Original Research
ZnT2-Mediated Zinc Import Into Paneth Cell Granules Is Necessary for Coordinated Secretion and Paneth Cell Function in Mice

https://doi.org/10.1016/j.jcmgh.2015.12.006Get rights and content
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Background & Aims

Defects in Paneth cell (PC) function are associated with microbial dysbiosis and intestinal inflammation. PC granules contain antimicrobial peptides, cytokines, and substantial stores of zinc (Zn). We hypothesized that Zn, transported into the granule through the Zn transporter (ZnT)2, is critical for signature PC functions.

Methods

ZnT2 was localized to PC granules using immunofluorescence and sucrose gradient fractionation in wild-type (wt) mice, and consequences of ZnT2 loss were characterized in ZnT2 knockout (ZnT2ko) mice. Terminal ilea were harvested for immunofluorescence, electron microscopy, and fluorescent imaging with the Zn reporter Zinpyr-1. Alterations in fecal microbiota were characterized using 16s ribosomal RNA sequencing. PC degranulation, bacterial translocation, cytokine response to Escherichia coli endotoxin lipopolysaccharide, crypt viability after exposure to the oxidant monochloramine (NH2Cl), and bactericidal activity of luminal contents of terminal ilea against enteropathogenic E coli were assessed.

Results

ZnT2 was localized to the membrane of PC granules. In ZnT2ko mice, spontaneous degranulation was observed more frequently than among wt mice. Secretory granules were hypodense with less active lysozyme, and there was evidence of autophagosome accumulation and granule degradation in PCs from ZnT2ko mice. Gut microbiota of ZnT2ko mice were enriched in Bacteroidales S24-7 and relatively depleted of species commonly found in wt mice. Evidence of PC dysfunction in ZnT2ko mice included impaired granule secretion and increased inflammatory response to lipopolysaccharide, less bactericidal activity, and greater susceptibility to cell death from NH2Cl.

Conclusions

ZnT2 is critical for Zn import into PC granules, and the inability to import Zn leads to profound defects in PC function and uncoordinated granule secretion.

Keywords

Small Intestine
Zinc Transporter
Microbiota

Abbreviations used in this paper

CFU
colony forming unit
EPEC
enteropathogenic Escherichia coli
ER
endoplasmic reticulum
IF
immunofluorescent
IL
interleukin
IP
intraperitoneal
ko
knockout
LPS
lipopolysaccharide
NEC
necrotizing enterocolitis
OTU
organizational taxonomic unit
PBS
phosphate-buffered saline
PC
Paneth cell
PCR
polymerase chain reaction
TNF
tumor necrosis factor
wt
wild-type
ZIP
ZRT, IRT-like protein
Zn
zinc
ZnT
zinc transporter

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Conflicts of interest The authors disclose no conflicts.

Funding Supported by intramural support from the Academic Enrichment Fund (S.L.K.) and David L. Nahrwold Endowment (D.I.S.) of the Penn State Hershey Department of Surgery, and work at Juniata College was supported by a grant from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program and a National Science Foundation award (DBI-1248096).