Inhibition of apoptosis in neuronal cells infected with Chlamydophila (Chlamydia) pneumoniae

Abstract

Background: Chlamydophila (Chlamydia) pneumoniae is an intracellular bacterium that has been identified within cells in areas of neuropathology found in Alzheimer disease (AD), including endothelia, glia, and neurons. Depending on the cell type of the host, infection by C. pneumoniae has been shown to influence apoptotic pathways in both pro- and anti-apoptotic fashions. We have hypothesized that persistent chlamydial infection of neurons may be an important mediator of the characteristic neuropathology observed in AD brains. Chronic and/or persistent infection of neuronal cells with C. pneumoniae in the AD brain may affect apoptosis in cells containing chlamydial inclusions.

Results: SK-N-MC neuroblastoma cells were infected with the respiratory strain of C. pneumoniae, AR39 at an MOI of 1. Following infection, the cells were either untreated or treated with staurosporine and then examined for apoptosis by labeling for nuclear fragmentation, caspase activity, and membrane inversion as indicated by annexin V staining. C. pneumoniae infection was maintained through 10 days post-infection. At 3 and 10 days post-infection, the infected cell cultures appeared to inhibit or were resistant to the apoptotic process when induced by staurosporine. This inhibition was demonstrated quantitatively by nuclear profile counts and caspase 3/7 activity measurements.

Conclusion: These data suggest that C. pneumoniae can sustain a chronic infection in neuronal cells by interfering with apoptosis, which may contribute to chronic inflammation in the AD brain.

Figure 1

Alzheimer diseased brain tissue from the hippocampal region. Tissue sections were labeled with an anti-chlamydial specific antibody revealing chlamydial inclusions within neurons (arrows, panels A and B). (A & B) Mag. bars = 20 μm.

Figure 2

SK-N-MC neuroblastoma cells immunolabeled with neuronal specific antibodies. In panels A and B, neuronal phenotype was determined by labeling with anti-βIII-tubulin (red). In panel C, the cells were incubated in 0.1% DMSO in growth media for 4 hr and Hoechst-stained to highlight nuclear profiles (blue). Mag. bars = 10 μm.

Figure 3

Infection of neuronal cells followed by staurosporine incubation. SK-N-MC neuroblastoma cells infected with Chlamydia pneumoniae for 3 days (A) and 10 days (B), revealing chlamydial inclusions (green). In panel A, Imagen anti-chlamydial specific antibody was used. In panel B, anti-chlamydial antibodies 60C19 and Imagen were used in combination to detect prolonged or persistent infection. Nuclei were identified by Hoechst stain (blue), and the cytoplasm was stained with Evans blue (red). Mag. bar = 10 μm.

Figure 4

Annexin labeling of SK-N-MC neruoblastoma cells infected with Chlamydia pneumoniae following staurosporine incubation. SK-N-MC neuroblastoma cells uninfected (A, B) and infected with Chlamydia pneumoniae for 3 days (C, D), untreated cells (A, C) and cells treated with 1 μM staurosporine for 4 hours (B, D), labeled with annexin V (green) and DAPI (blue). Mag. bar = 10 μm.

Figure 5

Analysis of the nuclear profiles of Chlamydia pneumoniae-infected neuroblastoma cells following apoptotic induction with staurosporine. Nuclear profiles were revealed with Hoechst stain and counted as normal or apoptotic depending on fragmentation. Conditions in panels A-F are as follows: A, uninfected cells; B, uninfected cells treated with 1 μM staurosporine for 4 hours; C,: cells infected for 3 days; D, cells infected for 3 days treated with 1 μM staurosporine for 4 hours; E, cells infected for 10 days; F, cells infected for 10 days treated with 1 μM staurosporine for 4 hours. Mag. bar = 20 μm.

Figure 6

Quantitative analysis of nuclear profile data. Lanes A-F correspond to the conditions in panels A-F of figure 5. Data are from 3 independent infections with 20 random fields counted for each condition. Total cell counts are: A, 793; B, 436; C, 512; D, 412; E, 402; F, 395. Chi-squared analysis of these data indicates that the difference between the uninfected result and the infected results (either 3 or 10 days) is highly statistically significant (p < 0.0001), however the infected results were not significantly different from each other (p = 0.36)

Figure 7

SK-N-MC neuroblastoma cells immunolabeled for active caspase 3 and C. pneumoniae inclusions following induction of apoptosis with staurosporine. Anti-caspase 3 specific for activated caspase 3 (red); DAPI to reveal the nuclear profile (blue); anti-chlamydial antibody specific for chlamydial inclusions (green). Shown are uninfected (A, B) and infected cells for 24 (C, D), 48 (E, F) and 72 (G, H) hours post infection, untreated cells (A, C, E, G) and cells treated with 1 μM staurosporine for 4 hours (B, D, F, H). Mag. bar = 20 μm.

Figure 8

Caspase 3/7 activity detected by the Apo-ONE assay. Plotted relative to the increase in activity in uninfected cells induced by staurosporine, at three timepoints after infection with C. pneumoniae. Lanes A-H correspond to the conditions in panels A-H of figure 7. Lanes C, E and G depict decreased activity relative to uninfected control. Error bars are s.e.m. for 9 samples of 10⁴ cells from 3 cell culture preparations.