TLR4-induced Inflammatory Responses Research Articles

DcR3 suppresses LPS-Induced Aggresome-like induced structures in Macrophages via inhibition of ROS and p38 MAPK

Abstract Decoy receptor 3 (DcR3), also known as tumor necrosis factor receptor (TNFR) superfamily member 6b (TNFRSF6B), is a pleiotropic factor which modulates cell functions via decoy and non-decoy actions. DcR3 has been shown its anti-apoptotic and anti-inflammatory effects on cancers and inflammatory diseases. Toll-like receptors (TLRs), which are the members of pattern recognition receptors (PRRs), trigger inflammatory response against invading pathogens. However, it remains unclear whether DcR3 plays a role in TLR4-induced inflammatory response. In this study, we demonstrated that in bone marrow-derived macrophages (BMDMs) DcR3 did not affect lipopolysaccharide (LPS)-induced COX-2, iNOS, NLRP3 and pro-IL-1b protein expression. It has been reported that aggresome-like induced structures (ALIS) are formed in response to LPS in macrophages. ALIS, which containing ubiquitinated protein aggregates, are stress-induced response involved in MHC class I antigen presentation. Our findings illustrated that DcR3 can decrease LPS-induced ALIS formation via inhibiting cellular ROS production and p38 MAPK phosphorylation. In addition to LPS, ZnPP-, bafilomycin A1- and MG132-induced ubiquitinated protein expression were reduced by DcR3. Confirming the notion that ALIS is controlled by autophagy, we found that rapamycin can decrease LPS-induced ALIS formation, while bafilomycin A1 can increase LC3-II accumulation with similar extent in WT and DcR3 macrophages. Nevertheless, DcR3 expression does not change p62 and HO-1 expression induced by LPS. Taken together, despite the fact that DcR3 does not alter LPS-induced inflammatory response, our data suggest a novel role of DcR3 in suppression of ALIS formation. MOST 111-2320-B-002-028

Pharmacological inhibition of TLR4-NF-κB signaling by TAK-242 attenuates hydrocephalus after intraventricular hemorrhage

Compelling evidence has confirmed that inflammatory pathways involving TLR4-regulated cytokines and immune cells are vitallyimportant for the pathogenesis of posthemorrhagic hydrocephalus (PHH), hinting that pharmacological prevention of PHH is feasible. TAK-242, as a toll-like receptor 4 (TLR4) inhibitor, downregulates TLR4-induced inflammatory responses and becomes a potent and noveltherapeuticdrugcandidatefor PHH. In the present study, we investigate whether TAK-242 protects against hydrocephalus and improves the prognosis of intraventricular hemorrhage (IVH). We also explore the possible role of TAK-242 for the regulation of TLR4-NF-κB signaling pathway. A model of PHH was conducted in 6-week-old Male Sprague-Dawley (SD) rats. The rats were divided into four main groups, including the sham, IVH + vehicle, IVH + TAK-242 and IVH groups. Magnetic resonance imaging (MRI) was applied to measure the lateral ventricle volume. Western blot (WB) and immunofluorescence (IF) were applied to detect the expression of TLR4, NF-κB, fibronectin and laminin. A combined scoring system and Morris water maze were employed to evaluate neurological functions after IVH. We found that IVH induced heightened activation of TLR4-NF-κB signaling pathway. We observed the increased lateral ventricular volume, elevation of NF-κB in choroidplexus, as well as fibronectin and laminin in the subarachnoid space (SAS) and ventricular wall after IVH. Obviously, TAK-242 treatment effectively inhibited the up-regulation of NF-κB, fibronectin, laminin and significantly alleviated ventriculomegaly after IVH. Importantly, TAK-242 improved neurocognitive deficits after PHH. In conclusion, TAK-242 attenuated IVH-induced hydrocephalus and improved the prognosis of PHH. The underlying mechanism involved the TAK-242-mediated downregulation of TLR4-NF-κB signaling pathway.

Single-cell secretion analysis reveals a dual role for IL-10 in restraining and resolving the TLR4-induced inflammatory response.

Following Toll-like receptor 4 (TLR4) stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine interleukin-10 (IL-10) limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterize the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enables evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data reveals three tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate interferon (IFN)-β signaling, while TNF likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.

Pharmacological Inhibition of TLR4-NF-κB Signaling by TAK-242 Attenuates Hydrocephalus after Intraventricular Hemorrhage

Compelling evidence has confirmed that inflammatory pathways involving TLR4-regulated cytokines and immune cells are vitally important for the pathogenesis of posthemorrhagic hydrocephalus (PHH), hinting that pharmacological prevention of PHH is feasible. TAK-242, as a toll-like 4 receptor (TLR4) inhibitor, downregulates TLR4-induced inflammatory responses and becomes a potent and novel therapeutic drug candidate for PHH. In the present study, we investigate whether TAK-242 protects against hydrocephalus and improves the prognosis of intraventricular hemorrhage (IVH). We also explore the possible role of TAK-242 for the regulation of TLR4-NF-κB signaling pathway. A model of PHH was conducted in 6-week-old Male Sprague-Dawley rats. The rats were divided into four main groups, including the sham, IVH+ vehicle, IVH+ TAK-242 and IVH groups. Magnetic resonance imaging was applied to measure the lateral ventricle volume. Western blot (WB) and immunofluorescence (IF) were applied to detect the expression of TLR4, NF-κB, fibronectin and laminin. A combined scoring system and Morris water maze were employed to evaluate neurological functions after IVH. We found that IVH induced heightened activation of TLR4-NF-κB signaling pathway. We observed the increased lateral ventricular volume, elevation of NF-κB in choroid plexus, as well as fibronectin and laminin in the subarachnoid space (SAS) and ventricular wall after IVH. Obviously, TAK-242 treatment effectively inhibited the up-regulation of NF-κB, fibronectin, laminin and significantly alleviated ventriculomegaly after IVH. Importantly, TAK-242 improved neurocognitive deficits after PHH. In conclusion, TAK-242 attenuated IVH-induced hydrocephalus and improved the prognosis of PHH. The underlying mechanism involved the TAK-242-mediated downregulation of TLR4-NF-κB signaling pathway.

Single-Cell Secretion Analysis Reveals a Dual Role for IL-10 in Restraining and Resolving the TLR4-Induced Inflammatory Response

Following TLR4 stimulation of macrophages, negative feedback mediated by the anti-inflammatory cytokine IL-10 limits the inflammatory response. However, extensive cell-to-cell variability in TLR4-stimulated cytokine secretion raises questions about how negative feedback is robustly implemented. To explore this, we characterized the TLR4-stimulated secretion program in primary murine macrophages using a single-cell microwell assay that enabled evaluation of functional autocrine IL-10 signaling. High-dimensional analysis of single-cell data revealed three distinct tiers of TLR4-induced proinflammatory activation based on levels of cytokine secretion. Surprisingly, while IL-10 inhibits TLR4-induced activation in the highest tier, it also contributes to the TLR4-induced activation threshold by regulating which cells transition from non-secreting to secreting states. This role for IL-10 in restraining TLR4 inflammatory activation is largely mediated by intermediate IFN-β signaling, while TNF-α likely mediates response resolution by IL-10. Thus, cell-to-cell variability in cytokine regulatory motifs provides a means to tailor the TLR4-induced inflammatory response.

Ellagic acid protects mice against sleep deprivation-induced memory impairment and anxiety by inhibiting TLR4 and activating Nrf2.

Sleep disorder has become a prevalent issue in current society and is connected with the deterioration of neurobehaviors such as mood, cognition and memory. Ellagic acid (EA) is a phenolic phytoconstituent extracted from grains and fruits that has potent neuroprotective properties. This research aimed to study the alleviative effect and mechanism of EA on memory impairment and anxiety caused by sleep deprivation (SD). EA ameliorated behavioral abnormalities in SD mice, associated with increased dendritic spine density, and reduced shrinkage and loss of hippocampal neurons. EA reduced the inflammatory response and oxidative stress injury caused by SD, which may be related to activation of the Nrf2/HO-1 pathway and mitigation of the TLR4-induced inflammatory response. In addition, EA significantly reduced the mortality and ROS levels in glutamate (Glu)-induced hippocampal neuron injury, and these effects of EA were enhanced in TLR4 siRNA-transfected neurons. However, knockdown of Nrf2 dramatically restrained the protective impact of EA on Glu-induced toxicity. Taken together, EA alleviated memory impairment and anxiety in sleep-deprived mice potentially by inhibiting TLR4 and activating Nrf2. Our findings suggested that EA may be a promising nutraceutical ingredient to prevent cognitive impairment and anxiety caused by sleep loss.

TAOK1 positively regulates TLR4-induced inflammatory responses by promoting ERK1/2 activation in macrophages

Thousand and one amino acid kinase 1 (TAOK1) is a member of Ste20-like kinases, but its function in regulating inflammatory responses remains largely unknown. In this study, we identify TAOK1 as a positive regulator of TLR4-triggered inflammatory responses in macrophages. TAOK1 increases LPS-induced production of pro-inflammatory cytokine such as IL-6, TNF-α and IL12p40 in macrophages. TAOK1 deficient mice showed decreased susceptibility to endotoxin shock, with less pro-inflammatory cytokine production than control mice. TAOK1 promotes LPS-induced activation of ERK1/2 by constitutively interacting with TRAF6 and TPL2. These finding unravel the important role of TAOK1 as a positive regulator of TLR4-induced inflammatory responses.

Shanxi Aged Vinegar Protects against Alcohol-Induced Liver Injury via Activating Nrf2-Mediated Antioxidant and Inhibiting TLR4-Induced Inflammatory Response.

Shanxi aged vinegar (SAV) is a typical fermented and antioxidant food, which has various health-promoting effects. This work aimed to explore the effects of SAV on alcohol-induced liver injury. A mice model of alcoholic liver injury was established to illuminate its potential mechanisms. All mice pretreated with SAV and then received an ethanol solution (50% w/v, 4.8 g/kg b.w.). The results showed that SAV ameliorated alcohol-induced histological changes and elevation of liver enzymes. SAV attenuated alcohol-induced oxidative stress by declining levels of hepatic oxidants, and restoring depletion of antioxidant enzyme activities in mice livers. Moreover, SAV alleviated alcohol-induced oxidative damage by activating nuclear factor erythroid-2-related factor 2 (Nrf2)-mediated signal pathway. In addition, SAV prevented alcohol-induced inflammation by suppressing lipopolysaccharide (LPS) level and activities of pro-inflammatory enzymes, and regulating inflammatory cytokines. SAV inhibited alcohol-induced inflammation through down-regulating the expression of Toll-like receptor 4 (TLR4)-mediated inflammatory response. The findings provide crucial evidence for elucidating the hepatoprotective mechanisms of SAV and encourage the future application of SAV as a functional food for liver protection.

Simvastatin reduces the TLR4-induced inflammatory response in human aortic valve interstitial cells

BackgroundCalcific aortic stenosis is a chronic inflammatory disease. Proinflammatory stimulation via toll-like receptor 4 (TLR4) causes the aortic valve interstitial cell (AVIC) to undergo phenotypic change. The AVIC first assumes an inflammatory phenotype characterized by the production of inflammatory mediators such as intercellular adhesion molecule-1 (ICAM-1), interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1). This change has been linked with an osteogenic phenotypic response. Statins have recently been shown to have anti-inflammatory properties. We therefore hypothesized that statins may have an anti-inflammatory effect on human AVICs by downregulation of TLR4-stimulated inflammatory responses. Our purposes were (1) to determine the effect of simvastatin on TLR4-induced expression of inflammatory mediators in human AVICs and (2) to determine the mechanism(s) through which simvastatin exert this effect. Materials and methodsHuman AVICs were isolated from the explanted hearts of four patients undergoing cardiac transplantation. Cells were treated with simvastatin (50 μM) for 1 h before stimulation with TLR4 agonist lipopolysaccharide (LPS, 0.2 μg/mL). Immunoblotting (IB) was used to analyze cell lysates for ICAM-1 expression, and enzyme-linked immunosorbent assay was used to detect IL-8 and MCP-1 in cell culture media. Likewise, lysates were analyzed for TLR4 and nuclear factor-kappa B activation (IB). After simvastatin treatment, lysates were analyzed for TLR4 levels (IB). Statistics were by analysis of variance (P < 0.05). ResultsSimvastatin reduced TLR4-induced ICAM-1, IL-8, and MCP-1 expression in AVICs. Simvastatin down-regulated TLR4 levels and suppressed TLR4-induced phosphorylation of nuclear factor-kappa B. ConclusionsThese data demonstrate the potential of a medical therapy (simvastatin) to impact the pathogenesis of aortic stenosis.

The ubiquitin ligase ZNRF1 promotes caveolin-1 ubiquitination and degradation to modulate inflammation

Caveolin-1 (CAV1), the major constituent of caveolae, plays a pivotal role in various cellular biological functions, including cancer and inflammation. The ubiquitin/proteasomal pathway is known to contribute to the regulation of CAV1 expression, but the ubiquitin ligase responsible for CAV1 protein stability remains unidentified. Here we reveal that E3 ubiquitin ligase ZNRF1 modulates CAV1 protein stability to regulate Toll-like receptor (TLR) 4-triggered immune responses. We demonstrate that ZNRF1 physically interacts with CAV1 in response to lipopolysaccharide and mediates ubiquitination and degradation of CAV1. The ZNRF1–CAV1 axis regulates Akt–GSK3β activity upon TLR4 activation, resulting in enhanced production of pro-inflammatory cytokines and inhibition of anti-inflammatory cytokine IL-10. Mice with deletion of ZNRF1 in their hematopoietic cells display increased resistance to endotoxic and polymicrobial septic shock due to attenuated inflammation. Our study defines ZNRF1 as a regulator of TLR4-induced inflammatory responses and reveals another mechanism for the regulation of TLR4 signalling through CAV1.

PLAP-1/Asporin Regulates TLR2- and TLR4-induced Inflammatory Responses

Periodontal ligament–associated protein 1 (PLAP-1)/asporin is an extracellular matrix protein preferentially expressed in periodontal ligaments. PLAP-1/asporin inhibits the cytodifferentiation and mineralization of periodontal ligament cells and has important roles in the maintenance of periodontal tissue homeostasis. However, the involvement of PLAP-1/asporin in inflammatory responses during periodontitis is poorly understood. This study hypothesized that PLAP-1/asporin might affect the pathogenesis of periodontitis by regulating periodontopathic bacteria-induced inflammatory responses. Proinflammatory cytokine expression induced by Toll-like receptor 2 (TLR2) and TLR4 was significantly downregulated when PLAP-1/asporin was overexpressed in periodontal ligament cells. Similarly, recombinant PLAP-1/asporin inhibited TLR2- and TLR4-induced proinflammatory cytokine expression in macrophages. We also confirmed that NF-κB activity induced by TLR2 and TLR4 signaling was suppressed by the addition of recombinant PLAP-1/asporin. Furthermore, IκB kinase α degradation induced by TLR4 was reduced by PLAP-1/asporin. Immunoprecipitation assays demonstrated the binding abilities of PLAP-1/asporin to both TLR2 and TLR4. Taken together, PLAP-1/asporin negatively regulates TLR2- and TLR4-induced inflammatory responses through direct molecular interactions. These findings indicate that PLAP-1/asporin has a defensive role in periodontitis lesions by suppressing pathophysiologic TLR signaling and that the modulating effects of PLAP-1/asporin might be useful for periodontal treatments.

Toll-like receptor 4-induced inflammatory responses contribute to the tumor-associated macrophages formation and infiltration in patients with diffuse large B-cell lymphoma.

To evaluate the expression of tumor-associated macrophages (TAMs) and Toll-like receptor 4 (TLR4) in diffuse large B-cell lymphoma (DLBCL) and their correlation with patient clinical characteristics, we detected using immunohistochemistry in 81 specimens of patients with DLBCL. The correlation between protein expression levels and clinical parameters, as well as the association between CD68 and TLR4 were analyzed. The number of CD68 TAMs was closely related to β2-microglobulin (P = .028 and P < .05), whereas there was no significant correlation between the number of CD68 TAMs and other clinical factors. Toll-like receptor 4 was related to tumor size and peripheral blood lymphocyte to monocyte ratio. The Spearman correlation coefficient indicated a significant positive correlation between CD68 TAMs and TLR4 expression (r = 0.240; P = .038, P = .05). These results, on one hand, indicated that TLR4-induced inflammatory responses may affect TAM infiltration and accumulation, and that TAMs and TLR4 may interact to play important roles in DLBCL microenvironment regulating the tumor growth, but, on the other hand demonstrated that both of TAMs and TLR4 had not only one side on DLBCL growth.

The helminth Trichuris suis suppresses TLR4-induced inflammatory responses in human macrophages

Recent clinical trials in patients with inflammatory diseases like multiple sclerosis (MS) or inflammatory bowel disease (IBD) have shown the beneficial effects of probiotic helminth administration, although the underlying mechanism of action remains largely unknown. Potential cellular targets may include innate immune cells that propagate inflammation in these diseases, like pro-inflammatory macrophages. We here investigated the effects of the helminth Trichuris suis soluble products (SPs) on the phenotype and function of human inflammatory (granulocyte-macrophage colony-stimulating factor (GM-CSF)-differentiated) macrophages. Interestingly, we here show that T. suis SPs potently skew inflammatory macrophages into a more anti-inflammatory state in a Toll-like receptor 4 (TLR4)-dependent manner, and less effects are seen when stimulating macrophages with TLR2 or -3 ligands. Gene microarray analysis of GM-CSF-differentiated macrophages further revealed that many TLR4-induced inflammatory mediators, including interleukin (IL)-12B, CCL1 and CXCL9, are downregulated by T. suis SPs. In particular, we observed a strong reduction in the expression and function of P2RX7, a purinergic receptor involved in macrophage inflammation, leading to reduced IL-1β secretion. In conclusion, we show that T. suis SPs suppress a broad range of inflammatory pathways in GM-CSF-differentiated macrophages in a TLR4-dependent manner, thereby providing enhanced mechanistic insight into the therapeutic potential of this helminth for patients with inflammatory diseases.

Human Resistin Promotes Neutrophil Proinflammatory Activation and Neutrophil Extracellular Trap Formation and Increases Severity of Acute Lung Injury

Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies also suggested that resistin has proinflammatory properties. We examined whether the human-specific variant of resistin affects neutrophil activation and the severity of LPS-induced acute lung injury. Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using humanized resistin mice that exclusively express human resistin (hRTN(+/-)(/-)) but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn(+/-/-) neutrophils compared with control Rtn(-/-/-) neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase, a major sensor and regulator of cellular bioenergetics that also is implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin enhanced neutrophil extracellular trap formation. In LPS-induced acute lung injury, humanized resistin mice demonstrated enhanced production of proinflammatory cytokines, more severe pulmonary edema, increased neutrophil extracellular trap formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4-induced inflammatory responses, and it may be a target for future therapies aimed at reducing the severity of acute lung injury and other inflammatory situations in which neutrophils play a major role.

Cross-Talk Between the Toll-Like Receptor 4 and Notch1 Pathways Augments the Inflammatory Response in the Interstitial Cells of Stenotic Human Aortic Valves

Calcific aortic stenosis is a chronic inflammatory disease, and aortic valve interstitial cells (AVIC) play an important role in valvular inflammation. Whereas AVIC from stenotic aortic valves exhibit an augmented response to Toll-like receptor 4 (TLR4) stimulation, the underlying mechanism is unclear. This study tested the hypothesis that an excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the inflammatory response to lipopolysaccharide (LPS) in AVIC of stenotic valves. Human AVIC were isolated from normal and stenotic leaflets. Nuclear factor kappa-B (NF-κB) activation and production of interleukin-8, monocyte chemoattactrant protein-1, and intercellular adhesion molecule-1 were analyzed after treatment with LPS. The role of Notch1 in the inflammatory response was determined using inhibitor, siRNA, and specific ligand. Cells from diseased valves produced greater levels of chemokines and intercellular adhesion molecule-1 that are associated with enhanced NF-κB activation. Interestingly, diseased cells exhibited augmented Jagged1 release and Notch1 activation after TLR4 stimulation. Inhibition and silencing of Notch1 each resulted in greater suppression of the TLR4-induced inflammatory response in diseased cells. Conversely, activation of Notch1 with a specific ligand, Jagged1, enhanced the LPS-induced inflammatory response in normal AVIC. Further, Notch1 intracellular domain was coimmunoprecipited with the inhibitor of NF-κB kinase after LPS stimulation, and inhibition of Notch1 abrogated the difference in the level of NF-κB activation between diseased and normal cells. Notch1 enhances the inflammatory response to TLR4 stimulation in human AVIC through modulating NF-κB activation. Excessive cross-talk between the TLR4 and Notch1 pathways is responsible for augmentation of the TLR4 response in AVIC of stenotic valves.

Development of β-amino alcohol derivatives that inhibit Toll-like receptor 4 mediated inflammatory response as potential antiseptics.

Toll-like receptor 4 (TLR4) induced proinflammatory signaling has been directly implicated in severe sepsis and represents an attractive therapeutic target. Herein, we report our investigations into the structure-activity relationship and preliminary drug metabolism/pharmacokinetics study of β-amino alcohol derivatives that inhibit the TLR4 signaling pathway. Lead compounds were identified from in vitro cellular examination with micromolar potency for their inhibitory effects on TLR4 signaling and subsequently assessed for their ability to suppress the TLR4-induced inflammatory response in an ex vivo whole blood model. In addition, the toxicology, specificity, solubility, brain-blood barrier permeability, and drug metabolism of several compounds were evaluated. Although further optimizations are needed, our findings lay the groundwork for the future drug development of this class of small molecule agents for the treatment of severe sepsis.

Heme oxygenase-1 upregulation improves lipopolysaccharide-induced acute lung injury involving suppression of macrophage migration inhibitory factor

Although studies have demonstrated that heme oxygenase-1 (HO-1) prevents leukocyte infiltration and organ damage following LPS challenge, the mechanisms involved in this protection are incompletely understood. Macrophage migration inhibitory factor (MIF) is thought to play a pivotal role in modulation of inflammatory and immune response through upregulation of TLR4 expression. Activation of TLR4 results in the production of proinflammatory mediators including MIF, which induce neutrophils recruitment and subsequent tissue insults. We hypothesized that HO-1 mediates its salutary effects in lipopolysaccharide (LPS)-induced inflammatory lung injury via downregulation of MIF through modulation of TLR4-induced proinflammatory mediator production. Compared with wild-type cells, MIF-knockdown macrophages in vitro are hyporesponsive to LPS stimulation, as shown by a profound reduction in TLR4 expression and TNF-α production. In the murine model of LPS-induced acute lung injury, administration of CoPP, a potent HO-1 inducer, leaded to a significant reduction in LPS-induced pulmonary edema, leucocytes influx, myeloperoxidase activity as well as histopathologic insults. Most strikingly, pretreatment with CoPP markedly decreased the expression of TLR4 and MIF in lung tissues in response to LPS challenge. These findings herein suggest that the cytoprotective functions of HO-1 in LPS-induced lung injury are associated with negative regulation of lung MIF and TLR4-induced inflammatory response.

The inositol 3-phosphatase PTEN negatively regulates Fc gamma receptor signaling, but supports Toll-like receptor 4 signaling in murine peritoneal macrophages.

Fc gamma R clustering in macrophages activates signaling events that result in phagocytosis. Phagocytosis is accompanied by the generation harmful byproducts such as reactive oxygen radicals and production of inflammatory cytokines, which mandate that the phagocytic process be subject to a tight regulation. The molecular mechanisms involved in this regulation are not fully understood. In this study, we have examined the role of the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in Fc gamma R-induced macrophage function. We demonstrate that in ex vivo murine peritoneal macrophages that are deficient in PTEN expression, Fc gamma R-induced Akt and extracellular signal-regulated kinase phosphorylation are enhanced. Notably, PTEN(-/-) macrophages showed constitutively high phosphorylation of Akt. However, PTEN did not seem to influence tyrosine phosphorylation events induced by Fc gamma R clustering. Furthermore, PTEN(-/-) macrophages displayed enhanced phagocytic ability. Likewise, Fc gamma R-induced production of TNF-alpha, IL-6, and IL-10 was significantly elevated in PTEN(-/-) macrophages. Surprisingly, LPS-induced TNF-alpha production was down-regulated in PTEN(-/-) macrophages. Analyzing the molecular events leading to PTEN influence on LPS/Toll-like receptor 4 (TLR4) signaling, we found that LPS-induced activation of mitogen-activated protein kinases is suppressed in PTEN(-/-) cells. Previous reports indicated that LPS-induced mitogen-activated protein kinase activation is down-regulated by phosphatidylinositol 3-kinase through the activation of Akt. Our observation that Akt activation is basally enhanced in PTEN(-/-) cells suggests that PTEN supports TLR4-induced inflammatory responses by suppressing the activation of Akt. Thus, we conclude that PTEN is a negative regulator of Fc gamma R signaling, but a positive regulator of TLR4 signaling. These findings are the first to demonstrate a role for PTEN in Fc gamma R- and TLR4-mediated macrophage inflammatory response.