Macrophages are mediators of gastritis in acute Helicobacter pylori infection in C57BL/6 mice

Abstract

Helicobacter pylori is the etiological agent of human chronic gastritis, a condition seen as a precursor to the development of gastrointestinal ulcers or gastric cancer. This study utilized the murine model of chronic H. pylori infection to characterize the role of macrophages in the induction of specific immune responses and gastritis and in the control of the bacterial burden following H. pylori infection and vaccination. Drug-loaded liposomes were injected intravenously to deplete macrophages from C57BL/6 mice, and effective removal of CD11b+ cells from the spleens and stomachs of mice was confirmed by immunofluorescence microscopy. Transient elimination of macrophages from C57BL/6 mice during the early period of infection reduced the gastric pathology induced by H. pylori SS1 but did not affect the bacterial load in the stomach. These data suggest that macrophages are important to the severity of gastric inflammation during H. pylori infection.

FIG. 1.

Absence of CD11b⁺ cells in the spleens and stomachs of Cl₂MDP-liposome-treated mice. C57BL/6 mice received Cl₂MDP-liposomes or PBS via i.v. injection every 4 or 5 days from 8 days prior to the first dose of H. pylori until 10 days after the final dose of H. pylori. Three days after the final dose of bacteria, mice were euthanized and their spleens and stomachs were frozen for analysis by histology. Shown are cryostat sections of the spleens and stomachs of PBS-treated or Cl₂MDP-liposome-treated, H. pylori-infected and naïve animals. Tissues were stained with rat anti-CD11b and anti-rat-fluorescein isothiocyanate (green) and rabbit anti-H. pylori antibodies and anti-rabbit-Texas Red (red, H. pylori-infected stomach only), and nuclei are stained with DAPI. Negative control slides were stained with fluorochrome-conjugated second antibody only. Representative sections from each treatment group are shown (n = 5 for each group). Data are representative of two independently performed experiments. Original magnifications: spleens, ×20; stomachs, ×10.

FIG. 2.

Cl₂MDP-liposome treatment during H. pylori infection does not affect colonization levels or H. pylori-specific antibody responses. C57BL/6 mice were treated with either PBS (white symbols) or Cl₂MDP-liposomes (black symbols) from 8 days prior to the first dose of H. pylori until 10 days after the final dose of H. pylori. (A) At 10 days (n = 5) and 3 months (n = 10) after infection, mice were euthanized and stomach homogenates were plated onto selective media to determine the gastric colonization levels. (B) The H. pylori-specific serum endpoint titer was determined by ELISA 3 months after infection. The median levels of the groups in panels A and B are indicated by dashes, and each symbol represents an individual animal. Data are representative of two independently performed experiments.

FIG. 3.

Cl₂MDP-liposome treatment during H. pylori infection reduces gastric inflammation. C57BL/6 mice were treated with either PBS (white symbols) or Cl₂MDP-liposomes (black symbols) from 8 days prior to the first dose of H. pylori until 10 days after the final dose of H. pylori. (A) Sections of gastric tissue from mice infected H. pylori for 3 months were stained with hematoxylin and eosin and examined for gastritis by light microscopy. Values for mononuclear cell infiltration, described as chronic inflammation, were estimated by using a six-point scale. Shown are representative images of PBS-treated (a to d) and Cl₂MDP-liposome-treated (e to h) mice showing inflammatory infiltrate (arrows) in the upper-body (a [score = 2.5] and e [score = 1.0]), mid-body (b [score = 2.0] and f [score = 1.5]), lower-body (c [score = 4.0] and g [score = 2.5]), and antrum (d [score = 3.0] and h [score = 2.0]) regions of the stomach. Original magnification, ×20. (B) Box plot showing the gastric inflammatory scores of Cl₂MDP-liposome (n = 10, hatched boxes) and PBS-treated (n = 10, white boxes), H. pylori-infected animals determined by histology 3 months after infection. Data are representative of two independently performed experiments.