Suppression of cell division-associated genes by Helicobacter pylori attenuates proliferation of RAW264.7 monocytic macrophage cells

Abstract

Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.

Figure 1. H. pylori-infected RAW264.7 monocytic macrophage cells.

RAW264.7 cells were seeded at 5 × 10⁵ /ml and infected with the indicated MOIs of H. pylori for 24 h. (a) Representative pictures of control and infected cells viewed under light microscope. Objective 200×. (b) Flow cytometry analysis of the control and infected cells. Intensities of forward scatter (FCS) and side scatter (SSC) indicate the cell size and complexity, respectively. Numbers represent the percentages of cells in the gated area. Shown are representative data of three independent experiments.

Figure 2. Microarray analysis of H. pylori-infected RAW264.7 cells.

(a and b) Scatter plots show the expressions of total probes (a) and significant probes (b), in the non-infected control versus H. pylori (MOI 10, 24 h)-infected cells. Significant probes were selected based on FC < –2 or FC > 2, P < 0.05. X and Y axis show normalized log₂ values. Number represents the number of probes in each quadrant. Blue and red dots show the intensities of up- or down-regulated probes, respectively based on the normalized values on X-axis. Three green lines demarcate the probes with FC of –2, 0 and 2. (c) Hierarchical clustering (HCL) for significant 5122 probes (2932 genes) was executed with Pearson Correlation distance metric and average linkage. (d) KEGG pathway analysis. Bar chart showing the FE of significantly modulated pathways in the H. pylori-infected RAW264.7 cells relative to control (FE < –2 or FE > 2, P < 0.05). In total, 8 pathways showed induction while 16 pathways were reduced.

Figure 3. H. pylori infection up-regulates genes encoded for immune reactions.

(a) Heatmap of significant genes encoded for cytokines, surface markers, chemokines, and intergrins. Color intensity reflects the normalized log₂ values of the RNA abundance. Yellow: increase, blue: decrease, dark: no change. (b) Flow cytometrical analysis of cell surface markers on the control and H. pylori (MOI 10, 24 h)-infected cells. Fluorescence intensities for different markers were as shown. Numbers represent the percentages of cell in the gated area.

Figure 4. H. pylori infection down-regulates genes encoded for DNA synthesis and cell cycle molecules.

(a) Heatmap of significant genes encoded for DNA synthesis and cell cycle molecules. Color intensity reflects the normalized log₂ values of the RNA abundance. Yellow: increase, blue: decrease, dark: no change. (b) qRT-PCR analysis. Relative fold change shows expression of each gene relative to internal control β-actin. –: Non-infected control; +: H. pylori (MOI 10, 24 h)-infected cells. Data were shown as mean ± SD, from one experiment run in triplicate. Statistical significance was analyzed with unpaired Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5. H. pylori infection blocks cell cycle progress.

(a) Cell cycle analysis of control and H. pylori (MOI 1, 5 or 10)-infected RAW264.7 cells. Cells were harvested at 24 hpi, fixed and stained with propidium iodide (PI) to detect DNA contents. Numbers represent the percentages of cells at G0/G1, S or G2/M phases. Data were shown as mean ± SD from one experiment run in duplicate, and were representative data of two independent experiments. (b) Immunoblot analysis of cell lysates prepared from control and H. pylori-infected RAW264.7 cells for 24 or 48 h. Antibodies against AurkB, Cenp-a, phospho-Cdk1 (Cdc2) or Cyclin D1 were used. β-actin was used as loading control. *All gels were run under same experimental condition. Images were cropped from full length blots (Supplementary Figure S4). Shown are representative data of two independent experiments.

Figure 6. Effect of H. pylori infection on the cell proliferation of RAW264.7 cells.

(a) Bar chart shows absolute cell count of the non-infected and H. pylori-infected cells at 12 or 24 hpi. Data were shown as mean ± SD, from one experiment run in triplicate. Statistical significance were analyzed with unpaired Student’s t-test (**P < 0.01, ***P < 0.001). (b and c) Flow cytometrical analysis of intranuclear expression of Ki-67 cell proliferation marker in the control and H. pylori-infected cells. (b) RAW264.7 cells were infected with 1, 5, 10 and 100 MOIs of H. pylori SS1 strain for 24 h. (c) RAW264.7 cells were infected with different strains of H. pylori including SS1, J99 and 298 at MOI 100 for 24 h. Numbers represent the percentages of cells in the gated area. Shown were representative data of two independent experiments.

Figure 7. Effect of H. pylori on the cell proliferation of primary macrophage cells.

BMDM cells were prepared by stimulating the C57BL/6 mice bone marrow cells with M-CSF (20 ng/ml) for 7 days. BMDM cells were infected with MOI 10 of H. pylori SS1 strain for 24 h. (a) Photos of the non-infected and H. pylori SS1 (MOI 10)-infected BMDM cells. (b) Flow cytometrical analysis of forward scatter (FSC), side scatter (SSC) and intranuclear expression of Ki-67 cell proliferation marker in the control and H. pylori-infected cells. Numbers represent the percentages of cells in the gated area. Shown were representative data of two independent experiments.