AIF And EndoG Mediated Mitochondria-associated Pathway Involye In Leptospira Interrogans-induced Macrophage Apoptosis

AIM AND GROUNDOur previous study found that Leptospira infection could induce monocyte-macrophage-like cells (J774A.1) apoptosis, but the specific mechanism of apoptosis has not been clarified. The purpose of this study is to investigate mitochondria-related pathway of L. interrogans-induced macrophages apoptosis.MATERIAL AND METHODS(1) Establish pathogenic leptospires infected macrophages apoptosis model in vitro: murine peritoneal macrophages (MPMs), murine monocytic macrophage-like cells (J774A.1) and human monocyte-like cells (THP-1) were infected with L. interrogans serovar Lai strain Lai with different MOIs for different times, respectively. Detect apoptosis of the macrophages by Annexin V-FITC/PI double staining method; analyze apoptosis rates by flow cytometry (2) Detect morphological change and the mitochondria impairment by transmission electron microscopy of leptospiral induced apoptotic macrophages. (3) Caspase-8 and caspase-9 activity was measured with assay kits. To detect mitochondrial potential with JC-1 staining by flow cytometry and fluorescence microscopy, simultaneously detect cells of reactive oxygen species (ROS) levels by DCFH-DA fluorescent probe. (4) Expression of cytochrome C, Smac, EndoG and AIF in macrophages were determined in the mitochondrial and cytosol component after infection by Western Blot, and translocation of AIF and EndoG were examined by immunofluorescence staining. (5) Apoptosis-related Bcl-2 family mRNA expression levels was detected by microarray and confirmed by real-time PCR.RESULTS(1) Pathogenic L. interrogans serogroup Lai strain Lai could induce apoptosis of MPMs, J774A.1 and THP-1 cells with a time- and dose-dependent manner. However, with the increase of MOI, apoptosis ratio firstly rose, and then declined, while cell necrosis ratio increased. (2) Transmission Electron Microscopy results showed that all three types macrophages demonstrated apoptotic morphological changes, such as chromatin condensation and marginalization and cell vacuolization, mitochondria swelling and disappearance of mitochondrial ridges. (3) The activity of caspase-8 of the L. interrogans strain Lai-infected macrophages increased, but activity of caspase-9 did not increase obviously. However, pan-caspase inhibitors could not completely block macrophages apoptosis. Mitochondrial membrane potential (MPT) in infected macrophages declined gradually as infected time; while the cells showed a visible color change under the fluorescence microscope. At the same time, reactive oxygen species (ROS) levels increased in the infected macrophages. (4) The results of Western Blot showed that it was not detectable of cytochrome C and Smac release from mitochondria to cytoplasm in the L. interrogans strain Lai induced-apoptotic macrophages. However, pro-apoptotic proteins, AIF and EndoG, releasing from mitchondria to cytosol was detectable in the infected MPMs and J774A. 1 cells, and AIF releasing was detectable in THP-1 cells. Results of immunofluorescent staining confirmed that AIF and/or EndoG translocation from mitochondria to the nucleus, thereby inducing cell apoptosis. MPT blocker could completely block the release of AIF or EndoG, and partly blocked the macrophages apoptosis. (5) The results of microarray and real-time PCR showed that Bcl-2 family mRNA levels changed in the L. interrogans strain Lai induced apoptotic macrophages. Pro-apoptotic genes (Bid, Bik1, Bnip3, Bcor, Bcl-2l1 and Bclaf1) were significantly up-regulated in the infected MPMs and J774A. 1 cells, pro-apoptotic genes (Bid, Bik1, Bnip3, Bcl-2l1 and Bclaf1) were lightly up-regulated in the infected THP-1 cells, other proapoptotic genes were unchanged. CONCLUSIONS(1) L. interrogans serovar Lai strain Lai induced apoptosis and necrosis in macrophages in vitro; apoptosis rates in primary macrophages was higher than that of continuous cell lines, apoptosis rates in murine macrophage was higher than people originate macrophages. The leptospires induced apoptosis in a certain range was time-and dose-dependent.(2) L. interrogans strain Lai -induced macrophages apoptosis via caspase-8 dependent pathway and mitochondrial related caspase-independent pathway.(3) In the process of Leptospiral stain Lai -induced macrophages apoptosis, mRNA expression level of some pro-apoptotic genes of Bcl-2 family were up-regulated, which may be involved in the process of apoptosis.