TLR4 Signaling In Macrophages Research Articles

The anti-TLR4 monoclonal antibody Sa15-21 enhances inflammatory cytokine production in LPS-stimulated macrophages.

Sa15-21, a monoclonal antibody against mouse Toll-like receptor (TLR) 4, can protect mice from lipopolysaccharide (LPS)/D-galactosamine-induced acute lethal hepatitis. Herein, we investigated the molecular mechanisms underlying Sa15-21-mediated regulation of TLR4 signaling in macrophages. Results showed that Sa15-21 enhanced the production of proinflammatory cytokines and attenuated the production of anti-inflammatory cytokines in LPS-stimulated macrophages. Western botting analysis revealed that Sa15-21 pretreatment had no effect on NF-κB and MAPK signaling in LPS-stimulated macrophages; however, Sa15-21 treatment alone led to a weak and delayed activation of NF-κB and MAPK signaling without any effect on proinflammatory cytokine production. In contrast, Sa15-21 failed to induce the activation of interferon regulatory factor 3. Taken together, our results indicate that Sa15-21 sensitizes macrophages to facilitate the inflammatory response via TLR signaling.

Activation of estrogen receptor α inhibits TLR4 signaling in macrophages and alleviates the instability of atherosclerotic plaques in the postmenopausal stage

Acute cardiovascular events increase significantly in postmenopausal women. The relationship between estrogen receptor (ER) and plaque stability in the postmenopausal stage remains to be elucidated. We aimed to explore whether ERα activation improves plaque instability in the postmenopausal stage. Here, we report that postmenopausal women showed increased macrophage activation and plaque instability with increased MCP-1, MMP9, TLR4, MYD88 and NF-κB p65 and decreased ERα and TIMP1 expression in the vascular endothelium. Moreover, ovariectomy in LDLR-/- mice resulted in a significant increase in plaque area and necrotic core area, as well as a significant decrease in collagen content and an increase in macrophage accumulation in the artery. Ovariectomy also reduced serum estrogen levels and ERα expression and upregulated TLR4 and MMP9 expression in arteries in LDLR-/- mice. Estrogen or phytoestrogen therapy upregulated the expression level of ERα in ovariectomized mice and increased plaque stability by inhibiting macrophage accumulation and TLR4 signaling. In vitro, LPS incubation of RAW264.7 cells resulted in a significant decrease in ERα and TIMP1 expression and an increase in TLR4 activation, and estrogen or phytoestrogen treatment increased ERα and TIMP1 expression and inhibited TLR4 activation and MMP9 expression in LPS-treated RAW264.7 cells. Compared to control siRNA transfected RAW264.7 cells, TLR4 siRNA promoted TIMP1 expression in RAW264.7 cells with LPS incubation, but did not affect ERα expression in RAW264.7 cells with or without LPS treatment. The ERα inhibitor MPP abolished the regulatory effect of estrogen or phytoestrogen on LPS-induced RAW264.7 cells. In conclusion, the present study demonstrates that decreased ERα expression promotes macrophage infiltration and plaque instability in the postmenopausal stage, and activation of ERα in the postmenopausal stage alleviates atherosclerotic plaque instability by inhibiting TLR4 signaling and macrophage-related inflammation.

Regulation of SIRT1-TLR2/TLR4 pathway in cell communication from macrophages to hepatic stellate cells contribute to alleviates hepatic fibrosis by Luteoloside

Regulating macrophage-hepatic stellate cells (HSCs) crosstalk through SIRT1-TLR2/TLR4 has contributed to the essence of new pharmacologic strategies to improve hepatic fibrosis. We investigated how Luteoloside (LUT), one of the flavonoid monomers isolated from Eclipta prostrata (L.) L., modulates macrophage-HSCs crosstalk during hepatic fibrosis. HSC-T6 or rat peritoneal macrophages were activated by TGF-β or LPS/ATP, and then treated with LUT or Sirtinol (SIRT1 inhibitor) for 6 h. Further, HSCs were cultured with the conditioned medium from the LPS/ATP activated peritoneal macrophages. In HSC-T6 or peritoneal macrophages, LUT could decrease the expressions of α-SMA, Collagen-I, the ratio of TIMP-1/MMP-13. LUT also significantly increased the expressions of SIRT1 and ERRα. And LUT significantly suppressed the releases of pro-inflammatory cytokines, including NLRP3, ASC, caspase-1, IL-1β, and regulated signaling TLR2/TLR4-MyD88 activation. The expressions of TLR2, TLR4, NLRP3, caspase-1, IL-1β, α-SMA were increased and the expression of ERRα was decreased by Sirtinol, indicated that LUT might mediate SIRT1 to regulate TLR4 expression and further alleviate inflammation and fibrosis. LUT could regulate SIRT1-mediated TLR4 and ECM in HSCs was reduced, when HSCs were cultured with conditioned medium. Hence, LUT could decrease the expressions of fibrosis markers, reduce the releases of inflammatory cytokines in activated HSCs or macrophages. In conclusion, LUT might be a promising candidate that regulating SIRT1-TLR2/TLR4 signaling in macrophages interacting with HSCs during hepatic fibrosis.

RANKL Impairs the TLR4 Pathway by Increasing TRAF6 and RANK Interaction in Macrophages.

High serum levels of osteoprotegerin (OPG) are found in patients with obesity, type 2 diabetes, sepsis, or septic shock and are associated with a high mortality rate in stroke. The primary known function of OPG is to bind to the receptor activator of NF-κB ligand (RANKL), and by doing so, it inhibits the binding between RANKL and its receptor (RANK). TLR4 signaling in macrophages involves TRAF6 recruitment and contributes to low-grade chronic inflammation in adipose tissue. LPS is a classical activator of the TLR4 pathway and induces the expression of inflammatory cytokines in macrophages. We have previously observed that in the presence of RANKL, there is no LPS-induced activation of TLR4 in macrophages. In this study, we investigated the crosstalk between RANK and TLR4 pathways in macrophages stimulated with both RANKL and LPS to unveil the role of OPG in inflammatory processes. We found that RANKL inhibits TLR4 activation by binding to RANK, promoting the binding between TRAF6 and RANK, lowering TLR4 activation and the expression of proinflammatory mediators. Furthermore, high OPG levels aggravate inflammation by inhibiting RANKL. Our findings elect RANKL as a candidate for drug development as a way to mitigate the impact of obesity-induced inflammation in patients.

Effects of Adipose-Derived Biogenic Nanoparticle-Associated microRNA-451a on Toll-like Receptor 4-Induced Cytokines.

Extracellular vesicles (EVs) are cell-released nanoparticles that transfer biomolecular content between cells. Among EV-associated biomolecules, microRNAs (miRNAs/miRs) represent one of the most important modulators of signaling pathways in recipient cells. Previous studies have shown that EVs from adipose-derived mesenchymal stromal cells (MSCs) and adipose tissue modulate inflammatory pathways in macrophages. In this study, the effects of miRNAs that are abundant in adipose tissue EVs and other biogenic nanoparticles (BiNPs) were assessed in terms of altering Toll-like receptor 4 (TLR4)-induced cytokines. TLR-4 signaling in macrophages is often triggered by pathogen or damage-induced inflammation and is associated with several diseases. This study demonstrates that miR-451a, which is abundant in adipose tissue BiNPs, suppresses pro-inflammatory cytokines and increases anti-inflammatory cytokines associated with the TLR4 pathway. Therefore, miR-451a may be partially responsible for immunomodulatory effects of adipose tissue-derived BiNPs.

CD38-mediated Inhibition of Bruton's Tyrosine Kinase in Macrophages Prevents Endotoxemic Lung Injury.

TLR4 signaling via endotoxemia in macrophages promotes macrophage transition to the inflammatory phenotype through NLRP3 inflammasome activation. This transition event has the potential to trigger acute lung injury (ALI). However, relatively little is known about the regulation of NLRP3 and its role in the pathogenesis of ALI. Here we interrogated the signaling pathway activated by CD38, an ectoenzyme expressed in macrophages, in preventing ALI through suppressing NLRP3 activation. Wild-type and Cd38-knockout (Cd38-/-) mice were used to assess inflammatory lung injury, and isolated macrophages were used to delineate underlying TLR4 signaling pathway. We showed that CD38 suppressed TLR4 signaling in macrophages by inhibiting Bruton's tyrosine kinase (Btk) through the recruitment of Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) and resulting in the dephosphorylation of activated Btk. Cd38-/- mice show enhanced lung polymorphonuclear leukocyte extravasation and severe lung injury. LPS- or polymicrobial sepsis-induced mortality in Cd38-/- mice were markedly augmented compared with wild types. CD38 in macrophages functioned by inhibiting Btk activation through activation of SHP2 and resulting dephosphorylation of Btk, and thereby preventing activation of downstream targets NF-κB and NLRP3. Cd38-/- macrophages displayed markedly increased activation of Btk, NF-κB, and NLRP3, whereas invivo administration of the Btk inhibitor ibrutinib (a Food and Drug Administration-approved drug) prevented augmented TLR4-induced inflammatory lung injury seen in Cd38-/- mice. Our findings together show upregulation of CD38 activity and inhibition of Btk activation downstream of TLR4 activation as potential strategies to prevent endotoxemic ALI.

IRF1 governs the differential interferon-stimulated gene responses in human monocytes and macrophages by regulating chromatin accessibility

SUMMARYMyeloid lineage cells use TLRs to recognize and respond to diverse microbial ligands. Although unique transcription factors dictate the outcome of specific TLR signaling, whether lineage-specific differences exist to further modulate the quality of TLR-induced inflammation remains unclear. Comprehensive analysis of global gene transcription in human monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells stimulated with various TLR ligands identifies multiple lineage-specific, TLR-responsive gene programs. Monocytes are hyperresponsive to TLR7/8 stimulation that correlates with the higher expression of the receptors. While macrophages and monocytes express similar levels of TLR4, macrophages, but not monocytes, upregulate interferon-stimulated genes (ISGs) in response to TLR4 stimulation. We find that TLR4 signaling in macrophages uniquely engages transcription factor IRF1, which facilitates the opening of ISG loci for transcription. This study provides a critical mechanistic basis for lineage-specific TLR responses and uncovers IRF1 as a master regulator for the ISG transcriptional program in human macrophages.

MiR-146a is a negative regulator of IL-33-stimulated mouse mast cells

Abstract Interleukin-33 (IL-33) is an inflammatory cytokine that promotes allergic disease by activating ILC2, Th2, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. The IL-33 receptor, ST2, shares signaling cascades with the TLR family, but homeostatic control of ST2 function is poorly understood. MicroRNA-146a (miR-146a) is induced by LPS and suppresses TLR4 signaling in macrophages. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow derived mast cells (BMMC). Induction required MyD88, Akt, and NFkB, since antagonizing these pathways decreased miR-146 expression. BMMC transfected with a miR-146a antagomir or derived from miR-146a KO mice showed enhanced cytokine expression in response to IL-33, suggesting that miR-146a is a negative regulator of IL-33-ST2 signaling. Our data further suggest that miR-146a may act by targeting IRAK proteins, because mir-146a KO BMMC have increased TRAF6, IRAK1, and IRAK4 expression. In vivo, miR-146a expression in plasma exosomes was elevated after intraperitoneal injection of IL-33. Also mast cell deficient c-Kitw-sh mice acutely reconstituted with miR-146a KO BMMC intraperitoneally had elevated plasma IL-6 levels in comparison to their WT counterparts after IL-33 challenge. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

MicroRNA-146a is a negative regulator of IL-33 stimulated mouse mast cells.

Abstract Interleukin 33 (IL-33) is an inflammatory cytokine that promotes allergic disease by activating ILC2, Th2 cells, and mast cells. IL-33 is increased in asthmatics, and its blockade suppresses asthma-like inflammation in mouse models. The IL-33 receptor, ST2, shares signaling cascades with the TLR family, but homeostatic control of ST2 function is poorly understood. MicroRNA-146a (miR-146a) is induced by LPS and suppresses TLR4 signaling in macrophages. Therefore, we explored whether miR-146a has a role in IL-33 signaling. IL-33 induced cellular and exosomal miR-146a expression in mouse bone marrow derived mast cells (BMMC). Induction required MyD88, Akt, and NFκB, since antagonizing these pathways decreased miR-146 expression. BMMC transfected with a miR-146a antagomir or derived from miR-146a KO mice showed enhanced cytokine expression in response to IL-33, suggesting that mir46a is a negative regulator of IL-33-ST2 signaling. Our data further suggest that miR-146a may act by targeting IRAK proteins, because mir146a KO BMMC have increased IRAK1 and IRAK4 expression. These results support the hypothesis that miR-146a is a feedback regulator of IL-33-mediated mast cell functions associated with allergic disease.

The mechanisms of graphene-based materials-induced programmed cell death: a review of apoptosis, autophagy, and programmed necrosis.

Graphene-based materials (GBMs) are widely used in many fields, including biomedicine. To date, much attention had been paid to the potential unexpected toxic effects of GBMs. Here, we review the recent literature regarding the impact of GBMs on programmed cell death (PCD). Apoptosis, autophagy, and programmed necrosis are three major PCDs. Mechanistic studies demonstrated that the mitochondrial pathways and MAPKs (JNK, ERK, and p38)- and TGF-β-related signaling pathways are implicated in GBMs-induced apoptosis. Autophagy, unlike apoptosis and necroptosis which are already clear cell death types, plays a vital pro-survival role in cell homeostasis, so its role in cell death should be carefully considered. However, GBMs always induce unrestrained autophagy accelerating cell death. GBMs trigger autophagy through inducing autophagosome accumulation and lysosome impairment. Mitochondrial dysfunction, ER stress, TLRs signaling pathways, and p38 MAPK and NF-κB pathways participate in GBMs-induced autophagy. Programmed necrosis can be activated by RIP kinases, PARP, and TLR-4 signaling in macrophages after GBMs exposure. Though apoptosis, autophagy, and necroptosis are distinguished by some characteristics, their numerous signaling pathways comprise an interconnected network and correlate with each other, such as the TLRs, p53 signaling pathways, and the Beclin-1 and Bcl-2 interaction. A better understanding of the mechanisms of PCD induced by GBMs may allow for a thorough study of the toxicology of GBMs and a more precise determination of the consequences of human exposure to GBMs. These determinations will also benefit safety assessments of the biomedical and therapeutic applications of GBMs.

Anti-WASP intrabodies inhibit inflammatory responses induced by Toll-like receptors 3, 7, and 9, in macrophages

Wiskott-Aldrich syndrome protein (WASP) is an adaptor molecule in immune cells. Recently, we showed that the WASP N-terminal domain interacted with the SH3 domain of Bruton's tyrosine kinase (Btk), and that the complex formed by WASP and Btk was important for TLR2 and TLR4 signaling in macrophages. Several other studies have shown that Btk played important roles in modulating innate immune responses through TLRs in immune cells. Here, we evaluated the significance of the interaction between WASP and Btk in TLR3, TLR7, and TLR9 signaling. We established bone marrow–derived macrophage cell lines from transgenic (Tg) mice that expressed intracellular antibodies (intrabodies) that specifically targeted the WASP N-terminal domain. One intrabody comprised the single-chain variable fragment and the other comprised the light-chain variable region single domain of an anti-WASP N-terminal monoclonal antibody. Both intrabodies inhibited the specific interaction between WASP and Btk, which impaired the expression of TNF-α, IL-6, and IL-1β in response to TLR3, TLR7, or TLR9 stimulation. Furthermore, the intrabodies inhibited the phosphorylation of both nuclear factor (NF)-κB and WASP in response to TLR3, TLR7, or TLR9 stimulation, in the Tg bone marrow-derived macrophages. These results suggested that WASP plays important roles in TLR3, TLR7, and TLR9 signaling by associating with Btk in macrophages.

Differential role for p120-catenin in regulation of TLR4 signaling in macrophages.

Activation of TLR signaling through recognition of pathogen-associated molecular patterns is essential for the innate immune response against bacterial and viral infections. We have shown that p120-catenin (p120) suppresses TLR4-mediated NF-кB signaling in LPS-challenged endothelial cells. In this article, we report that p120 differentially regulates LPS/TLR4 signaling in mouse bone marrow-derived macrophages. We observed that p120 inhibited MyD88-dependent NF-κB activation and release of TNF-α and IL-6, but enhanced TIR domain-containing adapter-inducing IFN-β-dependent IFN regulatory factor 3 activation and release of IFN-β upon LPS exposure. p120 silencing diminished LPS-induced TLR4 internalization, whereas genetic and pharmacological inhibition of RhoA GTPase rescued the decrease in endocytosis of TLR4 and TLR4-MyD88 signaling, and reversed the increase in TLR4-TIR domain-containing adapter-inducing IFN-β signaling induced by p120 depletion. Furthermore, we demonstrated that altered p120 expression in macrophages regulates the inflammatory phenotype of LPS-induced acute lung injury. These results indicate that p120 functions as a differential regulator of TLR4 signaling pathways by facilitating TLR4 endocytic trafficking in macrophages, and support a novel role for p120 in influencing the macrophages in the lung inflammatory response to endotoxin.

LPS induced circadian phase shift in macrophages

The circadian clock regulates the expression of a subset of genes important in cellular function. Recent studies have revealed that the recognition of pathogens and subsequent initiation of inflammatory response is strongly regulated by a macrophage‐intrinsic clock. We hypothesized that the circadian pattern of gene expression can be influenced by inflammatory stimuli and that changes in circadian function in inflammatory cells can influence cardiovascular disease. To investigate circadian rhythms in inflammatory cells, peritoneal macrophage were isolated from Per2‐luciferase transgenic mice. Luminescence was measured following exposure of Per2‐Luc macrophages to various stimuli. Surprisingly we found that the LPS significantly lengthened the circadian period in macrophages. Real‐time PCR data suggests that the expressions of core circadian genes (Bmal1 and Per2) are suppressed following LPS treatment. To investigate the mechanism by which LPS impacts circadian oscillation, we targeted TLR4 signaling. The TLR4 antagonist (LPS‐RS) which directs under‐acylated LPS competitively binding on MD‐2 reversed the phase‐shift effect caused by LPS, while CLI‐095, which blocks the intracellular domain of TLR4 showed no effect on circadian gene oscillation. To confirm the role of TLR4 in circadian phase‐shift by LPS, we plan to genetically manipulate TLR4 signaling in macrophages and in other cell lines.

Effect of PAR-2 Deficiency in Mice on KC Expression after Intratracheal LPS Administration

Protease activated receptors (PAR) have been shown to play a role in inflammation. PAR-2 is expressed by numerous cells in the lung and has either proinflammatory, anti-inflammatory, or no effect depending on the model. Here, we examined the role of PAR-2 in a model of LPS-induced lung inflammation. We found that PAR-2-deficient mice had significantly less KC expression in bronchial lavage fluid compared with wild-type mice but there was no difference in MIP-2 or TNF-α expression. We also found that isolated alveolar and resident peritoneal macrophages lacking PAR-2 showed a similar deficit in KC after LPS stimulation without differences in MIP-2 or TNF-α. Infiltration of neutrophils and macrophages into the lung following LPS administration was not affected by an absence of PAR-2. Our results support the notion that PAR-2 plays a role in LPS activation of TLR4 signaling in macrophages.

Group X Secretory Phospholipase A2 Enhances TLR4 Signaling in Macrophages

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.

Glia Maturation Factor-Gamma Negatively Modulates TLR4 Signaling In Macrophages Induced by Lipopolysaccharide (LPS).

AbstractAbstract 1482Toll-like receptor 4 (TLR4) plays a critical role in innate immunity that recognize pathogenic molecules and trigger inflammatory response. However, excessive activation of TLR4 activation may contribute to pathogenesis of autoimmune and inflammatory diseases. Therefore, the negative regulation of TLR4-triggered inflammatory response attracts much attention in recent years. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response in macrophages. Glia maturation factor gamma (GMFG), a member of the ADF/cofilin family of proteins that regulate actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in macrophages immune response remains unclear. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction to LPS in macrophages by knockdown of GMFG using small-interfering RNA approach. We show here that knockdown of GMFG significantly enhanced LPS-induced production of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-8, and MCP-1 in human peripheral blood monocytes-derived macrophage as determined by quantitative real time-PCR and confirmed by enzyme-linked immunosorbent assay. Silencing of GMFG expression potentiates LPS-induced activation of p38, ERK1/2 and NF-kappaB signaling pathways by Western blot analysis. Moreover, luciferase assay revealed that gene silencing of GMFG promoted LPS-induced NF-kappaB activity for ∼2.5- to 4-fold. Furthermore, we found that TLR4 protein expression level were higher in GMFG-silenced macrophage compared with that of the control siRNA-transfected macrophages after stimulated with LPS for 1 hour. These results suggest that GMFG negatively regulation of TLR4 signaling-induced inflammatory cytokines by modulation of TLR4 expression levels and its down-stream NF-kappaB and p38 MAPK signaling pathway. In summary, we report that GMFG, in macrophage, function as a novel negative regulator that participates in the regulation of TLR4-signaling pathway, implicating that macrophage-specific modulation of GMFG may be beneficial in the treatment of inflammation as well as autoimmune disease. Disclosures:No relevant conflicts of interest to declare.

Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

Beta-arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that beta-arrestin-1 (beta-arr-1) and -2 knockout (KO) mice are protected from TLR4-mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild-type (WT) and beta-arr-1 and -2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS-induced inflammatory cytokine levels in the plasma were markedly decreased in both beta-arr-1 and -2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11(b+) and CD11(b-) populations) from WT, beta-arr-1, and -2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS-induced inflammatory cytokines were significantly blocked in both splenocyte populations from the beta-arr-2 KO compared to the WT mice. This effect in the beta-arr-1 KO mice, however, was restricted to the CD11(b-) splenocytes. Our studies further indicate that regulation of cytokine production by beta-arrestins is likely independent of MAPK and IkappaBalpha-NFkappaB pathways. Our results, however, suggest that LPS-induced chromatin modification is dependent on beta-arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by beta-arrestins in vivo. Taken together, these results indicate that beta-arr-1 and -2 mediate LPS-induced cytokine secretion in a cell-type specific manner and that both beta-arrestins have overlapping but non-redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice.

Regulation of TLR4 signaling in macrophages by Macrophage Stimulating Protein, the ligand for the Ron receptor tyrosine kinase (134.20)

Abstract The Ron receptor tyrosine kinase regulates the balance between classical (M1) and alternative (M2) macrophage activation. The ligand for Ron, macrophage stimulating protein (MSP), inhibits the expression of iNOS, while promoting the expression of arginase I in primary murine macrophages. Ron-/- mice express elevated levels of IL-12 following endotoxin administration, resulting in increased serum IFNγ levels and enhanced susceptibility to septic shock. Here, we demonstrate that MSP inhibits LPS-induced IL-12p40 expression in primary macrophages. MSP does not inhibit the MyD88-independent activation of IRF3 and production of IFNβ in response to LPS, but limits the IFNβ-dependent phosphorylation of Stat1 following LPS stimulation. While MSP does not inhibit LPS-induced Tak1 phosphorylation or Map kinase activation in primary macrophages, the induction of IκB kinase (IKK) activity, IκB degradation and DNA binding of NFκB following LPS stimulation are delayed in the presence of MSP. In addition, Ron inhibits serine phosphorylation of p65 and NFκB transcriptional activity induced by LPS stimulation of TLR4. Finally, MSP inhibits the NFκB-dependent upregulation of the nuclear IκB family member, IκBζ, while inducing expression of the Stat3-regulated nuclear IκB family member, Bcl-3. LPS also induces expression of Ron and Sf-Ron in primary macrophages, suggesting that the upregulation of Ron by LPS could play a role in limiting subsequent LPS responses in macrophages.

TLR2 and TLR4 signaling in macrophages is negatively regulated by a Lyn-PI3K module and promoted by SHIP1

We demonstrate here that the Src family kinase Lyn negatively regulates Toll-like receptor (TLR) signaling in bone marrow-derived macrophages (BMMΦs) and in vivo. Lyn-/- BMMΦs produced and secreted significantly more IL-6, TNF-α, and IFN-α/β compared to WT BMMΦs, indicating that Lyn is able to control both MyD88- and TRIF-dependent signaling pathways downstream of TLR4. CD14 was not involved in this type of regulation. Moreover, Lyn attenuated proinflammatory cytokine production in BMMΦs in response to the TLR2 ligand, FSL-1. In agreement with these in vitro experiments, Lyn-deficient mice produced higher amounts of proinflammatory cytokines than WT mice after i. v. injection of LPS or lipopeptide. Though Lyn clearly acted as a negative regulator downstream of TLR4, it did not, different to what was proposed previously, alter the process of LPS tolerance. Stimulation with a low dose of LPS resulted in reduced production of proinflammatory cytokines after a subsequent stimulation with a high dose of LPS in both WT and Lyn-/- BMMΦs as well as in vivo. Mechanistically, Lyn interacted with PI3K and in correlation, PI3K inhibition resulted in increased LPS-triggered cytokine production. In this line, SHIP1-deficient BMMΦs, exerting enhanced PI3K-pathway activation, produced less cytokines compared to WT BMMΦs. In conclusion, Lyn is a negative regulator of TLR-induced cytokine production in vitro and in vivo and acts, at least in part, via PI3K.

Lactate Boosts TLR4 Signaling and NF-κB Pathway-Mediated Gene Transcription in Macrophages via Monocarboxylate Transporters and MD-2 Up-Regulation

It has been shown that lactate induces insulin resistance. However, the underlying mechanisms have not been well understood. Based on our observation that lactate augments LPS-stimulated inflammatory gene expression, we proposed that lactate may enhance TLR4 signaling in macrophages, which has been shown to play an important role in insulin resistance in adipocytes. In this study, we demonstrated that lactate stimulated MD-2, a coreceptor for TLR4 signaling activation, NF-kappaB transcriptional activity, and the expression of inflammatory genes in human U937 histiocytes (resident macrophages). Similar enhancement of the inflammatory gene expression by lactate was also observed in human monocyte-derived macrophages. The essential role of MD-2 in lactate-augmented TLR4 signaling was confirmed by observation that the suppression of MD-2 expression by small interfering RNA led to significant inhibition of inflammatory gene expression. To further elucidate how lactate treatment enhances TLR4 activation, we showed that the augmentation of inflammatory gene expression by lactate was abrogated by antioxidant treatment, suggesting a critical role of reactive oxygen species in the enhancement of TLR4 activation by lactate. Finally, we showed that alpha-cyano-4-hydroxycinnamic acid, a classic inhibitor for monocarboxylate transporters, blocked lactate-augmented inflammatory gene expression and nuclear NF-kappaB activity, indicating that lactate transport through monocarboxylate transporters is required for lactate-enhanced TLR4 activation. Collectively, this study documents that lactate boosts TLR4 activation and NF-kappaB-dependent inflammatory gene expression via monocarboxylate transporters and MD-2 up-regulation.