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Case Reports
. 2020;9(2):239-255.
doi: 10.1016/j.jcmgh.2019.10.006. Epub 2019 Oct 23.

Novel Human NKCC1 Mutations Cause Defects in Goblet Cell Mucus Secretion and Chronic Inflammation

Rainelli Koumangoye  1 , Salma Omer  1 , Mustafa H Kabeer  2 , Eric Delpire  3
Affiliations
Case Reports

Novel Human NKCC1 Mutations Cause Defects in Goblet Cell Mucus Secretion and Chronic Inflammation

Rainelli Koumangoye et al. Cell Mol Gastroenterol Hepatol. 2020.

Abstract

Background & aims: Infections resulting from intestinal yeast and bacteria affect a large number of patients with deficits in absorptive or secretory epithelial transport mechanisms. The basolateral Na+-K+-2Cl- cotransporter (NKCC1) has been implicated in intestinal epithelial fluid secretion. Two patients with deleterious heterozygous (NKCC1-DFX, DFX for Asp-Phe-stop codon) or homozygous (Kilquist) mutations in SLC12A2 (NKCC1) suffered from gastrointestinal deficits. Because of chronic infections, the colon and the small intestine of the NKCC1-DFX patient were resected surgically.

Methods: To investigate how NKCC1 affects the integrity and function of the gut epithelia, we used a mouse model recapitulating the NKCC1-DFX patient mutation. Electron microscopy and immunostaining were used to analyze the integrity of the colonic mucus layers and immune cell infiltration. Fluorescence in situ hybridization was performed on the distal colon sections to measure bacteria translocation to the mucosa and submucosa. Citrobacter rodentium was used to measure mouse ability to clear enteric infection. A multiplex cytokine assay was used to analyze mouse inflammatory response to infection.

Results: We show that NKCC1-DFX expression causes defective goblet cell mucus granule exocytosis, leading to secretion of intact granules into the lumen of the large intestine. In addition, NKCC1-DFX colon submucosal glands secrete mucus that remained attached to the epithelium. Importantly, expression of the mutant NKCC1 or complete loss of NKCC1 function leads to aggravated inflammatory response to C rodentium infection. Compared with wild-type, NKCC1-DFX mice showed decreased expression of claudin-2, a tight junction protein involved in paracellular Na+ and water transport and enteric infection clearance.

Conclusions: Our data indicate that NKCC1-DFX impairs gut barrier function by affecting mucus secretion and immune properties.

Keywords: Bacterial Infection; Goblet Cell; Mucus Secretion; NKCC1.

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Figures

None
Graphical abstract
Figure 1
Figure 1
Deletion of NKCC1 results in death in C57BL/6J. (A) NKCC1DFX/DFX mice in C57BL/6J and mixed C57BL/6J:DBA/2J background were maintained in a pathogen-free environment and survival was determined. The B6:D2 KO mice that survived the periweaning period lived for several months until use. (B) Gross anatomy of the cecum and colon of NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX in C57BL/6J background around weaning time.
Figure 2
Figure 2
Abnormal water and intestinal transit are observed in NKCC1-DFX mutant mice. (A) Water content of fecal pellets collected from WT NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX mice. Whisker plot data presents the median, lower and upper quartiles, and minimum and maximum for 9–12 pellets from 3–4 animals per group. (B) Gastrointestinal transit time measured by subtracting time of oral gavage with 6% carmine red solution from time of appearance of red fecal pellets (n = 3–4 mice per group). (C and D) The number of pellets increases in the colon of NKCC1DFX/DFX mice. (A, B, and D) P values were obtained from 1-way analysis of variance with follow-up Tukey posttests.
Figure 3
Figure 3
NKCC1 is required for goblet cells mucus granule exocytosis. Representative transmission electron microscopy images of (A and B) NKCC1WT/WT, (C and D) NKCC1WT/DFX, and (E and F) NKCC1DFX/DFX mouse colon sections. In this experiment, 3 mice per group and 10–20 micrographs per mouse section were analyzed. Stars show the proper release of mucus from goblet cell granules. Red circles highlight improper release of intact mucus granules. Scale bars: (A, C, and E) 2 μm, and (B, D, and F) 500 nm. (G–I) Representative immunostaining of mucus granules with specific protein CLCA1 (green), counterstained with DAPI (blue). Arrowheads indicate accumulation of CLCA1 in the lumen of NKCC1WT/DFX and NKCC1DFX/DFX mouse colons. Dotted lines mark the surface of the epithelium. Fg, floating granules.
Figure 4
Figure 4
Mucus remains attached on the luminal side of the colon in both NKCC1-DFX mutant mice and the NKCC1-DFX patient. (A–C) Representative AB/PAS staining of NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX colon sections. (D) Quantitation of the number of PAS-positive goblet cells per crypt in intestinal sections (3 mice, 8 micrographs per mouse). One-way analysis of variance showed no significant differences between WT and DFX (P = .092) and between DFX and KO (P = .066), but a small difference between WT and KO (P = .029). (E–I) messenger RNA expression of Hes1, Klf4, Spdef1, Gfi1, and TFF3 quantitated by quantitative PCR is compared among genotypes. There were no differences between groups (P > .05). (J–L) Representative immunostaining of NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX mouse colon sections with anti-Muc2 and anti-NKCC1 antibodies. Scale bars: 20 μm.
Figure 5
Figure 5
Evidence for mucus plugging colonic crypts in NKCC1-DFX patient. (A–C) H&E-stained sections of control colon showing clear crypts. (D–F) Evidence for dense material plugging the colonic crypts in sections of NKCC1-DFX patient. AB/PAS staining of (G) normal colon section and (H and I) patient colon sections showing abnormal mucus deposition at the surface of NKCC1-DFX patient colonic epithelial cells. Scale bars: 100 μm.
Figure 6
Figure 6
Loss of NKCC1 function impairs the organization of the outer and inner mucus layers. (A–C) UEA-1 lectin staining of colon section from (A and D) WT, (B and E) heterozygous, and (C and F) homozygous NKCC1-DFX mice. The inner mucus layer is thinner in the NKCC1WT/DFX and NKCC1DFX/DFX mice. Scale bars: 20 μm. (D–F) High magnification from indicated regions of panels A–C, respectively. (G) Quantification of the inner mucus layer thickness in the distal colon (n = 3 mice per group, 8 micrographs per mouse section). Data are shown as whisker boxes and statistical significance was calculated by 1-way analysis of variance followed by Tukey posttests. ***P < .001.
Figure 7
Figure 7
Bacteria penetrate the inner mucus layer in NKCC1WT/DFXand NKCC1DFX/DFXmice. Fluorescence in situ hybridization (FISH) analysis using the general bacterial probe EUB338-Cy3 (red) on (A) NKCC1WT/WT, (B) NKCC1WT/DFX, and (C) NKCC1DFX/DFX mouse colon sections. (D–F) The micrographs were counterstained with UEA-1 lectin (green) (n = 3 mice per group, 10–20 micrographs per mouse section). Scale bars: 20 μm. (G) Average number of bacteria per field in NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX mouse colon sections (n = 3 mice per genotype, 8–12 micrographs per mouse section were counted). *P < .01, ***P < .001, and ****P < .0001; 1-way analysis of variance followed by Tukey posttests. (H–J) Electron micrographs of extensively washed colon from NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX mice show bacteria (indicated by + signs) near the epithelium in mutant mice. Scale bars: 2 μm.
Figure 8
Figure 8
Loss of NKCC1 function increases CD3+ lymphocytes infiltration in the colon. (A–C) Representative H&E-stained micrographs of colon sections from 8-week-old NKCC1-WT, NKCC1-DFX, and NKCC1-KO mouse colon sections showing overall normal anatomy. (D–G) Sections of same genotypes stained with anti-CD3 antibody. Scale bars: 20 μm. (G) Quantification of the number of infiltrating CD3+ cells per genotype (N = 3 mice per group; 8 micrographs per section were taken and counted). Data are shown as whisker box plots, and statistical significance was calculated by 1-way analysis of variance followed by Tukey posttests. *P < .05, **P < .01.
Figure 9
Figure 9
NKCC1 is required for enteric C rodentium infection clearance. NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX mice were infected with 109 colony forming units of a kanamycin-resistant strain of C rodentium. Feces were collected every day, eluted, plated, and bacteria were enumerated. (A) Percentage of NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX mice with positive C rodentium shedding in feces over the 9-day period. (B) Bacteria plated on a kanamycin-containing plate show colonization or delay clearance in NKCC1WT/DFX and NKCC1DFX/DFX mice. (C) Inflammatory cytokine production in the serum of NKCC1WT/WT, NKCC1WT/DFX, and NKKCC1DFX/DFX mice after 9 days postinfection (n = 3–5 mice per group, the experiment was repeated twice). hets, heterozygotes; homos, homozygotes; IL, interleukin; INF, interferon.
Figure 10
Figure 10
Claudin-2 expression is limited to the base colon of crypt in NKCC1WT/DFXand NKCC1DFX/DFXmice. Representative IF images of NKCC1WT/WT, NKCC1WT/DFX, and NKCC1DFX/DFX mouse colon sections stained with (A–C) claudin-2 and (D–F) claudin-1 antibodies (red). Sections also show DAPI (blue) or nuclei staining. (D–F) In contrast, claudin-1 expression at the lateral membrane was similar in all 3 genotypes. Scale bars: 20 μm. Number of claudin-2–positive cells per crypt were counted on 5 fields, 11 crypts per genotype. There was a significant decrease in the number of cells expressing claudin-2 in the mutant mice. P = .043 for NKCC1–DFX and P = .019 for KO. There was no difference between NKCC1-DFX and NKCC1 KO mice (P = .93). (G) Data are shown as whisker boxes and statistical significance was calculated by 1-way analysis of variance followed by Tukey posttests. CLDN2, claudin-2.
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