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. 2017 Dec 14;130(24):2664-2677.
doi: 10.1182/blood-2017-05-782102. Epub 2017 Sep 7.

Cytoprotective activated protein C averts Nlrp3 inflammasome-induced ischemia-reperfusion injury via mTORC1 inhibition

Sumra Nazir  1 , Ihsan Gadi  1 , Moh'd Mohanad Al-Dabet  1 , Ahmed Elwakiel  1 , Shrey Kohli  1 , Sanchita Ghosh  1 , Jayakumar Manoharan  1 , Satish Ranjan  1 , Fabian Bock  1   2 , Ruediger C Braun-Dullaeus  3 , Charles T Esmon  4   5 , Tobias B Huber  6 , Eric Camerer  7 , Chris Dockendorff  8 , John H Griffin  9 , Berend Isermann  1 , Khurrum Shahzad  1   10
Affiliations

Cytoprotective activated protein C averts Nlrp3 inflammasome-induced ischemia-reperfusion injury via mTORC1 inhibition

Sumra Nazir et al. Blood. .

Abstract

Cytoprotection by activated protein C (aPC) after ischemia-reperfusion injury (IRI) is associated with apoptosis inhibition. However, IRI is hallmarked by inflammation, and hence, cell-death forms disjunct from immunologically silent apoptosis are, in theory, more likely to be relevant. Because pyroptosis (ie, cell death resulting from inflammasome activation) is typically observed in IRI, we speculated that aPC ameliorates IRI by inhibiting inflammasome activation. Here we analyzed the impact of aPC on inflammasome activity in myocardial and renal IRIs. aPC treatment before or after myocardial IRI reduced infarct size and Nlrp3 inflammasome activation in mice. Kinetic in vivo analyses revealed that Nlrp3 inflammasome activation preceded myocardial injury and apoptosis, corroborating a pathogenic role of the Nlrp3 inflammasome. The constitutively active Nlrp3A350V mutation abolished the protective effect of aPC, demonstrating that Nlrp3 suppression is required for aPC-mediated protection from IRI. In vitro aPC inhibited inflammasome activation in macrophages, cardiomyocytes, and cardiac fibroblasts via proteinase-activated receptor 1 (PAR-1) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Accordingly, inhibiting PAR-1 signaling, but not the anticoagulant properties of aPC, abolished the ability of aPC to restrict Nlrp3 inflammasome activity and tissue damage in myocardial IRI. Targeting biased PAR-1 signaling via parmodulin-2 restricted mTORC1 and Nlrp3 inflammasome activation and limited myocardial IRI as efficiently as aPC. The relevance of aPC-mediated Nlrp3 inflammasome suppression after IRI was corroborated in renal IRI, where the tissue protective effect of aPC was likewise dependent on Nlrp3 inflammasome suppression. These studies reveal that aPC protects from IRI by restricting mTORC1-dependent inflammasome activation and that mimicking biased aPC PAR-1 signaling using parmodulins may be a feasible therapeutic approach to combat IRI.

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Conflict of interest statement

Conflict-of-interest disclosure: C.D. is an inventor on a patent (WO2012/040636) describing parmodulin-2 (ML161). The remaining authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
aPC ameliorates inflammasome activation after myocardial IRI. (A) Experimental design. (B-C) aPC treatment reduces infarct size. Representative heart sections showing infarcted area detected by triphenyl tetrazolium chloride staining (area encircled by dashed line; size bar, 20 µm) (B) and dot plot summarizing data (C). (D-I) aPC pretreatment significantly reduces cardiac Nlrp3 expression and cleavage of caspase-1 (cl-Casp1) and IL-1β (cl–IL-1β). Representative immunoblots (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]: loading control [cont]) (D) and bar graph summarizing data (E). Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β (D). The active form was quantified (E). aPC treatment reduces caspase-1 activity within infarcted area. Representative images of frozen sections incubated with FLICA-Casp1 probes (size bar, 20 µm) (F) and dot plot summarizing data (G). aPC reduces plasma IL-1β (H) and IL-18 levels (I) after myocardial IRI; dot plots summarizing data. Sham-operated mice (sham) or mice with myocardial IRI without (cont; PBS) or with aPC pretreatment (aPC). Data shown represent mean ± SEM of at least 6 mice per group. *P < .05, **P < .01; analysis of variance (C,E,H,I) or Mann-Whitney U test (G).
Figure 2.
Figure 2.
Pivotal function of Nlrp3 inflammasome in myocardial IRI. (A-D) Kinetic analyses of infarct size and markers of inflammasome and apoptosis activation in hearts isolated at various time points after IRI (1-24 hours) compared with sham-operated mice. Representative images of triphenyl tetrazolium chloride (TTC) staining (infarct area encircled by dashed line; size bar, 20 µm) (A), dot plot summarizing data of infarct size (individual data points and mean ± SEM) (B), and representative immunoblots of inflammasome (cl-Casp1, cl–IL-1β) or apoptosis activation (cl-Casp3, cl-Casp7, BAX expression) markers, with glyceraldehyde-3-phosphate dehydrogenase [GAPDH] as loading control (C). (D) Line graph summarizing immunoblot data (mean ± SEM). (E) Experimental design. (F-L) Constitutively active Nlrp3A350V abolishes the protective effect of aPC in myocardial IRI. (F) Induction of Nlrp3A350V expression after tamoxifen injection into Nlrp3V-ER mice compared with PBS-treated mice; representative immunoblots, with GAPDH as loading control. (G-H) aPC treatment fails to protect against myocardial IRI in Nlrp3V-ER mice. Representative heart sections showing infarcted area detected by TTC staining (area encircled by dashed line; size bar, 20 µm) (G) and dot plot summarizing data (H). (I-L) aPC fails to reduce Nlrp3 expression, cl-Casp1 or cl–IL-1β, or plasma IL-1β and IL-18 levels in Nlrp3V-ER mice after myocardial IRI. Representative immunoblots (I) and bar graphs (J) summarizing results, with GAPDH as loading control; arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β (I). (J) The active form was quantified. (K-L) Dot plots of plasma IL-1β and IL-18 levels. Sham-operated mice (sham) or mice with myocardial IRI without (cont; PBS) or with aPC pretreatment (aPC). Data shown represent mean ± SEM of at least 6 mice per group. *P < .05, **P < .01; Student t test (B,D) or analysis of variance (H,J-L).
Figure 3.
Figure 3.
aPC prevents inflammasome activation in cardiac resident cells and macrophages in vitro. (A-F) In mouse BMDMs (A-B), mouse neonatal cardiomyocytes (C-D), and mouse neonatal cardiac fibroblasts (E-F), inflammasome activation was induced by priming with LPS (500 ng/mL, 3 hours) followed by ATP (10 µM, 3 hours; control [cont]: PBS). Concomitant treatment with aPC (20 nM; added once 30 minutes before ATP stimulation) markedly reduced the LPS/ATP-mediated induction of Nlrp3 and cl-Casp1 and cl–IL-1β; representative immunoblots (A,C,E) and corresponding bar graphs summarizing results (B,D,F). Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β (A,C,E). (B,D,F) The active form was quantified. (G-H) Nlrp3 expression and cl-Casp1 and cl–IL-1β are increased in mouse neonatal cardiomyocytes subjected to 6 hours of hypoxia (H; 1% oxygen) and serum and glucose deprivation (Hanks balanced salt solution medium), followed by 12 hours of reoxygenation (R; 21% oxygen) in complete medium. H/R induces inflammasome activation, which is prevented by aPC (20 nM; added once at the time of R); representative immunoblots of whole-cell lysates (G) and bar graph summarizing results (H), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. (G) Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β, respectively. (H) The active form was quantified. Data shown represent mean ± SEM. Data obtained from at least 3 independent experiments, each with at least 2 technical replicates (A-H); GAPDH as loading control (A,C,E,G). **P < .01; analysis of variance (B,D,F,H).
Figure 4.
Figure 4.
aPC restricts inflammasome by suppressing mTORC1. (A-C) Expression of Raptor, and HK1 and phosphorylation (phospho) of ribosomal p70-S6 kinase (pS6K70) were analyzed in LPS-primed and ATP-challenged BMDMs (A) and mouse neonatal cardiac fibroblasts (B) or mouse neonatal cardiomyocytes subjected to hypoxia/reoxygenation (H/R) (C). aPC inhibits expression of Raptor and HK1 and p70S6K phosphorylation levels in LPS-primed and ATP-stimulated cells (A-B) or in H/R-injured primary cardiomyocytes (C). (D-E) Treatment of mice with aPC inhibits mTORC1 signaling; representative immunoblots showing cardiac Raptor and HK1 expression as well as total and phosphorylated pS6K70. Representative immunoblots (D) and bar graph summarizing results (E). (F-J) BMDMs from TSC1LoxP/LoxP mice were transiently transfected with GFP- or Cre-expressing plasmids, resulting in loss of TSC1 expression after 48 hours. Representative immunoblots of TSC1, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (cont) (F). (G-H) In TSC1-deficient BMDM cells, aPC treatment fails to reduce Raptor or HK1 expression or phosphorylation of p70S6K (TSC1LoxP/LoxP+Cre+aPC) when compared with PBS-treated cells (TSC1LoxP/LoxP+Cre+PBS) Representative immunoblots (G) and bar graph summarizing results (H). (I-J) Likewise, aPC fails to reduce Nlrp3 expression or cl-Casp1 or cl–IL-1β in TSC1-deficient BMDMs. Representative immunoblots (I) and bar graph summarizing results (J). (I) Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β. (J) The active form was quantified. Data shown represent mean ± SEM. Data obtained from at least 3 independent experiments each with at least 2 technical replicates (A-J); GAPDH as loading control (A-D,F-G,I). **P < .01; analysis of variance (E,H,J).
Figure 5.
Figure 5.
aPC restricts inflammasome activation via PAR-1 after myocardial IRI. (A-C) The effect of aPC on inflammasome activation was analyzed after receptor inhibition. BMDMs (A), neonatal cardiomyocytes (B), or neonatal cardiac fibroblasts (C) were isolated from wild-type (WT), PAR-1−/−, PAR-2−/−, or PAR-3−/− mice. aPC fails to supress LPS/ATP- (A,C) or hypoxia/reoxygenation-induced (B) Nlrp3 expression and cl-Casp1 and cl–IL-1β in the absence of PAR-1 in all cell types, whereas loss of other receptors had no effect; representative immunoblots, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (A-C). (D) Experimental design. (E-F) Treatment of mice with aPC-HAPC1573 complex or 3K3A-aPC reduces the infarct size as efficiently as aPC, and blocking PAR-1 signaling (pepducin P1pal-12S) abolishes the inhibitory effect of aPC. Representative heart sections showing infarcted area detected by triphenyl tetrazolium chloride staining (area encircled by dashed line; size bar, 20 µm) (E) and dot plots summarizing data (F). (G-J) Treatment of mice with aPC-HAPC1573 complex or 3K3A-aPC reduces markers of inflammasome activation as efficiently as aPC, and blocking PAR-1 signaling (pepducin P1pal-12S) abolishes the inhibitory effect of aPC. (G) Representative immunoblots of cardiac Nlrp3 expression and cl-Casp1 and cl–IL-1β, with GAPDH as loading control (cont); arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β. (H) Representative images of active caspase-1 within the infarcted tissue (frozen sections incubated with FLICA-Casp1 probes; size bar, 20 µm). Dot plots summarizing plasma IL-1β (I) and IL-18 (J) levels. Sham-operated (sham) or mice with myocardial IRI without (PBS; cont), with aPC (aPC), with aPC-HAPC1573 complex (aPC+HAPC1573), with an aPC variant specifically lacking anticoagulant function (3K3A-aPC), or with aPC and PAR-1 pepducin P1pal-12S (aPC+P1pal-12S) pretreatment. Data shown represent mean ± SEM. Data obtained from at least 3 independent experiments each with at least 2 technical replicates (A-C) or from at least 6 mice per group (D-J). **P < .01; analysis of variance (F,I-J).
Figure 6.
Figure 6.
Par-1–specific parmodulin ameliorates inflammasome activation in myocardial IRI. (A) Experimental design. (B-C) The biased PAR-1 antagonist parmodulin-2 (5 mg/kg) reduces the infarcted area to the same extent as aPC. Representative heart sections showing infarcted area detected by TTC staining (infarcted area encircled by dashed line; size bar, 20 µm) (B) and dot plot summarizing data (C). (D-H) Treatment of mice with parmodulin-2 reduces markers of inflammasome activation and mTORC1 signaling as efficiently as aPC. Representative immunoblots showing cardiac Nlrp3 expression and cl-Casp1 and cl–IL-1β (D) and bar graph summarizing results, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (cont) (E). (D) Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β. (E) The active form was quantified. Dot plots summarizing plasma IL-1β (F) and representative immunoblots showing Raptor and HK1 expression and total and phosphorylated p70S6K (G) and bar graph summarizing results, with GAPDH as loading control (H). Sham-operated mice (sham) or mice with myocardial IRI without (cont; PBS) or with aPC (aPC) or parmodulin-2 (parmodulin) pretreatment. Data shown represent mean ± SEM of at least 6 mice per group (B-H). **P < .01; analysis of variance (C,E-F,H).
Figure 7.
Figure 7.
aPC restricts Nlrp3 inflammasome activation in renal IRI. (A) Experimental design. aPC treatment reduces plasma BUN (B) and creatinine (C) levels in mice with bilateral renal pedicle occlusion (30 minutes) and reperfusion for 24 hours. (D-I) aPC reduces renal IRI-induced inflammasome activation. Representative immunoblots of renal Nlrp3 expression and cl-Casp1 and cl–IL-1β (D) and bar graph summarizing results, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control (cont) (E). (D) Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β. (E) The active form was quantified. Representative images of active caspase-1 within renal medullary tubular cells (frozen sections antibody specific for cl-Casp1; size bar, 20 µm) (F) and dot plot summarizing data (G). Dot plots summarizing plasma IL-1β (H) and IL-18 levels (I). (J) Experimental design. (K-P) aPC fails to reduce Nlrp3 expression, cl-Casp1 or cl–IL-1β, or plasma IL-1β or IL-18 levels in Nlrp3V-ER mice after renal IRI. Plasma BUN (K) and creatinine (L) levels (dot plots); representative immunoblots of renal Nlrp3 expression and cl-Casp1 and cl–IL-1β) (M) and bar graph summarizing results (N). Arrowheads indicate inactive (white) and active (black) forms of caspase-1 or IL-1β (M); only the active form was quantified (N); GAPDH as loading control; dot plots of plasma IL-1β and IL-18 levels (O-P). Sham-operated mice (sham) or mice with renal IRI without (cont; PBS) or with aPC pretreatment (aPC) treatment. Data shown represent mean ± SEM of at least 6 different mice per group (B-I,K-P). **P < .01; analysis of variance (B-C,E,G-I,K-L,N-P).
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