Wasaporn Chanput

THP-1 cell line: An in vitro cell model for immune modulation approach

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions.

Transcription profiles of LPS-stimulated THP-1 monocytes and macrophages: a tool to study inflammation modulating effects of food-derived compounds

An assay was developed to study inflammation-related immune responses of food compounds on monocytes and macrophages derived from THP-1 cell line. First strategy focused on the effects after stimulation with either lipopolysaccharide (LPS) or Concanavalin A (ConA). Gene expression kinetics of inflammation-related cytokines (IL-1β, IL-6, IL-8, IL-10 and TNF-α), inflammation-related enzymes (iNOS and COX-2), and transcription factors (NF-κB, AP-1 and SP-1) were analyzed using RT-PCR. Time dependent cytokine secretion was investigated to study the inflammation-related responses at protein level. LPS stimulation induced inflammation-related cytokine, COX-2 and NF-κB genes of THP-1 monocytes and THP-1 macrophages with the maximum up-regulation at 3 and 6 h, respectively. These time points, were subsequently selected to investigate inflammation modulating activity of three well known immuno-modulating food-derived compounds; quercetin, citrus pectin and barley glucan. Co-stimulation of LPS with either quercetin, citrus pectin, or barley glucan in THP-1 monocytes and macrophages showed different immuno-modulatory activity of these compounds. Therefore, we propose that simultaneously exposing THP-1 cells to LPS and food compounds, combined with gene expression response analysis are a promising in vitro screening tool to select, in a limited time frame, food compounds for inflammation modulating effects.

Polysaccharides from Agaricus bisporus and Agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells

BackgroundMushroom polysaccharides have traditionally been used for the prevention and treatment of a multitude of disorders like infectious illnesses, cancers and various autoimmune diseases. Crude mushroom extracts have been tested without detailed chemical analyses of its polysaccharide content. For the present study we decided to chemically determine the carbohydrate composition of semi-purified extracts from 2 closely related and well known basidiomycete species, i.e. Agaricus bisporus and A. brasiliensis and to study their effects on the innate immune system, in particular on the in vitro induction of pro-inflammatory cytokines, using THP-1 cells.MethodsMushroom polysaccharide extracts were prepared by hot water extraction and precipitation with ethanol. Their composition was analyzed by GC-MS and NMR spectroscopy. PMA activated THP-1 cells were treated with the extracts under different conditions and the production of pro-inflammatory cytokines was evaluated by qPCR.ResultsSemi-purified polysaccharide extracts of A. bisporus and A. brasiliensis (= blazei) were found to contain (1→6),(1→4)-linked α-glucan, (1→6)-linked β-glucan, and mannogalactan. Their proportions were determined by integration of 1H-NMR signs, and were considerably different for the two species. A. brasiliensis showed a higher content of β-glucan, while A. bisporus presented mannogalactan as its main polysaccharide. The extracts induced a comparable increase of transcription of the pro-inflammatory cytokine genes IL-1β and TNF-α as well as of COX-2 in PMA differentiated THP-1 cells. Pro-inflammatory effects of bacterial LPS in this assay could be reduced significantly by the simultaneous addition of A. brasiliensis extract.ConclusionsThe polysaccharide preparations from the closely related species A. bisporus and A. brasiliensis show major differences in composition: A. bisporus shows high mannogalactan content whereas A. brasiliensis has mostly β-glucan. Semi-purified polysaccharide extracts from both Agaricus species stimulated the production of pro-inflammatory cytokines and enzymes, while the polysaccharide extract of A. brasiliensis reduced synthesis of these cytokines induced by LPS, suggesting programmable immunomodulation.

β-Glucans are involved in immune-modulation of THP-1 macrophages.

SCOPE We aimed to examine different immunological aspects of β-glucans derived from different food sources (oat, barley and shiitake) on phorbol myristate acetate (PMA)-differentiated THP-1 macrophages. Commercially purified barley β-glucan (commercial BG) and lentinan were included to compare β-glucans from the same origin but different degree of purity and processing. METHODS AND RESULTS Chemical composition and molecular weight distribution of β-glucan samples were determined. Inflammation-related gene expression kinetics (IL-1β, IL-8, nuclear factor kappa B [NF-κB] and IL-10) after 3, 6 and 24 h of stimulation with 100 μg/mL β-glucan were investigated. All tested β-glucans mildly upregulated the observed inflammation-related genes with differential gene expression patterns. Similar gene expression kinetics, but different fold induction values, was found for the crude β-glucan extracts and their corresponding commercial forms. Pre-incubation of THP-1 macrophages with β-glucans prior to lipopolysaccharide (LPS) exposure decreased the induction of inflammation-related genes compared to LPS treatment. No production of nitric oxide (NO) and hydrogen peroxide (H₂O₂) was detected in β-glucan stimulated THP-1 macrophages. Phagocytic activity was not different after stimulation by β-glucan samples. CONCLUSION Based on these in vitro analyses, it can be concluded that the analysed β-glucans have varying levels of immunomodulating properties, which are likely related to structure, molecular weight and compositional characteristic of β-glucan.

Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs).

In this study, two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIPs (rFIPs) were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to the well-known FIPs, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced rFIPs: rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity.

Anti-oxidative assays as markers for anti-inflammatory activity of flavonoids

The complexity of in vitro anti-inflammatory assays, the cost and time consumed, and the necessary skills can be a hurdle to apply to promising compounds in a high throughput setting. In this study, several antioxidative assays i.e. DPPH, ABTS, ORAC and xanthine oxidase (XO) were used to examine the antioxidative activity of three sub groups of flavonoids: (i) flavonol: quercetin, myricetin, (ii) flavanone: eriodictyol, naringenin (iii) flavone: luteolin, apigenin. A range of flavonoid concentrations was tested for their antioxidative activities and were found to be dose-dependent. However, the flavonoid concentrations over 50ppm were found to be toxic to the THP-1 monocytes. Therefore, 10, 20 and 50ppm of flavonoid concentrations were tested for their anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated THP-1 monocytes. Expression of inflammatory genes, IL-1β, IL-6, IL-8, IL-10 and TNF-α was found to be sequentially decreased when flavonoid concentration increased. Principle component analysis (PCA) was used to investigate the relationship between the data sets of antioxidative assays and the expression of inflammatory genes. The results showed that DPPH, ABTS and ORAC assays have an opposite correlation with the reduction of inflammatory genes. Pearson correlation exhibited a relationship between the ABTS assay and the expression of three out of five analyzed genes; IL-1β, IL-6 and IL-8. Our findings indicate that ABTS assay can potentially be an assay marker for anti-inflammatory activity of flavonoids.

Introduction of New Computer Softwares for Classification and Prediction Purposes of Bioactive Peptides: Case Study in Antioxidative Tripeptides

Antioxidative tripeptides were successfully classified according to their structure by using Sequence Principal Component Similarity (SPCS) software. During SPCS computation, hydrophobicity was the main characteristic of peptide residues in aspect of antioxidative activity to inhibit lipid peroxidation in linoleic acid model system. The prediction made by Homology Similarity Search (HSS)-BIOPEP combined software indicated that C-hordein fraction from barley protein showed the greatest potential to be antioxidative protein source followed by prolamin fraction from rice protein. Moreover, these homology segments of C-hordein were resistant against the digestion of mixed gastrointestinal enzymes in BIOPEP digestive model system.

Effect of genetic and climatic variability on the metabolic profiles of black gram (Vigna mungo L.) seeds and sprouts

BACKGROUND Black gram is becoming increasingly of interest for consumers worldwide. The metabolomics have been conducted to reflect the life history of each individual plant. The metabolic pattern of black gram seeds and sprouts was profiled to investigate genetic and climatic influences on a broad range of chemical constituents. RESULTS Distinct differences in metabolite profiles among three black gram varieties for both intact seeds and sprouts were observed. The differential impact of climate on metabolite profiles of the variety Chai Nat 80 during both dry and rainy seasons was investigated. Univariate statistical analysis demonstrated that greater maturity due to adequate moisture in the rainy season led to a higher content of nutritionally relevant polar metabolites, whereas the dry season resulted in a high relative amount of storage lipid because of immaturity due to insufficient rain and water supply. CONCLUSION The investigation confirmed the potential of metabolite profiling to assist in breeding and farming practices.

THP-1 and U937 Cells

Monocytes are circulatory precursor cells from myeloid origin that can develop into macrophages or dendritic cells upon migration from the blood stream to tissues. Both macrophages and dendritic cells are professional antigen-presenting cells. Monocytes and their macrophage and dendritic-cell progeny serve three main functions in the immune system. These are phagocytosis, antigen presentation, and cytokine production. THP-1 and U937 are (pro-) monocytic cell lines that can, also in vitro, be differentiated into either various types of macrophages or into dendritic cells.

Immunomodulatory effects of mushroom B-glucans

: Mushroom-derived dietary β-glucans have been shown to elicit diverse immunomodulatory effects in human and animal tissues, including the blood, gastrointestinal tract and spleen. In controlled human trials, β-glucan intake stimulated the immune system in the blood of healthy adults, dampened the allergic response to a respiratory inflammatory agent and improved survival in cancer patients. Additional randomized controlled trials are warranted to enable a more complete understanding of the immunomodulatory effects and specific applications of orally administered β-glucans.