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 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 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

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

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

Heightened ISR in <i>EIF2B4</i>^<i>A392D</i> cells.

<p>(A) Flow cytometry analysis of <i>CHOP</i>::<i>GFP</i> and <i>XBP1</i>::<i>Turquoise</i> dual reporter-containing parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells. The cells were untreated (UT) or stimulated with 250 nM thapsigargin (Tg) or 0.5 mM histidinol (His) for 24 hours. Note the enhanced response of the <i>CHOP</i>::<i>GFP</i> ISR reporter. (B) Bar diagram of the median ± S.D. of the reporter gene activity from experiments as shown in “A”. N = 3, *P = 0.0057 for Tg, *P = 0.037 for His, Unpaired t test. (C) Experimental design for tracking <i>EIF2B4</i>^<i>A392D</i> mutations. A fluorescent protein-marked sgRNA/Cas9 plasmid targeting <i>EIF2B4</i> and a wildtype or <i>EIF2B4</i>^<i>A392D</i> mutant repair template marked by a silent <i>Spe</i>I mutation were co-transfected into CHO-S21 cells. Transfected cells (selected by FACS), were treated with histidinol and divided into four bins (Bin #1 to #4) by level of <i>CHOP</i>::<i>GFP</i> expression. After recovery, genomic DNA was isolated from cells in each bin and the targeted region of <i>EIF2B4</i> was amplified by PCR and digested with <i>Spe</i>I to reveal frequency of targeting by either repair template. (D) PCR fragments digested with <i>Spe</i>I from genomic DNA of the indicated bins, visualized on an agarose gel. Shown is an image of a representative experiment reproduced twice. (E) Plot of the distribution of <i>Spe</i>I digested fragments in the four bins of transduced cells from the experiment in “D”. The band intensities of the digested fragments (reporting on recombination of the wildtype or mutant repair template) were normalized to total PCR product intensity and the distribution of the relative frequency of recombination in the different bins was plotted. Note the enrichment for recombination of the <i>EIF2B4</i>^<i>A392D</i> mutant repair template in the ISR^High bin.</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

Stress-resistance of wildtype and VWM cells.

<p>(A) Schema of experiments to compare the effect of thapsigargin in parental CHO-S21 and VWM mutant cells. Cells were treated with thapsigargin (Tg; 250 nM) for the indicated time, washed free of compounds and allowed to recover before assay. W = WST-1 assay, P = Puromycin labeling. (B) Immunoblot of puromycinylated proteins following a brief pulse of puromycin, reporting on levels of translation under the indicated experimental conditions. Shown is a representative experiment reproduced four times. P and E indicate parental CHO-S21 and <i>EIF2B4</i>^<i>A392D</i> mutant cells, respectively. (C) Quantification of “B”. Signal intensities of puromycinylated proteins were normalized by eIF2α. Shown are means ± SEM of four independent experiments. (D) Cell viability measured by the WST-1 assay in the experiment described in “A”. Shown are the mean ± SEM of four replicates of a representative experiment repeated three (R484W, R468W) to six (A392D) times.</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

Severe VWM mutant cells are unable to tolerate a second <i>EIF2S1</i>^<i>S51A</i> mutation.

<p>(A) Experimental design for tracking <i>EIF2S1</i>^<i>S51A</i> mutant cells. A fluorescent-tagged sgRNA/Cas9 plasmid targeting <i>EIF2S1</i> was co-transfected alongside wild type (WT) or <i>EIF2S1</i>^<i>S51A</i> (Mut) templates into CHO-S21 dual reporter cells. Following FACS selection for the transfected cells they were treated with 250 nM thapsigargin (Tg) for 24 hours and reporter expression was analyzed. (B) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) CHO-S21 cells from the experiment outlined in “A”. Note the emergence of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated cells in the pool offered an <i>EIF2S1</i>^<i>S51A</i> repair template. (C) Flow cytometry analysis of reporter activity in untreated (UT) and thapsigargin-treated (Tg) parental CHO-S21 or indicated VWM mutant cells following targeting of the <i>EIF2S1</i> locus with an <i>EIF2S1</i>^<i>S51A</i> repair template (as described in “A”). Note the lack of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>A392D</i> or <i>EIF2S1</i>^<i>S51A</i><i>; EIF2B4</i>^<i>R484W</i> double mutant cells (lower right panel). (D) Percentage of <i>CHOP</i>::<i>GFP</i> negative, <i>XBP1</i>::<i>turquoise</i> positive thapsigargin-treated putative <i>EIF2S1</i>^<i>S51A</i> mutant cells in the indicated population from experiments as in “C”. Shown are means ± S.D. N = 6 (Parent), 5 (<i>EIF2B4</i>^<i>A392D</i>), and 3 (<i>EIF2B4</i>^<i>R484W</i> and <i>EIF2B4</i>^<i>R468W</i>). *** P<0.001, ** P<0.01, n.s. not significant, One way ANOVA followed by Dunnett’s multiple comparisons test.</p