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

Abstract

Sepsis is an often deadly complication of infection in which systemic inflammation damages the vasculature, leading to tissue hypoperfusion and multiple organ failure. Currently, the standard of care for sepsis is predominantly supportive, with few therapeutic options available. Because of increased sepsis incidence worldwide, there is an urgent need for discovery of novel therapeutic targets and development of new treatments. The recently discovered function of the endoplasmic reticulum (ER) in regulation of inflammation offers a potential avenue for sepsis control. Here, we identify the ER-resident protein sigma-1 receptor (S1R) as an essential inhibitor of cytokine production in a preclinical model of septic shock. Mice lacking S1R succumb quickly to hypercytokinemia induced by a sublethal challenge in two models of acute inflammation. Mechanistically, we find that S1R restricts the endonuclease activity of the ER stress sensor IRE1 and cytokine expression but does not inhibit the classical inflammatory signaling pathways. These findings could have substantial clinical implications, as we further find that fluvoxamine, an antidepressant therapeutic with high affinity for S1R, protects mice from lethal septic shock and dampens the inflammatory response in human blood leukocytes. Our data reveal the contribution of S1R to the restraint of the inflammatory response and place S1R as a possible therapeutic target to treat bacterial-derived inflammatory pathology.

Figure 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, then stimulated for 24 hours with 100 ng/mL LPS in the presence of 80μM biotin. (C) Densitometric quantification of B (N=4, *P<0.05, repeated measures one-way ANOVA with post-hoc Sidak test). (D) Activity modulators of IRE1 and experimental parameters used in the study. XBP1 (US): Unspliced XBP1 transcript; XBP1 (S): Spliced XBP1 transcript. (E) XBP1 splicing ratio (i.e. GAPDH-normalized spliced XBP1 transcript/GAPDH-normalized unspliced XBP1 transcript) in S1R WT or KO BMDM stimulated for 6 hours with DMSO, 100 ng/mL LPS or 100 ng/mL LPS + IRE1 inhibitor (5μM 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 BMDM stimulated with DMSO or IRE1 activator (5μM or 10μM APY29) for 6 hours (N= 3, each dot represents one individual experiment, n.s. not significant, two-way ANOVA with post-hoc Sidak test).

Figure 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 BMDM stimulated for 6 hours with vehicle (NT) or 100ng/mL LPS (Representative of 4 independent experiments, one mouse is shown, **P<0.01, two-way ANOVA with post-hoc Tukey test). (B) IL-6 ELISA on supernatant from BMDM stimulated for 6 hours with 1μg/mL LPS (Representative of 4 independent experiments, one mouse 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 100ng/mL LPS (N=3, each dot pair represents one independent experiment, *p<0.05, paired t-test). (D) qPCR on BMDM stimulated for 6 hours with vehicle (DMSO), 100 ng/mL LPS, or 100 ng/mL LPS + IRE1 inhibitor (5μM 4μ8C) (Representative of 3 independent experiments, one mouse is shown, n.s. not significant, ***P<0.001, two-way ANOVA with post-hoc Tukey test). (E) IL-6 ELISA on supernatant from BMDM stimulated for 6 hours with 1μg/mL LPS with either <1% DMSO, NF-κB inhibitor (20μM JSH-23), ERK inhibitor (20μM PD98059), JNK inhibitor (1μM SP600125), or IRE1 inhibitor (5μM 4μ8C) (Representative of 3 independent experiments, one mouse is shown, n.s. not significant, **P<0.01, ***P<0.001, two-way ANOVA with post-hoc Tukey test).

Figure 3:. S1R is protective in murine models of inflammation and septic shock.

(A) Experimental design. (B) Survival curve of WT and S1R KO mice following LPS administration (n=11-13 mice/ group, LPS=5mg/kg, **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<0.05, 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 following administration of fecal content (n=10-13 mice/ group, fecal slurry =1g/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= 10-13 mice/group, **p<0.01, ***p<0.001, two-way repeated measures ANOVA with post-hoc Sidak test). (H-K) Mice were injected with 5mg/kg LPS or 1mg/kg fecal slurry 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=10-12 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.).

Figure 4:. Pharmacological modulation of S1R and IRE1 function in sepsis models.

(A) XBP1 splicing ratio from liver homogenate of mice challenged with 5mg/kg LPS 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 (30mg/kg) following administration of LPS as indicated in B (n=15-16 mice/ group, LPS=2mg/kg, **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, two-way ANOVA with post-hoc Sidak test. (E) Survival curve of WT and S1R KO mice treated with vehicle or fluvoxamine (20mg/kg) following administration of LPS as indicated in E (n=13-17 mice/group, LPS = 6mg/kg *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, n.s. not significant, **P<0.01, two-way ANOVA with post-hoc Sidak test. Genotype-treatment interaction: *P<0.05, two-way ANOVA).

Figure 5:. Therapeutic administration of the S1R agonist fluvoxamine is protective during models of inflammation and sepsis.

(A-B) Experimental design for (A) LPS challenge or (B) FIP with therapeutic S1R agonist treatment. (C) Rectal temperature of mice measured immediately before LPS injection and one hour after (each dot represents one mouse, ***p<0.001, 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/group, ***p<0.001, two-way ANOVA). (G) Rectal temperatures of mice 24 hours after I.P. 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 6mg/kg LPS and given therapeutic FLV or saline as indicated in A (n=16-20, ***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 1.5 g/kg fecal slurry and given therapeutic FLV or saline as indicated in I (n=14 mice/group **p<0.01, log-rank test). (K) Experimental design for FIP challenge with FLV and the antibiotic ceftriaxone (CRO) treatment. (L) Survival of C57/Bl6 mice treated with fluvoxamine (20mg/kg) i.p. and/or ceftriaxone (100mg/kg s.c.) following administration of fecal slurry at 2 g/kg (n=10-12 mice/group, **p<0.01, ***p<0.001, log-rank test).

Figure 6:. The S1R agonist fluvoxamine is anti-inflammatory in human cells.

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