The “Genomic Storm” Induced by Bacterial Endotoxin Is Calmed by a Nuclear Transport Modifier That Attenuates Localized and Systemic Inflammation

Abstract

Lipopolysaccharide (LPS) is a potent microbial virulence factor that can trigger production of proinflammatory mediators involved in the pathogenesis of localized and systemic inflammation. Importantly, the role of nuclear transport of stress responsive transcription factors in this LPS-generated “genomic storm” remains largely undefined. We developed a new nuclear transport modifier (NTM) peptide, cell-penetrating cSN50.1, which targets nuclear transport shuttles importin α5 and importin β1, to analyze its effect in LPS-induced localized (acute lung injury) and systemic (lethal endotoxic shock) murine inflammation models. We analyzed a human genome database to match 46 genes that encode cytokines, chemokines and their receptors with transcription factors whose nuclear transport is known to be modulated by NTM. We then tested the effect of cSN50.1 peptide on proinflammatory gene expression in murine bone marrow-derived macrophages stimulated with LPS. This NTM suppressed a proinflammatory transcriptome of 37 out of 84 genes analyzed, without altering expression of housekeeping genes or being cytotoxic. Consistent with gene expression analysis in primary macrophages, plasma levels of 23 out of 26 LPS-induced proinflammatory cytokines, chemokines, and growth factors were significantly attenuated in a murine model of LPS-induced systemic inflammation (lethal endotoxic shock) while the anti-inflammatory cytokine, interleukin 10, was enhanced. This anti-inflammatory reprogramming of the endotoxin-induced genomic response was accompanied by complete protection against lethal endotoxic shock with prophylactic NTM treatment, and 75% protection when NTM was first administered after LPS exposure. In a murine model of localized lung inflammation caused by direct airway exposure to LPS, expression of cytokines and chemokines in the bronchoalveolar space was suppressed with a concomitant reduction of neutrophil trafficking. Thus, calming the LPS-triggered “genomic storm” by modulating nuclear transport with cSN50.1 peptide attenuates the systemic inflammatory response associated with lethal shock as well as localized lung inflammation.

Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells

“…Karyopherin alpha is involved in the NF-kappaB p50/p65 heterodimer translocation upon TNF-alpha stimulation [45]. Together with Importin beta 1, Karyopherin alpha activates a large number of genes encoding pro-inflammatory cytokines and chemokines, and other proteins critical in host responses to infection [46]. Down-regulation of karyopherin alpha in response to Yersinia infection is interesting, as Yersinia evades immune response via inhibition of key inflammatory regulators by its virulence factors [47], some of which might be also regulated by karyopherin alpha [46]…”

Decoding inflammation, its causes, genomic responses, and emerging countermeasures

“…The suppressed genes encode the mediators of inflammation encompassing cytokines and growth factors, chemokines and their receptors. Importantly, NTM did not suppress the expression of five housekeeping genes nor altered cell viability . In systemic severe microbial inflammation exemplified by LPS‐induced lethal shock, the cSN50.1 peptide afforded 75% protection when administered after LPS exposure.…”

“…At this junction, we emphasize that the SN50 peptide is not exclusively an “NF‐κB inhibitor,” as inaccurately advertised by commercial peptide manufacturers and reported by their customers in numerous publications. Rather, SN50 and its congeners target a common nuclear transport shuttle, importin α5 also named karyopherin α1 that recognizes NLS on multiple SRTFs . The next‐generation NTM, the cSN50.1 peptide, has the highest solubility (100 mg/mL) compared to 13 mg/mL of the prototypical SN50 peptide .…”

“…The second type of intracellular anti‐inflammatory agents, NTMs, dismantle the nuclear transport of four stress‐responsive transcription factors signalling cascades, including NFĸB, AP‐1, NFAT and STAT1, and two metabolic transcription factors signalling pathways SREBPs and CHREBPs (Figure A,C). NTM (cSN50.1 peptide) reduced plasma level of 23 out of 26 LPS‐induced proinflammatory cytokines and chemokines . It also suppressed the expression of nine genes that encode the key proteins involved in the synthetic pathways of cholesterol, triglycerides and fatty acids …”

Translating dosages from animal models to human clinical trials—revisiting body surface area scaling

“…Although the BSA conversion equation is accepted for one purpose in pharmacology ( i.e. , as a potential scaling method for selecting a safe starting dosage), it has been inappropriately used to justify the clinical potential for multiple investigational therapeutics undergoing animal trials from this misinterpretation of the FDA recommendations by Reagan‐Shaw et al These examples include protease inhibitors for hepatitis C (32), serotonin reuptake inhibitor for Alzheimer's disease (33), nuclear transport modifier peptide to treat inflammation (34), and antibody‐mediated delivery of IL‐2 for the treatment of breast cancer (33). If the misinterpretation that human trials can proceed simply by scaling dosage according to BSA were to somehow move forward unchecked, it could lead to in‐efficacy due to underdosing or dangerous outcomes for first‐in‐human volunteers due to poor clearance or unforeseen reaction to biologic therapeutics due interspecies differences in physiology and toxicology (1, 4, 31, 36).…”