Jozef Zienkiewicz

Suppression of Acute Lung Inflammation by Intracellular Peptide Delivery of a Nuclear Import Inhibitor

Acute lung inflammation is a potentially life-threatening complication of infections due to community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), a worldwide emerging pathogen, which causes necrotizing pneumonia and acute respiratory distress syndrome (ARDS). MRSA virulence factors encompass immunotoxins termed superantigens that contribute to lung inflammation. In this study, we demonstrate that staphylococcal enterotoxin B (SEB)-induced lung inflammation is attenuated by a cell-penetrating peptide nuclear import inhibitor of nuclear factor (NF)-kappaB and other stress-responsive transcription factors (SRTFs). This inhibitor suppressed production of a wide spectrum of cytokines and chemokines induced by direct SEB airway exposure. Consequently, trafficking of neutrophils, monocytes/macrophages, and lymphocytes to the bronchoalveolar space was significantly reduced while vascular injury, manifested by increased permeability and protein leakage, was attenuated. Moreover, induction of systemic proinflammatory cytokines and chemokines in response to direct SEB airway exposure was reduced. Thus, intracellular delivery of a nuclear import inhibitory peptide suppresses respiratory and systemic expression of key mediators of lung inflammation evoked by SEB.

New paradigms in sepsis: from prevention to protection of failing microcirculation.

Sepsis, also known as septicemia, is one of the 10 leading causes of death worldwide. The rising tide of sepsis due to bacterial, fungal and viral infections cannot be stemmed by current antimicrobial therapies and supportive measures. New paradigms for the mechanism and resolution of sepsis and consequences for sepsis survivors are emerging. Consistent with Benjamin Franklins dictum ‘an ounce of prevention is worth a pound of cure’, sepsis can be prevented by vaccinations against pneumococci and meningococci. Recently, the NIH NHLBI Panel redefined sepsis as ‘severe endothelial dysfunction syndrome in response to intravascular and extravascular infections causing reversible or irreversible injury to the microcirculation responsible for multiple organ failure’. Microvascular endothelial injury underlies sepsis‐associated hypotension, edema, disseminated intravascular coagulation, acute respiratory distress syndrome and acute kidney injury. Microbial genome products trigger ‘genome wars’ in sepsis that reprogram the human genome and culminate in a ‘genomic storm’ in blood and vascular cells. Sepsis can be averted experimentally by endothelial cytoprotection through targeting nuclear signaling that mediates inflammation and deranged metabolism. Endothelial ‘rheostats’ (e.g. inhibitors of NF‐κB, A20 protein, CRADD/RAIDD protein and microRNAs) regulate endothelial signaling. Physiologic ‘extinguishers’ (e.g. suppressor of cytokine signaling 3) can be replenished through intracellular protein therapy. Lipid mediators (e.g. resolvin D1) hasten sepsis resolution. As sepsis cases rose from 387 330 in 1996 to 1.1 million in 2011, and are estimated to reach 2 million by 2020 in the US, mortality due to sepsis approaches that of heart attacks and exceeds deaths from stroke. More preventive vaccines and therapeutic measures are urgently needed.

In vivo islet protection by a nuclear import inhibitor in a mouse model of type 1 diabetes.

Background Insulin-dependent Type 1 diabetes (T1D) is a devastating autoimmune disease that destroys beta cells within the pancreatic islets and afflicts over 10 million people worldwide. These patients face life-long risks for blindness, cardiovascular and renal diseases, and complications of insulin treatment. New therapies that protect islets from autoimmune destruction and allow continuing insulin production are needed. Increasing evidence regarding the pathomechanism of T1D indicates that islets are destroyed by the relentless attack by autoreactive immune cells evolving from an aberrant action of the innate, in addition to adaptive, immune system that produces islet-toxic cytokines, chemokines, and other effectors of islet inflammation. We tested the hypothesis that targeting nuclear import of stress-responsive transcription factors evoked by agonist-stimulated innate and adaptive immunity receptors would protect islets from autoimmune destruction. Principal Findings Here we show that a first-in-class inhibitor of nuclear import, cSN50 peptide, affords in vivo islet protection following a 2-day course of intense treatment in NOD mice, which resulted in a diabetes-free state for one year without apparent toxicity. This nuclear import inhibitor precipitously reduces the accumulation of islet-destructive autoreactive lymphocytes while enhancing activation-induced cell death of T and B lymphocytes derived from autoimmune diabetes-prone, non-obese diabetic (NOD) mice that develop T1D. Moreover, in this widely used model of human T1D we noted attenuation of pro-inflammatory cytokine and chemokine production in immune cells. Conclusions These results indicate that a novel form of immunotherapy that targets nuclear import can arrest inflammation-driven destruction of insulin-producing beta cells at the site of autoimmune attack within pancreatic islets during the progression of T1D.

Smectic polymorphism of the 4-butyl-4-alkoxyazobenzenes and 4-pentyl-4-alkoxyazobenzenes

Two families of liquid crystalline compounds have been synthesised, the 4-butyl-4- alkoxyazobenzenes and the 4-pentyl-4-alkoxyazobenzenes; for the second family results are presented for the first time for alkoxy chains longer than butyl. The results for both families have been obtained up to the octadecyl homologues. In both families, on the basis of DSC, polarizing microscopy and thermo-optical analysis, a rich polymorphism has been detected (maximum tetramorphism). The smectic properties start with the hexyl derivative (for the butyl family) and with the heptyl derivative (for the pentyl family). Strong odd-even effects for the temperatures of clearing in both groups of compounds were detected. Our results are compared with those of de Jeu et al. and of Adomenas et al. for the 4-butyl-4- alkoxyazobenzenes, for which only one smectic modification was described.

Targeting nuclear import shuttles, importins/karyopherins alpha by a peptide mimicking the NFκB1/p50 nuclear localization sequence.

Background We recently reported that a bifunctional nuclear transport modifier (NTM), cSN50.1 peptide, reduced atherosclerosis, plasma cholesterol, triglycerides, and glucose along with liver fat and inflammatory markers, in a murine model of familial hypercholesterolemia. We determined that cSN50.1 improved lipid homeostasis by modulating nuclear transport of sterol regulatory element‐binding proteins through interaction with importin β. Previous studies established that cSN50.1 and related NTMs also modulate nuclear transport of proinflammatory transcription factors mediated by binding of their nuclear localization sequences (NLSs) to importins/karyopherins α. However, selectivity and specificity of NTMs for importins/karyopherins α were undetermined. Methods and Results We analyzed interaction of the NTM hydrophilic module, N50 peptide, derived from the NLS of NFκB1/p50, with endogenous human importins/karyopherins α to determine the mechanism of NTM modulation of importin α‐mediated nuclear transport. We show that N50 peptide forms stable complexes with multiple importins/karyopherins α. However, only interaction with importin α5 (Imp α5) displayed specific, high‐affinity binding. The 2:1 stoichiometry of the N50‐Imp α5 interaction (KD1=73 nmol/L, KD2=140 nmol/L) indicated occupancy of both major and minor NLS binding pockets. Utilizing in silico 3‐dimensional (3‐D) docking models and comparative structural analysis, we identified a structural component of the Imp α5 major NLS binding pocket that may stabilize N50 binding. Imp α5 also displayed rapid stimulus‐induced turnover, which could influence its availability for nuclear transport during the inflammatory response. Conclusions These results provide direct evidence that N50 peptide selectively targets Imp α5, encouraging further refinement of NLS‐derived peptides as new tools to modulate inflammatory disorders.

Nuclear transport modulation reduces hypercholesterolemia, atherosclerosis, and fatty liver.

Background Elevated cholesterol and triglycerides in blood lead to atherosclerosis and fatty liver, contributing to rising cardiovascular and hepatobiliary morbidity and mortality worldwide. Methods and Results A cell‐penetrating nuclear transport modifier (NTM) reduced hyperlipidemia, atherosclerosis, and fatty liver in low‐density lipoprotein receptor‐deficient mice fed a Western diet. NTM treatment led to lower cholesterol and triglyceride levels in blood compared with control animals (36% and 53%, respectively; P<0.005) and liver (41% and 34%, respectively; P<0.05) after 8 weeks. Atherosclerosis was reduced by 63% (P<0.0005), and liver function improved compared with saline‐treated controls. In addition, fasting blood glucose levels were reduced from 209 to 138 mg/dL (P<0.005), and body weight gain was ameliorated (P<0.005) in NTM‐treated mice, although food intake remained the same as that in control animals. The NTM used in this study, cSN50.1 peptide, is known to modulate nuclear transport of stress‐responsive transcription factors such as nuclear factor kappa B, the master regulator of inflammation. This NTM has now been demonstrated to also modulate nuclear transport of sterol regulatory element‐binding protein (SREBP) transcription factors, the master regulators of cholesterol, triglyceride, and fatty acid synthesis. NTM‐modulated translocation of SREBPs to the nucleus was associated with attenuated transactivation of their cognate genes that contribute to hyperlipidemia. Conclusions Two‐pronged control of inflammation and dyslipidemia by modulating nuclear transport of their critical regulators offers a new approach to comprehensive amelioration of hyperlipidemia, atherosclerosis, fatty liver, and their potential complications.

Lethality in a murine model of pulmonary anthrax is reduced by combining nuclear transport modifier with antimicrobial therapy.

Background In the last ten years, bioterrorism has become a serious threat and challenge to public health worldwide. Pulmonary anthrax caused by airborne Bacillus anthracis spores is a life- threatening disease often refractory to antimicrobial therapy. Inhaled spores germinate into vegetative forms that elaborate an anti-phagocytic capsule along with potent exotoxins which disrupt the signaling pathways governing the innate and adaptive immune responses and cause endothelial cell dysfunction leading to vascular injury in the lung, hypoxia, hemorrhage, and death. Methods/Principal Findings Using a murine model of pulmonary anthrax disease, we showed that a nuclear transport modifier restored markers of the innate immune response in spore-infected animals. An 8-day protocol of single-dose ciprofloxacin had no significant effect on mortality (4% survival) of A/J mice lethally infected with B. anthracis Sterne. Strikingly, mice were much more likely to survive infection (52% survival) when treated with ciprofloxacin and a cell-penetrating peptide modifier of host nuclear transport, termed cSN50. In B. anthracis-infected animals treated with antibiotic alone, we detected a muted innate immune response manifested by cytokines, tumor necrosis factor alpha (TNFα), interleukin (IL)-6, and chemokine monocyte chemoattractant protein-1 (MCP-1), while the hypoxia biomarker, erythropoietin (EPO), was greatly elevated. In contrast, cSN50-treated mice receiving ciprofloxacin demonstrated a restored innate immune responsiveness and reduced EPO level. Consistent with this improvement of innate immunity response and suppression of hypoxia biomarker, surviving mice in the combination treatment group displayed minimal histopathologic signs of vascular injury and a marked reduction of anthrax bacilli in the lungs. Conclusions We demonstrate, for the first time, that regulating nuclear transport with a cell-penetrating modifier provides a cytoprotective effect, which enables the hosts immune system to reduce its susceptibility to lethal B. anthracis infection. Thus, by combining a nuclear transport modifier with antimicrobial therapy we offer a novel adjunctive measure to control florid pulmonary anthrax disease.

The "genomic storm" induced by bacterial endotoxin is calmed by a nuclear transport modifier that attenuates localized and systemic inflammation.

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.

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

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.

Clinical assessment of HNF1A and GCK variants and identification of a novel mutation causing MODY2

A child with impaired fasting glucose was found to be heterozygous for a novel variant at c.659G>A in GCK and a variant at c.1663C>T in HNF1A. Structural modeling and clinical correlation suggests that the GCK variant causes monogenic diabetes while the variant in HNF1A is unlikely to be pathogenic.