ZnT2-Mediated Zinc Import Into Paneth Cell Granules Is Necessary for Coordinated Secretion and Paneth Cell Function in Mice.
OBJECTIVE
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 toEscherichia coliendotoxin lipopolysaccharide, crypt viability after exposure to the oxidant monochloramine (NH2Cl), and bactericidal activity of luminal contents of terminal ilea against enteropathogenicE coliwere assessed.


RESULTS
ZnT2 was localized to the membrane of PC granules. InZnT2komice, spontaneous degranulation was observed more frequently than amongwtmice. Secretory granules were hypodense with less active lysozyme, and there was evidence of autophagosome accumulation and granule degradation in PCs fromZnT2komice. Gut microbiota ofZnT2komice were enriched inBacteroidalesS24-7 and relatively depleted of species commonly found inwtmice. Evidence of PC dysfunction inZnT2komice 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.
Department of Surgery, Penn State Hershey College of Medicine, Hershey, Pennsylvania; Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, Pennsylvania.
Abigail B
AB
Podany
Department of Biology, Juniata College, Huntingdon, Pennsylvania.
Justin
J
Wright
Department of Biology, Juniata College, Huntingdon, Pennsylvania.
Regina
R
Lamendella
Department of Surgery, Penn State Hershey College of Medicine, Hershey, Pennsylvania; Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, Pennsylvania.
David I
DI
Soybel
Department of Surgery, Penn State Hershey College of Medicine, Hershey, Pennsylvania; Department of Cellular and Molecular Physiology, Penn State Hershey College of Medicine, Hershey, Pennsylvania; Department of Pharmacology, Penn State Hershey College of Medicine, Hershey, Pennsylvania.
Shannon L
SL
Kelleher
CFU, colony forming unit; EPEC, enteropathogenic Escherichia coli; ER, endoplasmic reticulum; IF, immunofluorescent; IL, interleukin; IP, intraperitoneal; LPS, lipopolysaccharide; Microbiota; NEC, necrotizing enterocolitis; OTU, organizational taxonomic unit; PBS, phosphate-buffered saline; PC, Paneth cell; PCR, polymerase chain reaction; Small Intestine; TNF, tumor necrosis factor; ZIP, ZRT, IRT-like protein; Zinc Transporter; Zn, zinc; ZnT, zinc transporter; ko, knockout; wt, wild-type