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. 2019 Sep 18;3(9):440-446.
doi: 10.4049/immunohorizons.1900064.

Protection from Endotoxin Shock by Selective Targeting of Proinflammatory Signaling to the Nucleus Mediated by Importin Alpha 5

Yan Liu  1   2 , Ruth Ann Veach  2   3 , Jozef Zienkiewicz  4   2 , Kelli L Boyd  5 , Taylor E Smith  1   2 , Zhi-Qi Xu  1 , Lukasz S Wylezinski  1 , Jacek Hawiger  4   2   6
Affiliations

Protection from Endotoxin Shock by Selective Targeting of Proinflammatory Signaling to the Nucleus Mediated by Importin Alpha 5

Yan Liu et al. Immunohorizons. .

Abstract

Endotoxin shock is induced by LPS, one of the most potent virulence factors of the Gram-negative bacteria that cause sepsis. It remains unknown if either proinflammatory stress-responsive transcription factors (SRTFs), ferried to nucleus by importin α5, or lipid-regulating sterol regulatory element binding proteins (SREBPs), transported to the nucleus by importin β1, mediate endotoxin shock. A novel cell-penetrating peptide targeting importin α5 while sparing importin β1 protected 80% of animals from death in response to a high dose of LPS. This peptide suppresses inflammatory mediators, liver glycogen depletion, endothelial injury, neutrophil trafficking, and apoptosis caused by LPS. In d-galactosamine-pretreated mice challenged by 700-times lower dose of LPS, rapid death through massive apoptosis and hemorrhagic necrosis of the liver was also averted by the importin α5-selective peptide. Thus, using a new tool for selective suppression of nuclear transport, we demonstrate that SRTFs, rather than SREBPs, mediate endotoxin shock.

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Conflict of interest statement

DISCLOSURES

Y.L., R.A.V., J.Z., and J.H. are coinventors of patents assigned to Vanderbilt University and the US Department of Veterans Affairs. The other authors have no financial conflicts of interest.

Figures

FIGURE 1.
FIGURE 1.. Conceptual depiction of the action of biselective NTM.
Biselective NTM simultaneously inhibits nuclear import of stress-responsive transcription factors (SRTFs) mediated jointly by Imp α5 and Imp β1, and nuclear translocation of metabolic transcription factors SREBP1 and -2 solely mediated by Imp β1. Biselective NTM suppresses expression of genes that encode mediators of inflammation and proteins participating in synthetic pathways of cholesterol, triglycerides, and fatty acids responsible for hyperlipidemia.
FIGURE 2.
FIGURE 2.. Imp α5–selective NTM impedes nuclear translocation of proinflammatory transcription factor NF-κB RelA while sparing Imp β1–mediated nuclear translocation of metabolic transcription factor SREBP2.
(A) Immunoblot analysis of nuclear NF-κB RelA in RAW 264 cells. Cells were treated with 30 μM NTM peptides as indicated for 30 min then stimulated with 10 ng/ml LPS for 6 h. (B) Immunoblot analysis of nuclear SREBP2 in HEK 293T cells. Cells were depleted of lipids by treatment with HPCD and treated with 30 μM NTM peptides as indicated for 2 h. (C and D) Quantitative representation of immunoblots shown in (A) and (B), respectively. Data presented in this figure represent two independent in vitro experiments completed in duplicates or triplicates (as indicated). All signals were normalized to Histone 3 and expressed as percent inhibition ± SEM. Significance was determined by one-way ANOVA. **p < 0.005, ***p < 0.005.
FIGURE 3.
FIGURE 3.. Imp α5–selective NTM (cSN50.1α) increases survival in endotoxin shock in two models.
(A) High-dose LPS model. Mice were challenged with a high dose of LPS (35 mg/kg). (B) Low-dose LPS model. Mice were primed with d-galactosamine (1 g/kg) and challenged with a nonlethal amount of LPS (50 μg/kg). Mice were treated with either seven (A) or five (B) doses of NTM (0.66 mg/dose) or saline. Data presented in this figure denote at least two independent in vivo experiments. Number of mice per condition group is indicated on the graph. Kaplan–Meier survival plot with p value calculated by log-rank analysis. ***p < 0.0005.
FIGURE 4.
FIGURE 4.. Imp α5–selective NTM, cSN50.1α prevents cytokine/chemokine expression in endotoxin shock.
Blood plasma levels of cytokines, TNF-α, IL-6, INF-γ, and chemokine MCP-1 in a high-dose LPS model (35 mg/kg LPS) (A) and a low-dose LPS model (1 g/kg D-Gal then 50 μg/kg LPS) (B). Mice were treated with either seven (A) or five (B) doses of NTM (0.66 mg/dose) or saline. Data presented in this figure denote two independent in vivo experiments completed with five mice per condition group. Data are expressed as a mean ± SEM (n = 10 mice per group; p values calculated by two-way repeated measures ANOVA). ***p < 0.0005, ****p < 0.0001.
FIGURE 5.
FIGURE 5.. Imp α5–selective NTM protects liver from LPS-induced glycogenolysis (PAS), endothelial injury (VCAM-1), and neutrophil infiltration in a high-dose LPS model and from massive apoptosis (Caspase 3) in a low-dose LPS model of endotoxin shock.
Representative images (original magnification ×40) of liver sections in unchallenged mice (mock control) or mice challenged with LPS. Data presented in this figure denote two independent in vivo experiments completed with five mice per condition group [(A) 35 mg/kg LPS; (B) 1 g/kg D-Gal then 50 μg/kg LPS] and treated with saline or cSN50.1 or cSN50.1α peptides (both at 0.66 mg/dose). See Materials and Methods for details.
See this image and copyright information in PMC

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