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. 2014 Nov 14;289(46):31736-31750.
doi: 10.1074/jbc.M114.609396. Epub 2014 Oct 6.

A role for stefin B (cystatin B) in inflammation and endotoxemia

Katarina Maher  1 , Barbara Jerič Kokelj  2 , Miha Butinar  2 , Georgy Mikhaylov  2 , Mateja Manček-Keber  3 , Veronika Stoka  2 , Olga Vasiljeva  4 , Boris Turk  5 , Sergei A Grigoryev  6 , Nataša Kopitar-Jerala  7
Affiliations

A role for stefin B (cystatin B) in inflammation and endotoxemia

Katarina Maher et al. J Biol Chem. .

Abstract

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1). In this study we demonstrated that stefin B-deficient (StB KO) mice were significantly more sensitive to the lethal LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1β and IL-18 in the serum. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase-1 and -11 activation determined in StB KO bone marrow-derived macrophages resulted in enhanced IL-1β processing. Pretreatment of macrophages with the cathepsin inhibitor E-64d did not affect secretion of IL-1β, suggesting that the increased cathepsin activity determined in StB KO bone marrow-derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS-induced sepsis in StB KO mice is dependent on caspase-11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.

Keywords: Caspase; Inflammasome; Mitochondria; Reactive Oxygen Species (ROS); Sepsis.

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Figures

FIGURE 1.
FIGURE 1.
LPS-induced lethality and LPS-triggered pro-inflammatory cytokine release in vivo is stefin B-dependent. A, Kaplan-Meier plot showing the percentage of survival over time of age-matched wild type (WT) (n = 13) and StB KO mice (n = 11). *, p < 0.05 (log-rank (Mantel-Cox) test). Mice were injected intraperitoneally with 30 mg/kg LPS, and survival was monitored six times daily for a total of 4 days. B, for serum cytokine analysis, mice were injected intraperitoneally with 10 mg/kg LPS, and blood was taken 0, 2, 4, and 6 h after LPS administration. Levels of indicated cytokines in serum were quantified by bead-based multiplex for the flow cytometer. Statistical significance between WT and StB KO mice was determined in serum for IL-1β at 4 and 6 h, for IL-18 at 0, 2, 4, and 6 h, and for TNF-α at 2 h post-LPS injection. Data were obtained from three independent biological experiments performed in triplicate, and the results are presented as means ± S.D. *, p < 0.05; **, p < 0.01.
FIGURE 2.
FIGURE 2.
Transcription of caspase-11 is up-regulated in LPS-stimulated StB KO BMDMs. A, BMDMs were stimulated with LPS and total RNA was isolated 4 h post-stimulation. Relative mRNA expression was determined. RNA expression was normalized to reference genes Gapdh and B2m and presented as fold increase. Data were obtained from three independent experiments performed in triplicate, and the results are presented as means ± S.D. **, p < 0.01. B, increased expression of stefin B diminished NF-κB activation. RAW-blue cells (InvivoGen) were transfected with empty (control) pcDNA3 vector or pcDNA3/Stefin B-HisTag to induce stefin B overexpression. SEAP activity was assessed by reading the OD at 655 nm. C, fold enrichment (ChIP/Input) of H3K9me2 immunoprecipitated nucleosomes at the promoter and the open reading frame (ORF) downstream of the transcriptional start of the mouse caspase-11 gene (Casp4) IL-1β (IL-1β) gene.
FIGURE 3.
FIGURE 3.
Decreased IFN-β expression and signaling in StB KO BMDMs. A, BMDMs from WT and StB KO mice were treated with inhibitory anti IFNAR1 antibody and stimulated for 4 h with LPS (100 ng/ml). Cell lysates were immunoblotted with anti-caspase-11 antibodies recognizing pro-caspase (casp)-11 (inactive zymogen) and subunits generated by proteolytic cleavages. Lysates were probed with anti-β-actin antibody as a loading control. Data shown are representative of three independent experiments. B, BMDMs were stimulated with LPS, and total RNA was isolated 2 h post-stimulation. Relative mRNA expression was determined. RNA expression was normalized to reference gene B2m and presented as fold increase. Data were obtained from three independent experiments performed in triplicate. C, fold enrichment (ChIP/Input) of H3K9me2-immunoprecipitated nucleosomes at the promoter and the open reading frame (ORF) downstream of the transcriptional start of the mouse IFN-β (IFN-β). D, input DNA normalized to genome average. E, immunoblotting of p-STAT1 (pY-701) and total STAT1 in WT and StB KO BMDM at the indicated times following stimulation with IFN-β. Data shown are representative of three independent experiments.
FIGURE 4.
FIGURE 4.
Enhanced processing and secretion of IL-1β and IL-18 in stefin B-deficient BMDMs is caspase-1-dependent. BMDMs from WT and StB KO mice were primed for 4 h with LPS (100 ng/ml), washed, and stimulated with ATP for 20 min (5 mm) to activate the Nlrp3 inflammasome. Cell lysates and culture supernatants were immunoblotted with the indicated antibodies. Anti-caspase-1 and anti-caspase-11 antibodies recognized pro-caspase (inactive zymogen) and subunits generated by proteolytic cleavages. Anti-IL-1β and anti-IL-18 antibodies recognized inactive pro-form in cell extracts and mature secreted cytokines in cell supernatants. Lysates were immunoblotted with anti-β-actin as a loading control. Data shown are representative of three independent experiments.
FIGURE 5.
FIGURE 5.
Increased inflammasome activation in StB KO BMDMs is independent of lysosomal cysteine cathepsins. A, biosynthesis and integrity of lysosomes were studied by LysoTracker Green probe, as described under “Experimental Procedures.” The lysosomal fluorescence intensity was quantified upon LPS and ATP stimulations. Lysosomal destabilization was increased by ATP addition and was comparable between genotypes. Data were obtained from three independent biological experiments performed in triplicate, and the results are presented as means of geometric mean fluorescence intensity ± S.D. B, release of cathepsins into the cytosol and their activity were measured after indicated stimulations using fluorogenic substrates specific for cathepsins. Incubation of BMDMs with E-64d completely prevented cathepsin activity. C, BMDMs from WT and StB KO mice were primed for 4 h with LPS (100 ng/ml), washed, and stimulated with ATP for 20 min (5 mm) or primed with broad spectrum cysteine cathepsin inhibitor E-64d for 1 h (15 μm), followed by LPS and ATP stimulation. Cell lysates were immunoblotted with indicated antibodies. Lysates were immunoblotted with anti-β-actin as a loading control. Incubation of BMDMs with E-64d did not affect the processing of caspase-1 and pro-inflammatory cytokine release. Data shown are representative of three independent experiments. D, BMDMs were seeded into 96-well plates and stimulated as above. IL-1β release was quantified in the media by FlowCytomixTM. StB KO BMDMs secreted higher amounts of IL-1β into the media compared with the WT; however, priming with E-64d did not affect IL-1β release (n.s., nonsignificant difference). Data were obtained from four independent experiments performed in triplicate, and the results are presented as means ± S.D. *, p < 0.05; **, p < 0.01. E, viability of BMDMs was assessed by LDH release into the cell culture media upon inflammasome activation. The cytotoxicity was expressed as the percent of the total LDH release. Data were obtained from three independent experiments performed in triplicate, and the results are presented as means ± S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 6.
FIGURE 6.
Stefin B is localized into mitochondria upon LPS stimulation. WT BMDMs were left untreated (A) or LPS (100 ng/ml)-primed (B). The localization of stefin B was determined by immunofluorescence. The images were merged to visualize co-localization of stefin B and mitochondria. Scale bar, 10 μm.
FIGURE 7.
FIGURE 7.
Mitochondrial disruption resulted in increased superoxide generation in StB KO BMDMs. A, MitoTracker Red CMXRos uptake was quantified by flow cytometry. The decrease in red fluorescence correlates with the loss of mitochondrial membrane potential. Mitochondria of StB KO BMDMs turned to be more susceptible to LPS and ATP stimulation. Results are representative of three independent experiments performed in duplicate and presented as histogram plots and as columns of geometric mean fluorescence intensity of three independent experiments performed in duplicate and presented as means ± S.D. *, p < 0.05. B, BMDMs were stimulated and probed with 5 μm MitoSOX Red fluorogenic dye as indicated under “Experimental Procedures.” Red fluorescence was measured by flow cytometry. LPS and LPS/ATP stimulations induced significant increase in superoxide (mtROS) generation in StB KO compared with WT BMDMs. Results are representative of three independent experiments performed in duplicate and presented as histogram plots and as columns of geometric mean fluorescence intensity of three independent experiments performed in duplicate and presented as means ± S.D. *, p < 0.05; **, p < 0.01.
FIGURE 8.
FIGURE 8.
Schematic of proposed model for the role of stefin B in Nlrp3 inflammasome activation. Step 1, upon LPS stimulation stefin B translocates into mitochondria and protects mitochondrial integrity. Step 2, mitochondrial ROS up-regulate NF-κB activation. Step 3, in the nucleus, stefin B deficiency resulted in a decreased level of H3K9me2-repressive histone variant at caspase-11 promoter and increased level of caspase-11 mRNA expression.
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