Volume 138, Issue 7, Pages 2399-2409.e1 (June 2010) CD206-Positive M2 Macrophages That Express Heme Oxygenase-1 Protect Against Diabetic Gastroparesis in Mice  Kyoung Moo Choi, Purna C. Kashyap, Nirjhar Dutta, Gary J. Stoltz, Tamas Ordog, Terez Shea Donohue, Anthony J. Bauer, David R. Linden, Joseph H. Szurszewski, Simon J. Gibbons, Gianrico Farrugia  Gastroenterology  Volume 138, Issue 7, Pages 2399-2409.e1 (June 2010) DOI: 10.1053/j.gastro.2010.02.014 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Gastric emptying and glucose levels of the mice studied. Panel A shows individual mean ± SEM T1/2 values for each mouse and the grouped data (*P < .001, one-way ANOVA with Tukey post-test). The 2 horizontal dashed lines indicate the age-adjusted normal range of gastric emptying. Bars represent means ± SEM, whereas the symbols represent individual mice. Panel B shows glucose levels in the 4 groups of mice. Boxes are medians with interquartile ranges. *P < .001, Kruskal–Wallis with Dunn post-test (n = 4). Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Number of macrophages is increased in diabetic mice. Panel A shows a representative Z-axis image from whole mount tissue labeled with antibodies to F4/80 in a nondiabetic mouse. CM, circular muscle layers; LM, longitudinal muscle layers. Panel B shows representative XY field images from whole mount tissues labeled with antibodies to F4/80. Left to right panels are from nondiabetic mice, diabetic mice with normal gastric emptying, diabetic mice with delayed gastric emptying, and diabetic mice treated with hemin after development of delayed gastric emptying. Panel C shows the number of F4/80 positive macrophages counted in stacked confocal images from 9 defined regions from each tissue (expressed as cells/mm3). Bars are means ± SEM, one-way ANOVA with Tukey post-test, *P < .001 (n = 4). Scale bar, 100 μm for all images. Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 CD206-positive macrophages in diabetic mice. Panel A shows the representative images from whole mount tissues labeled with antibodies for F4/80, CD206, and the merged images. Panel B shows the percentage and panel C the number of CD206-positive macrophages counted in stacked confocal images from 9 defined regions from each tissue (expressed as cells/mm3). Bars for panel B are medians with interquartile ranges (Mann–Whitney test, *P < .05, n = 4). Bars for panel C are means ± standard error of mean, t test, *P < .05 (n = 4). Scale bar, 100 μm for all images. Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Expression of CD206 and HO1 after development of delayed gastric emptying and after hemin treatment to reverse the delayed gastric emptying. Panel A shows the representative images from whole mount tissues and labeled with HO1 and CD206 antibodies using tissue from diabetic mice with normal gastric emptying (left), diabetic mice with delayed gastric emptying (middle), and delayed gastric emptying after hemin treatment (right). Panel B shows the number of CD206-positive macrophages and panel C HO1-positive macrophages counted from stacked confocal images from 9 defined regions of the tissue (expressed as cells/mm3). Development of delayed gastric emptying was associated with loss of HO1, CD206-positive macrophages, and this loss was reversed after treatment with HO1. Bars are means ± standard error mean, ANOVA with Tukey post-test, *P < .001 (n = 4). Scale bar, 100 μm for all images. Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 HO1 expression in CD206 positive macrophages. Panel A shows representative images from whole mount tissues labeled with antibodies to CD206 (left), HO1 (right), from a diabetic mouse with normal gastric emptying (top), and a diabetic mouse with delayed gastric emptying (bottom). Panel B shows the percentages of HO1-positive and CD206-positive macrophages in the 2 groups. Development of delayed gastric emptying was associated with loss of CD206 macrophages, and the residual macrophages had decreased HO1 expression. The white ovals show 2 residual CD206 macrophages that did not express HO1. Bars are medians with interquartile ranges, Mann–Whitney test, *P < .001 (n = 4). Scale bar, 100 μm for all images. Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 CD206-positive macrophages in db/db mice. Representative images from sections labeled with an antibody to CD206 (top) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (bottom). Panel A shows a representative image from a nondiabetic control, panel B a representative image from a diabetic mouse with normal gastric emptying, panel C a representative image from a diabetic mouse with delayed gastric emptying, and panel D a representative image from a diabetic mouse that initially had delayed gastric emptying and gastric emptying was normalized with hemin treatment. Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Figure 7 Expression levels of arginase-1, IL-10, CD163, and iNOS mRNA. Panel A shows mRNA expression level of arginase-1, panel B IL-10, panel C CD163, and panel D iNOS in each of the 4 groups of NOD mice. The data are expressed as the fold difference of levels of the target mRNA over the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Bars are means ± SEM, one-way ANOVA with Tukey post-test, *P < .05 and **P < .001 (n = 6). Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 Heme oxygenase 1 (HO1)-positive macrophages are CD206-positive M2 macrophages. Panel A shows representative images from whole mount tissues. Top panels are from a nondiabetic control and bottom panels from a diabetic mouse with normal gastric emptying. Tissue was triply labeled with antisera raised against F4/80, HO1, and CD206, and the merged images are on the far right. The white oval tracks a macrophage labeled with different antibodies showing that HO1 is expressed in CD206-positive M2 macrophages. Panel B shows the percentage and panel C the number of HO1-positive macrophages counted in stacked confocal images from 9 defined regions of the tissue (expressed as cells/mm3). Bars for panel B are medians with interquartile ranges (Mann–Whitney test, *P < .05, n = 4). Bars for panel C are means ± standard error of mean, t test, *P < .05 (n = 4). Scale bar, 100 μm for all images. Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 CD206-positive macrophages were decreased in CrMP-treated nonobese diabetic (NOD) mice. Representative images from sections labeled with an antibody to CD206 (top) and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (bottom). Panel A shows a representative image from a diabetic mouse treated with vehicle, whereas panel B shows a representative image from a diabetic mouse treated with with chromium mesoparphyrin (CrMP) to inhibit heme oxygenase activity. Scale bar, 100 μm for all images. Gastroenterology 2010 138, 2399-2409.e1DOI: (10.1053/j.gastro.2010.02.014) Copyright © 2010 AGA Institute Terms and Conditions