L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai, the dominant pathogenic strain in China (Hu et al., 2014), was provided by the Chinese National Institute for the Control of Pharmaceutical and Biological Products in Beijing, China, and was cultivated at 28°C in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid medium (Hu et al., 2013). The cell lines J774A.1 and THP-1 were provided by the Cell Bank in the Institute of Cytobiology, Chinese Academy of Science in Shanghai, China. The cells were cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal calf serum (FCS, Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma, USA) in an atmosphere of 5% CO2 at 37°C. In particular, THP-1 cells should be induced to differentiate into macrophages before use by stimulation with 10 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) at 37°C for 24 h (Hu et al., 2013).

Freshly cultured leptospires in EMJH medium was centrifuged at 17,200 × g for 15 min (15°C). After washing twice with autoclaved PBS, the precipitated leptospires were suspended in antibiotic-free RPMI-1640 medium with 2.5% FCS. The leptospires in the suspension were counted under the dark-field microscope with a Petroff-Hausser counting chamber (Fisher Scientific, USA) (Hu et al., 2013). J774A.1 or THP-1 cells were seeded in 6-well or 12-well culture plates (Corning, USA) and incubated for 12-h in an atmosphere of 5% CO₂ at 37°C. After washing with PBS, the cell monolayers were infected with the spirochete at a multiplicity of infection (MOI) of 10, 50, 100, or 500 leptospires per host cell, and incubated for 1, 2, 4, or 8 h at 37°C.

Annexin V-FITC apoptosis detection kit (BioVision, USA) was used to detect the apoptotic ratios of the leptospire-infected cells. According to the manufacturer's protocol, the harvested cells were resuspended in annexin V binding buffer containing annexin V and PI dyes and incubated in the dark for 15 min at room temperature (Hu et al., 2013). The stained cells were immediately analyzed by using a flow cytometer (FC500 MCL, Beckman) to distinguish the cells in apoptosis (annexin V⁺/PI⁻) from necrosis (annexin V⁺/PI⁺). In this experiment, healthy cells before infection were used as the controls.

The leptospire-infected macrophages were collected by centrifugation at 1,000 × g for 10 min at 4°C, fixed in 2.5% glutaraldehyde at 4°C for 2 h and then washed three times with PBS. The harvested pellets were post-fixed with 1% osmium tetroxide, and then rinsed, dehydrated and embedded in epoxy resin (Sigma). Ultrathin sections on 100–150 mesh nickel grids (Plano, Germany) were stained with lead citrate and uranyl acetate (Jin et al., 2009). Finally, the morphological changes in each of the types of leptospire-infected macrophages were observed under a TECNAI-10 transmission electron microscope (Philips, Holland).

The changes in mitochondrial membrane potential (MMP) was detected by using JC-1 (Molecular Probes, USA) (St-Louis and Archambault, 2007). In healthy mitochondria with a high MMP JC-1 shows red fluorescence, while the color shifts from red to green when the MMP is lost. Briefly, leptospire-infected macrophages were stained with 10 μg/mL JC-1 at 37°C for 15 min, washed three times with PBS and then observed using a fluorescence microscope (Zeiss, Germany) as well as measured by flow cytometry.

For blockage test, the cell monolayers were pretreated with 10 μM cyclosporine A (CSA, a blocker of mitochondrial permeability transition (MPT), Sigma) for 30 min, 50 μM Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhitor, Sigma) for 1 h at 37°C (Jin et al., 2009; Hu et al., 2013), 25 μM LY294002 (PI3K/Akt pathway inhibitor, Sigma) for 1 h at 37°C, or 10 mM N-acetyl-cysteine (NAC, an anti-oxidant, Sigma) for 1 h at 37°C. In these tests, macrophages without blocker pretreatment were used as the controls. For RNA interference test, small interfering RNAs (siRNAs) specific to silence the mouse or human bid, aif, and endoG genes were designed and synthesized by Invitrogen Co. (Shanghai, China). The sequences of siRNAs are 5′-CGACUGUCAACUUUAUUAATT-3′ in J774A.1 and 5′-GGCCACUGUUUGGAAAUAATT-3′ in THP-1 (targeting bid); 5′-GGAAAUAUGGGAAAGAUCCTT-3′ in J774A.1 and 5′-GGCUACGUCCAGGAGCGCACC-3′ in THP-1 (targeting aif); 5′-GGACCGAGGCTGATGGGAA-3′ in J774A.1 and 5′-AAGAGCCGCGAGUCGUACGUG-3′ in THP-1 (targeting endoG). The transfection was performed using Lipofectamine-2000 (Invitrogen), according to the manufacturer's protocol.

The activity of caspase-8 or−9 in the leptospire-infected macrophages was measured using a caspase fluorometric assay kit (BioVision) according to the manufacturer's protocol (Jin et al., 2009). Briefly, the leptospire-infected macrophages were trypsinized and subsequently collected by centrifugation as described above. After washing with cold PBS, the cell pellets were suspended with 50 μL chilled lysis buffer for 10 min on ice. The lysates were clarified by 10,000 × g centrifugation for 10 min at 4°C and the supernatants were collected for determination of protein concentration using a BCA protein quantitation kit (Thermo Scientific, USA). Equal amounts of each of the supernatants with the same protein concentration were incubated with 10 mM dithiothreitol and 50 μM caspase fluorometric substrates (Ac-IETD-AFC for caspase-8 and Ac-LEHD-AFC for caspase-9) at 37°C for 1 h (Jin et al., 2009). Fluorescence intensity changes due to the release of AFC (reflecting caspase activity) were measured using a microplate reader (Bio-Rad, Hercules, CA).

Fractions of mitochondria and cytosol from the leptospire-infected J774A.1 or THP-1 cells (MOI 100) were prepared using a mitochondria/cytosol fractionation kit (BioVision) following the manufacturer's protocol. Equivalent amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then electron-transferred onto a polyvinylidene difluoride membrane (Millipore, USA). Using 1:1000 diluted rabbit anti-mouse or human-CytC (Cell Signaling Technology, CST, USA), -AIF (CST), -EndoG-IgG (Absin, China), -Bid (CST), or -p-Akt (Ser-473, CST) as the primary antibody, and 1:3,000 diluted HRP-conjugated goat anti-rabbit-IgG as the secondary antibody (CST), several western blot assays were performed to detect the target proteins in the mitochondrial or cytosolic fractions. In this study, the β-actin were used as the controls.

J774A.1 or THP-1 cells (1 × 10⁵ per well) were seeded in 12-well culture plates containing 12 × 12 mm coverslip and incubated at 37°C overnight. After washing with PBS, the cell monolayers were infected with L. interrogans strain Lai (MOI 100) for 1, 2, 4, or 8 h. Subsequently, cells were loaded with 100 nM MitoTracker Red CMXRos (Invitrogen) for 15 min in culture medium. Then the cells were washed thoroughly with PBS and fixed in 4% paraformaldehyde at 4°C overnight. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, and then incubated with 5% BSA for 4 h at 4°C. Subsequently, the cells were incubated with 1:500 diluted rabbit anti-mouse or human-AIF- (CST), or -EndoG-IgG (Absin), followed by incubation with 1:1,000 diluted Alexa FluorTM488-conjugated anti-rabbit-IgG (CST) for 1 h at 37°C. After washing with PBS, DAPI (0.1 μg/ml; CST) was added for 5-min incubation at room temperature to stain nuclei. Finally, the nuclear translocation of AIF or EndoG in the leptospire-infected macrophages was observed using a laser confocal microscope (Olympus IX81-FV1000, Japan).

DNA fragmentation assays were performed with a Cellular DNA fragmentation ELISA kit (Roche) according to the manufacturer's protocol (Onaran et al., 2008). Cells were firstly incubated for 2 h at 37°C with 5′-Bromo-2′-deoxy-uridine (BrdU), which can be incorporated into genomic DNA. Subsequently, cells were infected with leptospires for 1, 2, or 4 h. After that, cells were lysed, transferred to a microtiter plate coated with an anti-DNA antibody, and incubated for 1 h at room temperature. After washing with washing solution, anti-BrdU-peroxidase conjugate solution was added, and the plate was incubated for 90 min at room temperature (Onaran et al., 2008). After washing three times, substrate solution was added, and the plate was incubated in the dark on a shaker until color development was sufficient. After stopping the reaction by adding stop solution, a microplate reader (Bio-Rad, Hercules, CA) was used to measure absorbance at 450 nm for each well.

The intracellular ROS levels in leptospire-infected cells were detected by Dichlorofluorescein diacetate (DCFH-DA) (Sigma), a ROS specific fluorescent dye (Hu et al., 2013). Briefly, the leptospire-infected cells were incubated in medium containing 5 mM DCFH-DA for 30 min at 37°C, and then the fluorescence intensity reflecting ROS levels was detected by laser confocal microscopy (LSM510-Meta, Zeiss, Germany).

Data from at least three independent experiments were averaged to present as mean ± SD (standard deviation). Statistical analysis of the differences between the groups was performed using the Student's t-test with P ≤ 0.05 considered as statistically significant.

Cells of the mouse macrophage cell line J774A.1 and human monocyte cell line THP-1 displayed apoptosis after infection with L. interrogans strain Lai. An MOI of 100 caused maximal early apoptosis in both J774A.1 and THP-1 cells (Figure 1A), and when the macrophages were infected with an MOI of 100, apoptosis was maximal at 4 h (Figure 1B). After infection with L. interrogans strain Lai at MOI of 100 for 4 h, all the macrophages displayed typical apoptotic morphological changes such as chromatin condensation and margination (Figure 1C,b). Compared to healthy mitochondrial control (Figure 1Cc), the cristae of mitochondria in the leptospire-infected macrophages had disappeared (Figure 1Cd). In addition, JC-1-based fluorescence microscopy and flow cytometry showed a gradual but significant decrease of MMP in both of leptospire-infected macrophag cell lines (Figures 1D–F). Pretreatment of the macrophages with CSA, a MPT blocker, significantly decreased the leptospire-induced apoptotic ratios (Figure 1G). These results suggested that Leptospira infection causes mitochondrial changes in mouse and human macrophages, which is consistent with mitochondrion-mediated apoptosis.

CytC release from mitochondria and subsequent caspase-9 activation are necessary steps to induce apoptosis through the mitochondrion-related caspase-dependent pathway (Green and Kroemer, 2004). However, in all the macrophages infected with L. interrogans strain Lai, only caspase-8 was activated and caspase-9 was not (Figure 2A). When the macrophages were pretreated with Z-VAD-FMK (pan-caspase inhibitor) or Z-IETD-FMK (caspase-8 inhibitor), the apoptotic ratios of leptospire-infected macrophages were substantially decreased, while no significant decrease in apoptotic ratios were observed in Z-LEHD-FMK (caspase-9 inhitor) pretreated cells (Figure 2B). Furthermore, no mitochondrial release of CytC from the infected macrophages was detected (Figure 2C). These data suggested that the typical CytC-caspase-9 pathway is not involved in mitochondrion-related apoptosis of macrophages caused by infection with Leptospira.

AIF and EndoG have been shown to mediate mitochondrion-related apoptosis when they are released from mitochondria (Li et al., 2001; Cregan et al., 2004; Green and Kroemer, 2004). In leptospire-infected J774A.1 cells, both AIF and EndoG were released from mitochondria into the cytosol, whereas in infected THP-1 cells, only the mitochondrial release of AIF was detectable (Figure 2C). When the macrophages were pretreated with CSA, the mitochondrial release of both AIF and EndoG was completely inhibited, while pretreatment with Z-VAD-FMK only decreased mitochondrial release of AIF and EndoG (Figure 2D). These results indicated that infection with leptospire induces mouse and human macrophages to selectively release AIF and/or EndoG from mitochondria, and that AIF and/or EndoG are involved in Leptospira-induced mitochondrion-related macrophage apoptosis in both caspase-dependent and caspase-independent ways.

Previous studies showed that AIF and EndoG that are released from mitochondria into the cytosol further translocate into the nucleus, resulting in chromatin condensation and DNA fragmentation (Li et al., 2001; Hong et al., 2004). In the leptospire-infected macrophages, AIF and/or EndoG proteins released from mitochondria into the cytosol were also translocated into the nucleus (Figure 3A). In addition, nuclear DNA fragmentation was detected by using a cellular DNA fragmentation ELISA. We subsequently used AIF RNAi (si AIF), EndoG RNAi (si EndoG), or a combination of both AIF and EndoG RNAi (si AIF/EndoG) to decrease the expression of AIF and EndoG in macrophages (Figure 3B). We found that knockdown of either AIF or EndoG alone could not efficiently decrease DNA fragmentation in J774A.1 cells infected with leptospire, while AIF and EndoG double knockdown substantially decreased DNA fragmentation (Figure 3C). However, since there is no EndoG released from mitochondria, knockdown of AIF alone could efficiently decrease DNA fragmentation in THP-1 cells (Figure 3C). These data demonstrated that the release of mitochondrial AIF and/or EndoG induces DNA fragmentation, which is required for leptospire to induce apoptosis during an infection.

Bid is a pro-apoptotic member of the Bcl-2 family. Previous studies have demonstrated that the activated Bid can mediate the release of AIF and EndoG (Li et al., 2001; Landshamer et al., 2008; Guo et al., 2015). Our results confirmed that Bid was cleaved into tBid, the active truncated form of Bid, during the time course of leptospire infection (Figure 4A). Simultaneously, tBid translocated from the cytosol into the mitochondria, which induced the release of mitochondrial proteins (Figure 4B). When the expression of Bid was knocked down using siRNA, the release and nuclear translocation of AIF and/or EndoG was affected (Figures 4C,D). In addition, Bid knockdown also substantially decreased nuclear DNA fragmentation and apoptosis in macrophages infected with leptospire (Figures 4E,F). These results demonstrate that Bid is able to induce the release of AIF and/or EndoG and consequently apoptosis.

Bid usually remains in an inactive form in the cytosolic fraction of living cells and is cleaved and activated by caspase-8 in response to TNF alpha or Fas ligand (Kantari and Walczak, 2011). Our data have already demonstrated that caspase-8 was activated during leptospire-induced apoptosis (Figure 2A). Subsequently, Z-IETD-FMK was used to inactivate caspase-8, which efficiently decreased activation of Bid and the release of AIF or EndoG (Figure 5A). These results indicate that activated caspase-8 mediates the cleavage and activation of Bid and the release of AIF and/or EndoG.

Akt activation is able to inhibit Bid processing and Bid-induced apoptosis (Majewski et al., 2004; Goncharenko-Khaider et al., 2010). Moreover, our previous study showed that phosphorylated (activated) Akt (p-Akt) was decreased during the time course of an infection, and that leptospire-induced apoptosis through the Akt-inactivation pathway in macrophages (Hu et al., 2013). Thus, we speculated that Akt is the upstream apoptotic regulator in Bid-induced apoptosis. Our data revealed that LY294002, an Akt inhibitor, efficiently increased the activation of Bid and the release of AIF and/or EndoG (Figure 5A). These results indicate that Akt inactivation mediates Bid-induced downstream apoptotic events instigated by leptospire-infection. It has been reported that ROS play critical roles in the regulation of diverse functional pathways involved in proliferation, apoptosis, and cellular transformation (Simon et al., 2000; Blomgran et al., 2007). The ROS specific fluorescent staining assay showed that ROS levels were rapidly increased in J774A.1 and THP-1 cells after infection with leptospire, and this increase could be significantly blocked by anti-oxidant NAC (Figure 5B). Furthermore, the decreased p-Akt protein levels observed after leptospire-infection was reversed after the removal of intracellular ROS by NAC, while the level of total Akt was not changed (Figure 5C). These results indicate that ROS induced Akt inactivation mediates Bid-induced release of AIF and EndoG during apoptosis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.