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Enhanced M1 macrophage polarization in human helicobacter pylori-associated atrophic gastritis and in vaccinated mice.

Quiding-Järbrink M, Raghavan S, Sundquist M - PLoS ONE (2010)

Bottom Line: Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination.Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages.However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden.

ABSTRACT

Background: Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined.

Methodology/principal findings: By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion.

Conclusions/significance: These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis.

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M1 polarization of gastric macrophages after H. pylori infection.(A) Expression of genes associated with macrophage polarization in the gastric mucosa of naïve and infected mice was determined by real-time PCR. Symbols represent individual mice, and horizontal bars indicate the medians. *, P<0.05 **, P<0.01 compared to the expression level in uninfected mice via Mann-Whitney U test. (B) Sorting of gastric lamina propria macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+) from pooled (n = 3) mice infected for 26 weeks. Contour plots show expression of CD11b versus Gr1 on gated live cells, and expression of Siglec-F versus MHC-II on gated CD11b+Gr1− cells before and after sorting. The gates for sorting (R1–R4) are shown in the top contour plots. Numbers by gates depict cell frequencies. Note that in this experiment the few CD103+ DCs that fall into the macrophage gate (∼2%, see Fig. 1A) were not gated out. (C) Real-time PCR analysis of iNOS and CXCL11 mRNA expression by total gastric lamina propria cells pooled from naïve (n = 3) or infected (n = 3, 26 weeks) mice before sorting as well as expression by sorted macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+, 9×105 cells), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−, 6×105 cells) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+, 3×106 cells) from infected mice. Bars are the mean of two to three replicates, and dots represent each replicate. Undet., undetected. MΦ, macrophages. Eos, eosinophils.
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pone-0015018-g004: M1 polarization of gastric macrophages after H. pylori infection.(A) Expression of genes associated with macrophage polarization in the gastric mucosa of naïve and infected mice was determined by real-time PCR. Symbols represent individual mice, and horizontal bars indicate the medians. *, P<0.05 **, P<0.01 compared to the expression level in uninfected mice via Mann-Whitney U test. (B) Sorting of gastric lamina propria macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+) from pooled (n = 3) mice infected for 26 weeks. Contour plots show expression of CD11b versus Gr1 on gated live cells, and expression of Siglec-F versus MHC-II on gated CD11b+Gr1− cells before and after sorting. The gates for sorting (R1–R4) are shown in the top contour plots. Numbers by gates depict cell frequencies. Note that in this experiment the few CD103+ DCs that fall into the macrophage gate (∼2%, see Fig. 1A) were not gated out. (C) Real-time PCR analysis of iNOS and CXCL11 mRNA expression by total gastric lamina propria cells pooled from naïve (n = 3) or infected (n = 3, 26 weeks) mice before sorting as well as expression by sorted macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+, 9×105 cells), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−, 6×105 cells) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+, 3×106 cells) from infected mice. Bars are the mean of two to three replicates, and dots represent each replicate. Undet., undetected. MΦ, macrophages. Eos, eosinophils.

Mentions: Since our results suggested that the gastric macrophages might not be fully activated during H. pylori infection, we characterized these cells further by investigating their M1/M2 polarization. To determine macrophage polarization during H. pylori infection, we used real-time PCR to measure the expression of genes associated with M1 or M2 polarization of macrophages in the gastric tissue [8], [9]. We also measured the expression of IL-10, which can be produced by regulatory macrophages [9]. None of the markers analyzed were differentially expressed four weeks after H. pylori infection relative to naïve mice (Fig. 4A). At eight weeks after infection the expression of the M1 markers iNOS and CXCL11 was significantly increased, and these markers were further upregulated at 26 weeks of infection (Fig. 4A). In addition, the expression of IL-10 was upregulated at eight and 26 weeks of infection relative to naïve mice (Fig. 4A). In contrast, the M2 markers found in inflammatory zone 1 (FIZZ1) and arginase-1 were not differentially expressed in the stomach at four, eight or 26 weeks of infection compared to naïve mice (Fig. 4A).


Enhanced M1 macrophage polarization in human helicobacter pylori-associated atrophic gastritis and in vaccinated mice.

Quiding-Järbrink M, Raghavan S, Sundquist M - PLoS ONE (2010)

M1 polarization of gastric macrophages after H. pylori infection.(A) Expression of genes associated with macrophage polarization in the gastric mucosa of naïve and infected mice was determined by real-time PCR. Symbols represent individual mice, and horizontal bars indicate the medians. *, P<0.05 **, P<0.01 compared to the expression level in uninfected mice via Mann-Whitney U test. (B) Sorting of gastric lamina propria macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+) from pooled (n = 3) mice infected for 26 weeks. Contour plots show expression of CD11b versus Gr1 on gated live cells, and expression of Siglec-F versus MHC-II on gated CD11b+Gr1− cells before and after sorting. The gates for sorting (R1–R4) are shown in the top contour plots. Numbers by gates depict cell frequencies. Note that in this experiment the few CD103+ DCs that fall into the macrophage gate (∼2%, see Fig. 1A) were not gated out. (C) Real-time PCR analysis of iNOS and CXCL11 mRNA expression by total gastric lamina propria cells pooled from naïve (n = 3) or infected (n = 3, 26 weeks) mice before sorting as well as expression by sorted macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+, 9×105 cells), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−, 6×105 cells) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+, 3×106 cells) from infected mice. Bars are the mean of two to three replicates, and dots represent each replicate. Undet., undetected. MΦ, macrophages. Eos, eosinophils.
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Related In: Results  -  Collection

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pone-0015018-g004: M1 polarization of gastric macrophages after H. pylori infection.(A) Expression of genes associated with macrophage polarization in the gastric mucosa of naïve and infected mice was determined by real-time PCR. Symbols represent individual mice, and horizontal bars indicate the medians. *, P<0.05 **, P<0.01 compared to the expression level in uninfected mice via Mann-Whitney U test. (B) Sorting of gastric lamina propria macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+) from pooled (n = 3) mice infected for 26 weeks. Contour plots show expression of CD11b versus Gr1 on gated live cells, and expression of Siglec-F versus MHC-II on gated CD11b+Gr1− cells before and after sorting. The gates for sorting (R1–R4) are shown in the top contour plots. Numbers by gates depict cell frequencies. Note that in this experiment the few CD103+ DCs that fall into the macrophage gate (∼2%, see Fig. 1A) were not gated out. (C) Real-time PCR analysis of iNOS and CXCL11 mRNA expression by total gastric lamina propria cells pooled from naïve (n = 3) or infected (n = 3, 26 weeks) mice before sorting as well as expression by sorted macrophages (Gate R1 and R2, CD11b+Gr1−Siglec-F−MHC-II+, 9×105 cells), eosinophils (Gate R1 and R3, CD11b+Gr1−Siglec-F+MHC-II−, 6×105 cells) and remaining cells after gating out macrophages and eosinophils (Gate R4, CD11b− and CD11b+Gr1+, 3×106 cells) from infected mice. Bars are the mean of two to three replicates, and dots represent each replicate. Undet., undetected. MΦ, macrophages. Eos, eosinophils.
Mentions: Since our results suggested that the gastric macrophages might not be fully activated during H. pylori infection, we characterized these cells further by investigating their M1/M2 polarization. To determine macrophage polarization during H. pylori infection, we used real-time PCR to measure the expression of genes associated with M1 or M2 polarization of macrophages in the gastric tissue [8], [9]. We also measured the expression of IL-10, which can be produced by regulatory macrophages [9]. None of the markers analyzed were differentially expressed four weeks after H. pylori infection relative to naïve mice (Fig. 4A). At eight weeks after infection the expression of the M1 markers iNOS and CXCL11 was significantly increased, and these markers were further upregulated at 26 weeks of infection (Fig. 4A). In addition, the expression of IL-10 was upregulated at eight and 26 weeks of infection relative to naïve mice (Fig. 4A). In contrast, the M2 markers found in inflammatory zone 1 (FIZZ1) and arginase-1 were not differentially expressed in the stomach at four, eight or 26 weeks of infection compared to naïve mice (Fig. 4A).

Bottom Line: Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination.Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages.However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Gothenburg, Gothenburg, Sweden.

ABSTRACT

Background: Infection with Helicobacter pylori triggers a chronic gastric inflammation that can progress to atrophy and gastric adenocarcinoma. Polarization of macrophages is a characteristic of both cancer and infection, and may promote progression or resolution of disease. However, the role of macrophages and their polarization during H. pylori infection has not been well defined.

Methodology/principal findings: By using a mouse model of infection and gastric biopsies from 29 individuals, we have analyzed macrophage recruitment and polarization during H. pylori infection by flow cytometry and real-time PCR. We found a sequential recruitment of neutrophils, eosinophils and macrophages to the gastric mucosa of infected mice. Gene expression analysis of stomach tissue and sorted macrophages revealed that gastric macrophages were polarized to M1 after H. pylori infection, and this process was substantially accelerated by prior vaccination. Human H. pylori infection was characterized by a mixed M1/M2 polarization of macrophages. However, in H. pylori-associated atrophic gastritis, the expression of inducible nitric oxide synthase was markedly increased compared to uncomplicated gastritis, indicative of an enhanced M1 macrophage polarization in this pre-malignant lesion.

Conclusions/significance: These results show that vaccination of mice against H. pylori amplifies M1 polarization of gastric macrophages, and that a similar enhanced M1 polarization is present in human H. pylori-induced atrophic gastritis.

Show MeSH
Related in: MedlinePlus