Cytokine Concentrations In Culture Supernatants Research Articles

Adipose-Derived Mesenchymal Stem Cells from Arthritis Patients: Differential Modulation of CD4⁺ T Cell Activation and Cytokine Production.

BACKGROUND Adipose-derived stem cells (ASCs) from intra-articular adipose tissue of osteoarthritis (OA) and rheumatoid arthritis (RA) patients similarly regulate the proliferation of activated CD4⁺ T lymphocytes and exhibit comparable differentiation potential. This study aimed to assess the impact of ASCs from RA patients on CD4⁺ T cell activation and differentiation into Th17 and T regulatory (Treg) cells. MATERIAL AND METHODS Intra-articular adipose tissue samples were obtained from patients with RA and OA, who underwent knee replacement surgery. ASCs were isolated and cultured either with isolated CD4⁺ cells or with peripheral blood mononuclear cells. After culture, CD4⁺ T cell phenotype was evaluated by flow cytometry, and cytokine concentrations in culture supernatants were analyzed via ELISA. Blocking experiments were conducted to identify the soluble agents responsible for the immunomodulatory effects of ASCs. RESULTS RA- and OA-derived ASCs effectively modulated CD25 and CD69 expression on CD4⁺ cells. RA-derived ASCs failed to induce Tregs, decreased HLA-DR expression, and increased IL-35 production. RA- and OA-derived ASCs reduced TNF and IFN-γ production but increased IL-17 production. The immunomodulatory activities of ASCs were linked to the kynurenine pathway and prostaglandin E2. CONCLUSIONS This study indicates that ASCs modulate the phenotype of CD4⁺ T cells and influence the production of both pro-inflammatory and anti-inflammatory cytokines. However, ASCs from RA patients appear to have impaired immunomodulatory abilities, raising concerns about their therapeutic potential. Further research is needed to enhance our understanding of ASCs biology and their therapeutic utility.

Abstract 1020: Anti-oxidant amino acid L-ergothioneine exhibits immunomodulatory effect by regulating immunosuppressive cytokine production of tumor cells

Abstract Ergothioneine (ERG) is a natural antioxidant nutrient containing in edible golden oyster mushroom, Pleurotus cornucopiae (Tamogitake). In previous AACR meeting, we reported that administration of ERG rich water extracts of p. cornucopiae to tumor-bearing mice could suppress the growth of subcutaneous tumors and reduce the number of regulatory T cells (Treg) in lymph node and spleen. However, the molecular events inducing antitumor and immunomodulatory effects are still unknown. Our aim is to identify the mechanism of ERG in tumor cells which secrete various immunomodulatory cytokines. We cocultured human tumor cell lines with ERG and tested cytokine concentration in culture supernatants and expression of immune checkpoint molecules on tumors. ERG could significantly inhibit the production of VEGF, CCL2 and TGF-β1 which are important in not only angiogenesis and tumor invasion but also Treg induction. Importantly, ERG also reduced the expression of some immune checkpoint molecules on tumor cells and prolonged the survival of IL-2-activated lymphocytes in vitro. Overall, ERG rich water extract of p. citrinopileatus might inhibit the induction of Treg in tumor-bearing host via suppression of VEGF and TGF-β1, indicating a novel immunomodulatory antioxidant nutrient in mushrooms Citation Format: Kazunori Kato, Katsuhiro Kuroki, Yui Shimizu. Anti-oxidant amino acid L-ergothioneine exhibits immunomodulatory effect by regulating immunosuppressive cytokine production of tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1020.

Abstract B80: Natural killer and dendritic cells collaborate in the immune response induced by Cetuximab and IL-15 against triple negative breast cancer

Abstract Background: While the majority of Triple Negative Breast Cancer (TNBC) overexpress epidermal growth factor receptor (EGFR), EGFR inhibition as a monotherapy with Cetuximab is not effective. The study of its mechanism of action is important to find out potentially synergistic combinations. Previously, we demonstrated that Cetuximab can trigger antibody-dependent NK cell-mediated cytotoxicity against TNBC cells. The aim of this study was to investigate the effect of NK cell activation by Cetuximab-coated TNBC cells on Dendritic Cell (DC) function, and the result of its combination with IL-15. Methods: Human monocyte-derived DCs were co-cultured with autologous isolated NK cells, TNBC cells, Cetuximab and/or IL-15 for 24hs. Costimulatory molecule and activation marker expression in DC and NK cells were analyzed by flow cytometry, and cytokine concentration in culture supernatants was assessed by ELISA. DCs obtained after co-cultures were incubated for 24hs with CD40 ligand transfected fibroblasts to mimic the interaction with CD40L expressing Th cells, and IL-12 was analyzed in culture supernatants by ELISA. Results: Cetuximab treatment of TNBC cells enhanced DC maturation only when NK cells were present in the co-culture (%CD83+: 6.6±0.7 vs 61.0±17, p<0.001; CD86 MIF: 121±36 vs 1868±833, p<0.01). In concert, NK cells were activated by Cetuximab showing increased CD25 expression and IFN-γ production, a cytokine known to induce DC activation. After subsequent CD40 ligand stimulation, DCs that where co-cultured with Cetuximab activated NK cells had an increased ability to produce IL-12p70. Furthermore, IL-15 combination with Cetuximab not only promoted NK cell cytotoxic activity against TNBC cells, but also induced IFN-γ production and DC maturation. Conclusions: Our results suggest that besides its anti-proliferative mechanism of action, Cetuximab has immunological effects in an in vitro model of TNBC, promoting NK cell activation and DC maturation, which could be potentiated by the combination with IL-15. This could ultimately result in the priming of T cell-based immunity, which will be evaluated in further studies. Citation Format: Estefanía P. Juliá, María Betina Pampena, Yamila S. Rocca, José Mordoh, Estrella M. Levy. Natural killer and dendritic cells collaborate in the immune response induced by Cetuximab and IL-15 against triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B80.

A6.2 Purified microbial capsular polysaccharide Galactoxylomannan (GalXM) inhibits the effector T lymphocyte arm and potentiates the regulatory counterpart in rheumatoid arthritis

Background and ObjectivesWe previously demonstrated that the purified microbial polysaccharide Galactoxylomannan (GalXM) from the opportunistic fungus Cryptococcus neoformans is able to impair T-cell proliferation and to induce T lymphocyte apoptosis...

Abnormalities in cytokine secretion from mesenchymal stem cells in psoriatic skin lesions

Psoriasis is a chronic inflammatory and proliferative skin disease associated with immune abnormalities. In our preliminary studies, it was found that bone marrow mesenchymal stem cells (BMSCs) were abnormal in patients with psoriasis. Mesenchymal stem cells (MSCs) are not only present in the bone marrow, but can also be separated from the skin.We reasoned that an investigation into the biological characteristics of MSCs in psoriatic skin lesions could more realistically reflect the actual conditions for the pathogenesis of psoriasis. To reveal whether MSCs in psoriatic skin lesions are abnormal. We obtained MSCs from psoriatic skin lesions and healthy human skin and identified these cells using flow cytometry and a cell differentiation assay; cytokine concentrations in the culture supernatants were measured with the ELISA assay. We compared cytokine concentrations in culture supernatants of MSCs derived from psoriatic skin lesions as well as those from healthy human skin. Compared with healthy control skintissue, in psoriatic skin lesions, MSC secretion of EGF, SCF, and IL-11 increased; secretion of bFGF, IL-3, IL-6, IL-8, and HGF decreased (p<0.05); and secretion of VEGF, M-CSF, G-CSF, GM-CSF, LIF, IL-1, IL-7, IL-10, and TNF-α showed no significant difference (p>0.05). Surface markers and the differentiation capacity of cells from the two sources were similar. This study demonstrated that MSCs derived from psoriatic skin lesions had abnormalities, which may be one of the pathogenic mechanisms of psoriasis.

Suppression of collagen-induced arthritis by intra-articular lentiviral vector-mediated delivery of Toll-like receptor 7 short hairpin RNA gene

Knockdown of Toll-like receptors (TLRs) is a novel therapeutic strategy in treating patients with rheumatoid arthritis (RA). We examined the effects of lentiviral vector-mediated delivery of TLR7 short hairpin RNA gene (Lt.shTLR7) on collagen-induced arthritis (CIA). After being immunized on days 0 and 7, Sprague-Dawley rats received intra-articular (i.a.) injection of Lt.shTLR7 or scramble control vector on days 7 and 10. The therapeutic effects were evaluated by measuring ankle circumferences, articular index, and radiographic and histological scores on killing on day 16. Microvessel densities, vascular endothelial growth factor (VEGF) levels, pro-inflammatory cytokine concentrations and T-cell numbers within the synovial tissues were measured. Moreover, VEGF and pro-inflammatory cytokine concentrations in culture supernatants from TLR7-transfected synovial fibroblasts (SFs) stimulated with imiquimod or endogenous ligands were examined. There were significant reduction in ankle circumferences, articular indexes, and radiographic and histological scores. Microvessel densities, VEGF concentrations, interleukin (IL)-1β and IL-6 levels and T-cell densities within synovial tissues were significantly lower. Induction of VEGF, IL-1β and IL-6 production from stimulated SFs was significantly suppressed. Taken together, these data demonstrate the effects of i.a. lentiviral vector-mediated delivery of shTLR7 RNA gene on inhibition of CIA, and implicate the manipulation of TLR7 as a potential therapeutic strategy in RA patients.

Inhibition of synovial hyperplasia, rheumatoid T cell activation, and experimental arthritis in mice by sulforaphane, a naturally occurring isothiocyanate

To investigate whether sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables such as broccoli, regulates synoviocyte hyperplasia and T cell activation in rheumatoid arthritis (RA). Synoviocyte survival was assessed by MTT assay. The levels of Bcl-2, Bax, p53, and pAkt were determined by Western blot analysis. Cytokine concentrations in culture supernatants from mononuclear cells were analyzed by enzyme-linked immunosorbent assay. The in vivo effects of SFN were examined in mice with experimentally induced arthritis. SFN induced synoviocyte apoptosis by modulating the expression of Bcl-2/Bax, p53, and pAkt. In addition, nonapoptotic doses of SFN inhibited T cell proliferation and the production of interleukin-17 (IL-17) and tumor necrosis factor alpha (TNFalpha) by RA CD4+ T cells stimulated with anti-CD3 antibody. Anti-CD3 antibody-induced increases in the expression of retinoic acid-related orphan receptor gammat and T-bet were also repressed by SFN. Moreover, the intraperitoneal administration of SFN to mice suppressed the clinical severity of arthritis induced by injection of type II collagen (CII), the anti-CII antibody levels, and the T cell responses to CII. The production of IL-17, TNFalpha, IL-6, and interferon-gamma by lymph node cells and spleen cells from these mice was markedly reduced by treatment with SFN. Anti-CII antibody-induced arthritis in mice was also alleviated by SFN injection. SFN was found to inhibit synovial hyperplasia, activated T cell proliferation, and the production of IL-17 and TNFalpha by rheumatoid T cells in vitro. The antiarthritic and immune regulatory effects of SFN, which were confirmed in vivo, suggest that SFN may offer a possible treatment option for RA.

Anti‐inflammatory effects of atorvastatin on peripheral blood mononuclear cells and synovial fibroblasts in rheumatoid arthritis

Objective: Statins, such as atorvastatin (ATV), are 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitors known to exert lipid‐lowering but also anti‐inflammatory, effects. In this study, we analysed the in vitro effects of ATV on peripheral blood mononuclear cells (PBMCs) and fibroblast‐like synoviocytes (FLS) in rheumatoid arthritis (RA), a chronic inflammatory joint disease.Methods: PBMCs isolated from 25 RA patients and 20 healthy blood donors were stimulated in vitro with 0.1 µM ATV for 24 h. PBMC cultures were analysed for cell surface markers to characterize T‐cell subtypes (CD4, CD8, CD69, HLA‐DR) by flow cytometry and for T helper cell type 1 (Th1) and type 2 (Th2) cytokines [interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4), IL‐10] in culture supernatants by enzyme‐linked immunosorbent assay (ELISA). Furthermore, RNA isolated from ATV‐stimulated RA‐FLS pre‐ and post‐ATV stimulation was analysed by microarray and quantitative reverse transcription polymerase chain reaction (RT‐PCR).Results: Flow cytometric analysis of T‐cell subsets revealed no significant differences for CD4, CD8, CD69, and HLA‐DR surface marker expression of PBMCs in RA patients and healthy controls after ATV stimulation. However, the proportion of IFN‐γ expressing CD4+ T cells and the IFN‐γ cytokine concentrations in culture supernatants were significantly reduced in T‐cell cultures from RA patients. In ATV‐stimulated FLS a significant downregulation of proinflammatory cytokine (IL‐6) and chemokine (IL‐8) expression was detected (p<0.001).Conclusions: Our study demonstrates a marked in vitro anti‐inflammatory activity of ATV in RA including a systemic effect on a pathogenic CD4+ T‐cell population (Th1) and a local effect on FLS. These findings may provide a scientific rationale for statins as add‐on therapy in RA.

Cytokine production and modulation: Comparison of patients with chronic fatigue syndrome and normal controls

We studied cytokine production in 15 patients with chronic fatigue syndrome (CFS) and 23 controls. CFS patients' peripheral blood mononuclear cells were cultured with lipopolysaccharide or phytohemagglutinin. Enzymatic immunoassay indicated cytokine concentration in culture supernatants. CFS patients showed significantly lower mRNA levels and transforming growth factor-beta1 (TGF- β1) production. Cytokine dysregulation affects CFS pathogenesis. TGF- β1 may aid treatment because it affects CFS inflammatory characteristics.

Activation of Monocytes by Aspergillus Fumigatus Hyphae and Conidia.

Invasive aspergillosis is a life-threatening infection in immunocompromised patients. Activation of innate immune cells is crucial for the successful control of invasive aspergillosis. Monocytes play an important role in the cellular defense against Aspergillus fumigatus by attacking defined differentiation states of the fungus and by the release of pro-inflammatory cytokines and chemokines. In order to get a better understanding on pathways involved in these defense mechanism, genome-wide expression profiling was performed after incubation of CD14+ selected monocytes with A. fumigatus conidia and hyphae for 3, 6 and 9 hours using the Affymetrix HG-U133A array. Moreover, phagocytosis of conidia was analyzed by immunofluorescence. Expression of defined cytokines and chemokines was confirmed by Real-time PCR and on protein level by ELISA. Phagocytosis of conidia was observed during the first 3 hours (up to 2–3 conidia/monocyte). The conidia became swollen after 6 hours. By genome-wide expression profiling, ~600 genes were differentially regulated after 3 hours of incubation with hyphae and ~200 after incubation with conidia. At 6 and 9 hours of incubation, the number of differentially regulated genes showed no difference (~400 genes by both stimuli). Hyphae but not conidia strongly induced the expression of chemokines attracting neutrophils and T-lymphocytes as well as their receptors already after 3 hours, indicating that phagocytosis of conidia does not immediately induce expression of inflammatory genes. Pentraxin-3 was upregulated after 3 but not after 6 hours by both conidia and hyphae. PTX-3 binds to A. fumigatus and plays a role in initiating phagocytosis. Genes encoding for inflammatory cytokines and chemokines as well as their receptors were found to be up-regulated at 6 and 9 hours by both stimuli. Urokinase plasminogen activator (uPA), which plays an essential role in regulating fibrinolysis and thereby monocyte/macrophage recruitment to sites of inflammation, was also found to be upregulated after stimulation with both stimuli. ELISA experiments confirmed these results on protein level, demonstrating a steady increase of chemokine and cytokine concentrations in culture supernatants from 3 to 9 hours.In conclusion, A. fumigatus differentially regulates a wide number of genes expressed by human monocytes, which play a crucial role in immune response during infection. The expression of these genes is dependent on the differentiation and germination state of the fungus and the incubation period.

In vitro effects of eicosanoids derived from different 20-carbon fatty acids on T helper type 1 and T helper type 2 cytokine production in human whole-blood cultures.

Different series prostaglandins (PGs) and leukotrienes (LTs) are synthesized from different 20 carbon fatty acid precursors. The effects of the different series of PGs and LTs on production of T helper type 1 (Th1) and Th2 cytokines by human cells are not well established. To characterize the influence of PGs and LTs produced from different fatty acid precursors on the Th1 and Th2 cytokine profile in mitogen-stimulated human whole-blood cultures. Blood from healthy adult males was diluted and cultured with concanavalin A in the presence or absence of a range of concentrations of various PGs or LTs. Cytokine concentrations in culture supernatants were analysed by enzyme-linked immunosorbent assay (ELISA). PGE1, PGE2 and PGE3 significantly and dose-dependently decreased the concentrations of the Th1 cytokines IL-2 and IFN-gamma by up to 50% and 70%, respectively. The three PGs exhibited similar potency towards IFN-gamma production. At the highest concentration used (10-6 m) PGE1, but not PGE2 or PGE3, increased the concentration of the Th2 cytokine IL-4 by about 70%. IL-10 production was not affected by PGs. The ratio of the concentrations of IFN-gamma to IL-4 was significantly decreased at PGE concentrations of 10-7 and 10-6 M with all three PGEs having similar effects. LTB4, LTC4 and LTC5 did not significantly affect production of the cytokines studied. PGE produced from different fatty acids significantly decrease Th1 cytokine production resulting in a shift in the Th1, Th2 balance in favour of a Th2 response. PGE produced from different fatty acid precursors are equipotent in their effects on human T lymphocytes. Thus, although changes in the pattern of dietary fatty intakes may contribute to the increased prevalence of atopic disease, this would probably not be mediated through substitution of one PGE with another from a different series. It may, however, be mediated through a change in the total amount of PGE produced at the site of antigen presentation.

Differential modulation of surface and intracellular protein expression by T cells after stimulation in the presence of monensin or brefeldin A.

Intracellular cytokine staining is an increasingly popular analytical tool that can be used to define the profile of cytokines in various disease states. One important requirement for this assay is the inclusion of a protein transport inhibitor in stimulated cell cultures to trap the cytokine, thus allowing a brighter signal. Two compounds commonly used for this purpose are brefeldin A (BFA) and monensin (MN). Flow cytometry was used to assess the differential effects of BFA and MN on surface CD3, -4, -8, and -69 expression and the intracellular expression of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) following stimulation with phorbol myristate acetate and ionomycin. We found that BFA blocked the majority of CD3(+) cells from expressing surface CD69, but BFA did not inhibit intracellular CD69 expression. MN did not significantly inhibit surface CD69 expression. With regard to lymphocyte marker expression following activation, surface CD4 expression was significantly downregulated; however, less downregulation was observed with BFA treatment than with MN treatment. Analyzing intracellular cytokine expression, BFA trapped a greater percentage of TNF-alpha inside activated cells than MN. An analysis of the cytokine concentration in culture supernatants indicated that cells treated with MN released TNF-alpha and IFN-gamma from the cells, while the BFA-treated cells released IFN-gamma only. With prolonged (18-h) stimulation, the cells treated with MN were less viable than those treated with BFA. We conclude that the choice of a protein transport inhibitor is an important variable in this assay. When developing this method as a tool for clinical immunology laboratory analysis, investigators should consider the differential effects of BFA and MN on results.

Mechanisms of Lipoplysaccharide-induced Lipopolysaccharide Tolerance in the Expression of TNF-alpha and IL-8 in Peripheral Blood Monocytes

Background : Monocytes/macrophages play a central role in determining the host response during Gram-negative infection through secretion of a variety of mediators after stimulation of LPS. Even though cytokine production has been shown to play an important role in host defense during sepsis, cytokine release may also lead to tissue injury. Thus, regulation of macrophage response to LPS is critical for host survival during Gram-negative sepsis. In animals exposed to nonlethal doses of endotoxin, a characteristic hyporesponsiveness to subsequent administration of endotoxin has been observed. This phenomenon was known as 'LPS tolerance'. However, little information is available regarding the underlying mechanism of LPS tolerance. Method : Peripheral blood monocyte(PBMC) was isolated from peripheral blood of normal volunteers by adhesion purification method. To evaluate the conditions to obtain LPS tolerance, preculture was carried out with LPS at 10ng/ml for 24 hours. For stimulation, culture plates were washed two times and were stimulated with LPS at for 4, 6 and 26 hours. To assess the underlying mechanisms of LPS tolerance, autologous serum, PMA, anti-CD14 Ab, Indomethacin or were added to preculture solution respectively. Cytokine concentrations in culture supernatants were measured using ELISA for TNF- and IL-8 and mRNA of TNF- and IL-8 were determined by Northern blot analysis. Results : The exposure of PBMC to low dose of LPS suppressed the cytokine production and mRNA expression of TNF-, but not IL-8. Anti-CD14 Ab partially recovered production of TNF- which was suppressed by preculture with low dose LPS. The preculture with PMA induces LPS tolerance, as preculture with low dose LPS. Conclusion : LPS tolerance to TNF- is regulated pretranslationally and is influenced by protein kinase C pathway and CD14.