1. Volume 133, Issue 1, Pages 150-163.e3 (July 2007)
Toll-Like Receptor–Dependent Activation of Antigen-Presenting Cells Affects Adaptive Immunity to Helicobacter pylori 
Roland Rad, Lena Brenner, Anne Krug, Petra Voland, Jörg Mages, Roland Lang, Susanne Schwendy, Wolfgang Reindl, Anar Dossumbekova, Wibke Ballhorn, Hermann Wagner, Roland M. Schmid, Stefan Bauer, Christian Prinz 
Gastroenterology 
Volume 133, Issue 1, Pages e3 (July 2007)
DOI: /j.gastro
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2. Figure 1
Interaction of H pylori with DCs. (A) Adherence of GFP-expressing H pylori to BMDCs. Day 7 mouse BMDCs were cultured overnight on tissue culture slides and incubated with GFP-transformed H pylori for 15 minutes. Combined transmission and confocal microscopic image of BMDCs stained with TOPRO 3 (blue nuclear staining) and Nile Red (red lipid staining; original magnification, 400×). (B) Confocal microscopic image showing the uptake of H pylori into BMDCs after 6 hours of incubation. DCs were stained with a hamster anti-mouse CD11c monoclonal antibody. The arrows indicate intracellularly localized bacteria (original magnification, 400×). (C) CD11c-positive DCs in the mouse gastric mucosa in close contact to a gastric gland (original magnification, 1000×). (D and E) Cytokine secretion from day 7 BMDCs in response to H pylori SS1 lysate and E coli LPS (100 ng/mL). IL-12p70 and IL-10 protein levels were detected after 24 hours in the supernatant by ELISA. Bars indicate the arithmetic mean and SEM from 4–6 samples per group. Basal IL-10 secretion was not detectable (n.d.). P values were calculated by a Mann–Whitney U test. One of 3 experiments yielding similar results is shown. *P < .05; **P < .01; ***P < (F–H) FACS analysis of MHC class II (IAb) and costimulatory molecule expression on DCs. Day 7 BMDCs were untreated (black open histograms) or stimulated with 1 μg (blue open histograms) or 10 μg H pylori SS1 lysate (red shaded histograms) for 24 hours. Histograms show expression of (F) CD80, (G) CD86, and (H) IAb on gated CD11c-positive BMDCs. One of 3–6 experiments yielding similar results is shown in panels D–H.
Gastroenterology  , e3DOI: ( /j.gastro )
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3. Figure 2
TLR signaling affects up-regulation of MHC II and costimulatory molecules in response to H pylori. FACS analysis of CD80, CD86, and IAb expression on BMDCs from wild-type, Myd88−/−, IL-1R−/−, and IL-18−/− mice. Day 7 BMDCs were untreated (□) or stimulated with 10 μg H pylori SS1 lysates (grey shaded histograms) or 100 ng E coli LPS (dark grey shaded histograms) for 24 hours. Histograms show expression of surface CD80, CD86, and IAb on gated CD11c+ DCs. One of 2–6 representative experiments yielding similar results is shown. By using living H pylori, the up-regulation of IAb and costimulatory molecules also was reduced significantly in Myd88-deficient mice, but was not abrogated completely (data not shown).
Gastroenterology  , e3DOI: ( /j.gastro )
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4. Figure 3
Cytokine expression in DCs is controlled by signals from TLRs. Day 7 BMDCs from wild-type, Myd88−/−, IL-1R−/−, and IL-18−/− mice were either untreated (■) or stimulated with H pylori SS1 (MOI 50; grey bars) for 6 hours. Expression of proinflammatory cytokines was analyzed by real-time reverse-transcription PCR and normalized to β-actin expression. Shown are pooled data from 3 similar experiments. Bars indicate the arithmetic mean and SEM from 6–8 samples per group. Statistically significant differences between values from stimulated Myd88−/− DCs and values from stimulated wild-type, IL-18−/−, or IL-1R−/− mice were statistically significant in all cases (*P < .05; **P < .001).
Gastroenterology  , e3DOI: ( /j.gastro )
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5. Figure 4
The H pylori–induced DC transcriptome is largely dependent on Myd88 signaling. (A) Hierarchic clustering of significantly regulated genes that were induced or repressed more than 3-fold in wild-type and/or Myd88−/− BMDCs. Average expression values were gene-wise normalized using Z-scores. Day 7 BMDCs were either untreated or incubated with H pylori SS1 (MOI, 50) for 6 hours. (B) Venn diagrams illustrating transcriptional differences between wild-type and Myd88−/− DCs. A total of 178 genes were regulated more than 3-fold in wild-type and/or Myd88−/− DCs. The green and red circles represent genes up-regulated more than 3-fold in wild-type and Myd88−/− DCs, respectively. A total of 126 genes (70.8%) were up-regulated uniquely in wild-type DCs, 4 genes (2.2%) only in Myd88−/− DCs, and 48 genes (27%) in both. Main functional gene groups with a predominant presence in one of the clusters are listed below the venn diagram.
Gastroenterology  , e3DOI: ( /j.gastro )
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6. Figure 5
Activation of B cells and macrophages from wild-type and Myd88-deficient mice by H pylori. (A–D) B cells and (E and F) day 7 bone marrow–derived macrophages from wild-type and Myd88−/− mice were either untreated (grey bars) or stimulated with H pylori SS1 (MOI, 50; ■) for 24 hours. (A, B, E, and F) Cytokine secretion was measured by ELISA from the supernatants. Shown are pooled data from 2 similar experiments (*P < .05; ***P < .001). For FACS analysis, B cells were stained with CD19-FITC, B220-APC, and CD86-hycoerythrin. CD86 expression in gated CD19+/B220+ B cells is shown. The purity of B cells was 90%–95%. nd, not detected.
Gastroenterology  , e3DOI: ( /j.gastro )
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7. Figure 6
Gastric inflammation and cytokine expression in wild-type and Myd88−/− mice infected with the H pylori strain SS1. Representative H&E-stained stomach sections from a mouse with no signs of inflammation (A and B, grade 0 inflammation) and 1 animal with dense leukocytic infiltration (C and D, grade 4 inflammation). Arrowheads and arrows indicate leukocytes in the lamina propria and between the glands, respectively. (E) Inflammatory scores in H pylori–infected wild-type and Myd88−/− mice 4 months postinfection. Each rhombus represents 1 mouse. Horizontal lines show mean values. One of 3 similar experiments is shown. Photographs were taken at magnifications of (A and C) 100× or (B and D) 200×. (F) Cytokine expression in the stomachs of mice 4 months postinfection with the H pylori strain SS1. Mucosal mRNA expression of cytokines was determined by real-time reverse-transcription PCR and normalized to β-actin expression. Bars indicate the arithmetic mean and SEM from 1 experiment including 30 mice. Similar results were obtained in 2 similar experiments (*P < .05).
Gastroenterology  , e3DOI: ( /j.gastro )
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8. Figure 7
H pylori–specific IgG2c and IgG1 levels in wild-type and Myd88-deficient mice. H pylori–specific immunoglobulin titers were determined in 1:1000 diluted sera of H pylori SS1–infected wild-type (grey bars; n = 20) and Myd88−/− mice (■; n = 10) 4 month postinfection with the H pylori strain SS1. Similar results were obtained in 2 separate infection experiments (n = 4–10 mice per group per experiment). The 2-sided P value was calculated by a Mann–Whitney U test. Asterisks indicate significant differences: ***P < .001.
Gastroenterology  , e3DOI: ( /j.gastro )
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9. Figure 8
Increased bacterial colonization in Myd88-deficient mice. Colonization density of the H pylori strain SS1 was determined in the stomachs of wild-type and Myd88-deficient mice 4 months postinfection by (A) quantitative culture or (B) quantification of bacterial ureB DNA. In panel A, values are expressed as colony forming units (cfus) per gram of stomach tissue. Each square represents one animal. Quantification of bacterial ureB DNA in panel B was performed by real-time PCR and normalized to β-actin DNA amounts. P values were calculated by a Mann–Whitney U test. One of 3 experiments yielding similar results is shown (*P < .05; **P < .01).
Gastroenterology  , e3DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions