A missense variant in SLC39A8 confers risk for Crohn's disease by disrupting manganese homeostasis and intestinal barrier integrity

Abstract

Common genetic variants interact with environmental factors to impact risk of heritable diseases. A notable example of this is a single-nucleotide variant in the Solute Carrier Family 39 Member 8 (SLC39A8) gene encoding the missense variant A391T, which is associated with a variety of traits ranging from Parkinson's disease and neuropsychiatric disease to cardiovascular and metabolic diseases and Crohn's disease. The remarkable extent of pleiotropy exhibited by SLC39A8 A391T raises key questions regarding how a single coding variant can contribute to this diversity of clinical outcomes and what is the mechanistic basis for this pleiotropy. Here, we generate a murine model for the Slc39a8 A391T allele and demonstrate that these mice exhibit Mn deficiency in the colon associated with impaired intestinal barrier function and epithelial glycocalyx disruption. Consequently, Slc39a8 A391T mice exhibit increased sensitivity to epithelial injury and pathological inflammation in the colon. Taken together, our results link a genetic variant with a dietary trace element to shed light on a tissue-specific mechanism of disease risk based on impaired intestinal barrier integrity.

Keywords: Crohn’s disease; barrier function; glycocalyx; inflammatory bowel disease; manganese.

Fig. 1.

Slc39a8 A391T variant is associated with reduced Mn levels and impaired intestinal barrier function. (A) Mn levels in the colon by ICP-MS (n = 6 male mice per genotype). Similar results were obtained in at least three independent experiments. (B) Representative TEM images of colon epithelial glycocalyx. (Scale bars, 200 nm.) (C) Measurement of glycocalyx thickness. At least 45 glycocalyxes were measured per mouse (n = 3 per genotype). (D) Alcian blue-periodic acid-Schiff staining of the Carnoy’s fixed colon. Arrows show the inner mucus layer. (Scale bars, 50 µm.) (E) FISH using EUB338 (green) and UEA-1 (red) staining of the Carnoy’s fixed colon. DAPI (blue) was used for the nuclear staining. (Scale bars, 100 µm.) Positive signals (white arrow) were detected in the inner mucus layer and the apical side of enterocytes in A391T colons. Yellow bars show the thickness of the bacteria-free zone. (F) Bacteria-free zone thickness (n = 4 mice per genotype). Each dot represents the average of four points of measurement. (D–F) Similar results were obtained in at least two independent experiments. (G) Dextran permeability assay. Mice (10 to 16 wk old) were used for the experiment. Data were pooled from two independent experiments (n = 8 per group). A391T male mice show significantly increased permeability. *P < 0.05, **P < 0.01, ***P < 0.001 using a two-tailed unpaired t test. All data represent mean ± SEM.

Fig. 2.

Development of mature isolated lymphoid follicles driven by barrier dysfunction. Whole-colon tissues were collected from WT and Slc39a8 A391T male mice and whole-mount staining was performed with B220 and CD21/CD35 antibodies. (A) Representative images of ILFs. (Scale bars, 500 µm [Upper] and 200 µm [Lower].) (A, Lower) Magnified version of Upper images. Red arrowheads indicate mature ILFs and white arrowheads indicate immature ILFs. (B) Representative images of immature and mature ILFs stained with B220 (green) and CD21/CD35 (red) antibodies and DAPI (blue) in cryosections. (C) The total number of ILFs per genotype. (D) The number of mature ILFs (B220+ and CD21/CD35+) per genotype. (E) Percent of mILFs per genotype. (F) The number of ILFs (B220+) per size. (G) The number of multifollicular structures per genotype. n = 7 mice per genotype from two independent experiments. *P < 0.05, ***P < 0.001 using a two-tailed unpaired t test; ns, not significant. All data represent mean ± SEM.

Fig. 3.

Exacerbated colitis in A391T mice following DSS treatment. (A) Weight was measured daily and expressed as the percent change from the initial weight. (B) Clinical disease activity index was scored daily as described in Materials and Methods. (C and D) Colon length (C) and cecum length (D) were measured on day 10. (E) Representative images of colon sections stained with hematoxylin and eosin. (E, Lower) Magnified version of Upper images. (Scale bars, 500 µm [Upper] and 100 µm [Lower].) (F) Histological scores were determined as described in Materials and Methods. (A–F) Data pooled from two independent experiments (n = 11 or 12 per genotype). (G) Inflammatory cytokine expression in the distal colons of mice on day 10 was determined by qPCR. Gapdh was used for normalization (n = 6 per genotype). Data are representative of two independent experiments. (H) Fecal occult blood score was measured daily until day 4 (n = 8 to 10 per genotype). (I) Fecal calprotectin levels on day 1 were measured by ELISA (n = 8 to 10 per genotype). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using a two-tailed unpaired t test. All data represent mean ± SEM.

Fig. 4.

Mn deficiency disrupts intestinal mucus barrier function. Samples were collected after a 6-wk Mn-depleted diet, unless otherwise noted. (A–C) Whole blood (A), liver (B), and colon (C) were collected and the levels of trace elements (Mn, Zn, Fe) were measured by ICP-MS. C57BL/6 male mice were used (n = 8 or 9 per group) from two independent experiments. (D) FITC-dextran gavage was performed. n = 11 or 12 per group were used from three independent experiments. (E) FISH using EUB338 (green) in the Carnoy’s fixed colon. UEA-1 (red) was used for goblet cell staining. DAPI (blue) was used for nuclear staining. Positive signals (white arrow) were observed in the epithelia of the mice fed the Mn-depleted diet. (Scale bars, 100 µm.) (F) Average bacterial free zone (n = 3 or 4 mice per condition). Each dot represents the average of four points of measurement. (G) Representative TEM images of colon epithelial glycocalyx (n = 4 per group). (Scale bars, 200 nm.) (H) Glycocalyx measurement of the Mn-depleted colon. *P < 0.05, ****P < 0.0001 using a two-tailed unpaired t test. All data represent mean ± SEM.

Fig. 5.

Schematic model of SLC39A8 A391T-induced intestinal barrier disruption. Slc39a8 A391T-induced Mn deficiency impairs intestinal barrier integrity at the level of glycoprotein barrier structures, including the glycocalyx and inner mucus layer. Bacterial invasion into the inner mucus layer of the Slc39a8 A391T colon leads to indolent inflammation that can prime inflammation driven by epithelial injury.