NTM treatment enhances survival from lethal endotoxic shock.

<p>Survival curves for mice challenged i.p. with high-dose LPS (A and B) or low-dose LPS+D-Gal (C). In (A) and (C) mice were administered the first NTM (cSN50.1 peptide) treatment 30 min before LPS challenge (prophylactic protocol), while in (B), the first treatment was administered 15 min after LPS challenge (therapeutic protocol). Saline injections were administered to control mice challenged with LPS following the same treatment schedule in each protocol, as described in “Materials and Methods”. ***<i>p</i><0.0005 by log rank test.</p

Survival is increased by combining NTM treatment with antibiotic therapy.

<p>Mice were infected with CS and treated with vehicle or NTM (cSN50.1), both supplemented by antibiotic therapy with meropenem (<i>n</i> = 20 mice/group; Kaplan-Meier survival plot with <i>p</i> value calculated by log rank analysis).</p

NTM treatment reduces plasma levels of multiple cytokines, chemokines and growth factors induced by LPS.

<p>(A) Wild type C57BL/6 mice were challenged i.p. with a lethal dose of LPS (800 µg) and treated with i.p. injections of NTM (cSN50.1 peptide) or diluent (saline) following a prophylactic protocol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110183#pone-0110183-g004" target="_blank">Figure 4A</a>. Blood was collected at baseline and 2 or 6 h after LPS challenge and a multiplex assay was used to measure 32 analytes in plasma. Twenty-four analytes were significantly altered by NTM treatment, as determined by repeated measures two-way analysis of variance with Sidak’s post-test. Twenty-three were reduced, while anti-inflammatory IL-10 was increased by NTM treatment. Results are shown as the % inhibition or increase by NTM compared to saline control set to 100% at the time point demonstrating maximal expression for that analyte. <i>n</i> = 10 animals/group. (B) Comparison of prophylactic and therapeutic NTM treatment protocols on selected plasma cytokine and chemokine levels in the high-dose LPS model of endotoxic shock. Data are presented as mean ± standard error, <i>n</i> = 5 −10 animals/group.</p

Amino acid sequence of cSN50.1 peptide and its congeners SN50 and cSN50.

<p>Fragment-linked peptides comprising the Signal Sequence Hydrophobic Region of Fibroblast Growth Factor 4 (bolded) and the NLS region of NFκB1/p50 (italicized) were analyzed for their solubility in water. In cSN50 and cSN50.1, an intra-molecular disulfide bond is formed between the two cysteines, which cyclizes the NLS motif.</p><p>Amino acid sequence of cSN50.1 peptide and its congeners SN50 and cSN50.</p

LPS-induced cellular trafficking to lungs is reduced in NTM-treated mice.

<p>Cell counts in BAL fluid collected from unchallenged mice (naïve) and 6 h after direct airway exposure to LPS, with i.p. NTM peptide (cSN50.1) or diluent control (saline) treatment. The LPS-induced increase in total cells is comprised primarily of neutrophils. Neutrophil trafficking to BAL is significantly reduced by NTM treatment while monocytes/macrophages and lymphocytes are not affected. Data are presented as mean ± standard error, <i>n</i> = 4 naïve and 5–7 NTM- or saline-treated animals/group from two independent experiments. *<i>p</i><0.05, **<i>p</i><0.005 by Mann-Whitney <i>U</i> test comparing LPS-challenged groups.</p

Bacterial clearance is improved in infected mice treated with NTM.

<p>CFU determined by serial dilution of whole blood and organ homogenates collected 12 h after infection from sham- or CS-infected mice treated with NTM (cSN50.1) or vehicle. Bars represent median values from 4–5 mice/group (<i>p</i> values determined by Mann-Whitney test).</p

LPS-induced expression of chemokines, cytokines, and growth factors in the lung is suppressed by NTM.

<p>Fourteen cytokines, chemokines and growth factors elevated in BAL after direct airway exposure to LPS are significantly suppressed by NTM (cSN50.1 peptide) treatment. Data are presented as mean ± standard error, <i>n</i> = 4 naïve and 8–9 NTM- or saline-treated animals/group from 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.005, and ***<i>p</i><0.0005 by Mann-Whitney <i>U</i> test comparing LPS-challenged groups.</p

NTM modulates blood and vascular responses to sepsis.

<p> (<b>A</b>) Cytokines/chemokine measured in blood plasma by cytometric bead array before and at 2 h, 6 h, 12 h, and 24 h after CS infection. Antibiotic therapy with meropenem began at 12 h post-infection. (<i>n</i> = 20 mice/group; <i>p</i> values calculated by two-way repeated measures ANOVA). IFN-γ measured by ELISA in blood plasma before and at 2 h, 6 h and 12 h after CS infection. (vehicle + antibiotic, <i>n</i> = 8; cSN50.1 + antibiotic, <i>n</i> = 13; <i>p</i> values calculated by two-way repeated measures ANOVA). (<b>B</b>) Total WBC, lymphocytes, neutrophils, monocytes, and platelets in whole blood collected 12 h after infection from sham-infected or CS-infected mice treated with NTM (cSN50.1) or vehicle. Bars represent median values from 5 mice/group (<i>p</i> values determined by Mann-Whitney test). (<b>C</b>) Soluble E-selectin, L-selectin, and P-selectin measured in blood plasma before and at 12 h and 24 h after CS infection. Antibiotic therapy with meropenem began at 12 h post-infection. (<i>p</i> values for sE-selectin and sL-selectin calculated by two-way repeated measures ANOVA, <i>p</i> value for sP-selectin determined by Mann-Whitney test at 24 h only, n = 9/group). (<b>D</b>) Representative images (400x magnification) and quantification of neutrophils (indicated by arrows) in the hepatic parenchyma of livers collected 12 h after infection from sham-infected or CS-infected mice treated with NTM (cSN50.1) or vehicle. The percentage of positive brown (DAB-positive) stained cells was calculated as a total analyzed number of positive cells divided by total number cells in the section of liver. Bars represent median values from 5 mice/group (<i>p</i> values determined by Mann-Whitney test).</p

Nuclear transport is a pivotal checkpoint in genomic regulation of microbial inflammation.

<p>Inflammatory signaling cascades initiated by cell responses to microbial virulence factors and cytokines culminate in nuclear translocation of stress-responsive transcription factors (SRTFs) that upregulate inflammatory gene networks. Unchecked, this genomic reprogramming (genomic storm) leads to endothelial dysfunction, multi-organ failure and ultimately fatal shock. Inhibiting nuclear transport at a common “checkpoint” located downstream of TLRs and cytokine receptors globally suppresses expression of inflammatory genes thereby calming the genomic storm and averting multiple organ injury. Legend: TLRs (Toll-like receptors); Ca²⁺ (calcium ions); IL-1R and IL-18R (interleukin 1 and 18 receptors, respectively); IFN-γ (Interferon-gamma); IFNGR (Interferon-gamma receptor); SFKs (Src family of protein tyrosine kinases); MyD88 (Myeloid Differentiation Primary Response 88); IRAK (interleukin-1 receptor-associated kinase); JAK (Janus kinase); P (Phosphate group); Ub (Ubiquitin); CaM (Calmodulin); TRAF6 (TNF receptor-associated factor 6); IKK (I kappa B kinase); IκBα (NF-κB inhibitor alpha); JNK (c-Jun N-terminal kinase); MAPKs (mitogen-activated protein kinases); NFAT (nuclear factor of activated T cells); AP-1 (Activator protein 1); NF-κB (Nuclear factor kappa B); NPC (nuclear pore complex); STAT1 (signal transducer and activator of transcription 1); Imp α5 (Importin alpha 5); Imp β1 (Importin beta 1); TNFα (tumor necrosis factor alpha); IL-1, IL-6, IL-10 and IL-17 (interleukin 1, 6, 10 and 17, respectively); MCP-1 (Monocyte Chemoattractant Protein-1).</p

Genes encoding mediators of inflammation are regulated by transcription factors that require nuclear transport shuttles targeted by NTM.

<p>Analysis was performed <i>in silico</i> using The UCSC Genome Browser (The UCSC Genome Bioinformatics). mNLS - classical monopartite Nuclear Localization Sequence; bNLS - nonclassical bipartite Nuclear Localization Sequence; uNLS - unconventional, structural or dimer-specific Nuclear Localization Sequence; bHLH - basic helix-loop-helix motif; “+” - the transcription factor specific binding site is present in the gene promoter.</p><p>Genes encoding mediators of inflammation are regulated by transcription factors that require nuclear transport shuttles targeted by NTM.</p