Survival, bacterial clearance and thrombocytopenia are improved in polymicrobial sepsis by targeting nuclear transport shuttles

Abstract

The rising tide of sepsis, a leading cause of death in the US and globally, is not adequately controlled by current antimicrobial therapies and supportive measures, thereby requiring new adjunctive treatments. Severe microvascular injury and multiple organ failure in sepsis are attributed to a “genomic storm” resulting from changes in microbial and host genomes encoding virulence factors and endogenous inflammatory mediators, respectively. This storm is mediated by stress-responsive transcription factors that are ferried to the nucleus by nuclear transport shuttles importins/karyopherins. We studied the impact of simultaneously targeting two of these shuttles, importin alpha 5 (Imp α5) and importin beta 1 (Imp β1), with a cell-penetrating Nuclear Transport Modifier (NTM) in a mouse model of polymicrobial sepsis. NTM reduced nuclear import of stress-responsive transcription factors nuclear factor kappa B, signal transducer and activator of transcription 1 alpha, and activator protein 1 in liver, which was also protected from sepsis-associated metabolic changes. Strikingly, NTM without antimicrobial therapy improved bacterial clearance in blood, spleen, and lungs, wherein a 700-fold reduction in bacterial burden was achieved while production of proinflammatory cytokines and chemokines in blood plasma was suppressed. Furthermore, NTM significantly improved thrombocytopenia, a prominent sign of microvascular injury in sepsis, inhibited neutrophil infiltration in the liver, decreased L-selectin, and normalized plasma levels of E-selectin and P-selectin, indicating reduced microvascular injury. Importantly, NTM combined with antimicrobial therapy extended the median time to death from 42 to 83 hours and increased survival from 30% to 55% (p = 0.022) as compared to antimicrobial therapy alone. This study documents the fundamental role of nuclear signaling mediated by Imp α5 and Imp β1 in the mechanism of polymicrobial sepsis and highlights the potential for targeting nuclear transport as an adjunctive therapy in sepsis management.

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

“…Thus, prolonged NTM administration did not seem to increase the susceptibility to environmental pathogens. These observations are consistent with the enhancement of bacterial clearance by NTM in a polymicrobial sepsis model …”

“…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 .…”

“…Until now, over 100 trials in sepsis, including anti‐TNFα monoclonal antibody failed . In a clinically relevant, polymicrobial sepsis model, a combination of antimicrobial therapy with NTM increased survival to 55% as compared to 30% with antimicrobial therapy alone . Thus, NTM prevents a localized and systemic expression of mediators associated with a runaway microbial inflammation, while also enhancing the innate immunity–mediated clearance of bacteria that produced 700‐fold reduction of the bacterial burden on the lungs of septic animals prior to the onset of antimicrobial therapy.…”

“…The encounter between microbial agents (such as LPS) or host endogenous irritants (also named "danger signals" such as heat shock proteins, nucleic acids, nuclear proteins) and their pattern recognition receptors (such as TLRs) and cytosolic DNA sensors evokes robust signalling to the nucleus mediated by signal transducers. [51][52][53] These signalling cascades originate at the extracellular and intracellular sensors and "splash" onto genes' regulome ( Figure 2A,C). Intracellular sensors, NOD receptors, inflammasomes and STING receptors are activated.…”

“…Rather, SN50 and its congeners target a common nuclear transport shuttle, importin α5 125 also named karyopherin α1 that recognizes NLS on multiple SRTFs. 22,51,52,81,125 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. 51 The binding of the NTM NLS module to importin α5 was specific and of high affinity (K D1 = 73 and K D2 = 140 nmol/L) with a 2:1 stoichiometry, suggesting that NTMs interact with both, major and minor NLS binding pockets, on importin α5.…”