"Candidatus Helicobacter heilmannii" from a cynomolgus monkey induces gastric mucosa-associated lymphoid tissue lymphomas in C57BL/6 mice

Abstract

Both Helicobacter pylori and "Candidatus Helicobacter heilmannii" infections are associated with peptic ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas. However, good animal models of H. pylori clinical diseases are rare. In this study, we aimed to establish an animal model of "Candidatus Helicobacter heilmannii" gastric MALT lymphoma. We used a urease-positive gastric mucosal and mucus homogenate from a cynomolgus monkey maintained in C57BL/6 mouse stomachs. The bacterium in the homogenate was identified as "Candidatus Helicobacter heilmannii" based on a DNA sequence analysis of the 16S rRNA and urease genes. Mucosal and mucus homogenates were used to inoculate C57BL/6 mice, which were then examined for 24 months. We observed a gradual increase in the surface area of protrusive lesions in almost all infected C57BL/6 mouse fundic stomachs 6 months after infection. Light microscopic observations revealed an accumulation of B lymphocytes along with destruction of glandular elements and the presence of lymphoepithelial lesions consistent with low-grade MALT lymphomas. Electron microscopic observation revealed numerous "Candidatus Helicobacter heilmannii" bacilli in the fundic glandular lumen, the intracellular canaliculi, and the cytoplasm of intact cells, as well as damaged parietal cells. In conclusion, "Candidatus Helicobacter heilmannii" induced gastric MALT lymphomas in almost 100% of infected C57BL/6 mice after a 6-month period associated with the destruction of parietal cells.

FIG. 1.

Macroscopic observation of control and “Candidatus Helicobacter heilmannii”-infected mouse gastric mucosal surfaces. (a) Uninfected surface. (b) Three months after infection, several round protrusive lesions were observed in the fundic area. (c) Six months after infection, the number of protrusive lesions increased. (d) Twelve months after infection, almost all fundic mucosa samples contained round, protrusive lesions. (e) Eighteen months after infection, the lesions united to form larger lesions.

FIG. 2.

Time course of occupancy of the protrusive lesions in the entire fundic mucosa. The surface area occupied by round, protrusive lesions gradually increased. *, P < 0.05 for a comparison with the control group; #, P < 0.05 for a comparison with the 3-month group; &, P < 0.05 for a comparison with the 12-month group; @, P < 0.05 for a comparison with the 18-month group.

FIG. 3.

Time course showing the copy numbers of “Candidatus Helicobacter heilmannii,” as determined by real-time PCR. At 3, 6, 12, and 18 months after infection, the number of “Candidatus Helicobacter heilmannii” obtained from the gastric homogenate was about 10⁵ CFU per stomach, and this number decreased until it reached approximately 5 × 10³ CFU at 24 months after infection. The asterisk indicates that the P value is <0.01 for a comparison with the data for 18 months.

FIG. 4.

Light microscopic observation of hematoxylin- and eosin-stained, “Candidatus Helicobacter heilmannii”-infected fundic mucosa. (a) Lymphocytic accumulation is recognized in the lamina propria mucosa (arrow) just above the muscularis mucosa 3 months after infection. Magnification, ×60. (b) Lymphoid follicle in the gastric body mucosa, with some accumulation of lymphocytes in the adjacent submucosal layer of mice infected for 6 months. Magnification, ×400. (c) Mouse infected for 12 months with a low-grade MALT lymphoma involving the submucosal layer. Multiple follicles are spanned by profuse bridging aggregates of the hypochromic marginal zone. Magnification, ×80. (d) At higher magnification, clear cell variety is predominant, with larger CCL cells with predominant nucleoli. Magnification, ×1,000.

FIG. 5.

Light and electron microscopic observations of lymphoid follicles and lymphoepithelial lesions. (a) Lymphoid follicle (L) observed in the fundic mucosa of a mouse infected for 6 months. Toluidine blue-stained 1-μm Epon section. Magnification, ×100. (b) Lymphoepithelial lesion in the stomach of a mouse infected for 6 months. Toluidine blue-stained 1-μm Epon section. Magnification, ×600. (c) Invasion of a lymphocyte between fundic glandular cells in a mouse infected for 6 months. Magnification, ×3,000. (d) Accumulation of centrocyte-like cells in a lymphoid follicle. Magnification, ×2,000.

FIG. 6.

Localization of “Candidatus Helicobacter heilmannii” by in situ hybridization, immunohistochemistry, and electron microscopic cytochemistry. (a and b) Many reactive bacilli were recognized by in situ hybridization at the luminal side of the body of the fundic gland (a). No reaction was detected with a sense probe (b). Magnification, ×800. (c and d) Indirect fluorescent immunohistochemistry using anti-H. pylori polyclonal antibody revealed immunoreactive bacilli at the luminal side of the body of the fundic gland (c), and Alexa-phalloidin fluorescence (d) revealed that the localization of bacilli coincided approximately with the localization of f-actin-rich parietal cells. Magnification, ×800. (e, f, and g) Electron microscopy revealed the presence of extremely tortuous bacilli (arrows) in the fundic glandular lumen near the parietal cells (e and f) and in the intracellular canaliculi (e and g). (e) Magnification, ×1,500; (f and g) Magnification, ×6,000. (h and i) Some bacilli (white arrow) were detected in the lamina propria mucosae. An adjacent parietal cell (P) was destroyed. (h) Magnification, ×2,000; (i) Magnification, ×6,000. Just adjacent to the disrupted nucleus of the cell (arrowheads), a substance (arrow) having the same electron density as the bacilli was recognized.

FIG. 7.

Immunohistochemistry and in situ hybridization of MALT lymphomas. (a and b) In situ hybridization revealed a positive reaction in a lymphoid follicle that reacted with primer Hhe1. (a) Magnification, ×400; (b) Magnification, ×1,000. (c) Positive B220 immunoreactivity was recognized in the lymphoid tissue (L). Magnification, ×400. (d) Only a few CD3-immunoreactive cells were detected in the lymphoid follicle (L). Magnification, ×400. (e) Some of the lymphocytes in and near the lymphoid follicle (L) were immunoreactive with Bcl-2. Magnification, ×400. (f) Most of the lymphocytes in the lymphoid follicle (L) were immunoreactive with VEGF-A. Magnification, ×400.