Highly soluble cell-penetrating NTM peptide cSN50.1 (Table 1), was synthesized, purified, and filter-sterilized as described elsewhere [6], [9].

Human genes encoding the cytokines, chemokines, receptors and growth factors studied in this manuscript, were analyzed for specific binding sites of transcription factors whose nuclear translocation was previously shown to be modulated by NTM (Table 2). The prediction process was conducted based on the presence of a binding site in the promoter region of the targeted gene. To accomplish this task we employed the UCSC Genome Browser publicly available on the website of the Center for Biomolecular Science and Engineering at the University of California Santa Cruz (UCSC Genome Bioinformatics, http://genome.ucsc.edu).

Bone marrow cells were isolated from femurs and tibias of C57BL/6 mice and suspended in Dulbecco's Modified Eagle Medium supplemented with 10% FBS, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 20% L929-conditioned medium. Non-adherent cells were removed and culture media replaced every three days. Cells were used in experiments after 10 days of culture for up to 2 weeks after maturation. Prior to use in experiments, culture purity of adherent cells was verified by fluorescence-activated cell sorting where ≥95% were MAC3⁺, CD11b⁺, CD3^–, CD11c^– and B220^–. Viability was ≥80% as determined by trypan blue exclusion.

BMDMs were left unstimulated for preparation of control RNA, or stimulated with 2 ng/ml LPS from E. coli O127:B8 (Sigma) and concurrently treated with cSN50.1 (30 µM) or saline (diluent). After incubation at 37°C for 6 h, RNA was prepared from cells using the Qiagen RNeasy Kit (Qiagen) and converted to cDNA using the RT² First Strand Kit, then analyzed using the RT² Profiler PCR array system (Qiagen) according the manufacturer’s instructions.

All animal handling and experimental procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Vanderbilt University Animal Care and Use Program (Permit Number: A3227-01), which has been accredited by the American Association of Accreditation of Laboratory Animal Care International (file #000020). Animals were housed in groups of five in the animal care facility of Vanderbilt University in a 12 hour light/dark cycle. Regular rodent chow and water were provided ad libitum. After administration of inflammatory agonists, mice are carefully monitored and any that exhibit end−stage symptoms consistent with acute toxic shock are euthanized as soon as it is apparent they will not recover.

Randomized groups of five female C57BL/6 mice (The Jackson Laboratory) 8–12 weeks of age (∼20 g weight) and LPS from E. coli O127:B8 (Sigma) were used in all animal experiments. To evaluate the protective efficacy of NTM (cSN50.1 peptide) against systemic inflammation, two models of lethal endotoxic shock were used: high-dose LPS; or low-dose LPS under conditions of metabolic stress imposed by 2-amino-2-deoxy-D-galactosamine (D-Gal), which sensitizes mice to the proinflammatory action of LPS [17]. In the high-dose LPS model, 800 µg LPS in 0.2 ml saline was administered by intraperitoneal (i.p.) injection. In the LPS+D-Gal model, mice were injected i.p. with 1 µg of LPS and 20 mg of D-Gal, each in 0.2 ml saline. Two NTM treatment protocols, prophylactic and therapeutic, were tested in the high-dose LPS model. In the prophylactic protocol, mice were given NTM (cSN50.1 peptide, 0.66 mg/injection), or diluent (saline) by i.p. injections of 0.2 ml at 30 min before and 30, 90, 150, 210, 360 and 720 min after LPS challenge, while in the therapeutic protocol, NTM was administered at 15, 90, 150, 210, 360, and 720 min post-LPS challenge. Blood samples (∼40 µL) were collected from the saphenous vein in heparinized tubes (Sarstedt) before and at 2, 4, 6 and 24 h post-LPS challenge. All injected reagents were sterile and prepared in pyrogen-free saline. These experiments are based on the death of animals as an experimental endpoint, so mice were allowed to progress to a moribund state before being euthanized by isoflurane asphyxiation. Since multiple organ systems are affected in the mechanism of systemic inflammation, any pain medication may inadvertently interfere with the progression of endotoxic shock. Therefore, we could not use agents that alleviate pain. However, we attempted to minimize the amount of pain experienced by the animals by closely monitoring mice, at least hourly for the first 24 hours and three times a day thereafter, and euthanizing any mice that exhibit end-stage symptoms consistent with acute toxic shock (lack of reaction to cage motion, or any of the following signs: ataxia, paralysis, cyanosis, or severe respiratory distress) as soon as it is apparent they will not recover. Any surviving mice were euthanized after 72 h.

For induction of localized acute lung inflammation, intranasal instillations of 50 ng of LPS in 50 µL saline (25 µL/nostril) were performed under ketamine/xylazine anesthesia (0.2 ml of 6.7 mg/ml ketamine and 1.3 mg/ml xylazine administered by i.p. injection). Mice were treated with NTM (cSN50.1 peptide, 0.66 mg/injection) or diluent (saline) administered by i.p. injections of 0.2 ml at 30 min before and at 30, 90, and 120 min after LPS challenge. Six hours post-LPS challenge, mice were euthanized by isoflurane asphyxiation. Bronchoalveolar lavage (BAL) collection and differential cell counts were performed as previously described [18]. For comparison, BAL was also collected from naïve mice not exposed to any intranasal instillation before euthanasia.

The effect of cSN50.1 on expression of chemokines, cytokines and growth factors was analyzed in cell-free BAL fluid or blood plasma from mice by cytometric bead array (BD BioSciences) or a 32-analyte Milliplex mouse panel (Millipore) according to the manufacturers’ instructions. Analytes included eotaxin, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), interleukin (IL) −1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IFN-γ-induced protein 10 (IP-10), keratinocyte chemoattractant (KC), leukemia inhibitory factor (LIF), LPS-induced CXC chemokine (LIX), macrophage colony-stimulating factor (M-CSF), monocyte chemoattractant protein-1 (MCP-1), monokine induced by gamma interferon (MIG), macrophage inflammatory protein (MIP) −1α, MIP-1β, MIP-2, regulated upon activation normal T-cell expressed, and presumably secreted (RANTES), tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor (VEGF).

Threshold cycle values from qRT-PCR were exported to Excel and analyzed using the Qiagen web-based PCR Array Data Analysis Software. Genes showing a >0.5 log fold change versus control were considered significant. Other data analysis and statistical calculations were performed using Prism (GraphPad). Cell counts and chemokine, cytokine, and growth factor levels in BAL were compared using the non-parametric Mann–Whitney U test. Survival data were analyzed by the log-rank test. Cytokine, chemokine and growth factor levels in plasma collected from the same animals at different time points were evaluated by repeated measures two-way analysis of variance with Sidak’s post-test. A p value of <0.05 was considered significant.

A fragment-linked peptide strategy to analyze signal-dependent nuclear transport is based on fusing a motif from the nuclear localization sequence (NLS) region of the NF-κB1/p50 subunit with the signal sequence hydrophobic region (SSHR) of human fibroblast growth factor 4 [7], [19]. NLS- and SSHR-containing NTMs (SN50, cSN50 and cSN50.1; see Table 1 for their composition and solubility) were designed to function at the nuclear transport level by binding to importins α during stimulus-initiated signaling and thereby modulate nuclear import of NLS-bearing SRTFs. The SSHR motif allows peptides to cross the plasma membrane of cells in culture or in experimental animals through an ATP- and endosome-independent mechanism [20]. In this study, we used cSN50.1, an improved version of our previously described cell-penetrating NTM peptides [6], [9], [21], [22] that shows increased solubility (100 mg/ml) in water, compared to cSN50 (38 mg/ml) and SN50 (13 mg/ml) (see Table 1), thereby increasing its potency [6], [21]. The functional utility of NTMs has been reported in numerous preclinical models of inflammation caused by microbial and autoimmune insults [18], [21]–[26]. Most recently, and unexpectedly, we found that cSN50.1 not only modulates nuclear transport of SRTFs such as NF-κB, but also sterol regulatory element-binding protein (SREBP) transcription factors that regulate lipid homeostasis [6].

Recently, it was shown that injection of bacterial endotoxin into healthy adult volunteers was associated with a robust genomic response in combined blood leukocyte populations [10]. Among the genes reported in that study, several encoded cytokines, chemokines, and their receptors as reflected by transcriptome analysis. We used a publicly available database to match genes encoding mediators of inflammation with transcription factors containing nuclear targeting motifs known to require nuclear transport shuttles recognized by cSN50.1 [6] (Table 2). We found that all 46 proinflammatory genes are potentially regulated by NF-κB1, 34 genes by STAT-1, 32 genes by c-Jun, and 19 genes by SREBP1. Most of these genes are combinatorially regulated by two or more transcription factors that are transported to the nucleus as monomers or homodimers/heterodimers by importins α and β. Their transport function is modulated by NTMs that encompass cell penetrating SN50, cSN50, and cSN50.1 peptides [6], [9] (Table 1). It is thus plausible that cSN50.1 peptide can suppress expression of multiple proinflammatory genes by modulating nuclear transport of transcription factors analyzed in Table 2.

We tested this hypothesis through analysis of the proinflammatory transcriptome in bone marrow-derived macrophages. These primary cells are one of the main myeloid lineage targets of LPS [27]. A mouse inflammatory cytokine/chemokine and receptor PCR array allowed us to assess the effects of NTM on 84 genes compared to untreated controls. Remarkably, NTM modified expression of 37 of the 84 genes tested. While NTM suppressed gene expression in LPS-activated inflammatory pathways (Figure 1), it did not alter gene expression of five housekeeping genes (Gusb, Hprt1, Hsp90ab1, Gapdh, Actb, <0.5 fold change versus control, not shown). Genes encoding most cytokines and CC chemokines were down-regulated by NTM whereas those for most CC chemokine receptors, cytokine receptors, and CX chemokines and their receptors were not affected. There were a few notable exceptions: LIX/CXCL5, MIG/CXCL9, and IP-10/CXCL10. Importantly, this suppression of genomic changes by NTM in primary macrophages challenged with LPS was not associated with changes in cell viability (data not shown), indicating that NTM has no adverse effect on cell growth. Thus, targeting nuclear transport pathways for LPS-activated SRTFs with cSN50.1 peptide prevented a “genomic storm” by reducing transcription of a wide array of genes that encode mediators of inflammation in primary macrophages.

After analysis of the primary macrophage response to LPS modulated by NTM, we studied its effect on two models of LPS-induced systemic inflammation exemplified by lethal shock. In a high-dose LPS model, both prophylactic and therapeutic NTM treatment protocols were employed. In the prophylactic protocol, the first dose of NTM was administered before LPS challenge while in the therapeutic protocol, NTM treatment was begun after LPS administration. As shown in Figure 2A, animals treated with NTM by the prophylactic protocol in the high-dose LPS model were completely protected, compared to only 10% survival in saline-treated control animals (p<0.0001). Strikingly, when a therapeutic NTM treatment protocol was employed in the high-dose LPS model, 75% of mice survived, compared to 100% mortality in the saline-treated control group (Figure 2B, p<0.0001). Thus, NTM was highly effective against high-dose LPS with both prophylactic and therapeutic protocols.

To further evaluate the effectiveness of the NTM, we tested its anti-inflammatory effect under conditions of metabolic stress imposed by D-Gal, which sensitizes mice to LPS [17]. In contrast to the high-dose LPS model, which requires at least 800 µg of LPS per 20 g mouse for induction of lethal shock, only 1 µg of LPS per 20 g mouse is required for lethality when mice are metabolically stressed with D-Gal, and death occurs much more rapidly (5–7 h after low-dose LPS+D-gal compared to 16–20 h post-LPS in the high-dose model). Consistent with results from the high-dose LPS shock model, prophylactic NTM treatment afforded robust protection (90%) in mice challenged with LPS+D-Gal. In contrast, no mice survived in the control group treated with saline (Figure 2C, p<0.0005).

We analyzed the striking gain in survival of NTM-treated mice that were challenged with LPS in the context of systemic proinflammatory cytokine and chemokine production. As expected, in the prophylactic protocol, NTM treatment engendered significant inhibition of 11 out of 13 proinflammatory cytokines whose plasma levels were increased by LPS challenge (Figure 3A, upper). An exception was the anti-inflammatory cytokine, IL-10, which was elevated >2 fold in NTM-treated animals. In parallel, a wide array of LPS-elevated chemokines and growth factors was suppressed in NTM-treated mice (Figure 3A, lower). Overall, of 26 cytokines, chemokines and growth factors elevated in plasma after LPS challenge, 23 were reduced by NTM treatment, two were unchanged (eotaxin/CCL11 and IL-5, not shown) and one was increased (IL-10). Plasma levels of the remaining six of the 32 analytes tested were not increased by administration of LPS (not shown). These results are consistent with the genomic response of primary macrophages in the qRT-PCR analysis (see Figure 1), indicating that NTM, under the conditions of these in vivo experiments, suppresses expression of multiple mediators of inflammation. A comparable trend in suppression of proinflammatory cytokines and chemokines TNF-α, IL-6, IFN-γ and MCP-1 was observed in mice treated with NTM when employing the therapeutic protocol, albeit to a lesser degree. Interestingly in the therapeutic protocol, IL-10 is suppressed instead of enhanced by NTM (Figure 3B), consistent with suppression of the Il10 gene in primary macrophages (see Figure 1).

Cumulatively, our results indicate that NTM affords robust protection of mice from LPS-induced systemic inflammation in two distinct models of lethal shock. Concurrently, we noted a striking reprogramming of the inflammatory response that entails suppression of many proinflammatory cytokines and chemokines in plasma.

We next sought to correlate our transcriptome analysis of primary macrophages with the production of inflammatory mediators in the bronchoalveolar space in a murine model of LPS-induced ALI. After challenging mice intranasally with LPS, we analyzed inflammatory mediators and cell populations in BAL fluid. We determined that i.p. administration of NTM suppressed the LPS-induced increase of 14 out of 32 proinflammatory chemokines, cytokines and growth factors analyzed in BAL fluid (Figure 4). Consistent with genomic reprogramming in primary macrophages (see Figure 1), NTM treatment effectively reduced chemokines linked to lung inflammation by their roles in mediating inflammatory cell migration to the lung: MCP-1/CCL2, MIP-1α/CCL3, and LIX/CXCL5. Suppressed production of cytokines IL-1α, IL-1β, IFN-γ, and IL-13 also correlated with results from BMDMs. Conversely, some inflammatory mediators, such as MIG, were not induced by direct airway exposure to LPS, while TNF-α, MIP-1β/CCL4, RANTES/CCL5 and IP-10/CXCL10 were induced by LPS but not suppressed by NTM treatment. However, these discrepancies between the qRT-PCR assay and BAL analysis can be attributed to the different cell populations present in each assay. NTM did reduce BAL levels of a number of other inflammatory mediators that were not analyzed by qRT-PCR: cytokines LIF, IL-9, and IL-12p70; chemokine MIP-2/CXCL2; and growth factors G-CSF, M-CSF, and VEGF, which is implicated in vascular permeability [28]. Overall, analysis of BAL documents the effectiveness of NTM in suppressing mediators of ALI after direct airway exposure to LPS.

Suppression of chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, and LIX/CXCL5 in the inflammatory transcriptome analysis of NTM-treated primary macrophages and in BAL following direct airway exposure to LPS, led us to postulate that NTM would reduce leukocyte trafficking to the lung. As shown in Figure 5, we observed a 5-fold reduction in total cell count in BAL fluid from mice treated with NTM in comparison to the saline-treated control group (p<0.005), reducing the total cell count in BAL from NTM-treated mice to that of naive mice. A reduction in neutrophils (∼80%, p<0.05) accounted for most of the difference in BAL cellularity. Trafficking of monocytes/macrophages, the primary cell type detected in BAL fluid from naïve mice was moderately reduced (30%), but not significantly. Lymphocytes represented less than 1% of cells detected in BAL and their numbers were unchanged by NTM treatment (Figure 5). Thus, migration of neutrophils to the bronchoalveolar space in response to direct airway exposure to LPS, which evokes a robust expression of chemokine genes, is profoundly reduced by systemic administration of NTM. As neutrophils are the main source of oxidative stress to the delicate structure of the blood-air barrier [29], NTM’s suppression of chemokines responsible for massive neutrophil trafficking to the bronchoalveolar space may engender a new approach to lung cytoprotection. Thus, LPS-induced respiratory and systemic blood inflammatory responses and their lethal outcomes are averted.