Lipopolysaccharide Endotoxemia Research Articles

Lipopolysaccharide, arbiter of the gut-liver axis, modulates hepatic cell pathophysiology in alcoholism.

Over the last four decades, clinical research and experimental studies have established that lipopolysaccharide (LPS)-a component of the outer membrane of gram-negative bacteria-is a potent hepatotoxic molecule in humans and animals. Alcohol abuse is commonly associated with LPS endotoxemia. This review highlights LPS molecular structures and modes of release from bacteria, plasma LPS concentrations, induction of microbiota dysbiosis, disruption of gut epithelial barrier, and translocation of LPS into the portal circulation impacting the pathophysiology of hepatic cells via the gut-liver axis. We describe and illustrate the portal vein circulation and its distributaries draining the gastrointestinal tract. We also elaborate on the gut-liver axis coupled with enterohepatic circulation that represents a bidirectional communication between the gut and liver. The review also updates the data on how circulating LPS is cleared in a coordinated effort between Kupffer cells, hepatocytes, and liver sinusoidal endothelial cells. Significantly, the article reviews and updates the modes/mechanisms of action by which LPS mediates the diverse pathophysiology of Kupffer cells, hepatocytes, sinusoidal endothelial cells, and hepatic stellate cells primarily in association with alcohol consumption. Specifically, we review the intricate linkages between ethanol, microbiota dysbiosis, LPS production, gut-liver axis, and pathophysiology of various hepatic cells. The maintenance of the gut barrier structural and functional integrity and microbiome homeostasis is essential in mitigating alcoholic liver disease and improving liver health.

Myeloid-specific ferritin light chain deletion does not exacerbate sepsis-associated AKI.

Sepsis-associated acute kidney injury (SA-AKI) is a key contributor to the life-threatening sequelae attributed to sepsis. Mechanistically, SA-AKI is a consequence of unabated myeloid cell activation and oxidative stress that induces tubular injury. Iron mediates inflammatory pathways directly and through regulating the expression of myeloid-derived ferritin, an iron storage protein comprising ferritin light (FtL) and ferritin heavy chain (FtH) subunits. Previous work revealed that myeloid FtH deletion leads to a compensatory increase in intracellular and circulating FtL and is associated with amelioration of SA-AKI. We designed this study to test the hypothesis that loss of myeloid FtL and subsequently, circulating FtL will exacerbate the sepsis-induced inflammatory response and worsen SA-AKI. We generated a novel myeloid-specific FtL knockout mouse (FtLLysM-/-) and induced sepsis via cecal ligation and puncture or lipopolysaccharide endotoxemia. As expected, serum ferritin levels were significantly lower in the knockout mice, suggesting that myeloid cells dominantly contribute to circulating ferritin. Interestingly, although sepsis induction led to a marked production of pro- and anti-inflammatory cytokines, there was no statistical difference between the genotypes. There was a similar loss of kidney function, as evidenced by a rise in serum creatinine and cystatin C and renal injury identified by expression of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Finally, RNA sequencing revealed upregulation of pathways for cell cycle arrest and autophagy postsepsis, but no significant differences were observed between genotypes, including in key genes associated with ferroptosis, an iron-mediated form of cell death. The loss of FtL did not impact sepsis-mediated activation of NF-κB or HIF-1a signaling, key inflammatory pathways associated with dysregulated host response. Taken together, while FtL overexpression was shown to be protective against sepsis, the loss of FtL did not influence sepsis pathogenesis.NEW & NOTEWORTHY Hyperferritinemia in sepsis is often associated with a proinflammatory phenotype and poor prognosis. We previously showed the myeloid deletion of FtH results in a compensatory increase in FtL and is associated with reduced circulating cytokines and decreased rates of SA-AKI in animal sepsis models. Here, we show that myeloid deletion of FtL does not impact the severity of SA-AKI following CLP or LPS, suggesting that FtH plays the predominant role in propagating myeloid-induced proinflammatory pathways.

Platelet Factor 4 Enhances Antimicrobial Function of the Endothelium and Improves Outcome in a Murine Model of Sepsis

Introduction: In this study, we investigated mechanism(s) by which the platelet-specific chemokine platelet factor 4 (PF4) interacts with bacteria to enhance host-defense function and improve outcome in sepsis. Sepsis, a life-threatening dysregulated response to infection, is the leading cause of mortality worldwide for which there are no targeted treatments. Upon sensing pathogens, platelets release high concentrations of PF4, a positively charged chemokine with high affinity for polyanions including polymers in the bacterial cell wall, the endothelial glycocalyx and cell-free DNA. We previously found that PF4 improves outcomes in the murine lipopolysaccharide endotoxemia and enhances in vitro bacterial capture by neutrophil extracellular traps. We now show that PF4-coating of bacteria reduces coagulopathy, limits bacterial dissemination, and improves survival in a murine model of polymicrobial sepsis, in part through the novel mechanism of enhancing the antimicrobial function of endothelial cells (ECs). Methods: Effects of PF4 on bacterial internalization by ECs was assessed by incubating heat-inactivated Staphylococcus (S) aureus conjugated to pHrodo, a dye that only fluoresces following cellular internalization, with PF4 (0-100mg/mL) and then expose those complexes to human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor a (TNFa) to simulate inflammation. Internalized S aureus fluorescent signal was visualized 16hr post-exposure. HUVECs were stained for von Willebrand factor (VWF) release to indicate bacteria-induced endothelial activation. Effects of PF4 on EC killing of bacteria was studied by exposing HUVECs to live Escherichia (E) coli pre-treated with PF4 (0-25mg/mL) for 1hr. ECs were washed to remove excess unbound bacteria, and cell lysates were plated on agar plates for 24hr to count colony forming units (CFUs). To investigate the effects of PF4-bacteria interactions on sepsis survival outcomes, contents from the cecum (cecal slurry, CS) of C57BL6 donor mice (100mg/mL) were pre-treated with PF4 (100mg) for 20min and injected intravenously (IV) into wildtype (WT) or PF4 -/-recipient mice to induce immediate bacteremia. Animals were evaluated for sepsis severity for up to 72hr, and survival analysis was performed using the Mantel-Cox test. A separate cohort of WT mice was given IV CS (400mg/mL) pre-incubated with PF4 (8mg). Blood was drawn at 3 to 24hr post-CS infusion and subjected to complete blood count. Thrombin anti-thrombin (TAT) and VWF levels were measured by ELISAs in plasma as indicators of intravascular coagulopathy and endothelial activation. To assess bacterial load, blood and liver and spleen homogenates were plated on agar plates to count for CFUs. Results: PF4 coating of bacteria enhanced bacterial uptake by HUVECs, peaking at 20-50 µg/mL of PF4. TNFa-stimulated HUVECs more readily internalized PF4-coated bacteria at 1-20 µg/mL of PF4. Although PF4 promoted S aureus uptake, it limited bacteria-induced VWF secretion by HUVECs. PF4 also enhanced EC killing of E coli upon bacterial internalization. In the CS model of murine polymicrobial sepsis, PF4 prevented CS-induced mortality in both WT and PF4 -/- mice ( Fig. 1). PF4-pretreatment of CS also reduced leukopenia and thrombocytopenia 3-6hr post-CS challenge in WT mice. Mice given PF4-treated CS exhibited lower TAT and VWF levels as compared to untreated-CS animals. Lastly, PF4 reduced bacterial load in blood, liver, and spleen 24hr post-CS inoculation ( Fig. 2). Conclusion: PF4 enhances bacterial uptake and killing by the endothelium in vitro, promotes bacterial clearance while preventing bacteria-induced coagulopathy and endothelial dysfunction leading to improved survival in the murine CS model of polymicrobial sepsis. These studies suggest that PF4 binding to bacteria may be an important host defense mechanism. Further studies are underway to define the contribution of EC antimicrobial activity to the observed outcomes. The ability of PF4-based therapeutics to enhance clearance of microbes merits further translational studies in sepsis.

Resolvin D5 (RvD5) Reduces Renal Damage Caused by LPS Endotoxemia in Female Mice.

In self-revolving gram-negative Escherichia coli infection, Resolvin D5 (RvD5) was found to enhance bacteria phagocytosis and reduce the production of inflammatory mediators, contributing to the resolution of infection. LPS (lipopolysaccharide) is a gram-negative bacterial structure product which activates the immune system and, at high doses, leads to endotoxemia. To our knowledge, the effect of RvD5 against LPS endotoxemia has not been investigated to date. Female Swiss mice received an i.p. treatment with RvD5 (0.1, 1 or 10 ng/animal). After 1 h, they were stimulated with LPS (10 mg/kg, i.v.), and samples were collected after additional 6 h. The resulting data demonstrated that RvD5 protected the kidneys (urea and creatinine serum levels) from tissue injury. These effects were related to an improvement in histopathological parameters and a reduction of enzymatic markers of leukocyte infiltration, pro-inflammatory cytokine (IL-1β, TNF-α, and IL-6) production, and oxidative stress. Antioxidant markers were also increased by RvD5, but IL-10 (an anti-inflammatory cytokine) levels were unaltered. We also observed that RvD5 reduced the infiltration of CD45+ hematopoietic cells into the kidneys, reduced the activation of NFκB and promoted the Nrf2 pathway by reducing Keap-1 levels. Our data indicate that RvD5 may be a therapeutic possibility to reduce kidney lesions in LPS endotoxemia.

Amelioration of Non-Alcoholic Steatohepatitis by Atractylodes macrocephala Polysaccharide, Chlorogenic Acid, and Geniposide Combination Is Associated With Reducing Endotoxin Gut Leakage.

Gut-derived lipopolysaccharide (LPS) leaking through the dysfunctional intestinal barrier contributes to the onset of non-alcoholic steatohepatitis (NASH) by triggering inflammation in the liver. In the present study, a combination consisting of Atractylodes macrocephala polysaccharide (A), chlorogenic acid (C), and geniposide (G) (together, ACG), was shown to ameliorate NASH in mice and reduce hepatic LPS signaling and endotoxemia without decreasing the abundance of identified Gram-negative bacteria through restoring the intestinal tight junctions. Our data indicated that inhibition of LPS gut leakage by the ACG combination contributed to its amelioration of NASH.

Hepatic pannexin‐1 controls interleukin‐33 synthesis and release via the ATP‐P2X7 pathway with clinical implications for the resolution of hyperinflammation in sepsis

Pannexin-1-dependent differences in hepatic damage and resolution of inflammation during lipopolysaccharide endotoxemia as described in Wang et al.7

Effects of pre-treatment with inulin on the kidney in lipopolysaccharide-induced endotoxemia in rats.

This study was aimed to evaluate the effects of inulin used as prebiotic on the kidney in lipopolysaccharide (LPS)-induced endotoxemia model. Wistar Albino rats were divided into four groups: Control group, LPS (endotoxemia) group, Inulin + LPS group in which LPS (1.5 mg/kg, E. coli, Serotype 0111: B4) was treated after inulin (500 mg/kg) given by gavage for 21 days and Inulin group. The animals were sacrificed 24 h after the last LPS injection. Kidney samples were taken for biochemical and immunohistochemical analyses. Total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), malondialdehyde (MDA) and myeloperoxidase (MPO) values were determined. In addition, kidney sections were stained for inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and interleukine-6 (IL-6) expression, and leukocyte infiltration. LPS caused oxidative stress and inflammation. Inulin administration could prevent oxidative stress and lipid peroxidation. Moreover, inulin decreased iNOS, TNF-α and IL-6 expression. However, it did not change the distribution of leukocytes in kidney tissues. These results suggest to promising benefits of inulin as prebiotic in reducing the effects of endotoxemia. Further studies should be conducted to evaluate the capacity of prebiotics in endotoxemia.

Immunotherapy-on-Chip Against an Experimental Sepsis Model.

Lipopolysaccharide (LPS) is commonly used in murine sepsis models, which are largely associated with immunosuppression and collapse of the immune system. After adapting the LPS treatment to the needs of locally bred BALB/c mice, the present study explored the protective role of Micrococcus luteus peptidoglycan (PG)-pre-activated vaccine-on-chip technology in endotoxemia. The established protocol consisted of five daily intraperitoneal injections of 0.2μg/g LPS, allowing longer survival, necessary for a therapeutic treatment application. A novel immunotherapy technology, the so-called vaccine-on-chip, consists of a 3-dimensional laser micro-textured silicon (Si) scaffold loaded with macrophages and activated in vitro with 1μg/ml PG, which has been previously shown to exert a mild immunostimulatory activity upon subcutaneous implantation. The LPS treatment significantly decreased CD4 + and CD8 + cells, while increasing CD11b + , Gr1 + , CD25 + , Foxp3 + , and class II + cells. These results were accompanied by increased arginase-1 activity in spleen cell lysates and C-reactive protein (CRP), procalcitonin (PCT), IL-6, TNF-a, IL-10, and IL-18 in the serum, while acquiring severe sepsis phenotype as defined by the murine sepsis scoring. The in vivo application of PG pre-activated implant significantly increased the percentage of CD4 + and CD8 + cells, while decreasing the percentage of Gr1 + , CD25 + , CD11b + , Foxp3 + cells, and arginase-1 activity in the spleen of LPS-treated animals, as well as all serum markers tested, allowing survival and rescuing the severity of sepsis phenotype. In conclusion, these results reveal a novel immunotherapy technology based on PG pre-activated micro-texture Si scaffolds in LPS endotoxemia, supporting thus its potential use in the treatment of septic patients.

Microbiome and osteoarthritis: New insights from animal and human studies.

Osteoarthritis (OA) is a common cause of disability, especially among the elderly. With an ageing and increasingly obese population, OA will become more prevalent. Obesity and metabolic syndrome are risk factors for OA and have been implicated in its pathogenesis. The gut microbiome may shed light on this possible common pathogenesis. Recent animal and human studies have gained important insights into the relationship between OA, obesity, and the gut microbiome. Animal studies have demonstrated links between obesity and increased severity of OA and altered gut microbial DNA profile. Use of prebiotics and probiotics in animal trials provides proof-of-concept that interventional options to the gut microbiome can modulate the progression of OA favorably. Current evidence in human studies is limited. Shifts in gut microbial profile and reduced gut microbial diversity have been associated with people with OA, as well as blood and synovial fluid lipopolysaccharide endotoxemia. Linkages between microbiome dysbiosis and host responses may help in the understanding of OA pathogenesis and the discovery of therapeutic targets. This narrative review provides a summary of up-to-date animal and human studies on the gut microbiome and its link with OA.

Mitofusin 2 in Macrophages Links Mitochondrial ROS Production, Cytokine Release, Phagocytosis, Autophagy, and Bactericidal Activity

Mitofusin 2 (Mfn2) plays a major role in mitochondrial fusion and in the maintenance of mitochondria-endoplasmic reticulum contact sites. Given that macrophages play a major role in inflammation, we studied the contribution of Mfn2 to the activity of these cells. Pro-inflammatory stimuli such as lipopolysaccharide (LPS) induced Mfn2 expression. The use of the Mfn2 and Mfn1 myeloid-conditional knockout (KO) mouse models reveals that Mfn2 but not Mfn1 is required for the adaptation of mitochondrial respiration to stress conditions and for the production of reactive oxygen species (ROS) upon pro-inflammatory activation. Mfn2 deficiency specifically impairs the production of pro-inflammatory cytokines and nitric oxide. In addition, the lack of Mfn2 but not Mfn1 is associated with dysfunctional autophagy, apoptosis, phagocytosis, and antigen processing. Mfn2floxed;CreLysM mice fail to be protected from Listeria, Mycobacterium tuberculosis, or LPS endotoxemia. These results reveal an unexpected contribution of Mfn2 to ROS production and inflammation in macrophages.

Physical Exercise Exacerbates Acute Kidney Injury Induced by LPS via Toll-Like Receptor 4.

Introduction: Lipopolysaccharide (LPS) is a systemic response-triggering endotoxin, which has the kidney as one of its first targets, thus causing acute injuries to this organ. Physical exercise is capable of promoting physiological alterations and modulating inflammatory responses in the infectious process through multiple parameters, including the toll-like receptor (TLR)-4 pathway, which is the main LPS signaling in sepsis. Additionally, previous studies have shown that physical exercise can be both a protector factor and an aggravating factor for some kidney diseases. This study aims at analyzing whether physical exercise before the induction of LPS endotoxemia can protect kidneys from acute kidney injury.Methods: C57BL/6J male mice, 12 weeks old, were distributed into four groups: (1) sedentary (control, N = 7); (2) sedentary + LPS (N = 7); (3) trained (N = 7); and (4) trained + LPS (N = 7). In the training groups, the animals exercised 5×/week in a treadmill, 60 min/day, for 4 weeks (60% of max. velocity). Sepsis was induced in the training group by the application of a single dose of LPS (5 mg/kg i.p.). Sedentary animals received LPS on the same day, and the non-LPS groups received a saline solution instead. All animals were euthanized 24 h after the administration of LPS or saline.Results: The groups receiving LPS presented a significant increase in serum urea (p < 0.0001) and creatinine (p < 0.001) concentration and renal gene expression of inflammatory markers, such as tumor necrosis factor alpha and interleukin-6, as well as TLRs. In addition, LPS promoted a decrease in reduced glutathione. Compared to the sedentary + LPS group, trained + LPS showed overexpression of a gene related to kidney injury (NGAL, p < 0.01) and the protein levels of LPS receptor TLR-4 (p < 0.01). Trained + LPS animals showed an expansion of the tubulointerstitial space in the kidney (p < 0.05) and a decrease in the gene expression of hepatic AOAH (p < 0.01), an enzyme involved in LPS clearance.Conclusion: In contrast to our hypothesis, training was unable to mitigate the renal inflammatory response caused by LPS. On the contrary, it seems to enhance injury by accentuating endotoxin-induced TLR-4 signaling. This effect could be partly due to the modulation of a hepatic enzyme that detoxifies LPS.

Protection Against Lipopolysaccharide-Induced Immunosuppression by IgG and IgM.

Lipopolysaccharide (LPS) is commonly used in murine sepsis models, which are largely associated with immunosuppression and collapse of the immune system. After adapting the LPS treatment to the needs of locally bred BALB/c mice, the present study explored the potential role of IgG and IgM in reversing LPS endotoxemia. The established protocol consisted of five daily intraperitoneal injections of 0.2 μg/g LPS, which was tolerable by half of the manipulated animals. Such a protocol allowed longer survival, necessary in the prospect of therapeutic treatment application. This treatment significantly decreased CD4+, CD8+, CD3z+, and CD19+ cells, while increasing myeloid-derived suppressor cells (MDSCs; CD11b+Gr1+), CD25+ and Foxp3+ cells. These results were accompanied by increased arginase-1 activity in spleen cell lysates and production of IL-6, TNF-α, IL-18, and C-reactive protein (CRP) in the serum. The applied LPS protocol did not alter serum procalcitonin levels. MDSCs isolated from the spleen of LPS-treated animals (LPS-MDSCs) decreased proliferation of naive T cells in coculture experiments. The application of IgG and IgM to the naive T cell/LPS-MDSCs cocultures significantly decreased CD25+, Foxp3+, and CD3z+ cells, indicating an anti-suppressive effect of immunoglobulins. The in vivo application of IgG and IgM significantly decreased the percent of CD11b+Gr1+, CD25+, Foxp3+ cells, and arginase-1 activity in the spleen of LPS-treated animals, while decreasing IL-6, TNF-α, and CRP levels in the serum, allowing survival to all animals tested. In conclusion, these results reveal a novel mode of action of IgG/IgM in LPS endotoxemia, strengthening thus the use of immunoglobulin treatment is septic patients.

Endotoxemia-mediated inflammation potentiates aminoglycoside-induced ototoxicity.

The ototoxic aminoglycoside antibiotics are essential to treat severe bacterial infections, particularly in neonatal intensive care units. Using a bacterial lipopolysaccharide (LPS) experimental model of sepsis, we tested whether LPS-mediated inflammation potentiates cochlear uptake of aminoglycosides and permanent hearing loss in mice. Using confocal microscopy and enzyme-linked immunosorbent assays, we found that low-dose LPS (endotoxemia) greatly increased cochlear concentrations of aminoglycosides and resulted in vasodilation of cochlear capillaries without inducing paracellular flux across the blood-labyrinth barrier (BLB) or elevating serum concentrations of the drug. Additionally, endotoxemia increased expression of both serum and cochlear inflammatory markers. These LPS-induced changes, classically mediated by Toll-like receptor 4 (TLR4), were attenuated in TLR4-hyporesponsive mice. Multiday dosing with aminoglycosides during chronic endotoxemia induced greater hearing threshold shifts and sensory cell loss compared to mice without endotoxemia. Thus, endotoxemia-mediated inflammation enhanced aminoglycoside trafficking across the BLB and potentiated aminoglycoside-induced ototoxicity. These data indicate that patients with severe infections are at greater risk of aminoglycoside-induced hearing loss than previously recognized.

Lymph node fibroblastic reticular cell transplants show robust therapeutic efficacy in high-mortality murine sepsis.

Sepsis is an aggressive inflammatory syndrome and a global health burden estimated to kill 7.3 million people annually. Single-target molecular therapies have not addressed the multiple disease pathways triggered by septic injury. Cell therapies might offer a broader set of mechanisms of action that benefit complex, multifocal disease processes. We describe a population of immune-specialized myofibroblasts derived from lymph node tissue, termed fibroblastic reticular cells (FRCs). Because FRCs have an immunoregulatory function in lymph nodes, we hypothesized that ex vivo-expanded FRCs would control inflammation when administered therapeutically. Indeed, a single injection of ex vivo-expanded allogeneic FRCs reduced mortality in mouse models of sepsis when administered at early or late time points after septic onset. Mice treated with FRCs exhibited lower local and systemic concentrations of proinflammatory cytokines and reduced bacteremia. When administered 4 hours after induction of lipopolysaccharide endotoxemia, or cecal ligation and puncture (CLP) sepsis in mice, FRCs reduced deaths by at least 70%. When administered late in disease (16 hours after CLP), FRCs still conveyed a robust survival advantage (44% survival compared to 0% for controls). FRC therapy was dependent on the metabolic activity of nitric oxide synthase 2 (NOS2) as the primary molecular mechanism of drug action in the mice. Together, these data describe a new anti-inflammatory cell type and provide preclinical evidence for therapeutic efficacy in severe sepsis that warrants further translational study.

CD40 ligation by T cells downregulates macrophage TNF production by IRAK1-mediated IL-10 expression and controls innate hyperinflammation (INM9P.462)

Abstract Endotoxemia is caused by excessive inflammation, but immune system has various mechanisms to avoid collateral organ damages in endotoxemia. A handful of reports have shown that innate immune responses are suppressed by the adaptive immune system. However, the molecular mechanism by which adaptive immune cells suppress innate inflammatory responses is not clear. Here, we found that T cell-macrophage interaction prolonged survival of mice in lipopolysaccharide (LPS)-induced enodotoxemia through controlling TNF levels, demonstrated by T cell reconstitution in Rag2-deficient recipients. Downregulation of macrophage TNF expression was achieved by crosstalk between CD40 and TLR4 signaling pathways, which mediated IRAK1 nuclear translocation and its binding to the Il10 promoter in macrophages, without interfering the NFκB pathway. IL-10 is then detected by macrophages in an autocrine fashion to destabilize Tnfa mRNA, resulting in attenuated TNF levels. Simultaneous stimulation of CD40 and TLR4 signaling pathways was essential to induce IRAK1-mediated IL-10 expression; because CD40 signaling licenses IRAK1 nuclear translocation by IRAK1 sumoylation in the presence of TRAF2 and intracellular isoform of osteopontin (iOPN), while TLR4 signaling provides IRF5 as a chaperone of sumoylated IRAK1 for its nuclear translocation. Our study demonstrates a mechanistic basis of the immunosuppressive role of macrophage CD40 in LPS endotoxemia.

A Key Regulatory Role for Vav1 in Controlling Lipopolysaccharide Endotoxemia via Macrophage-Derived IL-6

Macrophages are centrally involved in the pathogenesis of acute inflammatory diseases, peritonitis, endotoxemia, and septic shock. However, the molecular mechanisms controlling such macrophage activation are incompletely understood. In this article, we provide evidence that Vav1, a member of the RhoGEF family, plays a crucial role in macrophage activation and septic endotoxemia. Vav1-deficient mice demonstrated a significantly increased susceptibility for LPS endotoxemia that could be abrogated by anti-IL-6R Ab treatment. Subsequent studies showed that Vav1-deficient macrophages display augmented production of the proinflammatory cytokine IL-6. Nuclear Vav1 was identified as a key negative regulator of macrophage-derived IL-6 production. In fact, Vav1 formed a nuclear DNA-binding complex with heat shock transcription factor 1 at the HSE2 region of the IL-6 promoter to suppress IL-6 gene transcription in macrophages. These findings provide new insights into the pathogenesis of endotoxemia and suggest new avenues for therapy.

Tyrosol exhibits negative regulatory effects on LPS response and endotoxemia

Tyrosol, a phenolic compound, was isolated from wine, olive oil and other plant-derived products. In the present study, we first investigated the negative regulatory effects of tyrosol on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that tyrosol reduced tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) secretion. This inspired us to further study the effects of tyrosol in vivo. Tyrosol significantly attenuated TNF-α, IL-1β and IL-6 production in serum from mice challenged with LPS, and consistent with the results in vitro. In the murine model of endotoxemia, mice were treated with tyrosol prior to or after LPS challenge. The results showed that tyrosol significantly increased mice survival. We further investigated signal transduction ways to determine how tyrosol works. The data revealed that tyrosol shocked LPS-induced mitogen activated protein kinases (MAPKs) and nuclear transcription factor-κB (NF-κB) signal transduction pathways in RAW 264.7 macrophages. These observations indicated that tyrosol exerted negative regulatory effects on LPS response in vitro and in vivo through suppressing NF-κB and p38/ERK MAPK signaling pathways.

In vivo profile of the human leukocyte microRNA response to endotoxemia

To gain insight into microRNAs (miRNAs) involved in the regulation of the human innate immune response, we screened for differentially expressed miRNAs in circulating leukocytes in an in vivo model of acute inflammation triggered by Escherichia coli lipopolysaccharide (LPS) infusion. Leukocyte RNA was isolated from venous blood samples obtained from healthy male volunteers before and 4 h after LPS-infusion. After fluorescence labeling, RNA samples were hybridized to microarrays containing capture probes for measuring the abundance of more than 600 human miRNAs. Target genes were predicted for differentially expressed miRNAs and then compared to changes in genome-wide expression levels, which had been established in a previous study. Data analysis revealed that five miRNAs consistently responded to LPS-infusion, four of which were down-regulated (miR-146b, miR-150, miR-342, and let-7g) and one was up-regulated (miR-143). The miR-150 and mir-342 response was confirmed by real-time PCR. By correlating to measured LPS-induced changes of the leukocyte transcriptome, we next searched for predicted target genes, whose stability might be under (co-) control by these miRNAs. We found that the rapid transcriptional activation during acute inflammation of select genes, such as the gene encoding interleukin-1 receptor-associated kinase 2 ( IRAK2) might be facilitated by decreased levels of LPS-responsive miRNAs. The increased level of miR-143 might be associated with the pronounced down-regulation of the B-cell CLL/lymphoma 2 ( BCL2) gene expression during LPS endotoxemia, and could further be involved in the translational silencing of several other predicted inflammation-related target genes. This is the first in vivo study to demonstrate relative abundance of miRNA levels in peripheral blood leukocytes during acute LPS-induced inflammation. The miRNAs and their potential target genes identified herein contribute to the understanding of the complex transcriptional program of innate immunity initiated by pathogens.

In vivo profile of the human leukocyte microRNA response to endotoxemia

To gain insight into microRNAs (miRNAs) involved in the regulation of the human innate immune response, we screened for differentially expressed miRNAs in circulating leukocytes in an in vivo model of acute inflammation triggered by Escherichia coli lipopolysaccharide (LPS) infusion. Leukocyte RNA was isolated from venous blood samples obtained from healthy male volunteers before and 4h after LPS-infusion. After fluorescence labeling, RNA samples were hybridized to microarrays containing capture probes for measuring the abundance of more than 600 human miRNAs. Target genes were predicted for differentially expressed miRNAs and then compared to changes in genome-wide expression levels, which had been established in a previous study. Data analysis revealed that five miRNAs consistently responded to LPS-infusion, four of which were down-regulated (miR-146b, miR-150, miR-342, and let-7g) and one was up-regulated (miR-143). The miR-150 and mir-342 response was confirmed by real-time PCR. By correlating to measured LPS-induced changes of the leukocyte transcriptome, we next searched for predicted target genes, whose stability might be under (co-) control by these miRNAs. We found that the rapid transcriptional activation during acute inflammation of select genes, such as the gene encoding interleukin-1 receptor-associated kinase 2 (IRAK2) might be facilitated by decreased levels of LPS-responsive miRNAs. The increased level of miR-143 might be associated with the pronounced down-regulation of the B-cell CLL/lymphoma 2 (BCL2) gene expression during LPS endotoxemia, and could further be involved in the translational silencing of several other predicted inflammation-related target genes. This is the first in vivo study to demonstrate relative abundance of miRNA levels in peripheral blood leukocytes during acute LPS-induced inflammation. The miRNAs and their potential target genes identified herein contribute to the understanding of the complex transcriptional program of innate immunity initiated by pathogens.

Oral insulin up-regulates Toll-like receptor 4 expression and enhances intestinal recovery following lipopolysaccharide-induced gut injury in a rat.

In the present study, we evaluated the protective effect of oral insulin (OI) on intestinal mucosa following lipopolysaccharide-induced intestinal damage in a rat. Male Sprague-Dawley rats were divided into three experimental groups: Sham rats, LPS-rats that were treated with lipopolysaccharide (LPS), and LPS-INS rats that were treated with OI given in drinking water 72 h before and following injection of LPS. Intestinal structural changes, enterocyte proliferation, enterocyte apoptosis, and mucosal expression of Toll-like receptor 4 (TLR4) were determined 24 h after the last LPS injection. LPS-INS animals showed a significantly greater bowel and mucosal weight in jejunum and ileum, mucosal DNA and protein in jejunum and ileum, villus height in ileum, crypt depth in jejunum and ileum, cell proliferation rates in jejunum, and significantly lower apoptotic index in ileum compared to LPS- animals. LPS rats demonstrated 50% increase in TLR4 expression in jejunum compared to sham animals. Treatment with OI resulted in a three-fold increase in TLR4 expression in jejunum, compared to LPS animals. In conclusion, OI improves intestinal recovery after LPS endotoxemia in a rat.