Figure 3.
LysM-Atg5−/− mice have splenic and enterocytic iron depletion, ferroportin membrane localization, and body iron overload. (A) Histologic examination of nonheme iron deposits in the spleen, duodenum, and liver in LysM-Atg5−/− and WT mice. Nonheme iron deposition was visualized by Perl’s staining (blue) of spleen and duodenum sections and after DAB enhancement (brown) for liver sections (representative images are shown from n = 12 per group; NanoZoomer scan imaging; original magnification, 10× for spleen and liver and 20× for duodenum; scale bars, 100 μm for spleen and liver and 40 μm for duodenum; enlarged inset images, 40×; scale bars, 20 μm). The duodenum images show the proximal (top) part where food was no longer in transit and the more distal (down) part where food was in transit. Iron was localized in the apical compartment of WT enterocytes in both parts of the duodenum, whereas in LysM-Atg5−/− enterocytes, no iron was seen in the proximal duodenum, but iron was localized in both the apical and basal compartments of the distal duodenum. Enlarged liver images show Kupffer cells stained for iron in WT mice and hepatocyte staining in LysM-Atg5−/− mice. (B) Tissue iron content in the spleen (i top) and liver (iii top) in LysM-Atg5−/− and WT mice, as determined by a ferrozine assay and reported as ng of iron per mg of dried tissue. Data are presented as histograms of the mean and standard error values with dot plots (n = 10 in each group). Representative Fpn immunoblotting from duodenal membrane protein extracts (ii top) from LysM-Atg5−/− and WT mice (n = 2 samples per genotype are shown from 6 mice). Representative staining of ferric iron loaded in ferritin (Fe-Ft) and immunoblotting of ferritin H (Ft-H) in the spleen (i bottom), duodenal (ii bottom), and liver (iii bottom) extracts. For ferric iron–loaded ferritin, 25 ng of native proteins from cytosolic lysates was loaded in each lane of a nondenaturing polyacrylamide gel electrophoresis (PAGE) gel, and after electrophoresis, the gel was directly stained with Perl’s staining solution and enhanced with DAB. The gel was then immunoblotted for Ft-H as a control for the band specificity (representative images from n = 4 experiments are shown). For Ft-H western blot, 25 ng of cytoplasmic extracts was loaded on a denaturing PAGE gel, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is shown as the loading control. Densitometric analysis was performed on the immunoblot shown as well as on immunoblots with lysates from 3 additional LysM-Atg5−/− and WT mice; after normalization to Gapdh in each lane, LysM-Atg5−/− results were normalized to the WT control group average. Data are presented as means ± standard errors. (C) Calcein fluorescence quenching assay in spleen cells from LysM-Atg5−/− and WT mice for measurement of the labile iron pool. Immediately after recovery, cells dissociated from the spleen were stained simultaneously with calcein (CA-AM) and anti-mouse PerCP-conjugated CD11b and phycoerythrin-Cy7–conjugated F4/80 antibodies and were then analyzed by flow cytometry. Gates were set on FSC, CD45, and TCRβ. Representative calcein fluorescence profiles of unstained cells (FMO-calcein) and Calcein-AM–stained cells (WT and LysM-Atg5−/−) with respect to cell markers are shown, and histograms with the mean and standard error values from 3 independent experiments are shown as dot plots (from n = 3 independent experiments). (D) Detection of ferroportin by immunofluorescence in spleen tissues, isolated cells from the spleen, and proximal duodenal villi from LysM-Atg5−/− and WT mice. Representative images are composites from ferroportin (red) and 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei (blue) (n = 4 animals per group; for spleen and spleen cell smear: Leica fluorescence microcopy imaging; original magnification, 20×; scale bars, 20 μm; enlarged inset images, magnification 100×; scale bars, 5 μm; for liver: Zeiss inverted microscopy imaging; original magnification, 20×; scale bars, 20 μm). *P < .05, ****P < 10−4. MFI, mean fluorescence intensity.

LysM-Atg5−/− mice have splenic and enterocytic iron depletion, ferroportin membrane localization, and body iron overload. (A) Histologic examination of nonheme iron deposits in the spleen, duodenum, and liver in LysM-Atg5−/− and WT mice. Nonheme iron deposition was visualized by Perl’s staining (blue) of spleen and duodenum sections and after DAB enhancement (brown) for liver sections (representative images are shown from n = 12 per group; NanoZoomer scan imaging; original magnification, 10× for spleen and liver and 20× for duodenum; scale bars, 100 μm for spleen and liver and 40 μm for duodenum; enlarged inset images, 40×; scale bars, 20 μm). The duodenum images show the proximal (top) part where food was no longer in transit and the more distal (down) part where food was in transit. Iron was localized in the apical compartment of WT enterocytes in both parts of the duodenum, whereas in LysM-Atg5−/− enterocytes, no iron was seen in the proximal duodenum, but iron was localized in both the apical and basal compartments of the distal duodenum. Enlarged liver images show Kupffer cells stained for iron in WT mice and hepatocyte staining in LysM-Atg5−/− mice. (B) Tissue iron content in the spleen (i top) and liver (iii top) in LysM-Atg5−/− and WT mice, as determined by a ferrozine assay and reported as ng of iron per mg of dried tissue. Data are presented as histograms of the mean and standard error values with dot plots (n = 10 in each group). Representative Fpn immunoblotting from duodenal membrane protein extracts (ii top) from LysM-Atg5−/− and WT mice (n = 2 samples per genotype are shown from 6 mice). Representative staining of ferric iron loaded in ferritin (Fe-Ft) and immunoblotting of ferritin H (Ft-H) in the spleen (i bottom), duodenal (ii bottom), and liver (iii bottom) extracts. For ferric iron–loaded ferritin, 25 ng of native proteins from cytosolic lysates was loaded in each lane of a nondenaturing polyacrylamide gel electrophoresis (PAGE) gel, and after electrophoresis, the gel was directly stained with Perl’s staining solution and enhanced with DAB. The gel was then immunoblotted for Ft-H as a control for the band specificity (representative images from n = 4 experiments are shown). For Ft-H western blot, 25 ng of cytoplasmic extracts was loaded on a denaturing PAGE gel, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) is shown as the loading control. Densitometric analysis was performed on the immunoblot shown as well as on immunoblots with lysates from 3 additional LysM-Atg5−/− and WT mice; after normalization to Gapdh in each lane, LysM-Atg5−/− results were normalized to the WT control group average. Data are presented as means ± standard errors. (C) Calcein fluorescence quenching assay in spleen cells from LysM-Atg5−/− and WT mice for measurement of the labile iron pool. Immediately after recovery, cells dissociated from the spleen were stained simultaneously with calcein (CA-AM) and anti-mouse PerCP-conjugated CD11b and phycoerythrin-Cy7–conjugated F4/80 antibodies and were then analyzed by flow cytometry. Gates were set on FSC, CD45, and TCRβ. Representative calcein fluorescence profiles of unstained cells (FMO-calcein) and Calcein-AM–stained cells (WT and LysM-Atg5−/−) with respect to cell markers are shown, and histograms with the mean and standard error values from 3 independent experiments are shown as dot plots (from n = 3 independent experiments). (D) Detection of ferroportin by immunofluorescence in spleen tissues, isolated cells from the spleen, and proximal duodenal villi from LysM-Atg5−/− and WT mice. Representative images are composites from ferroportin (red) and 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei (blue) (n = 4 animals per group; for spleen and spleen cell smear: Leica fluorescence microcopy imaging; original magnification, 20×; scale bars, 20 μm; enlarged inset images, magnification 100×; scale bars, 5 μm; for liver: Zeiss inverted microscopy imaging; original magnification, 20×; scale bars, 20 μm). *P < .05, ****P < 10−4. MFI, mean fluorescence intensity.

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