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM (n=5). One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#p<0.05 vs. SBH.

Fig. 9.

Effects of calprotectin and a sterile bacterial homogenate on Zn²⁺ metabolism in closed organoids. Calprotectin (CP, 10 μM) and/or SBH (5 mg/l) were added to the culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM. n=10 from 2 separate experiments. One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#<0.05 vs. SBH.

Fig. 10.

Effect of calprotectin and a sterile bacterial homogenate on the expression of genes involved in proliferative response. A. Organoid monolayers. B. Closed organoids. Calprotectin (CP, 10 μM), and/or SBH (5 mg/l) were added to the culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM (n=5). One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#p<0.05 vs. SBH.

Fig. 11.

Effect of calprotectin and a sterile bacterial homogenate on the expression of genes involved in intestinal epithelial cell differentiation in organoid monolayers. Calprotectin (CP, 10 μM) and/or SBH (5 mg/l) were added to the culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM (n=5). One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#p<0.05 vs. SBH.

Fig. 12.

Effects of calprotectin and a sterile bacterial homogenate on cell differentiation in closed organoids. Calprotectin (CP, 10 μM), and/or SBH (5 mg/l) were added to the culture medium and incubated for 24 hours. RT-qPCR results are shown. Data are expressed as mean ± SEM. n=10 from 2 separate experiments. One way ANOVA: *p<0.05 vs. CONTROL; ⁺p<0.05 vs. CP; ^#p<0.05 vs. SBH.

Fig. 13.

Effects of calprotectin in colonic samples of DSS colitis mice. RT-qPCR results are shown. Data are expressed as Media±SEM (n=8). One way ANOVA was performed: *p<0.05 vs. CONTROL; ⁺<0.05 vs. CP; #p<0.05 vs. DSS.