Activation Of Human Monocytes Research Articles

Photocatalytic Degradation of Bacterial Lipopolysaccharides by Peptide-Coated TiO2 Nanoparticles.

In this study, we report the degradation of smooth and rough lipopolysaccharides (LPS) from Gram-negative bacteria and of lipoteichoic acid (LTA) from Gram-positive bacteria by peptide-coated TiO2 nanoparticles (TiO2 NPs). While bare TiO2 NPs displayed minor binding to both LPS and LTA, coating TiO2 NPs with the antimicrobial peptide LL-37 dramatically increased the level of binding to both LPS and LTA, decorating these uniformly. Importantly, peptide coating did not suppress reactive oxygen species generation of TiO2 NPs; hence, UV illumination triggered pronounced degradation of LPS and LTA by peptide-coated TiO2 NPs. Structural consequences of oxidative degradation were examined by neutron reflectometry for smooth LPS, showing that degradation occurred preferentially in its outer O-antigen tails. Furthermore, cryo-TEM and light scattering showed lipopolysaccharide fragments resulting from degradation to be captured by the NP/lipopolysaccharide coaggregates. The capacity of LL-37-TiO2 NPs to capture and degrade LPS and LTA was demonstrated to be of importance for their ability to suppress lipopolysaccharide-induced activation in human monocytes at simultaneously low toxicity. Together, these results suggest that peptide-coated photocatalytic NPs offer opportunities for the confinement of infection and inflammation.

Mycophenolate mofetil directly modulates myeloid viability and pro-fibrotic activation of human macrophages.

Mycophenolate mofetil (MMF) is an immunosuppressant used to treat rheumatological diseases, including Systemic Sclerosis (SSc). While MMF is an established inhibitor of lymphocyte proliferation, recent evidence suggests MMF also mediates effects on other cell types. The goal of this study was to determine the effect of MMF on monocytes and macrophages, which have been implicated in SSc pathogenesis. Human monocyte-derived macrophages were cultured with the active MMF metabolite, mycophenolic acid (MPA), and assessed for changes in viability and immuno-phenotype. Guanosine supplementation studies were performed to determine whether MPA-mediated effects were dependent on de novo purine synthesis. The ability of MPA-treated macrophages to induce fibroblast activation was evaluated, and dermal myeloid expression signatures were analyzed in MMF-treated SSc patients. MPA reduced viability and induced apoptosis in monocytes and macrophages at doses (average IC50 = 1.15 µg/ml) within the target serum concentration of MMF patients (1-3µg/ml). These effects were reversed by guanosine supplementation. Low-dose MPA (0.5 µg/ml) attenuated IL-4 or SSc plasma-mediated macrophage activation, and inhibited the ability of SSc plasma-activated macrophages to induce SSc fibroblast activation. Gene expression studies demonstrated significant reductions in dermal myeloid signatures in MMF-responsive SSc patients. For the first time, we demonstrate that MMF inhibits the viability and pro-fibrotic activation of human monocytes and macrophages, which is dependent on de novo purine synthesis. Coupled with myeloid gene expression attenuation following MMF treatment in patients, these results suggest that fibrotic inhibition observed with MMF may be attributable, at least in part, to direct effects on myeloid cells.

Multiple Mechanisms of Action of Sulfodyne®, a Natural Antioxidant, against Pathogenic Effects of SARS-CoV-2 Infection.

Few therapeutic options are available to treat COVID-19. The KEAP1/NRF2 pathway, the major redox-responsive pathway, has emerged as a potential therapeutic target for COVID-19 as it regulates redox homeostasis and inflammation that are altered during SARS-CoV-2 infection. Here, we characterized the effects of NRF2-agonist Sulfodyne®, a stabilized natural Sulforaphane, in cellular and animal models of SARS-CoV-2 infection. In pulmonary or colonic epithelial cell lines, Sulfodyne® elicited a more efficient inhibition of SARS-CoV-2 replication than NRF2-agonists DMF and CDDO. This antiviral activity was not dependent on NRF2 but was associated with the regulation of several metabolic pathways, including the inhibition of ER stress and mTOR signaling, which are activated during SARS-CoV-2 infection. Sulfodyne® also decreased SARS-CoV-2 mediated inflammatory responses by inhibiting the delayed induction of IFNB1 and type I IFN-stimulated genes in infected epithelial cell lines and by reducing the activation of human by-stander monocytes recruited after SARS-CoV-2 infection. In K18-hACE2 mice infected with SARS-CoV-2, Sulfodyne® treatment reduced both early lung viral load and disease severity by fine-tuning IFN-beta levels. Altogether, these results provide evidence for multiple mechanisms that underlie the antiviral and anti-inflammatory activities of Sulfodyne® and pinpoint Sulfodyne® as a potent therapeutic agent against pathogenic effects of SARS-CoV-2 infection.

FAM3D regulation of head and neck squamous cell carcinoma behaviour through the EMT pathway.

Head and neck squamous cell carcinoma present challenges in effective treatment, with 50%-60% of cases exhibiting recurrence or metastasis, often resistant to surgery alone. Immunotherapy, a promising approach, does not guarantee benefits for all patients. Thus, the imperative lies in identifying reliable biomarkers for predicting immunotherapy efficacy. FAM3D, a protein-coding gene known for its potent chemotactic activity in human peripheral blood monocytes and neutrophils, plays a crucial role in regulating tumour immune responses and holds promise as an immune biomarker. We employed comprehensive database analysis to scrutinise FAM3D, evaluating its gene expression, mutation profiles and prognostic implications in head and neck squamous cell carcinoma, along with its associations with clinical characteristics and immune cell infiltration. Complementary functional experiments were conducted to delve into the potential mechanisms governed by FAM3D. Our findings establish a significant correlation between low FAM3D expression and the invasiveness and metastatic potential of head and neck squamous cell carcinoma. FAM3D likely exerts its influence through the regulation of epithelial-mesenchymal transition. FAM3D emerges as a valuable biomarker for predicting the responsiveness of patients with head and neck squamous cell carcinoma to immunotherapy, holding substantial clinical diagnostic and therapeutic relevance.

Grape/Blueberry Anthocyanins and Their Gut-Derived Metabolites Attenuate LPS/Nigericin-Induced Inflammasome Activation by Inhibiting ASC Speck Formation in THP-1 Monocytes.

Inflammasomes are multi-protein complexes, which are formed in response to tissue injury, infections, and metabolic stress. However, aberrant inflammasome activation has been linked to several inflammatory diseases. Anthocyanins have been reported to attenuate NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation, but the influence of grape/blueberry anthocyanins and especially their gut-derived metabolites on NLRP3 inflammasome activation in human monocytes remains unclear. Therefore, human leukemic monocytes (THP-1 cells, Tohoku Hospital Pediatrics-1 cells) were preincubated with different concentrations of grape/blueberry anthocyanins, homovanillyl alcohol, or 2,4,6-trihydroxybenzaldehyde (THBA) before the NLRP3 inflammasome was activated by lipopolysaccharide and/or nigericin. Apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, as well as ASC and NLRP3 protein expression, were determined using flow cytometry. Caspase-1 activity was measured in cultured cells, and pro-inflammatory cytokine secretion was determined using enzyme-linked immunosorbent assays. Anthocyanins and their metabolites had no effect on ASC or NLRP3 protein expression. However, THBA significantly inhibited ASC speck formation in primed and unprimed THP-1 monocytes, while caspase-1 activity was significantly declined by grape/blueberry anthocyanins. Furthermore, reduced inflammasome activation resulted in lower pro-inflammatory cytokine secretion. In conclusion, our results show for the first time that grape/blueberry anthocyanins and their gut-derived metabolites exert anti-inflammatory effects by attenuating NLRP3 inflammasome activation in THP-1 monocytes.

Biorefining of legume and grass biomasses: Technological properties and bioactivities of the green juice

Five legume species (fodder galega, green pea, faba bean, red clover, and white clover) and one grass (timothy) grown under field conditions were investigated as potential future foods. Technological characteristics, such as emulsifying and foaming properties and nitrogen solubility indices, were determined from the green juices pressed from the whole plants. Secondly, the juices were evaluated for their antioxidative, antibacterial and anti-inflammatory properties. Faba bean green juice showed the highest emulsion stability and capacity, 66.0 ± 3.3 % and 18.0 ± 2.7 g/mg respectively, and second highest emulsion activity (58.4 ± 0.92 %). Considering the foaming properties, the highest activity was shown by white clover (433 ± 0.0 %) and green pea juices (411 ± 19.2 %). At the same time, the foam stability was remarkable for green pea juice (1078 ± 181.9 min) when compared to the other juices studied. The juices showed nitrogen solubility indices of about 50 % at pH 8, which were rather similar to those shown for the seeds of the same species in the literature. The antioxidant activities correlated with the total phenolic content. The highest antioxidant activity was shown by faba bean green juice both in the oxygen radical absorbance capacity (ORAC) and the ferric reducing antioxidant power (FRAP) method. In addition, a weak positive correlation was observed between the total phenolic content and the antibacterial properties using Escherichia coli and Staphylococcus aureus strains, although mainly in the case of the latter strain, high inhibition readings were observed for all other plants than white clover. All juices showed some anti-inflammatory activity on human monocyte activation: the faba bean and red clover were the most efficient materials (IC50 concentrations 49 ± 41 and 206 ± 68 mg/L, respectively).- These results suggest that the green juices tested may provide novel health-promoting food and feed ingredients in the future.

Identification, characterization, and molecular docking of immunomodulatory oligopeptides from bioavailable hempseed protein hydrolysates

Promoting dietary patterns in which the content of vegetables is higher than the current consumption of them is one of the strategies to achieve a sustainable food system while promoting health in humans. Hemp (Cannabis sativa L.) protein contains bioactive peptides that can be released via enzymatic hydrolysis. These peptides must reach the target organ in order to potentially exert bioactivity and regulate specific metabolic pathways. The peptides contained in two bioavailable hempseed protein hydrolysates (bioHPHs) showing anti-inflammatory activity were identified using a transwell system employing CACO-2 cell culture as absorption model and subjected to in silico analysis to select 10 unique peptides. These sequences were chemically synthetized to verify their activity in primary human monocytes (assessing gene expression of IL-1β, IL-6, TNF-α, IL-4, IL-10, and TLR4), in addition to evaluate the interaction with TRL4/MD2 by molecular docking. Six peptides (DDNPRRF, SRRFHLA, RNIFKGF, VREPVFSF, QADIFNPR and SAERGFLY) showed high immunomodulatory activity in in vitro and the mechanisms of interaction with TLR4/MD2 were described. Bioavailable anti-inflammatory hempseed-derived peptides were identified, and their activity verified, suggesting the health benefits that the ingestion of HPHs could exert in humans. These findings open new opportunities for developing nutritional strategies with hemp as a dietary source of biopeptides to prevent the development and progression of inflammatory-related diseases.

Tumor Regression upon Intratumoral and Subcutaneous Dosing of the STING Agonist ALG-031048 in Mouse Efficacy Models.

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

The Influence of Permafrost Microorganisms on Monocytes Differentiation In Vitro.

Metabolites of fossil microorganisms of dispersed watered rocks that have passed into a frozen state (Bacillus sp. strains 2/09 and 9/08, Bacillus megaterium 8-75) can modulate the differentiation activity of human peripheral blood monocytes into phenotypically different subpopulations when cultured in vitro for 7 days. This effect is largely determined by the type of metabolites that depends on the temperature of their production: cold (collected after bacterium culturing at 5°C), medium (at 22°C), and warm temperature (at 37°C). All three types of metabolites had a weak negative influence on the level of classical (CD14hiCD16-) monocytes and stimulated the differentiation of intermediate (CD14+CD16+) and non-classical (CD14loCD16+) monocytes. The monocytes differentiation into the subpopulation of intermediate (CD14+CD16+) was stimulated to a greater extent by medium-temperature metabolites of the strain 8/75 and into the subpopulation of non-classical (CD14loCD16+) monocytes by warm metabolites of the strains 8/75 and 2/09 throughout culturing of mononuclear cells (days 1, 3, 7). Bearing in mind the anti-infection activity of intermediate and non-classical monocytes, we can consider strains 8/75 Bacillus megaterium and 2/09 Bacillus sp. promising for their in-depth testing.

Investigating DAGLβ expression and activity in human monocytes via proteomic and lipidomic profiling

Abstract Lipid-metabolizing enzymes are important targets for small-molecule inhibitor discovery as the regulation of inflammation responses have been linked to lipid metabolism cascades controlled by these enzymes. Non-steroidal anti-inflammatory drugs inhibit enzymes in the COX pathway preventing conversion of arachidonic acid (AA) into inflammation-inducing prostaglandins. An upstream precursor to AA is the endocannabinoid 2-arachidonoyl-glycerol (2-AG), which is biosynthesized from the cleavage of arachidonate-containing diacylglycerols (DAGs) by diacylglycerol lipase beta (DAGLβ). Previous work to identify DAGLβ’s role in inflammatory function led to the creation of KT109, a DAGLβ-specific 1,2,3-triazole urea inhibitor. Primary mouse macrophages treated with KT109 showed reductions in AA and 2-AG and an increase in arachidonate-containing DAGs, demonstrating that DAGLβ is key in proinflammatory lipid regulation and signaling. These studies focused on the effects of inhibiting mouse DAGLβ, but information regarding KT109 inhibition of human DAGLβ is still lacking. Herein, we applied activity-based protein profiling (ABPP) and quantitative proteomic and lipidomic methods to determine the effectivity of KT109 inhibition in human monocytic cell line THP1. Potency (IC 50values) for DAGLβ inhibition by KT109 in human THP1 cells were comparable to the magnitude of inhibition in mouse cells. Lipidomic analysis of KT109-treated monocytic and macrophage-differentiated THP1s did not demonstrate an accumulation of arachidonate-containing DAGs. These findings show a potential difference in the expression and activity of human DAGLβ when compared to mouse DAGLβ and additional targets of KT109 in human cells. Funding and disclosures: This work was supported by the National Institute on Drug Abuse grant no. R01 DA043571.

The calcium channel modulator 2-APB hydrolyzes in physiological buffers and acts as an effective radical scavenger and inhibitor of the NADPH oxidase 2

2-aminoethoxydiphenyl borate (2-APB) is commonly used as a tool to modulate calcium signaling in physiological studies. 2-APB has a complex pharmacology and acts as activator or inhibitor of a variety of Ca2+ channels and transporters. While unspecific, 2-APB is one of the most-used agents to modulate store-operated calcium entry (SOCE) mediated by the STIM-gated Orai channels. Due to its boron core structure, 2-APB tends to readily hydrolyze in aqueous environment, a property that results in a complex physicochemical behavior. Here, we quantified the degree of hydrolysis in physiological conditions and identified the hydrolysis products diphenylborinic acid and 2-aminoethanol by NMR. Notably, we detected a high sensitivity of 2-APB/diphenylborinic acid towards decomposition by hydrogen peroxide to compounds such as phenylboronic acid, phenol, and boric acid, which were, in contrast to 2-APB itself and diphenylborinic acid, insufficient to affect SOCE in physiological experiments. Consequently, the efficacy of 2-APB as a Ca2+ signal modulator strongly depends on the reactive oxygen species (ROS) production within the experimental system. The antioxidant behavior of 2-APB towards ROS and its resulting decomposition are inversely correlated to its potency to modulate Ca2+ signaling as shown by electron spin resonance spectroscopy (ESR) and Ca2+ imaging. Finally, we observed a strong inhibitory effect of 2-APB, i.e., its hydrolysis product diphenylborinic acid, on NADPH oxidase (NOX2) activity in human monocytes. These new 2-APB properties are highly relevant for Ca2+ and redox signaling studies and for pharmacological application of 2-APB and related boron compounds.

ROCK2 interacts with p22phox to phosphorylate p47phox and to control NADPH oxidase activation in human monocytes

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox invitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

Effects of Magnetic Nanoparticles on the Functional Activity of Human Monocytes and Dendritic Cells.

The use of nanoparticles in medicine is sometimes hampered by their potential to activate immune cells, eliciting inflammation or allergy. We investigated whether magnetic nanoparticles (MNPs) or biomimetic magnetic nanoparticles (BMNPs) affect relevant activities of human monocytes. We found that the nanoparticles neither elicited the production of pro-inflammatory mediators IL-6 and TNFα by resting monocytes (when BMNP dose < 300 μg/mL) nor enhanced their secretion induced by R848, a molecule engaging virus-recognizing receptors, or bacterial lipopolysaccharide (LPS). MNPs and BMNPs neither induced the generation of reactive oxygen species (ROS), nor affected the ROS production elicited by the NADPH oxidase activator phorbol myristate acetate (PMA) or the fungal derivative β-glucan. BMNPs, but not MNPs, caused an up-regulation of the maturation markers CD80, CD83, and CD86 in immature monocyte-derived dendritic cells (DCs), whereas both nanoparticles did not affect the LPS-induced expression of these markers. Moreover, the nanoparticles were greedily ingested by monocytes and DCs without altering their viability. Therefore, these nanoparticles are candidates for medical applications because they do not activate pro-inflammatory activities of monocytes. Furthermore, their ability to stimulate DC maturation could be used for the design of vaccines. Moreover, harmlessly engulfed nanoparticles could be vehicles to carry molecules inside the immune cells to regulate the immune response.

ValCitGlyPro-dexamethasone antibody conjugates selectively suppress the activation of human monocytes.

Glucocorticoids (GCs) are effective in treating autoimmune and inflammatory disorders but come with significant side effects, many of which are mediated by non-immunological cells. Therefore, there is rapidly growing interest in using antibody drug conjugate (ADC) technology to deliver GCs specifically to immune cells, thereby minimizing off-target side effects. Herein, we report the study of anti-CD11a, anti-CD38, and anti-TNFα ADCs to deliver dexamethasone to monocytes. We found that anti-CD11a and anti-CD38 were rapidly internalized by monocytes, while uptake of anti-TNFα depended on pre-activation with LPS. Using these antibodies were attached to a novel linker system, ValCitGlyPro-Dex (VCGP-Dex), that efficiently released dexamethasone upon lysosomal catabolism. This linker relies on lysosomal cathepsins to cleave after the ValCit sequence, thereby releasing a GlyPro-Dex species that undergoes rapid self-immolation to form dexamethasone. The resulting monocyte-targeting ADCs bearing this linker payload effectively suppressed LPS-induced NFκB activation and cytokine release in both a monocytic cell line (THP1) and in human PBMCs. Anti-TNFα_VCGP-Dex and anti-CD38_VCGP-Dex were particularly effective, suppressing ∼60-80% of LPS-induced IL-6 release from PBMCs at 3-10 μg mL-1 concentrations. In contrast, the corresponding isotype control ADC (anti-RSV) and the corresponding naked antibodies (anti-CD38 and anti-TNFα) resulted in only modest suppression (0-30%) of LPS-induced IL-6. Taken together, these results provide further evidence of the ability of glucocorticoid-ADCs to selectively suppress immune responses, and highlight the potential of two targets (CD38 and TNFα) for the development of novel immune-suppressing ADCs.

Kinin B1 receptor blockade attenuates hepatic fibrosis and portal hypertension in chronic liver diseases in mice

Background and aimsKinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl4) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs).MethodsFibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl4) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study.ResultsB1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl4 and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFβ, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl4- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-β and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway.ConclusionsB1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.

The Organogermanium Compound 3-(Trihydroxygermyl) Propanoic Acid (THGP) Suppresses Inflammasome Activation Via Complexation with ATP.

Inflammasome activity is a key indicator of inflammation. The inflammasome is activated by pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), which activate the p38-NF-κB pathway and promote IL-1β transcription (signaling step 1). Next, extracellular adenosine triphosphate (ATP) activates the inflammasome (a protein complex consisting of a signal recognition protein, an adapter protein, and Caspase-1) and secretion of inflammatory cytokines such as IL-1β (signaling step 2). Inflammasome activation causes excessive inflammation, leading to inflammasome-active diseases such as atherosclerosis and type 2 diabetes. A hydrolysate of the organogermanium compound Ge-132, 3-(Trihydroxygermyl) propanoic acid (THGP) can form a complex with a cis-diol structure. We investigated the inhibitory effect of THGP on inflammasome activity in human THP-1 monocytes. THGP inhibited IL-1β secretion and caspase-1 activation (signaling step 2) in an ATP-dependent manner. On the other hand, THGP did not suppress IL-1β secretion induced by only lipopolysaccharide (LPS) stimulation. In addition, as IL-6 is an ATP-independent inflammatory cytokine, THGP did not decrease its secretion. THGP also suppressed pyroptosis, which is a caspase-1 activity-dependent form of cell death. Therefore, THGP is expected to become a new therapeutic or prophylactic agent for inflammasome-associated diseases.

Glycocalyx Disruption Triggers Human Monocyte Activation in Acute Heart Failure Syndromes.

Acute heart failure (AHF) syndromes manifest increased inflammation and vascular dysfunction; however, mechanisms that integrate the two in AHF remain largely unknown. The glycocalyx (GAC) is a sugar-based shell that envelops all mammalian cells. Much GAC research has focused on its role in vascular responses, with comparatively little known about how the GAC regulates immune cell function. In this study, we sought to determine if GAC degradation products are elevated in AHF patients, how these degradation products relate to circulating inflammatory mediators, and whether the monocyte GAC (mGAC) itself modulates monocyte activation. Inflammatory markers and GAC degradation products were profiled using ELISAs. Flow cytometry was used to assess the mGAC and RNA-seq was employed to understand the role of the mGAC in regulating inflammatory activation programs. In a cohort of hospitalized AHF patients (n = 17), we found that (1) the GAC degradation product heparan sulfate (HS) was elevated compared with age-matched controls (4396 and 2903ng/mL; p = 0.01) and that (2) HS and soluble CD14 (a marker of monocyte activation) levels were closely related (Pearson's r = 0.65; p = 0.002). Mechanistically, Toll-like receptor (TLR) activation of human monocytes results in GAC remodeling and a decrease in the mGAC (71% compared with no treatment; p = 0.0007). Additionally, we found that ex vivo enzymatic removal of HS and disruption of the mGAC triggers human monocyte activation and amplifies monocyte inflammatory responses. Specifically, using RNA-seq, we found that enzymatic degradation of the mGAC increases transcription of inflammatory (IL6, CCL3) and vascular (tissue factor/F3) mediators. These studies indicate that the mGAC is dynamically remodeled during monocyte activation and that mGAC remodeling itself may contribute to the heightened inflammation associated with AHF.

Inflammatory and diabetic conditions trigger SHP2 tyrosine phosphatase expression and subsequent aberrant activation of primary human monocytes

Abstract Purpose Atherosclerosis is an inflammatory process that is particularly accelerated in diabetics, leading to increased incidence of cardiovascular diseases such as CAD and PAD in diabetic patients. Monocytes are the main component of atherosclerosis development. SHP-2 tyrosine phosphatase has been identified as an important regulator of monocyte function. The present study therefore aims to investigate the regulation of SHP-2 in inflammatory and diabetic conditions. Methods Primary human monocytes were isolated from the peripheral blood of type 2 Diabetes mellitus (T2DM) patients and healthy individuals. Monocytes were incubated with pro-inflammatory cytokine TNFa. For diabetic conditions, monocytes were incubated with methylglyoxal (MG), a highly reactive side product of glycolysis, or Receptor for advanced glycation end product (RAGE) ligand AGE-bovine serum (AGE-BSA). Monocyte migration was studied with Transwell migration assays. Expression of important molecules was investigated with Western Blot, RT-qPCR or FACS. Pharmacological inhibitors for SHP2, RAGE or NFκB were used. Results First, we could detect a significant correlation between SHP-2 mRNA and TNFa levels in T2DM monocytes in comparison to monocytes from healthy individuals. In line with that, incubation of monocytes with TNFa lead to an enhanced expression of SHP-2. Co-incubation with NFκB-inhibitor blocked TNFa-induced SHP-2 upregulation. Interestingly, incubation of monocytes with methylglyoxal caused increased release of TNFa and also augmented expression of SHP-2, indicating a pro-inflammatory effect of diabetic conditions. Moreover, AGE-BSA treatment induced enhanced SHP-2 expression, reflecting an inflammatory-independent pathway which regulates SHP-2 additionally. This could be supported by the observation that pharmacological inhibition of RAGE attenuated both AGE-BSA and MG-induced SHP-2 activation. On a functional level, increased expression of SHP-2 in each treatment resulted in a pro-migratory phenotype that could be completely reversed by inhibition of RAGE, respectively. Fittingly, monocytes from T2DM patients showed increased migration, which normalized to an ordinary level after application of a SHP-2 inhibitor. Conclusions The present results reveal a new mechanism for accelerated atherosclerosis development in diabetic patients. MG and advanced glycated end products, as crucial components of the diabetic milieu, lead to increased expression of SHP-2 via the RAGE-NFkB signalling axis. Interestingly, this diabetic environment causes an increased inflammatory response through the release of TNFa cytokine, which itself leads to enhanced SHP-2 expression through activation of the NFkB transcription factor. Finally, by pharmacological inhibition of each component in this outlined SHP-2 regulatory pathway, we were able to prevent the pro-migratory activation of monocytes, offering a new approach to the treatment of diabetes-induced atherosclerosis. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): IZKF SEED Project 14/20

Identification of immunomodulatory drugs that inhibit multiple inflammasomes and impair SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces mild or asymptomatic COVID-19 in most cases, but some patients develop an excessive inflammatory process that can be fatal. As the NLRP3 inflammasome and additional inflammasomes are implicated in disease aggravation, drug repositioning to target inflammasomes emerges as a strategy to treat COVID-19. Here, we performed a high-throughput screening using a 2560 small-molecule compound library and identified FDA-approved drugs that function as pan-inflammasome inhibitors. Our best hit, niclosamide (NIC), effectively inhibits both inflammasome activation and SARS-CoV-2 replication. Mechanistically, induction of autophagy by NIC partially accounts for inhibition of NLRP3 and AIM2 inflammasomes, but NIC-mediated inhibition of NAIP/NLRC4 inflammasome are autophagy independent. NIC potently inhibited inflammasome activation in human monocytes infected in vitro, in PBMCs from patients with COVID-19, and in vivo in a mouse model of SARS-CoV-2 infection. This study provides relevant information regarding the immunomodulatory functions of this promising drug for COVID-19 treatment.

In vitro inflammatory and immune response to uncrosslinked hyaluronic acid (HA) and HA fillers

IntroductionDelayed-onset nodules are a complication associated with soft tissue filler products, including hyaluronic acid (HA) fillers; however, the etiology of these events remains unclear. The role of uncrosslinked HA and crosslinked HA fillers in the response of immune cells (dendritic cells, B cells, and T cells) was evaluated with and without concurrent bacterial stimulation. MethodsUncrosslinked HA of varying molecular weights and crosslinked HA fillers were tested in a series of in vitro assays to evaluate 1) human monocyte activation; 2) activation, maturation, and migration of human monocyte-derived dendritic cells (MoDC); 3) T-cell activation and associated skin damage; and 4) T-cell–mediated B-cell response. For the latter assay, the HA test articles were also evaluated following stimulation by bacteria. ResultsWhen treated with lipopolysaccharide (LPS) as a positive control, inflammatory cytokines (interleukin [IL]-1β, IL-6, IL-10, IL-12p70, and tumor necrosis factor [TNF]-α) released by MoDC were significantly upregulated. Treatment of the MoDC with HA (0.776, 150, and 485 kDa) did not result in stimulation of IL-1β, IL-6, IL-10, and IL-12p70, although increased TNF-α was observed with all HA test samples. The TNF-α increase was significantly lower for HA samples (1.5–2.64 pg/mL) compared with LPS (966.96–4834.18 pg/mL). Similarly, treatment of MoDC with HA samples did not result in expression of surface markers indicative of dendritic cell maturation. MoDC treated with 0.776 and 150 kDa HA, but not with 485 kDa HA, had significantly increased migration toward fetal bovine serum, but not C–C motif chemokine ligand 21 (CCL21). In the T-cell activation and skin damage assay, VYC-15L, degraded VYC-15L, HYC-24L+, and oligosacharride HA samples were negative in the T-cell proliferation and interferon-γ assays at all tested concentrations. The degraded HYC-24L + sample was borderline at the highest concentration and negative at the middle and low concentrations. Skin damage was determined to be negative for all HA samples at all concentrations. In the human monocyte activation assay, all tested uncrosslinked HA samples (5, 150, and 731 kDa) were determined to be non-sensitizing (i.e., did not activate monocyte cells). In the T-cell–mediated B-cell activation assay, treatment with HA oligosaccharides, uncrosslinked HA, and crosslinked HA fillers did not result in any consistent stimulation of any cytokines measured. A molecular weight–dependent decrease in the secreted immunoglobulin G (IgG) was observed, and this effect was more pronounced with the crosslinked HA fillers. Addition of heat-inactivated Cutibacterium acnes bacteria (HIB) to uncrosslinked HA and crosslinked HA fillers resulted in increased cytokine production similar to when HIB was tested alone. The decrease in secreted IgG observed for crosslinked HA filler samples without HIB was maintained with addition of HIB to VYC-20, VYC-25, SGD-30XP, and HYC-24+ samples. The reduction of secreted IgG observed for RSQ and VYC-15 alone was no longer apparent when HIB was added to these cultures (i.e., returned to control levels but were not upregulated). ConclusionThe results from this study suggest that uncrosslinked HA and HA fillers themselves do not stimulate an inflammatory/immune response, regardless of molecular weight or crosslinking. However, the immune response following HIB stimulation may be impacted by the presence of HA fillers, suggesting a potential initiating role of contaminating factors such as bacteria and a modulating role of HA.