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. 2014 Feb 15;192(4):1806-14.
doi: 10.4049/jimmunol.1300835. Epub 2014 Jan 8.

Hydrogen Sulfide [Corrected] Increases Survival During Sepsis: Protective Effect of CHOP Inhibition

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Free PMC article

Hydrogen Sulfide [Corrected] Increases Survival During Sepsis: Protective Effect of CHOP Inhibition

Marcella Ferlito et al. J Immunol. .
Free PMC article

Erratum in

  • J Immunol. 2014 Apr 15;192(8):3991

Abstract

Sepsis is a major cause of mortality, and dysregulation of the immune response plays a central role in this syndrome. H2S, a recently discovered gaso-transmitter, is endogenously generated by many cell types, regulating a number of physiologic processes and pathophysiologic conditions. We report that H2S increased survival after experimental sepsis induced by cecal ligation and puncture (CLP) in mice. Exogenous H2S decreased the systemic inflammatory response, reduced apoptosis in the spleen, and accelerated bacterial eradication. We found that C/EBP homologous protein 10 (CHOP), a mediator of the endoplasmic reticulum stress response, was elevated in several organs after CLP, and its expression was inhibited by H2S treatment. Using CHOP-knockout (KO) mice, we demonstrated for the first time, to our knowledge, that genetic deletion of Chop increased survival after LPS injection or CLP. CHOP-KO mice displayed diminished splenic caspase-3 activation and apoptosis, decreased cytokine production, and augmented bacterial clearance. Furthermore, septic CHOP-KO mice treated with H2S showed no additive survival benefit compared with septic CHOP-KO mice. Finally, we showed that H2S inhibited CHOP expression in macrophages by a mechanism involving Nrf2 activation. In conclusion, our findings show a protective effect of H2S treatment afforded, at least partially, by inhibition of CHOP expression. The data reveal a major negative role for the transcription factor CHOP in overall survival during sepsis and suggest a new target for clinical intervention, as well potential strategies for treatment.

Conflict of interest statement

The authors have no conflicting financial interests.

Figures

Figure 1
Figure 1. H2S increases survival after sepsis
A) Survival study of mice monitored for 8 days after CLP. A group of mice received sodium hydrosulphide (NaHS, 100 mmol/kg) as an H2S donor subcutaneously (CLP+H2S) or saline and after 1 h CLP was carried out (CLP n=21, CLP+H2S n=22. * P=0.0001). Data are pooled from three different experiments). B) Sepsis was induced in mice by CLP, and 2h later, H2S was administrated as described above (both groups n=6. * P=0.01).
Figure 2
Figure 2. H2S decreases inflammation
A) Plasma TNFα and IL-10 levels were measured at 5 h and 18 h after CLP by an ELISA (Sham n=3–5, CLP and CLP+H2S n=6–11) Data are from individual mice from two to three experiments. B) Levels of cytokines were measured in the peritoneal lavage (Sham n=3–4, CLP and CLP+H2S n=6–8). C) Left panel: MPO activity from lung homogenized measured at 18 h after CLP. Right panel: AST measured from plasma at 18 h after CLP (Sham n=2, CLP and CLP+H2S n=4–8). Data shown are from individual mice from two different experiments (* P≤0.05).
Figure 3
Figure 3. Apoptosis is reduced by H2S treatment
A) Activated Caspase-3 immunoperoxidase staining was performed on spleen sections 18h after CLP (40 X objective, bar 20 mm). B) Lysates from spleens 18 h after CLP were subjected to Western blotting assay to detect activated Caspase-3 (CAS-3). Tubulin (TUB) was used as a loading control. Samples represent individual mouse spleen from 2 sham, 5 CLP and 5 CLP+H2S.
Figure 4
Figure 4. H2S increases bacterial clearance
Whole blood (upper panels) and peritoneal lavage (lower panels) were assessed for viable bacteria by colony-forming unit (CFU) assay at 5h and 18h after CLP. Samples from single mice were streaked on brain and heart infusion agar plates for 24 h and then colonies were counted (n=4–6). Data are a representative from two independent experiments with similar results. (* P≤0.05).
Figure 5
Figure 5. H2S decreases CHOP expression in vivo
A) Spleens, lungs and livers harvested 18h after CLP were homogenized and analyzed by Western blot for expression of CHOP. Tubulin (Tub) was used as a loading control. Samples analyzed are from single mice (Sham n=2–3, CLP and CLP+H2S n=5). Data are a representative of two independent experiments with similar results. B) Representative immunohystochemistry of CHOP expression in sham and CLP mice.
Figure 6
Figure 6. H2S decreases other ER stress response proteins expression in vivo
Spleens harvested 18h after CLP were homogenized and analyzed by Western blot for expression of ATF4 and ATF6. Tubulin (Tub) was used as a loading control. Samples analyzed are from single mice.
Figure 7
Figure 7. Genetic deletion of Chop protects against LPS induced mortality
The survival rate of WT and CHOP KO mice (n=8) was observed for 8 days after intraperitoneal injection of 25 mg/kg of LPS (n=8, * P=0.001).
Figure 8
Figure 8. Genetic deletion of Chop protects against CLP induced mortality
The survival rate of WT and CHOP KO mice (8 mice per group) was observed for 8 days after induction of sepsis by CLP (n=8–10 *, P=0.01). A group of CHOP KO mice was also treated with H2S 2h prior to CLP (CHOP KO vs CHOP KO+H2S P=0.9).
Figure 9
Figure 9. Septic CHOP KO mice display reduced apoptosis
Representative activated Caspase-3 immunoperoxidase staining performed on spleen sections 18 h after CLP.
Figure 10
Figure 10. Decreased TNFα and IL-10 and increased bacterial clearance in CHOP KO mice
A) TNFα and IL-10 levels were measured by ELISA in the plasma of sham operated and septic (CLP) mice from WT and CHOP KO groups 18h after CLP (n=4–6). B) Levels of cytokines in the peritoneum of septic WT and CHOP KO mice (n=4–6). C) Colony-forming unit (CFU) assay was performed on blood and peritoneum lavage at 18h after CLP. Samples from single mice were streaked on brain and heart infusion agar plates for 24 h and then colonies were counted (n=4–5, * P≤0.05).
Figure 11
Figure 11. H2S treatment decreases CHOP expression in macrophages in a Nrf2 dependent manner
A) Representative Western blot of CHOP. RAW cells were incubated for 18h with LPS (L, 10 mg/ml) or PGN (P, 10 mg/ml) with or without H2S (200 mM). Densitometric analysis is shown in the graph below and it was calculated by ratio of CHOP over tubulin (Tub) and normalized to LPS (n=3, p≤0.01, * vs LPS alone; ** vs PGN alone). B) CHOP mRNA is reduced after incubation of cells with LPS and/or H2S for 6h (n=2). C) Representative confocal image of peritoneal macrophages incubated with LPS and/or H2S for 18h and then stained for expression of CHOP. (n=2) D) RAW cells were treated with tunicamicyn (TN) for 3h with or without H2S (n=3). E) Cells were incubated for the time indicates with and Nrf2 was then evaluated (n=2). F) RAW cells were transfected with scrambled oligo (siCon) or siRNA for Nrf2 (siNrf2) for 24h and then incubated with H2S for 18h (n≥3). G) After transfection with siCon or siNrf2, RAW cells were incubated with LPS with (L+H) or without H2S for 12h. Densitometric analysis is shown in the graph (n=3, p≤0.01, #vs LPS, *vs Bas and **vs LPS+H2S of siCon group).

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