Research ArticleSepsis

Modulation of the sigma-1 receptor–IRE1 pathway is beneficial in preclinical models of inflammation and sepsis

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Science Translational Medicine  06 Feb 2019:
Vol. 11, Issue 478, eaau5266
DOI: 10.1126/scitranslmed.aau5266
  • Fig. 1 S1R is an inhibitor of IRE1 during inflammation.

    (A) Experimental design and principle of proximity ligation assay. HA, hemagglutinin. (B) Western blots on input lysates and biotinylated (streptavidin pulldown) proximity ligation samples of HEK293 transfected with BirA or S1R-BirA and then stimulated for 24 hours with LPS (100 ng/ml) in the presence of 80 μM biotin. AU, arbitrary units. (C) Densitometric quantification of (B) [N = 4; *P < 0.05, repeated measures one-way analysis of variance (ANOVA) with post hoc Sidak test]. (D) Activity modulators of IRE1 and experimental parameters were used in the study. XBP1 (US), unspliced XBP1 transcript; XBP1 (S), spliced XBP1 transcript. (E) XBP1 splicing ratio [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH)–normalized spliced XBP1 transcript/GAPDH-normalized unspliced XBP1 transcript] in S1R WT or KO BMDMs stimulated for 6 hours with dimethyl sulfoxide (DMSO), LPS (100 ng/ml), or LPS (100 ng/ml) + IRE1 inhibitor (5 μM 4μ8C) (N = 3; n.s., not significant; *P < 0.05, two-way ANOVA with post hoc Sidak test). (F) XBP1 splicing ratio in S1R WT or KO BMDMs stimulated with DMSO or IRE1 activator (5 μM or 10 μM APY29) for 6 hours (N = 3, each dot represents one individual experiment, two-way ANOVA with post hoc Sidak test).

  • Fig. 2 S1R controls the production of inflammatory cytokines by inhibiting IRE1.

    (A) Relative quantity (RQ) of IL-6 was determined by qPCR on S1R WT or KO BMDMs stimulated for 6 hours with vehicle (NT) or LPS (100 ng/ml; representative of N = 4 independent experiments is shown; **P < 0.01, two-way ANOVA with post hoc Tukey test). (B) IL-6 enzyme-linked immunosorbent assay (ELISA) on supernatant from BMDMs stimulated for 6 hours with LPS (1 μg/ml; n = 1, representative of N = 4 independent experiments is shown; ***P < 0.001, t test). (C) qPCR on TLR4-MD2-CD14–expressing HEK293 cells that were transfected with empty vector (EV) or S1R and stimulated for 6 hours with LPS (100 ng/ml; N = 3, each dot pair represents one independent experiment; *P < 0.05, paired t test). (D) qPCR on BMDMs stimulated for 6 hours with vehicle (DMSO), LPS (100 ng/ml), or LPS (100 ng/ml) + IRE1 inhibitor (5 μM 4μ8C) (n = 1, representative of N = 3 independent experiments is shown; ***P < 0.001, two-way ANOVA with post hoc Tukey test). (E) IL-6 ELISA on supernatant from BMDMs stimulated for 6 hours with LPS (1 μg/ml) with either <1% DMSO, nuclear factor κB (NF-κB) inhibitor (20 μM JSH-23), mitogen-activated protein kinase (ERK) inhibitor (20 μM PD98059), c-Jun N-terminal kinase (JNK) inhibitor (1 μM SP600125), or IRE1 inhibitor (5 μM 4μ8C) (n = 1, representative of N = 3 independent experiments is shown; ***P < 0.001, two-way ANOVA with post hoc Tukey test).

  • Fig. 3 S1R is protective in murine models of inflammation and septic shock.

    (A) Experimental design. (B) Survival curve of WT and S1R KO mice after LPS (5 mg/kg) administration (n = 11 to 13 mice per group; **P < 0.01, log-rank test). (C) ELISA for TNF-α in serum 1.5 hours after LPS injection (each dot represents one mouse; **P < 0.01, t test). (D) ELISA for IL-6 in serum collected 3 hours after LPS injection (each dot represents one mouse; *P < 0.05, t test). (E) Survival curve of WT and S1R KO mice after administration of fecal content (n = 10 to 13 mice per group, fecal slurry = 1 g/kg; *P < 0.05, log-rank test). (F) ELISA for IL-6 in serum collected 3 hours after fecal slurry injection (each dot represents one mouse; *P < 0.05, t test). (G) Rectal temperature of animals presented in (E) (n = 10 to 13 mice per group; **P < 0.01, ***P < 0.001, two-way repeated measures ANOVA with post hoc Sidak test). (H to K) Mice were injected with LPS (5 mg/kg) or fecal slurry (1 mg/kg), and 24 hours later, serum was analyzed for amount of (H) alanine aminotransferase (ALT), (I) aspartate aminotransferase (AST), (J) creatinine, (K) and creatine kinase (CK) (each dot represents one mouse, n = 10 to 12 per group; *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA; outliers have been removed from visualization and are available in data file S1).

  • Fig. 4 Pharmacological modulation of S1R and IRE1 function in sepsis models.

    (A) XBP1 splicing ratio from liver homogenate of mice challenged with LPS (5 mg/kg) for 3 hours. Data shown are ratio of XBP1 spliced transcript/XBP1 unspliced transcript (each dot represents one mouse; **P < 0.01, two-way ANOVA with post hoc Sidak test). (B) Experimental design. (C) Survival curve of WT and S1R KO mice treated with vehicle (33% Kolliphor in saline) or STF (30 mg/kg) after administration of LPS (2 mg/kg) as indicated in (B) (n = 15 to 16 mice per group; **P < 0.01, ***P < 0.001, log-rank test). (D) IL-6 peritoneal exudate 3 hours after LPS injection in mice (each dot represents one mouse; *P < 0.05, ***P < 0.001, two-way ANOVA with post hoc Sidak test). (E) Survival curve of WT and S1R KO mice treated with vehicle or FLV (20 mg/kg) after administration of LPS (6 mg/kg) as indicated in (E) (n = 13 to 17 mice per group; *P < 0.05, **P < 0.01, log-rank test). (F) Serum IL-6 ELISA 3 hours after LPS injection in mice challenged as shown in (E) (each dot represents one mouse; **P < 0.01, two-way ANOVA with post hoc Sidak test; genotype-treatment interaction: *P < 0.05, two-way ANOVA).

  • Fig. 5 Therapeutic administration of the S1R agonist FLV is protective during models of inflammation and sepsis.

    (A and B) Experimental design for (A) LPS challenge or (B) FIP with therapeutic S1R agonist treatment. Intraperitoneally, ip. (C) Rectal temperature of mice measured immediately before LPS injection and 1 hour after (each dot represents one mouse; **P < 0.01, paired t test). (D) Rectal temperature of mice measured immediately before FIP induction and 0.5 hour after (each dot represents one mouse; ***P < 0.001, paired t test). (E) Clinical score, expressed as total murine sepsis score, of mice treated as in (A) (**P < 0.01, ***P < 0.001, repeated measures two-way ANOVA with post hoc Sidak test). (F) Clinical score, expressed as total murine sepsis score, of mice treated as in (B) (n = 14 mice per group; ***P < 0.001, two-way ANOVA). (G) Rectal temperatures of mice 24 hours after intraperitoneal LPS injection, treated with saline vehicle or FLV as indicated in (A) (each dot represents one mouse; *P < 0.05, t test). (H) Survival curve of mice challenged with LPS (6 mg/kg) and given therapeutic FLV or saline as indicated in (A) (n = 16 to 20; ***P < 0.001, log-rank test). (I) Rectal temperatures of mice 24 hours after FIP induction, treated with saline vehicle or FLV as indicated in (B) (each dot represents one mouse; **P < 0.01, t test). (J) Survival curve of mice challenged with fecal slurry (1.5 g/kg) and given therapeutic FLV or saline as indicated in (I) (n = 14 mice per group; **P < 0.01, log-rank test). (K) Experimental design for FIP challenge with FLV and the antibiotic CRO treatment. (L) Survival of C57/Bl6 mice treated with FLV (20 mg/kg, ip) and/or CRO (100 mg/kg, subcutaneously) after administration of fecal slurry at 2 g/kg (n = 10 to 12 mice per group; **P < 0.01, ***P < 0.001, log-rank test).

  • Fig. 6 The S1R agonist FLV is anti-inflammatory in human cells.

    (A to D) Multiplex ELISA on serum from human blood. Heparinized whole blood was stimulated ex vivo with LPS (10 ng/ml) and vehicle (RPMI 1640) or 20 μM FLV for 4 hours (n = 4, each dot pair represents serum from one participant; *P < 0.05, **P < 0.01, paired t test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/478/eaau5266/DC1

    Fig. S1. S1R deletion influences a subset of LPS-induced processes without causing global perturbation.

    Fig. S2. Canonical TLR4 signaling in macrophages is unperturbed by S1R deletion.

    Fig. S3. Gating strategy and quantification of immunophenotyping on blood.

    Fig. S4. Gating strategy and quantification of immunophenotyping on peritoneal contents.

    Fig .S5. Gating strategy and quantification of immunophenotyping on spleen and lymph node.

    Fig. S6. S1R controls inflammation in primary fibroblasts.

    Fig. S7. STF affects cytokine production in vitro and in vivo.

    Fig. S8. Anti-inflammatory effect of FLV does not globally suppress cytokine production from human blood.

    Fig. S9. Proposed mechanism of action of S1R during LPS-mediated inflammatory response.

    Data file S1. Primary data (Excel file).

  • The PDF file includes:

    • Fig. S1. S1R deletion influences a subset of LPS-induced processes without causing global perturbation.
    • Fig. S2. Canonical TLR4 signaling in macrophages is unperturbed by S1R deletion.
    • Fig. S3. Gating strategy and quantification of immunophenotyping on blood.
    • Fig. S4. Gating strategy and quantification of immunophenotyping on peritoneal contents.
    • Fig .S5. Gating strategy and quantification of immunophenotyping on spleen and lymph node.
    • Fig. S6. S1R controls inflammation in primary fibroblasts.
    • Fig. S7. STF affects cytokine production in vitro and in vivo.
    • Fig. S8. Anti-inflammatory effect of FLV does not globally suppress cytokine production from human blood.
    • Fig. S9. Proposed mechanism of action of S1R during LPS-mediated inflammatory response.

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    Other Supplementary Material for this manuscript includes the following:

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