WT mouse macrophages effectively activate and release caspase-1 in response to L. pneumophila infection. This is accompanied by the fusion of the L. pneumophila-containing phagosome with the lysosome, bacterial degradation, and elimination (Amer et al., 2006; Akhter et al., 2009). Consistent with that, caspase-1 knockout (^−/−) macrophages are permissive to L. pneumophila replication. Asc is an adaptor molecule involved in caspase-1 activation in response to a variety of agents. However, its role during L. pneumophila infection is not well established. To characterize the role of Asc during L. pneumophila infection, Asc was down regulated in both caspase-1^−/− (Figure 1A) and WT macrophages (Figure 1B) using Asc specific siRNA. After transfection, Asc protein level declined in both sets of cells, while other components of the inflammasome, such as Nlrc4 and pro-caspase-1, were not affected (Figures 1A,B). After depletion of Asc, cells were infected with L. pneumophila and the bacterial replication was assessed by counting colony-forming units (CFU). Depletion of Asc in caspase-1^−/− cells supported significantly more L. pneumophila replication compared to caspase-1^−/− cells treated with control siRNA (Figure 1C). In WT mouse macrophages, depletion of Asc did not have an effect on the bacterial growth compared to that of cells treated with control siRNA or untreated cells (Figure 1D). Therefore, Asc controls L. pneumophila replication in murine macrophages in the absence of caspase-1.

It has been shown by several reports that L. pneumophila triggers NF-κB activation in TLR5 dependent and independent manner depending on the stage of the infection (Bartfeld et al., 2009; Losick et al., 2010). Nevertheless, the role of Asc in NF-κB modulation depends on its protein levels and its location within the cells (Stehlik et al., 2002; Sarkar et al., 2006; Yu et al., 2006; Bedoya et al., 2007). Our results showed that Asc controls L. pneumophila replication in a caspase-1-independent manner. To determine if the mechanism by which Asc restricts L. pneumophila (independently of caspase-1) involves the NF-κB pathway, Asc was depleted in caspase-1^−/− macrophages by siRNA against Asc. Then, cells were infected with L. pneumophila and NF-κB activation was assessed in nuclear extracts by electrophoretic mobility shift assay (EMSA). We found that caspase-1^−/− macrophages depleted for Asc allowed more NF-κB activation at 1, 4, and 8 h of infection compared with cells treated with control siRNA (Figure 2A). These results suggest that Asc decreased NF-κB during L. pneumophila infection. To confirm these findings, Asc^−/− and WT macrophages were infected with L. pneumophila and EMSA assay was performed. Initially, WT and Asc^−/− macrophages showed NF-κB activation within 1 h after infection. This activation declined in WT macrophages by 8 h infection whereas NF-κB pathway remained activated in Asc^−/− 8 h after L. pneumophila infection (Figure 2B). Thus, in the context of L. pneumophila infection, Asc hinders NF-κB activation, and decreases cell survival.

The involvement of Asc in Nlrc4 inflammasome is still unclear (Zamboni et al., 2006). To discern the role of Asc in L. pneumophila-mediated caspase-1 activation, Asc^−/− macrophages were left untreated or infected with L. pneumophila at a low multiplicity of infection (MOI). Then, cleaved caspase-1 was examined in cellular extracts and culture supernatants of Asc^−/− macrophages and of their WT counterparts. Notably, cells lacking Asc still allowed the cleavage of caspase-1 within their cytosols when infected with L. pneumophila (Figure 3A). The amount of cleaved caspase-1 in Asc^−/− cell lysates was less than that of WT macrophages (Figure 3A). Cleaved caspase-1 was detected in culture supernatants of infected WT macrophages but not that of infected Asc^−/− macrophages (Figure 3B). This data indicates that in the absence of Asc, a fraction of caspase-1 is cleaved by a yet unknown mechanism.

To further investigate the role of Asc in caspase-1 activation during L. pneumophila of WT macrophages, Asc was depleted in WT murine macrophages, as in Figure 1B and then infected with L. pneumophila. The amount of cleaved caspase-1 detected in extracts of cells treated with Asc specific siRNA was less than that in cells treated with control siRNA (Figure 4A). Accordingly, the release of active caspase-1 from siAsc treated cells was diminished (Figure 4B). In support of this data, total cleaved caspase-1 in combined cell extracts and culture supernatants was less in Asc heterozygote macrophages than that of WT ones (Figure A1 of Appendix). Furthermore, IL-1β release from macrophages was impeded when Asc was depleted (Figure 4C). Therefore, Asc contributes to the activation of a portion of pro-caspase-1, while the rest of caspase-1 pro-form is cleaved independently of Asc.

The activation of caspase-1 requires flagellin, thereby promoting the restriction of L. pneumophila infection (Amer et al., 2006; Ren et al., 2006; Zamboni et al., 2006; Lamkanfi et al., 2007; Akhter et al., 2009; Case et al., 2009; Amer, 2010; Kang et al., 2010). Thus, we next examined the growth of L. pneumophila in WT, caspase-1^−/−, and Asc^−/− macrophages. As previously reported by our group and by others, caspase-1^−/− macrophages were permissive, whereas WT and Asc^−/− macrophages restricted L. pneumophila replication (Amer et al., 2006; Ren et al., 2006; Zamboni et al., 2006; Lamkanfi et al., 2007; Akhter et al., 2009; Case et al., 2009; Amer, 2010; Kang et al., 2010). However, our data demonstrate that caspase-1 is cleaved in the cytosol of Asc^−/− macrophages, therefore, we examined if this cytosolic cleavage required flagellin and if it is sufficient for restriction of L. pneumophila infection. WT macrophages were infected with the L. pneumophila mutant lacking flagellin (Fla), then, caspase-1 activation in cell extracts was examined by Western blots. Figure 5 shows that the Fla mutant did not activate caspase-1 within the cytosols of macrophages (Figure 5A). Because caspase-1 activation requires flagellin and is accompanied with restriction to L. pneumophila infection, we examined the growth of L. pneumophila mutants lacking flagellin (Fla) in macrophages lacking Asc in comparison to WT cells, since both cells lack caspase-1 activation in response to Fla (data not shown). Specifically, Figure 5B demonstrates that the growth of Fla mutants in Asc^−/− macrophages exceeds that exhibited in WT cells. Yet, NF-κB activation during Fla was similar in WT and Asc^−/− macrophages (Figure 5C). Therefore, there must be another pathway mediated by Asc that maintains the replication of Fla mutant under control in WT macrophages and is missing in Asc^−/− macrophages. To answer this question, we next determined the levels of LDH release in culture supernatants of WT and Asc^−/− macrophages infected with L. pneumophila or the Fla mutant. We found that the absence of Asc allows more host cell survival during Fla infection. To further understand the mechanism by which Asc modulates cell survival, we examined the activation of caspase-3, a caspase involved in apoptosis and know to be activated during L. pneumophila infection (Molmeret et al., 2004). First, we tested if the Fla mutant activates caspase-3 in wild type macrophages. Until 4 h post infection only WT L. pneumophila activated caspase-3 (Figure 6A). However, at 5 h post infection, the Fla mutant activated caspase-3 in WT macrophages but not in Asc^−/− macrophages (Figure 6B). Taken together, these data suggest that Asc is involved in caspase-3 activation which mediates cell death in WT macrophages at later stages of Fla infection.

In WT murine macrophages L. pneumophila flagellin is detected by Nlrc4 with subsequent activation of caspase-1 (Amer et al., 2006; Ren et al., 2006; Zamboni et al., 2006; Lamkanfi et al., 2007; Akhter et al., 2009; Case et al., 2009; Amer, 2010; Kang et al., 2010). This response is completely lacking in human phagocytes. In contrast to murine phagocytes, human phagocytes which are permissive to L. pneumophila do not activate caspase-1 in response to the pathogen (Abdelaziz et al., 2011). This lack of activation is due to the down regulation of ASC in human cells upon infection with L. pneumophila (Abdelaziz et al., 2011). To characterize the effect of L. pneumophila on the expression of the Nlrc4 inflammasome components in the mouse, murine WT macrophages were infected with L. pneumophila and the expression of caspase-1, IL-1β, Nlrc4, and Asc was assessed on both mRNA and protein levels. The expression of both caspase-1 and IL-1β proteins were induced significantly within 4 h after infection and remained up regulated for 24 h of infection (Figures A2A,B of Appendix). Subsequently, both caspase-1 and IL-1β were activated early upon infection (Figures A2A,B lower panel of Appendix) and their active forms were released into the supernatant (Data not shown). Remarkably, in contrast to mouse macrophages, the expression of Asc was enhanced 24 h after infection (Figure A2C). As for Nlrc4, mRNA, and protein levels were decreased later in infection (Figure A2D lower panel of Appendix). We found that this decrease in Nlrc4 protein levels is due to its release with the rest of the inflammasome components into the media (data not shown). Therefore, L. pneumophila differentially modulates the expression of Asc in murine and in human phagocytes.

Wild type C57BL/6 (B6) and Asc^−/− mice were previously described (Mariathasan et al., 2004). Caspase-1^−/− mice were from Dr. Amy Hise at Case Western University. All knockout mice were in a C57BL/6 background. Bone marrow-derived macrophages (BMDMs) were isolated from femurs of 6- to 12-week-old mice and were cultured in IMDM containing 10% heat-inactivated FBS, 20% L cell-conditioned medium, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. After 5 days of incubation, cells were collected and plated in 6-well plates or in 24-well plates in IMDM containing 10% heat-inactivated FBS (Stanley, 1997; Amer et al., 2005, 2006; Akhter et al., 2009).

Legionella pneumophila strain Lp02, is a thymine auxotrophic derivative of Philadelphia-1 (Brenner et al., 1979; McDade and Shepard, 1979). L. pneumophila flagellin (Fla) mutant was previously described (Albert-Weissenberger et al., 2010). L. pneumophila was cultured as described previously (Sturgill-Koszycki and Swanson, 2000; Akhter et al., 2009) All experiments were performed at a low MOI of 0.5, followed by centrifugation and rinsing of the wells after 30 min except when otherwise indicated (Derre and Isberg, 2004).

All experiments were performed at an MOI ranging 0.1–1, followed by centrifugation and rinsing of the wells after 30 min except when otherwise indicated (Derre and Isberg, 2004). All experiments were performed in the absence of ferric nitrate and l-cysteine from the monocytes or macrophage culture medium, to allow L. pneumophila multiplication only intracellularly. At designated time points, macrophages were lysed and plated on AYE plates for CFUs (Amer et al., 2006; Abdelaziz et al., 2010). The quantification of the CFU in vitro was performed more than four independent times as described (Amer et al., 2006; Abdelaziz et al., 2010).

Cell extracts of macrophages were prepared and immunoblotted with an antibody that recognizes Nlrc4, Asc (Alexis Biochemicals), caspase-1 (Santa Cruz), IL-1β (National Cancer Institute), caspase-3 (Cell Signalling), actin (Abcam), followed by appropriate secondary antibody as described (Amer et al., 2006; Abdelaziz et al., 2010).

Nuclear extracts of L. pneumophila treated or untreated BMDMs from WT and Asc^−/− mice were prepared as previously described (Akhter et al., 2009). EMSA was used to measure NF-κB DNA binding activity as described (Sarkar et al., 2006).

Total RNA was extracted from cells lysed in Trizol (Invitrogen Life Technologies) and 1–2 μg of the RNA was converted to cDNA by ThermoScript RNase H⁻ Reverse Transcriptase (Invitrogen, Life Technologies). 20–60 ng of the converted cDNA was then used for quantitative PCR with SYBR Green I PCR Master Mix using the StepOne Plus Real Time PCR System (Applied Biosystems). The target gene C_t values were normalized to the C_t values of two housekeeping genes (mouse GAPDH and CAP-1) and expressed as relative copy number (RCN), as described earlier (Gavrilin et al., 2006; Zakharova et al., 2010). Primers used in RT-PCR are presented in Table A1 of Appendix. We also evaluated expression of an around 600 genes with Open Array Mouse Inflammatory Panel (BioTrove, Life Technologies). All individual C_t values were normalized to the average of 18 housekeeping genes used in this array, and also expressed as RCN.

Active caspase-3 was determined by the AFC assay, as previously described (Gonzalez-Mejia et al., 2010). Lysates were incubated in a cyto-buffer (10% glycerol, 50 mM Pipes, pH 7.0, 1 mM EDTA, containing 1 mM DTT) containing 20 mM of the tetrapeptide substrate DEVD-AFC. The tetrapeptide was obtained from Enzyme Systems Products (Livermore, CA, USA). Release of free AFC was determined using a Cytofluor 4000 fluorometer (Perseptive Company, Framingham, MA, USA; Filters: excitation; 400 nm, emission; 508 nm).

LDH release into cell culture medium was used as an indicator of cell death using NAD+ reduction assay, according to the manufacturer (Roche Applied Science). Cells were plated in 12-well plate at the density 0.5 × 10⁶, and 0.5 MOI of both wild type L. pneumophila (Leg) and its flagellin mutant (Fla) were added. Cell culture medium was collected 24 h post infection; clarified from floating bacteria by centrifugation; and used for LDH assay. To determine spontaneous cell death, referred as a negative control, we collected medium from cells incubated the same time without bacteria. To measure total LDH content in cells, referred as a positive control, cells were lysed by adding TritonX-100 (1% final concentration) to the well. Media alone was used as a blank. LDH concentration in the medium was detected at OD 490 nm. Cell death was calculated by the following formula: cytotoxicity (%) = (sample−blank/positive control−blank) × 100.

Data are displayed as mean of three independent experiments ± SD. P value ≤0.05 was considered significant.