CD206-positive M2 macrophages that express heme oxygenase-1 protect against diabetic gastroparesis in mice - PubMed (original) (raw)

CD206-positive M2 macrophages that express heme oxygenase-1 protect against diabetic gastroparesis in mice

Kyoung Moo Choi et al. Gastroenterology. 2010 Jun.

Abstract

Background & aims: Gastroparesis is a well-recognized complication of diabetes. In diabetics, up-regulation of heme oxygenase-1 (HO1) in gastric macrophages protects against oxidative stress-induced damage. Loss of up-regulation of HO1, the subsequent increase in oxidative stress, and loss of Kit delays gastric emptying; this effect is reversed by induction of HO1. Macrophages have pro- and anti-inflammatory activities, depending on their phenotype. We investigated the number and phenotype of gastric macrophages in NOD/ShiLtJ (nonobese diabetic [NOD]) mice after onset of diabetes, when delayed gastric emptying develops, and after induction of HO1 to reverse delay.

Methods: Four groups of NOD and db/db mice were studied: nondiabetic, diabetic with normal emptying, diabetic with delayed gastric emptying, and diabetic with delayed gastric emptying reversed by the HO1 inducer hemin. Whole mount samples from stomach were labeled in triplicate with antisera against F4/80, HO1, and CD206, and macrophages were quantified in stacked confocal images. Markers for macrophage subtypes were measured by quantitative polymerase chain reaction.

Results: Development of diabetes was associated with an increased number of macrophages and up-regulation of HO1 in CD206(+) M2 macrophages. Onset of delayed gastric emptying did not alter the total number of macrophages, but there was a selective loss of CD206(+)/HO1(+) M2 macrophages. Normalization of gastric emptying was associated with repopulation of CD206(+)/HO1(+) M2 macrophages.

Conclusions: CD206(+) M2 macrophages that express HO1 appear to be required for prevention of diabetes-induced delayed gastric emptying. Induction of HO1 in macrophages might be a therapeutic option for patients with diabetic gastroparesis.

Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

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Figures

Figure 1

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<0.001, oneway ANOVA with Tukey's post-test). The two horizontal dashed lines indicate the age-adjusted normal range of gastric emptying. Bars represent means ± SEM while the symbols represent individual mice. Panel B shows glucose levels in the four groups of mice. Boxes are medians with IQRs. * P<0.001, Kruskal-Wallis with Dunn's post test (n = 4).

Figure 2

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 non-diabetic mouse. Panel B shows representative XY field images from whole mount tissues labeled with antibodies to F4/80. Left to right panels are from non-diabetic 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's post-test, * P<0.001, (n = 4). Scale bar 100 _μ_m for all images.

Figure 3

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 IQRs, (Mann Whitney test, * P < 0.05, n =4). Bars for panel C are means + SEM, t test, * P<0.05, (n = 4). Scale bar 100 _μ_m for all images

Figure 4

Figure 4

Expression of CD206 and HO1 after development of delayed gastric emptying and after hemin treatment to reverse the delayed gastric emptying. The representative images from whole mount tissues are 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 + SEM, ANOVA with Tukey's post test * P<0.001, (n = 4). Scale bar 100 _μ_m for all images.

Figure 5

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 mice with normal gastric emptying (top) and a diabetic mice with delayed gastric emptying (bottom). Panel B shows the percentages of HO1 positive and CD206 positive macrophages in the two 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 two residual CD206 macrophages that did not express HO1. Bars are medians with IQRs, Mann-Whitney test, * P<0.001, (n = 4). Scale bar 100 _μ_m for all images.

Figure 6

Figure 6

CD206 positive macrophages in db/db mice. Representative images from sections labeled with an antibody to CD206 (top) and DAPI (bottom). Panel A shows a representative image from a non-diabetic 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 with that initially had delayed gastric emptying and gastric emptying was normalized with hemin treatment.

Figure 7

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 GAPDH mRNA. Bars are means + SEM, one-way ANOVA with Tukey's post-test, *P<0.05 and ** P<0.001, (n = 6).

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