Surface TLR4 Research Articles

Hyperoxia/Hypoxia Exposure Primes a Sustained Pro-Inflammatory Profile of Preterm Infant Macrophages Upon LPS Stimulation.

Preterm infants are highly susceptible to sustained lung inflammation, which may be triggered by exposure to multiple environmental cues such as supplemental oxygen (O2) and infections. We hypothesized that dysregulated macrophage (MФ) activation is a key feature leading to inflammation-mediated development of bronchopulmonary dysplasia (BPD) in preterm infants. Therefore, we aimed to determine age-dependent differences in immune responses of monocyte-derived MФ comparing cord blood samples derived from preterm (n=14) and term (n=19) infants as well as peripheral blood samples from healthy adults (n=17) after lipopolysaccharide (LPS) exposure. Compared to term and adult MФ, LPS-stimulated preterm MФ showed an enhanced and sustained pro-inflammatory immune response determined by transcriptome analysis, cytokine release inducing a RORC upregulation due to T cell polarization of neonatal T cells, and TLR4 surface expression. In addition, a double-hit model was developed to study pulmonary relevant exposure factors by priming MФ with hyperoxia (O2 = 65%) or hypoxia (O2 = 3%) followed by lipopolysaccharide (LPS, 100ng/ml). When primed by 65% O2, subsequent LPS stimulation in preterm MФ led to an exaggerated pro-inflammatory response (e.g. increased HLA-DR expression and cytokine release) compared to LPS stimulation alone. Both, exposure to 65% or 3% O2 together with subsequent LPS stimulation, resulted in an exaggerated pro-inflammatory response of preterm MФ determined by transcriptome analysis. Downregulation of two major transcriptional factors, early growth response gene (Egr)-2 and growth factor independence 1 (Gfi1), were identified to play a role in the exaggerated pro-inflammatory response of preterm MФ to LPS insult after priming with 65% or 3% O2. Preterm MФ responses to LPS and hyperoxia/hypoxia suggest their involvement in excessive inflammation due to age-dependent differences, potentially mediated by downregulation of Egr2 and Gfi1 in the developing lung.

Red blood cells join the ranks as immune sentinels.

New research shows that red blood cells express surface TLR9 that binds cell-free DNA, leading to erythrophagocytosis and innate immune activation.

Vernal keratoconjunctivitis activity induces decrease of ocular surface CD14, TLR-4 and TLR-9 expression.

CD14 is involved in the modulation of immune reaction via toll-like receptors (TLR) and may influence the development of allergic diseases. The role of CD14 in vernal keratoconjunctivitis (VKC) has not yet been investigated. The aim of this study is to evaluate changes of tear soluble sCD14 and conjunctival CD14, TLR-4 and 9 expression in patients with VKC in the active and quiescent phases. Eighteen patients with VKC during active inflammation (group A, N = 9), in the quiescent phase (group Q, N = 5) and after recovery (group R, N = 4) and 10 healthy subjects were included. Expression of sCD14 in tears and of CD14, TLR-4, and TLR-9 by conjunctival epithelium were evaluated by Western Blot in all groups. Expression of tear sCD14 and of conjunctival CD14, TLR-4, and TLR-9 was significantly decreased in group A when compared with healthy subjects and with VKC group Q and R. Lower expression of sCD14, CD14, TLR-4, and TLR-9 were significantly correlated with the severity of papillary reaction, while the lower sCD14 was correlated with severity of conjunctival hyperemia. Tear sCD14, and conjunctival CD14, TLR4, and TLR-9 decreased during ocular surface inflammatory reaction in patients with VKC. CD14 and TLRs ocular surface evaluation may represent biomarkers of VKC activity and novel therapeutic target.

Extraction, Structural Characterisation, and Immunomodulatory Properties of Edible Amanitahemibapha subspecies javanica (Corner and Bas) Mucilage Polysaccharide as a Potential of Functional Food.

This research aimed to extract mucilage polysaccharides (MP) from Amanita hemibapha subspecies javanica (Corner and Bas), and further fractionate them using anion-exchange chromatography, yielding two fractions (MPF1 and MPF2). The crude extract, and fractions mainly consisted of carbohydrates (83.5–93.2%) with minor amounts of proteins (5.40–7.20%), and sulphates (1.40–9.30%). Determination of the monosaccharide composition revealed that glucose was the major unit, followed by galactose, mannose, rhamnose, and arabinose. The average molecular weight (MW) of the crude extract and fractions was in the range 104.0–479.4 × 103 g/mol. Interestingly, the crude extract, and fractions did not cause any toxic effect in RAW264.7 cells. However, they stimulated the RAW264.7 cells to release nitric oxide and cytokines through the activation of nuclear factor-kappa B (NF-κB), and mitogen-activated protein kinase (MAPK) pathways via cell surface TLR4. Structural analysis of the most immunestimulating extract fraction, MPF2, revealed that the main backbone consisted of α-D-(1→6)-glucopyranoside. These results suggest that the MPs derived from A. hemibapha subspecies javanica (Corner and Bas) are potent in enhancing immunity; hence, they can be used as a functional ingredient in food products.

Erlotinib protests against LPS-induced parthanatos through inhibiting macrophage surface TLR4 expression

Sepsis is a life-threatening cascading systemic inflammatory response syndrome on account of serve infection. In inflamed tissues, activated macrophages generate large amounts of inflammatory cytokines reactive species, and are exposed to the damaging effects of reactive species. However, comparing with necroptosis and pyroptosis, so far, there are few studies focusing on the overproduction-related cell death, such as parthanatos in macrophage during sepsis. In LPS-treated macrophage, we observed PARP-1 activation, PAR formation and AIF translocation. All these phenomena could be inhibited by both erlotinib and 3-AB, indicating the presence of parthanatos in endotoxemia. We further found that LPS induced the increase of cell surface TLR4 expression responsible for the production of ROS and subsequent parthanatos in endotoxemia. All these results shed a new light on how TLR4 regulating the activation of PARP-1 by LPS in macrophage.

TLR4 and TNFR1 blockade dampen M1 macrophage activation and shifts them towards an M2 phenotype.

The Gram-negative bacterial lipopolysaccharide (LPS)-induced sepsis has emerged as major concern worldwide due to the pressing need to develop its effective treatment strategies which is not available yet. LPS is the major causative agent in the pathogenesis of septic shock. In macrophages, LPS interacts with cell surface TLR4 leading to reactive oxygen species (ROS), TNF-α, IL-1β production, oxidative stress and markedly activated the MAPKs and NF-kB pathway. Post cell isolation, the macrophages were subjected to administration with neutralizing antibodies to TLR4 and TNFR1 either alone or in combination prior to LPS challenge. Subsequently, we performed flow cytometric analysis along with Western blots, reactive oxygen species production, and TNF-α, IL-1β release. Outcomes suggested that the dual blockade of TLR4 and TNFR1 was indeed beneficial in shifting the LPS-induced M1 polarization towards M2. Both TLR4 and TNFR1 exhibited dependency during LPS stimulation. Furthermore, the switch towards the M2 phenotype might be responsible for the decreased levels of TNF-α, IL-1β, NO, and superoxide anion and the simultaneous elevation in the activity level of anti-oxidant enzymes like SOD, CAT (catalase), and GSH content in the isolated peritoneal macrophages. Simultaneous blocking of both TLR4 and TNFR1 also showed reduced expression of NF-kB, JNK, and COX-2 by promoting TNFR2-mediated TNF-α signaling. The increased arginase activity further confirmed the polarization towards M2. Thus it may be inferred that dual blockade of TLR4 and TNFR1 might be an alternative therapeutic approach for regulating of sepsis in future.

Matrix lumican endocytosed by immune cells controls receptor ligand trafficking to promote TLR4 and restrict TLR9 in sepsis

Infections and inflammation are profoundly influenced by the extracellular matrix (ECM), but their molecular underpinnings are ill defined. Here, we demonstrate that lumican, an ECM protein normally associated with collagens, is elevated in sepsis patients' blood, while lumican-null mice resolve polymicrobial sepsis poorly, with reduced bacterial clearance and greater body weight loss. Secreted by activated fibroblasts, lumican promotes Toll-like receptor (TLR) 4 response to bacterial lipopolysaccharides (LPS) but restricts nucleic acid-specific TLR9 in macrophages and dendritic cells. The underlying mechanism involves lumican attachment to the common TLR coreceptor CD14 and caveolin 1 (Cav1) in lipid rafts on immune cell surfaces via two epitopes, which may be cryptic in collagen-associated lumican. The Cav1 binding epitope alone is sufficient for cell surface enrichment of Cav1, while both are required for lumican to increase cell surface TLR4, CD14, and proinflammatory cytokines in response to LPS. Endocytosed lumican colocalizes with TLR4 and LPS and promotes endosomal induction of type I interferons. Lumican-null macrophages show elevated TLR9 in signal-permissive endolysosomes and increased response, while wild types show lumican colocalization with CpG DNA but not TLR9, consistent with a ligand sequestering, restrictive role for lumican in TLR9 signaling. In vitro, lumican competes with CD14 to bind CpG DNA; biglycan, a lumican paralog, also binds CpG DNA and suppresses TLR9 response. Thus, lumican and other ECM proteins, synthesized de novo or released from collagen association during ECM remodeling, may be internalized by immune cells to regulate their transcriptional programs and effector responses that may be harnessed in future therapeutics.

Impact of Coffee Containing Medium-Chain Triglyceride Oil and Ghee on Markers of Cellular Inflammation in Young Healthy Humans

ObjectivesLow-carbohydrate, high-fat “ketogenic” food supplements have become increasingly popular in recent years with claims of improving body composition and cognition, while reducing hunger. However, acute consumption of high-fat foods has been shown to promote dietary endotoxemia; the release of bacterial lipopolysaccharide from the gut into the blood, which is linked to proinflammatory responses through activating toll like receptor (TLR) 4 on circulating monocytes. Bulletproof Coffee is a popular high-fat beverage consisting of coffee, medium chain triglyceride (MCT) oil, and grass-fed ghee. The purpose of this study is to determine whether consuming this high-fat coffee beverage would impact cellular inflammation assessed by increases in the number of circulating monocytes and monocyte surface TLR4 expression. We hypothesize that consuming one high-fat “Bulletproof Coffee” will elevate concentrations of circulating monocytes and increase TLR4 expression when compared to a black coffee comparator drink. MethodsThis study is a single-blind (researcher), randomized crossover design wherein participants consume either a freshly prepared coffee (1 pod with 12oz water; ∼1 kcal), or high-fat bulletproof coffee (1 pod with 12 oz water containing 1 tbsp MCT oil and 1 tbsp ghee; 27 g fat; ∼250 kcal) separated by ∼7 days. Participants provided blood samples in the fasted state and at 60- and 180-minutes following beverage consumption. Blood samples were analyzed by flow cytometry. ResultsSix healthy adults (n = 5 females) aged 25 ± 8 years who consume coffee regularly have completed both conditions. Preliminary statistical analysis using a linear mixed model has shown no significant time x condition interaction (P = 0.184) or main effect of time (P = 0.211) for the concentration of circulating monocytes. Similarly, no interaction (P = 0.675) or main effects of time (P = 0.337) were observed for monocyte surface TLR4 expression. ConclusionsPreliminary data suggests that consuming a single high-fat bulletproof coffee does not appear to increase circulating monocyte concentrations or monocyte TLR4 expression. Further research will be required to determine whether acute consumption of a high-fat coffee beverage impacts inflammation in humans. Funding SourcesThis study is funded by a Natural Sciences and Engineering Research Council (NSERC) of Canada grant.

Acute Alcohol Intoxication Modulates Monocyte Subsets and Their Functions in a Time-Dependent Manner in Healthy Volunteers.

BackgroundExcessive alcohol intake is associated with adverse immune response-related effects, however, acute and chronic abuse differently modulate monocyte activation. In this study, we have evaluated the phenotypic and functional changes of monocytes in acutely intoxicated healthy volunteers (HV).MethodsTwenty-two HV consumed individually adjusted amounts of alcoholic beverages until reaching a blood alcohol level of 1‰ after 4h (T4). Peripheral blood was withdrawn before and 2h (T2), 4h (T4), 6h (T6), 24h (T24), and 48h (T48) after starting the experiment and stained for CD14, CD16 and TLR4. CD14brightCD16-, CD14brightCD16+ and CD14dimCD16+ monocyte subsets and their TLR4 expression were analyzed by flow cytometry. Inflammasome activation via caspase-1 in CD14+ monocytes was measured upon an ex vivo in vitro LPS stimulation. Systemic IL-1β and adhesion capacity of isolated CD14+ monocytes upon LPS stimulation were evaluated.ResultsThe percentage of CD14+ monocyte did not change following alcohol intoxication, whereas CD14brightCD16- monocyte subset significantly increased at T2 and T24, CD14brightCD16+ at T2, T4 and T6 and CD14dimCD16+ at T4 and T6. The relative fraction of TLR4 expressing CD14+ monocytes as well as the density of TLR4 surface presentation increased at T2 and decreased at T48 significantly. TLR4+CD14+ monocytes were significantly enhanced in all subsets at T2. TLR4 expression significantly decreased in CD14brightCD16- at T48, in CD14brightCD16+ at T24 and T48, increased in CD14dimCD16+ at T2. IL-1β release upon LPS stimulation decreased at T48, correlating with TLR4 receptor expression. Alcohol downregulated inflammasome activation following ex vivo in vitro stimulation with LPS between T2 and T48 vs. T0. The adhesion capacity of CD14+ monocytes decreased from T2 with significance at T4, T6 and T48. Following LPS administration, a significant reduction of adhesion was observed at T4 and T6.ConclusionsAlcohol intoxication immediately redistributes monocyte subsets toward the pro-inflammatory phenotype with their subsequent differentiation into the anti-inflammatory phenotype. This is paralleled by a significant functional depression, suggesting an alcohol-induced time-dependent hyporesponsiveness of monocytes to pathogenic triggers.

CpG ODN as an adjuvant arouses the vigor of B cells by relieving the negative regulation of surface TLR9 to enhance the antibody response to vaccine.

The surface Toll-like receptor 9 (sTLR9) has been identified on the surface of the B cells and was presumed to be a negative regulator of B cell responses. CpG ODN, a TLR9 agonist, has been successfully used as an adjuvant of hepatitis B vaccine to enhance antibody responses. However, it is unknown whether the sTLR9 is involved in regulating the activation and maturation of B cells in the antibody responses induced by CpG ODN-adjuvanted vaccines. In this study, we immunized mice with hepatitis B vaccine adjuvanted by CpG ODN (CpG 5805) and found that CpG 5805 enhanced the antibody response to vaccine and meanwhile down-regulated the sTLR9 levels on B cells. With antibody feeding assay and flow cytometry analysis, we further found that CpG 5805 induced a movement of the sTLR9 in B cells, internalized first and then mobilized to endosomes. Accompanied with the movement, CD80, CD86, CD40, and MHC II molecules were significantly up-regulated on the B cells. Interestingly, the B cells with internalized sTLR9 enlarged morphologically, and the sTLR9 levels were obviously lower and CD40 levels were obviously higher on the enlarged B cells. Together, the data presented here uncover that CpG ODN can induce the mobilization and relocation of sTLR9 in B cells, thereby triggering the B cell vigor by relieving the negative regulatory effect of sTLR9 on B cells, which may be one of the mechanisms for CpG ODN acting as a vaccine adjuvant to enhance the antibody response.Key points• CpG ODN-enhanced antibody response positively associates with B cell sTLR9 reduction.• CpG ODN reduces the sTLR9 levels by relocating it from B cell surface to endosomes.• sTLR9 reduction arouses B cell vigor via promoting B cell maturation and activation. Graphical Abstract.

The Role of TLR2 and TLR4 in Recognition and Uptake of the Apicomplexan Parasite Eimeria bovis and Their Effects on NET Formation.

Background: Bovine polymorphonuclear neutrophils (PMN) constitutively express the Toll-like receptors (TLRs) TLR2 and TLR4 and have been shown to generate Neutrophil extracellular traps (NETs) upon exposure to Eimeria bovis. The present work investigated the role of TLR2 and TLR4 in the recognition and uptake of E. bovis sporozoites, IL-8 production and neutrophil extracellular trap (NET) formation. Methods: TLR expression was performed by flow cytometric analysis on PMN exposed to live carboxyfluorescein succinimidyl ester (CFSE)-stained sporozoites. Supernatants of PMN exposed to different E. bovis sporozoite preparations and antigens in the absence or presence of TLR antibodies were assessed for IL-8 secretion. Cells were exposed to sporozoite preparations and assessed for the activation of transcription factor NF-κB using a luciferase reporter assay. Immunofluorescence analysis was done to investigate TLR2 and TLR4 surface expression and NET formation on bovine PMN exposed to vital sporozoites. Results: we observed significantly increased TLR2 and TLR4 expression with a mean increase in expression that was greater for TLR2 than TLR4. This upregulation neither inhibited nor promoted sporozoite phagocytosis by bovine PMN. Live sporozoites together with anti-TLR2 mAb resulted in a significant enhancement of IL-8 production. NF-κB activation was more strongly induced in TLR2-HEK cells than in TLR4/MD2-HEK cells exposed to heat-killed sporozoites and antigens. Immunofluorescence analysis showed TLR-positive signals on the surface of PMN and concomitant NET formation. Conclusions: This is the first report on E. bovis-induced concomitant TLR2 and TLR4 expression during bovine PMN-derived NETosis.

Intracellular TLRs of Mast Cells in Innate and Acquired Immunity.

Mast cells (MCs) distribute to interface tissues with environment, such as skin, airway, and gut mucosa, thereby functioning as the sentinel against invading allergens and pathogens. To respond to and exclude these external substances promptly, MCs possess granules containing inflammatory mediators, including heparin, proteases, tumor necrosis factor, and histamine, and produce these mediators as a consequence of degranulation within minutes of activation. As a delayed response to external substances, MCs de novo synthesize inflammatory mediators, such as cytokines and chemokines, by sensing pathogen- and damage-associated molecular patterns through their pattern recognition receptors, including Toll-like receptors (TLRs). A substantial number of studies have reported immune responses by MCs through surface TLR signaling, particularly TLR2 and TLR4. However, less attention has been paid to immune responses through nucleic acid-recognizing intracellular TLRs. Among intracellular TLRs, human and rodent MCs express TLR3, TLR7, and TLR9, but not TLR8. Some virus infections modulate intracellular TLR expression in MCs. MC-derived mediators, such as histamine, cysteinyl leukotrienes, LL-37, and the granulocyte-macrophage colony-stimulating factor, have also been reported to modulate intracellular TLR expression in an autocrine and/or paracrine fashion. Synthetic ligands for intracellular TLRs and some viruses are sensed by intracellular TLRs of MCs, leading to the production of inflammatory cytokines and chemokines including type I interferons. These MC responses initiate and facilitate innate responses and the subsequent recruitment of additional innate effector cells. MCs also associate with the regulation of adaptive immunity. In this overview, the expression of intracellular TLRs in MCs and the recognition of pathogens, including viruses, by intracellular TLRs in MCs were critically evaluated.

Naringenin suppresses Toll-like receptor 2-mediated inflammatory responses through inhibition of receptor clustering on lipid rafts.

Toll‐like receptors (TLRs) are important innate immune receptors that sometimes cause excessive inflammatory responses and a perpetuated inflammatory loop that can be involved in inflammatory and autoimmune diseases. TLR2 recognizes bacterial lipoproteins in association with TLR1 or TLR6, and triggers inflammatory responses through activation of the transcription factor NF‐κB. Naringenin, a type of citrus flavonoid, has been shown to possess anti‐inflammatory properties, but its detailed action against TLR2 remains to be fully elucidated. The present study was designed to determine whether naringenin affects the inflammatory responses triggered by TLR2. Naringenin inhibited proinflammatory cytokine production and attenuated NF‐κB activation in cells stimulated with a synthetic triacylated‐type lipopeptide known as a TLR2/TLR1 ligand, as well as a synthetic diacylated‐type lipopeptide known as a TLR2/TLR6 ligand. Moreover, a similar inhibitory effect was observed in cells stimulated with a crude lipophilic fraction extracted from Staphylococcus aureus cell walls and in cells stimulated with S. aureus cells. Furthermore, we showed that such an effect is caused by inhibition of TLR2 clustering in lipid rafts on the cell membrane. These results suggest that naringenin suppresses the inflammatory responses induced by TLR2 signal transduction. Our findings indicate a novel anti‐inflammatory property of naringenin, mediated through the regulation of cell surface TLR2 functioning.

Astragalin Retards Atherosclerosis by Promoting Cholesterol Efflux and Inhibiting the Inflammatory Response via Upregulating ABCA1 and ABCG1 Expression in Macrophages.

Lipid metabolism disorder and inflammatory response are considered to be the major causes of atherosclerogenesis. Astragalin, the most important functional component of flavonoid obtained from persimmon leaves, has the hypolipidemic effects. However, it is unknown, how astragalin protects against atherosclerosis. The aim of this study was to observe the effects of astragalin on cholesterol efflux and inflammatory response and to explore the underlying mechanisms. Our results showed that astragalin upregulated the expression of ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1), promoted cholesterol efflux, and suppressed foam cell formation. Inhibition of the PPARγ/LXRα pathway abrogated the promotive effects of astragalin on both transporter expression and cholesterol efflux. In addition, treatment of astragalin markedly decreased the secretion of inflammatory factors, including interleukin 6, monocyte chemotactic protein 1, tumor necrosis factor α, and interleukin 1β. Mechanistically, astragalin upregulated ABCA1 and ABCG1 expression, which in turn reduced TLR4 surface levels and inhibited NF-κB nuclear translocation. Consistently, astragalin reduced atherosclerotic plaque area in apoE-/- mice. Taken together, these findings suggest that astragalin protects against atherosclerosis by promoting ABCA1- and ABCG1-mediated cholesterol efflux and inhibiting proinflammatory mediator release.

An alternative model for type I interferon induction downstream of human TLR2

Surface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines, including type I interferons (IFN-I). Downstream of TLR4, IFNβ secretion is only vigorously triggered in macrophages when the receptor undergoes endocytosis and switches signaling adaptor; surface TLR4 engagement predominantly induces proinflammatory cytokines via the signaling adaptor MyD88. It is unclear whether this dichotomy is generally applicable to other TLRs, cell types, or differentiation states. Here, we report that diverse TLR2 ligands induce an IFN-I response in human monocyte-like cells, but not in differentiated macrophages. This TLR2-dependent IFN-I signaling originates from the cell surface and depends on MyD88; it involves combined activation of the transcription factors IRF3 and NF-κB, driven by the kinases TBK1 and TAK1-IKKβ, respectively. TLR2-stimulated monocytes produced modest IFNβ levels that caused productive downstream signaling, reflected by STAT1 phosphorylation and expression of numerous interferon-stimulated genes. Our findings reveal that the outcome of TLR2 signaling includes an IFN-I response in human monocytes, which is lost upon macrophage differentiation, and differs mechanistically from IFN-I-induction through TLR4. These findings point to molecular mechanisms tailored to the differentiation state of a cell and the nature of receptors activated to control and limit TLR-triggered IFN-I responses.

S100A8 and S100A9 Promote Apoptosis of Chronic Eosinophilic Leukemia Cells.

S100A8 and S100A9 function as essential factors in inflammation and also exert antitumor or tumorigenic activity depending on the type of cancer. Chronic eosinophilic leukemia (CEL) is a rare hematological malignancy having elevated levels of eosinophils and characterized by the presence of the FIP1L1-PDGFRA fusion gene. In this study, we examined the pro-apoptotic mechanisms of S100A8 and S100A9 in FIP1L1-PDGFRα+ eosinophilic cells and hypereosinophilic patient cells. S100A8 and S100A9 induce apoptosis of the FIP1L1-PDGFRα+ EoL-1 cells via TLR4. The surface TLR4 expression increased after exposure to S100A8 and S100A9 although total TLR4 expression decreased. S100A8 and S100A9 suppressed the FIP1L1-PDGFRα-mediated signaling pathway by downregulating FIP1L1-PDGFRα mRNA and protein expression and triggered cell apoptosis by regulating caspase 9/3 pathway and Bcl family proteins. S100A8 and S100A9 also induced apoptosis of imatinib-resistant EoL-1 cells (EoL-1-IR). S100A8 and S100A9 blocked tumor progression of xenografted EoL-1 and EoL-1-IR cells in NOD-SCID mice and evoked apoptosis of eosinophils derived from hypereosinophilic syndrome as well as chronic eosinophilic leukemia. These findings may contribute to a progressive understanding of S100A8 and S100A9 in the pathogenic and therapeutic mechanism of hematological malignancy.

Interplay between RAGE and TLR4 Regulates HMGB1-Induced Inflammation by Promoting Cell Surface Expression of RAGE and TLR4.

Receptor for advanced glycation end-products (RAGE) and TLR4 play an important role in the inflammatory response against High-mobility group box 1 protein (HMGB1), a late proinflammatory cytokine and a damage-associated molecular pattern. As cell surface receptors, both RAGE and TLR4 are constantly trafficking between the cytoplasm and plasma membrane. However, whether TLR4 is related to the intracellular transport of RAGE in HMGB1-induced inflammation remains unknown. In this study, we demonstrated that HMGB1 not only increased RAGE expression in both the cytoplasm and plasma membrane but also upregulated the expression of TLR4 in the plasma membrane. Knocking out of RAGE led to decreased MAPK activation, TLR4 cellular membrane expression, and corresponding inflammatory cytokine generation. Meanwhile, inhibiting MAPK activation also decreased TLR4 surface expression. These results indicated that HMGB1 may bind to cell surface RAGE receptors on the cell surface, leading to MAPK activation, thus promoting TLR4 translocation on the cell surface, but does not regulate its transcription and translation. In contrast, TLR4 can increase the transcription and translation of RAGE, which translocates to the cell surface and is able to bind to more HMGB1. The cell surface receptors TLR4 and RAGE bind to HMGB1, leading to the transcription and secretion of inflammatory cytokines. Finally, we also observed these results in the mice pseudofracture model, which is closely related to HMGB1-induced inflammatory response. All these results demonstrated that the interplay between RAGE and TLR4 are critical for HMGB1-induced inflammatory response.

Addition of High Molecular Weight Hyaluronic Acid to Fibroblast-Like Stromal Cells Modulates Endogenous Hyaluronic Acid Metabolism and Enhances Proteolytic Processing and Secretion of Versican.

We have examined the effect of exogenous linear chain high molecular weight hyaluronic acid (HMW HA) on endogenously synthesized hyaluronic acid (HA) and associated binding proteins in primary cultures of fibroblast-like stromal cells that were obtained by collagenase digestion of the murine peripatellar fat pad. The cultures were expanded in DMEM that was supplemented with fetal bovine serum and basic fibroblast growth factor (bFGF) then exposed to macrophage-colony-stimulating factor (MCSF) to induce macrophage properties, before activation of inflammatory pathways using E. coli lipopolysaccharide (LPS). Under all culture conditions, a significant amount of endogenously synthesized HA localized in LAMP1-positive lysosomal vesicles. However, this intracellular pool was depleted after the addition of exogenous HMW HA and was accompanied by enhanced proteolytic processing and secretion of de novo synthesized versican, much of which was associated with endosomal compartments. No changes were detected in synthesis, secretion, or proteolytic processing of aggrecan or lubricin (PRG4). The addition of HMW HA also modulated a range of LPS-affected genes in the TLR signaling and phagocytosis pathways, as well as endogenous HA metabolism genes, such as Has1, Hyal1, Hyal2, and Tmem2. However, there was no evidence for association of endogenous or exogenous HMW HA with cell surface CD44, TLR2 or TLR4 protein, suggesting that its physiochemical effects on pericelluar pH and/or ionic strength might be the primary modulators of signal transduction and vesicular trafficking by this cell type. We discuss the implications of these findings in terms of a potential in vivo effect of therapeutically applied HMW HA on the modification of osteoarthritis-related joint pathologies, such as pro-inflammatory and degradative responses of multipotent mesenchymal cells residing in the synovial membrane, the underlying adipose tissue, and the articular cartilage surface.

All-trans retinoic acid increases NF-κB activity in PMA-stimulated THP-1 cells upon unmethylated CpG challenge by enhancing cell surface TLR9 expression.

An active metabolite of vitamin A, all-trans retinoic acid (ATRA), is known to exert immunomodulatory functions. This study investigates the possible immune potentiating effect of ATRA on NF-κB activity in human monocytic THP-1 cells after exposure to unmethylated CpG DNA ODN2006. We observed that challenge with ODN2006 significantly enhanced the NF-κB activity of PMA-differentiated THP-1 cells. ATRA synergistically enhanced NF-κB activity of cells, in a concentration- and time-dependent manner. The enhanced NF-κB activity of PMA-differentiated THP-1 cells after ODN2006 challenge was dependent on the RAR/RXR pathway. To determine the mechanism involved in increasing in the NF-κB activity of stimulated THP-1 cells, we examined the effects of PMA and ATRA on the expression of TLR9 (a receptor of ODN2006) in THP-1 cells. PMA treatment significantly enhanced both the intracellular and cell surface expression of TLR9, while ATRA alone showed no effect. However, ATRA synergistically enhanced the cell surface TLR9 expression of PMA-differentiated cells. To determine whether the ATRA-enhanced NF-κB activity is due to the enhanced cell surface TLR9 expression, we examined NF-κB activity after treatment with anti-TLR9 blocking antibody. Results revealed that the anti-TLR9 antibody treatment almost completely reverses the ATRA-enhanced NF-κB activity, suggesting that ATRA enhances NF-κB activity through upregulation of the cell surface TLR9 expression in PMA-differentiated and unmethylated CpG challenged THP-1 cells.

Single dose of anti-high mobility group box 1 protein (HMGB-1) neutralizing antibody ameliorates dysregulated inflammation and restores fracture healing in a polytrauma rat (Rattus norvegicus) model

Abstract Polytrauma (PT) is associated with dysregulated inflammation (DI) and complicated/delayed fracture healing. We recently reported delayed fracture healing and increased circulating levels of HMGB-1 in a rat model of PT compared to osteotomy (OST). Since HMGB-1 binds to and activates TLR2, TLR4 and RAGE surface receptors to induce pro-inflammation, we investigated this protein’s role in delayed healing. We hypothesized that neutralization of cell-free HMGB-1 immediately after PT would resolve DI and restore fracture healing in PT. Anti-HMGB-1 polyclonal antibody (2mg/kg) was administered via IP injection following PT (PT-Ab) and bone/tissue volume (BV/TV) of fractures was evaluated by micro-CT imaging at 5 weeks post PT. As hypothesized, by neutralizing HMGB-1, fracture healing was improved with significantly higher BV/TV ratio in PT-Ab compared to PT rats. Changes to circulating CD68+ CD86+ (M1) and CD68+CD163+ (M2) monocyte/macrophage populations, as well as alterations to CD4+ and CD8+ T cells and their RAGE and TLR4 surface expression, were assessed on 1 and 3 days post-injury (d). While circulating M1 and M2 populations are scarce in naïve rats, these populations elevated post trauma. M1 was significantly higher in PT than in OST and PT-Ab at 1d but not at 3d. M2 was significantly high in only OST at 1d. In contrast to OST, we observed decreased RAGE and TLR4 expression on circulating T cells at 1d in PT and PT-Ab and at 3d in PT but not in OST and PT-Ab. Our observations suggest that elevated levels of HMGB-1 following severe trauma initiate immune dysregulation causing delayed fracture repair and the data presented herein indicates that HMGB-1 may be a therapeutic target to restore normal immune response and normal fracture healing in PT.