Murine Endotoxin Research Articles

H2S as a Therapeutic Adjuvant Against COVID-19: Why and How?

H2S as a Therapeutic Adjuvant Against COVID-19: Why and How?

TLR4 induced Wnt3a-Dvl3 restrains the intensity of inflammation and protects against endotoxin-driven organ failure through GSK3β/β-catenin signaling

BackgroundAccumulating evidence suggests a regulatory role of Wnt proteins in innate immune responses. However, the effects of Wnt3a signaling on TLR4-mediated inflammatory responses are controversial and the signaling crosstalk between TLR4 and Wnt3a remains uncertain. MethodsGain- and Loss- of function approaches were utilized to determine the function of Wnt3a signaling in TLR4-mediated inflammatory responses. Cytokine production at protein and mRNA levels and phosphorylation of signaling molecules were measured by ELISA, qRT-PCR, and Western Blot, respectively. Endotoxemia mouse model was employed to assess the effect of Wnt3a on systemic inflammatory cytokine levels and neutrophil infiltration. ResultsLPS stimulation leads to an increase of Wnt3a expression and its downstream molecule, Dvl3, in primary monocytes. Inhibition or silence of Wnt3a or Dvl3 significantly increases the production of pro-inflammatory cytokines (IL-12, IL-6, TNFα), robustly reduces β-catenin accumulation, and enhances the phosphorylation of NF-κB P65 and its DNA binding activity. These results were confirmed by multiple gain- and loss- of function approaches including specific siRNA and ectopic expression of Dvl3, GSK3β, and β-catenin in monocytes. Moreover, in vivo relevance was established in a murine endotoxin model, in which Wnt3a inhibition enhances the inflammatory responses by augmenting the systemic pro-inflammatory cytokine levels and neutrophil infiltration. ConclusionsTLR4 activation promotes Wnt3a-Dvl3 signaling, which acts as rheostats to restrain the intensity of inflammation through regulating GSK3β-β-catenin signaling and NF-κB activity. General significanceWnt3a-Dvl3-β-catenin signaling axis could be a potential interventional target for manipulating the direction and intensity of inflammatory responses.

Whole body periodic acceleration improves survival and microvascular leak in a murine endotoxin model.

Sepsis is a life threatening condition which produces multi-organ dysfunction with profound circulatory and cellular derangements. Administration of E.Coli endotoxin (LPS) produces systemic inflammatory effects of sepsis including disruption of endothelial barrier, and if severe enough death. Whole body periodic acceleration (pGz) is the headward-footward motion of the body. pGz has been shown to induce pulsatile shear stress to the endothelium, thereby releasing vascular and cardio protective mediators. The purpose of this study was to determine whether or not pGz performed as a pre-treatment or post-treatment strategy improves survival in a lethal murine endotoxin model.This study was designed as a prospective randomized controlled study in mice. pGz was performed in mice as pre-treatment (pGz-LPS, 3 days prior to LPS), post-treatment (LPS- pGz, 30 min after LPS) strategies or Control (LPS-CONT), in a lethal murine model of endotoxemia. Endotoxemia was induced with intraperitoneal injection of E.Coli LPS (40mg/kg). In a separate group of mice, a nonspecific nitric oxide synthase inhibitor (L-NAME) was provided in their drinking water and pGz-LPS and LPS-pGz performed to determine the effect of nitric oxide (NO) inhibition on survival. In another subset of mice, micro vascular leakage was determined. Behavioral scoring around the clock was performed in all mice at 30 min intervals after LPS administration, until 48 hrs. survival or death. LPS induced 100% mortality in LPS-CONT animals by 30 hrs. In contrast, survival to 48 hrs. occurred in 60% of pGz-LPS and 80% of LPS-pGz. L-NAME abolished the survival effects of pGz. Microvascular leakage was markedly reduced in both pre and post pGz treated animals and was associated with increased tyrosine kinase endothelial-enriched tunica interna endothelial cell kinase 2 (TIE2) receptor and its phosphorylation (p-TIE2). In a murine model of lethal endotoxemia, pGz performed as a pre or post treatment strategy significantly improved survival, and markedly reduced microvascular leakage. The effect was modulated, in part, by NO since a non-selective inhibitor of NO abolished the pGz survival effect.

Anti-inflammatory activities of Aedes aegypti cecropins and their protection against murine endotoxin shock

BackgroundMosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown.ResultsBy searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: AeaeCec1, 2, 3, 4 and 5) were selected. AeaeCec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, AeaeCec5 had the strongest anti-inflammatory activity, and generated an additive effect with other AeaeCec peptides. In a mouse model of endotoxin shock, AeaeCec1-5 effectively reduced TNF-α, IL-1β and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with AeaeCec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa, administration of AeaeCec5 reduced the production of TNF-α, IL-1β and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti AeaeCec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS.ConclusionsThe current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided AeaeCec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy.

Abstract 389: The Impact of Phospholipid Composition of Synthetic High-Density Lipoprotein on Its Pharmacological Activity

Objective: Synthetic high-density lipoprotein (sHDL) is a nanoparticle that can mimic biological activities of endogenous HDL such as reverse cholesterol transport (RCT) and anti-inflammatory properties. We hypothesize that differences in the fluidity of sHDL phospholipids at body temperature effect plasma stability of sHDL, particle ability to efflux cholesterol and inhibit inflammation. Methods: sHDL particles with different membrane fluidities were prepared complexing phospholipids with different fatty acid chain length and saturation (POPC, DMPC, DPPC, and DSPC) with the apoA-I mimetic peptide, PVLDLFRELLNELLEALKQKLK (22A). The ability of various sHDL compositions to efflux cholesterol, inhibit NF-kB activation and cytokine release, and cause lipid raft disruption was examined in RAW264.7 macrophages. Various sHDL were administered to mice challenged by injection of 0.05 mg/kg LPS at 10 mg/kg dose and the levels of cytokine release were measured at 2 hours post-dose. Various sHDL were dosed to normal rats at 50 mg/kg and cholesterol mobilization and pharmacokinetics were examined. Results: 22A-POPC and 22A-DMPC sHDL have relatively fluid phospholipid layer at body temperature compared to 22A-DPPC and 22A-DSPC sHDL due to lower phospholipid transition temperature. 22A-POPC and 22A-DMPC sHDL inhibited NF-κB activation and cytokine release in a concentration-dependent manner. From murine endotoxin infusion studies, 22A-DMPC displayed the significant inhibition of cytokine release. Cholesterol efflux studies demonstrated that 22A-POPC and 22A-DMPC displayed highest cholesterol efflux. Interestingly, in vivo RCT study showed that 22A-DSPC had longest plasma residence time and resulted in greatest cholesterol mobilization. Conclusions: Phospholipid composition of sHDL particles has a significant effect on its cholesterol efflux and anti-inflammatory properties, yet the effect appears to be different in vitro and in vivo . in vitro effect is driven by the ability of fluid phospholipid bilayer to bind LPS, cholesterol, and LCAT, while in vivo effect is defined by particle stability in plasma and residence time in the body.

Soluble vimentin binds to P‐selectin and attenuates leukocytic infiltration into the lungs in murine endotoxin induced acute lung injury

BackgroundAcute lung injury (ALI) is a devastating condition that culminates as inflammation of the lungs mediated by leukocytic infiltration. In animal models, platelets and platelet P‐selectin are well described mediators of ALI and other forms of inflammation through enhancing capture and transmigration of neutrophils across vascular endothelium. Vimentin is an intermediate filament protein mainly thought to be involved in maintaining the cytoskeleton. Recently, there has been increasing evidence of vimentin's role during inflammation, although its precise role is unclear. We had recently observed that recombinant soluble vimentin (sVim) blocks neutrophil adhesion to platelets, endothelial cells, and P‐selectin monolayers; whether these observations translate into attenuation of ALI is unknown. Therefore, we hypothesize that soluble vimentin sVim blocks P‐selectin‐PSGL‐1 interactions and will decrease leukocytic infiltration into the lungs of endotoxin‐exposed mice.MethodsAll experiments were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine and the Michael E. DeBakey Veterans Affairs Medical Center. Recombinant sVim was expressed in E. coli and His‐purified using affinity chromatography. Binding kinetics of sVim to P‐selectin was determined using both enzyme‐linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). ELISA was performed by detecting sVim binding to immobilized P‐selectin/Fc or PSGL‐1/Fc protein. In SPR (BIACore), sVim was perfused over immobilized P‐selectin/Fc or IgG protein to determine the equilibrium dissociation constant KD. To determine whether sVim inhibits leukocytic infiltration into lungs after endotoxin exposure, C57BL/6 mice were treated with intraperitoneal injections of sVim (3 mg/kg) or vehicle control followed by endotoxin (O111:B4; 15 mg/kg) 1 hour later and then euthanized at 96 hours. Mice were evaluated by blinded investigators for both physical activity and degree of lung inflammation. Activity was determined using the mouse clinical assessment score for sepsis (M‐CASS) and lung inflammation was determined by alveolar septal thickening and leukocytic infiltration on hematoxylin and eosin stained sections.ResultssVim preferentially binds to P‐selectin/Fc and but not PSGL‐1/Fc in ELISA. Using SPR, sVim binds to P‐selectin/Fc with a KD of 125 ± 1.2 nM and not to IgG control. Endotoxin‐exposed mice treated with sVim were more active as determined by the M‐CASS (1.9 ± 0.2 vs 2.5 ± 0.2; 4‐point scale where lower is better; n=10/grp, p<0.05). Mice receiving sVim also had less alveolar septal thickening and leukocytic infiltration into the lungs after exposure to endotoxin.ConclusionSoluble vimentin binds to P‐selectin and not P‐selectin glycoprotein ligand‐1 and decreases leukocytic infiltration into the lungs of endotoxin‐exposed mice. Further studies need to take place to determine whether vimentin also improves gas exchange in the lungs. Finally, dose‐response studies need to be performed to determine the optimal dose and timing of sVim to improve outcomes in acute lung injury.Support or Funding InformationNIH/NHLBI HL‐116524, NIH/NIGMS GM‐112806

Anti-inflammatory effect of desoxo-narchinol-A isolated from Nardostachys jatamansi against lipopolysaccharide

We previously reported that Nardostachys jatamansi (NJ) exhibits anti-inflammatory activity against lipopolysaccharide (LPS). However, the active compound in NJ is unknown. Therefore, here, we examined the effects of desoxo-narchinol-A (DN) isolated from NJ against LPS-induced inflammation. To demonstrate the anti-inflammatory effect of DN against LPS, we used two models; murine endotoxin shock model for in vivo model, and peritoneal macrophage responses for in vitro. In endotoxin shock model, DN was administrated intraperitoneally 1h before LPS challenge, then we evaluated mice survival rates and organ damages. Pretreatment with DN (0.05mg/kg, 0.1mg/kg, or 0.5mg/kg) dramatically reduced mortality in a murine LPS-induced endotoxin shock model. Furthermore, DN inhibited tissue injury and production of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), in the liver and lung. In in vitro macrophage model, we examined the inflammatory mediators and regulatory mechanisms such as mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB). DN inhibited the production of inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and its derivative nitric oxide (NO), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), IL-1β, IL-6 and TNF-α and H3 protein acetylation in murine peritoneal macrophages. DN also inhibited p38 activation, but not extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and NF-κB. These results suggest that DN from NJ exhibits protective effects against LPS-induced endotoxin shock and inflammation through p38 deactivation.

Azithromycin inhibits IL-1 secretion and non-canonical inflammasome activation.

Deregulation of inflammasome activation was recently identified to be involved in the pathogenesis of various inflammatory diseases. Although macrolide antibiotics display well described immunomodulatory properties, presumably involved in their clinical effects, their impact on inflammasome activation has not been investigated. We compared the influence of macrolides on cytokine induction in human monocytes. The role of intracellular azithromycin-accumulation was examined by interference with Ca++-dependent uptake. We have also analysed the signalling cascades involved in inflammasome activation, and substantiated the findings in a murine sepsis model. Azithromycin, but not clarithromycin or roxithromycin, specifically inhibited IL-1α and IL-1β secretion upon LPS stimulation. Interference with Ca++-dependent uptake abolished the cytokine-modulatory effect, suggesting a role of intracellular azithromycin accumulation in the modulatory role of this macrolide. Azithromycin’s inhibiting effects were observed upon LPS, but not upon flagellin, stimulation. Consistent with this observation, we found impaired induction of the LPS-sensing caspase-4 whereas NF-κB signalling was unaffected. Furthermore, azithromycin specifically affected IL-1β levels in a murine endotoxin sepsis model. We provide the first evidence of a differential impact of macrolides on the inflammasome/IL-1β axis, which may be of relevance in inflammasome-driven diseases such as chronic obstructive pulmonary disease or asthma.

Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure.

Serum- and glucocorticoid-regulated kinase (SGK)1 is associated with several important pathologic conditions and plays a modulatory role in adaptive immune responses. However, the involvement and functional role of SGK1 in innate immune responses remain entirely unknown. In this study, we establish that SGK1 is a novel and potent negative regulator of TLR-induced inflammation. Pharmacologic inhibition of SGK1 or suppression by small interfering RNA enhances proinflammatory cytokine (TNF, IL-12, and IL-6) production in TLR-engaged monocytes, a result confirmed in Cre-loxP-mediated SGK1-deficient cells. SGK1 inhibition or gene deficiency results in increased phosphorylation of IKK, IκBα, and NF-κB p65 in LPS-stimulated cells. Enhanced NF-κB p65 DNA binding also occurs upon SGK1 inhibition. The subsequent enhancement of proinflammatory cytokines is dependent on the phosphorylation of TGF-β-activated kinase 1 (TAK1), as confirmed by TAK1 gene silencing. In vivo relevance was established in a murine endotoxin model, in which we found that SGK1 inhibition aggravates the severity of multiple organ damage and enhances the inflammatory response by heightening both proinflammatory cytokine levels and neutrophil infiltration. These findings have identified an anti-inflammatory function of SGK1, elucidated the underlying intracellular mechanisms, and establish, for the first time, that SGK1 holds potential as a novel target for intervention in the control of inflammatory diseases.

Tracking of CD8+ effector T cells in intact lung: analysis of environmental guidance (CAM4P.151)

Abstract Effector T lymphocytes patrolling peripheral tissues protect organisms against infection and cancer. Recent two-photon imaging studies have revealed that T cells move with high speed through the interstitium of inflammatory tissues. We are interested in assessing migration strategies of tissue-infiltrating T cells and determining how T cell motility affects the efficiency of target search. We perform two-photon imaging using a murine endotoxin lung model to define the rules that govern motility of CD8+ effector T cells. We track individual T cells moving in intact lung for up to three hours but we do not find any evidence for long-range guidance cues for T cells, consistent with observations in other environments. Nevertheless, computational analysis of trajectories with an automated image-analysis algorithm reveals a significant alignment of T cells moving in conjunction with lung-associated vessels. Our results suggest that the architecture of the lung actively guides the migration of effector T cells and may act to limit their movement within tissues. Long term tracking of T cells moving in live tissue can provide a better understanding of the underlying mechanisms driving the search-efficiency of T cells during the effector phase of an immune response.

Azithromycin inhibits IL-1 secretion and non-canonical inflammasome activation (VAC9P.1110)

Abstract Deregulation of inflammasome activation was recently identified to be involved in the pathogenesis of various inflammatory diseases. Although macrolide antibiotics have well-established immunomodulatory properties probably involved in their clinical effects, studies on their impact on this pathway are missing. Comparing the influence of macrolides on cytokine induction in human monocytes, we found that azithromycin, but not clarithromycin or roxithromycin, specifically inhibited IL-1β secretion upon LPS stimulation whereas no effects were observed after flagellin stimulation. Blockage of azithromycin’s transmembrane transport abolished the cytokine-modulatory effect, indicating intracellular azithromycin accumulation to be causative for diverging effects. Consistent with IL-1β inhibition restricted to LPS stimulation, we found impaired induction of the intracellular LPS-sensing caspase-4 whereas NF-κB signalling was unaffected. Substantiating our in vitro data, azithromycin specifically dampened IL-1β levels and enhanced survival in a murine endotoxin sepsis model. Overall, we provide first evidence of a differential impact of macrolides on the inflammasome/IL-1β axis, which might be of relevance in inflammasome driven diseases like COPD or asthma.

Human anti-microbial cathelicidin peptide LL-37 suppresses the LPS-induced apoptosis of endothelial cells

Sepsis is a systemic disease resulting from harmful host response to bacterial infections. During the exacerbation of severe sepsis or septic shock, apoptosis of endothelial cells is induced in susceptible organs such as the lung and liver and triggers microcirculatory disorder and organ dysfunction. LPS, an outer membrane component of Gram-negative bacteria, is one of the major virulence factors for the pathogenesis. We previously reported that LL-37, a human anti-microbial cathelicidin peptide, potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. However, the effect of LL-37 on the LPS-induced endothelial cell apoptosis remains to be clarified. In this study, to further elucidate the action of LL-37 on severe sepsis/endotoxin shock, we investigated the effects of LL-37 on the LPS-induced endothelial cell apoptosis in vitro and in vivo using lung-derived normal human microvascular blood vessel endothelial cells (HMVEC-LBls) and D-galactosamine hydrochloride (D-GalN)-sensitized murine endotoxin shock model. LL-37 suppressed the LPS-induced apoptosis of HMVEC-LBls. In addition, LL-37 inhibited the binding of LPS possibly to the LPS receptors (CD14 and toll-like receptor 4) expressed on the cells. Thus, LL-37 can suppress the LPS-induced apoptosis of HMVEC-LBls via the inhibition of LPS binding to the cells. Furthermore, LL-37 drastically suppressed the apoptosis of hepatic endothelial cells as well as hepatocytes in the liver of murine endotoxin shock model. Together, these observations suggest that LL-37 could suppress the LPS-induced apoptosis of endothelial cells, thereby attenuating lethal sepsis/endotoxin shock.

Antimicrobial cathelicidin polypeptide CAP11 suppresses the production and release of septic mediators in D-galactosamine-sensitized endotoxin shock mice

Endotoxin shock is a severe systemic inflammatory response that is caused by the augmented production and release of septic mediators. Among them, inflammatory cytokines such as tumor necrosis factor-alpha, IL-1beta and IL-6 play a pivotal role. In addition, anandamide, an endogenous cannabinoid and high-mobility group box-1 (HMGB1), a non-histone chromosomal protein has recently been recognized as members of septic mediators. We previously reported that cationic antibacterial polypeptide of 11-kDa (CAP11), an antimicrobial cathelicidin peptide (originally isolated from guinea pig neutrophils), potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. In this study, to clarify the protective mechanism of CAP11 against endotoxin shock, we evaluated the effects of CAP11 on the production and release of septic mediators in vitro and in vivo using a murine macrophage cell line RAW264.7 and a D-galactosamine-sensitized murine endotoxin shock model. LPS stimulation induced the production of inflammatory cytokines and anandamide and release of HMGB1 from RAW264.7 cells. Importantly, CAP11 suppressed the LPS-induced production and release of these mediators by RAW264.7 cells. Moreover, LPS administration enhanced the serum levels of HMGB1, anandamide and inflammatory cytokines in the endotoxin shock model. Of note, CAP11 suppressed the LPS-induced increase of these mediators in sera, and LPS binding to CD14-positive cells (peritoneal macrophages), accompanied with the increase of survival rates. Together these observations suggest that the protective action of CAP11 on endotoxin shock may be explained by its suppressive effect on the production and release of septic mediators by CD14-positive cells possibly via the inhibition of LPS binding to the targets.

P85. Nitric oxide makes the glucocorticoid treatment more efficient in a combined murine endotoxin and ovalbumin asthma model

P85. Nitric oxide makes the glucocorticoid treatment more efficient in a combined murine endotoxin and ovalbumin asthma model

An anti-endotoxin peptide that generates from the amino-terminal domain of complement regulatory protein C1 inhibitor

C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2 (18–30), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14 + cells, and suppressing production of tumor necrosis factor (TNF)-α by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2 (18–30) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2 (18–30) derived from the amino-terminal region of C1INH is anti-endotoxin.

Inhibitory Effects of 2,6-Di-O-methyl-3-O-acetyl-β-cyclodextrins with Various Degrees of Substitution of Acetyl Group on Macrophage Activation and Endotoxin Shock Induced by Lipopolysaccharide

The effects of 2,6-di-O-methyl-3-O-acetyl-β-cyclodextrins (DMA-β-CyD) with various degrees of substitution (DS) of an acetyl group of 1.5, 3.8, 6.3 and 7, which are abbreviated to DMA2-β-CyD, DMA4-β-CyD, DMA6-β-CyD and DMA7-β-CyD, respectively, on murine macrophage activation and endotoxin shock induced by lipopolysaccharide (LPS) were examined. Of four DMA-β-CyDs used in the present study, cytotoxicity of DMA-β-CyDs in RAW264.7 cells, a murine macrophage-like cell line, decreased with an increase in the DS values of DMA-β-CyD, and DMA7-β-CyD had no cytotoxicity on RAW264.7 cells up to 100 mM. DMA2-β-CyD and DMA7-β-CyD at the concentration of 5 mM had greater inhibitory effects on nitric oxide (NO) production in RAW264.7 cells stimulated with LPS than DMA4-β-CyD and DMA6-β-CyD. In addition, these inhibitory effects of DMA2-β-CyD and DMA7-β-CyD were concentration-dependent. In the in vivo study, all of the mice died within 12 h after intraperitoneal administration of the solution containing LPS and d-galactosamine. When 100 mM DMA7-β-CyD was concomitantly administered with both LPS and d-galactosamine intraperitoneally in mice, the survival rate significantly increased, but DMA4-β-CyD and DMA6-β-CyD did not. In conclusion, we revealed that DS values of DMA-β-CyDs strikingly affect not only the cytotoxic activity but also the inhibitory effects of LPS-induced NO production in RAW264.7 cells and fatality of endotoxin shock mice induced by LPS and d-galactosamine. These results suggest the potential use of DMA7-β-CyD as an antagonist of LPS-induced endotoxin shock.

Inhibitory effects of dimethylacetyl-β-cyclodextrin on lipopolysaccharide-induced macrophage activation and endotoxin shock in mice

The potential use of hydrophilic cyclodextrins (CyDs) as an inhibitor for lipopolysaccharide (LPS) was examined. Of the five CyDs used in this study, dimethylacetyl-β-cyclodextrin (DMA7-β-CyD) had greater inhibitory activity than other CyDs against the production of nitric oxide (NO) and various proinflammatory cytokines including tumor necrosis factor-α (TNF-α) in murine macrophages stimulated with two serotypes of LPS and lipid A. The inhibitory effect of DMA7-β-CyD on NO production was also observed in macrophages stimulated with lipoteichoic acid (LTA), but not peptidoglycan (PGN), polyinosinic–polycytidylic acid (poly I:C) or CpG oligonucleotide (CpG-ODN). Several studies have suggested that the inhibitory effects of DMA7-β-CyD could be ascribed to the interaction with LPS. Simultaneous administration of DMA7-β-CyD not only intraperitoneally but also intravenously and intraperitoneal injection of aqueous solution containing LPS and d-galactosamine in murine endotoxin shock model suppressed fatality. Also, DMA7-β-CyD decreased blood level of TNF-α as well as serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) in mice. In conclusion, DMA7-β-CyD may have promise as a new therapeutic agent for endotoxin shock induced by LPS.

Augmentation of the lipopolysaccharide-neutralizing activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by replacement with hydrophobic and cationic amino acid residues.

Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading microorganisms. Among these peptides, human cathelicidin CAP18/LL-37 (L(1) to S(37)) possesses not only potent antibacterial activity against gram-positive and gram-negative bacteria but also the ability to bind to gram-negative lipopolysaccharide (LPS) and neutralize its biological activities. In this study, to develop peptide derivatives with improved LPS-neutralizing activities, we utilized an 18-mer peptide (K(15) to V(32)) of LL-37 as a template and evaluated the activities of modified peptides by using the CD14(+) murine macrophage cell line RAW 264.7 and the murine endotoxin shock model. By replacement of E(16) and K(25) with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further replacement of Q(22), D(26), and N(30) with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK displayed the most powerful LPS-neutralizing activity: it was most potent at binding to LPS, inhibiting the interaction between LPS and LPS-binding protein, and attaching to the CD14 molecule, thereby suppressing the binding of LPS to CD14(+) cells and attenuating production of tumor necrosis factor alpha (TNF-alpha) by these cells. Furthermore, in the murine endotoxin shock model, 18-mer LLKKK most effectively suppressed LPS-induced TNF-alpha production and protected mice from lethal endotoxin shock. Together, these observations indicate that the LPS-neutralizing activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and that 18-mer LLKKK is the most potent of the peptide derivatives, with therapeutic potential for gram-negative bacterial endotoxin shock.

A novel anti-fibrotic agent pirfenidone suppresses tumor necrosis factor-α at the translational level

A new experimental drug pirfenidone (5-methyl-1-phenyl-2-1 H-pyridine-2-one) has been reported to have beneficial effects for the treatment of certain fibrotic diseases. Here, we studied the anti-inflammatory activities of pirfenidone by investigating the mechanism of its inhibitory effect on cytokine production. In RAW264.7 cells, a murine macrophage-like cell line, pirfenidone suppressed the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by a translational mechanism, which was independent of activation of the mitogen-activated protain kinase (MAPK) 2, p38 MAP kinase, and c- Jun N-terminal kinase (JNK). In the murine endotoxin shock model, pirfenidone potently inhibited the production of the proinflammatory cytokines, TNF-α, interferon-γ, and interleukin-6, but enhanced the production of the anti-inflammatory cytokine, interleukin-10. The in vivo model also showed that pirfenidone suppressed the cytokine production by a translational mechanism, though interleukin-10 transcription was activated by pirfenidone. These findings show that pirfenidone inhibits the production of the proinflammatory cytokine selectively at the translational level. Therefore, cytokine inhibitory activities play an important role in the anti-inflammatory activities of pirfenidone. Coupled with the fact that this inhibitory effect is selective, translational, and not for total protein synthesis, this drug may have a clinical effect on inflammation and fibrosis with very low toxicity.

LPS-neutralizing activities of cathelicidin family of antibacterial polypeptides LL-37 and CAP 11

Mammalian myeloid and epithelial cells express several kinds of antibacterial peptides (α-/β-defensins and cathelicidins), which contribute to the innate host defense by killing invaded microorganisms. Recently, we have evaluated the lipopolysaccharide (LPS) -neutralizing activities of cathelicidin peptides human LL-37 and guinea pig CAP 11, using CD 14+ murine macrophage cell line RAW 264.7 and murine endotoxin shock model. LL-37 and CAP 11 inhibited the binding of LPS to RAW 264.7 cells, and suppressed the LPS-induced TNF-α mRNA and protein expression by RAW 264.7 cells. Interestingly, LL-37 and CAP 11 possessed the LPS-binding activities, and they strongly suppressed the interaction of LPS with LPS-binding protein (LBP) that mediates the transport of LPS to CD 14. Moreover, LL 37 and CAP 11 bound to the cell surface CD 14 and inhibited LPS-binding to the cells. Furthermore, in murine endotoxin shock model, LL-37-or CAP 11 -administration inhibited the binding of LPS to CD 14+ cells (peritoneal macrophages) and suppressed the LPS-induced TNF-α expression by these cells. Together these observations indicate that cathelicidin peptides LL 37 and CAP 11 likely exert the protective actions in endotoxin shock by blocking the binding of LPS to CD 14+ cells, thereby suppressing the production of cytokines by these cells via their potent binding activities for LPS and CD 14.