PMC

Overproduction and ectopic placement of ileal intermediate cells in Irgm1 KO mice. Ileal tissue from Irgm1 KO mice was processed for hematoxylin-and-eosin staining (A; ×600 magnification), immunofluorescence staining with anti-Muc2 (green) and anti-Lyz (red) antibodies (B; ×600 magnification), and transmission electron microscopy [C; ×2,500 (left) and ×7,500 (right) magnification]. All modalities show numerous intermediate cells with properties of both goblet and Paneth cells (white arrows).

Paneth cell abnormalities in Irgm1 KO mice. A and B: histological staining with hematoxylin and eosin (A; ×400 magnification) and transmission electron microscopy (B; ×5,000 magnification) of ileal tissues from untreated WT and Irgm1 KO mice. C: lysozyme (Lyz) immunohistochemical staining of ileal tissue from control and DSS-treated WT and KO mice (×200 magnification). D: total number of lysozyme (Lyz)-positive cells (left) and Lyz-positive cells above the base of the crypt (ectopic, right). Values are means ± SE; data were collected from 3 individual mice for each group, with ≥10 crypt-villus units per mouse. *P < 0.05.

Altered autophagy in Irgm1 KO intestinal cells. Ileal tissue from untreated WT and Irgm1 KO mice was processed for immunofluorescence staining with anti-LC3 (green) and anti-Lyz (red) antibodies (A and C) or immunohistochemical staining with anti-LC3 (brown) followed by hematoxylin counterstain (purple; B and D). Images are representative of 8 mice examined per genotype. Magnification ×400. Quantitative analyses from control and DSS-treated WT and Irgm1 KO mice include average percentage of Paneth cells that were LC3-positive (E; 5 mice analyzed per genotype, with 150 cells examined per mouse) and total LC3-positive punctae per LC3-positive Paneth cell (F; 4 mice analyzed per genotype, with 40 LC3-positive cells examined per mouse, counted on ≥15 crypts per section). Values are means ± SE. *P < 0.05, **P < 0.001.

Altered antimicrobial peptide production in Irgm1 KO mice. Male WT and Irgm1 KO mice were given 3% DSS in drinking water (DSS) or maintained on standard drinking water (cont) for 7 days. Quantitative RT-PCR was used to measure transcript levels of selected molecules from several mouse antimicrobial peptide classes, including Lyz, Reg3γ, and Ang4 (A), representative α-defensin isoforms, including Defa3, Defa5, and Defa20 (B), and villin (Vil1, C), as an indicator of total epithelial cell mass. Values (means ± SE) are normalized to β-actin and expressed as fold change relative to the WT control group. Cohort sizes were as follows: n = 13 WT control, 11 KO control, 10 WT DSS, and 9 KO DSS. *P ≤ 0.05.

Transcript expression of selected genes altered in Atg16L1^HM mice. Atg16L1-deficient Paneth cells have been shown to exhibit increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) pathways, adipocytokine signaling, lipid metabolism, and acute-phase reactant responses. Ileal mRNA expression of specific transcripts enriched in Atg16L1-deficient Paneth cells, relevant to these pathways, was evaluated in untreated WT and Irgm1 KO mice by quantitative RT-PCR. A: stearoyl-coenzyme A desaturase 1 (Scd1), involved in lipid metabolism. B: adiponectin (Adipoq), involved in adipocytokine signaling. C: serum amyloid A1 (Saa1), involved in acute-phase reactant response. D: leptin (Lep), involved in adipocytokine signaling and the PPAR pathway. Values are means ± SE; n = 9 WT and KO. No significant differences were observed for any of the transcripts tested (P > 0.2).

Altered mitophagy in Irgm1 KO cells. A–C: representative transmission electron micrographs from WT mouse ileal enterocytes (A), Irgm1 KO ileal enterocytes (B), and Irgm1 KO goblet cells (C). Magnification ×20,000. Note swollen mitochondria (arrowheads) and tubular mitochondria (outlined with dashed line) in Irgm1 KO cells in B and C, respectively. These phenotypes were seen in multiple Irgm1 KO enterocytes and goblet and Paneth cells and were not cell-specific. D: representative WT mouse embryonic fibroblasts transfected with pMito to label mitochondria, exposed to IFN-γ for 24 h, and then processed for staining with anti-Irgm1 antibodies. Magnification ×1,000. E: representative WT and Irgm1 KO mouse embryonic fibroblasts exposed to IFN-γ for 24 h, stained with Mito Tracker Red to label mitochondria, and then imaged. Magnification ×1,000. F: quantified punctate, tubular, or mixed mitochondrial morphologies assessed blindly from >50 cells per treatment group per experiment, with the averages of 4 experiments shown. Values are means ± SE. *P < 0.05.

Enhanced ileal inflammation in Irgm1 KO mice receiving DSS. Male WT and Irgm1 KO mice were given 3% DSS in drinking water (DSS) or maintained on standard drinking water (control) for 7 days. A: representative histological tissue sections from ileum of WT control, WT DSS, KO control, and KO DSS mice (hematoxylin-eosin stain, ×100 magnification). B: average histological scores from ileum. C: results of quantitative RT-PCR measurement of ileal tissue TNF-α transcript levels in DSS-treated and control mice, normalized to Gapdh and expressed as fold change relative to WT control group. Values are means ± SE; data were combined from ≥3 experiments. For A and B, combined cohort sizes were as follows: n = 9 WT control and WT DSS and 7 KO control and KO DSS; for C, combined cohort sizes were n = 13 WT control, 11 KO control, 10 WT DSS, and 9 KO DSS. *P < 0.02, **P < 0.006.

Enhanced colonic inflammation in immunity-related GTPase (Irg) M (Irgm)-deficient (Irgm1 KO) mice receiving dextran sodium sulfate (DSS). Male wild-type (WT) and Irgm1 KO mice were given 3% DSS in drinking water (DSS) or maintained on standard drinking water (control) for 7 days. A–C: clinical indicators of inflammation [average body weight, average stool consistency score, and average bleeding (Hemoccult) index] at days 0–7. D: average colon length at necropsy. E: representative histological tissue sections from transverse colon (hematoxylin-eosin stain; ×100 magnification). F: average histological scores from proximal (Prox), transverse (Trans), and distal (Dist) colon and cecum. Values are means ± SE; data were combined from 3 separate studies. Combined cohort sizes were as follows: n = 9 WT control and WT DSS and 7 KO control and KO DSS. *P < 0.05, **P < 0.005 (KO DSS vs. WT DSS).