scholarly journals AB0062 CHARACTERIZATION OF IL-12 AND IL-23 REDUCTION BY TOFACITINIB IN MDCS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1332.2-1333
Author(s):  
N. Vincken ◽  
C. Angiolilli ◽  
S. Cardoso ◽  
A. Lopes ◽  
M. Olde-Nordkamp ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Psoriatic Arthritis ◽  
Mrna Expression ◽  
Intracellular Signaling ◽  
Magnetic Beads ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Myeloid Dendritic Cells ◽  
Ifn Γ

Background:Psoriatic arthritis (PsA) is a chronic inflammatory auto-immune disease characterized by an excessive production of pathogenic mediators that cause inflammation of the skin, peripheral joints, entheses and the spine. Among these, interleukin (IL)-23, IL-12, the IL-17 family and TNF constitute key players in PsA pathogenesis.1,2IL-23, consisting of IL23A (IL-23p19) and IL12B (IL-12p40) subunits, is predominantly produced by myeloid dendritic cells (mDCs). While the p19 subunit is unique to IL-23, the p40 subunit is shared with IL-12. Together, IL-12 and IL-23 play a crucial role in promoting the differentiation of naïve T lymphocytes into T helper (Th) interferon (IFN)-γ-producing Th1 or IL17-producing Th17 cells, respectively.3Small-molecule inhibitors, such as the JAK/STAT inhibitor Tofacitinib, have recently shown promising therapeutic potential in PsA clinical trials.4The inhibition of JAKs by Tofacitinib results in the direct suppression of multiple intracellular signaling pathways which constitute key hubs in the cytokine network.5However, whether Tofacitinib is able directly target IL-12/IL-23 production by mDCs has not yet been documented. Suppression of these canonical inflammatory pathways would provide further evidence that Tofacitinib is an effective drug in halting both innate and adaptive immune responses.Objectives:To evaluate the transcriptional and molecular events underlining IL-12 and IL-23 regulation by Tofacitinib in mDCs.Methods:Peripheral blood mononuclear cells from healthy donors were isolated by Ficoll gradient. Monocytes and myeloid dendritic cells (mDCs) were isolated by using magnetic beads on autoMACS. Monocytes were cultured for 6 days in the presence of IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate monocyte-derived dendritic cells (moDCs). MoDCs were harvested, washed and put to rest for 1 day prior to stimulation, while mDCs were stimulated on the same day of isolation. Both moDCs and mDCs were pre-treated with Tofacitinib and then stimulated with either lipopolysaccharide (LPS) or combination of LPS with IFN-γ for 4 hours. Cytokines were measured using enzyme-linked immunosorbent assay (ELISA) and gene expression was assessed using quantitative polymerase chain reaction (qPCR).Results:Treatment of both mDCs and moDCs with Tofacitinib led to a decreased mRNA expression of IL-12 p40 (IL12B) in the presence of TLR4 and IFNγ co-stimulation. The decreasedIL12BmRNA expression also resulted in lower production of IL-12 p40 and IL-23 proteins in mDCs.Conclusion:In this work, we demonstrated for the first time that Tofacitinib can suppress the production of IL-23/IL-12 p40 subunit in mDCs, upon the condition that an active type II IFN signalling is also present in these cells. This observation indicates that specific factors, such as endogenous IFN-γ levels in the serum of PsA patients, can possibly predict differential responses to Tofacitinib treatment.References:[1]Gaffen SL. et al. The IL-23-IL-17 immune axis: from mechanisms to therapeutic testing. Nat Rev Immunol. 2014 Sep;14(9):585-600[2]Bravo A, Kavanaugh A. Bedside to bench: defining the immunopathogenesis of psoriatic arthritis. Nat Rev Rheumatol. 2019 Nov;15(11):645-656[3]Floss DM. et al. Insights into IL-23 biology: From structure to function. Cytokine Growth Factor Rev. 2015 Oct;26(5):569-78[4]Berekmeri A. et al. Tofacitinib for the treatment of psoriasis and psoriatic arthritis. Expert Rev Clin Immunol. 2018 Sep;14(9):719-730[5]T Virtanen A. et al. Selective JAKinibs: Prospects in Inflammatory and Autoimmune Diseases. BioDrugs. 2019 Feb;33(1):15-32Disclosure of Interests:None declared

scholarly journals Up-Regulation of CD40 Ligand and Induction of a Th2 Response in Children Immunized with Pneumococcal Polysaccharide Vaccines

2001 ◽  
Vol 8 (2) ◽  
pp. 233-240 ◽  
Author(s):  
Lily E. Leiva ◽  
Boyd Butler ◽  
James Hempe ◽  
Alejandro P. Ortigas ◽  
Ricardo U. Sorensen
Keyword(s):  
Mrna Expression ◽  
Mononuclear Cells ◽  
Cd40 Ligand ◽  
Interleukin 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Th2 Response ◽  
Polysaccharide Antigens ◽  
Ifn Γ ◽  
Antibody Levels

ABSTRACT We wished to determine whether pneumococcal polysaccharide antigens induce mRNA expression of CD40 ligand (CD40L) and Th1 or Th2 cytokines in unimmunized individuals in vitro and whether immunization with the 23-valent pneumococcal polysaccharide vaccine induces changes in CD40L and cytokine mRNA expression. Children with recurrent respiratory infections were studied before and 4 to 6 weeks after receiving the pneumococcal vaccine. One patient who failed to respond to the polysacharide vaccine subsequently received a single dose of the experimental 7-valent pneumococcal conjugate vaccine. Unimmunized healthy adults were included as controls. Quantification of mRNA expression of CD40L, interleukin-4 (IL-4), IL-12p40, and gamma interferon (IFN-γ) was performed by reverse transcription-PCR and enzyme-linked immunosorbent assay (ELISA)-PCR with resting and stimulated peripheral blood mononuclear cells. Serum immunoglobulin G (IgG) anti pneumococcal antibody levels were measured by ELISA. The results showed a significant increase in the expression of mRNAs for CD40L and IL-4, but not IL-12p40 or IFN-γ, in stimulated cultures from unimmunized individuals. CD40L and IL-4 mRNA expression was significantly higher in postimmunization than in preimmunization samples stimulated with the individual pneumococcal serotypes. These results suggest that pneumococcal polysaccharide antigens specifically up-regulate CD40L expression and induce a Th2 response in vitro which parallels the increase in IgG antipneumococcal antibody levels in serum.

scholarly journals Effect of interleukin (IL)-35 on IL-17 expression and production by human CD4+ T cells

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e2999 ◽  
Author(s):  
Kosuke Okada ◽  
Takeki Fujimura ◽  
Takeshi Kikuchi ◽  
Makoto Aino ◽  
Yosuke Kamiya ◽  
...  
Keyword(s):  
T Cells ◽  
Mrna Expression ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Quantitative Polymerase Chain Reaction ◽  
Orphan Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Controls ◽  
Cytokine Cocktail

Background Interleukin (IL)-17 produced by mainly T helper 17 (Th17) cells may play an important destructive role in chronic periodontitis (CP). Thus, anti-inflammatory cytokines, such as IL-35, might have a beneficial effect in periodontitis by inhibiting differentiation of Th17 cells. Th17 differentiation is regulated by the retinoic acid receptor-related orphan receptor (ROR) α (encoded by RORA) and RORγt (encoded by RORC). However, the role of IL-35 in periodontitis is not clear and the effect of IL-35 on the function of Th17 cells is still incompletely understood. Therefore, we investigated the effects of IL-35 on Th17 cells. Methods Peripheral blood mononuclear cells (PBMCs) were sampled from three healthy volunteers and three CP patients and were analyzed by flow cytometry for T cell population. Th17 cells differentiated by a cytokine cocktail (recombinant transforming growth factor-β, rIL-6, rIL-1β, anti-interferon (IFN)-γ, anti-IL-2 and anti-IL-4) from PBMCs were cultured with or without rIL-35. IL17A (which usually refers to IL-17), RORA and RORCmRNA expression was analyzed by quantitative polymerase chain reaction, and IL-17A production was determined by enzyme-linked immunosorbent assay. Results The proportion of IL-17A+CD4+ slightly increased in CP patients compared with healthy controls, however, there were no significant differences in the percentage of IL-17A+CD4+ as well as IFN-γ+CD4+ and Foxp3+CD4+ T cells between healthy controls and CP patients. IL17A, RORA and RORC mRNA expression was significantly increased in Th17 cells induced by the cytokine cocktail, and the induction was significantly inhibited by addition of rIL-35 (1 ng/mL). IL-17A production in Th17 cells was significantly inhibited by rIL-35 addition (1 ng/mL). Discussion The present study suggests that IL-35 could directly suppress IL-17 expression via RORα and RORγt inhibition and might play an important role in inflammatory diseases such as periodontitis.

scholarly journals The role of CXCR3 and its ligands expression in Brucellar spondylitis

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Xin Hu ◽  
Xiaoqian Shang ◽  
Liang Wang ◽  
Jiahui Fan ◽  
Yue Wang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Inflammatory Infiltration ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Inflammatory Damage ◽  
Ifn Γ ◽  
Mrna Expression Levels

Abstract Aim Brucellar spondylitis (BS) is one of the most serious complications of brucellosis. CXCR3 is closely related to the severity of disease infection. This research aimed to study the degree of BS inflammatory damage through analyzing the expression levels of CXCR3 and its ligands (CXCL9 and CXCL10) in patients with BS. Methods A total of 29 BS patients and 15 healthy controls were enrolled. Real-Time PCR was used to detect the mRNA expression levels of IFN-γ, CXCR3, CXCL9 and CXCL10 in peripheral blood mononuclear cells (PBMCs) of BS patients and healthy controls. Hematoxylin-Eosin staining was used to show the pathological changes in BS lesion tissues. Immunohistochemistry staining was used to show the protein expression levels of Brucella-Ab, IFN-γ, CXCR3, CXCL9 and CXCL10 in BS lesion tissues. At the same time, ELISA was used to detect the serum levels of IFN-γ, CXCL9 CXCL10 and autoantibodies against CXCR3 in patients with BS. Results In lesion tissue of BS patients, it showed necrosis of cartilage, acute or chronic inflammatory infiltration. Brucella-Ab protein was abundantly expressed in close lesion tissue. And the protein expression levels of IFN-γ, CXCR3 and CXCL10 were highly expressed in close lesion tissue and serum of BS patients. At the same time, the mRNA expression levels of IFN-γ, CXCR3 and CXCL10 in PBMCs of BS patients were significantly higher than those in controls. Conclusion In our research, the expression levels of IFN-γ, CXCR3 and its ligands were significantly higher than those in controls. It suggested that high expression levels of IFN-γ, CXCR3 and its ligands indicated a serious inflammatory damage in patients with BS.

scholarly journals 2109

2017 ◽  
Vol 1 (S1) ◽  
pp. 1-1
Author(s):  
Stephanie Davis ◽  
Jeffrey Huang
Keyword(s):  
Mononuclear Cells ◽  
Age Of Onset ◽  
Quantitative Polymerase Chain Reaction ◽  
Study Groups ◽  
Interleukin 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peripheral Blood Mononuclear ◽  
Repair Capacity ◽  
Relapsing Remitting ◽  
Inflammatory Profile

OBJECTIVES/SPECIFIC AIMS: The overall objective of this proposal is to establish and modulate the inflammatory profile of individuals across the spectrum of multiple sclerosis (MS), with a focus on determining the potential of interleukin 4-induced protein 1 (IL4I1) as a possible marker of progression and modulator of inflammation in human blood samples. METHODS/STUDY POPULATION: The proposed experimental approach involves isolating plasma and peripheral blood mononuclear cells (PBMCs) from individuals across the spectrum of MS phenotypes, and analyzing these samples primarily by quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) methods. Specifically, study groups include: (1) actively relapsing-remitting MS (a-RRMS), (2) non-actively relapsing-remitting MS (n-RRMS), (3) non-active secondary-progressive MS (SPMS), (4) other autoimmune diseases (OAD), (5) healthy controls (HC). RESULTS/ANTICIPATED RESULTS: We expect that IL4I1 treatment increases regulatory cytokine (eg, IL10, TGFb) expression while decreasing Th1 and Th17-derived cytokines (IFNg, IL17), as well as increasing relative composition of regulatory cells (Th2, Treg, M2) as compared with Th1, Th17, M1 (aim 1). Preliminary data on healthy control cells support this prediction. Our central hypothesis is that IL4I1 level indicates the body’s ability to repair itself. As such, we anticipate that all MS groups are deficient in IL4I1, to varying degrees, such that HC>n-RRMS>a-RRMS>SPMS. HC have full repair capacity. RRMS>SPMS as remission indicates existent repair capacity, which is lost in SPMS. n-RRMS>a-RRMS since both, as RRMS, capable of repair response, but a-RRMS triggered this response more recently in response to more recent relapse. In all groups, we expect IL4I-treatment to mitigate inflammation (aim 2). Finally, we expect that H2O2 production by IL4I1 is a key player in IL4I1 function, and that H2O2 will preferentially induce oxidative stress to pro-inflammatory subsets of PBCMs (aim 3). DISCUSSION/SIGNIFICANCE OF IMPACT: MS is a chronic inflammatory neurodegenerative disease of the central nervous system that, with an average age of onset of 34, afflicts over 2.3 million individuals worldwide during many of the most productive years of their lives. The pathogenesis of MS, which involves autoimmune destruction of myelin, is poorly understood. Accurate biomarkers, which could predict disease progression, are yet to be identified and would provide valuable information to patients and their treating clinicians. Likewise, effective treatments are few and in high demand. IL4I1 is a promising candidate for both roles.

scholarly journals A potential inflammatory role of IL-31 in psoriatic arthritis: A correlation with Th17 cytokine profile

2020 ◽  
Vol 34 ◽  
pp. 205873842090718
Author(s):  
LA Bautista-Herrera ◽  
U De la Cruz-Mosso ◽  
IV Román-Fernández ◽  
I Parra-Rojas ◽  
JG Soñanez-Organis ◽  
...  
Keyword(s):  
Gene Expression ◽  
Psoriatic Arthritis ◽  
Mrna Expression ◽  
Close Association ◽  
Quantitative Polymerase Chain Reaction ◽  
Serum Levels ◽  
Cytokine Profile ◽  
Orphan Receptor ◽  
Cross Sectional ◽  
Th17 Cytokine

The goals of our study were to determine the possible association of interleukin (IL)-31 with Th17 cytokine profile in serum and to quantify retinoic acid-related orphan receptor C ( RORC) mRNA expression in psoriatic arthritis (PsA) patients. This cross-sectional study was conducted in 50 patients with PsA and 30 control subjects (CS) matched by age and gender. The cytokine serum levels were quantified by magnetic bead–based assay using the Bio-Plex MAGPIX system, and RORC mRNA expression was determined by quantitative polymerase chain reaction (qPCR). As a result, significant differences in IL-31 were observed between study groups (77.23 pg/mL in PsA vs 64.4 pg/mL in CS, P < 0.001) and Th17 cytokine profile serum levels (IL-17A: 6.36 pg/mL in PsA vs 2.97 pg/mL in CS, P = 0.02; IL-17F: 44.15 pg/mL in PsA vs 23.36 pg/mL in PsA, P = 0.01; IL-17E: 3.03 pg/mL in PsA vs 0.82 pg/mL in CS, P < 0.001; IL-21: 36.45 pg/mL in PsA vs 12.44 pg/mL in CS, P = 0.02); however, significant differences were not observed for IL-23 (31.2 pg/mL in PsA vs 53.26 pg/mL in CS, P = 0.58). Furthermore, positive correlations between IL-31 and Th17 cytokine profile serum levels were found (IL-17A: rs = 0.64, P < 0.001; IL-17F: rs = 0.73, P < 0.001; IL-17E: rs = 0.70, P < 0.001; IL-21: rs = 0.54, P = 0.002; IL-23: rs = 0.5, P < 0.01). Regarding RORC gene expression, the PsA group showed an increase of 6.85-fold compared to the CS group. We did not find any association between the serum levels of cytokines and RORC gene expression. In conclusion, in PsA, there are increased serum levels of IL-31, IL-17A, IL-17F, IL-17E, and IL-21, but not IL-23. Moreover, there was a positive correlation of IL-31 with the Th17 cytokine profile and a high RORC gene expression. Altogether, these findings suggest a proinflammatory contribution of IL-31 in close association with the Th17 cytokine profile in PsA.

scholarly journals Constitutive Changes in Circulating Follicular Helper T Cells and Their Subsets in Patients with Graves’ Disease

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Yan Liu ◽  
Xinwang Yuan ◽  
Xiaofang Li ◽  
Dawei Cui ◽  
Jue Xie
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
Mrna Expression ◽  
Mononuclear Cells ◽  
Plasma Concentrations ◽  
Enzyme Linked Immunosorbent Assay ◽  
Graves Disease ◽  
Mrna Levels ◽  
Tfh Cells ◽  
Follicular Helper

Background. Follicular helper T (Tfh) cells are critical for high-affinity antibody generation and B cell maturation and differentiation, which play important roles in autoimmune diseases. Graves’ disease (GD) is one prototype of common organ-specific autoimmune thyroid diseases (AITD) characterized by autoreactive antibodies, suggesting a possible role for Tfh cells in the pathogenesis of GD. Our objective was to explore the role of circulating Tfh cell subsets and associated plasma cells (PCs) in patients with GD. Methods. Thirty-six patients with GD and 20 healthy controls (HC) were enrolled in this study. The frequencies of circulating Tfh cell subsets and PCs were determined by flow cytometry, and plasma cytokines, including interleukin- (IL-) 21, IL-4, IL-17A, and interferon- (IFN-) γ, were measured using an enzyme-linked immunosorbent assay (ELISA). The mRNA expression of transcription factors (Bcl-6, T-bet, GATA-3, and RORγt) in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time quantitative PCR. Results. Compared with HC, the frequencies of circulating CD4+CXCR5+CD45RA−Tfh (cTfh) cells with ICOS and PD-1 expression, the Tfh2 subset (CXCR3−CCR6−Tfh) cells, and PCs (CD19+CD27highCD38high) were significantly increased in the GD patients, but the frequencies of Tfh1 (CXCR3+CCR6−Tfh) and Tfh17 (CXCR3−CCR6+Tfh) subset cells among CD4+T cells were significantly decreased in GD patients. The plasma concentrations of IL-21, IL-4, and IL-17A were elevated in GD patients. Additionally, a positive correlation was found between the frequency of PD-1+Tfh cells (Tfh2 or PCs) and plasma IL-21 concentration (or serum TPO-Ab levels). The mRNA levels of transcription factors (GATA-3 and RORγt) were significantly increased, but T-bet and Bcl-6 mRNA expression was not obviously varied in PBMCs from GD patients. Interestingly, Tfh cell subsets and PCs from GD patients were partly normalized by treatment. Conclusion. Circulating Tfh cell subsets and PCs might play an important role in the pathogenesis of GD, which are potential clues for GD patients’ interventions.

scholarly journals Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on T Cell Differentiation in Primary Biliary Cholangitis

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Chunhui She ◽  
Jing Wang ◽  
Ning Tang ◽  
Zhaoyang Liu ◽  
Lishan Xu ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Autoimmune Disease ◽  
Mononuclear Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
T Cell Differentiation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Primary Biliary Cholangitis ◽  
Tetrachlorodibenzo P Dioxin

Exposure to dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is reported to affect the autoimmune system and increase the risk of autoimmune disease. Generally, dioxin exerts its toxicity via aryl hydrocarbon receptor (AhR). Primary biliary cholangitis (PBC) is a chronic autoimmune disease, and its pathogenesis involves the interplay between immune and environmental factors. This study showed the effect of dendritic cells (DCs) activated by TCDD on naïve CD4+ T cell differentiation in patients with PBC. CD14+ mononuclear cells were isolated from peripheral blood mononuclear cells (PBMCs) of patients with PBC and healthy people by magnetic cell separation and introduced into DCs. Two days after stimulation by TCDD, DCs were cocultured with naïve CD4+ T cells in a ratio of 1 : 2 for 3 days. Then, differentiation-related factors for naïve CD4+ T cells were detected by real-time fluorescence quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, and flow cytometry. The results showed that TCDD-activated DCs could promote Th1 and Th17 differentiation in patients with PBC. Therefore, this study demonstrated TCDD as an AhR agonist in regulating naïve CD4+ T cell differentiation in patients with PBC.

scholarly journals Development and Evaluation of a Real-Time Reverse Transcription-PCR Assay for Quantification of Gamma Interferon mRNA To Diagnose Tuberculosis in Multiple Animal Species

2007 ◽  
Vol 14 (12) ◽  
pp. 1563-1571 ◽  
Author(s):  
Noel P. Harrington ◽  
Om P. Surujballi ◽  
W. Ray Waters ◽  
John F. Prescott
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Reverse Transcription ◽  
Skin Testing ◽  
Gamma Interferon ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sequence Information ◽  
Tuberculin Skin Testing ◽  
Reverse Transcription Pcr ◽  
Ifn Γ

ABSTRACT Tuberculosis of free-ranging and captive wildlife, including species implicated in the maintenance and transmission of Mycobacterium bovis, is a difficult disease to diagnose and control. Historically, diagnosis of tuberculosis has relied largely upon assays of cell-mediated immunity (CMI), such as tuberculin skin testing. This approach, however, is problematic or impractical for use with many wildlife species. Increasingly, in vitro diagnostic tests, including gamma interferon (IFN-γ)-based assays, are replacing or complementing skin testing of cattle and humans. Analogous assays are unavailable for most wildlife because of a lack of species-specific immunological reagents. This report describes the development and validation of a whole-blood assay to quantify antigen-specific IFN-γ mRNA expression by quantitative real-time reverse transcription-PCR. Oligonucleotide primers and probes were designed and tested for reactivity towards several susceptible species of interest with respect to tuberculosis infection. The assay was subsequently optimized to quantify the IFN-γ mRNA expression in elk and red deer (Cervus elaphus) and was evaluated for its ability to detect mycobacterial antigen-specific responses of experimentally tuberculosis-infected animals. The assay was a simple, rapid, and sensitive measure of antigen-specific CMI. The IFN-γ mRNA responses correlated well with IFN-γ protein production and showed performance in determining an animal's infection status superior to that of either lymphocyte proliferation or IFN-γ protein enzyme-linked immunosorbent assay methods. An additional advantage is the ease with which the assay can be modified to reliably quantify IFN-γ expression by using consensus sequences of closely related species or of other species for which IFN-γ sequence information is available.

Superantigen immune stimulation activates epithelial STAT-1 and PI 3-K: PI 3-K regulation of permeability

2000 ◽  
Vol 279 (5) ◽  
pp. G1094-G1103 ◽  
Author(s):  
Derek M. McKay ◽  
Fernando Botelho ◽  
Peter J. M. Ceponis ◽  
Carl D. Richards
Keyword(s):  
Epithelial Cells ◽  
Conditioned Medium ◽  
Intracellular Signaling ◽  
Mononuclear Cells ◽  
Interferon Γ ◽  
Transepithelial Resistance ◽  
Mobility Shift ◽  
Peripheral Blood Mononuclear ◽  
Ussing Chambers ◽  
Ifn Γ

Signal transducers and activators of transcription (STATs) are critical intracellular signaling molecules for many cytokines. We compared the ability of T84 epithelial cells to activate STATs in response to cytokines [interferon-γ (IFN-γ), interleukin (IL)-4, IL-10, and tumor necrosis factor-α (10 ng/ml)] and conditioned medium from superantigen [ Staphylococcus aureus enterotoxin B (SEB)]-activated peripheral blood mononuclear cells (PBMC) using electrophoretic mobility shift assays (EMSA). Of the cytokines tested, only IFN-γ caused a STAT-1 response. Exposure to SEB-PBMC-conditioned medium resulted in STAT-1 or STAT-1/3 activation, and inclusion of anti-IFN-γ antibodies in the conditioned medium abolished the STAT-1 signal. Cells treated with transcription factor decoys, DNA oligonucleotides bearing the STAT-1 recognition motif, and then SEB-PBMC-conditioned medium displayed a reduced STAT-1 signal on EMSA, yet this treatment did not prevent the drop in transepithelial resistance (measured in Ussing chambers) caused by SEB-PBMC-conditioned medium. In contrast, the phosphatidylinositol 3′-kinase (PI 3-K) inhibitor LY-294002 significantly reduced the drop in transepithelial resistance caused by SEB-PBMC-conditioned medium. Thus data are presented showing STAT-1 (±STAT-3) and PI 3-K activation in epithelial cells in response to immune mediators released by superantigen immune activation. Although the involvement of STAT-1/-3 in the control of barrier function remains a possibility, PI-3K has been identified as a regulator of T84 paracellular permeability.

Environmentally Relevant Level of Aflatoxin B1 Dysregulates Human Dendritic Cells Through Signaling on Key Toll-Like Receptors

2014 ◽  
Vol 33 (3) ◽  
pp. 175-186 ◽  
Author(s):  
Azam Mohammadi ◽  
Jalil Mehrzad ◽  
Mahmoud Mahmoudi ◽  
Marion Schneider
Keyword(s):  
Dendritic Cells ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Immune Surveillance ◽  
Toll Like Receptors ◽  
Myeloid Dendritic Cells ◽  
Protein Levels ◽  
Tlr4 Mrna ◽  
Relevant Level

Aflatoxins (AFs) are highly hazardous fungal biometabolites usually present in feeds and foods. Aflatoxin B1 (AFB1) is the most toxic and a known carcinogen. Toll-like receptors (TLRs), highly expressed by myeloid dendritic cells (DC), are key innate immune-surveillance molecules. Toll-like receptors not only sense pathogen-associated molecular patterns but also contribute to infections and cancer. To assess AFB1–TLR interactions on human myeloid DC, pure CD11c+ DC were generated from monocytes isolated from healthy individuals and then exposed to relevant level of AFB1 for 2 hours. Both quantitative polymerase chain reaction and flow cytometric assays were used to quantify, respectively, expression of TLR2 and TLR4 at the messenger RNA (mRNA) and protein levels in these DC. Levels of interleukin (IL) 1β, IL-6, and IL-10 were also analyzed in AFB1- and mock-treated DC. Compared to nontreated CD11c+ DC, expression levels of both TLR2 and TLR4 mRNA and proteins were significantly upregulated in AFB1-treated cells. Further, although IL-10 levels in AFB1-treated DC were similar to those in the mock-treated DC, the AFB1-exposed DC secreted higher amounts of IL-1β and IL-6. Dendritic cells are sensitive to environmentally relevant level of AFB1, and TLR2 and TLR4 are involved in sensing AFB1. Considering the broad roles of TLR2, TLR4, and DC in immunity and infections, our novel findings open a new door to understanding the molecular mechanisms and functional consequences of AFB1 in inducing immunodysregulation, immunotoxicity, and thus (non)infectious diseases in humans.

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