Monocytic Cell Line THP-1 Research Articles

Immunopathological Inflammation in the Evolution of Mucositis and Peri-Implantitis

The purpose of this study was to provide an immuno-mediated substantiation of the etiopathogenesis of mucositis and peri-implantitis based on the results of experimental, laboratory and clinical studies. The biopsy material was studied to identify impregnated nanoscale and microscale particles in the structure of pathological tissues by using X-ray microtomography and X-ray fluorescence analyses. Electron microscopy with energy-dispersive analysis identified the composition of supernatants containing nanoscale metal particles obtained from the surfaces of dental implants. The parameters of the nanoscale particles were determined by dynamic light scattering. Flow cytometry was used to study the effect of nanoscale particles on the ability to induce the activation and apoptosis of immunocompetent cells depending on the particles' concentrations during cultivation with the monocytic cell line THP-1 with the addition of inductors. An analysis of the laboratory results suggested the presence of dose-dependent activation, as well as early and late apoptosis of the immunocompetent cells. Activation and early and late apoptosis of a monocytic cell line when THP-1 was co-cultured with nanoscale metal particles in supernatants were shown for the first time. When human venous blood plasma was added, both activation and early and late apoptosis had a dose-dependent effect and differed from those of the control groups.

Trifluoromethylcinnamanilide Michael Acceptors for Treatment of Resistant Bacterial Infections

A series of thirty-two anilides of 3-(trifluoromethyl)cinnamic acid (series 1) and 4-(trifluoromethyl)cinnamic acid (series 2) was prepared by microwave-assisted synthesis. All the compounds were tested against reference strains Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and resistant clinical isolates of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecalis (VRE). All the compounds were evaluated in vitro against Mycobacterium smegmatis ATCC 700084 and M. marinum CAMP 5644. (2E)-3-[3-(Trifluoromethyl)phenyl]-N-[4-(trifluoromethyl)phenyl]prop-2-enamide (1j), (2E)-N-(3,5-dichlorophenyl)-3-[3-(trifluoromethyl)phenyl]prop-2-enamide (1o) and (2E)-N-[3-(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)-phenyl]prop-2-enamide (2i), (2E)-N-[3,5-bis(trifluoromethyl)phenyl]-3-[4-(trifluoromethyl)phenyl]-prop-2-enamide (2p) showed antistaphylococcal (MICs/MBCs 0.15-5.57 µM) as well as anti-enterococcal (MICs/MBCs 2.34-44.5 µM) activity. The growth of M. marinum was strongly inhibited by compounds 1j and 2p in a MIC range from 0.29 to 2.34 µM, while all the agents of series 1 showed activity against M. smegnatis (MICs ranged from 9.36 to 51.7 µM). The performed docking study demonstrated the ability of the compounds to bind to the active site of the mycobacterial enzyme InhA. The compounds had a significant effect on the inhibition of bacterial respiration, as demonstrated by the MTT assay. The compounds showed not only bacteriostatic activity but also bactericidal activity. Preliminary in vitro cytotoxicity screening was assessed using the human monocytic leukemia cell line THP-1 and, except for compound 2p, all effective agents did show insignificant cytotoxic effect. Compound 2p is an interesting anti-invasive agent with dual (cytotoxic and antibacterial) activity, while compounds 1j and 1o are the most interesting purely antibacterial compounds within the prepared molecules.

Effects of Klebsiella pneumoniae Bacteriophages on IRAK3 Knockdown/Knockout THP-1 Monocyte Cell Lines.

Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression.

Comparison of monocytic cell lines U937 and THP-1 as macrophage models for in vitro studies

Understanding macrophage biology can improve comprehension of diverse biological processes and provide insights into novel therapeutic immunomodulatory strategies. Due to limited yield and technical difficulty in isolating primary macrophages, in vitro studies commonly use monocytes as precursor cells. Monocytic cell lines are a virtually unlimited source of macrophage precursors and two of the most frequently used cell lines are THP-1 and U937. Besides a great variability in macrophage differentiation protocols there is scarce information on possible differences in the biological responses of these cell lines. In this study, we used a standardized differentiation protocol using PMA and compared the response of macrophages derived from THP-1 and U937 cells to M1-and M2-polarizing conditions. THP-1-derived macrophages are more responsive to M1 stimuli and skewed towards M1 phenotype, whereas U937-derived macrophages were more responsive to M2 stimuli and skewed towards M2 phenotype. THP-1-derived macrophages also had greater production of ROS and phagocytic activity. Under M1-polarizing conditions, macrophages derived from both THP-1 and U937 reduced phagocytosis activity and the increased production of ROS. This information should be considered to make an informed choice on the cell line used as in vitro macrophage model, according to the experimental goals and biological context.

Efficient Knock-out of Serine Protease Cathepsin G Enhances Chemosensitivity to Cytarabine By Modulating the Cytarabine Metabolic Pathway Genes in Acute Myeloid Leukemia

Relapse and refractoriness to chemotherapy may exist prior to the exposure to chemotherapeutic agents or can develop and increase during the treatment leading to acquired resistance. Despite considerable progress in developing new therapeutic agents to be administered singly or in combination with the old regimen, drug resistance poses a serious concern preventing adequate response to Acute Myeloid Leukemia (AML) treatment with cytarabine (AraC) and daunorubicin (DNR). Differentially expression analysis of the RNA sequencing data between the naive and the AraC-resistant human monocytic MLL-AF9 cell line THP1 revealed cathepsin G (CTSG) as one of the top hits among the upregulated genes with Log2Fc 4.2. This was further validated in the AML cell line by q-RT-PCR with a fold change ~5 and immunoblot, confirming increased CTSG expression in THP1 Ara-C resistant cell line. Although the role of (CTSG) in host defence, granulopoiesis has been intensively investigated, its role in acquired cytarabine resistance has not been explored yet. To investigate the functional role of CTSG in the acquired resistant model, we performed CRISPR/Cas9-based editing of CTSG in THP1 Ara-C resistant cell line using dual gRNAs, which resulted in a robust deletion of the targeted region and complete loss of protein expression as measured by immunoblotting. CTSG KO cells showed a reduced cell proliferation rate with a significant reduction at 72hrs (compared to the scrambled gRNA (ScrG) transduced cells doubling time CTSG KO=30.3 vs ScrG=22.39, p= 0.0049). We also checked the sensitivity of the CTSG KO cells to AraC and other chemo drugs (DNR, ATO and Venetoclax). In vitro cytotoxicity assay (72hrs) for the CTSG KO cells treated with AraC showed sensitivity to Ara-C with a reduction in IC50 as low as the parental IC50 (IC50 ScrG: 1457 µM; IC50 CTSG KO: 38.13 µM; IC50 Parental: 56.6 µM). CTSG KO cells also sensitized the cells to DNR (IC50 ScrG: 288nM vs and CTSG KO: 58nM but not to ATO and venetoclax. Further, we measured the intracellular DNR by flow cytometry in the CTSG KO cells. There was a considerable increase in the intracellular DNR at the end of the treatment compared to the ScrG cells (~1.5-fold, p <0.0001). Our results show that CTSG KO re-sensitized the resistant cells to cytarabine and showed significant sensitivity to DNR. To further understand the molecular mechanism contributing to the increased sensitivity of the CTSG KO cells to AraC, we checked our basal RNA sequencing data for the expression of AraC metabolizing genes. The THP1 Ara-C R cell line showed decreased expression of DCK and increased expression of NT5C2 and CDA compared to the parental cell line. We checked the RNA expression of Ara-C metabolizing genes post CTSG KO by RT-q PCR. There was a 6-fold increase in DCK and a 5-fold decrease in NT5C2 with a further increase in SAMHD1 and CDA genes with no changes in the RRM1 and ENT1 genes, suggesting that CTSG KO increased the sensitivity to by rewiring the AraC metabolic pathway. Further studies are ongoing to assess the in-vivo efficacy of molecular and pharmacological inhibition of CTSG in reversing AraC resistance in the AML mouse model.

Chinese Herbal Medicine 'Diwu' Suppresses Myeloma Bone Disease Via ERK/MAPK Signaling Pathway By Targeting CCL3

Background： Multiple myeloma (MM) associated bone disease (MBD) is largely dependent on the bone marrow microenvironment (BME). The cross-talk between MM cells, bone marrow mesenchymal cells and myeloid progenitors affects the osteoclastogenesis. Traditional Chinese medicine 'Diwu' is effective for patients with arthritis and inflammation, also occasionally uses as one ingredient treating MM. But the effects of 'Diwu' in MBD are largely unknown. The aim of this study is to investigate the role and underlying mechanism of 'Diwu' in regulating BME and osteoclast differentiation. Methods： Myeloma cell lines RPMI-8226, NCI-H929, U266 and MM1 were treated with 'Diwu' extract. Killing effects was evaluated by the half inhibitory dose (IC50) value and proportion of apoptotic MM cells. RNA-seq was applied in 'Diwu' treated myeloma cells to identify MBD associated signaling pathways. RPMI-8226 was co-cultured with mesenchymal stromal cell (MSC) line HS5. Human monocyte cell line THP-1 was treated with CCL3 and M-CSF to form osteoclasts determined by tartrate-resistant acid phosphatase (TRACP) staining. Primary human MSCs were cultured into osteogenic or adipogenic differentiation, determined by alizarin red staining and oil red staining, respectively. RNAi technology was applied to knock down CCL3. The expression of osteoclast-specific genes TRACP and MAPK pathway-related genes was tested using quantitative real-time PCR. Phosphorylation and expression of MEK1, ERK, FOS were measured by Western Blot. The myeloma-bearing NOD/SCID mice were treated with intragastric 'Diwu'. Serum C-terminal telopeptide of type 1 collagen (CTXI) and propeptide of type I procollagen (PINP) was tested by ELISA. The tibia of mice was fixed and stained to analyze the differentiation and maturation of osteoblasts and osteoclasts. Micro-CT was used for bone destruction measurement. Results： 'Diwu' inhibited MM cells proliferation (IC50, 29.22-364.7μg/ml) in a dose-dependent manner and promoted apoptosis by upregulating protein expression of CASP3, BCL2. 'Diwu' decreased the secretion of chemokine (C-C motif) ligand 3 (CCL3) and receptor activator of nuclear factor-kappa B ligand (RANKL) in myeloma-MSC co-cultured system by diminishing the direct attachment and interaction of these two cells. The mRNA and protein levels of osteoclast-specific genes such as TRACP, and the phosphorylation and expression of MEK1, ERK, FOS in osteoclast were all reduced after 'Diwu' treatment. We observed improved osteogenic activation rather than lipid formation in hMSC differentiation. The level of TRACP isoform 5b (TRACP-5b) was significantly downregulated in mouse serum after 'Diwu' treatment for 14 days. Conclusions: Altogether, our results demonstrated that effective extract of Chinese herbal medicine 'Diwu' could simultaneously inhibit myeloma cells and suppress osteoclast formation, indicating an important role of 'Diwu' in regulating BM microenvironment. It is possible to translate 'Diwu' into clinical application as an oral agent for myeloma bone disease. Key words： 'Diwu'; Myeloma Bone Disease; Bone Marrow Microenvironment; CCL3; ERK/MAPK

Lipoxin A4, Maresin 1, and Resolvin D1 As Key Factors for the Actions of Plasminogen/Plasmin on Inflammation Resolution

The plasminogen/plasmin (Plg/Pla) system plays a fundamental role in modulating fibrinolysis and many other biological processes, including extracellular matrix degradation, cell migration, and tissue repair. Recent studies highlight the role of the Plg/Pla system in key steps of inflammation resolution, which is mediated, at least in part, by the glucocorticoid-induced protein annexin A1. However, it is unclear whether Plg/Pla might also modulate specialized proresolving mediators (SPMs) derived from omega-3 and omega-6 fatty acids, including lipoxin A4 (LXA4), maresin 1 (MaR1), and resolvin D1 (RvD1). Therefore, we investigated the ability of Plg/Pla to modulate the synthesis of LXA4, MaR1, and RvD1, as well as the involvement of these SPMs in the proresolving effects elicited by Plg or Pla using both in vitro and in vivo strategies. Treatment of bone marrow-derived macrophages (BMDMs) with Plg/Pla (4 μg/mL) induced the release of LXA4, MaR1, and RvD1 as well as increased mRNA levels of their lipoxygenase (LO) synthesizing enzymes 5-LO and 15-LO. Plg treatment also increased the levels of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokine TNF-α in BMDMs. Moreover, Plg decreased the levels of TNF-α and IL-1β induced by lipopolysaccharide (LPS) stimulus of the human monocytic cell line THP-1. Interestingly, LXA4 was able to induce Plg release in THP-1 cells. Notably, Pla (4 μg, i.p.) was able to increase the efferocytosis of apoptotic neutrophils in wild-type (SV129) mice, but not in 5-LO deficient mice (5-LO-/-). Taken together, these findings suggest that Plg and Pla increase the levels of SPMs and IL-10, and modulate the levels of LPS-elicited pro-inflammatory cytokines. Mechanistically, the action of Plg/Pla on efferocytosis was 5-LO dependent, suggesting that SPMs mediate this proresolving effect of Plg/Pla. Moreover, the reciprocal modulation between the Plg/Pla system and SPMs pathways elucidated should be explored in future studies on candidate targets for therapies for inflammatory diseases. Keywords: Inflammation resolution; plasminogen/plasmin system; pro-resolving lipid mediators. Financial support: FAPEMIG, CNPq, CAPES.

Extracellular vesicles produced by the human gut commensal bacterium Bacteroides thetaiotaomicron elicit anti-inflammatory responses from innate immune cells.

Bacterial extracellular vesicles (BEVs) produced by gut commensal bacteria have been proposed to play an important role in maintaining host homeostasis via interactions with the immune system. Details of the mediators and pathways of BEV-immune cell interactions are however incomplete. In this study, we provide evidence for the anti-inflammatory and immunomodulatory properties of extracellular vesicles produced by the prominent human gut commensal bacterium Bacteroides thetaiotaomicron (Bt BEVs) and identify the molecular mechanisms underlying their interaction with innate immune cells. Administration of Bt BEVs to mice treated with colitis-inducing dextran sodium sulfate (DSS) ameliorates the symptoms of intestinal inflammation, improving survival rate and reducing weight loss and disease activity index scores, in association with upregulation of IL-10 production in colonic tissue and in splenocytes. Pre-treatment (conditioning) of murine bone marrow derived monocytes (BMDM) with Bt BEVs resulted in higher ratio of IL-10/TNFα production after an LPS challenge when compared to LPS pre-conditioned or non-conditioned BMDM. Using the THP-1 monocytic cell line the interactions between Bt BEVs and monocytes/macrophages were shown to be mediated primarily by TLR2. Histone (H3K4me1) methylation analysis showed that Bt BEVs induced epigenetic reprogramming which persisted after infectious challenge, as revealed by increased levels of H3K4me1 in Bt BEV-conditioned LPS-challenged BMDM. Collectively, our findings highlight the important role of Bt BEVs in maintaining host immune homeostasis and raise the promising possibility of considering their use in immune therapies.

Small Extracellular Vesicles Loaded with Immunosuppressive miRNAs Leads to an Inhibition of Dendritic Cell Maturation

In particular conditions, inhibition of an immune response is required to prevent tissue damage. Among these conditions are diseases caused by an over-reactive immune response, such as autoimmune or allergic disorders, or imminent organ rejection after transplantation. To avoid tissue damage, drug-mediated systemic immune suppression is an option, but it comes with high costs in the form of susceptibility to viral and bacterial infections. Thus, the induction of antigen-specific tolerance is preferable. Extracellular vesicles (EVs) are capable of delivering antigen together with immunosuppressive signals and may be used to specifically induce antigen-specific tolerance. However, naturally occurring EVs are heterogeneous and not all of them show immunosuppressive character. In our trials to engineer cell culture derived EVs to increase their tolerogenic potential, we equipped them with immunosuppressive miRNA mimics. Small EVs (sEVs) were isolated and purified from the human monocytic THP-1 cell line or from healthy donor peripheral blood mononuclear cells, and electroporated with miR-494 and miR-146a mimics. The acquired immunosuppressive potential of the modified sEVs was demonstrated by their ability to alter the major histocompatibility complex molecules and co-stimulatory receptors present on dendritic cells (DCs). To avoid allogeneic responses, the same cells that produced the sEVs served also as recipient cells. In contrast to the treatment with unmodified sEVs, the tolerogenic sEVs impeded lipopolysaccharide-induced maturation and kept DCs in a more immature developmental stage. Our experiments show that simple manipulations of sEVs using immunosuppressive cargo can lead to the inhibition of DC maturation.

Selection and validation of reference genes for qPCR analysis of differentiation and maturation of THP-1 cells into M1 macrophage-like cells.

For cell-based assays studying monocytes and macrophages, the immortalized monocyte cell line THP-1 is widely used and stimulated with phorbol 12-myristate 13-acetate, lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ), after which it differentiates and polarizes into proinflammatory M1-like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription-quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP-1 cells stimulated with LPS and IFN-γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), UBC (ubiquitin C), B2M (0β2-microbulin), ACTB (β-actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP-1 monocyte model system.

Conjugation of Listeriolysin O to Anti-CD47 mAb: Harnessing Listeria Toxin to Weaponize Macrophages against Tumor Cells

<h3>Purpose/Objective(s)</h3> CD47 is expressed on various tumor types including breast, prostate, glioblastoma, non-Hodgkin lymphoma, and hepatocellular carcinoma. Interrupting the CD47-SIRP alpha axis by monoclonal antibodies fosters macrophage-mediated phagocytosis of solid and hematopoietic tumor cells. The pore-forming toxin listeriolysin O (LLO) is activated in the acidic vacuoles of bone marrow-derived macrophages. We hypothesize that LLO conjugation to the anti-CD47 mAb enhances the release of autophagic cargo within macrophages thereby augmenting cGAS-STING activation, antigen presentation, and tumor phagocytosis. <h3>Materials/Methods</h3> The anti-CD47 mAb was conjugated to the LLO toxin using a water-soluble SPDP crosslinker and purified by affinity chromatography. The cytotoxicity of anti-CD47-LLO mAb relative to anti-CD47 mAb was tested in the mouse breast cancer cell line E0771, the human breast cancer cell line MDA-MB-468, the mouse lung cancer cell line LLC, and the human leukemia monocytic cell line THP-1. C57B6 mouse bone marrow-derived macrophages (BMDM) co-cultured with tumor cell lines were analyzed in the presence of anti-CD47-LLO mAb and anti-CD47 mAb to determine the efficiency of BMDM tumor cell phagocytosis, STING activation, and antigen presentation. Tumor growth of E0771-bearing wildtype mice was measured following intratumoral injection of anti-CD47 mAb and anti-CD47-LLO mAB. <h3>Results</h3> The anti-CD47-LLO mAb conjugate enhanced BMDM phagocytosis of LLC and E0771 tumor cells in a dose dependent manner that was significantly higher than cells treated with the anti-CD47 mAb alone. The anti-CD47-LLO mAb significantly increased BMDM levels of phosphorylated STING, interferon-stimulated genes, and antigen presentation relative to anti-CD47 mAb treatment. At doses compatible with preserved animal total body weight, red blood cell counts, and lymphocyte counts, intratumoral injection of anti-CD47-LLO mAb significantly suppressed tumor growth of E0771-bearing wildtype mice relative to the anti-CD47 mAb. <h3>Conclusion</h3> Conjugation of the LLO toxin to anti-CD47 mAb enhances macrophage cGAS-STING activation, antigen presentation, and tumor cell phagocytosis. This novel CD47 antibody builds on clinical interest in targeting macrophages for the treatment of malignancy and may be studied as a supplemental therapy for patients with tumors resistant or refractory to checkpoint therapy.

17β-neriifolin from unripe fruits of Cerbera manghas suppressed cell proliferation via the inhibition of HOXA9-dependent transcription and the induction of apoptosis in the human AML cell line THP-1.

Homeobox A9 (HOXA9) is a transcription factor that is overexpressed in acute myeloid leukemia (AML). It is associated with the pathogenesis and progression of AML, and is a factor responsible for a poor prognosis. Therefore, the development of HOXA9-targeting molecules may contribute to not only better understanding of the mechanism of HOXA9 regulation, but also the development of therapeutic applications. We constructed a reporter assay system using the promoter region of the KBTBD10 gene, to which HOXA9 directly binds and regulates transcription, in the human acute monocytic leukemia cell line THP-1. Using this luciferase gene assay, we screened 1120 plant extracts and a methanol extract of the unripe fruits of Cerbera manghas was found to suppress the reporter gene expression mediated by the KBTBD10 promoter. From the extract, five steroid-type compounds were identified as the active constituents: 7α-neriifolin (1), 17β-neriifolin (2), 17α-digitoxigenin β-D-glucosyl-(1 → 4)-α-L-thevetoside (3), 17β-digitoxigenin β-D-glucosyl-(1 → 4)-α-L-thevetoside (4), and acetylthevetin B (5). Among the five compounds, 17β-neriifolin most potently inhibited HOXA9-dependent gene expression without affecting the HOXA9 mRNA levels, and suppressed cell proliferation by inducing apoptosis. The findings on the structure-activity relationships of the compounds from C. manghas may contribute to the development of small molecule inhibitors of HOXA9.

Toxicity of nanomixtures to human macrophages: Joint action of silver and polystyrene nanoparticles

Increasing use of nano-enabled products provides many benefits in various industrial processes and medical applications, but it also raises concern about release of nanoparticles (NPs) into the environment and subsequent human exposure. While potential toxicity of individual NPs types has been well described in scientific literature, exposure and health-related effects of nanomixtures has been poorly described. This study aimed to evaluate the combined effect of silver (AgNP) and polystyrene NPs (PSNP) on the human macrophages. AgNP are one of the most commercialized NPs due to efficient antimicrobial activity, while PSNP are ubiquitous in terrestrial and aquatic environments due to plastic pollution and degradation of polystyrene-based products. Differentiated monocytic cell line THP-1 were used as an in vitro model of human macrophages. Multiple aspects of cellular response to AgNP-PSNP nanomixture were analyzed including cell death, induction of apoptosis, oxidative stress response, expression of pro- and anti-inflammatory cytokines, and nanomechanical properties of cells. NPs uptake was visualized by confocal microscopy and quantified using flow cytometry. Results show that nanomixture increased apoptosis and cell death, expression of IL-6, IL-8 and TNFa, oxidative stress and mitochondrial dysfunction in cells compared to AgNP and PSNP applied as single treatments, indicating mixture additive action. Anti-inflammatory cytokines IL1b, IL-4 and IL-10 were not affected by combined exposure compared to single NPs. Visualization of NPs uptake and internalization showed that AgNP and PSNP were localized mostly in cytoplasm, with small fraction of AgNP translocated into cell nuclei, which explain increased number of double-stranded DNA breaks following exposure of cells to AgNPs alone or in the mixture. Study outcomes represent clear warnings on the human co-exposure to AgNP and PSNP that needs to be implemented in risk assessment approaches towards toxic-free environment.

Immunomodulatory effect of IL-1RA in LPS-activated macrophage/dental pulp stem cells co-culture.

Lipopolysaccharides (LPS)-activated human dental pulp stem cells (hDPSCs) and macrophage co-cultures showed downregulated TNF-α secretion that is modulated by hDPSCs through IDO axis, whereas the secretory levels of IL-1β remained unchanged. Therefore, sustained production of IL-1β could contribute to progressive dental pulp inflammation. However, the role of interleukin-1 receptor antagonist (IL-1RA) in downregulating the secretion of IL-1β and TNF-α in LPS-activated M0/M1/M2 macrophage and hDPSCs co-culture has not been studied yet. Therefore, the aim of the present study was to determine the immunomodulatory role of blocking IL-1 receptors in DPSCs macrophage co-culture activated with LPS. Human monocytic cell line THP-1 was polarized to M0, M1 and M2 macrophages and co-cultured with hDPSCs. The viability of the co-cultured cells was assessed by apoptosis assay. Co-cultures were activated with LPS followed by the assessment of gene expression and protein levels of IL-1β and TNF-α with and without IL-1RA blocking via qRT-PCR and cytokine flex assay by flow cytometry. Data from three separate experiments were analysed using one-way anova followed by Tukey's post hoc test and a p-value of <.05 was considered statistically significant. THP-1-derived M0, M1 and M2 macrophages co-cultured with hDPSCs showed spindle and round-shaped cells, with >90% viability when assessed by apoptosis assay. Inflammatory TNF-α and IL-1β profiles in stimulated co-cultures showed upregulated IL-1β, whereas TNF-α was downregulated (p < .05). Anti-inflammatory gene expression levels of IL-10 and TGF-β were downregulated (p < .05). Blocking with IL-1RA resulted in a remarkable decrease in IL-1β at the gene expression and protein production levels whilst TNF-α levels remained low (p < .05). Levels of anti-inflammatory cytokine IL-10 showed no significant difference. Blocking the IL-1 receptor in hDPSCs and macrophage (M0, M1, M2) co-cultures activated with LPS resulted in downregulation of inflammatory cytokines IL-1β and TNF-α. These findings highlight the immunomodulatory effect of IL-1RA in inflammatory conditions of dental pulp infections.

Proteins from Modern and Ancient Wheat Cultivars: Impact on Immune Cells of Healthy Individuals and Patients with NCGS.

In non-celiac gluten sensitivity (NCGS), the elimination of wheat results in a clear symptom improvement, but gluten has still not been proven as (the sole) trigger. Due to the increase in the prevalence of gluten-related diseases, the breeding of high-performance wheat cultivars is discussed as a trigger. To analyze the immune stimulation and signal pathways, the immune cells of healthy subjects and patients with NCGS were stimulated with gliadins from wheat, and the expression and secretion of interleukin 1ß (IL1ß) and interleukin 6 (IL6) were studied. To determine the impact of wheat breeding, the monocyte cell line THP1 and human immune cells were stimulated with gliadin, glutenin, and albumin/globulin fractions of ancient and modern cereals, and expression of inflammatory molecules was checked. Immune cells of patients with NCGS showed an increased expression of IL1ß and IL6 after stimulation with gliadins compared to immune cells of healthy controls. Gliadins caused a strong activation of P-STAT3 in immune cells of healthy controls, and inhibitors of JAK and NFκB pathways considerably reduced this response. In addition to gliadins, we further showed that glutenins and albumin/globulins from all wheat cultivars from the last century, and especially from einkorn and spelt, also markedly induced the expression of inflammatory genes in THP1 and human immune cells. There was no correlation between enhanced immune stimulation and ancient or modern cultivars. This does not support the hypothesis that modern wheat breeding is responsible for the increase in gluten-related diseases. An altered immune situation is suggested in patients with NCGS.

Therapeutic effect of chinese herbal medicine gu-ben-hua-shi (AESS) formula on atopic dermatitis through regulation of yes-associated protein

Background: Atopic dermatitis (AD) is a chronic and recurrent skin disease. At present, there is a lack of sufficiently effective and safe medicines that can be used for a prolonged time and reduce the recurrence of AD. The Gu-Ben-Hua-Shi (AESS) formula has been used for many years with a good clinical effect on AD but its specific treatment mechanism is unknown. Methods: The main components of AESS were analyzed using ultra-high performance liquid chromatography (UPLC). The composition of AESS compounds in the serum from rats was analyzed using ultra-high performance liquid chromatography-mass spectrometry. An AD mouse model was constructed using 2,4-dinitrofluorobenzene stimulation in Balb/C mice and the effect on the reduction of skin lesions and Th1/Th2/Th17/Treg balance after AESS administration were measured. The effects of AESS serum on the proliferation and apoptosis of keratinocyte cell line HaCaT and adhesion of HaCaT to human monocyte cell line THP-1 were detected in an IFN-γ/TNF-α stimulated AD-like inflammatory cell model. The effects of Yes-associated protein (YAP) expression on the therapeutic effect and a related signaling pathway were also investigated. Results: In total, 10 components were confirmed using UPLC, namely five organic acids, three flavonoids, and two chromogenic ketones. Additionally, the similarity of the three batches of samples (S1–3) was above 0.98, indicating that the formula samples have good uniformity. These 10 compounds were also detected in rat serum, suggesting that they are absorbed into rat blood as prototype components. Furthermore, AESS effectively reduced the skin lesions in the AD mouse model, regulated the Th1/Th2/Th17/Treg imbalance, improved the proliferation ability of the AD-like cell model, and inhibited HaCaT apoptosis and adhesion to THP-1 cells. It also reduced the expression of YAP in Th17 and Treg cells of the mouse spleen and increased YAP expression in the skin. The change in YAP expression in keratinocytes weakened the curative effect of AESS, and AESS exerted its effects through the NF-κB signaling pathway. Conclusion: AESS may play a role in the treatment of AD by affecting the expression of YAP. These findings can be used to promote its use as an alternative medication for prolonged use with fewer side effects.

Suppressive Effects of 4-(Phenylsulfanyl) Butan-2-One on CCL-1 Production via Histone Acetylation in Monocytes.

The 4-(phenylsulfanyl) butan-2-one (4-PSB-2), a marine-derived compound from soft coral, was proven to have multiple biological activities including neuroprotection and potent anti-inflammatory effects. CC chemokine ligand (CCL)-1 belongs to T helper (Th)2-related chemokines that are involved in the recruitment of Th2 inflammatory cells. Histone acetylation has been recognized as a critical mechanism underlying the regulated cytokine and chemokine production. Our study tried to investigate the anti-inflammatory effect of 4-PSB-2 on CCL-1 production in human monocytes and explore possible underlying intracellular processes, including epigenetic regulation. To confirm our hypothesis, human monocyte THP-1 cell line and primary CD14+ cells were pretreated with various concentrations of 4-PSB-2 and then were stimulated with lipopolysaccharide (LPS). The CCL-1 concentration was measured by enzyme-linked immunosorbent assays, and the intracellular signaling pathways and epigenetic regulation of 4-PSB-2 were investigated by using Western blotting and chromatin immunoprecipitation analysis. In this study, we found that 4-PSB-2 had a suppressive effect on LPS-induced CCL-1 production. Moreover, this suppressive effect of 4-PSB-2 was mediated via intracellular signaling such as the mitogen-activated protein kinase and nuclear factor-κB pathways. In addition, 4-PSB-2 could suppress CCL-1 production by epigenetic regulation through downregulating histone H3 and H4 acetylation. In short, our study demonstrated that 4-PSB-2 may have a potential role in the treatment of allergic inflammation.

Study of Biological Activities and ADMET-Related Properties of Salicylanilide-Based Peptidomimetics.

A series of eleven benzylated intermediates and eleven target compounds derived from salicylanilide were tested against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 as reference strains and against three clinical isolates of methicillin-resistant S. aureus (MRSA) and three isolates of vancomycin-resistant E. faecalis. In addition, the compounds were evaluated against Mycobacterium tuberculosis H37Ra and M. smegmatis ATCC 700084. The in vitro cytotoxicity of the compounds was assessed using the human monocytic leukemia cell line THP-1. The lipophilicity of the prepared compounds was experimentally determined and correlated with biological activity. The benzylated intermediates were found to be completely biologically inactive. Of the final eleven compounds, according to the number of amide groups in the molecule, eight are diamides, and three are triamides that were inactive. 5-Chloro-2-hydroxy-N-[(2S)- 4-(methylsulfanyl)-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (3e) and 5-chloro-2-hydroxy-N-[(2S)-(4-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino)pentan-2-yl)benzamide (3f) showed the broadest spectrum of activity against all tested species/isolates comparable to the used standards (ampicillin and isoniazid). Six diamides showed high antistaphylococcal activity with MICs ranging from 0.070 to 8.95 μM. Three diamides showed anti-enterococcal activity with MICs ranging from 4.66 to 35.8 μM, and the activities of 3f and 3e against M. tuberculosis and M. smegmatis were MICs of 18.7 and 35.8 μM, respectively. All the active compounds were microbicidal. It was observed that the connecting linker between the chlorsalicylic and 4-CF3-anilide cores must be substituted with a bulky and/or lipophilic chain such as isopropyl, isobutyl, or thiabutyl chain. Anticancer activity on THP-1 cells IC50 ranged from 1.4 to >10 µM and increased with increasing lipophilicity.

Monocytes as suitable carriers for dissemination of dengue viral infection

Dengue viruses (DENVs) exploit monocytes and macrophages for tropism and replication, therefore, establishing a long-term reservoir. However, their roles in dengue pathogenesis remains unclear. Here, using the human monocytic cell line THP-1, human primary monocytes, and non-human primate models, we show that DENV-infected monocytes represent suitable carriers for circulatory viral dissemination. Monocyte-derived macrophages expressing M2 surface markers at the gene level efficiently replicated, while the productivity of monocyte replication was low. However, attachment of DENVs to the cellular surface of monocytes was similar to that of macrophages. Furthermore, after differentiation with type-2 cytokines, DENV-attached monocytes could replicate DENVs. Productive DENV infection was confirmed by intravenous injection of DENVs into nonhuman primate model, in which, DENV attachment to monocytes was positively correlated with viremia. These results provide insight into the role of circulating monocytes in DENV infection, suggesting that monocytes directly assist in DENV dissemination and replication during viremia and could be applied to design antiviral intervention.

Investigation of the effect of free fatty acids on cell growth and Cytokine secretion

Interactions between immune and inflammatory cells are mediated primarily through the interaction of proteins, termed interleukins (IL) that are able to promote cell growth, differentiation as well as functional activation. TNF-α, IL-1 and IL-6 are the most important cytokines produced by monocytes. The aim of this study was to investigate the effect of docosahexaenoic acid (DAH), on immune cell proliferation along with cytokine production. This was tested using the THP-1 monocytic cell line, which in turn allowed observing how surface protein cytokine expression was affected by docosahexaenoic acid treatment and whether the effect of incubation would reduce the stimulus-induced production of pro-inflammatory cytokines. The experimental method employed Linked Immunosorbent Assays, while both Cell Titer 96® AQueous One Solution Cell Proliferation Assay and Titer 96 AQueous One Solution were used to determine cell proliferation. THP-1 cells induce growth-promoting activity and, the concentration of DHA toxic effect began at around 100 µM and reached maximum toxicity at around 200 µM. The results demonstrate that DHA inhibited endogenous IL-8 release when compared to a control group where the inhibition was maintained for 3 days. The study revealed the effect of docosahexaenoic acid (DHA), on immune cell proliferation and cytokine production.