Ligand Lipopolysaccharide Research Articles

Sphinganine recruits TLR4 adaptors in macrophages and promotes inflammation in murine models of sepsis and melanoma

After recognizing its ligand lipopolysaccharide, Toll-like receptor 4 (TLR4) recruits adaptor proteins to the cell membrane, thereby initiating downstream signaling and triggering inflammation. Whether this recruitment of adaptor proteins is dependent solely on protein-protein interactions is unknown. Here, we report that the sphingolipid sphinganine physically interacts with the adaptor proteins MyD88 and TIRAP and promotes MyD88 recruitment in macrophages. Myeloid cell-specific deficiency in serine palmitoyltransferase long chain base subunit 2, which encodes the key enzyme catalyzing sphingolipid biosynthesis, decreases the membrane recruitment of MyD88 and inhibits inflammatory responses in in vitro bone marrow-derived macrophage and in vivo sepsis models. In a melanoma mouse model, serine palmitoyltransferase long chain base subunit 2 deficiency decreases anti-tumor myeloid cell responses and increases tumor growth. Therefore, sphinganine biosynthesis is required for the initiation of TLR4 signal transduction and serves as a checkpoint for macrophage pattern recognition in sepsis and melanoma mouse models.

An immune-adrenergic pathway induces lethal levels of platelet-activating factor in mice

Acute immune responses with excess production of cytokines, lipid/chemical mediators, or coagulation factors, often result in lethal damage. In addition, the innate immune system utilizes multiple types of receptors that recognize neurotransmitters as well as pathogen-associated molecular patterns, making immune responses complex and clinically unpredictable. We here report an innate immune and adrenergic link inducing lethal levels of platelet-activating factor. Injecting mice with toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS), cell wall N-glycans of Candida albicans, and the α2-adrenergic receptor (α2-AR) agonist medetomidine induces lethal damage. Knocking out the C-type lectin Dectin-2 prevents the lethal damage. In spleen, large amounts of platelet-activating factor (PAF) are detected, and knocking out lysophospholipid acyltransferase 9 (LPLAT9/LPCAT2), which encodes an enzyme that converts inactive lyso-PAF to active PAF, protects mice from the lethal damage. These results reveal a linkage/crosstalk between the nervous and the immune system, possibly inducing lethal levels of PAF.

In vitro analysis of the expression of inflammasome, antiviral, and immune genes in an Oreochromis niloticus liver cell line following stimulation with bacterial ligands and infection with tilapia lake virus.

The inflammasome is a multimeric protein complex that plays a vital role in the defence against pathogens and is therefore considered an essential component of the innate immune system. In this study, the expression patterns of inflammasome genes (NLRC3, ASC, and CAS-1), antiviral genes (IFNγ and MX), and immune genes (IL-1β and IL-18) were analysed in Oreochromis niloticus liver (ONIL) cells following stimulation with the bacterial ligands peptidoglycan (PGN) and lipopolysaccharide (LPS) and infection with TiLV. The cells were stimulated with PGN and LPS at concentrations of 10, 25, and 50 µg/ml. For viral infection, 106 TCID50 of TiLV per ml was used. After LPS stimulation, all seven genes were found to be expressed at specific time points at each of the three doses tested. However, at even higher doses of LPS, NLRC3 levels decreased. Following TiLV infection, all of the genes showed significant upregulation, especially at early time points. However, the gene expression pattern was found to be unique in PGN-treated cells. For instance, NLRC3 and ASC did not show any response to PGN stimulation, and the expression of IFNγ was downregulated at 25 and 50 µg of PGN per ml. CAS-1 and IL-18 expression was downregulated at 25 µg of PGN per ml. At a higher dose (50 µg/ml), IL-1β showed downregulation. Overall, our results indicate that these genes are involved in the immune response to viral and bacterial infection and that the degree of response is ligand- and dose-dependent.

Distinct metabolic responses to heme in inflammatory human and mouse macrophages – Role of nitric oxide

Activation of inflammation is tightly associated with metabolic reprogramming in macrophages. The iron-containing tetrapyrrole heme can induce pro-oxidant and pro-inflammatory effects in murine macrophages, but has been associated with polarization towards an anti-inflammatory phenotype in human macrophages. In the current study, we compared the regulatory responses to heme and the prototypical Toll-like receptor (TLR)4 ligand lipopolysaccharide (LPS) in human and mouse macrophages with a particular focus on alterations of cellular bioenergetics. In human macrophages, bulk RNA-sequencing analysis indicated that heme led to an anti-inflammatory transcriptional profile, whereas LPS induced a classical pro-inflammatory gene response. Co-stimulation of heme with LPS caused opposing regulatory patterns of inflammatory activation and cellular bioenergetics in human and mouse macrophages. Specifically, in LPS-stimulated murine, but not human macrophages, heme led to a marked suppression of oxidative phosphorylation and an up-regulation of glycolysis. The species-specific alterations in cellular bioenergetics and inflammatory responses to heme were critically dependent on the availability of nitric oxide (NO) that is generated in inflammatory mouse, but not human macrophages. Accordingly, studies with an inducible nitric oxide synthase (iNOS) inhibitor in mouse, and a pharmacological NO donor in human macrophages, reveal that NO is responsible for the opposing effects of heme in these cells. Taken together, the current findings indicate that NO is critical for the immunomodulatory role of heme in macrophages.

Production of codon-optimized Factor C fragment from Tachypleus gigas in the Pichia pastoris GS115 expression system for endotoxin detection

BackgroundFactor C (FC) is widely used as a standard material for endotoxin testing. It functions as a zymogenic serine protease and serve as a biosensor that detects lipopolysaccharides. Prior investigations involving molecular docking and molecular dynamics simulations of FC demonstrated an interaction between the C-type lectin domain (CLECT) and the ligand lipopolysaccharide (lipid A). In this study, our aim was to assess the stability of the interaction between fragment FC and the lipid A ligand using protein modeling approaches, molecular docking, molecular dynamics simulation, and gene construction into the pPIC9K expression vector. Methods and resultsThe FC structure was modelled by online tools. In this case, both molecular docking and MD simulations were applied to identify the interaction between protein and ligand (lipid A) including its complex stability. The FC structure model using three modeling websites has varied values, according to a Ramachandran plot study. When compared to other models, AlphaFold server modeling produced the best Ramachandran findings, with residues in the most advantageous area at 88.3%, followed by ERRAT values at 89.83% and 3D Verify at 71.93%. From the docking simulation of FC fragments with three ligands including diphosphoryl lipid A, FC-Core lipid A, and Kdo2 lipid A can be an activator of FC protein by binding to receptor regions to form ligand-receptor complexes. MD simulations were performed on all three complexes to assess their stability in water solvents showing that all complexes were stable during the simulation. The optimization of recombinant protein expression in Pichia pastoris was conducted by assessing the OD value and protease activity. Induction was carried out using 1% (v/v) methanol in BMMY media at 30°C for 72 h. ConclusionsProtein fragments of Factor C has been proven to detect endotoxins and serve as a potential biomarker. Molecular docking simulation and MD simulation were employed to study the complex formation of protein fragments FC with ligands. The expression of FC fragments was successfully achieved through heterologous expression. We propose optimizing the expression of FC fragments by inducing them with 1% methanol at 30°C and incubating them for 72 h. These optimized conditions are well-suited for upscaling the production of recombinant FC fragments using a bioreactor.

Expression profiling and antibacterial analysis of cd36 in mandarin fish, Siniperca chuatsi.

Cd36 is classified as a class B scavenger receptor and has also been identified as a pattern recognition receptor. In this study, we investigated the genomic structure and molecular characteristics of cd36 in mandarin fish (Siniperca chuatsi), examined its tissue distribution, and evaluated its antibacterial activity. Genomic structure analysis showed that Sccd36 consists of 12 exons and 11 introns. Sequencing analysis confirmed that the open reading frame of Sccd36 contains 1410 bp, encoding 469 amino acids. Sccd36 is deeply conserved with other vertebrates in terms of genomic structure, gene loci and molecular evolution, and the feature of two transmembrane was observed in ScCd36 through structural prediction. Sccd36 was constitutively expressed in all tissues tested, with the strongest expression in the intestine, followed by the heart and the kidney. Dramatic changes of Sccd36 mRNA were detected in mucosal tissues, including the intestine, gill and skin, when stimulated by the microbial ligands lipopolysaccharide and lipoteichoic acid. In addition, ScCd36 was identified as having strong binding ability to microbial ligands and antibacterial activity against the gram-negative bacteria Aeromonas hydrophila and the gram-positive bacteria Streptococcus lactis. Furthermore, we verified that the genetic ablation of cd36 impaired the resistance of fish to bacterial challenge by using zebrafish cd36 knockout line. In conclusion, our findings suggest that ScCd36 plays a crucial role in the innate immune response of mandarin fish against bacterial infections. This also sets the stage for further exploration into the antibacterial function of Cd36 in lower vertebrate species.

Lipopolysaccharide Tolerance in Human Primary Monocytes and Polarized Macrophages.

Innate immune memory allows macrophages to adequately respond to pathogens to which they have been pre-exposed. To what extent different pattern recognition receptors, cytokines and resolution signals influence innate immune memory needs further elucidation. The present study assessed whether lipopolysaccharide (LPS) tolerance in monocytes and macrophages is affected by these factors. Human CD14+ cells were isolated from peripheral blood, stimulated by LPS and re-stimulated after 3 days of resting. Hereafter, immune-responsive gene 1 (IRG-1), heme oxygenase 1 (HO-1), tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) expression were assessed. Our study revealed the following findings: (1) While pre-stimulation with the Toll-like receptor 4 ligand LPS inhibits the induction of IRG-1, TNF-α and IL-6 expression, pre-stimulation with TLR 1/2 ligands only affects cytokine production but not IRG-1 expression upon subsequent TLR4 engagement. (2) Prior TNF-α stimulation does not affect LPS tolerance but rather increases LPS-mediated cytokine expression. (3) Dimethyl itaconate (DMI) inhibits the expression of IRG-1 in a dose-dependent manner but does not affect TNF-α or IL-6 expression. (4) Docosahexaenoic acid (DHA) partly inhibits IRG-1 expression in monocytes but not in M(IFNγ) and M(IL-4) polarized macrophages. LPS tolerance is not affected in these cells by DHA. The data presented in this study partly corroborate and extend previous findings on innate immune memory and warrant further studies on LPS tolerance to gain a better understanding of innate immune memory at the molecular level.

RUNX1 is required in granulocyte-monocyte progenitors to attenuate inflammatory cytokine production by neutrophils.

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity.

Estrogen-sensitive activation of SGK1 induces M2 macrophages with anti-inflammatory properties and a Th2 response at the maternal–fetal interface

BackgroundDecidual macrophages participate in immune regulation at the maternal–fetal interface. Abnormal M1/M2 polarization of decidual macrophages might predispose immune maladaptation in recurrent pregnancy loss (RPL). However, the mechanism of decidual macrophage polarization is unclear. We explored the role of Estradiol (E2)-sensitive serum-glucocorticoid regulated kinase (SGK) 1 in promoting macrophage polarization and suppressing inflammation at the maternal–fetal interface.MethodsWe assessed serum levels of E2 and progesterone during first trimester of pregnancy in women with or without threatened miscarriages (ended in live birth, n = 448; or early miscarriages, n = 68). For detection of SGK1 in decidual macrophages, we performed immunofluorescence labeling and western blot analysis applying decidual samples from RPL (n = 93) and early normal pregnancy (n = 66). Human monocytic THP-1 cells were differentiated into macrophages and treated with Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS), E2, inhibitors or siRNA for in vitro analysis. Flow cytometry analysis were conducted to detect macrophages polarization. We also applied ovariectomized (OVX) mice with hormones exploring the mechanisms underlying the regulation of SGK1 activation by E2 in the decidual macrophages in vivo.ResultsSGK1 expression down regulation in the decidual macrophages of RPL was consistent with the lower concentration and slower increment of serum E2 from 4 to 12 weeks of gestation seen in these compromised pregnancies. LPS reduced SGK1 activities, but induced the pro-inflammatory M1 phenotype of THP-1 monocyte-derived macrophages and T helper (Th) 1 cytokines that favored pregnancy loss. E2 pretreatment promoted SGK1 activation in the decidual macrophages of OVX mice in vivo. E2 pretreatment amplified SGK1 activation in TLR4-stimulated THP-1 macrophages in vitro through the estrogen receptor beta (ERβ) and PI3K pathway. E2-sensitive activation of SGK1 increased M2 macrophages and Th2 immune responses, which were beneficial to successful pregnancy, by inducing ARG1 and IRF4 transcription, which are implicated in normal pregnancy. The experiments on OVX mice have shown that pharmacological inhibition of E2 promoted nuclear translocation of NF-κB in the decidual macrophages. Further more, pharmacological inhibition or knockdown of SGK1 in TLR4-stimulated THP-1 macrophages activated NF-κB by promoting its nuclear translocation, leading to increased secretion of pro-inflammatory cytokines involved in pregnancy loss.ConclusionOur findings highlighted the immunomodulatory roles of E2-activated SGK1 in Th2 immune responses by priming anti-inflammatory M2 macrophages at the maternal–fetal interface, resulting in a balanced immune microenvironment during pregnancy. Our results suggest new perspectives on future preventative strategies for RPL.

Type I Interferon-Dependent and -Independent TRIF Signaling are Required for Autoantibody Generation in an Induced Model of Systemic Lupus Erythematosus

Abstract Systemic lupus erythematosus (SLE) is an autoimmune condition characterized by the production of autoantibodies to cellular and nuclear self-antigens, with damage to multiple organs. Immunization with the SLE autoantigen β2-glycoprotein I (β2GPI) and TLR4 ligand lipopolysaccharide (LPS) induces a murine model of SLE characterized by the generation of autoantibodies to multiple SLE antigens. LPS induces both inflammatory cytokines and type I interferons (IFNs) by signaling through the adaptor proteins MyD88 and TRIF, respectively. We investigated the mechanisms of LPS-dependent signaling on the induction of murine SLE following β2GPI/LPS immunization. Immunization of mice deficient in MyD88 or TRIF with β2GPI/LPS revealed that LPS-dependent TRIF signaling preferentially promoted autoantibody production in this model, compared to MyD88 signaling. Furthermore, SLE-specific autoantibody production was induced by TLR3 or TLR4 agonists, in combination with β2GPI, but not by agonists of TRIF-independent TLRs. RNA-sequencing of dendritic cells and macrophages isolated from wildtype (WT) and TRIF-deficient mice immunized with β2GPI/LPS revealed differential expression of both type I IFN-dependent and -independent genes. Moreover, type I IFN receptor (IFNaR)-deficient mice immunized with β2GPI/LPS showed diminished autoantibody production compared to WT mice, but to a lesser extent than TRIF-deficient mice. We conclude that LPS-dependent TRIF signaling is required for the generation of autoantibody production in our induced SLE model, both through type I IFN-dependent and - independent effects. Supported by grants from CIHR (????)

Staphylococcus aureus induces tolerance in human monocytes accompanied with expression changes of cell surface markers.

Exposure of human monocytes to lipopolysaccharide (LPS) or other pathogen-associated molecular pattern (PAMPs) induces a temporary insensitivity to subsequent LPS challenges, a cellular state called endotoxin tolerance (ET), associated with the pathogenesis of sepsis. In this study, we aimed to characterize the cellular state of human monocytes from healthy donors stimulated with Staphylococcus aureus in comparison to TLR2-specific ligands. We analyzed S. aureus induced gene expression changes after 2 and 24 hours by amplicon sequencing (RNA-AmpliSeq) and compared the pro-inflammatory response after 2 hours with the response in re-stimulation experiments. In parallel, glycoprotein expression changes in human monocytes after 24 hours of S. aureus stimulation were analyzed by proteomics and compared to stimulation experiments with TLR2 ligands Malp-2 and Pam3Cys and TLR4 ligand LPS. Finally, we analyzed peripheral blood monocytes of patients with S. aureus bloodstream infection for their ex vivo inflammatory responses towards S. aureus stimulation and their glycoprotein expression profiles. Our results demonstrate that monocytes from healthy donors stimulated with S. aureus and TLR ligands of Gram-positive bacteria entered the tolerant cell state after activation similar to LPS treatment. In particular reduced gene expression of pro-inflammatory cytokines (TNF, IL1β) and chemokines (CCL20, CCL3, CCL4, CXCL2, CXCL3 and CXCL8) could be demonstrated. Glycoprotein expression changes in monocytes tolerized by the different TLR agonists were highly similar while S. aureus-stimulated monocytes shared some of the PAMP-induced changes but also exhibited a distinct expression profile. 11 glycoproteins (CD44, CD274, DSC2, ICAM1, LAMP3, LILRB1, PTGS2, SLC1A3, CR1, FGL2, and HP) were similarly up- or downregulated in all four comparisons in the tolerant cell state. Monocytes from patients with S. aureus bacteremia revealed preserved pro-inflammatory responsiveness to S. aureus stimulation ex vivo, expressed increased CD44 mRNA but no other glycoprotein of the tolerance signature was differentially expressed.

TNF promotes DECTIN2 family C-type lectin receptor expression in human macrophages.

TNF blockade constitutes an effective therapy for inflammatory bowel disease, yet increases the risk for infection, including active tuberculosis. The DECTIN2 family C-type lectin receptors MINCLE, MCL, and DECTIN2 sense mycobacterial ligands and activate myeloid cells. In mice, upregulation of DECTIN2 family C-type lectin receptor after stimulation with Mycobacterium bovis Bacille Calmette-Guérin requires TNF. Here, we investigated whether TNF controls inducible C-type lectin receptor expression in human myeloid cells. Monocyte-derived macrophages were stimulated with Bacille Calmette-Guérin and the TLR4 ligand lipopolysaccharide, and expression of C-type lectin receptor was analyzed. Bacille Calmette-Guérin and lipopolysaccharide strongly upregulated messenger RNA expression of DECTIN2 family C-type lectin receptor but not of DECTIN1. Bacille Calmette-Guérin and lipopolysaccharide also induced robust production of TNF. Recombinant TNF was sufficient to upregulate expression of DECTIN2 family C-type lectin receptor. Blocking TNF with the TNFR2-Fc fusion protein etanercept abrogated, as expected, the effect of recombinant TNF and impaired induction of DECTIN2 family C-type lectin receptor by Bacille Calmette-Guérin and lipopolysaccharide. Flow cytometry confirmed upregulation of MCL at the protein level by recombinant TNF and showed inhibition of Bacille Calmette-Guérin-induced MCL by etanercept. To investigate the impact of TNF on C-type lectin receptor expression in vivo, we analyzed peripheral blood mononuclear cells of patients with inflammatory bowel disease and observed downregulation of MINCLE and MCL expression after therapeutic TNF blockade. Together, TNF is sufficient to upregulate DECTIN2 family C-type lectin receptor in human myeloid cells and contributes to this process after encounter with Bacille Calmette-Guérin or lipopolysaccharide. Impaired C-type lectin receptor expression in patients receiving TNF blockade may dampen the sensing of microbes and defense against infection.

Abstract WMP112: Type 2 Diabetes Dampens Neuroprotection Against Ischemic Injury By Lipopolysaccharide-Induced Preconditioning

Introduction: Evidence suggests that lipopolysaccharide ( LPS ) preconditioning (PC) provides neuroprotection by inducing tolerance to subsequent lethal ischemia. Ischemic tolerance (IT) declines with age, yet it is unclear whether metabolic disease like type 2 diabetes (T2DM) also interfere with IT. The current study sought to determine whether T2DM reduces neuroprotection induced by LPS PC, and the molecular mechanisms underlying the dampened IT. Methods: Male and female diabetic db/db and control db/+ mice (8-10 months of age) were i.p. injected with Toll-like receptor (TLR)4 ligand LPS at 0.2 mg/kg or saline. Stroke was induced 3 days later by permanent distal occlusion of middle cerebral artery and temporary occlusion of ipsilateral common carotid artery for 60 min. Sensory and motor function was evaluated 1 day after stroke by a combination of tests. Infarct size was determined 3 days after stroke following TTC staining. The effect of LPS PC on the phenotypes of leukocytes in the brain parenchyma after stroke was quantified by Fluorescent activated cell sorter (FACS) 2 days after stroke with a panel of markers for subsets of myeloid cells. Transcriptome was determined by RNAseq of isolated monocytes from peripheral blood mononuclear cells. Differentially expressed genes were examined for distinct biological pathways via enrichment analysis. Results: LPS PC significantly reduced infarct in db/+ mice by one third and improved neurological scores, but it did not protect the db/db mice from stroke. FACS data showed that LPS PC reduced the number of Ly6G+ cells in db/+ but not db/db brains, suggesting that LPS PC curbed stroke-induced infiltration of neutrophils in the brain parenchyma. Pathway analysis suggests that diabetic mice exhibited down-regulated pathways in interferon signaling. The expression of Jak-Stat signaling (such as Stat1), MHC antigen presentation (H2A family), and Toll-like receptor (TLR) signaling (such as Irf7) were also reduced in diabetic mice after stroke. Conclusions: The dampened IT in the diabetic mice induced by LPS-PC is associated with increased neutrophil infiltration after stroke compared to control mice. Ongoing studies will seek toovercome the underlying defective molecular mechanisms in T2DM.

Olfactory receptors in macrophages and inflammation.

Olfactory receptors (ORs) that bind odorous ligands are the largest family of G-protein-coupled receptors. In the olfactory epithelium, approximately 400 and 1,100 members are expressed in humans and mice, respectively. Growing evidence suggests the extranasal functions of ORs. Here, we review OR expression and function in macrophages, specialized innate immune cells involved in the detection, phagocytosis, and destruction of cellular debris and pathogens as well as the initiation of inflammatory responses. RNA sequencing data in mice suggest that up to 580 ORs may be expressed in macrophages. Macrophage OR expression is increased after treatment with the Toll-like receptor 4 ligand lipopolysaccharide, which also induces the transcription of inflammasome components. Triggering human OR6A2 or its mouse orthologue Olfr2 with their cognate ligand octanal induces inflammasome assembly and the secretion of IL-1β, which exacerbates atherosclerosis. Octanal is positively correlated with blood lipids like low-density lipoprotein –cholesterol in humans. Another OR, Olfr78, is activated by lactate, which promotes the generation of tumor-associated macrophages that dampen the immune response and promote tumor progression. Olfactory receptors in macrophages are a rich source of untapped opportunity for modulating inflammation. It is not known which of the many ORs expressed in macrophages promote or modulate inflammation. Progress in this area also requires deorphanizing more ORs and determining the sources of their ligands.

Lipopolysaccharide reduces melanin synthesis in vitiligo melanocytes by regulating autophagy.

Vitiligo is an autoimmune-related disease with a complex aetiology that involves innate immunity. Toll-like receptors (TLRs) are important parts of innate immunity and are related to a variety of autoimmune diseases, including vitiligo, through an unknown mechanism. In this study, we found that the TLR4 gene expression was increased in blood samples of patients with advanced stage vitiligo, and then, we evaluated the effect of TLR4 ligand lipopolysaccharide (LPS) on melanin synthesis in a vitiligo melanocyte cell line PIG3V and along with its mechanism. LPS suppressed melanin synthesis, downregulated the expression of melanin synthesis-related proteins and activated autophagy in vitiligo melanocytes. Inhibiting autophagy with 3-methyladenine or chloroquine blocked these effects. This suggests that LPS inhibits skin pigmentation by modulating autophagy, thus providing novel insights into the pathogenesis of vitiligo.

ADP-dependent glucokinase (ADPGK) is not critical for the activation of human macrophages by LPS

Background: Activated immune cells show an enhanced glucose metabolism, suggesting that the inhibition of this pathway selective in immune cells could be a potential approach to combat inflammatory diseases. We studied here whether ADP-dependent glucokinase (ADPGK), a glucose-phosphorylating enzyme predominantly expressed in immune cells, could be a suitable target for the inhibition of macrophage activation. Methods: The regulation and role of ADPGK in human primary macrophages differentiated from blood monocytes was studied using Real-time quantitative PCR (RT-qPCR), gene silencing, whole-cell MALDI-mass spectrometry (MS) imaging as well as immune-based and enzymatic medium analyzes. Results: The expression of ADPGK was induced in response to the activation of toll-like receptors (TLRs). The most robust effect was observed with the TLR4 ligand Lipopolysaccharide (LPS) leading to an approximately 4-fold increase of ADPGK RNA levels. For this induction, the activation of p38 MAPK and IKKε was important. Silencing of ADPGK expression using siRNAs had neither an effect on LPS-induced expression and release of proinflammatory cytokines nor on cellular ATP levels and lactate production. Untargeted metabolic cell profiling by whole-cell MALDI-MS imaging did not reveal any metabolic regulations after ADPGK down-regulation suggesting no specific metabolic pathway involvement. Conclusions: ADPGK neither catalyzes a rate-limiting step of glucose metabolism in LPS-activated macrophages nor is required for the proinflammatory phenotype of these cells in vitro. Our data do not indicate that ADPGK inhibition could be a pharmacological approach to modulate immunometabolism.

Macrophage Differentiation and Polarization Regulate the Release of the Immune Checkpoint Protein V-Domain Ig Suppressor of T Cell Activation.

Recently, the V-domain immunoglobulin suppressor of T-cell activation (VISTA) was identified as a negative immune checkpoint regulator (NCR) that is mainly expressed in hematopoietic cells. Preclinical studies have shown that VISTA blockade results in impeded tumor growth and improved survival. Nevertheless, little is known about the physiological role of VISTA expression in macrophages. This study focused on the differential expression of VISTA in human monocytes and macrophages in order to elucidate a putative role of VISTA regulation upon macrophage polarization and activation. We observed that human peripheral monocytes constitutively release soluble VISTA, which was regulated via matrix metalloproteinases. However, monocyte stimulation with cytokines that induce macrophage differentiation, such as granulocyte-macrophage colony–stimulating (GM-CSF) and macrophage colony-stimulating factor (M-CSF), substantially reduced soluble VISTA release. VISTA release was further affected by various pro- and anti-inflammatory stimuli that led to macrophage polarization, where activated M1 macrophages generally released more VISTA than M2 macrophages. Additionally, we observed that stimulation of activated macrophages with the toll-like receptor 4 ligand lipopolysaccharide (LPS) led to a further decrease of soluble VISTA release. Moreover, we found that soluble VISTA impairs T cell cytotoxic activity but did not induce their programmed death. Our results suggest that VISTA is constantly produced and released in the peripheral blood where it may contribute to peripheral tolerance.

Proliferation of CD11b+ myeloid cells induced by TLR4 signaling promotes hepatitis B virus clearance

Backgrounds and aimsEffective immune response plays a key role in the clearance of hepatitis B virus (HBV). However, the specific role of innate immune response in the clearance of virus is still unclear. Here we investigated the effect of TLR4 signaling on the proliferation and differentiation of CD11b+ myeloid cells, which contributes to virus clearance. MethodsC57BL/6 mice were pretreated with TLR4 ligand lipopolysaccharide by intraperitoneal injection. Hydrodynamic injection (HI) was performed to establish HBV-replicated mice. The viremia was monitored. The immune cells were isolated from liver and spleen of the mice. The proliferation and differentiation of CD11b+ myeloid cells were analyzed by flow cytometry. The changes of CD11b+ myeloid cells and its role in virus clearance during HBV infection after LPS stimulation were analyzed. ResultsLPS stimulation induced the proliferation of CD11b+ myeloid cells which differentiated into neutrophils and inflammatory mononuclear macrophages. The expression of F4/80 protein on the surface of mononuclear macrophages in the liver of LPS-stimulated mice was significantly lower than that of control. It indicated that intrahepatic Kupffer cells were significantly decreased in the LPS-stimulated mice, which promoted the clearance of virus. ConclusionLPS stimulation induces the proliferation of CD11b+ myeloid cells that differentiate into inflammatory neutrophils and monocytes, which inhibits HBV replication. And the decrease of intrahepatic Kupffer cells also contributes to the clearance of HBV during HBV infection.

LPS/TLR4 Pathways in Breast Cancer: Insights into Cell Signalling.

Cancer cells are usually recognized as foreign particles by the immune cells. Mounting evidence suggest an important link between toll-like receptors (TLRs) and carcinogenesis. This review article focused on the role of TLRs, especially TLR4, in breast cancer. Research data on TLRs and cancer was explored in PubMed, Scopus, Google Scholar and reviewed. Although some pioneer works are referenced, papers published in the last ten years were mostly cited. TLRs are widely investigated pattern recognition receptors (PRR), and TLR4 is the most studied TLRs, implicated with the occurrence of several types of cancers, including breast cancer. TLR4 activation occurs via the binding of its ligand lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria. Upon LPS binding, TLR4 dimerizes and recruits downstream signalling and/or adapter molecules, leading to gene expression related to cancer cell proliferation, survival, invasion, and metastasis. Although LPS/TLR4 signalling seems a single signal transduction pathway, the TLR4 activation results in the activation of multiple diverse intracellular networks with huge cellular responses in both immune and cancer cells. The role of TLR4 in the growth, invasion, and metastasis of breast cancer is attracting huge attention in oncology research. Several clinical and preclinical studies utilize both TLR4 agonists and antagonists as a treatment option for cancer therapy, either as monotherapy or adjuvants for vaccine development. This review narrates the role of LPS/TLR4 signalling in breast cancer development and future prospects for targeting LPS/TLR4 axis in the treatment of breast cancer.

Promoting mechanism of serum amyloid a family expression in mouse intestinal epithelial cells

Serum amyloid A (SAA) is an acute phase inflammatory protein that we previously described as a robust biomarker of colorectal inflammation in patients with ulcerative colitis (UC) in clinical remission. However, what induces SAA expression in UC remains unclear. This study demonstrates that SAA is significantly expressed in the intestinal tract of UC mouse models when compared with C-reactive protein, another inflammatory biomarker. Moreover, interleukin-6 and tumor necrosis factor-α were found to promote SAA1 expression, as were Toll-like receptor ligands flagellin and lipopolysaccharide. Furthermore, results suggested that the nuclear factor-kappa B (NF-κB) pathway may be involved in the promotion of SAA1 expression by flagellin, which was inhibited by treatment with 5-aminosalicylic acid (5-ASA). Therefore, the flagellin/NF-κB/SAA1 axis may represent one of the mechanisms by which 5-ASA suppresses intestinal inflammation.