A nanoconjugate Apaf-1 inhibitor protects mesothelial cells from cytokine-induced injury

Abstract

Background: Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology: Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma. Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion: Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.

Figure 1. Cooperation of cytokines induces apoptosis in mesothelial cells.

A) Representative flow cytometry of DNA content diagrams. Note the increase in the hypodiploid, apoptotic population in HPMC cultured for 48 h in presence of TNFα/IFNγ. B) Quantification of apoptosis in HPMC by flow cytometry. The combination of TNFα/IFNγ induces apoptosis. When not specified, 300 U/mL IFNγ and 250 ng/mL TNFα were used, * p<0.04 vs each individual cytokine at 24 h. ** p<0.001 vs each individual cytokine at 48 h. & p<0.001 vs control. Mean±SEM of 6 different experiments. C) Dose-response in HPMC. Mean±SEM of 4 different experiments * p<0.05 vs control. D) Quantification of apoptosis in HOMC by flow cytometry. The combination TNFα/IFNγ for 48 h induces apoptosis. *p<0.005 vs. control. Mean±SEM of 4 different experiments.

Figure 2. Apoptosis induced by cooperation of cytokines is caspase-dependent.

A) Caspase activation in HPMC exposed to TNFα/IFNγ for 24 h. Apoptosis was decreased by zVAD (pan-caspase inhibitor), DEVD (caspase-3 inhibitor) and IETD (caspase-8 inhibitor). Note cytokeratin (CK) 8/18 cleavage by caspases as well as the nuclear apoptotic morphology in cells exposed to TNFα/IFNγ (arrowheads). Green: FITC-M30 cytodeath antibody, red: propidium iodide in permeabilized cells. Original magnification ×100. B) Representative diagram of detection of caspase-cleaved CK 8/18 by flow cytometry in HPMC. C) Quantification of caspase-cleaved CK 8/18 by flow cytometry in HPMC treated with TNFα and IFNγ for 24 h. * p<0.005 vs control and # p<0.05 vs TNFα/IFNγ. Mean±SEM of 4 different experiments. D) Flow cytometry diagrams of permeabilized, propidium iodide-stained cells. Note the decrement of hypodiploid cells (arrowhead) in the presence of caspase inhibitors in HPMC. E) Quantification of apoptosis by flow cytometry of DNA content in HPMC exposed to TNFα/IFNγ for 48 h. Apoptosis was decreased by zVAD, DEVD and IETD. *p<0.001 vs control and # p<0.0005 vs TNFα/IFNγ. Mean±SEM of 6 independent experiments.

Figure 3. Inflammatory cytokines retard remesothelization.

A) Wound healing in HPMC. Contrast phase microscopy. Cells were preincubated with caspase inhibitor or vehicle and cultured in presence of proinflammatory TNFα/IFNγ or control media. Original magnification 20×. B) Quantification of wound healing as number of HPMC cells filling the denuded area (cells/mm²). In presence of cytokines there is delayed wound healing that is not improved by zVAD.* p<0.03 control vs. TNFα/IFNγ and control vs TNFα/IFNγ/zVAD. Mean±SEM of 5 different experiments.

Figure 4. A nanoconjugate Apaf-1 inhibitor (PGA-peptoid QM56) inhibits caspase activation and apoptosis.

A) Quantification of apoptosis in HOMC by flow cytometry. HOMC were preincubated with different concentrations of inhibitors or vehicle and cultured with TNFα and IFNγ for 48 hours. Mean±SEM of 3 different experiments *p<0.001vs TNFα/IFNγ. B) QM56 prevents procaspase-3 processing and the appearance of active p17 and p19 caspase-3 fragments 24 h following exposure to TNFα/IFNγ in HOMC. zVAD stalled the process of caspase activation at the level of the p21 precursor as previously described , . Binding of zVAD.fmk to this p21 intermediate blocks its activity . Western blot, representative of 3 independent experiments. C) Representative flow cytometry of DNA content diagrams. Cells were preincubated with QM56 and then cultured with TNFα/IFNγ for 48 h. Note the decreased number of hypodiploid apoptotic HPMC in TNFα/IFNγ/QM56 treated cells (black arrow). Inset: nuclear apoptotic morphology in cells exposed to TNFα/IFNγ and stained with DAPI (white arrows). D) Quantification of apoptosis by flow cytometry of DNA content in HPMC. Mean±SEM of 6 different experiments. *p<0.002 vs cells treated with TNFα/IFNγ/QM56.

Figure 5. QM56 preserves remesothelization.

A) Wound healing in HPMC. Contrast phase microscopy. Cells were preincubated with Apaf-1 inhibitor and cultured in presence of proinflammatory medium (TNFα and IFNγ). Original magnification 20×. B) Quantification of wound healing in HPMC as number of cells filling the denuded area (cells/mm²). The delayed wound healing induced by TNFα/IFNγ is recovered in presence of QM56. * p<0.03 control vs. TNFα/IFNγ ** p<0.003 TNFα/IFNγ/QM56 vs. TNFα/IFNγ. Mean±SEM of 5 different experiments.

Figure 6. Caspase inhibition compromises long-term recovery from inflammatory injury while Apaf-1 inhibition does not.

A) HPMC plated on twelve-well plates were preincubated with inhibitors or vehicle and exposed to TNFα/IFNγ for 24 h. Cells were then trypsinized, washed, and seeded in Petri dishes in the presence of complete medium with 20% FCS for 5 days, but without cytokines or inhibitors, to allow for their recovery. Cells were stained with crystal violet after 5 days. Representative of 3 different experiments. B) Similar results were obtained with HOMC. HOMC were preincubated with inhibitors or vehicle and exposed to TNFα/IFNγ for 48 h. Then the cells were trypsinized, washed and seeded without inhibitors and cytokines for 5 days. Representative of 4 different experiments. C) Quantification of crystal violet staining in HOMC. Mean±SEM of 4 different experiments. *p<0.03 vs TNFα/IFNγ; #p<0.02 vs TNFα/IFNγ.

Figure 7. QM56 prevents mesothelial cell apoptosis induced by intraperitoneal cytokine administration in vivo.

Mice were injected ip with 250 ng/mL TNFα and 300 U/mL IFNγ at time 0 and sacrificed at 48 h. QM56 or vehicle was administrated 1 h before the cytokines and 5 h later. A) Representative images of Cytodeath (green)/propidium iodide co-staining. Note an increased number of cells with green cytoplasm indicative of caspase-mediated apoptosis in the sample from the mouse injected with TNFα/IFNγ. Magnification ×120 B) Quantification of positive cells (green) using Image Pro plus software in 5 fields per mouse (around 600 cells). Mean±SEM of 5 mice per group. * p<0.001 vs. control: # p<0.001vs.TNFα IFNγ.

Figure 8. QM56 prevents mesothelial cell apoptosis during S. aureus peritonitis in mice.

Mice were injected ip. with 5×10⁸ c.f.u. S. aureus at time 0 and sacrificed at 48 h. QM56 or vehicle was administrated 1 h before the S. aureus and 5 h later A) Representative images of Cytodeath (green)/propidium iodide co-staining. Note an increased number of cells with green cytoplasm (arrows) indicative of caspase-mediated apoptosis in the sample from the mouse injected with S. aureus. Apoptosis was prevented by QM56 Magnification ×120. B) Quantification of cytodeath positive cells (green) using Image Pro plus software in 5 fields per mouse (around 600 cells). Mean±SEM of 5 mice per group. * p<0.05 vs control.