The Dynamics of Circulating Heparin-Binding Protein: Implications for Its Use as a Biomarker

Heparin-binding protein (HBP) is a promising biomarker for the development and severity of sepsis. To guide its use, it is important to understand the factors that could lead to false-positive or negative results, such as inappropriate release and inadequate clearance of HBP. HBP is presumably released only by neutrophils, and the organs responsible for its elimination are unknown. In this study, we aimed to determine whether non-neutrophil cells can be a source of circulating HBP and which organs are responsible for its removal. We found that in two cohorts of neutropenic patients, 12% and 19% of patients in each cohort, respectively, had detectable plasma HBP levels. In vitro, three leukemia-derived monocytic cell lines and healthy CD14+ monocytes constitutively released detectable levels of HBP. When HBP was injected intravenously in rats, we found that plasma levels of HBP decreased rapidly, with a distribution half-life below 10 min and an elimination half-life of 1–2 h. We measured HBP levels in the liver, spleen, kidneys, lungs, and urine using both ELISA and immunofluorescence quantitation, and found that the majority of HBP was present in the liver, and a small amount was present in the spleen. Immunofluorescence imaging indicated that HBP is associated mainly with hepatocytes in the liver and monocytes/macrophages in the spleen. The impact of hematologic malignancies and liver diseases on plasma HBP levels should be explored further in clinical studies.

Extracellular CIRP Induces Macrophage Extracellular Trap Formation Via Gasdermin D Activation

Extracellular cold-inducible RNA-binding protein (eCIRP) is a damage-associated molecular pattern promoting inflammation and tissue injury. During bacterial or viral infection, macrophages release DNA decorated with nuclear and cytoplasmic proteins known as macrophage extracellular traps (METs). Gasdermin D (GSDMD) is a pore-forming protein that has been involved in extracellular trap formation in neutrophils. We hypothesized that eCIRP induces MET formation by activating GSDMD. Human monocytic cell line THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) and treated with recombinant murine (rm) CIRP. The MET formation was detected by three methods: time-lapse fluorescence microscopy (video imaging), colorimetry, and ELISA. Cleaved forms of GSDMD, and caspase-1 were detected by Western blotting. Treatment of THP-1 cells with rmCIRP increased MET formation as revealed by SYTOX Orange Staining assay in a time- and dose-dependent manner. METs formed by rmCIRP stimulation were further confirmed by extracellular DNA, citrullinated histone H3, and myeloperoxidase. Treatment of THP-1 cells with rmCIRP significantly increased the cleaved forms of caspase-1 and GSDMD compared to PBS-treated cells. Treatment of macrophages with caspase-1, and GSDMD inhibitors z-VAD-fmk, and disulfiram, separately, significantly decreased rmCIRP-induced MET formation. We also confirmed rmCIRP-induced MET formation using primary cells murine peritoneal macrophages. These data clearly show that eCIRP serves as a novel inducer of MET formation through the activation of GSDMD and caspase-1.

697 Binding of gamma-glutamyl transferase to toll-like receptor-4 allows tissue factor activation in monocytes

Aims Gamma-glutamyl transferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of tissue factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies carried out in human peripheral blood mononuclear cell (PBMC). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation site. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. To assess whether GGT-induced TF stimulation is a consequence of binding to toll-like receptor (TLR)-4 and activation of NFkB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from an acute monocytic leukaemia patient, were incubated with hrGGT (0.5 ng/μL for PBMCs and 1 ng/µL for THP-1). LPS-Rs (0.5 ng/µL for PBMCs and 1 ng/µL for THP-1), CLI-095 (3 × 10−6M) and BAY-11-7082 (10−5 M) were used to block TLR-4 receptors, TLR4 signalling and NFkB respectively. TF protein concentration was determined by western blot analysis. TF pro-coagulant activity (PCA) was assessed through the use of Start Max coagulometer and results were expressed in pg/mL after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110 ± 70 to 510 ± 43, n = 7, P < 0.01) and THP-1 cells (PCA from 170 ± 64 to 460 ± 80, n = 15, P < 0.001), result confirmed by western blot. In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72 ± 17% n = 4, P < 0.01) a TLR-4 antagonist, CLI-095 (PCA: −74 ± 34%, n = 7, P < 0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71 ± 32%, n = 7, P < 0.001), a NF-kB inhibitor. Similar results were obtained in THP-1 cells (LPS-Rs: −76 ± 15%, n = 6, P < 0.01; CLI-095: −100 ± 6,6 %, n = 6, P < 0.01; BAY-11-7082: −100 ± 2,1%, n = 6, P < 0.01). hrGGT activates NFkB in hTLR4-positive HEK cell lines while does not induces effect in TLR4-negative HEK cell lines. Conclusions These data confirm the cytokine-like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells. Furthermore, it is highlighted for the first time the possible role of TLR-4 as the receptor of GGT and NFkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Repopulating Kupffer Cells Originate Directly from Hematopoietic Stem Cells

Kupffer cells (KCs) originate from yolk sac progenitors before birth, but the origin of repopulating KCs in adult remains unclear. In current study, we firstly traced the fate of preexisting KCs and that of monocytic cells with tissue-resident macrophage-specific and monocytic cell-specific fate mapping mouse models, respectively, and found no evidences that repopulating KCs originate from preexisting KCs or MOs. Secondly, we performed genetic lineage tracing to determine the type of progenitor cells involved in response to KC depletion in mice, and found that in response to KC depletion, hematopoietic stem cells (HSCs) proliferated in the bone marrow, mobilized into the blood, adoptively transferred into the liver and differentiated into KCs. Finally, we traced the fate of HSCs in a HSC-specific fate-mapping mouse model, in context of chronic liver inflammation induced by repeated carbon tetrachloride treatment, and confirmed that repopulating KCs originated directly from HSCs. Taken together, these findings provided in vivo fate-mapping evidences that repopulating KCs originate directly from hematopoietic stem cells, which present a completely novel understanding of the cellular origin of repopulating Kupffer Cells and shedding light on the divergent roles of KCs in liver homeostasis and diseases.

The use of Ipratropium and Tiotropium as novel agents to reduce inflammation in in vitro macrophage models.

Background: Chronic Obstructive Pulmonary Disease (COPD) affects an estimated 330 million individuals worldwide. Approximately, 3 million individuals died of COPD in 2012 and it is predicted that COPD would be the third leading factor for deaths worldwide by 2020. In United Kingdom nearly one million individuals suffer from COPD. Purpose: There are no effective pharmacotherapies available for COPD. it is only managed by using bronchodilators and inhaled corticosteroids mostly. However, cardiovascular effects are associated with these drugs. Most importantly, there is an unmet need of COPD treatment worldwide. Our research aim was to identify Ipratropium and Tiotropium as novel anti-inflammatory agents in in vitro macrophage models. Aims: To investigate the LPS stimulated pro-inflammatory cytokines IL-6 and TNF-α levels in THP-1 cells. To investigate whether the drugs Ipratropium and Tiotropium are capable of decreasing LPS-induced inflammation in THP-1 cells. Materials: Human monocytic cell line THP-1 cells, Rosewell Park Memorial Institute RPMI 1640 with Glutamax I, 1% Penicillin Streptomycin (PenStrep) and 10% foetal bovine serum (FBS), Lipopolysaccharide 10μl/ml, 0.05% Tween20, 0.4% Trypan blue, Reagent diluent (10% Bovine Serum Albumin in PBS), Budesonide Fenoterol, Ipratropium and Tiotropium. Human IL-6 DuoSet ELISA, Human TNF-α ELISA, TMB ELISA Substrate solution and Stop solution. Methods: THP-1 cells were cultured and challenges with LPS to stimulate the IL-6 and TNF- α cytokines. The cells were treated with Budesonide, Fenoterol, Ipratropium and Tiotropium. ELISA was performed to determine the concentrations of cytokines. Results: The results suggested that Ipratropium and Tiotropium reduce IL-6 and TNF- α concentrations in the cells. However, Budesonide and Fenoterol were found to reduce cytokines more effectively than Ipratropium and Tiotropium. The data was considered significant only when P <0.05. Conclusions: The anti-inflammatory or cytokine reducing properties of Ipratropium and Tiotropium were acknowledged. The research hypothesis was found to be true. Budesonide and Fenoterol substantially reduce cytokine levels. The receptor interactions of Ipratropium and Tiotropium may be responsible for their duration of action. Overall, Ipratropium and Tiotropium display the characteristics of novel anti-inflammatories.

Investigating pathogenicity and virulence of Staphylococcus pettenkoferi: an emerging pathogen

Staphylococcus pettenkoferi is a coagulase-negative Staphylococcus identified in 2002 that has been implicated in human diseases as an opportunistic pathogenic bacterium. Its multiresistant character is becoming a major health problem, yet the pathogenicity of S. pettenkoferi is poorly characterized. In this study, pathogenicity of a S. pettenkoferi clinical isolate from diabetic foot osteomyelitis was compared to a Staphylococcus aureus strain in various in vitro and in vivo experiments. Growth kinetics were compared against S. aureus and bacteria survival was assessed in the RAW 264.7 murine macrophage cell line, the THP-1 human leukemia monocytic cell line and the HaCaT human keratinocyte cell line. Ex vivo analysis were performed in whole blood survival assays, and in vivo assays via the infection model of zebrafish embryos. Moreover, whole-genome analysis was performed. Our results showed that S. pettenkoferi was able to survive in human blood, human keratinocytes, murine macrophages, and human macrophages. S. pettenkoferi demonstrated its virulence by causing substantial embryo mortality in the zebrafish model. Genomic analysis revealed virulence factors such as biofilm- (e.g., icaABCD; rsbUVW) and regulator- (e.g., agr, mgrA, sarA, saeS) encoding genes well characterized in S. aureus. This study thus advances the knowledge of this under investigated pathogen and validates the zebrafish infection model for this bacterium

Interferon-γ Produced by EBV-Positive Neoplastic NK-Cells Induces Differentiation into Macrophages and Procoagulant Activity of Monocytes, Which Leads to HLH

Epstein–Barr virus (EBV)-positive T- or NK-cell neoplasms show progressive systemic inflammation and abnormal blood coagulation causing hemophagocytic lymphohistiocytosis (HLH). It was reported that inflammatory cytokines were produced and secreted by EBV-positive neoplastic T- or NK-cells. These cytokines can induce the differentiation of monocytes into macrophages leading to HLH. To clarify which products of EBV-positive neoplastic T- or NK-cells have effects on monocytes, we performed a co-culture assay of monocytes with the supernatants of EBV-positive T- or NK-cell lines. The expression of differentiation markers, the phagocytosis ability, and the mRNA expression of the inflammatory cytokines of THP-1, a monocytic cell line, clearly increased after culturing with the supernatants from EBV-NK-cell lines. Co-culturing with the supernatants promoted the expression of CD80 and CD206 as well as M1 and M2 macrophage markers in human monocytes. Co-culturing with the supernatants of EBV-NK-cell lines significantly enhanced the procoagulant activity and the tissue factor expression of monocytes. Interferon (IFN)-γ was elevated extremely not only in the supernatant of EBV-NK-cell lines but also in the plasma of EBV-positive NK-cell neoplasms patients accompanying HLH. Finally, we confirmed that IFN-γ directly enhanced the differentiation into M1-like macrophages and the procoagulant activity of monocytes. Our findings suggest that IFN-γ may potentially serve as a therapeutic target to regulate HLH in EBV-positive NK-cell neoplasms.

Binding of gamma-glutamyl transferase to TLR-4 allows the activation of tissue factor expression in human monocytes

Background Gamma-glutamyl transferase (GGT) plays a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the initiator of the clotting cascade. GGT may modulate TF expression, an assumption verified by previous studies carried out in human Peripheral Blood Mononuclear Cells (PBMCs). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic activity since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. Purpose To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of NF-κB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from a leukemia patient, were incubated with hrGGT (0.5 ng/μl for PBMCs and 1ng/μl for THP-1). LPS-Rs (0.5 ng/μl for PBMCs and 1 ng/μl for THP-1), CLI-095 (3x10–6 M) and BAY-11-7082 (10–5 M) were used to block TLR-4 receptors, TLR4 signaling and NF-κB respectively. TF pro-coagulant activity (PCA) was assessed using of StartMax coagulometer and results were expressed in pg/ml after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110±70 to 510±43, n=7, p&lt;0.01) and THP-1 cells (PCA from 170±64 to 460±80, n=15, p&lt;0.001).In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72±17% n=4, p&lt;0.01) a TLR-4 antagonist, CLI-095 (PCA:-74±34%, n=7, p&lt;0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71±32%, n=7, p&lt;0.001), a NF-κB inhibitor. Similar results were obtained in THP-1 cells [LPS-Rs: −76±15%, n=6, p&lt;0.01; CLI-095: −100±6,6%, n=6, p&lt;0.01; BAY-11-7082: −100±2,1%, n=6, p&lt;0.01]. hrGGT activates NF-κB in hTLR4-positive HEK cell lines while doesn't induces effect in TLR4-negative HEK cells. Conclusions Besides confirming the cytokine-Like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells, these data identify for the first time the possible role of TLR-4 as the receptor of GGT and NfkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque. FUNDunding Acknowledgement Type of funding sources: None.