Differentiated THP-1 Cells Research Articles

Surface chemistry mediated albumin adsorption, conformational changes and influence on innate immune responses

The surface chemistry of biomaterials plays a pivotal role in regulating the type, amount, and conformational changes of adsorbed proteins, which then modulates the subsequent innate immune responses. Albumin is the most abundant protein in serum and a major constituent of the protein corona that forms on biomaterial surfaces. Therefore, it is important to understand the role of surface chemistry in albumin adsorption, surface induced conformational changes, and how this affects the subsequent immune responses. To interrogate these events, we generated model substrata with four tailored surface chemistries rich in amines, oxazolines, carboxylic acid groups and pure hydrocarbons via plasma polymerization. The positively charged amine and oxazoline rich surface chemistries caused greatest albumin adsorption and protein conformational changes. Our data demonstrated that macrophages (differentiated THP-1 cells) were able to interact with unfolded albumin via their scavenger receptors. The innate inflammatory responses were studied by measuring the expression of pro- and anti- inflammatory markers. As an overall trend, the pre-adsorption of albumin resulted in a reduction in the level of expression of pro-inflammatory cytokines while the secretion of anti-inflammatory markers was stimulated. This study provides valuable information, which could aid in the design of future biomaterials that elicit predictable immune response.

FC020: The Uraemic Toxin Indoxyl Sulfate is an Activator Of Caspase-4 but not of Caspase-1 in Monocyte-Like THP-1 Cells

Abstract BACKGROUND AND AIMS Among the plethora of uraemic toxins the protein-bound indoxyl sulphate (IS) plays a prominent role in cardiovascular and renal disease progression. IS also appears to be involved in programmed cell death pathways. Regarding the high inflammatory pyroptosis rates in blood cells of haemodialysis patients (HD), there is the question of whether IS can induce caspase-1 and/or caspases-4/-5, thus triggering pyroptosis-related pathways and the release of proinflammatory interleukin IL-1ß in monocytes. METHOD IS-related effects were investigated in the monocytic cell line THP-1. Differentiated THP-1 (100 nM PMA) cells were used to study IS effects in macrophages. IS concentrations in the range of 1 to 4 mM were applied to reveal putative mechanistic effects mediated by IS. Caspase-1, -4 and -5 specific substrates were used to analyse the different caspase activities by flow-cytometry and luminometric assays. IL-1ß activity of THP-1 supernatants was measured in the HEK-Blue™ IL-1ß cell line, which produces secreted embryonic alkaline phosphatase (SEAP) upon induction with active IL-ß. RESULTS Flow cytometric analysis demonstrates that pyroptosis (AAD+/caspase-1+) is already increased by the stimulation of THP-1 cells at concentrations of 1 mM IS, a concentration which can be found in HD patients with high IS load. These data were confirmed by a second luminometric assay, which is based on a different caspase-1 specific substrate compared with the flow-cytometric assay (Ac-WEHD-aminoluciferin versus FAM-YVAD-FMK). Further, the caspase-1 activity could be enhanced by supplementing IS-stimulated (2 mM) samples with the caspase-1-dependent NLRP3 inflammasome activator nigericin (21 µM). Taking in mind that especially FAM-YVAD-FMK substrate can be cleaved by caspase-1 and caspase-4/-5, we examined the specificity of the tested substrate using inhibitor probes. Supplementing cells with the caspase-1 specific inhibitor Ac-YVAD-CHO (2 µM), we failed to inhibit IS-mediated effects, whereas the nigericin-driven effect could be blocked. However, the addition of the inhibitory substrate Ac-LEVD-CHO (2µM), which specifically blocks caspase-4 and -5 activities totally decreased IS-derived caspase activity (Control: 100%, IS: 128 ± 12%, IS + Ac-LEVD: 81 ± 22%, IS + AC-YVAD: 107 ± 15%; IS versus IS + Ac-LEVD, P < .001). It is known that IS has a stimulating impact on mRNA expression of pro-IL-1ß in macrophage-like THP-1 cells. When differentiated THP-1 cells were stimulated with 4 mM IS for 24-h we found a two-fold increase in IL-1ß mRNA expression. Further, we find a significant increase in caspase-1 and caspase-4 in these cells. However, IL-1ß protein levels seem to be only marginally elevated in IS stimulated samples. Analysing the biological activity of the THP-1 derived supernatants in the HEK-Blue™ IL-1ß cell line, we did not find a SEAP increase in samples stimulated with the IS-derived THP-1 supernatants. CONCLUSION Analysing the effects of indoxyl sulphate in the THP-1 model systems we provide evidence for the first time that the inflammatory caspase-4 and/or caspase-5 is activated in monocyte-like cells without increasing IL-1ß levels. Therefore, one can conclude that IS is not an archetypical NLRP3 inflammasome activator but may contribute to cell death via caspase-4 activation.

Macrophage Clearance of Neutrophil Extracellular Traps (NETs)

Abstract Neutrophil Extracellular Traps (NETs) are formed from dying neutrophils expelling their contents to capture pathogens and limit the spread of infection. While they are beneficial during infection, overproduction of NETs or the inability to degrade them can be pathogenic. Different inflammatory disorders and autoimmune diseases have been shown to be associated with an increase in NET production. Therefore, it is important to understand the method in which NETs are removed or broken down. Cayman Chemical has developed an imaging-based assay to sensitively assay NET uptake by phagocytic cells. Imaging was found to be an ideal method to monitor NET clearance in vitro, in part due to the ability to monitor the uptake of NETs kinetically. Both primary monocyte-derived macrophages and differentiated THP-1 cells, which are frequently used as phagocytic cell models, were shown to take up NETs. Putative modulators of NET uptake were tested in the assay. We aimed to develop a method in which the process of NET uptake can be monitored and quantified. Modulators of NET uptake can be tested in this assay, allowing for further studies toward treating autoimmune disorders that are driven by aberrant NETs.

Differential susceptibility to lipopolysaccharide affects the activation of toll-like-receptor 4 signaling in THP-1 cells and PMA-differentiated THP-1 cells.

Monocytes and macrophages that originate from common myeloid progenitors perform various crucial roles in the innate immune system. Stimulation with LPS combined with TLR4 drives the production of pro-inflammatory cytokines through MAPKs and NF-κB pathway in different cells. However, the difference in LPS susceptibility between monocytes and macrophages is poorly understood. In this study, we found that pro-inflammatory cytokines—IL-1β, IL-6 and TNFα showed greater induction in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells than in THP-1 cells. To determine the difference in cytokine expression, the surface proteins such as TLR4-related proteins and intracellular adaptor proteins were more preserved in PMA-differentiated THP-1 cells than in THP-1 cells. MyD88 is a key molecule responsible for the difference in LPS susceptibility. Moreover, MAPKs and NF-κB pathway-related molecules showed higher levels of phosphorylation in PMA-differentiated THP-1 cells than in THP-1 cells. Upon MyD88 depletion, there was no difference in the phosphorylation of MAPK pathway-related molecules. Therefore, these results demonstrate that the difference in LPS susceptibility between THP-1 cells and PMA-differentiated THP-1 cells occur as a result of gap between the activated MAPKs and NF-κB pathways via changes in the expression of LPS-related receptors and MyD88.

Enzyme-digested Colla Corii Asini (E'jiao) suppresses lipopolysaccharide-induced inflammatory changes in THP-1 macrophages and OP9 adipocytes.

Gut microbial lipopolysaccharides (LPS)-induced inflammatory responses in adipose tissue are associated with the dysfunction of adipocytes, insulin resistance and the development of metabolic syndrome. The aim of this study is to investigate (1) the effects of LPS on the differentiation and inflammatory responses of THP-1 monocytes and OP9 preadipocytes under serum free conditions and (2) the repressive effects of enzyme-digested Colla Corii Asini (CCAD) and fish gelatin (FGD) on LPS-induced inflammatory responses in THP-1 macrophages and OP9 adipocytes. Immunofluorescence and oil red O staining showed that a serum free medium supplied with phorbol 12-myristate 13-acetate (PMA) could induce differentiation and lipid accumulation in THP-1 cells as well as OP9 cells. ELISA showed that LPS significantly increased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) secretions in PMA-differentiated THP-1 macrophages in a dose-dependent manner. LPS significantly suppressed lipid accumulation and adiponectin secretions, and enhanced IL-6 secretions in OP9 adipocytes. Both CCAD and FGD significantly reduced the levels of both macrophages- and adipocytes-derived inflammatory cytokines and increased the level of OP9-secreted adiponectin. In conclusion, LPS could induce inflammatory responses in both THP-1 and OP9 cells and cause dysfunction of OP9 adipocytes under the serum free conditions. CCAD and FGD can repress LPS-induced inflammatory responses in both THP-1 macrophages and OP9 adipocytes, and increase the secretion of adiponectin in OP9 adipocytes. They could be used as health care supplements for improving metabolic syndrome.

Peptides Regulating Proliferative Activity and Inflammatory Pathways in the Monocyte/Macrophage THP-1 Cell Line.

This study evaluates the effects of five different peptides, the Epitalon® tetrapeptide, the Vilon® dipeptide, the Thymogen® dipeptide, the Thymalin® peptide complex, and the Chonluten® tripeptide, as regulators of inflammatory and proliferative processes in the human monocytic THP-1, which is a human leukemia monocytic cell line capable of differentiating into macrophages by PMA in vitro. These peptides (Khavinson Peptides®), characterized by Prof. Khavinson from 1973 onwards, were initially isolated from animal tissues and found to be organ specific. We tested the capacity of the five peptides to influence cell cultures in vitro by incubating THP-1 cells with peptides at certain concentrations known for being effective on recipient cells in culture. We found that all five peptides can modulate key proliferative patterns, increasing tyrosine phosphorylation of mitogen-activated cytoplasmic kinases. In addition, the Chonluten tripeptide, derived from bronchial epithelial cells, inhibited in vitro tumor necrosis factor (TNF) production of monocytes exposed to pro-inflammatory bacterial lipopolysaccharide (LPS). The low TNF release by monocytes is linked to a documented mechanism of TNF tolerance, promoting attenuation of inflammatory action. Therefore, all peptides inhibited the expression of TNF and pro-inflammatory IL-6 cytokine stimulated by LPS on terminally differentiated THP-1 cells. Lastly, by incubating the THP1 cells, treated with the peptides, on a layer of activated endothelial cells (HUVECs activated by LPS), we observed a reduction in cell adhesion, a typical pro-inflammatory mechanism. Overall, the results suggest that the Khavinson Peptides® cooperate as natural inducers of TNF tolerance in monocyte, and act on macrophages as anti-inflammatory molecules during inflammatory and microbial-mediated activity.

Eicosanoid production by macrophages during inflammation depends on the M1/M2 phenotype

Macrophages are important in inflammation, and are involved in many physiological and pathological processes. Additionally, macrophages are important producers of eicosanoids, lipids that influence the inflammatory response. Our study aimed to explore the role of eicosanoids in the inflammatory response by studying the production of eicosanoids by macrophages on different stages of inflammation. Murine peritoneal macrophages (MPMs) were obtained at different stages of inflammation, which were then cultured in vitro with polyunsaturated fatty acids. Eicosanoids in MPMs were then detected by liquid chromatography–mass spectrometry. The metabolites derived from the cyclooxygenase (COX) pathway were increased, whereas those from the lipoxygenase (LOX) pathway were reduced. Additionally, the ratio of arachidonic acid (AA)-derived and eicosapentaenoic acid (EPA)-derived eicosanoids was dependent on the stage of inflammation. Moreover, the composition of macrophages with different phenotypes changed. To clarify the relationship between the phenotypes of macrophages and eicosanoids metabolism, we detected the eicosanoids in M1 and M2 differentiated THP-1 cells. Overall, M1 preferred AA, whereas M2 preferred EPA as substrate, which was related to the expression of COX and LOX. In conclusion, this study demonstrates that the difference in macrophage eicosanoids metabolism during the inflammatory response is related to the macrophage polarisation.

Picroside II alleviates DSS-induced ulcerative colitis by suppressing the production of NLRP3 inflammasomes through NF-κB signaling pathway

Context Ulcerative colitis (UC) is a common acute or chronic intestinal disease with an imbalance of inflammation. Picroside II (P-II) exerts a protective role in various inflammation-related diseases. However, the effect of P-II on UC is still unclear. Objective To explore the effect of P-II on UC and its potential mechanism. Materials and methods Human monocytic leukemia cell line THP-1 was treated with phorbol ester (PMA) to differentiate into a macrophage. The differentiated THP-1 cells were hatched with LPS combined with ATP or Nigericin to activate the NLRP3 inflammasome in vitro. The UC model was constructed by injection of DSS into mice. Results The maximum nontoxic concentration of P-II on THP-1 cells was 60 μM. LPS combined with ATP or Nigericin stimulated the production of IL-1β, which was antagonized by P-II treatment. Meanwhile, P-II administration interfered with the aggregation of ASC and the assembly of NLRP3 inflammasomes. Also, P-II treatment reduced the LPS and ATP-induced elevation of the relative protein expression of NLRP3, pro-caspase-1, IL-1β and p-p65/p65, and the concentration of TNF-α and IL-6. Besides, the NF-κB specific inhibitor BAY-117082 notably repressed the LPS together with ATP-enhanced the relative protein expression of NLRP3, caspase-1 and IL-1β. Moreover, in vivo results showed that P-II relieved the DDS-induced UC, as evidenced by the improvement of mice weight, DAI and pathological scores. In addition, P-II treatment notably decreased DDS-promoted expression of NLRP3 inflammasomes and inflammatory factors in vivo. Conclusion P-II alleviated DSS-induced UC by repressing the production of NLRP3 inflammasomes via the NF-κB signaling pathway.

Regorafenib and reactive metabolite of sunitinib activate inflammasomes: Implications for multi tyrosine kinase inhibitor-Iiduced immune related adverse events.

Multi-targeted tyrosine kinase inhibitors have been developed for the treatment of various cancers, but they are associated with a significant incidence of idiosyncratic drug reactions (IDRs). There is compelling evidence that most IDRs are immune mediated. Activation of inflammasomes is often one of the early steps in the initiation of an immune response. This activation could involve the pharmacological effect of the drug, or it could involve the release of damage associated molecular patters (DAMPs) caused by a reactive metabolite. We tested whether sunitinib, regorafenib, lenvatinib and cabozantinib can directly activate inflammasomes in differentiated THP-1 cells. We found that regorafenib activated the inflammasome of differentiated THP-1 cells directly. We also found that the supernatant from the incubation of sunitinib with FLC-4 cells, which have a high capacity to metabolize drugs, led to activate the inflammasome of differentiated THP-1 cells. In the supernatant of FLC-4 cells with sunitinib, the heat shock protein (HSP) 90 was significantly increased. Sunitinib is known to be oxidized to generate a reactive, potentially toxic quinone imine. These results support the hypothesis that the reactive metabolite of sunitinib can cause the release of DAMPs from hepatocytes, which leads to activation of inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by regorafenib and sunitinib, which in some patients, can cause IDRs.

Elimination of Aicardi–Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi–Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR–Cas9 gene KO or lentiviral viral protein X–mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2–infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.

The Promoter of the Immune-Modulating Gene TIR-Containing Protein C of the Uropathogenic Escherichia coli Strain CFT073 Reacts to the Pathogen's Environment.

The TIR-containing protein C (TcpC) of the uropathogenic Escherichia coli strain CFT073 modulates innate immunity by interfering with the Toll-like receptor and NALP3 inflammasome signaling cascade. During a urinary tract infection the pathogen encounters epithelial and innate immune cells and replicates by several orders of magnitude. We therefore analyzed whether these cell types and also the density of the pathogen would induce the recently defined promoter of the CFT073 tcpC gene to, in time, dampen innate immune responses. Using reporter constructs we found that the uroepithelial cell line T24/83 and the monocytic cell line THP-1 induced the tcpC promoter. Differentiation of monocytic THP-1 cells to macrophages increased their potential to switch on the promoter. Cell-associated CFT073 displayed the highest promoter activity. Since potassium represents the most abundant intracellular ion and is secreted to induce the NLRP3 inflammasome, we tested its ability to activate the tcpC promoter. Potassium induced the promoter with high efficiency. Sodium, which is enriched in the renal cortex generating an antibacterial hypersalinity, also induced the tcpC promoter. Finally, the bacterial density modulated the tcpC promoter activity. In the search for promoter-regulating proteins, we found that the DNA-binding protein H-NS dampens the promoter activity. Taken together, different cell types and salts, present in the kidney, are able to induce the tcpC promoter and might explain the mechanism of TcpC induction during a kidney infection with uropathogenic E. coli strains.

SUMOylation regulates the number and size of promyelocytic leukemia-nuclear bodies (PML-NBs) and arsenic perturbs SUMO dynamics on PML by insolubilizing PML in THP-1 cells.

The functional roles of protein modification by small ubiquitin-like modifier (SUMO) proteins are not well understood compared to ubiquitination. Promyelocytic leukemia (PML) proteins are good substrates for SUMOylation, and PML-nuclear bodies (PML-NBs) may function as a platform for the PML SUMOylation. PML proteins are rapidly modified both with SUMO2/3 and SUMO1 after exposure to arsenite (As3+) and SUMOylated PML are further ubiquitinated and degraded by proteasomes. However, effects of As3+ on SUMO dynamics on PML-NBs are not well investigated. In the present study, we report that (1) the number and size of PML-NBs were regulated by SUMO E1-activating enzyme, (2) SUMO2/3 co-localized with PML irrespective of As3+ exposure and was restricted to PML-nuclear bodies (PML-NBs) via covalent binding in response to As3+, and (3) As3+-induced biochemical changes in PML were not modulated by ubiquitin-proteasome system (UPS) in THP-1 cells. Undifferentiated and differentiated THP-1 cells responded to As3+ similarly and PML proteins were changed from the detergent soluble to the insoluble form and further SUMOylated with SUMO2/3 and SUMO1. ML792, a SUMO E1 inhibitor, decreased the number of PML-NBs and reciprocally increased the size irrespective of exposure to As3+, which itself slightly decrease both the number and size of PML-NBs. TAK243, a ubiquitin E1 inhibitor, did not change the PML-NBs, while SUMOylated proteins accumulated in the TAK243-exposed cells. Proteasome inhibitors did not change the As3+-induced SUMOylation levels of PML. Co-localization and further restriction of SUMO2/3 to PML-NBs were confirmed by PML-transfected CHO-K1 cells. Collectively, SUMOylation regulates PML-NBs and As3+ restricts SUMO dynamics on PML by changing its solubility.

The influence of exposure approaches to in vitro lung epithelial barrier models to assess engineered nanomaterial hazard

Exposure to engineered nanomaterials (ENM) poses a potential health risk to humans through long-term, repetitive low-dose exposures. Currently, this is not commonplace within in vitro lung cell cultures. Therefore, the purpose of this study was to consider the optimal exposure approach toward determining the stability, sensitivity and validity of using in vitro lung cell mono- and co-cultures to determine ENM hazard. A range of exposure scenarios were conducted with DQ12 (previously established as a positive particle control) (historic and re-activated), TiO2 (JRC NM–105) and BaSO4 (JRC NM–220) on both monocultures of A549 cells as well as co-cultures of A549 cells and differentiated THP-1 cells. Cell cultures were exposed to either a single, or a repeated exposure over 24, 48- or 72-hours at in vivo extrapolated concentrations of 0–5.2 µg/cm2, 0-6 µg/cm2 and 0-1µg/cm2. The focus of this study was the pro-inflammatory, cytotoxic and genotoxic response elicited by these ENMs. Exposure to DQ12 caused pro-inflammatory responses after 48 hours repeat exposures, as well as increases in micronucleus frequency. Neither TiO2 nor BaSO4 elicited a pro-inflammatory response at this time point. However, there was induction of IL-6 after 24 hours TiO2 exposure. In conclusion, it is important to consider the appropriateness of the positive control implemented, the cell culture model, the time of exposure as well as the type of exposure (bolus or fractionated) before establishing if an in vitro model is appropriate to determine the level of response to the specific ENM of interest.

Protective effects of pentoxifylline on T-cell viability under inflammatory conditions

Introduction: Pentoxifylline (PTX) reduces the levels of pro-inflammatory cytokines; however, its effects on immune system is not well understood. The aim of this study was to investigate the effect of PTX on T cells under inflammatory conditions in co-culture with THP-1-derived macrophages.Methods: Toll-like receptor 4 (TLR4) and macrophage migration inhibitory factor (MIF) levels were measured after addition of PTX to lipopolysaccharide (LPS)-stimulated differentiated THP-1 cells. T cell viability and MIF levels were measured after PTX was added to prostaglandin E2(PGE2)-stimulated Jurkat T-cell leukemia line. Co-culture was conducted to determine the effect of LPS-stimulated differentiated THP-1 cells that are affected by PTX on Jurkat cells. To prevent the direct effects of LPS and PTX on Jurkat cells, LPS and PTX were washed from THP-1 cells before co-culture. T cell viability and interleukin-2 (IL-2) levels were determined in Jurkat cells. Results: Increase in the MIF concentration and TLR4expression level in differentiated THP-1 cells stimulated with LPS were reversed after PTX addition. However, PTX did not improve T cell viability in PGE2–stimulated Jurkat cells. Co-culturing Jurkat cell and LPS-stimulated differentiated THP-1 cells resulted in a decreased viability of T cells. The addition of PTX restored T cell viability to normal control levels and IL-2 expression level in Jurkat cells. Conclusion: LPS-stimulated THP-1-derived macrophages reduced the T cell viability under inflammation. However, PTX restored T cells viability and IL-2 back to normal levels. Therefore, the immunomodulatory action of PTX may be mediated by macrophage-T cell interactions.

CXCR4 Knockdown Via CRISPR/CAS9 in a Tumor-Associated Macrophage Model Decreases Human Breast Cancer Cell Migration.

IntroductionBreast cancer is the leading cause of cancer-related deaths in women worldwide with the majority of deaths due to metastasis. The development of metastasis is closely related to the tumor microenvironment where tumor-associated macrophages (TAMs) are the main immune cell component playing a crucial role in tumor migration. Key players in tumor progression, metastasis and survival are the receptor CXCR4 and its ligand CXCL12. CXCR4 is expressed in multiple cell types including macrophages and breast cancer cells. Many studies have focus on the role of CXCR4 expressed in breast cancer cells.MethodsIn this study, we investigated the role of CXCR4 expressed in TAMs on breast cancer cell migration by reducing CXCR4 expression via CRISPR-CAS9 system in differentiated THP-1 cells (a TAMs model).ResultsAccording to wound healing migration assay, MCF7 cancer cells co-cultured with genetically edited dTHP-1 cells have a lower migration rate as compared to MCF7 cancer cells co-cultured with unedited and dTHP-1 cells.ConclusionThe study demonstrates the role of CXCR4 on breast cancer cell migration through TAM-cancer cell crosstalk.

A DNA Topoisomerase II Inhibitor Results in Ex Vivo Differentiation of THP-1 Cells and Activation of Dendritic Cells.

Effective ex vivo maturation of dendritic cells (DCs) can increase the efficiency of cancer immunotherapy. We aimed to identify novel chemicals with the potential to differentiate and activate immature DCs (iDCs) to mature DCs (mDCs). The expression of surface markers on THP-1 monocytes treated with the screened compounds was analyzed using FACS. Subsequent DC subset analysis and secreted cytokine profiling were also performed. FACS analysis showed that THP-1 cells treated with amsacrine hydrochloride, a DNA topoisomerase II inhibitor, exhibited the typical phenotype of conventional DCs (cDCs). The expression of DC activation markers was also increased after amsacrine treatment. The profile of cytokines produced by THP-1 cells treated with amsacrine was similar to that of mDCs. Amsacrine has an ex vivo capability of differentiating THP-1 monocytes into cDCs. As amsacrine has been used as a stable chemotherapeutic agent in humans, it can be useful for producing mDCs for cancer immunotherapy.

Surface nanotopography mediated albumin adsorption, unfolding and modulation of early innate immune responses

Surface roughness plays an important role in regulating protein adsorption to biomaterial surfaces and modulating the subsequent inflammatory response. In this study, we examined the role of surface nanotopography on albumin adsorption, unfolding and subsequent immune responses. To achieve the objectives of the study, we create model surfaces of hill-like nanoprotrusions by covalently immobilizing gold nanoparticles (AuNPs) of predetermined sizes (16, 38, and 68 nm) on a functional plasma polymer layer. The amount of adsorbed albumin increased with the increase in surface area caused by greater surface nanotopography scales. Circular dichroism spectroscopy was used to evaluate albumin conformational changes and pointed to loss of α-helical structure on all model surfaces with the greatest conformational changes found on the smooth surface and the surface with largest nanotopography features. Studies with differentiated THP-1 cells (dTHP-1) demonstrated that immune cells interacted with surface adsorbed albumin via their scavenger receptors, which could bind to exposed peptide sequences caused by surface induced unfolding of the albumin. Pre-adsorption of albumin resulted in an overall decrease in the level of expression of pro-inflammatory cytokines from dTHP-1 cells. On the other hand, pre-adsorption of albumin led in an increase in the production of anti-inflammatory markers, which suggests a switch to the M2 pro-healing phenotype. The knowledge obtained from this study could instruct the design of healthcare materials where the generation of targeted surface nanotopography and pre-adsorption of albumin may enhance the biomaterial biocompatibility and lead to faster wound healing.

Curcumin Improves Epithelial Barrier Integrity of Caco-2 Monolayers by Inhibiting Endoplasmic Reticulum Stress and Subsequent Apoptosis.

Curcumin is a natural polyphenol and is supposed to possess antioxidant, anti-inflammatory, anticancer, and antiapoptotic properties. Although some studies have reported the therapeutic effects of curcumin on ulcerative colitis (UC), the specific mechanism remains unclear. An in vitro coculture model of Caco-2 and differentiated THP-1 cells was established. After administration of curcumin (10 μM), Western blot analysis was performed to evaluate the protein levels of tight junction (TJ) proteins zonula occludens- (ZO-) 1 and claudin-1. Annexin V-APC/7-AAD assays and flow cytometry were conducted to assess Caco-2 cell apoptosis. The expression levels of oxidative stress and endoplasmic reticulum stress- (ERS-) related molecules were determined by Western blot analysis. Curcumin administration significantly upregulated ZO-1 and claudin-1 protein levels and reduced Caco-2 cell apoptosis. The protein levels of oxidative stress markers inducible nitric oxide synthase (iNOS) and γH2AX and ERS-induced apoptosis-related molecules C/EBP homologous protein (CHOP) and cleaved caspase-12 were significantly downregulated upon curcumin treatment. Furthermore, curcumin administration greatly blocked the protein kinase-like endoplasmic reticulum kinase- (PERK-) eukaryotic translation initiation factor 2α- (eIF2α-) activating transcription factor 4- (ATF4-) CHOP signaling pathway. Curcumin enhanced intestinal epithelial barrier integrity in the in vitro coculture model by upregulating TJ protein expressions and reducing intestinal epithelial cell apoptosis. The potential mechanisms may be suppression of ERS and subsequent apoptosis.

Psoriasiform Inflammation Is Associated with Mitochondrial Fission/GDAP1L1 Signaling in Macrophages.

While psoriasis is known as a T cell- and dendritic cell-driven skin inflammation disease, macrophages are also reported to play some roles in its development. However, the signaling pathway of activated macrophages contributing to psoriasis is not entirely understood. Thus, we aimed to explore the possible mechanisms of how macrophages initiate and sustain psoriasis. The differentiated THP1 cells, stimulated by imiquimod (IMQ), were utilized as the activated macrophage model. IMQ was also employed to produce psoriasis-like lesions in mice. A transcriptomic assay of macrophages revealed that the expressions of pro-inflammatory mediators and GDAP1L1 were largely increased after an IMQ intervention. The depletion of GDAP1L1 by short hairpin (sh)RNA could inhibit cytokine release by macrophages. GDAP1L1 modulated cytokine production by activating the phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways. Besides GDAP1L1, another mitochondrial fission factor, Drp1, translocated from the cytosol to mitochondria after IMQ stimulation, followed by the mitochondrial fragmentation according to the immunofluorescence imaging. Clodronate liposomes were injected into the mice to deplete native macrophages for examining the latter’s capacity on IMQ-induced inflammation. The THP1 cells, with or without GDAP1L1 silencing, were then transplanted into the mice to monitor the deposition of macrophages. We found a significant THP1 accumulation in the skin and lymph nodes. The silencing of GDAP1L1 in IMQ-treated animals reduced the psoriasiform severity score from 8 to 2. After depleting GDAP1L1, the THP1 recruitment in the lymph nodes was decreased by 3-fold. The skin histology showed that the GDAP1L1-mediated macrophage activation induced neutrophil chemotaxis and keratinocyte hyperproliferation. Thus, mitochondrial fission can be a target for fighting against psoriatic inflammation.

Electrical Detection of Innate Immune Cells.

Accurately classifying the innate immune players is essential to comprehensively and quantitatively evaluate the interactions between the innate and the adaptive immune systems. In addition, accurate classification enables the development of models to predict behavior and to improve prospects for therapeutic manipulation of inflammatory diseases and cancer. Rapid development in technologies that provide an accurate definition of the type of cell in action, allows the field of innate immunity to the lead in therapy developments. This article presents a novel immunophenotyping technique using electrical characterization to differentiate between the two most important cell types of the innate immune system: dendritic cells (DCs) and macrophages (MACs). The electrical characterization is based on capacitance measurements, which is a reliable marker for cell surface area and hence cell size. We differentiated THP-1 cells into DCs and MACs in vitro and conducted electrical measurements on the three cell types. The results showed average capacitance readings of 0.83 µF, 0.93 µF, and 1.01 µF for THP-1, DCs, and MACs, respectively. This corresponds to increasing cell size since capacitance is directly proportional to area. The results were verified with image processing. Image processing was used for verification because unlike conventional techniques, especially flow cytometry, it avoids cross referencing and by-passes the limitation of a lack of specificity of markers used to detect the different cell types.