KLHDC10 deficiency selectively enhances necroptosis in inflammatory cells <i>in vitro</i>.

<p>(A) Schematic abstract of the experimental procedure. (B) RAW264.7 cells transfected with control or mKLHDC10 siRNAs were stimulated with the indicated L929 cell conditioned media. After 24 hours, LDH release was quantified as an indicator of cell death (n = 3). (C, E, G) RAW264.7 cells (C), immortalized MEF cells (E), and L929 cells (G) transfected with control or mKLHDC10 siRNAs were stimulated as indicated. After 24 hours, LDH release was quantified as an indicator of cell death (n = 3 each). (D, F, H) In RAW264.7 cells (C), immortalized MEF cells (E), and L929 cells (G), the knockdown efficiencies of KLHDC10 were determined by immunoblotting analysis after transfection with the indicated siRNAs. (I) Summary of this study (see the detailed explanation in the text). Data are represented as the mean ± SEM. *<i>P</i><0.05 versus control siRNA analyzed by Student’s <i>t</i> test (B). **<i>P</i><0.01 versus control siRNA analyzed by one-way ANOVA with Dunnette’s post-hoc test (C). T: mTNFα (20 ng/ml), S: Smac-mimetic (50 nM), Z: Z-VAD-fmk (10 μM), N: Necrostatin-1 (10 μM).</p

KLHDC10 Deficiency Protects Mice against TNFα-Induced Systemic Inflammation

<div><p>Systemic inflammatory response syndrome (SIRS) is a form of fatal acute inflammation for which there is no effective treatment. Here, we revealed that the ablation of Kelch domain containing 10 (KLHDC10), which we had originally identified as an activator of Apoptosis Signal-regulating Kinase 1 (ASK1), protects mice against TNFα-induced SIRS. The disease development of SIRS is mainly divided into two stages. The early stage is characterized by TNFα-induced systemic necroptosis, a regulated form of necrosis mediated by Receptor-interacting protein (RIP) 1/3 kinases. The later stage presents with an over-production of inflammatory cytokines induced by damage-associated molecular patterns (DAMPs), which are immunogenic cellular contents released from cells that underwent necroptosis. Analysis of TNFα-challenged mice revealed that KLHDC10-deficient mice show a reduction in the inflammatory response, but not in early systemic necroptosis. <i>In vitro</i> analysis suggested that the reduced inflammatory response observed in KLHDC10-deficient mice might be caused, in part, by enhanced necroptosis of inflammatory cells encountering DAMPs. Interestingly, the enhancement of necroptosis induced by KLHDC10 deficiency was selectively observed in inflammatory cells. Our results suggest that KLHDC10 is a cell-type specific regulator of necroptosis that ultimately contributes to the development of TNFα-induced SIRS.</p></div

KLHDC10 knockout (KO) mice are resistant against TNFα-induced SIRS.

<p>(A) Survival period of WT, KLHDC10 KO, and ASK1 KO mice intravenously injected with 5 μg of mTNFα. (B) Body temperature of WT, KLHDC10 KO, and ASK1 KO mice intravenously injected with 5 μg of mTNFα. Each dot indicates the number of surviving mice at that time point. (C) Body temperature of WT, KLHDC10 KO, and ASK1 KO mice after 0 and 6 hours from the intravenous injection of 5 μg of mTNFα. Data are represented as the mean ± SEM. **<i>P</i><0.01 versus WT mice and *<i>P</i><0.05 versus ASK1 KO mice analyzed by Log-rank test followed with Gehan-Breslow-Wilcoxon test (A). ***<i>P</i><0.001 (blank line) versus WT mice and *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 (red line) versus ASK1 KO mice. *<i>P</i><0.05 (green line) versus WT mice. All samples were analyzed by two-way ANOVA with the Bonferroni post-hoc test (B). ***<i>P</i><0.001 (blue line) versus WT mice and *<i>P</i><0.05 (green line) versus ASK1 KO mice analyzed by the Student’s <i>t</i> test (C).</p

The MB gains a toxic function, rather than loses a protective function, in response to HS in the absence of dPGAM5.

<p>(<b>A</b>) Ablation of the MB. Ablation of the MB in adult flies pretreated with hydroxyurea (HU) at the larval stage was confirmed by MB-specific expression of mCD8::GFP. Scale bar = 50 µm. The genotype is <i>c739-GAL4/+; UAS-mCD8::GFP/+</i>. (<b>B</b>) Ablation of the MB attenuates the vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of adult male <i>PGAM5¹</i> flies (<i>y¹</i>, <i>PGAM5¹/Y</i>) untreated (−) or treated (+) with HU are shown (<i>n</i> = 110). <i>p</i><0.0001 by the log-rank test. (<b>C</b>) Ablation of the MB does not affect the response of control flies to HS. Survival curves of adult male control (<i>y¹w¹/Y</i>) flies untreated (−) or treated (+) with HU are shown (<i>n</i> = 110). (<b>D</b>) Suppression of neurotransmission does not decrease vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of the indicated adult male flies (day 9–15) subjected to HS are shown. <i>c739-GAL4</i> flies (<i>+/Y; c739-GAL4/+; Sb/+</i>, <i>n</i> = 91), <i>c739>shi^TS</i> flies (<i>+/Y; c739-GAL4/+; UAS- shi^TS/+</i>, <i>n</i> = 100), <i>PGAM5¹, c739-GAL4</i> flies (<i>PGAM5¹/Y; c739-GAL4/+; Sb/+</i>, <i>n</i> = 100) and <i>PGAM5¹, c739>shi^TS</i> flies (<i>PGAM5¹/Y; c739-GAL4/+; UAS-shi^TS/+</i>, <i>n</i> = 100) were examined.</p

Flies deficient in dPGAM5 are vulnerable to HS.

<p>(<b>A</b>) <i>PGAM5¹</i> flies are vulnerable to HS. Survival curves of adult male control (<i>y¹w¹/Y</i>) and <i>PGAM5¹</i> flies (<i>y¹</i>, <i>PGAM5¹/Y</i>) subjected to oxidative stress (0.1% H₂O₂, <i>n</i> = 66; left), starvation (<i>n</i> = 180; middle), or HS (37°C, <i>n</i> = 190; right) are shown. Control vs. <i>PGAM5¹</i> in HS, <i>p</i><0.0001 by the log-rank test. (<b>B</b>) dPGAM5 and dPINK1 act independently in HS response. Survival curves of the indicated adult male flies (day 5–15) subjected to HS are shown (control, <i>n</i> = 120; <i>PGAM5¹</i>, <i>n</i> = 112; <i>PINK1^B9</i>, <i>n</i> = 105; <i>PINK1^B9, PGAM5¹</i>, <i>n</i> = 116). <i>PINK1^B9</i> vs <i>PGAM5¹, PINK1^B9</i>, p<0.0001 by the log-rank test. The genotypes are revertant <i>PINK1^REV/Y</i> (Control), <i>PGAM5¹/Y</i> (<i>PGAM5¹</i>), <i>PINK1^B9/Y</i> (<i>PINK1^B9</i>) and <i>PGAM5¹, PINK1^B9/Y</i> (<i>PGAM5¹, PINK1^B9</i>). (<b>C</b>) Efficiency of IR-mediated <i>dPGAM5</i> knockdown. Protein expression of dPGAM5 in <i>daughterless (da)>LacZ IR</i> flies (<i>da-GAL4/+; UAS-LacZ IR/+</i>) and <i>da>dPGAM5 IR</i> flies (<i>da-GAL4/+</i>; <i>UAS-dPGAM5 IR/+</i>) was detected by immunoblotting with dPGAM5 antibody. Actin was also detected as a loading control. (<b>D</b>) Knockdown of <i>dPGAM5</i> in the whole body induces vulnerability of flies to HS. Survival curves of adult male <i>da>LacZ IR</i> flies and <i>da>dPGAM5 IR</i> flies subjected to HS are shown (<i>n</i> = 110). <i>p</i><0.0001 by the log-rank test. (<b>E</b>) Transient knockdown of <i>dPGAM5</i> prior to HS is sufficient to induce vulnerability of flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown. IR RNA was induced two days prior to sustained HS by two cycles of HS pretreatment, each of which was composed of 37°C for 30 min, 25°C for 5 hr, 37°C for 30 min and 25°C for 18 hr. “Pre-HS +” and “pre-HS −” indicate flies expressing and not expressing IR RNA, respectively, when they were subjected to sustained HS. The genotypes are <i>hs-GAL4/UAS-LacZ-IR</i> (<i>hs>LacZ-IR</i>) and <i>hs-GAL4/UAS-dPGAM5-IR</i> (<i>hs>dPGAM5-IR</i>). <i>hs>LacZ-IR</i> (pre-HS +) flies (<i>n</i> = 79), <i>hs>dPGAM5-IR</i> (pre-HS +) flies (<i>n</i> = 68), <i>hs>LacZ-IR</i> (pre-HS −) flies (<i>n</i> = 50) and <i>hs>dPGAM5-IR</i> (pre-HS −) flies (<i>n</i> = 45) were examined. <i>hs>LacZ-IR</i> (pre-HS +) vs <i>hs>dPGAM5-IR</i> (pre-HS +), p<0.0001 by the log-rank test.</p

Locomotor activity is reduced in dPGAM5-deficient flies.

<p><i>PGAM5¹</i> flies are less active than control flies under both HS (37°C; <b>A</b>) and unstressed (25°C; <b>B</b>) conditions. Locomotor activity was monitored using the <i>Drosophila</i> activity monitor (DAM) system as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030265#s4" target="_blank">Materials and Methods</a>. Data are shown as the mean counts ± s.e.m of total flies examined for each genotype [Control, <i>n</i> = 77 and 79; <i>PGAM5¹</i>, n = 76 and 78; at 37°C and 25°C, respectively]. The genotypes are <i>y¹w¹/Y</i> (Control) and <i>PGAM5¹/Y</i> (<i>PGAM5¹</i>).</p

HS induces a rapid degeneration of the nuclei of the MB neurons in the absence of dPGAM5.

<p>The number of cell bodies of the KCs decreases in HS-treated <i>PGAM5¹</i> flies. Representative images of lobes (<b>A</b>–<b>D</b>) and calyxes (<b>E</b>–<b>H</b>) visualized by mCD8::GFP are shown. The genotypes are <i>c739-GAL4/+; UAS-mCD8::GFP/+</i> (Control) and <i>PGAM5¹</i>/<i>Y; c739</i>-<i>Gal4</i>/+; <i>UAS</i>-<i>mCD8::GFP</i>/+ (<i>PGAM5¹</i>). Scale bar (<b>A</b>–<b>H</b>) = 50 µm. Representative images of the KC nuclei visualized by Histone2B::ECFP (<b>I</b>–<b>L</b>; scale bar = 20 µm) and the total number of KC nuclei (<b>M</b>) are shown. The latter was calculated by summing up the number counted manually in every tenth section, and the results were shown as the mean cell number per fly ± s.e.m. [<i>n</i> = 6, Control HS (−) and (+) and <i>PGAM5¹</i> HS (−); <i>n</i> = 8, <i>PGAM5¹</i> HS (+)]. ** P<0.01, unpaired t-test (<b>M</b>). The genotypes are <i>c739-GAL4</i>/<i>UAS-Histone2B::ECFP</i> (Control) and <i>PGAM5¹</i>/<i>Y</i>; <i>c739-GAL4</i>/<i>UAS-Histone2B::ECFP</i> (<i>PGAM5¹</i>).</p

dPGAM5 exerts its protective effect against HS by preventing apoptosis in the MB.

<p>(<b>A–F</b>) TUNEL-positive KCs are detected in the MB of HS-treated <i>PGAM5¹</i> flies, but not in that of control flies. TUNEL staining of the MB of control flies (<b>A</b>) and <i>PGAM5¹</i> flies (<b>B</b>) treated with HS for 75 min is shown. Counter nuclear staining with Hoechst33258 (<b>C</b> and <b>D</b>) and merged images of TUNEL and Hoechst 33258 staining (<b>E</b> and <b>F</b>) are also shown. Scale bar (<b>A</b>–<b>F</b>) = 20 µm. The genotypes are <i>c739-GAL4/UAS-Histone2B::ECFP</i> (Control) and <i>PGAM5¹/Y; c739-GAL4/UAS-Histone2B::ECFP</i> (<i>PGAM5¹</i>). (<b>G</b>) Expression of p35 in the MB attenuates the vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 105). <i>PGAM5¹</i> vs. <i>PGAM5¹; c739>p35</i>, <i>p</i><0.0001 by the log-rank test. The genotypes are <i>c739-GAL4/+</i> (Control), <i>PGAM5¹/Y; c739-GAL4/+</i> (<i>PGAM5¹</i>) and <i>PGAM5¹/Y; c739-GAL4/+; UAS-p35/+</i> (<i>PGAM5¹; c739>p35</i>). (<b>H</b>) Expression of p35 in the MB does not affect the response of control flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 130). The genotypes are <i>c739-GAL4/+</i> (Control) and <i>c739-GAL4/+; UAS-p35/+</i> (Control; <i>c739>p35</i>).</p

Identification of a conserved redox-sensitive surface-exposed cysteine in the TRAF C-terminal domain of TRAF2, 3, and 6.

<p>(A) Structure-based sequence alignment of a region of the TRAF domain of human and murine TRAF2, 3, 4, and 6. Secondary structures in the region and TRAF-motif interacting residues are indicated in the figure. (B) <i>Traf6-/-</i> MEFs were left untreated or treated with HSV-2 (3×10⁶ pfu/ml, MOI 3, 5 h) or poly(I∶C) (25 µg/ml, 2 h). Total cellular lysates we generated and phospho-IκBα was measured by Luminex. (C–F) Traf6-/- MEFs were transfected with empty vector (pCMV), WT TRAF6, or C390S TRAF6. The cells were treated with HSV-2 (3×10⁶ pfu/ml, MOI 3, 5 h), poly(I∶C) (25 µg/ml, 2 h), IL-1β (10 ng/ml), and LNAC (3.2 mM, 30 min prior to further treatment) as indicated. Total RNA and total cell lysates were isolated and analyzed for IFN-β mRNA and P-IκBα by RT-qPCR and Luminex, respectively. Data is shown as means of triplicates +/- st.dev. (G) <i>Traf3-/-</i> MEFs were transfected with empty vector (pRK5), WT TRAF3, or C455S TRAF3. The cells were treated with HSV-2 (3×10⁶ pfu/ml, MOI 3) and LNAC (3.2 mM, 30 min prior to further treatment) as indicated. Total RNA was isolated 5 h post-infection and analyzed for IFN-β mRNA by RT-qPCR. Data is shown as means of triplicates +/- st.dev. RU, relative units; NR, normalized ratio.</p

Intracellular ROS produced following HSV infection is essential for cytokine expression.

<p>(A) RAW264.7 cells were seeded in chambered-cover slides, either pre-treated with 6.4mM LNAC or complete media for 30 min, and subsequently infected with HSV-2 for 1, 2, or 3 h. CM-H₂DCFDA (5 µM) was added immediately before imaging. ROS production was monitored in z-stack images (1 µm) and images were acquired with a 40x objective every 30 seconds by confocal microscopy. Images represent maximum intensity projections. Amount of ROS was determined and represented as mean fluorescence per cell with a minimum of 100 cells counted per condition. Data is presented as mean of 3 experiments for the untreated samples and of 2 experiments for the LNAC samples +/- sem. (B–D) Peritoneal macrophages were seeded and either left uninfected or infected with HSV-2 (3×10⁶ pfu/ml, MOI 3); each in the absence or presence of 10 µM hydrogen peroxide. Supernatants were harvested after 16 h for measurement of IFN-α/β, CXCL10, and CCL5. (E, F) Peritoneal macrophages were pre-incubated with increasing concentrations of PDTC or LNAC and subsequently infected with HSV-2 (3×10⁶ pfu/ml, MOI 3). Supernatants were harvested 12 h post infection and CCL5 protein was measured by ELISA. The data is shown as mean of 3 replicates +/- st. dev. and is representative of 3 experiments. CCL5 levels significantly different from those induced by HSV-2 alone (p<0.05) are indicated with *.</p