Galectin-3 Plays an Important Role in Innate Immunity to Gastric Infection by Helicobacter pylori - PubMed (original) (raw)
Galectin-3 Plays an Important Role in Innate Immunity to Gastric Infection by Helicobacter pylori
Ah-Mee Park et al. Infect Immun. 2016.
Abstract
We studied the role of galectin-3 (Gal3) in gastric infection by Helicobacter pylori We first demonstrated that Gal3 was selectively expressed by gastric surface epithelial cells and abundantly secreted into the surface mucus layer. We next inoculated H. pylori Sydney strain 1 into wild-type (WT) and Gal3-deficient mice using a stomach tube. At 2 weeks postinoculation, the bacterial cells were mostly trapped within the surface mucus layer in WT mice. In sharp contrast, they infiltrated deep into the gastric glands in Gal3-deficient mice. Bacterial loads in the gastric tissues were also much higher in Gal3-deficient mice than in WT mice. At 6 months postinoculation,H. pylori had successfully colonized within the gastric glands of both WT and Gal3-deficient mice, although the bacterial loads were still higher in the latter. Furthermore, large lymphoid clusters mostly consisting of B cells were frequently observed in the gastric submucosa of Gal3-deficient mice.In vitro, peritoneal macrophages from Gal3-deficient mice were inefficient in killing engulfed H. pylori Furthermore, recombinant Gal3 not only induced rapid aggregation of H. pylori but also exerted a potent bactericidal effect on H. pylori as revealed by propidium iodide uptake and a morphological shift from spiral to coccoid form. However, a minor fraction of bacterial cells, probably transient phase variants of Gal3-binding sugar moieties, escaped killing by Gal3. Collectively, our data demonstrate that Gal3 plays an important role in innate immunity to infection and colonization of H. pylori.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Figures
FIG 1
Localization of Gal3 in the mouse stomach. (A) Immunohistochemistry (IHC) for Gal3. IHC was performed for Gal3 using tissue sections from formalin-fixed and paraffin-embedded gastrointestinal tracts obtained from WT and Gal3-deficient mice. Representative results from four different anatomical locations are shown (n = 7). (B) PAS staining. Tissue sections from formalin-fixed and paraffin-embedded gastric tissues were stained with PAS. Representative results are shown (n = 7). (C) Immunoblot analysis for Gal3. Lysates were prepared from gastric tissues and surface mucus layer samples obtained from WT and Gal3-deficient mice. Immunoblot analysis was performed for Gal3. Representative results from three separate experiments are shown.
FIG 2
Role of Gal3 in H. pylori infection. WT and Gal3-deficient mice were inoculated with 5 × 107H. pylori cells daily for 5 days using a stomach tube. Two weeks after the last inoculation, mice were sacrificed and samples were taken. (A) Quantitation of H. pylori cells in the stomach. After washing in PBS was performed, excised stomach tissues were finely minced and vigorously shaken in PBS for 10 min. Supernatants were plated on brucella agar plates and cultured at 37°C for 4 days under microaerobic conditions. Colonies were counted by using Image J software. Data are shown as means ± standard deviations (SD) (n = 7). *, P < 0.05. (B) Measurement of serum anti-H. pylori IgG levels. An ELISA was used to determine serum anti-H. pylori IgG contents. Data are shown as means ± SD (n = 7). *, P < 0.05. (C) IHC of H. pylori. The bacterial cells were stained by IHC in formalin-fixed and paraffin-embedded gastric tissue sections (brown dots). Nuclei were counterstained with hematoxylin (blue). Magnified images of the squared regions are shown below. Representative results from three separate experiments are shown. (D) Immunofluorescence staining of Gal3 and H. pylori. Tissue sections were stained for Gal3 (red) and H. pylori (green) using secondary antibodies conjugated with Alexa 555 and Alexa 488, respectively. Nuclear DNA was counterstained with TO-PRO-3 (blue). The white dotted lines indicate the mucosal surface of the stomach. Representative results from four separate experiments are shown.
FIG 3
Long-term outcome of H. pylori infection. WT and Gal3-deficient mice were inoculated with 5 × 107H. pylori cells daily for 5 days using a stomach tube. Six months after the last inoculation, body weights were measured and samples were taken. (A) Average body weights. Data are shown as means ± standard errors (SE) (n = 8). *, P < 0.05. (B) Quantitation of H. pylori cells. After washing in PBS was performed, excised gastric tissues were finely minced and vigorously shaken in PBS for 10 min. Supernatants were plated on brucella agar plates and cultured at 37°C for 4 days under microaerobic conditions. Formed colonies were counted using Image J software. Data are shown as means ± SE (n = 8). *, P < 0.05. (C) IHC of H. pylori. Formalin-fixed and paraffin-embedded gastric tissue sections were immunohistochemically stained for H. pylori (brown dots). Nuclei were counterstained with hematoxylin (blue). Magnified images of the squared regions are shown on the right. Representative results are shown (n = 8). (D) IHC of gastric tissue. IHC was performed for CD45R and CD3 using formalin-fixed and paraffin-embedded gastric tissue sections. Representative results are shown (n = 8). (E) Immunofluorescent staining of gastric tissue. Double immunofluorescent staining was performed for IgG (H+L) (red) and CD45R (green). Nuclei were counterstained with TO-PRO-3 (blue). Representative results are shown (n = 6).
FIG 4
Tissue infiltration of macrophages. WT and Gal3-deficient mice were inoculated with 5 × 107H. pylori cells daily for 5 days using a stomach tube. Two weeks after the last inoculation, mice were sacrificed and samples were taken. (A) Detection of macrophages. IHC was performed using formalin-fixed and paraffin-embedded gastric tissue sections. Macrophages were detected as F4/80+ cells. Magnified images of the squared regions are shown below. Representative results are shown (n = 7). (B) Enumeration of macrophages. F4/80+ cells were counted using Image J software. Data are shown as means ± SE (n = 7). *, P < 0.05.
FIG 5
Role of Gal3 in macrophage function. Peritoneal macrophages were obtained from WT and Gal3-deficient mice. (A) Phagocytosis assay. Macrophages were incubated with FITC beads (2 μm) or H. pylori cells at 37°C. At the indicated time points, macrophages were fixed. For detection of FITC beads (green), fixed macrophages were stained with TO-PRO-3 for nuclei (blue) and with rhodamine-phalloidin for cytoskeleton (red). For detection of intracellular H. pylori, fixed macrophages were first stained for H. pylori (green) and then stained with TO-PRO-3 for nuclei (blue) and with rhodamine-phalloidin for cytoskeleton (red). Cells containing FITC beads or H. pylori were counted using Image J software. Data are shown as means ± SE (n = 5). (B) Intracellular killing assay. Macrophages were incubated with H. pylori or E. coli at 37°C for 4 h. After washing with PBS was performed, cell lysates were prepared with 0.1% saponin, spread on agar plates, and cultured at 37°C. Colonies were counted using Image J software. Data are shown as means ± SE (n = 6). *, P < 0.05. (C) NO2− production assay. Macrophages were incubated with H. pylori at 37°C for 2 days. Culture supernatants were collected and mixed with 1% Griess-Romijn nitrite reagent. Absorbance at 550 nm was read. Data are shown as means ± SE (n = 6). (D) Immunofluorescence staining. Macrophages were incubated with H. pylori at 37°C for 30 min. After washing was performed, macrophages were fixed and stained for H. pylori (red) and Gal3 (green). Nuclei were counterstained with TO-PRO-3 (blue). Fluorescent images were taken on a confocal laser microscope. Representative results from four separate experiments are shown.
FIG 6
Effect of recombinant Gal3 on H. pylori. H. pylori and E. coli in the mid-logarithmic growth phase were used. Bacterial cells were suspended in PBS (1 × 103 cells/10 μl), mixed with recombinant human Gal3, and incubated at 37°C in a 5% CO2 incubator. (A) Colony-forming assay. H. pylori and E. coli bacteria were suspended in PBS and treated with indicated concentrations of recombinant human Gal3 at 37°C for 1 h. Viable bacteria were quantitated by colony formation on agar. Data are shown as means ± SD (n = 6). *, P < 0.05. (B) Effect of lactose. H. pylori bacteria were incubated with 2 μM Gal3 with or without 2 mM lactose at 37°C for 1 h. Viable bacteria were quantitated by colony formation on agar. Data are shown as means ± SD (n = 6). *, P < 0.05. (C) Microscopic observation. H. pylori and E. coli bacteria were suspended in PBS and treated with 2.5 μM Gal3 for 10 min. Microscopic images were taken. Representative results from six separate experiments are shown. (D) Bactericidal assay. H. pylori cells were suspended in PBS and treated with 2 μM Gal3 without or with 2 mM lactose for 10 min in the presence of CFDA (the live-bacterium staining reagent) (green). After incubation, killed cells were stained with PI (red). Representative results from four separate experiments are shown. (E) Scanning electron microscopic observation. H. pylori and E. coli bacteria were suspended in PBS and treated with 2.5 μM Gal3 for 1 h. Images were taken on a scanning electron microscope. Bar, 1 μm. Representative results from three separate experiments are shown. (F) Measurement of cellular ATP content. H. pylori and E. coli bacteria were suspended in HEPES-buffered saline (pH 7.4) and treated with indicated concentrations of Gal3 at 37°C for 1 h. Cellular ATP contents were determined by using luciferase-catalyzed luciferin chemiluminescence. Data are shown as means ± SD (n = 3). *, P < 0.05. (G) Analysis of Gal3-resistant clones. H. pylori bacteria were incubated with 2 μM Gal3 at 37°C for 1 h and spread on agar plates. Formed colonies were individually isolated and expanded in liquid culture. The expanded clones were then treated with 2 μM Gal3 at 37°C for 1 h. Viable bacteria were quantitated by colony formation on agar. Data are shown as means ± SD (n = 5). *, P < 0.05.
References
- Murray E, Khamri W, Walker MM, Eggleton P, Moran AP, Ferris JA, Knapp S, Karim QN, Worku M, Strong P, Reid KB, Thursz MR. 2002. Expression of surfactant protein D in the human gastric mucosa and during Helicobacter pylori infection. Infect Immun 70:1481–1487. doi: 10.1128/IAI.70.3.1481-1487.2002. -DOI -PMC -PubMed
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