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.

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.

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.

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.

Differential Expression of Human MicroRNAs During Dengue Virus Infection in THP-1 Monocytes

Dengue virus (DENV) is the most widespread arbovirus, responsible for a wide range of clinical manifestations, varying from self-limited illness to severe hemorrhagic fever. Dengue severity is associated with host intense proinflammatory response and monocytes have been considered one of the key cell types involved in the early steps of DENV infection and immunopathogenesis. To better understand cellular mechanisms involved in monocyte infection by DENV, we analyzed the expression levels of 754 human microRNAs in DENV-infected THP-1 cells, a human monocytic cell line. Eleven human microRNAs showed differential expression after DENV infection and gene ontology and enrichment analysis revealed biological processes potentially affected by these molecules. Five downregulated microRNAs were significantly linked to cellular response to stress, four to cell death/apoptosis, two to innate immune responses and one upregulated to vesicle mediated, TGF-β signaling, phosphatidylinositol mediated signaling, lipid metabolism process and blood coagulation.

Beta-agonist Exposure Preferentially Impacts Lung Macrophage Cyclic AMP Related Gene Expression in Asthma and Asthma COPD Overlap Syndrome

Bronchoalveolar lavage (BAL) samples from Severe Asthma Research Program (SARP) patients display suppression of a module of genes involved in cAMP-signaling pathways (BALcAMP) correlating with severity, therapy, and macrophage constituency. We sought to establish if gene expression changes were specific to macrophages and compared gene expression trends from multiple sources. Datasets included Single-cell RNA sequencing (scRNA-seq) from lung specimens including a fatal exacerbation of severe Asthma COPD Overlap Syndrome (ACOS) after intense therapy and controls without lung disease, Bulk RNA sequencing from cultured macrophage (THP1) cells after acute or prolonged beta-agonist exposure, SARP datasets, and data from the Immune Modulators of Severe Asthma (IMSA) cohort). THP monocytes suppressed BALcAMP network gene expression after prolonged relative to acute beta agonist exposure, corroborating SARP observations. scRNA-seq from healthy and diseased lung tissue revealed 13 cell populations enriched for macrophages. In severe ACOS, BALcAMP gene network expression scores were decreased in many cell populations, most significantly for macrophage populations (p < 3.9e-111). NK cell and type II alveolar epithelial cells displayed less robust network suppression (p < 9.2e-8). Alveolar macrophages displayed the most numerous individual genes affected and the highest amplitude of modulation. Key BALcAMP genes demonstrate significantly decreased expression in severe asthmatics in the IMSA cohort. We conclude that suppression of the BALcAMP gene module identified from SARP BAL samples is validated in the IMSA patient cohort with physiologic parallels observed in a monocytic cell line and in a severe ACOS patient sample with effects preferentially localizing to macrophages.

Bioengineered Nanoparticles Loaded-Hydrogels to Target TNF Alpha in Inflammatory Diseases

Rheumatoid Arthritis (RA) is an incurable autoimmune disease that promotes the chronic impairment of patients’ mobility. For this reason, it is vital to develop therapies that target early inflammatory symptoms and act before permanent articular damage. The present study offers two novel therapies based in advanced drug delivery systems for RA treatment: encapsulated chondroitin sulfate modified poly(amidoamine) dendrimer nanoparticles (NPs) covalently bonded to monoclonal anti-TNF α antibody in both Tyramine-Gellan Gum and Tyramine-Gellan Gum/Silk Fibroin hydrogels. Using pro-inflammatory THP-1 (i.e., human monocytic cell line), the therapy was tested in an inflammation in vitro model under both static and dynamic conditions. Firstly, we demonstrated effective NP-antibody functionalization and TNF-α capture. Upon encapsulation, the NPs were released steadily over 21 days. Moreover, in static conditions, the approaches presented good anti-inflammatory activity over time, enabling the retainment of a high percentage of TNF α. To mimic the physiological conditions of the human body, the hydrogels were evaluated in a dual-chamber bioreactor. Dynamic in vitro studies showed absent cytotoxicity in THP-1 cells and a significant reduction of TNF-α in suspension over 14 days for both hydrogels. Thus, the developed approach showed potential for use as personalized medicine to obtain better therapeutic outcomes and decreased adverse effects.

Effects of Extracellular Vesicles from Blood-Derived Products on Osteoarthritic Chondrocytes within an Inflammation Model

Osteoarthritis (OA) is hallmarked by a progressive degradation of articular cartilage. One major driver of OA is inflammation, in which cytokines such as IL-6, TNF-α and IL-1β are secreted by activated chondrocytes, as well as synovial cells—including macrophages. Intra-articular injection of blood products—such as citrate-anticoagulated plasma (CPRP), hyperacute serum (hypACT), and extracellular vesicles (EVs) isolated from blood products—is gaining increasing importance in regenerative medicine for the treatment of OA. A co-culture system of primary OA chondrocytes and activated M1 macrophages was developed to model an OA joint in order to observe the effects of EVs in modulating the inflammatory environment. Primary OA chondrocytes were obtained from patients undergoing total knee replacement. Primary monocytes obtained from voluntary healthy donors and the monocytic cell line THP-1 were differentiated and activated into proinflammatory M1 macrophages. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis and Western blot. Gene expression analysis of chondrocytes by RT-qPCR revealed increased type II collagen expression, while cytokine profiling via ELISA showed lower TNF-α and IL-1β levels associated with EV treatment. In conclusion, the inflammation model provides an accessible tool to investigate the effects of blood products and EVs in the inflammatory context of OA.