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.

Deficiency of gp91phox inhibits allergic airway inflammation.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multienzyme complex, is the major source for production of reactive oxygen species (ROS). ROS are increased in allergic diseases, such as asthma, but the role of ROS in disease pathogenesis remains uncertain. We hypothesized that mice unable to generate ROS via the NADPH oxidase pathway would have decreased allergic airway inflammation. To test this hypothesis, we studied gp91phox(-/-) mice in a model of allergic airway inflammation after sensitization and challenge with ovalbumin. Serum, bronchoalveolar lavage fluid, and lungs were then examined for evidence of allergic inflammation. We found that mice lacking a functional NADPH oxidase complex had significantly decreased ROS production and allergic airway inflammation, compared with wild-type (WT) control animals. To determine the mechanism by which allergic inflammation was inhibited by gp91phox deficiency, we cultured bone marrow-derived dendritic cells from WT and gp91phox(-/-) mice and activated them with LPS. IL-12 expression was significantly increased in the gp91phox(-/-) bone marrow-derived dendritic cells, suggesting that the cytokine profile produced in the absence of gp91phox enhanced the conditions leading to T helper (Th) type 1 differentiation, while inhibiting Th2 polarization. Splenocytes from sensitized gp91phox(-/-) animals produced significantly less IL-13 in response to ovalbumin challenge in vitro compared with splenocytes from sensitized WT mice, suggesting that NADPH oxidase promotes allergic sensitization. In contrast, inflammatory cytokines produced by T cells cultured from WT and gp91phox(-/-) mice under Th0, Th1, Th2, and Th17 conditions were not significantly different. This study demonstrates the importance of NADPH oxidase activity and ROS production in a murine model of asthma.

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 host's 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.

Differential immune responses and pulmonary pathophysiology are induced by two different strains of respiratory syncytial virus.

In this study we performed comparisons of pulmonary responses between two different respiratory syncytial virus (RSV) antigenic subgroup A strains, A2 and Line 19. Line 19 strain induced significant dose-responsive airway hyperreactivity (AHR) in BALB/c mice at days 6 and 9 after infection, whereas the A2 strain induced no AHR at any dose. Histological examination indicated that A2 induced no goblet cell hyper/metaplasia, whereas the Line 19 induced goblet cell expansion and significant increases in gob5 and MUC5AC mRNA and protein levels in vivo. When examining cytokine responses, A2 strain induced significant interleukin (IL)-10 expression, whereas Line 19 strain induced significant IL-13 expression. When IL-13-/- mice were infected with Line 19 RSV, the AHR responses were abrogated along with gob5 gene expression. There was little difference in viral titer throughout the infection between the line 19- and A2-infected mice. However, the A2 strain grew to significantly higher titers than the Line 19 strain in HEp-2 cells in vitro. Thus, RSV Line 19-induced airway dysfunction does not correlate with viral load in vivo. These data demonstrate that different RSV strains of the same antigenic subgroup can elicit differential immune responses that impact the phenotypic expression of RSV-induced illness.

Increased and prolonged pulmonary fibrosis in surfactant protein C-deficient mice following intratracheal bleomycin.

Recent reports have linked mutations in the surfactant protein C gene (SFTPC) to familial forms of pulmonary fibrosis, but it is uncertain whether deficiency of mature SP-C contributes to disease pathogenesis. In this study, we evaluated bleomycin-induced lung fibrosis in mice with genetic deletion of SFTPC. Compared with wild-type (SFTPC+/+) controls, mice lacking surfactant protein C (SFTPC-/-) had greater lung neutrophil influx at 1 week after intratracheal bleomycin, greater weight loss during the first 2 weeks, and increased mortality. At 3 and 6 weeks after bleomycin, lungs from SFTPC-/- mice had increased fibroblast numbers, augmented collagen accumulation, and greater parenchymal distortion. Furthermore, resolution of fibrosis was delayed. Although remodeling was near complete in SFTPC+/+ mice by 6 weeks, SFTPC-/- mice did not return to baseline until 9 weeks after bleomycin. By terminal dUTP nick-end labeling staining, widespread cell injury was observed in SFTPC-/- and SFTPC+/+ mice 1 week after bleomycin; however, ongoing apoptosis of epithelial and interstitial cells occurred in lungs of SFTPC-/- mice, but not SFTPC+/+ mice, 6 weeks after bleomycin. Thus, SP-C functions to limit lung inflammation, inhibit collagen accumulation, and restore normal lung structure after bleomycin.

Nuclear import of proinflammatory transcription factors is required for massive liver apoptosis induced by bacterial lipopolysaccharide.

Stimulation of macrophages with lipopolysaccharide (LPS) leads to the production of cytokines that elicit massive liver apoptosis. We investigated the in vivo role of stress-responsive transcription factors (SRTFs) in this process focusing on the precipitating events that are sensitive to a cell-permeant peptide inhibitor of SRTF nuclear import (cSN50). In the absence of cSN50, mice challenged with LPS displayed very early bursts of inflammatory cytokines/chemokines, tumor necrosis factor alpha (1 h), interleukin 6 (2 h), interleukin 1 beta (2 h), and monocyte chemoattractant protein 1 (2 h). Activation of both initiator caspases 8 and 9 and effector caspase 3 was noted 4 h later when full-blown DNA fragmentation and chromatin condensation were first observed (6 h). At this time an increase of pro-apoptotic Bax gene expression was observed. It was preceded by a decrease of anti-apoptotic Bcl2 and BclX(L) gene transcripts. Massive apoptosis was accompanied by microvascular injury manifested by hemorrhagic necrosis and a precipitous drop in blood platelets observed at 6 h. An increase in fibrinogen/fibrin degradation products and a rise in plasminogen activator inhibitor 1 occurred between 4 and 6 h. Inhibition of SRTFs nuclear import with the cSN50 peptide abrogated all these changes and increased survival from 7 to 71%. Thus, the nuclear import of SRTFs induced by LPS is a prerequisite for activation of the genetic program that governs cytokines/chemokines production, liver apoptosis, microvascular injury, and death. These results should facilitate the rational design of drugs that protect the liver from inflammation-driven apoptosis.

Suppression of Staphylococcal Enterotoxin B-induced Toxicity by a Nuclear Import Inhibitor.

Staphylococcal enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, tissue injury, and death. Genes that encode mediators of inflammation can be globally inhibited by blocking the nuclear import of stress-responsive transcription factors. Here we show that cell-permeant peptides targeting Rch1/importin alpha/karyopherin alpha 2, a nuclear import adaptor protein, are delivered to T cells where they inhibit the staphylococcal enterotoxin B-induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo. The systemic production of tumor necrosis factor alpha, interferon gamma, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor kappa B in T cells. The extent of liver apoptosis and hemorrhagic necrosis was also reduced, which correlated with significantly decreased mortality rates. These findings highlight nuclear import inhibitors as a potentially useful countermeasure for staphylococcal enterotoxin B and other toxins that trigger harmful systemic inflammatory responses.