Hyperlipidemic hypersensitivity to lethal microbial inflammation and its reversal by selective targeting of nuclear transport shuttles

Hyperlipidemia, the hallmark of Metabolic Syndrome that afflicts millions of people worldwide, exacerbates life-threatening infections. We present a new evidence for the mechanism of hyperlipidemic hypersensitivity to microbial inflammation caused by pathogen-derived inducer, LPS. We demonstrate that hyperlipidemic animals succumbed to a non-lethal dose of LPS whereas normolipidemic controls survived. Strikingly, survival of hyperlipidemic animals was restored when the nuclear import of stress-responsive transcription factors (SRTFs), Sterol Regulatory Element-Binding Proteins (SREBPs), and Carbohydrate-Responsive Element-Binding Proteins (ChREBPs) was impeded by targeting the nuclear transport checkpoint with cell-penetrating, biselective nuclear transport modifier (NTM) peptide. Furthermore, the burst of proinflammatory cytokines and chemokines, microvascular endothelial injury in the liver, lungs, heart, and kidneys, and trafficking of inflammatory cells were also suppressed. To dissect the role of nuclear transport signaling pathways we designed and developed importin-selective NTM peptides. Selective targeting of the importin α5, ferrying SRTFs and ChREBPs, protected 70–100% hyperlipidemic animals. Targeting importin β1, that transports SREBPs, was only effective after 3-week treatment that lowered blood triglycerides, cholesterol, glucose, and averted fatty liver. Thus, the mechanism of hyperlipidemic hypersensitivity to lethal microbial inflammation depends on metabolic and proinflammatory transcription factors mobilization, which can be counteracted by targeting the nuclear transport checkpoint.

Remarkably, similar to the chow diet-fed ldlr −/− animals, the 80% survival was observed in HFD-fed ldlr −/− mice when treated with the bi-selective cell-penetrating NTM, cSN50.1 peptide 30 min before LPS challenge and thereafter for 60 h (see "Short-Term protocol" below) (Fig. 1D). This biselective peptide (Fig. S2A) controls the nuclear transport checkpoint by simultaneously targeting two nuclear transport shuttles, importin α5 (Imp α5) and importin β1 (Imp β1) 21,26 . Importin α5 ferries to the cell's nucleus the proinflammatory SRTFs, whereas importin β1 is responsible for nuclear translocation of the MTFs, SREBPs 11,27 . To dissect the contribution of each importin-mediated nuclear transport pathway to lethal microbial inflammation in hyperlipidemia, we designed and developed pathway-selective NTM peptides that would target separately importin α5 and importin β1.
Design, development, and testing of importin pathways-selective nuclear transport modifier peptides. As depicted in Supplementary Fig. 2A, the biselective NTM peptide denoted cSN50.1 is a twofragment construct comprising the membrane translocating motif based on the Signal Sequence Hydrophobic Region (SSHR), and the Nuclear Localization Sequence (NLS) that is cyclized by the insertion of two cysteines forming the intrachain disulfide bond. The two fragments are derived from the highly conserved human Fibroblast Growth Factor 4 SSHR and human transcription factor NF-κB1 NLS motif, respectively 28 . The importin α5-selective, cell-penetrating peptide termed cSN50.1α (Fig. S2B), has an intact NLS fragment responsible for the interaction with Imp α5 NLS binding pockets 26 . The SSHR fragment has amino acid substitutions, thereby incapacitating its hydrophobic interaction with Imp β1 responsible for translocation of SREBPs to the nucleus 21 . In contrast, the monoselective NTM, cSN50.1β peptide, designed to target Imp β1, has amino acid substitutions within the NLS sequence disabling its docking to Imp α5 26 (Fig. S2C). The control peptide, Null cSN50.1, contains amino acid substitutions in both fragments, within the SSHR and the NLS motifs, which disabled Null cSN50.1 from targeting Imp β1 and Imp α5 (Fig. S2D). Importantly, the biselective peptide as well as both monoselective and control peptides penetrate the cell membrane. This non-invasive peptide delivery process consists of direct (receptor-and energy-independent) crossing of membrane phospholipid bilayer and bypassing endosomal compartment 29 .
First, we compared the effect of treatment with two monoselective NTM peptides, cSN50.1α and cSN50.1β, to the effect of treatment with the biselective NTM, cSN50.1 peptide on nuclear translocation of SRTFs and MTFs in cell-based essays. Targeting Imp α5-mediated nuclear import by the monoselective NTM, cSN50.1α peptide, inhibited the nuclear translocation of NF-κB RelA, consistent with previous result in different models of microbial inflammation 30 (Fig. 2A).
Moreover, we extended this analysis of Imp α5-selective NTM, cSN50.1α peptide inhibition of the nuclear translocation of NF-κB RelA to two other members of the SRTFs set, the phosphorylated form of STAT1, and cFos ( Fig. 2B,C). The latter transcription factor is a component of the AP-1 complex comprising cFos and cJun (Fig. S1). In turn, the nuclear translocation of nSREBP2 was spared by selective targeting of Imp α5-mediated nuclear import by cSN50.1α peptide (Fig. 2D). However, somehow surprisingly we found that the monoselective NTM peptide targeting importin α5 also strongly inhibited the nuclear import of ChREBPs in comparison to the lesser effect of cSN50.1β peptide (Fig. 2E). ChREBPs also display bipartite NLS located near phosphorylation site, Ser-196 31 . Thus, we present the new evidence that importin α5 also plays a significant role in the nuclear translocation of ChREBPs, a key regulator of glucose, glycogen, and triglycerides metabolism 32 .
Cumulatively, the selective targeting of the nuclear transport checkpoint in cultured cells indicates that Imp α5 ferries SRTFs and ChREBPs to the nucleus while Imp β1 is solely responsible for the nuclear translocation of SREBPs. The Imp β1-mediated pathway is also involved, albeit to a lesser degree, in ChREBPs nuclear translocation (see Fig. 2E).
We also designed and tested the control "loss of function" NTM, Null cSN50.1 peptide, with disabled binding sites for Imp α5 and Imp β1 (see Fig.S2D). The Null cSN50.1 peptide was tested along with active NTMs in in vivo model of lethal LPS-induced microbial inflammation. Normolipidemic, chow diet-fed, C57BL/6 female mice were challenged with a single lethal dose of LPS (700 μg) administered through IP injection. Mice were treated with active biselective and monoselective NTMs (cSN50.1, cSN50.1α, and cSN50.1β) and two controls (vehicle/saline and Null cSN50.1 peptide) also administered through IP injection. As documented in Fig.S3, mice challenged with the lethal dose of LPS and treated with saline (vehicle) or the control NTM, Null cSN50.1 peptide, died within 24 h. Likewise, Imp β1-selective NTM, cSN50.1β peptide, was not protective in this lethal microbial inflammation-mediated model of endotoxemia (Fig. S3A). In a striking contrast, the 80% survival was observed in these normolipidemic mice when treated with the bi-selective NTM, cSN50.1 peptide or Imp α5-selective NTM, cSN50.1α peptide. Consistent with survival data, blood levels of cytokines TNF-α and IL-6,  www.nature.com/scientificreports/ and chemokine MCP-1 were suppressed in survivors treated with cSN50.1 and cSN50.1α peptides. Contrariwise, these proinflammatory biomarkers remained elevated in mice treated with the control NTM, Null cSN50.1 peptide, and comparable with the levels observed in mice treated with saline (vehicle). As expected, plasma levels of TNF-α, IL-6, and MCP-1 in mice treated with Imp β1-selective NTM, cSN50.1β peptide, were similar to those treated with vehicle and control Null NTM peptide.
Importin pathway-selective NTM peptides prevent and/or reverse lethal microbial inflammation in hyperlipidemic ldlr −/− mice. After having established the survival-enhancing action of NTM peptides in hyperlipidemic (Fig. 1D) and normolipidemic ( Fig. S3) animals, we explored the transcriptional mechanism of hyperlipidemic hypersensitivity to LPS. To this end, we tested pathway-selective inhibitors of the nuclear transport checkpoint in the high fat diet-fed ldlr −/− mice using two in vivo protocols comprising the long-and short-term treatments (Fig. 3).
In the Long-Term Treatment protocol (Fig. 3A), ldlr −/− mice fed HFD for 3 weeks were simultaneously treated with NTM peptides while control animals received saline (diluent control). All control mice died of LPS shock within 40 h (Fig. 4A). In striking contrast, 100% mice treated with the Imp α5-selective NTM peptide, cSN50.1α, survived along with the mice treated with the biselective NTM, cSN50.1 peptide. This indicates that the Imp α5-mediated signaling pathway to the nucleus contributes to death in LPS-induced microbial inflammation superimposed on preexisting metabolic inflammation caused by HFD.
Importantly, 60% survival was also observed in HFD-fed ldlr −/− mice treated with Imp β1-selective NTM, cSN50.1β peptide for 3 weeks. These results indicate that the Imp β1-mediated signaling pathway to the nucleus also contributes to death in LPS-induced microbial inflammation superimposed on preexisting metabolic inflammation caused by HFD. Consequently, targeting Imp β1-mediated transport of SREBPs 21 should improve metabolic markers in the HFD-fed ldlr −/− mice. Treatment with nuclear import pathway-selective NTMs improves metabolic markers in the blood and reduces nuclear content of proinflammatory transcription factor, NF-κB RelA, in the liver. Indeed, hypertriglyceridemia was precipitously reduced by the Imp β1-selective NTM, cSN50.1β peptide, producing a 2.5-fold decline in blood triglycerides after a 3-week treatment. Notably, this new monoselective inhibitor of the Imp β1-mediated signaling pathway also reduced the elevated blood glucose level (Fig. 4B). Hypercholesterolemia was significantly albeit moderately lowered by the biselective and two monoselective NTM peptides in hyperlipidemic mice before their challenge with the inducer of microbial inflammation, LPS. Liver transaminases (AST and ALT) were also moderately reduced. Altogether, selective targeting of Imp β1 improves metabolic profile of HFD-fed mice after the 3-week treatment.
We focused further mechanistic analysis of hyperlipidemic hypersensitivity to LPS on the nuclear translocation of the main proinflammatory SRTFs, NF-κB RelA, in the liver cells. The liver nuclear extracts of control hyperlipidemic ldlr −/− mice treated with saline displayed abundant content of this prominent proinflammatory transcription factor (Fig. 4C). In contrast, the 3-week treatment with biselective or two monoselective NTM peptides significantly reduced the nuclear transport of NF-κB RelA (Fig. 4C). The paucity of the NF-κB RelA in the liver nuclear extracts of animals treated with Imp β1-selective NTM, cSN50.1β peptide was striking. This presumably paradoxical result (cSN50.1β peptide is not targeting Imp α5-mediated nuclear transport of NF-κB RelA) (see Fig. 2A) offers a proof for the activation of the NF-κB RelA signaling pathway by elevated neutral lipids in the liver cells. When accumulation of these noxious metabolites is prevented by the 3-week treatment with Imp β1-selective NTM, the NF-κB RelA is not mobilized. Thus, the very low levels of nuclear NF-κB RelA, the transcriptional vanguard of inflammation in the liver cells' nuclei, coincided with the effectiveness of the life-saving treatment with biselective and nuclear import pathway-selective NTM peptides.
Suppression of inflammatory cytokines and chemokines, fatty liver, glycogen depletion, microvascular endothelial injury, and trafficking of neutrophils by differential action of nuclear import pathway-selective NTM peptides. Consistent with the survival data, LPS-induced burst of proinflammatory cytokines [TNF-α, Interferon (IFN)-γ, Interleukin (IL)-6, and chemokine MCP-1] was suppressed by the long-term treatment protocol (3-week) with the biselective and the Imp α5-selective NTM peptides (Fig. 4D). In agreement with the prior studies, the blood level of anti-inflammatory cytokine, IL-10, remained elevated in all treatment groups 33 . The IL-10, also known as cytokine synthesis inhibitory factor (CSIF), has a potent anti-inflammatory properties contributing to amelioration of tissue damage by suppressing activity of immune cells such as Th1 cells, NK cells or macrophages 34 .
The remarkable immunometabolic improvements due to the modulation of nuclear import pathways for SRTFs and MTFs were reaffirmed by an immunocytochemistry analysis of the liver and other organs (Fig. 5). The accumulation of liver neutral lipids stained with Oil-Red-O (ORO) 35 was reduced by both the biselective NTM, cSN50.1, and the Imp β1-selective NTM, cSN50.1β, peptides. The latter result supports our mechanistic analysis of nuclear transport regarding the paucity of nuclear translocation of the NF-κB RelA in the liver cells of animals treated with the Imp β1-selective NTM (see above). Glycogen breakdown in the liver is detrimental in bacterial and viral infections 36 . LPS-induced glycogenolysis is linked to the hyperglycemia and hypertriglyceridemia mediated by the nuclear import of ChREBP transcription factors involved in the development of MetS 32 . Indeed, we demonstrated an almost total depletion of the glycogen stained with PAS in the saline-treated control mice. This LPS-induced loss of liver glycogen was prevented by the biselective NTM, cSN50.1 peptide, whereas  Figure 3. Graphic depiction of two treatment protocols. (A) Long-term treatment protocol. 12-week-old ldlr −/− female mice were placed on HFD and treated with saline (IP, twice a day (BID), 0.2 mL) or NTM peptides (IP, BID, 33 μg/g in 0.2 mL saline) for three weeks before non-lethal dose of LPS (10 μg/g in 0.2 mL saline) was administered through IP injection. HFD feeding and treatment with saline or NTM were continued for additional week. (B) Short-term treatment protocol. 15-week-old ldlr −/− female mice fed HFD for three weeks were challenged with a non-lethal dose of LPS (10 μg/g in 0.2 mL saline, IP) and threated with 15 doses of saline (0.2 mL) or NTM peptides (IP, 33 μg/g in 0.2 mL saline) given 30 min before LPS challenge (10 μg/g in 0.2 mL saline) and for three days thereafter. HFD feeding continued for another week. www.nature.com/scientificreports/ the Imp α5-and Imp β1-selective NTMs, cSN50.1α and cSN50.1β peptides were partially protective indicating that both nuclear transport pathways contribute to the glycogen breakdown (Fig. 5).
Severe microvascular endothelial injury is the main mechanism of lethal LPS shock, the end stage of LPSinduced microbial inflammation 11 . It was manifested by increased expression of Vascular Cell Adhesion Molecule (VCAM)-1, regulated by SRTFs (e.g., NF-κB and STAT1). VCAM-1 was prominently expressed in the endothelial cells of the liver, lungs, heart, and kidney of control animals fed HFD as compared to those on chow diet after LPS challenge (Fig. 5). In contrast, VCAM-1 expression was dramatically suppressed in cSN50.1-and cSN50.1αtreated groups in the 3-week treatment protocol. The cSN50.1β-treated group shows a diminished albeit not completely suppressed expression of VCAM-1.
The activation and trafficking of neutrophils and other myeloid cells into the organs' parenchyma, such as seen in the liver and lungs, is also associated with microvascular endothelial injury 37 . These inflammatory cells contribute to the oxidant stress 11 . Their trafficking was suppressed in animals treated with biselective, and Imp α5-selective NTMs, cSN50.1 and cSN50.1α peptides (Fig. 5). Notably, targeting Imp β1-mediated nuclear import of SREBPs by cSN50.1β peptide also resulted in a significant reduction of neutrophils migration, consistent with increased survival data (see Fig. 4A). Thus, it is apparent that a significant gain in survival of LPS-challenged hyperlipidemic mice, following a 3-week treatment with the pathway-selective NTM peptides, was linked to their anti-inflammatory and cytoprotective action in the liver and other organs.
Contrariwise, the short-term treatment (see Fig. 3B), with Imp β1-monoselective NTM peptide (see Fig. S2C) administered shortly before the LPS challenge and 3 days thereafter, during the acute stage of LPS shock was largely ineffective (10% survival) indicating that this lipid-lowering treatment requires more chronic regimen (Fig. 6A).
Only animals treated with NTMs targeting Imp α5-mediated nuclear import, i.e. cSN50.1 and cSN50.1α peptides, displayed inhibition of glycogenolysis, suppressed VCAM-1 expression in endothelial cells, and the arrest of neutrophil trafficking in the liver and lungs, In striking contrast, short-term administration of Imp β1-selective NTM, cSN50.1β peptide, was ineffective in protecting organs from devastating action of LPS shock in hyperlipidemic mice (Fig. 7), consistent with the survival data (Fig. 6A).

Discussion
Cumulatively, these results support the concept that hyperlipidemia, the key component of MetS, increases the susceptibility to lethal microbial inflammation. Further, they demonstrate that selective inhibition of Imp α5-mediated nuclear transport of SRTFs and Imp β1-mediated nuclear transport of MTFs counteracts lethal microbial inflammation in hyperlipidemic animals. The nuclear transport checkpoint is an attractive target for therapeutic intervention in this context because NTM peptides administered parenterally significantly offset the lethal microbial inflammation. Imp α5-mediated nuclear import plays a key role in hyperlipidemic hypersensitivity to lethal microbial inflammation since its targeting with the cSN50.1 and cSN50.1α peptides was protective in both long-term and short-term treatment protocols. In contrast, Imp β1-mediated pathway could be effectively suppressed by the long-term treatment with the Imp β1-selective NTM, cSN50.1β peptide whereas short-term administration was not effective. Thus, the transcriptional signaling cascades mediated by importin α5 and importin β1 provide the new nexus for hyperlipidemic hypersensitivity to potentially lethal microbial diseases. . HFD-induced hypersensitivity to lethal microbial inflammation induced by LPS is suppressed by targeting nuclear import pathways of SRTFs and MTFs. ldlr −/− female mice were treated according to the long-term treatment protocol (see Fig. 3A). Briefly, 15-week-old mice fed with HFD and treated BID with saline, biselective NTM, cSN50.1 peptide, or monoselective NTMs, cSN50.1α or cSN50.1β peptides for three weeks were challenged with non-lethal dose of LPS (10 μg/g) and observed for 7 days. (A) All mice treated with biselective, and Imp α5-selective NTMs were protected from death caused by LPS shock. Treatment with Imp β1-selective NTMs resulted in 60% survival. Data is presented as Kaplan-Meier survival plot with p value calculated by log rank analysis, **p < 0.005, ***p < 0.0005. (B) Levels of metabolic markers (cholesterol, triglycerides, glucose) and liver transaminases (AST, ALT) were measured in blood plasma collected at the end of week 3 of HFD feeding and NTM treatment, before LPS challenge. (C) Nuclear translocation of NF-κB RelA, was determined by quantitative immunoblotting of liver nuclear extracts obtained at the time of sacrifice. Western blots were analyzed using LI-COR Odyssey infrared imaging system (unedited fulllength immunoblots are presented in Supplementary Figure S5A). Data in (B,C) is presented as a mean ± SEM (n = 10). Statistical significance was determined by ordinary One-way ANOVA with Holm-Sidak test for multiple comparison, *p < 0.05, **p < 0.005, ****p < 0.0001. (D) Cytokines (TNF-α, IFN-γ, IL-6, and IL-10) and chemokine MCP-1, levels were determined in blood plasma collected from saphenous vein before and 2, 6, and 12 h after LPS challenge. Data is presented as a mean ± SEM (n = 10). Statistical significance was determined by repeated measures two-way ANOVA analysis of variance with Holm-Sidak test for multiple comparison, **p < 0.005, ***p < 0.0005, ****p < 0.0001. www.nature.com/scientificreports/ The major step in understanding the mechanism of HFD-induced metabolic inflammation "sensitizing" animals to lethal LPS-induced microbial inflammation is the outcome of the long-term administration of monoselective NTM targeting Imp β1, which ferries SREBPs, during the 3-week feeding HFD. Notably, this longterm protocol was sufficient to abrogate LPS shock in the majority of HFD diet-fed mice. These mice displayed a precipitous decline in blood triglycerides and glucose while the reduction of cholesterol levels was smaller albeit significant. Hence, the genes encoding proteins involved in the synthesis of triglycerides, cholesterol, and glucose were not activated. We reported previously 21 that biselective NTM, cSN50.1 peptide suppressed the expression of the genes encoding HMG-CoA reductase (a target of statins), ATP citrate lyase, fatty acid synthase, and Nieman-Pick C1-like 1 protein (a key enterohepatic cholesterol absorption receptor), among over 30 genes regulated by SREBPs 38 . Since SREBPs are translocated to the nucleus by Imp β1, its long-term targeting by Imp β1-selective peptide was sufficient to protect 60% of the ldlr −/− mice fed HFD from lethal microbial inflammation.
Paradoxically, we discovered the striking paucity of NF-κB RelA in the nuclei of animals fed HFD and treated for 3 weeks with Imp β1-selective peptide (Fig. 4C). How the long-term treatment with Imp β1-selective peptide suppresses nuclear translocation of NF-κB RelA, the master proinflammatory SRTF? As we documented above, the Imp β1-selective peptide does not directly inhibit the nuclear translocation of NF-κB RelA (see Fig. 2A Figure 6. Short-term treatment of LPS-induced lethal microbial inflammation in hyperlipidemic mice with biselective and Imp α5-selective NTMs is effective while Imp β1-selective NTM is ineffective. ldlr −/− female mice were treated according to the short-term treatment protocol (see Fig. 3B). Briefly, 15-week-old female mice fed with HFD for three weeks were challenged with non-lethal dose of LPS (IP, 10 μg/g in 0.2 mL saline) followed by treatment with 15 doses of saline (control; IP, 0.2 mL), biselective NTM, cSN50.1 peptide, or monoselective NTMs, cSN50.1α or cSN50.1β peptides (33 μg/g/injection in 0.2 mL saline) administered 30 min before LPS challenge and over 3 days thereafter.
(A) Mice treated with biselective, and Imp α5-selective NTMs display 80% and 70% survival, respectively. Treatment with Imp β1-selective NTMs did not protect the majority of hyperlipidemic mice from death caused by LPS-induced microbial inflammation (10% survival). Data is presented as Kaplan-Meier survival plot with p value calculated by log rank analysis, **p < 0.005, ***p < 0.0005. (B) Cytokines (TNF-α, IFN-γ, IL-6, and IL-10) and chemokine MCP-1, levels were determined in blood plasma collected from saphenous vein of animals before and 2, 6, and 12 h after LPS challenge. Data is presented as a mean ± SEM (n = 10). Statistical significance was determined by repeated measures two-way ANOVA analysis of variance with Holm-Sidak test for multiple comparison, *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001. (C) Nuclear translocation of SRTFs NF-κB RelA was determined by quantitative immunoblotting of liver nuclear extracts obtained at the time of sacrifice. Western blots were analyzed using LI-COR Odyssey Infrared Imaging System (unedited full-length immunoblots are presented in Supplementary Figure S5B). Data is presented as a mean ± SEM (n = 10). Statistical significance was determined by ordinary one-way ANOVA with Holm-Sidak test for multiple comparison, ***p < 0.0005, ****p < 0.0001. Hence, their activation of the main proinflammatory signaling pathway mediated by NF-κB RelA was averted. Inhibition of SREBPs nuclear import during the long-term (3 weeks) treatment protocol (see Fig. 3A) counteracted the metabolic derangements caused by the HFD thereby warding off signs of steatohepatitis and hyperlipidemic hypersensitivity to LPS shock. Thus, the long-term treatment of HFD-induced hyperlipidemia and hyperglycemia with the Imp β1-selective, cSN50.1β peptide offers a promising strategy to prevent fatty liver and subsequent steatohepatitis thereby reducing the susceptibility to the end-stage microbial inflammation such as LPS shock. In contrast, the short-term ("just-in-time") treatment protocol (see Fig. 3B), with the importin β1-selective NTM, administered coincidently with the LPS challenge, was not effective in reducing the lethal outcome (see Fig. 6A). We infer that, SREBPs do not seem to contribute to the LPS shock in the acute phase of microbial inflammation.

Scientific Reports
The discovery of the nuclear transport pathway mediated by importin α5 for the Carbohydrate-Responsive Element-Binding Proteins (ChREBP) family 39 , is of particular significance. It explains why selective targeting of the importin α5-mediated pathway prevented glycogen depletion observed in the livers of hyperlipidemic animals succumbing to the LPS shock. Hence, the resulting hyperglycemia and hypertriglyceridemia were reduced. This metabolic duo comprises "the deadly combination" underlying MetS 40 . As ChREBPs, the regulators of glucose homeostasis, are translocated to the nucleus through the same pathway as proinflammatory SRTFs, targeting the Imp α5-mediated pathway by selective NTM peptide (cSN50.1α) was equally protective in comparison to the biselective NTM in hyperlipidemia-aggravated LPS shock. We submit that hyperlipidemic hypersensitivity to LPS shock is chiefly mediated by the proinflammatory SRTFs along with ChREBPs in the short-term treatment protocol. The documented outcome of inhibiting ChREBPs access to the nucleus in our study, is in agreement with previous reports showing that ChREBPs deficiency results in glycogen synthesis while triglyceride formation is reduced 32 . Another evidence for the ChREBPs involvement in metabolic syndrome, is their cooperation with SREBP1c, in induction of glycolytic and lipogenic genes 32,41 . The nuclear transport checkpoint inhibition of importin α5 and importin β1 would keep these genes silent.
As depicted in Fig. 8, different causes of microbial diseases (bacteria, viruses, fungi, protozoa) activate in immune and non-immune cells the proinflammatory signaling pathway mediated by transcription factors, SRTFs. In hyperlipidemic and hyperglycemic host with MetS, the metabolic transcription factors, ChREBPs, and SREBPs, Neutr.

Figure 7.
Multi-organ injury in the LPS-induced microbial inflammation in hyperlipidemic mice is prevented only by treatment with NTMs targeting nuclear import pathways of SRTFs and ChREBPs mediated by Imp α5 pathway whereas targeting Imp β1-mediated pathway with cSN50.1β peptide is ineffective in the Short-Term treatment protocol. LPS-induced glycogenolysis (PAS) in the liver, microvascular endothelial injury (VCAM-1; in the liver, and lungs) and inflammatory cells infiltration (neutrophils; in the liver, and lungs) are attenuated by treatment targeting nuclear import pathway mediated by Imp α5 with monoselective NTM cSN50.1α and biselective NTM cSN50.1. Targeting  www.nature.com/scientificreports/ are mobilized. The products of the genes regulated by these transcription factors (cholesterol, triglycerides, glucose) also activate proinflammatory signaling pathway mediated by the Imp α5, thereby producing more severe and difficult to control stage of microbial inflammation. This advanced stage of microbial inflammation comprises acute respiratory distress syndrome (ARDS), septic cardiomyopathy, microvascular thrombosis (known as Disseminated Intravascular Coagulation), and Acute Kidney Injury. These life-threatening disorders result from the microvascular endothelial injury due to genomic storm that underlies septic shock, the end stage of microbial inflammation 10 . The proinflammatory and metabolic pathways involved in the end stage of lethal microbial inflammation depend on the nuclear transport shuttles, importins α5 and β1. The pathway-selective cell-penetrating NTM peptides control the nuclear transport checkpoint staffed by these importins. The NTM peptides modulate the cross-talk between metabolic and microbial signaling pathways thereby suppressing the potentially lethal inflammatory response to infections aggravated by hyperlipidemia and hyperglycemia. The NTM peptides also enhance the innate immunity-mediated clearance of bacteria in a polymicrobial septic shock model 37 . Thus, targeting of the nuclear transport checkpoint offers a new and potentially effective countermeasure (as an adjunct to the pathogen-specific antimicrobial therapies) for microbial diseases in a host compromised by underlying metabolic syndrome. Our study explains the transcriptional mechanism of hyperlipidemic hypersensitivity to lethal microbial inflammation caused by microbial agents. We provide new experimental evidence that the immunometabolic axis mediated by importin α5 and importin β1 underlies hyperlipidemic hypersensitivity to lethal microbial inflammation. This immunometabolic axis can be dismantled by selective targeting of the nuclear transport checkpoint.  Figure 8. Microbial inflammation is interwoven with metabolic inflammation. Polymicrobial causes (viruses, bacteria, fungi, and protozoa) evoke two signaling pathways (proinflammatory and metabolic). These pathways can be independently targeted by new, pathway selective NTMs, cSN50.1α and cSN50.1β, that respectively bind nuclear transport shuttles, Imp α5 and Imp β1, to modulate Imp α5-mediated nuclear import of stress responsive transcription factors (SRTFs), and Imp β1-mediated nuclear import of metabolic transcription factors (MTFs) such as SREBPs. Transcription factors ChREBPs can be imported to the nucleus either by heterodimeric complex Imp α5/Imp β1 or by Imp β1 alone. Inhibition of nuclear transport of SRTFs reduces expression of genes encoding mediators of inflammation, cytokines, chemokines, signal transducers, and cell adhesion molecules (see Fig. S1). Inhibition of ChREBPs, that regulate genes encoding proteins involved in glucose homeostasis, reduces hyperglycemia and hypertriglyceridemia, inhibition of nuclear transport of SREBPs reduces expression of genes encoding proteins involved in synthesis of cholesterol, triglycerides, and fatty acids. The accumulation of these metabolites can activate proinflammatory pathway mediated by NF-κB RelA and other SRTFs thereby aggravating microbial inflammation (see text for details; NPS nuclear pore complex). www.nature.com/scientificreports/ In summary, we successfully enabled two selective inhibitors of nuclear transport to dissect the transcriptional mechanism of hyperlipidemic hypersensitivity to microbial inflammation. Our findings are of significant relevance to individuals displaying signs of metabolic syndrome that predisposes them to life-threatening microbial diseases, including recent outbreaks of COVID-19 [6][7][8][9] as well as autoimmune and allergic disorders 3,42 . The biselective NTM peptide (AMTX-100 CF) is undergoing Phase I/II treatment trial for inflammatory skin diseases (NCT04313400).

Nuclear translocation of SRTFs and
Human hepatocyte carcinoma. HepG2 cells (ATCC) were cultured according to the supplier's instruction in 10 cm dishes until confluent. Cells were starved for 24 h in DMEM containing 5.5 mM glucose then refed with 25 mM glucose (HG) with addition of 100 nM insulin (In) and 30 µM cSN50.1, cSN50.1α or cSN50.1β for 24 h. HG/In-stimulated cells not treated with NTM peptides and unstimulated cells served as positive and negative controls, respectively. Nuclear extracts were obtained as described above. Viability of cells used in cell-based assays was greater than or equal to 80%.
Animal studies. Animal experiments were carried out in compliance with the ARRIVE guidelines and in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and submitted protocols were approved by the Vanderbilt University Institutional Animal Care and Use Ethics Committee (Permit Number: A3227-01). Mice were closely monitored during the course of experiments and euthanized by Isoflurane inhalation followed by cervical dislocation upon expression of the signs of moribund state. Survivors were euthanized at the experimental end point.
Mouse model of endotoxin shock. We used this model to compare biselective and monoselective NTM peptides with the control "loss of function" Null cSN50.1 peptide (see Fig. S3). 15-week-old female C57BL/6 mice (The Jackson Laboratory, 20 g body weight) selected into five experimental groups (5 mice/group) using double blinded randomization method. Briefly, mice ear tag's numbers were written down on 25 pieces of paper, which were folded, placed in a receptacle, and shaken. Similarly, another 25 pieces of paper were marked with the numbers corresponding to the treatment groups, five of each [1--vehicle (saline); 2-cSN50.1; 3-cSN50.1α; 4-cSN50.1β, 5-Null cSN50.1]. Paper was folded, placed in a separate receptacle and well shaken. Selection was completed by drawing ear tag numbers and pairing them with the treatment group number drawn from second receptacle. Each experiment was performed twice to assure statistical significance and experimental reproducibility. Mice were challenged by IP injection of lethal dose of LPS (700 µg in 200 µL saline; E. coli strain O127:B8 Mouse model of hyperlipidemia-induced susceptibility to microbial inflammation. An 8-week-old ldlr −/− female mice (B6.129S7-Ldlr tm1Her /J on C57BL/6J genetic background; The Jackson Laboratory) were placed on nonirradiated regular chow diet for 4 weeks. Four experimental groups, vehicle, cSN50.1, cSN50.1α and cSN50.1β (5 mice/group) were selected using double blinded randomization method described above. We used saline as a control representing vehicle for all NTM peptides used in this study. This type of control is used in the FDAapproved clinical studies of human subjects in which tested new drug (including therapeutic peptides) is administered. Each experiment was performed twice to assure statistical significance and experimental reproducibility. Metabolic challenge was instituted in 12-week-old ldlr −/− female mice fed a high-fat, high-cholesterol diet (HFD) for three weeks before LPS (10 μg/g, E. Coli O127:B8; Sigma) induction of microbial inflammation. Survivors were fed HFD for another week until the end of experiment. Body weight and food consumption was monitored weekly. Blood samples (~ 50 µL) were collected from the saphenous vein of 6-h-fasting mice in EDTA-coated tubes (Sarstedt) before HFD, before LPS administration and at 2, 6, and 12 h after LPS injection.
Long-term NTM treatment protocol (see Fig. 3A). Mice were treated with the intraperitoneal injection (IP) of NTM peptides, biselective cSN50.1, and monoselective cSN50.1α and cSN50.1β (33 μg/g/injection in 200 μL saline) two times a day (BID) for the course of HFD feeding (3 weeks before LPS challenge and 1 week thereafter). Control group of animals received intraperitoneal injection of saline (200 μL). Survivors were euthanized by overdosed inhalation of isoflurane following cervical dislocation at 168 h post LPS challenge.
Preparation of nuclear extracts from the liver cells. Nuclear extracts were prepared from frozen livers as previously described 21 . Briefly, liver pieces were disrupted in a Dounce hand homogenizer on ice in hypotonic lysis buffer 26 containing 2% NP-40, protease and phosphatase inhibitors (Roche), vortexed, and left on ice for 20 min. Nuclei were pelleted at 10,000×g and washed twice in hypotonic lysis buffer. Nuclear proteins were extracted with high salt solution (450 mM) by shaking nuclei at 2000 rpm for 1 h at 4 °C. Extracts were analyzed by quantitative immunoblotting using rabbit monoclonal anti-NF-κB RelA (p65) antibody (Cell Signaling, Cat# 8242). Mouse monoclonal anti-TATA binding protein antibody (TBP, Abcam, Cat# ab818) was used as nuclear loading control for normalization. Immunoblots were analyzed on a LI-COR Odyssey Infrared Imaging System.

Histology.
Organ samples (liver, kidney, heart, and lungs) were collected and fixed overnight in 10% formalin, routinely processed, embedded in paraffin, sectioned at 5 μm and stained with Hematoxylin and Eosin (H&E) or Periodic Acid-Schiff-hematoxylin to assess injury, hemorrhage, and liver glycogen stores, respectively. Separately, pieces of liver were embedded in OCT buffer and frozen on dry ice. Cryostat sections were stained with Oil-red-O to determine accumulation of neutral lipids. Immunohistochemistry analyses with antibodies against VCAM-1 (Abcam) and neutrophils (Abcam) were performed on the Leica Bond Max following standard protocols in the Translational Pathology Shared Resource at Vanderbilt University Medical Center.
Cytokine/chemokine and E-selectin assays. Blood plasma levels of cytokines (TNFα, IL-6, IL-10, IFNγ), chemokine MCP-1, and E-Selectin were determined using Cytometric Bead Array (BD BioSciences) following manufacturer's protocol and analyzed in the Vanderbilt University Medical Center Flow Cytometry Shared Resource as previously described 33 .
Statistical analysis. Statistical analysis was performed using tools built-in Prism 6 software (GraphPad).
Cytokine (TNFα, IL-6, IL-10, IFN-γ) and chemokine MCP-1 levels in plasma collected from the same animals at different time points were evaluated by repeated measures two-way ANOVA analysis of variance with Holm-Sidak's post-test for multiple comparison. Survival data were plotted as Kaplan-Meier survival curves and analyzed by the log-rank test. Immunoblots of SRTFs and MTFs in nuclear extracts, and blood chemistry (Chol., Trig., Glu., ALT, AST) from Long-Term NTM Treatment Protocol were analyzed by ordinary One-way ANOVA with Holm-Sidak test for multiple comparison. Blood chemistry (Chol., Trig., Glu., ALT, AST) and E-Selectin levels from Short-Term NTM Treatment Protocol were analyzed by nonparametric t test with Mann-Whitney rank comparison. Data are presented as the means ± SEM and p values of < 0.05 were considered significant.