Involvement of Lysosome Membrane Permeabilization and Reactive Oxygen Species Production in the Necrosis Induced by Chlamydia muridarum Infection in L929 Cells원문보기
Chlamydiae, obligate intracellular bacteria, are associated with a variety of human diseases. The chlamydial life cycle undergoes a biphasic development: replicative reticulate bodies (RBs) phase and infectious elementary bodies (EBs) phase. At the end of the chlamydial intracellular life cycle, EBs...
Chlamydiae, obligate intracellular bacteria, are associated with a variety of human diseases. The chlamydial life cycle undergoes a biphasic development: replicative reticulate bodies (RBs) phase and infectious elementary bodies (EBs) phase. At the end of the chlamydial intracellular life cycle, EBs have to be released to the surrounded cells. Therefore, the interactions between Chlamydiae and cell death pathways could greatly influence the outcomes of Chlamydia infection. However, the underlying molecular mechanisms remain elusive. Here, we investigated host cell death after Chlamydia infection in vitro, in L929 cells, and showed that Chlamydia infection induces cell necrosis, as detected by the propidium iodide (PI)-Annexin V double-staining flow-cytometric assay and Lactate dehydrogenase (LDH) release assay. The production of reactive oxygen species (ROS), an important factor in induction of necrosis, was increased after Chlamydia infection, and inhibition of ROS with specific pharmacological inhibitors, diphenylene iodonium (DPI) or butylated hydroxyanisole (BHA), led to significant suppression of necrosis. Interestingly, live-cell imaging revealed that Chlamydia infection induced lysosome membrane permeabilization (LMP). When an inhibitor upstream of LMP, CA-074-Me, was added to cells, the production of ROS was reduced with concomitant inhibition of necrosis. Taken together, our results indicate that Chlamydia infection elicits the production of ROS, which is dependent on LMP at least partially, followed by induction of host-cell necrosis. To our best knowledge, this is the first live-cell-imaging observation of LMP post Chlamydia infection and report on the link of LMP to ROS to necrosis during Chlamydia infection.
Chlamydiae, obligate intracellular bacteria, are associated with a variety of human diseases. The chlamydial life cycle undergoes a biphasic development: replicative reticulate bodies (RBs) phase and infectious elementary bodies (EBs) phase. At the end of the chlamydial intracellular life cycle, EBs have to be released to the surrounded cells. Therefore, the interactions between Chlamydiae and cell death pathways could greatly influence the outcomes of Chlamydia infection. However, the underlying molecular mechanisms remain elusive. Here, we investigated host cell death after Chlamydia infection in vitro, in L929 cells, and showed that Chlamydia infection induces cell necrosis, as detected by the propidium iodide (PI)-Annexin V double-staining flow-cytometric assay and Lactate dehydrogenase (LDH) release assay. The production of reactive oxygen species (ROS), an important factor in induction of necrosis, was increased after Chlamydia infection, and inhibition of ROS with specific pharmacological inhibitors, diphenylene iodonium (DPI) or butylated hydroxyanisole (BHA), led to significant suppression of necrosis. Interestingly, live-cell imaging revealed that Chlamydia infection induced lysosome membrane permeabilization (LMP). When an inhibitor upstream of LMP, CA-074-Me, was added to cells, the production of ROS was reduced with concomitant inhibition of necrosis. Taken together, our results indicate that Chlamydia infection elicits the production of ROS, which is dependent on LMP at least partially, followed by induction of host-cell necrosis. To our best knowledge, this is the first live-cell-imaging observation of LMP post Chlamydia infection and report on the link of LMP to ROS to necrosis during Chlamydia infection.
In conclusion, the production of ROS during Chlamydia infection resulted in host-cell necrosis. We show for the first time that LMP is associated with late induction of oxidative stress of ROS, culminating in necrosis during Chlamydia infection.
In addition, it has been shown that NADH-driven lysosomal respiration is a permanent source of cellular ROS release [20]. In this study, with the addition of CA-074-Me, Chlamydia-induced ROS production was dramatically attenuated, whereas it had no obvious effect on the control group.
We then tested whether the attenuation of ROS production can affect Chlamydia-induced necrosis by measuring the percentage of PI-positive cells. Our results showed that indeed, treatment of Chlamydia-infected L929 cells with DPI and BHA can inhibit necrosis post-infection compared with controls. These results suggest that ROS production during Chlamydia infection is essential for Chlamydia-induced necrosis.
First, to discriminate apoptosis from necrosis, we used a classic method of dual staining with fluorescent Annexin V and PI [24]. Our results showed that the percentage of PI-positive cells gradually increased, even up to about 30% after 48 h post infection, but with no effect on Annexin-V-positive cells. When Nec1, an inhibitor of necrosis, was added, the cell death decreased, whereas ZVAD-FMK, a pan caspase inhibitor, did not show obvious inhibitory effects.
후속연구
We show for the first time that LMP is associated with late induction of oxidative stress of ROS, culminating in necrosis during Chlamydia infection. Our findings might provide new insights into Chlamydia-host interplays and deepen our understandings of the relationship between mechanisms of cell death and Chlamydia infection, and as such provide novel clues for the development of effective prophylactic and therapeutic approaches to treat Chlamydia infection.
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