FBXO11 governs macrophage cell death and inflammation in response to bacterial toxins.

Staphylococcus aureus causes severe infections such as pneumonia and sepsis depending on the pore-forming toxin Panton-Valentine leukocidin (PVL). PVL kills and induces inflammation in macrophages and other myeloid cells by interacting with the human cell surface receptor, complement 5a receptor 1 (C5aR1). C5aR1 expression is tighly regulated and may thus modulate PVL activity, although the mechanisms involved remain incompletely understood. Here, we used a genome-wide CRISPR/Cas9 screen and identified F-box protein 11 (FBXO11), an E3 ubiquitin ligase complex member, to promote PVL toxicity. Genetic deletion of FBXO11 reduced the expression of C5aR1 at the mRNA level, whereas ectopic expression of C5aR1 in FBXO11-/- macrophages, or priming with LPS, restored C5aR1 expression and thereby PVL toxicity. In addition to promoting PVL-mediated killing, FBXO11 dampens secretion of IL-1ß after NLRP3 activation in response to bacterial toxins by reducing mRNA levels in a BCL-6-dependent and BCL-6-independent manner. Overall, these findings highlight that FBXO11 regulates C5aR1 and IL-1ß expression and controls macrophage cell death and inflammation following PVL exposure.

Panton-Valentine Leukocidin Induces Cytokine Release and Cytotoxicity Mediated by the C5a Receptor on Rabbit Alveolar Macrophages.

Necrotizing pneumonia caused by Panton-Valentine leukocidin (PVL)-positive community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has high mortality rates and is currently a serious clinical issue. PVL is a two-component toxin (LukS-PV and LukF-PV). It can cause necrosis in target cells by forming pores consisting of an octamer comprised of LukS-PV and LukF-PV. However, considering the specificity of PVL towards several target cells and species, the specific effect of PVL remains controversial. Therefore, we focused on necrotizing pneumonia caused by PVL-positive S. aureus and clarified the effect of PVL on alveolar macrophages, which play a central role in innate immunity in the alveolar space. We constructed recombinant PVL (rPVL) components and stimulated alveolar macrophages isolated from rabbits to evaluate cytotoxicity and pro-inflammatory cytokine release. Recombinant LukS-PV (rLukS-PV), but not recombinant LukF-PV (rLukF-PV), induced pro-inflammatory cytokine release. Specifically, tumor necrosis factor (TNF)-α release was mediated by the C5a receptor (C5aR) expressed on rabbit alveolar macrophages, and the toxicity of rPVL, consisting of rLukS-PV and rLukF-PV, towards rabbit alveolar macrophages was mediated by the same receptor. Overall, our findings shed light on the C5aR-mediated cytotoxic effect of PVL on alveolar macrophages, which may be useful for understanding the mechanism of necrotizing pneumonia caused by PVL.

Staphylococcal Panton-Valentine Leucocidin and Gamma Haemolysin Target and Lyse Mature Bone Marrow Leucocytes.

Staphylococcus aureus is a major human pathogen, inducing several infections ranging from the benign to the life-threatening, such as necrotising pneumonia. S. aureus is capable of producing a great variety of virulence factors, such as bicomponent pore-forming leucocidin, which take part in the physiopathology of staphylococcal infection. In necrotising pneumonia, Panton-Valentine leucocidin (PVL) induces not only lung injury and necrosis, but also leukopenia, regarded as a major factor of a poor prognosis. The aim of the present study was to evaluate the effect of bicomponent pore-forming leucocidin, PVL and gamma haemolysin on bone marrow leucocytes, to better understand the origin of leukopenia. Using multi-parameter cytometry, the expression of leucocidin receptors (C5aR, CXCR1, CXCR2, and CCR2) was assessed and toxin-induced lysis was measured for each bone marrow leucocyte population. We observed that PVL resulted in myeloid-derived cells lysis according to their maturation and their C5aR expression; it also induced monocytes lysis according to host susceptibility. Haemolysin gamma A, B, and C (HlgABC) displayed cytotoxicity to monocytes and natural killer cells, hypothetically through CXCR2 and CXCR1 receptors, respectively. Taken together, the data suggest that PVL and HlgABC can lyse bone marrow leucocytes. Nevertheless, the origin of leukopenia in severe staphylococcal infection is predominantly peripheral, since immature cells stay insensitive to leucocidins.

Leukotoxin of Bibersteinia trehalosi Contains a Unique Neutralizing Epitope, and a Non-Neutralizing Epitope Shared with Mannheimia haemolytica Leukotoxin.

Bibersteinia trehalosi and Mannheimia haemolytica, originally classified as Pasteurella haemolytica biotype T and biotype A, respectively, under Genus Pasteurella has now been placed under two different Genera, Bibersteinia and Mannheimia, based on DNA-DNA hybridization and 16S RNA studies. While M. haemolytica has been the predominant pathogen of pneumonia in ruminants, B. trehalosi is emerging as an important pathogen of ruminant pneumonia. Leukotoxin is the critical virulence factor of these two pathogens. While the leukotoxin of M. haemolytica has been well studied, the characterization of B. trehalosi leukotoxin has lagged behind. As the first step towards addressing this problem, we developed monoclonal antibodies (mAbs) against B. trehalosi leukotoxin and used them to characterize the leukotoxin epitopes. Two mAbs that recognized sequential epitopes on the leukotoxin were developed. One of them, AM113, neutralized B. trehalosi leukotoxin while the other, AM321, did not. The mAb AM113 revealed the existence of a neutralizing epitope on B. trehalosi leukotoxin that is not present on M. haemolytica leukotoxin. A previously developed mAb, MM601, revealed the presence of a neutralizing epitope on M. haemolytica leukotoxin that is not present on B. trehalosi leukotoxin. The mAb AM321 recognized a non-neutralizing epitope shared by the leukotoxins of B. trehalosi and M. haemolytica. The mAb AM113 should pave the way for mapping the leukotoxin-neutralizing epitope on B. trehalosi leukotoxin and the development of subunit vaccines and/or virus-vectored vaccines against this economically important respiratory pathogen of ruminants.

Receptor-Based Peptides for Inhibition of Leukotoxin Activity.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans, commonly associated with localized aggressive periodontitis (LAP), secretes an RTX (repeats-in-toxin) protein leukotoxin (LtxA) that targets human white blood cells, an interaction that is driven by its recognition of the lymphocyte function-associated antigen-1 (LFA-1) integrin. In this study, we report on the inhibition of LtxA-LFA-1 binding as an antivirulence strategy to inhibit LtxA-mediated cytotoxicity. Specifically, we designed and synthesized peptides corresponding to the reported LtxA binding domain on LFA-1 and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxic activity in human immune cells. We found that several of these peptides, corresponding to sequential ß-strands in the LtxA-binding domain of LFA-1, inhibit LtxA activity, demonstrating the effectiveness of this approach. Further investigations into the mechanism by which these peptides inhibit LtxA binding to LFA-1 reveal a correlation between toxin-peptide affinity and LtxA-mediated cytotoxicity, leading to a diminished association between LtxA and LFA-1 on the cell membrane. Our results demonstrate the possibility of using target-based peptides to inhibit LtxA activity, and we expect that a similar approach could be used to hinder the activity of other RTX toxins.

Aggregatibacter (Actinobacillus) actimycetemcomitans leukotoxin and human periodontitis - A historic review with emphasis on JP2.

Aggregatibacter (Actinobacillus) actimycetemcomitans (Aa) is a gram-negative bacterium that colonizes the human oral cavity and is causative agent for localized aggressive (juvenile) periodontitis (AgP). In the middle of 1990s, a specific JP2 clone of belonging to the cluster of serotype b strains of Aa with highly leukotoxicity (leukotoxin, LtxA) able to kill human immune cells was isolated. JP2 clone of Aa was strongly associated with in particularly in rapidly progressing forms of aggressive periodontitis. The JP2 clone of Aa is transmitted through close contacts. Therefore, AgP patients need intense monitoring of their periodontal status as the risk for developing severely progressing periodontitis lesions are relatively high. Furthermore, timely periodontal treatment, including periodontal surgery supplemented by the use of antibiotics, is warranted. More importantly, periodontal attachment loss should be prevented by early detection of the JP2 clone of Aa by microbial diagnosis testing and/or preventive means.

Exotoxins and endotoxins: Inducers of inflammatory cytokines.

Endotoxins and exotoxins are among the most potent bacterial inducers of cytokines. During infectious processes, the production of inflammatory cytokines including tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), gamma interferon (IFNγ) and chemokines orchestrates the anti-infectious innate immune response. However, an overzealous production, leading up to a cytokine storm, can be deleterious and contributes to mortality consecutive to sepsis or toxic shock syndrome. Endotoxins of Gram-negative bacteria (lipopolysaccharide, LPS) are particularly inflammatory because they generate auto-amplificatory loops after activation of monocytes/macrophages. LPS and numerous pore-forming exotoxins also activate the inflammasome, the molecular platform that allows the release of mature IL-1ß and IL-18. Among exotoxins, some behave as superantigens, and as such activate the release of cytokines by T-lymphocytes. In most cases, pre-exposure to exotoxins enhances the cytokine production induced by LPS and its lethality, whereas pre-exposure to endotoxin usually results in tolerance. In this review we recall the various steps, which, from the very early discovery of pyrogenicity induced by bacterial products, ended to the discovery of the endogenous pyrogen. Furthermore, we compare the specific characteristics of endotoxins and exotoxins in their capacity to induce inflammatory cytokines.

Influence of DPH1 and DPH5 Protein Variants on the Synthesis of Diphthamide, the Target of ADPRibosylating Toxins.

The diphthamide on eukaryotic translation elongation factor 2 (eEF2) is the target of ADPribosylating toxins and -derivatives that serve as payloads in targeted tumor therapy. Diphthamide is generated by seven DPH proteins; cells deficient in these (DPHko) lack diphthamide and are toxin-resistant. We have established assays to address the functionality of DPH1 (OVCA1) and DPH5 variants listed in dbSNP and cosmic databases: plasmids encoding wildtype and mutant DPHs were transfected into DPHko cells. Supplementation of DPH1 and DPH5 restores diphthamide synthesis and toxin sensitivity in DPH1ko and DPH5ko cells, respectively. Consequently, the determination of the diphthamide status of cells expressing DPH variants differentiates active and compromised proteins. The DPH1 frameshift variant L96fs* (with Nterminal 96 amino acids, truncated thereafter) and two splice isoforms lacking 80 or 140 amino acids at their N-termini failed to restore DPH1ko deficiency. The DPH1 frameshift variant R312fs* retained some residual activity even though it lacks a large C-terminal portion. DPH1 missense variants R27W and S56F retained activity while S221P had reduced activity, indicated by a decreased capability to restore diphthamide synthesis. The DPH5 nonsense or frameshift variants E60*, W136fs* and R207* (containing intact N-termini with truncations after 60, 136 or 207 amino acids, respectively) were inactive: none compensated the deficiency of DPH5ko cells. In contrast, missense variants D57G, G87R, S123C and Q170H as well as the frequently occurring DPH5 isoform delA212 retained activity. Sensitivity to ADP-ribosylating toxins and tumor-targeted immunotoxins depends on diphthamide which, in turn, requires DPH functionality. Because of that, DPH variants (in particular those that are functionally compromised) may serve as a biomarker and correlate with the efficacy of immunotoxin-based therapies.

Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus.

Exotoxin A (PE) from Pseudomonas aeruginosa is a bacterial ADP-ribosyltransferase, which can permanently inhibit translation in the attacked cells. Consequently, this toxin is frequently used in immunotoxins for targeted cancer therapies. In this study, we propose a novel modification to PE by incorporating the NLS sequence at its C-terminus, to make it a selective agent against fast-proliferating cancer cells, as a nucleus-accumulated toxin should be separated from its natural substrate (eEF2) in slowly dividing cells. Here, we report the cytotoxic activity and selected biochemical properties of newly designed PE mutein using two cellular models: A549 and HepG2. We also present a newly developed protocol for efficient purification of recombinant PE and its muteins with very high purity and activity. We found that furin cleavage is not critical for the activity of PE in the analyzed cell lines. Surprisingly, we observed increased toxicity of the toxin accumulated in the nucleus. This might be explained by unexpected nuclease activity of PE and its potential ability to cleave chromosomal DNA, which seems to be a putative alternative intoxication mechanism. Further experimental investigations should address this newly detected activity to identify catalytic residues and elucidate the molecular mechanism responsible for this action.

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity.

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aeruginosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process.

Inhibition of LtxA toxicity by blocking cholesterol binding with peptides.

The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans kills host immune cells, allowing the bacterium to establish an ecological niche in the upper aerodigestive tract of its human host. The interaction of LtxA with human immune cells is both complex and multifaceted, involving membrane lipids as well as cell-surface proteins. In the initial encounter with the host cell, LtxA associates with lymphocyte function-associated antigen-1, a cell surface adhesion glycoprotein. However, we have also demonstrated that the toxin associates strongly with the plasma membrane lipids, specifically cholesterol. This association with cholesterol is regulated by a cholesterol recognition amino acid consensus (CRAC) motif, with a sequence of (334) LEEYSKR(340), in the N-terminal region of the toxin. Here, we have demonstrated that removal of cholesterol from the plasma membrane or mutation of the LtxA CRAC motif inhibits the activity of the toxin in THP-1 cells. To inhibit LtxA activity, we designed a short peptide corresponding to the CRAC(336) motif of LtxA (CRAC(336WT)). This peptide binds to cholesterol and thereby inhibits the toxicity of LtxA in THP-1 cells. Previously, we showed that this peptide inhibits LtxA toxicity against Jn.9 (Jurkat) cells, indicating that peptides derived from the cholesterol-binding site of LtxA may have a potential clinical applicability in controlling infections of repeats-in-toxin-producing organisms.

Protective effects of bacterial osmoprotectant ectoine on bovine erythrocytes subjected to staphylococcal alpha-haemolysin.

Ectoine (ECT) is a bacterial compatible solute with documented protective action however no data are available on its effects on various cells against bacterial toxins. Therefore, we determined the in vitro influence of ECT on bovine erythrocytes subjected to staphylococcal α-haemolysin (HlyA). The cells exposed to HlyA alone showed a distinct haemolysis and reduced glutathione (GSH)/oxidised glutathione (GSSG) level, however the toxic effects were attenuated in the combinations of HlyA + ECT suggesting ECT-induced protection of erythrocytes from HlyA.

Staphylococcus Alpha-Toxin Action on the Rabbit Iris: Toxic Effects and Their Inhibition.

PURPOSE: Staphylococcus aureus infection of the anterior chamber can occur after cataract surgery, causing inflammation and extensive damage to the iris. Alpha-toxin, the most potent S. aureus corneal toxin, was tested as a possible mediator of damage to the iris, and alpha-toxin anti-serum and a chemical toxin inhibitor were tested as potential pathology-reducing agents. METHODS: The hemolytic activity of alpha-toxin and its inhibition by a chemical inhibitor or anti-serum were quantified in vitro. Purified alpha-toxin, heat-inactivated toxin, or alpha-toxin plus normal serum, alpha-toxin anti-serum, or the chemical inhibitor, methyl-ß-cyclodextrin-cholesterol (CD-cholesterol), was injected into the rabbit anterior chamber. Pathological changes were photographed, quantified by slit-lamp examination (SLE) scoring, and further documented by histopathological analysis. RESULTS: At five hours post-injection, eyes injected with alpha-toxin or heat-inactivated toxin had a mean SLE score of 7.3 ± 0.59 or 0.84 ± 0.19, respectively. Active toxin caused moderate to severe iris edema, severe erosion of the iris, and mild to moderate fibrin accumulation in the anterior chamber. Alpha-toxin plus anti-serum or CD-cholesterol, in contrast to alpha-toxin alone, caused less iris edema and epithelium sloughing as well as significantly lower SLE scores than eyes receiving alpha-toxin alone (p ≤ 0.019). CONCLUSION: Alpha-toxin caused extensive iris damage and inflammation, and either anti-alpha-toxin anti-serum or CD-cholesterol was able to significantly reduce toxin-mediated damage and inflammation.

Sequence diversity, cytotoxicity and antigenic similarities of the leukotoxin of isolates of Mannheimia species from mastitis in domestic sheep.

Species within the genus Mannheimia are among the most important causes of ovine mastitis. Isolates of these species can express leukotoxin A (LktA), a primary virulence factor of these bacteria. To examine the significance of variation in the LktA, the sequences of the lktA genes in a panel of isolates from cases of ovine mastitis were compared. The cross-neutralising capacities of rat antisera raised against LktA of one Mannheimia glucosida, one haemolytic Mannheimia ruminalis, and two Mannheimia haemolytica isolates were also examined to assess the effect that variation in the lktA gene can have on protective immunity against leukotoxins with differing sequences. The lktA nucleotide distance between the M. haemolytica isolates was greater than between the M. glucosida isolates, with the M. haemolytica isolates divisible into two groups based on their lktA sequences. Comparison of the topology of phylogenetic trees of 16S rDNA and lktA sequences revealed differences in the relationships between some isolates, suggesting horizontal gene transfer. Cross neutralisation data obtained with monospecific anti-LktA rat sera were used to derive antigenic similarity coefficients for LktA from the four Mannheimia species isolates. Similarity coefficients indicated that LktA of the two M. haemolytica isolates were least similar, while LktA from M. glucosida was most similar to those for one of the M. haemolytica isolates and the haemolytic M. ruminalis isolate. The results suggested that vaccination with the M. glucosida leukotoxin would generate the greatest cross-protection against ovine mastitis caused by Mannheimia species with these alleles.

Kineret®/IL-1ra blocks the IL-1/IL-8 inflammatory cascade during recombinant Panton Valentine Leukocidin-triggered pneumonia but not during S. aureus infection.

OBJECTIVES: Community-acquired Staphylococcus aureus necrotizing pneumonia is a life-threatening disease. Panton Valentine Leukocidin (PVL) has been associated with necrotizing pneumonia. PVL triggers inflammasome activation in human macrophages leading to IL-1ß release. IL-1ß activates lung epithelial cells to release IL-8. This study aimed to assess the relevance of this inflammatory cascade in vivo and to test the potential of an IL-1 receptor antagonist (IL-1Ra/Kineret) to decrease inflammation-mediated lung injury. METHODS: We used the sequential instillation of Heat-killed S. aureus and PVL or S. aureus infection to trigger necrotizing pneumonia in rabbits. In these models, we investigated inflammation in the presence or absence of IL-1Ra/Kineret. RESULTS: We demonstrated that the presence of PVL was associated with IL-1ß and IL-8 release in the lung. During PVL-mediated sterile pneumonia, Kineret/IL-1Ra reduced IL-8 production indicating the relevance of the PVL/IL-1/IL-8 cascade in vivo and the potential of Kineret/IL-1Ra to reduce lung inflammation. However, Kineret/IL-1Ra was ineffective in blocking IL-8 production during infection with S. aureus. Furthermore, treatment with Kineret increased the bacterial burden in the lung. CONCLUSIONS: Our data demonstrate PVL-dependent inflammasome activation during S.aureus pneumonia, indicate that IL-1 signaling controls bacterial burden in the lung and suggest that therapy aimed at targeting this pathway might be deleterious during pneumonia.

The direction and role of phenotypic transition between podocytes and parietal epithelial cells in focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a podocyte disease. Among the various histologies of FSGS, active epithelial changes, hyperplasia, as typically seen in the collapsing variant, indicates disease progression. Using a podocyte-specific injury model of FSGS carrying a genetic podocyte tag combined with double immunostaining by different sets of podocytes and parietal epithelial cell (PEC) markers [nestin/Pax8, Wilms' tumor-1 (WT1)/claudin1, and podocalyxin/Pax2], we investigated the direction of epithelial phenotypic transition and its role in FSGS. FSGS mice showed progressive proteinuria and renal dysfunction often accompanied by epithelial hyperplasia, wherein 5-bromo-4-chloro-3-indoyl ß-d-galactoside (X-gal)-positive podocyte-tagged cells were markedly decreased. The average numbers of double-positive cells in all sets of markers were significantly increased in the FSGS mice compared with the controls. In addition, the average numbers of double-positive cells for X-gal/Pax8, nestin/Pax8 and podocalyxin/Pax2 staining in the FSGS mice were comparable, whereas those of WT1/claudin1 were significantly increased. When we divided glomeruli from FSGS mice into those with FSGS lesions and those without, double-positive cells tended to be more closely associated with glomeruli without FSGS lesions compared with those with FSGS lesions. Moreover, the majority of double-positive cells appeared to be isolated and very rarely associated with FSGS lesions (1/1,997 glomeruli). This study is the first to show the incidence and localization of epithelial cells with phenotypical changes in FSGS using a genetic tag. The results suggest that the major direction of epithelial phenotypic transition in cellular FSGS is from podocytes to PECs and that these cells were less represented in the active lesions of FSGS.

Leukotoxin kills rodent WBC by targeting leukocyte function associated antigen 1.

Leukotoxin is a protein that is secreted by Aggregatibacter actinomycetemcomitans and that primarily targets the active form of leukocyte function associated antigen 1 (LFA1) on WBC. Because of its specificity for WBC, leukotoxin is being developed as a novel biologic treatment for hematologic malignancies and autoimmune-inflammatory diseases. Early studies indicated that leukotoxin is specific for WBC from humans and Old World primates. In the current study, we used in vivo and in vitro assays to show that leukotoxin has a wider host range than previously believed and can kill rodent WBC. Administration of leukotoxin to rats and mice resulted in a rapid drop in WBC number but had no effect on RBC or platelet counts. Using LFA1-knockout mice, we showed that leukotoxin-mediated depletion of WBC is dependent on LFA1. In addition, similar to its effect on human monocytes, leukotoxin kills murine myeloid leukemia via a lysosome-mediated pathway that is dependent on cathepsin D. This newly described broader host range of leukotoxin enables the biology of the protein to be studied in rodent species and offers the possibility of using rodent models for evaluating the therapeutic efficacy of leukotoxin in various diseases.

A peptide antagonist of CD28 signaling attenuates toxic shock and necrotizing soft-tissue infection induced by Streptococcus pyogenes.

Staphylococcus aureus and group A Streptococcus pyogenes (GAS) express superantigen (SAg) exotoxin proteins capable of inducing lethal shock. To induce toxicity, SAgs must bind not only to the major histocompatibility complex II molecule of antigen-presenting cells and the variable ß chain of the T-cell receptor but also to the dimer interface of the T-cell costimulatory receptor CD28. Here, we show that the CD28-mimetic peptide AB103 (originally designated "p2TA") protects mice from lethal challenge with streptococcal exotoxin A, as well as from lethal GAS bacterial infection in a murine model of necrotizing soft-tissue infection. Administration of a single dose of AB103 increased survival when given up to 5 hours after infection, reduced inflammatory cytokine expression and bacterial burden at the site of infection, and improved muscle inflammation in a dose-dependent manner, without compromising cellular and humoral immunity. Thus, AB103 merits further investigation as a potential therapeutic in SAg-mediated necrotizing soft-tissue infection.

Pretreatment with lipopolysaccharide ameliorates Pseudomonas exotoxin A-induced hepatotoxicity in rats.

CONTEXT: Liver injury can be induced by various hepatotoxicants, including Pseudomonas aeruginosa exotoxin A (PEA). Our previous study indicated that PEA-induced rat hepatotoxicity was T cells and Kupffer cells dependent. Several reports have demonstrated that non-toxic doses of bacterial lipopolysaccharide (LPS) can protect liver against the chemicals-induced toxicity such as acetaminophen and concanavalin-A. OBJECTIVE: This study aimed to investigate the protecting mechanisms of LPS on PEA-induced hepatotoxicity. RESULTS: Rats pretreated with LPS (40 µg/kg, 12 h before PEA admission) significantly decreased animal mortality, serum enzyme (ALT, AST and T-bil) activities, histopathological changes and hepatocytes apoptosis following challenge with PEA. The concentrations of tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin-2 (IL-2) were reduced, but IL-6 and IL-10 were increased in the serum. In addition, prior treatment of these LPS-pretreated rats with gadolinium chloride (GdCl3), a selective Kupffer cell depletion agent, markedly enhanced liver injury after PEA administration. In contrast, the pretreatment of LPS to T-cell deficient athymic nude rats still display significant attenuation of PEA-induced liver injury. This observation further confirmed our hypothesis that LPS ameliorate PEA-hepatotoxicity was through Kupffer cells but not T cells. Moreover, LPS-induced hepatoprotection ability was neutralized by co-treatment with anti-TNF-α antibodies, but not with anti-IFN-γ antibodies. Finally, replacement of LPS with RS-LPS (Rhodobacter sphaeroides LPS), a Toll like receptor-4 (TLR-4) antagonist, resulted in severe hepatotoxicity. CONCLUSION: These results suggested that Kupffer cells, TNF-α and TLR-4 play central mediator roles during the hepatoprotection against PEA-induced hepatotoxicity conferred by LPS.