Induction of the metal transporter ZIP8 by interferon gamma in intestinal epithelial cells: Potential role of metal dyshomeostasis in Crohn's disease

https://doi.org/10.1016/j.bbrc.2019.05.137Get rights and content

Highlights

  • SLC39A8 mRNA is increased in most severe forms of Crohn's disease.

  • Interferon gamma induces ZIP8 in human ileal enteroids.

  • Human enteroids provide a model system to study ZIP8 and metal homeostasis.

  • Functional experiments suggest ZIP8 A391T impairs negative regulation of NF-κB.

Abstract

Transition metals are required for intestinal homeostasis and provide essential nutrients for the resident microbiota. Abnormalities in metal homeostasis are common in Crohn's disease (CD), but remain poorly defined and causes appear multifactorial. There has been renewed interest in understanding these mechanisms with the discovery of an association between a coding variant in SLC39A8 (rs13107325; ZIP8 A391T) and increased CD risk. SLC39A8 encodes the protein ZIP8, a metal transporter that is induced under inflammatory stimuli; however, studies of its gut-specific functions are lacking. Here, we show that SLC39A8 mRNA is differentially expressed in active CD with a high positive correlation with markers of disease severity, including CXCL8, TNFα, IFNγ, and calprotectin. SLC39A8 expression exhibits a negative correlation with SLC39A4 and SLC39A5, two key zinc importers in absorptive enterocytes, and a lack of correlation with two manganese transporters, SLC39A14 and SLC11A2. Immunohistochemistry demonstrates ZIP8 expression in intestinal epithelial cells and immune cells of the lamina propria. Patients with CD exhibit variable patterns of ZIP8 subcellular localization within IECs. In ileal enteroids, SLC39A8 was induced by IFNγ and IFNγ + TNFα, but not by TNFα alone, independent of NF-κB activation. IFNγ also down-regulated SLC39A5. To explore the functional implications of disease-associated genetic variation, in over-expression experiments in HEK293A cells, ZIP8 A391T was associated with increased TNFα-induced NF-κB activation, consistent with a loss of negative regulation. Taken together, these results suggest a potential role for ZIP8 in intestinal inflammation, induced by IFNγ in the intestinal epithelial compartment, and that perturbations in negative regulation of NF-κB by ZIP8 A391T may contribute to CD pathogenesis.

Introduction

In the intestinal microenvironment, transition metals are required for intestinal homeostasis and used as a nutrient source by the resident microbiota. Interest in metal homeostasis in intestinal disease has been spurred by an exome sequencing study of Crohn's disease (CD) patients that identified a single nucleotide polymorphism (SNP) in SLC39A8 (rs13107325; ZIP8 A391T) associated with an increased risk of CD [1]. The SNP ranks in the top 10 most frequently identified SNPs in genome-wide association studies; the major allele is associated with hypertension and Parkinson's disease, while the minor allele is associated obesity, dyslipidemia, schizophrenia, and CD [2]. The mechanisms underlying these pleiotropic associations are unknown.

SLC39A8 encodes the plasma membrane protein ZIP8. ZIP proteins are best characterized as zinc influx transporters, although ZIP8 has been shown to transport multiple divalent cations, including zinc [3] and manganese [4,5]. SLC39A8 is induced by inflammatory stimuli in multiple cell types [[6], [7], [8]]. Acute induction of ZIP8 restrains NF-κB signaling by zinc binding of IKKβ to inhibit nuclear translocation [7], while chronic ZIP8 induction and increased intracellular zinc drives metallothionein expression to promote tissue destruction [8]. ZIP8-mediated manganese transport is critical for the activity of two manganese-dependent enzymes, arginase and β-1,4-galactosyltransferase [9], but less is known about the role of ZIP8-mediated manganese transport in inflammation. This foundation of known ZIP8 biology engenders hypotheses that perturbation of the constitutive and induced activities of ZIP8 by disease-associated genetic variation contribute to CD pathogenesis, and that better understanding of metal homeostasis in the gut may offer new approaches to diagnosis and treatment. Our results represent the first characterization of ZIP8 in the gut and CD.

Section snippets

Secondary data analysis

We analyzed publicly-available RNAseq data derived from ileal biopsies from newly diagnosed CD patients [10]. Co-expression analyses were performed in Excel (Microsoft) and Prism 8.0 (Graphpad).

Human specimens

Formalin-fixed, paraffin-embedded human ileal sections were obtained with patient consent and Johns Hopkins Hospital (JHH) institutional review board approval (NA_00038329). Human enteroids (3D spheroids) were prepared from biopsies taken at ileocolonoscopy from three healthy subjects with patient

SLC39A8 expression is increased in active ileal CD and expression correlates with inflammatory cytokine expression

Initially, we examined whether SLC39A8 is differentially expressed in active CD. Comparing transcriptional data from ileal biopsies from patients with CD versus controls [10], SLC39A8 was differentially expressed in patients with CD with the highest expression in the most severe inflammatory disease (Fig. 1a). The relative increase in expression paralleled CD severity (p-trend < 0.0001). There was significant inter-individual variation in SLC39A8 (coefficient of variation in most severe disease

Discussion

In this work, we found that SLC39A8 mRNA expression is increased in active CD. There is high inter-individual variation in SLC39A8 expression in severe disease compared to controls. This observation is in line with our previous study in which we showed that IBD-associated risk genes with the lowest variance in expression in controls demonstrated the highest variance in CD patients [19]. We hypothesized that IBD-associated risk genes are tightly regulated to maintain intestinal homeostasis in

Funding

This work was supported by the NIH/NIDDK Johns Hopkins Conte Gastrointestinal Core Center (P30 DK-089502); Johns Hopkins University School of Medicine Clinician Scientist Career Development Award; and the Ludwig-Bayless Discovery Award to J. Melia.

Acknowledgements

We wish to acknowledge Ms. Karen Fox-Talbot (Johns Hopkins Oncology Tissue Services), Dr. Nicholas Zachos, and Mr. George McNamara of the Hopkins Digestive Diseases Basic and Translational Research Core (P30 DK-089502).

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