Intestinal Metrnl released into the gut lumen acts as a local regulator for gut antimicrobial peptides

Abstract

Aim: Metrnl is a novel secreted protein, but its physiological roles remain elusive. In this study, we investigated the tissue expression pattern of Metrnl in humans and explored its possible physiological role in the tissues with most highly expressed levels.

Methods: A human tissue microarray containing 19 types of tissues from 69 donors was used to examine the tissue expression pattern of Metrnl, and the expression pattern was further verified in fresh human and mouse tissues. Intestinal epithelial cell-specific Metrnl knockout mice were generated, which were used to analyze the physiological roles of Metrnl.

Results: Metrnl was the most highly expressed in the human gastrointestinal tract, and was specifically expressed in the intestinal epithelium. Consistent with this, Metrnl mRNA was also most highly expressed in the mouse gastrointestinal tract among the 14 types of tissues tested. In the intestinal epithelial cell-specific Metrnl knockout mice, the Metrnl levels in the gut fluid were significantly reduced, whereas the Metrnl serum levels showed a trend towards a reduction, but this change was not statistically significant. This cell-specific deletion of Metrnl did not affect body weight, food intake, blood glucose, colon length and histology, intestinal permeability, mucus content or mucin 2 expression under physiological conditions, but statistically decreased the expression of antimicrobial peptides, such as regenerating islet-derived 3 gamma (Reg3g) and lactotransferrin.

Conclusion: Metrnl is highly expressed in the intestinal epithelial cells of humans and mice, which mainly contributes to the local gut Metrnl levels and affects the serum Metrnl level to a lesser extent. Metrnl plays a role in maintaining gut antimicrobial peptides.

Figure 1

High expression of Metrnl in the gastrointestinal tracts of humans and mice. (A) Immunohistochemical staining of representative human tissues. Strong positive immunostaining in the colon and small intestine, moderate positive immunostaining in the thyroid and kidney, weak positive immunostaining in the liver and adrenal glands, and nearly negative immunostaining in the brain and skeletal muscle were observed. Bar, 50 μm. Staining of Metrnl is indicated by a yellow arrow. (B) Metrnl expression in human intestinal epithelial cells. Bar, 200 μm. (C) Metrnl mRNA expression in 14 tissues from C57BL/6 mice, with the highest expression in the colon, followed by the small intestine and stomach.

Figure 2

Breeding and identification of Metrnl^loxp/loxp Villin-cre mice (Metrnl^−/−). (A) Breeding strategy for Metrnl conditional knockout mice. (B) Genotyping of Metrnl-floxed mice. The PCR product of 243 bp is amplified from the conditional floxed (loxp-flanked) Metrnl allele, and the PCR product of 131 bp is amplified from the wild-type Metrnl allele. (C) Genotyping of Cre-positive mice, including Metrnl^loxp/wt Villin-cre and Metrnl^loxp/loxp Villin-cre mice. (D) Metrnl mRNA expression in the tissues of Metrnl^−/− and wild-type mice. WAT, white adipose tissue. BAT, brown adipose tissue. n=6. The data are expressed as the mean±SEM. ^**P<0.01. (E) Immunohistochemical staining for Metrnl in the colons of Metrnl^−/− and wild-type mice. Bar, 100 μm.

Figure 3

Effects of intestinal epithelial Metrnl on body weight, food intake and blood glucose. (A) The body weight of Metrnl^−/− mice is comparable to that of wild-type mice fed a normal chow diet. (B) Food intake of Metrnl^−/− and wild-type mice fed a normal chow diet. n=12. (C) Blood glucose levels were measured at the indicated time points after fasting in Metrnl^−/− and wild-type mice fed a normal chow diet. n=7. Mean±SEM.

Figure 4

Effects of intestinal epithelial Metrnl on colon length, intestinal permeability and histology, serum Metrnl levels, and the level of Metrnl in the gut fluid. (A) Metrnl^−/− mice display no difference in colon length compared with wild-type mice. (B) H&E staining of the colon in Metrnl^−/− and wild-type mice. Bar, 200 μm. (C) The fluorescence intensity of FITC in the serum is comparable between Metrnl^−/− and wild-type mice after gavage with FITC-conjugated dextran at the indicated molecular weight. (D) The serum concentration of Metrnl tended towards a decrease in Metrnl^−/− mice compared to wild-type mice. n=7. (E) The Metrnl level in the gut fluid is significantly reduced in Metrnl^−/− mice. n=7. The data are expressed as the mean±SEM. ^*P<0.05.

Figure 5

Effects of intestinal epithelial Metrnl on mucus production and antimicrobial gene expression. (A) Periodic acid-Schiff staining of the colon in Metrnl^−/− and wild-type mice. Semiquantitative analysis was performed using ImageJ software. n=5. Bar, 200 μm. (B) Relative expression of mucin 2 and antimicrobial genes in the colons of Metrnl^−/− and wild-type mice. n=6. The data are expressed as the mean±SEM. ^*P<0.05.