Mitochondrial translocation of oxidized cofilin induces caspase-independent necrotic-like programmed cell death of T cells

Abstract

Oxidative stress leads to T-cell hyporesponsiveness or death. The actin-binding protein cofilin is oxidized during oxidative stress, which provokes a stiff actin cytoskeleton and T-cell hyporesponsiveness. Here, we show that long-term oxidative stress leads to translocation of cofilin into the mitochondria and necrotic-like programmed cell death (PCD) in human T cells. Notably, cofilin mutants that functionally mimic oxidation by a single mutation at oxidation-sensitive cysteins (Cys-39 or Cys-80) predominately localize within the mitochondria. The expression of these mutants alone ultimately leads to necrotic-like PCD in T cells. Accordingly, cofilin knockdown partially protects T cells from the fatal effects of long-term oxidative stress. Thus, we introduce the oxidation and mitochondrial localization of cofilin as the checkpoint for necrotic-like PCD upon oxidative stress as it occurs, for example, in tumor environments.

Figure 1

Long-term oxidative stress induces caspase-independent T-cell death in primary human T cells. (a) Primary human T cells (PBTs) were incubated with 100 μM H₂O₂ for the indicated time points. Cell death was quantified with Annexin V (y axis) and 7-AAD (x axis). The upper left quadrant shows the early apoptotic cells and the upper right quadrant shows the late apoptotic cells. (b) The graph shows a quantification of late apoptotic cells as shown in (a) (n=3; S.E.M.) (^***P<0.001; NS: not significant). (c) PBTs were treated with the indicated concentrations of H₂O₂ for 26 h in the presence (black columns) or absence (white columns) of the pan-caspase inhibitor Z-VAD-fmk (20 mM). Cell death was analyzed using Annexin V/7-AAD staining as described in (a) and (b) (n=3; S.E.M.). (d) Jurkat T cells were treated with 1 μg/ml CD95 antibody in the presence or absence of Z-VAD-fmk. Cell death was assessed as described above

Figure 2

Long-term oxidative stress leads to a necrotic-like cell death. (a, b) PBTs were incubated without (a, upper panel) or with 50 μM (a, lower panel) H₂O₂. A total of 10 000 cells were acquired using an ImageStream. The representative pictures show the dark field or side scatter (SSC, cyan), nuclear stain (green) and bright field. The dot plots of the cell populations on the right-hand side of (a) display the calculation of the area of the nuclear fragments (y axis) and bright detail intensity (x axis) on the single-cell level. Cells with nuclear fragments display higher nuclear bright detail intensity and smaller nuclear fragments area. The nuclear fragmentation is shown as percent of cells (for details, see Materials and Methods). A quantification of three independent experiments is shown in (b). (c, d) Jurkat T cells were treated with CD95 in the presence or absence of Z-VAD-fmk. The nuclear fragmentation was assessed for 10 000 cells as in (a) and (b). (e) Primary human T cells were incubated without (i and iv) or with (ii, iii, v, vi) 50 μM H₂O₂ for 26 h. Thereafter, cell morphology was analyzed using transmission electron microscopy (TEM). The lower lane shows pictures with higher magnifications of the respective cells in the upper lane. The bar in the lower lane corresponds to 500 and 1000 nm in the upper lane. The middle panel (ii and v) shows cells with a swollen phenotype of mitochondria. Shown are representative pictures taken from two independent experiments (M, mitochondria; N, nuclei; G, Golgi apparatus)

Figure 3

Mitochondrial disintegration upon oxidative stress. (a) Transmission electron microscopic pictures of the mitochondria from the control or oxidatively stressed (50 μM H₂O₂) T cells. Shown are representative pictures taken from two independent experiments (M, mitochondria). (b) The area (in μm²) of the mitochondria was analyzed using MIFC and is shown for untreated (gray histogram) and hydrogen peroxide-treated cells (black histogram). The graph shows the mean area of the mitochondria from untreated or hydrogen peroxide-treated T cells (n=3; S.E.; ^*P<0.05). (c, d) The mitochondrial membrane potential (ΔΨ_m) of unstressed (left histograms) or H₂O₂-stressed cells was assessed using TMRE. A decrease in the TMRE fluorescence corresponds to a loss of the membrane potential of the mitochondria. A quantification of three independent experiments is shown in (c). The black bars represent T cells that were pre-treated with Z-VAD-fmk (n=3; S.E.M.). Note that experiments using DilC₁(5) instead of TMRE gave essentially the same results (Supplementary Figure 1)

Figure 4

Cofilin translocates to the mitochondria upon oxidative stress. (a) Primary human T cells were incubated with (lower panel) or without (upper panel) 50 μM H₂O₂. Thereafter, cells were stained for cofilin (red) or mitochondria (MitoTracker, green) and analyzed via confocal laser scan microscopy. Merge displays the digital overlay of red and green fluorescence. The figure is representative of three independent experiments. (b) For cryo-immunogold electron microscopy, primary human T cells were either left untreated (i and iii) or treated with H₂O₂ (ii and iv) and subsequently fixed with 2% PFA for 10 min. Cells were stained with cofilin antiserum combined with protein A labeled with 15 nm gold particles. Shown are two example pictures taken from two independent experiments (M, mitochondria; N, nucleus). (c, d) The colocalization of cofilin and mitochondria was evaluated by the calculation of a similarity score of the two probes from untreated (gray histogram) and H₂O₂-treated (black lined histogram) PBT using MIFC. The histogram shows the distribution of the similarity within the whole-cell population as in conventional flow cytometry (up to 10 000 cells). A score of 1 indicates that the two probes are uncorrelated, whereas higher numbers indicate a higher degree of similarity. The mean similarity score of four independent experiments is shown in (d) (n=4; S.E.M.; ^*P<0.05)

Figure 5

Cofilin mutants translocate into the mitochondria and induce PCD in primary human T cells. (a) T cells expressing wt-, G39- or A39-cofilin were incubated in the absence (white bars) or presence (black bars) of H₂O₂. The percentages of dead cells were analyzed as described above (n=3; S.E.M.; ^#P<0.05 compared with wt-cofilin without H₂O₂; ^*P<0.05 compared with wt-cofilin with H₂O₂). (b) T cells expressing eGFP-tagged wt-cofilin, G39-cofilin or G80-cofilin were stained for Annexin V (y axis) and 7-AAD (x axis). The percentage of late apoptotic cells is depicted in the upper right corner of the dot plot. (c) The mitochondrial membrane potential was analyzed in wt-, G39- or G80-cofilin-expressing T cells using DilC₁(5) as described (n=3; S.E.; ^*P<0.05). Note that TMRE staining yielded similar results (not shown). (d, e) Wt-cofilin, G39-cofilin or G80-cofilin was incubated with (black bars) or without (white bars) Z-VAD-fmk for 26 h. The activation of caspase-3 (d) as well as cell death (e) was analyzed using flow cytometry (n=3; S.E.M.). (f) Primary human T cells were incubated with the indicated amounts of H₂O₂ for 26 h in the presence (black bars) or absence (white bar) of 500 nM PJ-34. Cell death was assessed by flow cytometry using 7-AAD and Annexin V. The bars represent the mean of three independent experiments and S.E.M. (g) Cofilin constructs (wt-, G39- or G80-Cofilin) were transfected into primary human T cells. The T cells were then incubated without (white bars) or with 500 nM (black bars) or with 1000 nM (gray bars) of the PARP inhibitor PJ-34. The percentage of dead cells was determined as described above (n=3; S.E.M.)

Figure 6

Oxidation-mimicking mutants of cofilin translocate into the mitochondria and induce programmed cell death. (a) Transfected T cells were stained for mitochondria (red) and analyzed by confocal laser scan microscopy. The eGFP-tagged cofilin is shown in green. The localization was analyzed for at least 500 cells per construct using MIFC. (b) The colocalization of mitochondria and cofilin was evaluated using the similarity score (MIFC analysis). The higher background colocalization originates from the high expression levels of the eGFP-tagged cofilin constructs as well as the tendency of eGFP to disperse throughout the cell. (c, d) The area and nuclear fragmentation of eGFP-positive cells was assessed as described above

Figure 7

Cofilin binds to HSC70 under oxidative stress conditions. (a) FLAG-tagged wt- or G39-cofilin was expressed in and then precipitated from Jurkat T cells. Lysates were subjected to PAGE and stained with Coomassie blue. The heavy (50 kDa) and light (ca. 25 kDa) chains of the precipitating antibody were found in each lane, including the control (untransfected Jurkat T cells). Bands that were found only in lane 2 (G39-cofilin) or lane 3 (wt-cofilin) were analyzed using mass spectrometry and identified as HSC70 (only present in lane 2) or actin (lanes 2 and 3). (b) Wt-, G39- or G80-cofilin was precipitated from Jurkat T cells. The corresponding western blot was stained for the FLAG-tag (lower part) or HSC70 (upper part). (c, d) PBTs were treated with 50 μM H₂O₂ for 7 or 24 h. Thereafter, cells were lysed and endogenous cofilin was immunoprecipitated. The precipitates were subjected to western blot analysis and stained for HSC70 (upper panel) or cofilin (lower panel). The graph in (d) shows a quantification of the HSC70 to cofilin ratios of three independent experiments (n=3; S.E.M.; ^*P<0.05). (e) The subcellular localization of HSC70 in long-term oxidatively stressed (lower part) or control T cells (upper part) was analyzed using confocal laser scan microscopy. The white color in the merge of the stressed cells shows the colocalization of cofilin and HSC70 with the mitochondria. The figure is representative of three experiments. (f) The colocalization of cofilin and mitochondria (upper graph), cofilin and HSC70 (lower graph) or HSC70 and mitochondria (central graph) was evaluated by the calculation of a similarity score of the corresponding probes (n=3; S.E.M.; ^*P<0.05)

Figure 8

Knockdown of cofilin prevents necrotic-like PCD upon oxidative stress conditions. (a) T cells were treated with control or cofilin-specific siRNA. The expression of cofilin and actin was assessed in the lysates of these cells. Note that the mock-transfected cells were undistinguishable from control siRNA-treated cells (data not shown). (b) A quantification of three independent experiments shows that cofilin was efficiently knocked down by the siRNA (n=3; S.E.; ^**P<0.01). (c) The induction of cell death (Annexin V/7-AAD staining) was analyzed in control siRNA or cofilin-specific siRNA-treated cells that were either incubated with (black bars) or without (white bars) H₂O₂ for 26 h (n=3; S.E.; ^**P<0.01)