Calcium Dynamics Regulate Protective Responses and Growth of Staphylococcus aureus in Macrophages

Calcium homeostasis regulates expression of cell surface molecules for T cell activation and function

To begin with, we first ensured that treating macrophages with calcium modulators induces changes in intracellular calcium levels. To that end macrophages loaded with calcium-sensitive dye FLUO-4-AM were treated with different calcium modulators for 1h with EGTA (for blocking extracellular calcium influx), TMB8 (for blocking calcium release from intracellular stores), BAYK8644 (for activating Voltage-Gated Calcium Channels; VGCC) and Amlodipine (for blocking VGCC). Cells were incubated with these modulators for 1h followed by stimulations of TLR2 (with Pam₃Csk₄) and TLR4 (with LPS) for 3 min. Fluorescence from these cells was measured using a fluorescence spectrometer. As shown in Supplementary Figure 1 treatment of cells with calcium modulators alone did not appreciably modulate basal calcium levels indicating that these blockers have minimal effects. However, stimulation of TLR2 and TLR4 with specific ligands in cells treated with calcium blockers does indeed differentially modulate intracellular calcium homeostasis.

Figure 1

Calcium differentially regulates expression of T cell co-stimulatory molecules on macrophages during S. aureus infection. Murine J774 macrophages were incubated with either EGTA or TMB-8 or BAYK8664 or amlodipine for 2h followed by infection with 20 MOI S. aureus infection for 24h. Cells were stained with anti-CD80-PE (Panel A) or anti-IL-12Rβ1-PE (Panel B) and analyzed by flow cytometry. Shaded histograms represent unstimulated control, dark shaded represents treatments with calcium modulators only, thin line represents infection with S. aureus and the thick line represents treatment with indicated calcium modulator followed by infection with S. aureus. Data from one of three independent experiments are shown. Bar graphs adjacent to the histograms show Mean Fluorescent Intensity (MFI) of individual groups. Controls represent unstimulated cells. Data represent the mean ± SD of three independent experiments. S indicates S. aureus infection. For Panel A (CD80), P-value between Control and S. aureus is p<0.0001; S. aureus vs S. aureus + EGTA is p <0.0001; S. aureus vs S. aureus + TMB 8 is p=0.006; S. aureus vs S. aureus + Amlodipine is p<0.0001; S. aureus vs S. aureus + BAY is p<0.0001. For Panel B (IL-12Rβ1), P-value between Control vs S. aureus is p=0.0129; S. aureus vs S. aureus + EGTA is p<0.0001; S. aureus vs S. aureus + TMB8 is p<0.0001; S. aureus vs S. aureus + Amlodipine is p<0.0001 and S. aureus vs S. aureus + BAYK8664 is p<0.0001.

We next monitored the expression levels of costimulatory molecule CD80 and the cytokine IL-12Rβ1 receptor on macrophages treated with different calcium modulators, followed by S. aureus infection. T cell activation occurs through the interaction of peptide presenting major histocompatibility complex (MHC) on antigen-presenting cells (APCs) (like macrophages and dendritic cells) and the T-cells receptor complex. For effective T cell activation and function, co-stimulatory molecules are critical. CD80 is an important costimulatory molecule that interacts with CD28 on the T cell surface and augments T cell activation and effector function [26, 27]. Studies also showed that CD80 promotes pro-inflammatory responses [27, 28]. As shown in Figure 1A, infection with S. aureus increased CD80 expression, which was further enhanced upon blocking extracellular calcium influx (with EGTA) or upon activating VGCC (with BAYK8644); while blocking VGCC (with amlodipine) also increased CD80 expression over and above S. aureus infection. No significant role of calcium from intracellular stores was noticeable (upon blocking with TMB8). This indicated a tight regulation of CD80 expression by calcium. While a role for extracellular calcium from different sources was evident, no apparent role for intracellular calcium was observed for CD80 expression.

The profile of cytokines at the time of APC: T cell interaction plays a determinant role in regulating Th1 or Th2 responses [29, 30, 31]. These cytokines bind to their receptors on APCs as well as T cells and regulate the quality of T cell responses [32]. A key cytokine that mediates Th1 responses is IL-12, which binds to IL-12R and induces signals that promote APC activation as well as Th1 polarization [33, 34]. We, therefore, investigated the role of IL-12Rβ1 on macrophages following calcium modulation and S. aureus infection. Interestingly, the expression of IL-12Rβ1 was regulated by all calcium modulators (Figure 1B). Inhibiting either route of calcium homeostasis increased IL-12Rβ1 expression over and above S. aureus infection, with maximal effects observed with inhibiting all calcium influx from extracellular medium (with EGTA). This indicated that unlike CD80 wherein calcium differentially regulated its expression, IL-12Rβ1 expression was more or less dependent on calcium influx irrespective of its route of entry. It has also been shown that IL-12R present on activated NK cells or T-cells also showed higher production of IFN-γ and enhanced pro-inflammatory responses [32, 34] and also skewed TGF-β and TGF-β dependent T-cells development towards a Th1 phenotype [35].

Calcium homeostasis regulated the expression of IFN-γ and TGF-β from S. aureus infected macrophages

Therefore, we next monitored the expression of IFN-γ and TGF-β from macrophages in the context of calcium modulation. Supplementary Figure 2 shows that calcium modulators on their own did not have any significant effects on the expression levels of either IFN-γ or TGF-β. Infection of macrophages with S. aureus showed a decrease in IFN-γ levels with no change in the expression of TGF-β (Figure 2). Blocking VGCC significantly decreased the expression of TGF-β without majorly affecting IFN-γ. In contrast, activating VGCC showed a different trend with a drastic decrease in IFN-γ and a significant reduction in TGF-β levels. Chelating calcium from the extracellular medium significantly increased the levels of IFN-γ without affecting TGF-β levels, while inhibiting calcium from intracellular stores did not affect IFN-γ levels but did significantly decrease TGF-β levels. Put together these results once again point towards calcium-mediated fine regulation of cytokine responses from macrophages during S. aureus infection.

Figure 2

Calcium differentially regulates cytokine profile from macrophages during S. aureus infection. Murine J774 macrophages were incubated with either EGTA or TMB-8 or BAYK8664 or amlodipine for 2h followed by infection with 20 MOI S. aureus infection for 24h. Culture supernatants were scored for the levels of IFN-γ or TGF-β. Data represent the mean ± SD of 3 independent experiments. S indicates S. aureus infection. For IFN-γ, P-value between Control and S. aureus is p=0.0069; S. aureus vs S. aureus + EGTA is p=0.0099 and S. aureus vs S. aureus + BAY is p=0.022. For TGF-β, P-value between-group S. aureus vs S. aureus + TMB8 is p<0.0001; S. aureus vs S. aureus + Amlodipine is p<0.0001 and S. aureus vs S. aureus + BAYK8664is p<0.0001.

Calcium homeostasis regulates induction of autophagy from S. aureus infected macrophages

In the next experiment, we investigated if the route of calcium dynamics regulates key defence responses from macrophages. Over the years it has been shown that many pathogens modulate cell survival for long-term persistence in macrophages. We earlier showed that the route of calcium entry regulated macrophage survival in an M. tb HIV co-infection model [14]. Likewise, we also showed that mycobacteria and its proteins regulate autophagy and other post-translational modifications to their advantage [20, 36]. Many of these processes are regulated by calcium [37, 38, 39].

We next investigated the role of calcium in regulating autophagy. S. aureus infection has been shown to modulate autophagic response for its growth [24]. This response appears necessary for replication, following destabilizing the autophagosome and leads to host cell death through apoptosis or necrosis. Studies revealed that α-hemolysin, a pore-forming toxin secreted from S. aureus is a key factor responsible for the autophagic response. This response appears to be independent of Beclin-1, although it does require ATG5 [40]. Further, many studies suggest elevated cytosolic calcium stimulates autophagy and chelation of intracellular calcium inhibits autophagy [3, 41, 42]. Therefore, to investigate the role of calcium in autophagy modulation by S. aureus, we monitored the expression levels of key proteins in the autophagy

pathway, namely, Beclin-1, ATG5 and Microtubule-associated protein 1A/1B-light chain 3 (LC3). While Beclin-1 is an initiator protein of autophagy, ATG5 feeds into the autophagosome pathway. The cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to form LC3-phosphatidylethanolamine conjugate (LC3-II), which is recruited to autophagosomal membranes [43, 44, 45, 46].

As shown in Figure 3A blocking VGCC with amlodipine significantly increased the expression of autophagy initiator Beclin-1, while activating VGCC also increased Beclin-1 although not to the same extent as blocking VGCC. This indicated a critical threshold for VGCC function in regulating Beclin-1, thereby initiating VGCC. This indicated that blocking VGCC had a dominant role over activating VGCC in regulating Beclin-1 expression. Calcium from either extracellular medium or intracellular stores had no significant effect on Beclin-1 expression. In contrast, calcium did not have a significant effect in regulating ATG5 expression. Interestingly, however, blocking calcium release from intracellular stores, or blocking VGCC significantly increased LC3-II expression, which is the critical step in the autophagy process of engulfment of cytosolic components. Once again, maximal effects were observed upon blocking VGCC with amlodipine. The above results clearly point towards a role for calcium modulation in regulating the initiation and final step of autophagy.

Figure 3

Calcium differentially regulates expression of autophagy pathway molecules in macrophages during S. aureus infection. Murine J774 macrophages were incubated with either EGTA or TMB-8 or BAYK8664 or amlodipine for 2h followed by infection with 20 MOI S. aureus infection for 2h. Cells were processed and stained with FITC tagged antibody to Beclin-1 (Panel A) or ATG5 (Panel B). Images were captured under a confocal microscope. Confocal imaging was performed with Nikon C2 laser scan confocal microscope with 60x magnification. Data represent the mean ± SD of 3 independent experiments. S indicates S. aureus infection. Bar graphs adjacent to confocal images show fluorescence intensity of Beclin-1 (Panel C) and ATG-5 (Panel D), respectively. For Panel C (Beclin-1), P-value between S. aureus vs S. aureus + Amlodipine is p<0.0001; S. aureus vs S. aureus + BAYK8664 is p=0.0017. For Panel E, J774 macrophages were incubated with either EGTA or TMB-8 or BAYK8664 or amlodipine for 2h followed by infection with 20 MOI S. aureus infection for 2h. Cytoplasmic extracts were Western blotted for the levels of LC3-I/II. The blot for GAPDH represents loading control.

Calcium homeostasis regulates phagosome-lysosome fusion and bacterial survival in S. aureus infected macrophages

Following internalization of the pathogen, the phagosome fuses with the lysosomes. This process is facilitated by calcium concentration inside of the cells [47]. Many pathogens devise multiple strategies to evade phago-lysosome fusion [1]. For example, mycobacteria prevent the acidification of phagosomes, retains Rab5 on the phagosome surface and prevents the upregulation of Rab7 [48]. In addition, mycobacteria also retain Coronin-1 on phagosomes that prevent its fusion with lysosomes [49]. Trypanosoma cruzi modulates intracellular calcium to regulate phagosome-lysosome fusion [50]. We have earlier shown a critical role for VGCC in mediating phagosome-lysosome fusion. Blocking VGCC promotes phago-lysosome fusion while activating VGCC prevents the same [13]. In addition, we identified M. tb protein Rv3529c that has similarity to MyD88 death domain of the TLR pathway in reducing calcium-mediated phagosome-lysosome fusion [25].

We, therefore, investigated the role of calcium modulations in regulating the fusion of phagosomes with lysosomes during S. aureus infection. As shown in Figure 4, blocking extracellular calcium influx or calcium release from intracellular stores decreased S. aureus induced phagosome-lysosome fusion, blocking VGCC maximally enhanced the phago-lysosome fusion. In contrast, activating VGCC dramatically inhibited the fusion of phagosomes with lysosomes. These results point towards a critical role for calcium routing towards the fusion of phagosomes with lysosomes.

Figure 4

Calcium differentially regulates fusion of phagosomes with lysosomes in macrophages during S. aureus infection. Murine J774 macrophages were incubated with either EGTA or TMB-8 or BAYK8664 or amlodipine for 2h followed by infection with FM4-64 labelled 20 MOI S. aureus infection for 2h. Following incubation cells were washed and processed and stained for Lysotracker green. Cells were fixed with 4% paraformaldehyde and mounted with DAPI. Images were analyzed using confocal microscopy. Confocal imaging was performed with Nikon C2 laser scan confocal microscope with 60x magnification. Merged images with DAPI (blue), S. aureus tagged with FM4-64 (red) and Lysotracker (green) staining are depicted. Bar graphs adjacent to confocal images show Region of Interest (ROI). Data represent mean ± SD of three independent experiments of 10 Fields of each group. P-value between-group S. aureus vs S. aureus + BAYK8664 is p=0.0002.

Finally, we investigated the role of calcium in regulating the growth/survival of S. aureus inside macrophages. As shown in Figure 5, all calcium modulators significantly attenuated growth of S. aureus inside macrophages. The data suggest that the route of calcium homeostasis played a crucial role in mediating the survival of Staphylococcus in macrophages. While blocking extracellular calcium influx seems to rely on IFN-γ production and IL-12Rβ receptor in reducing the bacterial survival. Increased signaling from the IFN-γ and IL12 receptors could mediate reduction in bacterial survival. On the other hand blocking either intracellular calcium release or via VGCC route seems to rely on enhanced autophagy mediated reduction of intracellular bacterial survival. These results indicate a complementarity of function by calcium homeostasis in regulating the survival of Staphylococcus in macrophages.

Figure 5

Calcium differentially regulates S. aureus survival in macrophages. Murine J774 macrophages were incubated with either EGTA or TMB-8 or amlodipine or BAYK8664 for 2h followed by 20 MOI S. aureus infection for 24h. After incubation, cells were lysed with 10% SDS and serial dilutions were plated on nutrient agar plates and scored for CFUs. Data represent mean ± SD of three independent experiments for each group. P value between group S. aureus vs S. aureus + EGTA is p=0.0023; S. aureus vs S. aureus + TMB-8 is p=0.0155, S. aureus vs S. aureus + Amlodipine is p=0.0104 and S. aureus vs S. aureus + BAYK8664 is p=0.0038.

Put together, the results presented in this report have identified critical and determinant roles for routes of calcium homeostasis in regulating various aspects of S. aureus immunity. We show a differential requirement and fine-tuning of calcium routes in each of the parameters tested. While some routes seem to regulate T cell activation and function, other routes seem to regulate innate defence responses like autophagy and phagosome-lysosome fusion. A conglomeration and coordinated efforts of all these processes by calcium finally regulate the survival of S. aureus inside macrophages. A central and a net negative role of VGCC was evident wherein its blocking mediated protective responses to this pathogen.