Fig 1.
Dithizone significantly depletes Paneth cells in immature intestine.
Paneth cells were quantified per crypt in normal tissue. (A) Mice at age P35 have significantly more Paneth cells than at P14 (n = 6 for all treatment groups, p < 0.0001). (B) Dithizone exposure significantly reduces Paneth cell counts acutely (20–30% reduction) and chronically (11%) compared to age matched controls (n = 6 for all treatment groups, p < 0.019). (C) In addition to reduction in cell counts, dithizone exposure also induces significant decreases in Paneth cell-specific genes (n = 5 for all treatment groups, p < 0.019 for all significant points).
Fig 2.
Dithizone induced-Paneth cell depletion causes initial non-significant disturbances to the cecal microbiome composition that develop into lasting significant alterations.
(A) The cecal microbial population significantly changes during normal aging in C57BL6 mice (n = 6 animals in each group, p < 0.006 for all significant points). (B) Dithizone-treated mice exhibited increased relative percentages of Bacteroidetes along with decreased relative percentages of Firmicutes and Proteobacteria compared to the intestinal microbiota of controls; however, none of the alterations reached statistical significance. (C) However, these non-significant population shifts in Bacteroidetes (26% sham vs 32% dithizone) and Firmicutes (40% vs 48%) persist up to 3 weeks following Paneth cell disruption, and the alterations in Proteobacteria become significantly different from sham controls (30% vs 14%, p < 0.0001). (n = 6 for all treatment groups).
Fig 3.
Paneth cell-depletion induced changes in the microbiome are not dependent on dithizone.
Normal cecal microbiomes were compared between P14 non-treated wild type C57Bl6 mice and PC-DTR mice (on C57Bl6 background). (A) PC-DTR mice had no significant differences in taxa compared to wild type mice of the same age n = 6 for each treatment. (B) Paneth cell depletion using DTX induces significant changes in the microbiota composition by 24 hours in Deferribacteres (7% in Sham vs 28% in DTX treated, p = 0.0004) and in Proteobacteria (46% in Sham vs 27% in DTX treated, p = 0.001), but these significant changes disappear and shift back towards normal floral composition after 72 hours.
Fig 4.
Dithizone and DTX work through different mechanisms to disrupt Paneth cells.
(A) Dithizone treatment reduces lysozyme containing Paneth cells by 33% while DTX treatment reduces Paneth cells by 60% using flow cytometry for anti-lysozyme (n = 3 per group). (B) Examination of Paneth cell histology by lysozyme following treatment with dithizone or DTX shows a loss of lysozyme stained cells compared to controls. However, examination of Paneth cell histology using the novel D1C2 antibody that appears to target Paneth cells shows a loss of cells following DTX, but not following dithizone (n = 3 per group, representative samples shown). (C) Serum levels of IL-10, IL-6, TNF and KC-GRO 16 hours following treatment with DTX show significantly increased levels compared to sham controls (n = 5, p < 0.0069 for all cytokines) while dithizone does not cause any significant changes.
Fig 5.
Dithizone induces autophagy-like changes in Paneth cells.
(A) Treatment with dithizone induces a significant increase in Atg10 (p = 0.03), Atg4a (p = 0.0004), and Atg12 (p = 0.000006), as well as a significant decrease in the beclin1 regulator Ambra1 (p = 0.0003). (n = 5 for each group). (B) Cellular examination using transmission electron microscopy shows presence of autophagosomes (red arrows) in animals treated with dithizone, but not in those treated with DTX or in sham controls. (n = 3 per group). EM sections were evaluated by a single blinded investigator.
Fig 6.
Paneth cell depletion-induced NEC results in Enterobacteriaceae blooms similar to patterns seen in human patients.
(A) Paneth cell depletion followed by Klebsiella exposure induces significant alterations to the cecal bacterial population in both dithizone and DTX models (n = 6 for all treatments at all time points, all significant p values are < 0.05). (B) Furthermore, Paneth cell depletion induced NEC induces significant blooms of Enterobacteriaceae which has been reported in human disease. This bloom corresponds with a significant decrease in Helicobacteraceae.
Fig 7.
Enterobacteriaceae blooms seen in Paneth cell depletion-induced NEC are independent of gavaged bacteria.
To determine if the bloom of Enterobacteriaceae was due to the Klebsiella gavage, tomato mice with constitutively expressed red fluorescent protein were gavaged with GFP labeled Klebsiella. Small intestinal samples were harvested and examined for presence of GFP-tagged Klebsiella. (A) As seen above, Klebsiella was gone from the small intestine by 5 hours, well before the bloom was seen in dithizone or DTX models (n = 3 animals at each time point, representative microscopy shown). (B) To determine bacterial load, cecal samples were measured by spectrophotometry. Bacterial levels returned to pre-gavage levels within 4 hours of gavage (n = 3).