LPS Exposure Increases Maternal Corticosterone Levels,Causes Placental Injury and Increases IL-1Β Levels in Adult Rat Offspring: Relevance to Autism

Maternal immune activation can induce neuropsychiatric disorders, such as autism and schizophrenia. Previous investigations by our group have shown that prenatal treatment of rats on gestation day 9.5 with lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally), which mimics infections by gram-negative bacteria, induced autism-like behavior in male rats, including impaired communication and socialization and induced repetitive/restricted behavior. However, the behavior of female rats was unchanged. Little is known about how LPS-induced changes in the pregnant dam subsequently affect the developing fetus and the fetal immune system. The present study evaluated the hypothalamic-pituitary-adrenal (HPA) axis activity, the placental tissue and the reproductive performance of pregnant Wistar rats exposed to LPS. In the adult offspring, we evaluated the HPA axis and pro-inflammatory cytokine levels with or without a LPS challenge. LPS exposure increased maternal serum corticosterone levels, injured placental tissue and led to higher post-implantation loss, resulting in fewer live fetuses. The HPA axis was not affected in adult offspring. However, prenatal LPS exposure increased IL-1β serum levels, revealing that prenatal LPS exposure modified the immune response to a LPS challenge in adulthood. Increased IL-1β levels have been reported in several autistic patients. Together with our previous studies, our model induced autistic-like behavioral and immune disturbances in childhood and adulthood, indicating that it is a robust rat model of autism.     

Role of pulmonary intravascular macrophages in endotoxin-induced lung inflammation and mortality in a rat model

Background

Bile-duct ligated (BDL) rats recruit pulmonary intravascular macrophages (PIMs) and are highly susceptible to endotoxin-induced mortality. The mechanisms of this enhanced susceptibility and mortality in BDL rats, which are used as a model of hepato-pulmonary syndrome, remain unknown. We tested a hypothesis that recruited PIMs promote endotoxin-induced mortality in a rat model.

Methods

Rats were subjected to BDL to induce PIM recruitment followed by treatment with gadolinium chloride (GC) to deplete PIMs. Normal and BDL rats were treated intravenously with E. coli lipopolysaccharide (LPS) with or without GC pre-treatment followed by collection and analyses of lungs for histopathology, electron microscopy and cytokine quantification.

Results

BDL rats recruited PIMs without any change in the expression of IL-1β, TNF-α and IL-10. GC caused reduction in PIMs at 48 hours post-treatment (P < 0.05). BDL rats treated intravenously with E. coli LPS died within 3 hours of the challenge while the normal LPS-treated rats were euthanized at 6 hours after the LPS treatment. GC treatment of rats 6 hours or 48 hours before LPS challenge resulted in 80% (1/5) and 100% (0/5) survival, respectively, at 6 hours post-LPS treatment. Lungs from BDL+LPS rats showed large areas of perivascular hemorrhages compared to those pre-treated with GC. Concentrations of IL-1β, TNF-α and IL-10 were increased in lungs of BDL+LPS rats compared to BDL rats treated with GC 48 hours but not 6 hours before LPS (P < 0.05).

Conclusion

We conclude that PIMs increase susceptibility for LPS-induced lung injury and mortality in this model, which is blocked by a reduction in their numbers or their inactivation.     

Role of Protein Tyrosine Phosphatase Non-Receptor Type 7 in the Regulation of TNF-α Production in RAW 264.7 Macrophages

Protein tyrosine phosphatases play key roles in a diverse range of cellular processes such as differentiation, cell proliferation, apoptosis, immunological signaling, and cytoskeletal function. Protein tyrosine phosphatase non-receptor type 7 (PTPN7), a member of the phosphatase family, specifically inactivates mitogen-activated protein kinases (MAPKs). Here, we report that PTPN7 acts as a regulator of pro-inflammatory TNF-α production in RAW 264.7 cells that are stimulated with lipopolysaccharide (LPS) that acts as an endotoxin and elicits strong immune responses in animals. Stimulation of RAW 264.7 cells with LPS leads to a transient decrease in the levels of PTPN7 mRNA and protein. The overexpression of PTPN7 inhibits LPS-stimulated production of TNF-α. In addition, small interfering RNA (siRNA) analysis showed that knock-down of PTPN7 in RAW 264.7 cells increased TNF-α production. PTPN7 has a negative regulatory function to extracellular signal regulated kinase 1/2 (ERK1/2) and p38 that increase LPS-induced TNF-α production in macrophages. Thus, our data presents PTPN7 as a negative regulator of TNF-α expression and the inflammatory response in macrophages.     

Differential Response of Primary Alveolar Type I and Type II Cells to LPS Stimulation

The alveolar epithelium serves as a barrier between organism and environment and functions as the first line of protection against potential respiratory pathogens. Alveolar type II (TII) cells have traditionally been considered the immune cells of the alveolar epithelium, as they possess immunomodulatory functions; however, the precise role of alveolar type I (TI) cells, which comprise ∼95% of the alveolar epithelial surface area, in lung immunity is not clear. We sought to determine if there was a difference in the response of TI and TII cells to lung injury and if TI cells could actively participate in the alveolar immune response. TI cells isolated via fluorescence activated cell sorting (FACS) from LPS-injured rats demonstrated greater fold-induction of multiple inflammatory mediators than TII cells isolated in the same manner from the same animals. Levels of the cytokines TNF-α, IL-6 and IL-1β from cultured primary rat TI cells after LPS stimulation were significantly increased compared to similarly studied primary rat TII cells. We found that contrary to published reports, cultured TII cells produce relatively small amounts of TNF-α, IL-6 and IL-1β after LPS treatment; the higher levels of cytokine expression from cultured TII cells reported in the literature were likely from macrophage contamination due to traditional non-FACS TII cell isolation methods. Co-culture of TII cells with macrophages prior to LPS stimulation increased TNF-α and IL-6 production to levels reported by other investigators for TII cells, however, co-culture of TI cells and macrophages prior to LPS treatment resulted in marked increases in TNF-α and IL-6 production. Finally, exogenous surfactant blunted the IL-6 response to LPS in cultured TI cells. Taken together, these findings advocate a role for TI cells in the innate immune response and suggest that both TI and TII cells are active players in host defense mechanisms in the lung.     

S100-β aggravates spinal cord injury via activation of M1 macrophage phenotype

Objectives:S100-β has been identified as a sensitive biomarker in central nervous system injuries. However, the functions and mechanisms of S100-β are unknown in spinal cord injury.Methods:Spinal cord injury (SCI) mouse model was generated by surgical operation, microglia activation model was established by inducing BV-2 cells with LPS. The SCI model was evaluated by Basso-Beattie-Bresnahan (BBB) behavioral score, HE staining, and Nissl staining. The expression level of S100-β was detected by qRT-PCR, western blot, and immunofluorescence. qRT-PCR and western blot were used to detect the expression of iNOS and CD16. Pro-inflammatory cytokines TNF-α and IL-1β levels were detected by qRT-PCR and ELISA.Results:The expression of IL-1β, TNF-α, iNOS, and CD16 increased at 3^rd day after SCI. In BV2 microglia, LPS treatment promoted the expression of S100-β, IL-1β, TNF-α, iNOS, and CD16. Knockdown of S100-β reduced the expression of iNOS stimulated by LPS. Over-expression of S100-β increased IL-1β and TNF-α, and S100-β inhibition suppressed IL-1β and TNF-α. In SCI mice, knockdown of S100-β attenuated the spinal cord injury and inhibited the expression of iNOS, IL-1β, and TNF-α.Conclusions:Down-regulation of S100-β could inhibit the pathogenesis of SCI and inhibit the activation of M1 macrophages. S100-β may be a useful diagnostic biomarker or therapeutic target for SCI.     

Anti-Inflammatory Effects of Acupuncture Stimulation via the Vagus Nerve

Although acupuncture therapy is widely used in traditional Asian medicine for the treatment of diverse internal organ disorders, its underlying biological mechanisms are largely unknown. Here, we investigated the functional involvement of acupuncture stimulation (AS) in the regulation of inflammatory responses. TNF-α production in mouse serum, which was induced by lipopolysaccharide (LPS) administration, was decreased by manual acupuncture (MAC) at the zusanli acupoint (stomach36, ST36). In the spleen, TNF-α mRNA and protein levels were also downregulated by MAC and were recovered by using a splenic neurectomy and a vagotomy. c-Fos, which was induced in the nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMV) by LPS and electroacupuncture (EAC), was further increased by focal administration of the AMPA receptor blocker CNQX and the purinergic receptor antagonist PPADS. TNF-α levels in the spleen were decreased by CNQX and PPADS treatments, implying the involvement of inhibitory neuronal activity in the DVC. In unanesthetized animals, both MAC and EAC generated c-Fos induction in the DVC neurons. However, MAC, but not EAC, was effective in decreasing splenic TNF-α production. These results suggest that the therapeutic effects of acupuncture may be mediated through vagal modulation of inflammatory responses in internal organs.     

Chronic Endotoxemia in Subjects with Type-1 Diabetes Is Seen Much before the Onset of Microvascular Complications

Background

Lipopolysaccharide (LPS)/Endotoxin is hypothesized to play an important role in chronic inflammation associated with Type-1 diabetes (T1DM) and its complications. Endotoxin core antibodies (EndoCAb), LPS binding protein (LBP) and soluble CD14 (sCD14) act as modulators of LPS induced activation of innate immune system in vivo. For the present study we estimated the levels of LPS and its translocation markers in T1DM subjects with and without microvascular complications (MVC) and correlate them with clinical parameters of T1DM and serum inflammatory cytokine levels (TNF-α, IL-6, IL-1β and GM-CSF).

Methods

A total of 197 subjects (64 normal glucose tolerance (NGT) subjects, 97 T1DM subjects without MVC and 36 with MVC) were included in this study and the levels of serum LPS, its translocation markers and cytokines measured by immunoassays.

Results

Compared to NGT, T1DM subjects (both with and without MVC) had significantly higher levels of LPS, reduced levels of LBP and EndoCAb along with significant increase in the levels of IL-1β, IL-6, TNF-α and GM-CSF (p<0.05). No significant change was seen in the levels of these biomarkers between T1DM subjects with and without MVC.

Conclusions

Decreased levels of EndoCAb and LBP suggest sustained endotoxin activity in T1DM subjects even before the onset of microvascular complications.     

Post-Natal Inhibition of NF-κB Activation Prevents Renal Damage Caused by Prenatal LPS Exposure

Prenatal exposure to an inflammatory stimulus has been shown to cause renal damage in offspring. Our present study explored the role of intra-renal NF-κB activation in the development of progressive renal fibrosis in offspring that underwent prenatal exposure to an inflammatory stimulus. Time-dated pregnant rats were treated with saline (control group) or 0.79 mg/kg lipopolysaccharide (LPS) through intra-peritoneal injection on gestational day 8, 10 and 12. At the age of 7 weeks, offspring from control or LPS group were treated with either tap water (Con+Ve or LPS+Ve group) or pyrollidine dithiocarbamate (PDTC, 120mg/L), a NF-κB inhibitor, via drinking water starting (Con+PDTC or LPS+PDTC group), respectively, till the age of 20 or 68 weeks. The gross structure of kidney was assessed by hematoxylin-eosin, periodic acid–Schiff staining and Sirius red staining. The expression levels of TNF-α, IL-6, α-smooth muscle actin (α-SMA) and renin-angiotensin system (RAS) genes were determined by real time polymerase chain reaction and/or immunohistochemical staining. Our data showed that post-natal persistent PDTC administration efficiently repressed intra-renal NF-κB activation, TNF-α and IL-6 expression. Post-natal PDTC also prevented intra-renal glycogen deposition and collagenous fiber generation as evident by the reduced expression of collagen III and interstitial α-SMA in offspring of prenatal LPS exposure. Furthermore, post-natal PDTC administration reversed the intra-renal renin-angiotensin system (RAS) over-activity in offspring of prenatal LPS exposure. In conclusion, prenatal inflammatory exposure results in offspring’s intra-renal NF-κB activation along with inflammation which cross-talked with excessive RAS activation that caused exacerbation of renal fibrosis and dysfunction in the offspring. Thus, early life prevention of NF-κB activation may be a potential preventive strategy for chronic renal inflammation and progressive renal damage.     

Regulation of MRP2/ABCC2 and BSEP/ABCB11 Expression in Sandwich Cultured Human and Rat Hepatocytes Exposed to Inflammatory Cytokines TNF-α, IL-6, and IL-1β

In the present study MRP2/ABCC2 and BSEP/ABCB11 expression were investigated in sandwich cultured (SC) human and rat hepatocytes exposed to the proinflammatory cytokines. The investigation was also done in lipopolysaccharide (LPS)-treated rats. In SC human hepatocytes, both absolute protein and mRNA levels of MRP2/ABCC2 were significantly down-regulated by TNF-α, IL-6, or IL-1β. In contrast to mRNA decrease, which was observed for BSEP/ABCB11, the protein amount was significantly increased by IL-6 or IL-1β. A discrepancy between the change in BSEP/ABCB11 mRNA and protein levels was encountered in SC human hepatocytes treated with proinflammatory cytokines. In SC rat hepatocytes, Mrp2/Abcc2 mRNA was down-regulated by TNF-α and IL-6, whereas the protein level was decreased by all three cytokines. Down-regulations of both Bsep/Abcb11 mRNA and protein levels were found in SC rat hepatocytes exposed to TNF-α or IL-1β. Administration of LPS triggered the release of the proinflammatory cytokines and caused the decrease of Mrp2/Abcc2 and Bsep/Abcb11 protein in liver at 24 h post-treatment; however, the Mrp2 and Bsep protein levels rebounded at 48 h post-LPS treatment. In total, our results indicate that proinflammatory cytokines regulate the expression of MRP2/Mrp2 and BSEP/Bsep and for the first time demonstrate the differential effects on BSEP/Bsep expression between SC human and rat hepatocytes. Furthermore, the agreement between transporter regulation in vitro in SC rat hepatocytes and in vivo in LPS-treated rats during the acute response phase demonstrates the utility of in vitro SC hepatocyte models for predicting in vivo effects.     

Yohimbine Enhances Protection of Berberine against LPS-Induced Mouse Lethality through Multiple Mechanisms

Sepsis remains a major cause of mortality in intensive care units, better therapies are urgently needed. Gram-negative bacterial lipopolysaccharide (LPS) is an important trigger of sepsis. We have demonstrated that berberine (Ber) protects against lethality induced by LPS, which is enhanced by yohimbine (Y) pretreatment, and Ber combined with Y also improves survival in septic mice. However, the precise mechanisms by which Y enhances protection of Ber against LPS - induced lethality remain unclear. The present study confirmed that simultaneously administered Y also enhanced protection of Ber against LPS-induced lethality. Ber or/and Y attenuated liver injury, but not renal injury in LPS-challenged mice. Ber or/and Y all inhibited LPS-stimulated IκBα, JNK and ERK phosphorylation, NF-κB activation as well as TNF-α production. Ber also increased IL-10 production in LPS-challenged mice, which was enhanced by Y. Furthermore, Ber or/and Y all suppressed LPS-induced IRF3, TyK2 and STAT1 phosphorylation, as well as IFN-β and IP-10 mRNA expression in spleen of mice at 1 h after LPS challenge. Especially, Y enhanced the inhibitory effect of Ber on LPS-induced IP-10 mRNA expression. In vitro experiments further demonstrated that Y significantly enhanced the inhibitory effect of Ber on TNF-α production in LPS-treated peritoneal macrophages, Ber combined with Y promoted LPS-induced IL-10 production and LPS-stimulated IκBα, JNK, ERK and IRF3 phosphorylation and NF-κB activation were also suppressed by Ber or/and Y pretreatment in peritoneal macrophages. Taken together, these results demonstrate that Y enhances the protection of Ber against LPS-induced lethality in mice via attenuating liver injury, upregulating IL-10 production and suppressing IκBα, JNK, ERK and IRF3 phosphorylation. Ber combined with Y may be an effective immunomodulator agent for the prevention of sepsis.     

Lactobacillus fermentum ZYL0401 Attenuates Lipopolysaccharide-Induced Hepatic TNF-α Expression and Liver Injury via an IL-10- and PGE2-EP4-Dependent Mechanism

Lipopolysaccharide (LPS) has essential role in the pathogenesis of D-galactosamine-sensitized animal models and alcoholic liver diseases of humans, by stimulating release of pro-inflammatory mediators that cause hepatic damage and intestinal barrier impairment. Oral pretreatment of probiotics has been shown to attenuate LPS-induced hepatic injury, but it is unclear whether the effect is direct or due to improvement in the intestinal barrier. The present study tested the hypothesis that pretreatment with probiotics enables the liver to withstand directly LPS-induced hepatic injury and inflammation. In a mouse model of LPS-induced hepatic injury, the levels of hepatic tumor necrosis factor-alpha (TNF-α) and serum alanine aminotransferase (ALT) of mice with depleted intestinal commensal bacteria were not significantly different from that of the control models. Pre-feeding mice for 10 days with Lactobacillus fermentum ZYL0401 (LF41), significantly alleviated LPS-induced hepatic TNF-α expression and liver damage. After LF41 pretreatment, mice had dramatically more L.fermentum-specific DNA in the ileum, significantly higher levels of ileal cyclooxygenase (COX)-2 and interleukin 10 (IL-10) and hepatic prostaglandin E2 (PGE₂). However, hepatic COX-1, COX-2, and IL-10 protein levels were not changed after the pretreatment. There were also higher hepatic IL-10 protein levels after LPS challenge in LF41-pretreaed mice than in the control mice. Attenuation of hepatic TNF-α was mediated via the PGE₂/E prostanoid 4 (EP4) pathway, and serum ALT levels were attenuated in an IL-10-dependent manner. A COX-2 blockade abolished the increase in hepatic PGE₂ and IL-10 associated with LF41. In LF41-pretreated mice, a blockade of IL-10 caused COX-2-dependent promotion of hepatic PGE₂, without affecting hepatic COX-2levels. In LF41-pretreated mice, COX2 prevented enhancing TNF-α expression in both hepatic mononuclear cells and the ileum, and averted TNF-α-mediated increase in intestinal permeability. Together, we demonstrated that LF41 pre-feeding enabled the liver to alleviate LPS-induced hepatic TNF-α expression and injury via a PGE₂-EP4- and IL-10-dependent mechanism.     

A Possible Link between Food and Mood: Dietary Impact on Gut Microbiota and Behavior in BALB/c Mice

Major depressive disorder is a debilitating disease in the Western World. A western diet high in saturated fat and refined sugar seems to play an important part in disease development. Therefore, this study is aimed at investigating whether saturated fat or sucrose predisposes mice to develop behavioral symptoms which can be interpreted as depression-like, and the possible influence of the gut microbiota (GM) in this. Fourty-two mice were randomly assigned to one of three experimental diets, a high-fat, a high-sucrose or a control diet for thirteen weeks. Mice on high-fat diet gained more weight (p = 0.00009), displayed significantly less burrowing behavior than the control mice (p = 0.034), and showed decreased memory in the Morris water maze test compared to mice on high-sucrose diet (p = 0.031). Mice on high-sucrose diet burrowed less goal-oriented, showed greater latency to first bout of immobility in the forced swim test when compared to control mice (p = 0.039) and high-fat fed mice (p = 0.013), and displayed less anxiety than mice on high-fat diet in the triple test (p = 0.009). Behavioral changes were accompanied by a significant change in GM composition of mice fed a high-fat diet, while no difference between diet groups was observed for sucrose preferences, LPS, cholesterol, HbA1c, BDNF and the cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12(p70), IL-17 and TNF-α. A series of correlations was found between GM, behavior, BDNF and inflammatory mediators. In conclusion, the study shows that dietary fat and sucrose affect behavior, sometimes in opposite directions, and suggests a possible association between GM and behavior.     

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background

Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).

Methods

We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.

Results

Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).

Conclusions

Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.

Key Messages

Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.

Trial Registration

Clinical Trials.gov NCT02284074     

A New IRAK-M-Mediated Mechanism Implicated in the Anti-Inflammatory Effect of Nicotine via α7 Nicotinic Receptors in Human Macrophages

Nicotine stimulation of α7 nicotinic acetylcholine receptor (α7 nAChR) powerfully inhibits pro-inflammatory cytokine production in lipopolysaccharide (LPS)-stimulated macrophages and in experimental models of endotoxemia. A signaling pathway downstream from the α7 nAChRs, which involves the collaboration of JAK2/STAT3 and NF-κB to interfere with signaling by Toll-like receptors (TLRs), has been implicated in this anti-inflammatory effect of nicotine. Here, we identifiy an alternative mechanism involving interleukin-1 receptor-associated kinase M (IRAK-M), a negative regulator of innate TLR-mediated immune responses. Our data show that nicotine up-regulates IRAK-M expression at the mRNA and protein level in human macrophages, and that this effect is secondary to α7 nAChR activation. By using selective inhibitors of different signaling molecules downstream from the receptor, we provide evidence that activation of STAT3, via either JAK2 and/or PI3K, through a single (JAK2/PI3K/STAT3) or two convergent cascades (JAK2/STAT3 and PI3K/STAT3), is necessary for nicotine-induced IRAK-M expression. Moreover, down-regulation of this expression by small interfering RNAs specific to the IRAK-M gene significantly reverses the anti-inflammatory effect of nicotine on LPS-induced TNF-α production. Interestingly, macrophages pre-exposed to nicotine exhibit higher IRAK-M levels and reduced TNF-α response to an additional LPS challenge, a behavior reminiscent of the ‘endotoxin tolerant’ phenotype identified in monocytes either pre-exposed to LPS or from immunocompromised septic patients. Since nicotine is a major component of tobacco smoke and increased IRAK-M expression has been considered one of the molecular determinants for the induction of the tolerant phenotype, our findings showing IRAK-M overexpression could partially explain the known influence of smoking on the onset and progression of inflammatory and infectious diseases.     

The Therapeutic Potential and Mechanisms of Action of Quercetin in Relation to Lipopolysaccharide-Induced Sepsis In Vitro and In Vivo

Sepsis caused by Gram-negative bacterial infection is characterized by extensive inflammatory cytokine production, which leads to multiple organ failure and a high lethality rate. Therefore, compounds that are able to alleviate profound inflammatory responses may have therapeutic potential in relation to sepsis. Quercetin, one of the flavonoids found widely in the human diet, has been reported to have many health benefits, but the mechanisms underlying its biological effects remain obscure. In the present study, our aim was to investigate the molecular mechanisms by which quercetin inhibits lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production and to evaluate the capacity of quercetin to attenuate the mortality rate in a mice model of lethal sepsis. Our results show that quercetin significantly attenuates LPS-induced production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 macrophages. The LPS-stimulated phosphorylations of the inhibitors of κB kinase (IKKs), Akt, and c-Jun N-terminal kinase (JNK) are also inhibited by quercetin. Quercetin causes a significant reduction in the phosphorylation and degradation of inhibitor of κBα (IκBα) and in the nuclear level of nuclear factor-κB (NF-κB), the latter being associated with decreased NF-κB binding activity. Most importantly, acute administration of quercetin reduces the lethality rate and circulating levels of TNF-α and IL-1β in C57BL/6J mice with endotoxemia induced by LPS, whereas chronic dietary supplementation with quercetin shows no inhibitory effect on serum TNF-α and IL-1β levels. These findings provide clues that quercetin may be a promising agent for the prevention of systemic inflammatory diseases such as sepsis.     

WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1) Negatively Regulates TLR4-Mediated TNF-α and IL-6 Production by Proteasomal Degradation of TNF Receptor Associated Factor 6 (TRAF6)

Background

Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.

Methodology/Results

Knocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6.

Conclusions/Significance

We identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6.