Human Monocytic THP-1 Cells Research Articles

Utilization of CRISPR interference to investigate the contribution of genes to pathogenesis in a macrophage model of Mycobacterium tuberculosis infection.

There is an urgent need for novel drugs that target unique cellular pathways to combat infections caused by Mycobacterium tuberculosis. CRISPR interference (CRISPRi)-mediated transcriptional repression has recently been developed for use in mycobacteria as a genetic tool for identifying and validating essential genes as novel drug targets. Whilst CRISPRi has been applied to extracellular bacteria, no studies to date have determined whether CRISPRi can be used in M. tuberculosis infection models. Using the human monocytic macrophage-like THP-1 cell line as a model for M. tuberculosis infection we investigated if CRISPRi can be activated within intracellular M. tuberculosis. The transcriptional repression of two candidate M. tuberculosis genes, i.e. mmpL3 and qcrB, leads to a reduction in viable M. tuberculosis within infected THP-1 cells. The reduction in viable colonies is dependent on both the level of CRISPRi-mediated repression and the duration of repression. These results highlight the utility of CRISPRi in exploring mycobacterial gene function and essentiality under a variety of conditions pertinent to host infection.

Monocytes secrete CXCL7 to promote breast cancer progression

Certain immune cells and inflammatory cytokines are essential components in the tumor microenvironment to promote breast cancer progression. To identify key immune players in the tumor microenvironment, we applied highly invasive MDA-MB-231 breast cancer cell lines to co-culture with human monocyte THP-1 cells and identified CXCL7 by cytokine array as one of the increasingly secreted cytokines by THP-1 cells. Further investigations indicated that upon co-culturing, breast cancer cells secreted CSF1 to induce expression and release of CXCL7 from monocytes, which in turn acted on cancer cells to promote FAK activation, MMP13 expression, migration, and invasion. In a xenograft mouse model, administration of CXCL7 antibodies significantly reduced abundance of M2 macrophages in tumor microenvironment, as well as decreased tumor growth and distant metastasis. Clinical investigation further suggested that high CXCL7 expression is correlated with breast cancer progression and poor overall survival of patients. Overall, our study unveils an important immune cytokine, CXCL7, which is secreted by tumor infiltrating monocytes, to stimulate cancer cell migration, invasion, and metastasis, contributing to the promotion of breast cancer progression.

Tyrosine kinase nonreceptor 1 (TNK1) knockdown ameliorates hemorrhage shock-induced kidney injury via inhibiting macrophage M1 polarization.

Hemorrhage shock (HS) is a major threat to patients with trauma and spontaneous bleeding, resulting in multi-organ failure including the kidney. Tyrosine kinase nonreceptor 1 (TNK1) has been shown to be upregulated in the kidney of experimental HS and patients with severe trauma. The study aims to investigate the role of TNK1 and the underlying mechanism in HS-induced kidney injury. A model of HS was established with femoral artery bloodletting, followed by resuscitation in Sprague-Dawley rats. Renal expression of TNK1 was abnormally induced by HS in rats. Knockdown of TNK1 alleviated HS-induced cell apoptosis and the level of proinflammatory cytokines (TNF-α, IL-6 and IL-1β) in the kidney. The expression of M1 macrophage markers (CD86 and iNOS) and the activation of STAT1 were inhibited by TNK1 knockdown in HS rats. In vitro, human monocyte THP-1 cells were treated with 20ng/mL interferon-gamma plus 100ng/mL lipopolysaccharide to induce M1 polarization. TNK1 knockdown exerted inhibitory effect on macrophage M1 polarization, M1-type inflammatory cytokine production and STAT1 activation in THP-1 cells. In conclusion, downregulation of TNK1 alleviates HS-induced kidney injury by suppressing macrophage M1 polarization, inflammation and kidney cell apoptosis, in which the deactivation of STAT1 signaling may be involved.

Biosynthesis of Silver Nanoparticles from Leaf Extract of Salvia coccinea and Its Effects of Anti-inflammatory Potential in Human Monocytic THP-1 Cells.

Inflammation is one of the basic pathophysiologically important components in many life-threatening diseases. Metallic nanoparticles play a crucial role in biomedical applications. The present study was aimed at investigating the ameliorative effect of biosynthesized silver nanoparticles (ScAgNPs) using Salvia coccinea leaf extracts and characterizing them using physical and chemical methods, followed by evaluation of their cytotoxic, anti-oxidant, and anti-inflammatory potentials in monocytic THP-1 cells. Luciferase reporter assays and qRT-PCRs were used for gene expression studies. As oxidative stress and inflammation are mutually induced by each other, inhibiting oxidative stress could subsequently lead to inhibition of inflammation. Spherical-shaped ScAgNPs with 24 nm average size were successfully synthesized. The DCF staining technique, in addition to DPPH and reducing power activity assays, showed that 100-400 μg/mL concentration of ScAgNPs decreased oxidative stress significantly, induced by high glucose, in THP-1 cells. Anti-inflammatory effects of ScAgNPs have corroborated inhibition of high-glucose-induced oxidative stress-sensitive transcription factor NF-κB-driven transcription of proinflammatory COX-2, MCP-1, IP-10, IL-17E, and IL-6 promoters significant in high-glucose-grown THP-1 cells, consistent with promoter inhibition, and the corresponding mRNA expression levels were also decreased, suggesting that ScAgNPs could be a potential anti-inflammatory agent, which could efficiently inhibit inflammation in THP-1 cells. Our initial in vitro studies suggested that ScAgNPs could serve as therapeutic candidates to alleviate inflammatory diseases by inhibiting oxidative stress and inflammation.

Mitochondrial MicroRNAs Contribute to Macrophage Immune Functions Including Differentiation, Polarization, and Activation.

A subset of microRNA (miRNA) has been shown to play an important role in mitochondrial (mt) functions and are named MitomiR. They are present within or associated with mitochondria. Most of the mitochondrial miRNAs originate from the nucleus, while a very limited number is encoded by mtDNA. Moreover, the miRNA machinery including the Dicer and Argonaute has also been detected within mitochondria. Recent, literature has established a close relationship between miRNAs and inflammation. Indeed, specific miRNA signatures are associated with macrophage differentiation, polarization and functions. Nevertheless, the regulation of macrophage inflammatory pathways governed specifically by MitomiR and their implication in immune-mediated inflammatory disorders remain poorly studied. Here, we propose a hypothesis in which MitomiR play a key role in triggering macrophage differentiation and modulating their downstream activation and immune functions. We sustain this proposition by bioinformatic data obtained from either the human monocytic THP1 cell line or the purified mitochondrial fraction of PMA-induced human macrophages. Interestingly, 22% of the 754 assayed miRNAs were detected in the mitochondrial fraction and are either exclusively or highly enriched cellular miRNA. Furthermore, the in silico analysis performed in this study, identified a specific MitomiR signature associated with macrophage differentiation that was correlated with gene targets within the mitochondria genome or with mitochondrial pathways. Overall, our hypothesis and data suggest a previously unrecognized link between MitomiR and macrophage function and fate. We also suggest that the MitomiR-dependent control could be further enhanced through the transfer of mitochondria from donor to target cells, as a new strategy for MitomiR delivery.

Surfactin reduces particulate matter-induced VCAM-1-dependent monocyte adhesion in human gingival fibroblasts by increasing Nrf2-dependent HO-1 expression.

The mechanisms of particulate matter (PM) toxicity involve the generation of ROS and upregulation of proinflammatory molecules. Nrf2 is a multifunctional cytoprotective transcription factor that regulates the expression of various antioxidant, anti-inflammatory, and detoxifying molecules, such as HO-1. As surfactin has potential to induce Nrf2 activation and HO-1 expression, this study aimed to investigate the anti-inflammatory effects of surfactin on PM-exposed human gingival fibroblasts (HGFs) and signaling pathways engaged by surfactin. Human gingival fibroblasts were challenged by PM with or without surfactin pretreatment. The expression of Nrf2, HO-1, VCAM-1, and other molecules was determined by western blot, real-time PCR, or ELISA. Human monocytic THP-1 cells labeled with fluorescent reagent were added to HGFs, and the cell adhesion was assessed. ROS generation and NADPH oxidase activity were also measured. The involvement of Nrf2/HO-1 and ROS signaling pathways was investigated by treating HGFs with specific pathway interventions, genetically or pharmacologically. One dose of surfactin was given to mice before PM treatment to explore its in vivo effect on VCAM-1 expression in gingival tissues. Particulate matter led to VCAM-1-dependent monocyte adhesion in HGFs, which was regulated by PKCα/NADPH oxidase/ROS/STAT1/IL-6 pathway. Surfactin could attenuate monocyte adhesion by disrupting this VCAM-1-dependent pathway. Additionally, surfactin promoted Nrf2-dependent HO-1 expression in HGFs, mitigating VCAM-1 expression. PM-treated mice exhibited the lower expression of IL-6 and VCAM-1 in gingival tissues if they previously received surfactin. Surfactin exerts anti-inflammatory effects against PM-induced inflammatory responses in HGFs by inhibiting VCAM-1-dependent pathway and inducing Nrf2/HO-1 axis.

In Vivo PET Imaging of Monocytes Labeled with [89Zr]Zr-PLGA-NH2 Nanoparticles in Tumor and Staphylococcus aureus Infection Models.

Simple SummaryImmune cells are increasingly used for therapy in cancer and other diseases. To better understand immune-cell kinetics, cell-tracking with highly sensitive imaging modalities is required. The aim of this study was to develop a new strategy for the in vivo tracking of a small number of cells, using positron emission tomography (PET). We labeled poly(lactic-co-glycolic acid) nanoparticles containing a primary endcap (PLGA-NH2) with the radionuclide zirconium-89. The nanoparticles were characterized for size, polydispersity index, zetapotential and radiolabel retention. Subsequently, they were used for the ex vivo radiolabeling of a monocyte cell line (THP-1). We demonstrated that these radiolabeled monocyte cells can be traced in vivo in mouse tumor and infection models.The exponential growth of research on cell-based therapy is in major need of reliable and sensitive tracking of a small number of therapeutic cells to improve our understanding of the in vivo cell-targeting properties. 111In-labeled poly(lactic-co-glycolic acid) with a primary amine endcap nanoparticles ([111In]In-PLGA-NH2 NPs) were previously used for cell labeling and in vivo tracking, using SPECT/CT imaging. However, to detect a low number of cells, a higher sensitivity of PET is preferred. Therefore, we developed 89Zr-labeled NPs for ex vivo cell labeling and in vivo cell tracking, using PET/MRI. We intrinsically and efficiently labeled PLGA-NH2 NPs with [89Zr]ZrCl4. In vitro, [89Zr]Zr-PLGA-NH2 NPs retained the radionuclide over a period of 2 weeks in PBS and human serum. THP-1 (human monocyte cell line) cells could be labeled with the NPs and retained the radionuclide over a period of 2 days, with no negative effect on cell viability (specific activity 279 ± 10 kBq/106 cells). PET/MRI imaging could detect low numbers of [89Zr]Zr-THP-1 cells (10,000 and 100,000 cells) injected subcutaneously in Matrigel. Last, in vivo tracking of the [89Zr]Zr-THP-1 cells upon intravenous injection showed specific accumulation in local intramuscular Staphylococcus aureus infection and infiltration into MDA-MB-231 tumors. In conclusion, we showed that [89Zr]Zr-PLGA-NH2 NPs can be used for immune-cell labeling and subsequent in vivo tracking of a small number of cells in different disease models.

ROS/TNF-α Crosstalk Triggers the Expression of IL-8 and MCP-1 in Human Monocytic THP-1 Cells via the NF-κB and ERK1/2 Mediated Signaling.

IL-8/MCP-1 act as neutrophil/monocyte chemoattractants, respectively. Oxidative stress emerges as a key player in the pathophysiology of obesity. However, it remains unclear whether the TNF-α/oxidative stress interplay can trigger IL-8/MCP-1 expression and, if so, by which mechanism(s). IL-8/MCP-1 adipose expression was detected in lean, overweight, and obese individuals, 15 each, using immunohistochemistry. To detect the role of reactive oxygen species (ROS)/TNF-α synergy as a chemokine driver, THP-1 cells were stimulated with TNF-α, with/without H2O2 or hypoxia. Target gene expression was measured by qRT-PCR, proteins by flow cytometry/confocal microscopy, ROS by DCFH-DA assay, and signaling pathways by immunoblotting. IL-8/MCP-1 adipose expression was significantly higher in obese/overweight. Furthermore, IL-8/MCP-1 mRNA/protein was amplified in monocytic cells following stimulation with TNF-α in the presence of H2O2 or hypoxia (p ˂ 0.0001). Synergistic chemokine upregulation was related to the ROS levels, while pre-treatments with NAC suppressed this chemokine elevation (p ≤ 0.01). The ROS/TNF-α crosstalk involved upregulation of CHOP, ERN1, HIF1A, and NF-κB/ERK-1,2 mediated signaling. In conclusion, IL-8/MCP-1 adipose expression is elevated in obesity. Mechanistically, ROS/TNF-α crosstalk may drive expression of these chemokines in monocytic cells by inducing ER stress, HIF1A stabilization, and signaling via NF-κB/ERK-1,2. NAC had inhibitory effect on oxidative stress-driven IL-8/MCP-1 expression, which may have therapeutic significance regarding meta-inflammation.

BEWO trophoblast cells and Toxoplasma gondii infection modulate cell death mechanisms in THP-1 monocyte cells by interference in the expression of death receptor and intracellular proteins

Crosstalk between trophoblast and monocytes is essential for gestational success, and it can be compromised in congenital toxoplasmosis. Cell death is one of the mechanisms involved in the maintenance of pregnancy, and this study aimed to evaluate the role of trophoblast in the modulation of monocyte cell death in the presence or absence of Toxoplasma gondii infection. THP-1 cells were stimulated with supernatants of BeWo cells and then infected or not with T. gondii. The supernatants were collected and analyzed for the secretion of human Fas ligand, and cells were used to determine cell death and apoptosis, cell death receptor, and intracellular proteins expression. Cell death and apoptosis index were higher in uninfected THP-1 cells stimulated with supernatants of BeWo cells; however, apoptosis index was reduced by T. gondii infection. Macrophage migration inhibitory factor (MIF) and transforming growth factor (TGF)-β1, secreted by BeWo cells, altered the cell death and apoptosis rates in THP-1 cells. In infected THP-1 cells, the expression of Fas/CD95 and secretion of FasL was significantly higher; however, caspase 3 and phosphorylated extracellular-signal-regulated kinase (ERK1/2) were downregulated. Results suggest that soluble factors secreted by BeWo cells induce cell death and apoptosis in THP-1 cells, and Fas/CD95 can be involved in this process. On the other hand, T. gondii interferes in the mechanism of cell death and inhibits THP-1 cell apoptosis, which can be associated with active caspase 3 and phosphorylated ERK1/2. In conclusion, our results showed that human BeWo trophoblast cells and T. gondii infection modulate cell death in human THP-1 monocyte cells.

Anti-Inflammatory Effect of Charantadiol A, Isolated from Wild Bitter Melon Leaf, on Heat-Inactivated Porphyromonas gingivalis-Stimulated THP-1 Monocytes and a Periodontitis Mouse Model.

Porphyromonas gingivalis has been identified as one of the major periodontal pathogens. Activity-directed fractionation and purification processes were employed to identify bioactive compounds from bitter melon leaf. Ethanolic extract of bitter melon leaf was separated into five subfractions by open column chromatography. Subfraction-5-3 significantly inhibited P. gingivalis-induced interleukin (IL)-8 and IL-6 productions in human monocytic THP-1 cells and then was subjected to separation and purification by using different chromatographic methods. Consequently, 5β,19-epoxycucurbita-6,23(E),25(26)-triene-3β,19(R)-diol (charantadiol A) was identified and isolated from the subfraction-5-3. Charantadiol A effectively reduced P. gingivalis-induced IL-6 and IL-8 productions and triggered receptors expressed on myeloid cells (TREM)-1 mRNA level of THP-1 cells. In a separate study, charantadiol A significantly suppressed P. gingivalis-stimulated IL-6 and tumor necrosis factor-α mRNA levels in gingival tissues of mice, confirming the inhibitory effect against P. gingivalis-induced periodontal inflammation. Thus, charantadiol A is a potential anti-inflammatory agent for modulating P. gingivalis-induced inflammation.

Increased LPS levels coexist with systemic inflammation and result in monocyte activation in severe COVID-19 patients

Mucosal barrier alterations may play a role in the pathogenesis of several diseases, including COVID-19. In this study we evaluate the association between bacterial translocation markers and systemic inflammation at the earliest time-point after hospitalization and at the last 72 h of hospitalization in survivors and non-survivors COVID-19 patients. Sixty-six SARS-CoV-2 RT-PCR positive patients and nine non-COVID-19 pneumonia controls were admitted in this study. Blood samples were collected at hospital admission (T1) (Controls and COVID-19 patients) and 0–72 h before hospital discharge (T2, alive or dead) to analyze systemic cytokines and chemokines, lipopolysaccharide (LPS) concentrations and soluble CD14 (sCD14) levels. THP-1 human monocytic cell line was incubated with plasma from survivors and non-survivors COVID-19 patients and their phenotype, activation status, TLR4, and chemokine receptors were analyzed by flow cytometry. COVID-19 patients presented higher IL-6, IFN-γ, TNF-α, TGF-β1, CCL2/MCP-1, CCL4/MIP-1β, and CCL5/RANTES levels than controls. Moreover, LPS and sCD14 were higher at hospital admission in SARS-CoV-2-infected patients. Non-survivors COVID-19 patients had increased LPS levels concomitant with higher IL-6, TNF-α, CCL2/MCP-1, and CCL5/RANTES levels at T2. Increased expression of CD16 and CCR5 were identified in THP-1 cells incubated with the plasma of survivor patients obtained at T2. The incubation of THP-1 with T2 plasma of non-survivors COVID-19 leads to higher TLR4, CCR2, CCR5, CCR7, and CD69 expression. In conclusion, the coexistence of increased microbial translocation and hyperinflammation in patients with severe COVID-19 may lead to higher monocyte activation, which may be associated with worsening outcomes, such as death.

Ergosta-7,9(11),22-trien-3β-ol Attenuates Inflammatory Responses via Inhibiting MAPK/AP-1 Induced IL-6/JAK/STAT Pathways and Activating Nrf2/HO-1 Signaling in LPS-Stimulated Macrophage-like Cells.

Chronic inflammation induces autoimmune disorders and chronic diseases. Several natural products activate nuclear factor erythroid 2-related factor 2 (Nrf2) signaling, attenuating inflammatory responses. Ergosta-7,9(11),22-trien-3β-ol (EK100) isolated from Cordyceps militaris showed anti-inflammatory and antioxidative activity, but those mechanisms are still unclear. This study is the first to investigate EK100 on antioxidant Nrf2 relative genes expression in LPS-stimulated macrophage-like cell lines. The results showed that EK100 reduced IL-6 (interleukin-6) and tumor necrosis factor-α production. EK100 also attenuated a mitogen-activated protein kinase/activator protein-1 (MAPK/AP-1) pathway and interleukin-6/Janus kinase/signal transducer and activator of transcription (IL-6/JAK/STAT) pathway in LPS-stimulated cells. Toll-like receptor 4 (TLR4) inhibitor CLI-095 and MAPK inhibitors can synergize the anti-inflammatory response of EK100 in LPS-stimulated cells. Moreover, EK100 activated Nrf2/HO-1 (heme oxygenase-1) signaling in LPS-stimulated murine macrophage-like RAW 264.7 cells, murine microglial BV2 cells, and human monocytic leukemia THP-1 cells. However, Nrf2 small interfering RNA (Nrf2 siRNA) reversed EK100-induced antioxidative proteins expressions. In conclusion, EK100 showed anti-inflammatory responses via activating the antioxidative Nrf2/HO-1 signaling and inhibiting TLR4 related MAPK/AP-1 induced IL-6/JAK/STAT pathways in the LPS-stimulated cells in vitro. The results suggest EK100 acts as a novel antioxidant with multiple therapeutic targets that can potentially be developed to treat chronic inflammation-related diseases.

Synthesis of new thiazole derivatives and evaluation of their antimicrobial and cytotoxic activities

Novel 2-heteroaryl-N-[4-(substituted aryl)thiazol-2-yl]propanamide derivatives (7a–7o) were synthesized and investigated for their antimicrobial activity. Among the tested compounds, 2-[(1H-Benzimidazol-2-yl)thio]-N-[4-(naphthalen-2-yl)thiazol-2-yl]propanamide (7e) and N-[4-(4-Chlorophenyl)thiazol-2-yl]-2-[(1-methyl-1H-tetrazol-5-yl)thio]propanamide (7f) showed the highest antibacterial activity, whereas 2-[Benzothiazol-2-ylthio]-N-[4-(4-chlorophenyl)thiazol-2-yl]propanamide (7i) and 2-[(1H-Benzimidazol-2-yl)thio]-N-[4-(4-chlorophenyl)thiazol-2-yl]propanamide (7j) displayed anticandidal effect against C. parapsilosis and C. glabrata. The cytotoxic activity of the compounds (7a–7o) was also tested against HL-60 human leukemia cells, THP-1 human monocytic leukemia cells, and NIH/3T3 mouse embryonic fibroblast cells. Compound N-[4-(4-Chlorophenyl)thiazol-2-yl]-2-[(1-methyl-1H-imidazol-2-yl)thio]propanamide (7 g) and compound 7j exhibited high cytotoxicity against HL-60; and compounds 2-[(1-Methyl-1H-imidazol-2-yl)thio]-N-[4-(naphthalen-2-yl)thiazol-2-yl]propanamide (7b), 7 g and N-[4-(4-Methoxyphenyl)thiazol-2-yl]-2-[(4-methyl-4H-1,2,4-triazol-3-yl)thio]propanamide (7 m) also had cytotoxic activity against THP-1 compared with standard drug with selective profile. Additionally, in silico physicochemical properties of the compounds were described.

Regulation of NADPH Oxidase-Mediated Superoxide Production by Acetylation and Deacetylation.

Oral treatment of apolipoprotein E-knockout (ApoE-KO) mice with the putative sirtuin 1 (SIRT1) activator resveratrol led to a reduction of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the heart. In contrast, the SIRT1 inhibitor EX527 enhanced the superoxide production in isolated human polymorphonuclear granulocytes. In human monocytic THP-1 cells, phorbol ester-stimulated superoxide production was enhanced by inhibitors of histone deacetylases (HDACs; including quisinostat, trichostatin A (TSA), PCI34051, and tubastatin A) and decreased by inhibitors of histone acetyltransferases [such as garcinol, curcumin, and histone acetyltransferase (HAT) Inhibitor II]. These results indicate that protein acetylation and deacetylation may represent crucial mechanisms regulating NADPH oxidase-mediated superoxide production. In cell-free systems, incubation of recombinant Rac1 with SIRT1 resulted in decreased Rac1 acetylation. Mass spectrometry analyses identified lysine 166 (K166) in Rac1 as a residue targeted by SIRT1. Deacetylation of Rac1 by SIRT1 markedly reduced the interaction of Rac1 with p67phox in in vitro assays. Computational modeling analyses revealed that K166 deacetylation of Rac1 led to a 5-fold reduction in its binding affinity to guanosine-5'-triphosphate, and a 21-fold decrease in its binding potential to p67phox. The latter is crucial for Rac1-mediated recruitment of p67phox to the membrane and for p67phox activation. In conclusion, both SIRT1 and non-sirtuin deacetylases play a role in regulating NADPH oxidase activity. Rac1 can be directly deacetylated by SIRT1 in a cell-free system, leading to an inhibition of Rac1-p67phox interaction. The downstream targets of non-sirtuin deacetylases are still unknown. The in vivo significance of these findings needs to be investigated in future studies.

Necroptosis Underlies Neutrophilic Inflammation Associated with the Chronic Rhinosinusitis with Nasal Polyps (CRSwNP).

BackgroundNecroptosis is an inflammatory cell death associated with a variety of chronic diseases. Chronic rhinosinusitis with nasal polyps (CRSwNP) is a chronic inflammatory disease accompanied by eosinophil and neutrophil infiltration. The role of necroptosis in the pathogenesis of CRSwNP remains elusive.MethodsCell death, including apoptosis, pyroptosis and necroptosis in control sinonasal mucosa and CRSwNP, were analyzed by immunoblotting, immunohistochemistry (IHC) and immunofluorescence (IF) staining for cleaved caspase 3, cleaved gasdermin D and p-MLKL, respectively. Correlations between necroptosis, inflammatory cytokines and neutrophil infiltration were assessed and a possible role of necroptosis in CRSwNP was evaluated. Primary nasal polyp cells (DNPCs) were stimulated with damage-associated molecular patterns (DAMPs) including ATP or IL-1α and their expression of inflammatory cytokines was analyzed using RT-PCR. The expression of TNF-α and IFNs in nasal polyps was measured by ELISA; human monocyte THP-1 cells were treated with TNF-α or IFN-γ and cell death was measured by LDH release.ResultsNecroptosis, rather than apoptosis or pyroptosis, was overtly activated in both eosinophilic and non-eosinophilic CRSwNP as evidenced by the presence of prominent phosphorylation of MLKL compared to controls. The abundance of DAMPs (IL-1α, HMGB1), inflammatory cytokines (IL-6) and chemokines (IL-8, CXCL-1) were all increased especially in non-eosinophilic CRSwNP. The extent of necroptosis was positively correlated with the abundance of DAMPs and cytokines, and neutrophil infiltration in CRSwNP. In DNPCs, ATP and IL-1α induced the expression of IL-8 and CXCL-1. Macrophage was found to be the predominant cell type positive for p-MLKL in CRSwNP. Concomitant treatment with TNF-α and IFN-γ, which were abundantly present in CRSwNP, triggered marked necroptosis in THP-1 cells.ConclusionNecroptosis induced by TNF-α and IFN-γ may facilitate the production and release of a myriad of proinflammatory cytokines and entailed neutrophil infiltration to exacerbate inflammation in CRSwNP.

Assessment of changes in autophagic vesicles in human immune cell lines exposed to nano particles

BackgroundSafe and rational development of nanomaterials for clinical translation requires the assessment of potential biocompatibility. Autophagy, a critical homeostatic pathway intrinsically linked to cellular health and inflammation, has been shown to be affected by nanomaterials. It is, therefore, important to be able to assess possible interactions of nanomaterials with autophagic processes.ResultsCEM (T cell), Raji (B lymphocyte), and THP-1 (human monocyte) cell lines were subject to treatment with rapamycin and chloroquine, known to affect the autophagic process, in order to evaluate cell line-specific responses. Flow cytometric quantification of a fluorescent autophagic vacuole stain showed that maximum observable effects (105%, 446%, and 149% of negative controls) were achieved at different exposure durations (8, 6, and 24 h for CEM, Raji, and THP-1, respectively). THP-1 was subsequently utilised as a model to assess the autophagic impact of a small library of nanomaterials. Association was observed between hydrodynamic size and autophagic impact (r2 = 0.11, p = 0.004). An ELISA for p62 confirmed the greatest impact by 10 nm silver nanoparticles, abolishing p62, with 50 nm silica and 180 nm polystyrene also lowering p62 to a significant degree (50%, 74%, and 55%, respectively, p < 0.05).ConclusionsThis data further supports the potential for a variety of nanomaterials to interfere with autophagic processes which, in turn, may result in altered cellular function and viability. The association of particle size with impact on autophagy now warrants further investigation.Graphic abstract

MKP-3 suppresses LPS-induced inflammatory responses in HUVECs via inhibition of p38 MAPK/NF-κB pathway

ABSTRACT Endothelial cell dysfunction and inflammatory responses play critical roles in the development of atherosclerosis. Recent data on the processes underlying atherogenesis indicate the substantial role of endotoxins (lipopolysaccharides; LPS) of the intestinal microflora in the initiation and progression of atherosclerosis. Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a cytoplasmic dual-specificity protein phosphatase that specifically binds to and inactivates MAP kinases in mammalian cells, but its biological function in endothelial cell dysfunction and inflammatory responses remains largely unknown. The aim of the present study was to investigate the role of MKP-3 in endotoxin-induced endothelial inflammation by western blotting, quantitative polymerase chain reaction, and immunofluorescence. The results of our study demonstrated that MKP-3 overexpression markedly inhibited the adhesion of human monocytic THP-1 cells to human umbilical vein endothelial cells (HUVECs) by downregulating the expression of vascular cell adhesion protein 1 (VCAM-1) and pro-inflammatory cytokines. In contrast, MKP-3-encoding gene knockdown by small interfering RNA (siRNA) exacerbated LPS-induced endothelial dysfunction. Additionally, we found that MKP-3 overexpression inhibited LPS-induced p38 MAPK phosphorylation and decreased the nuclear translocation of nuclear factor kappa B (NF-κB) after LPS treatment, suggesting its implication in the LPS/Toll-like receptor 4 (TLR4)/p38/NF-κB pathway. Overall, these observations suggest that MKP-3 plays a protective role in endothelial dysfunction and may be a therapeutic target.

Abstract 2693: Macrophage-derived endotrophin supports tumor migration potentials in thyroid cancer

Abstract Endotrophin (ETP), a cleaved fragment of the C5 domain of the Type VI collagen α3 (Col6α3), has been shown to play pro-tumorigenic roles in breast and liver cancers. However, the ETP actions in tumor microenvironment (TME) is still undetermined. This study aimed to investigate the role and the mechanism of ETP in macrophage-enriched thyroid cancer TMEs. First, the expression of ETP on various human thyroid tissues was studied. Immunohistochemical staining showed that the ETP was expressed in papillary (PTC) or anaplastic (ATC) thyroid cancer tissues but not in benign adenoma or autoimmune thyroiditis. Among 146 PTCs, tumors were divided into two groups according to their ETP expression, ETPlow and ETPHigh. Tumor size was bigger and LN metastasis rates were higher in ETPHigh than ETPlow group. Interestingly, the ETP expression showed positive correlations with the CD163-positive macrophages in thyroid tumors. To clarify the origin of ETP in TME of thyroid cancer, xenograft tumor from FRO (ATC) cells was co-stained with anti-ETP and anti-CD11b antibodies. Immunofluorescent staining showed that the expression of ETP and CD11b-postive macrophages were co-localized in the peritumoral area. Moreover, CD163+ cells, as a marker of M2-type macrophage, were co-localized with ETP+ cells. Additionally, human monocytic THP-1 cells were treated with conditioned media (CM) from FRO or BCPAP cells (designated FRO-CM or BCPAP-CM) and demonstrated that ETP expression was more up-regulated in the FRO-CM or BCPAP-CM treated group than in the control group. Collectively, ETP produced from M2-type macrophages in thyroid cancer microenvironments. Next, the pro-tumorigenic functions of ETP has been studied. CMs from co-cultures of FRO or BCPAP cells with THP-1 cells were harvested and treated to FRO or BCPAP cells. Treatment of CMs from co-cultures increased cell migration potentials than that of the single cell alone, and these effects were reduced by anti-ETP neutralizing antibody, indicating that ETPs play essential role in macrophage-induced tumor cell migration potentials. Moreover, MMP-9 and MMP-14, the candidates of Col6α3 protease to produce ETP were increased in THP-1 cells by treatment of CM. Additionally, treatment of CMs from MMP-9 or MMP-14 inhibitor-treated co-cultures of FRO and THP-1 cells decreased cancer cells migration potentials than the control CMs, and these effects were recovered by treatment of ETP. Finally, the RNA-seq dataset of human thyroid cancer showed that higher expressions of ETP positively correlated with macrophage-related genes such as CD163 and MMPs including MMP-9 and MMP-14. In conclusion, macrophage contributes ETP productions by modulating MMP expressions and ETP supports pro-metastatic potentials of human thyroid cancer cells in TMEs. Thus ETP can be a good therapeutic target for macrophage-enriched advanced thyroid cancers Citation Format: Hyo Shik Shin, Hana Kim, Young Shin Song, Hyun Jin Sun, Young Mi Whang, Jiyoung Park, Do Joon Park, Young Joo Park, Sun Wook Cho. Macrophage-derived endotrophin supports tumor migration potentials in thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2693.

Elucidation of Autoinflammatory Mechanism in Pigt-PNH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder caused by somatic mutations in PIGA gene (PIGA-PNH). PNH red blood cells are susceptible to complement-mediated intravascular hemolysis because of a loss of the glycosylphosphatidyl-inositol (GPI) anchored proteins including complement regulatory proteins. Recently, a PNH patient caused by the PIGT mutations was reported (PIGT-PNH) and had unusual symptoms similar to autoinflammatory syndrome. Whereas PIGA is essential for the first step in the biosynthesis of GPI anchors, PIGT is involved in transfer of GPI to proteins; thus its defect causes accumulation of free GPI, non-protein linked GPI, in affected cells. It is likely that the free GPI accumulated in affected cells together with activated complement contribute to inflammasome activation. Here, we aimed to investigate whether free GPI together with complement activation can induce inflammasome activation in human monocytic cells in PIGT-PNH.We generated THP-1 human monocytic cells in which PIGT or PIGA gene was deleted using the CRISPR/Cas9 system. Each knockout cell was rescued with a corresponding wild-type cDNA. Cells were differentiated into macrophages under RPMI-1640 medium containing PMA (100 ng/ml, 3h) in the presence of 10% fetal bovine serum. For induction of inflammasome activation, both of the priming signal and the stimulating signal are necessary. PMA-differentiated cells were primed for 5hr with Pam3CSK4 (200 ng/ml), which specifically activates the Toll-like receptor 2 (TLR 2), followed by ATP stimulation (5 mM, 1h), or cells were stimulated by acidified serum (21:1 of 0.4M HCl) alone, which activates alternative complement pathway. Heat-inactivated (56°C for 30 min) serum was used as a negative control. ELISA kits were used to measure the interleukin (IL) -1β levels in supernatants according to the manufacturers' instructions. Total RNA was extracted from cultured cells and quantitative Real-Time PCR (qRT-PCR) was performed for mRNA level of IL-1β and NLRP3.Results are expressed as means ± SD of three independent experiments. Priming of PMA-differentiated WT, PIGT deficient (PIGTKO), and PIGA deficient (PIGAKO) THP-1 cells with Pam3CSK4, followed by ATP stimulation induced similar levels of IL-1β secretion (167.9±61.5, 144.3±19.7, 200.3±48.8 pg/1.6x105 cells). In contrast, when cells were stimulated with acidified serum alone, PIGTKO cells significantly enhanced IL-1β secretion compared to WT and PIGAKO cells, in which PIGTKO cells showed about 50- and 2-fold increase, respectively (23.7±2.2, 1221.8±91.6, 568.2±101 pg/1.6x105 cells; P<0.01 PIGTKO compered to PIGAKO). This elevated levels of IL-1β were decreased comparably to WT cells by the treatments with heat-inactivated acidified serum or anti-C5 monoclonal antibody, indicating IL-1β secretion was enhanced due to complement activation in PIGTKO and PIGAKO cells. PIGT and PIGA rescued cells showed similar levels of IL-1β to WT cells. Interestingly, qPCR revealed that PMA treatment alone induced transcription of proIL-1β and NLRP3 and further incubation of PIGTKO cells with acidified serum showed similar elevation in transcription compered to WT and PIGAKO cells.Thus, our data demonstrated that complement activation works as both a priming signal and a stimulating signal for inflammasome activation in PIGAKO and PIGTKO THP-1 cells. The facts that the PIGT-PNH patients, but not PIGA-PNH patients, showed autoinflammatory symptoms and that PIGTKO THP-1 secreted higher level of L-1β than PIGAKO THP-1 cells upon stimulation with acidified serum, indicate that the free GPI accumulated in PIGTKO cells contribute to inflammasome activation together with complement activation. DisclosuresOsato:Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharma GK: Honoraria, Research Funding. Ueda:Alexion Pharma GK: Honoraria, Research Funding. Kinoshita:Alexion Pharmaceuticals: Honoraria. Kanakura:Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding.

A genome-wide CRISPR/Cas9 screen to identify phagocytosis modulators in monocytic THP-1 cells

Phagocytosis of microbial pathogens, dying or dead cells, and cell debris is essential to maintain tissue homeostasis. Impairment of these processes is associated with autoimmunity, developmental defects and toxic protein accumulation. However, the underlying molecular mechanisms of phagocytosis remain incompletely understood. Here, we performed a genome-wide CRISPR knockout screen to systematically identify regulators involved in phagocytosis of Staphylococcus (S.) aureus by human monocytic THP-1 cells. The screen identified 75 hits including known regulators of phagocytosis, e.g. members of the actin cytoskeleton regulation Arp2/3 and WAVE complexes, as well as genes previously not associated with phagocytosis. These novel genes are involved in translational control (EIF5A and DHPS) and the UDP glycosylation pathway (SLC35A2, SLC35A3, UGCG and UXS1) and were further validated by single gene knockout experiments. Whereas the knockout of EIF5A and DHPS impaired phagocytosis, knocking out SLC35A2, SLC35A3, UGCG and UXS1 resulted in increased phagocytosis. In addition to S. aureus phagocytosis, the above described genes also modulate phagocytosis of Escherichia coli and yeast-derived zymosan A. In summary, we identified both known and unknown genetic regulators of phagocytosis, the latter providing a valuable resource for future studies dissecting the underlying molecular and cellular mechanisms and their role in human disease.