10 research outputs found
Sulforaphane Inhibits Inflammatory Responses of Primary Human T-Cells by Increasing ROS and Depleting Glutathione
The activity and function of T-cells are influenced by the intra- and extracellular redox milieu. Oxidative stress induces hypo responsiveness of untransformed T-cells. Vice versa increased glutathione (GSH) levels or decreased levels of reactive oxygen species (ROS) prime T-cell metabolism for inflammation, e.g., in rheumatoid arthritis. Therefore, balancing the T-cell redox milieu may represent a promising new option for therapeutic immune modulation. Here we show that sulforaphane (SFN), a compound derived from plants of the Brassicaceae family, e.g., broccoli, induces a pro-oxidative state in untransformed human T-cells of healthy donors or RA patients. This manifested as an increase of intracellular ROS and a marked decrease of GSH. Consistently, increased global cysteine sulfenylation was detected. Importantly, a major target for SFN-mediated protein oxidation was STAT3, a transcription factor involved in the regulation of TH17-related genes. Accordingly, SFN significantly inhibited the activation of untransformed human T-cells derived from healthy donors or RA patients, and downregulated the expression of the transcription factor RORγt, and the TH17-related cytokines IL-17A, IL-17F, and IL-22, which play a major role within the pathophysiology of many chronic inflammatory/autoimmune diseases. The inhibitory effects of SFN could be abolished by exogenously supplied GSH and by the GSH replenishing antioxidant N-acetylcysteine (NAC). Together, our study provides mechanistic insights into the mode of action of the natural substance SFN. It specifically exerts TH17 prone immunosuppressive effects on untransformed human T-cells by decreasing GSH and accumulation of ROS. Thus, SFN may offer novel clinical options for the treatment of TH17 related chronic inflammatory/autoimmune diseases such as rheumatoid arthritis
Metastasis of prostate cancer and melanoma cells in a preclinical in vivo mouse model is enhanced by L-plastin expression and phosphorylation
BACKGROUND: Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. METHODS: We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. RESULTS: Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. CONCLUSIONS: Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable
A reducing milieu renders cofilin insensitive to phosphatidylinositol 4,5-bisphosphate (PIP2) inhibition
Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP(2)- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP(2). Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP(2) inhibition, resulting in enhanced actin dynamics
The actin remodeling protein cofilin is crucial for thymic αβ but not γδ T-cell development
<div><p>Cofilin is an essential actin remodeling protein promoting depolymerization and severing of actin filaments. To address the relevance of cofilin for the development and function of T cells in vivo, we generated knock-in mice in which T-cell–specific nonfunctional (nf) cofilin was expressed instead of wild-type (WT) cofilin. Nf cofilin mice lacked peripheral αβ T cells and showed a severe thymus atrophy. This was caused by an early developmental arrest of thymocytes at the double negative (DN) stage. Importantly, even though DN thymocytes expressed the TCRβ chain intracellularly, they completely lacked TCRβ surface expression. In contrast, nf cofilin mice possessed normal numbers of γδ T cells. Their functionality was confirmed in the γδ T-cell–driven, imiquimod (IMQ)-induced, psoriasis-like murine model. Overall, this study not only highlights the importance of cofilin for early αβ T-cell development but also shows for the first time that an actin-binding protein is differentially involved in αβ versus γδ T-cell development.</p></div
DN thymocytes of Cfl1<sup>nf/nf</sup> mice show a dramatically enhanced F-actin content and impaired migratory capacity as well as a lack of TCRβ surface expression.
<p>(A) CD3<sup>+</sup> splenocytes were analyzed for expression of TCRβ and TCRγδ. Shown are representative dot blots (left panels) and calculated absolute cell numbers (right panel) of TCRβ and TCRγδ expressing cells. Data is represented as mean ± SEM and summarizes 3 independent experiments with a total of 6 mice per group. (B) Total F-actin amount of DN thymocytes or γδ thymocytes was determined by SiR-actin staining (<i>n</i> = 3 independent experiments with a total ≥6 mice per group). (C) Migratory capacity of DN cells or γδ thymocytes was determined in a transwell assay (pore size 5 μm) in which SDF-1α (200 ng/ml) was used as chemotactic stimulus. Migration was carried out for 3 h (<i>n</i> = 3 independent experiments with ≥4 mice per group). (D) TCRβ surface (surface TCRβ) and intracellular (ic TCRβ) expression was analyzed in DN cells by flow cytometry. Representative dot plots from TCRβ versus TCRγδ staining on B6 and Cfl1<sup>nf/nf</sup> DN cells are shown (<i>n</i> = 4 independent experiments with a total of ≥7 mice per group). (E) Analysis of surface and ic expression of TCRβ in DN cells of B6 (grey bar) and Cfl1<sup>nf/nf</sup> mice (black bar) (left bar chart). Analysis of MFI of TCRβ of icTCRβ<sup>+</sup> DN cells of B6 (grey bar) and Cfl1<sup>nf/nf</sup> mice (black bar) (right bar chart). (F) Analysis of surface expression of TCRβ in DN thymocytes of B6 and Cfl1<sup>nf/nf</sup> mice before (-cytoD) and after cytochalasin D treatment (+cytoD). Data is represented as mean ± SEM. **** <i>p</i> < 0.0001; *** <i>p</i> < 0.001; ** <i>p</i> < 0.01; * <i>p</i> < 0.05; Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005380#pbio.2005380.s006" target="_blank">S1 Data</a>. cytoD, cytochalasin D; ic, intracellular; MFI, mean fluorescence intensity nf, nonfunctional; ns, not significant.</p
Mice expressing nf cofilin show a severe thymus atrophy and a developmental arrest at the DN3 stage.
<p>(A) Total T-cell number in spleen of B6 mice and nf cofilin knock-in mice (<i>n</i> = 6 independent experiments with a total of ≥8 mice per group). (B) Thymus was isolated and weighed, and the total cell number was determined from 4–5-weeks-old B6 or nf cofilin knock-in mice (<i>n</i> = 6 independent experiments with a total of ≥10 mice per group). (C) Flow cytometric analysis of thymocyte differentiation by CD4, CD8, CD25, and CD44 staining (<i>n</i> ≥ 8 mice per group). Exemplary dot blots from representative mice are shown on the left, whereas the statistical evaluation of summary data is shown in the middle (for DN, DP, and SP stages) and on the right (for DN cell stages). (D) Creation of mixed bone marrow chimera. Lethally irradiated B6 mice were reconstituted with equal numbers of CD3<sup>+</sup> cell–depleted BM cells from CD45.2<sup>+</sup> tester (Cfl1<sup>nf/nf</sup>) and CD45.1<sup>+</sup> competitor (B6) mice. Total chimerism was measured and CD4 versus CD8 plots show the developmental stage of thymocytes derived from CD45.1<sup>+</sup> or CD45.2<sup>+</sup> BM cells. Plots are representative of six mixed chimeras per group. Bar graphs show the average abundance of each major thymocyte population within the chimera from both tester (CD45.2<sup>+</sup>) and competitor (CD45.1<sup>+</sup>) donor cells. Data is represented as mean ± SEM. **** = p<0.0001; ** = p<0.01. Underlying Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005380#pbio.2005380.s006" target="_blank">S1 Data</a>. BM, bone marrow; DN, double negative; DP, double positive; SP, single positive.</p
Cfl1<sup>nf/nf</sup> mice show normal γδ T-cell subsets which remain functional.
<p>(A) Analysis of the surface expression of Vγ1, Vγ2, and Vγ3 of γδ T-cells isolated from the spleen, skin, lung, and thymus of B6 (grey bar) or Cfl1<sup>nf/nf</sup> mice (black bar) (<i>n</i> = 4 independent experiments with ≥4 mice per group). (B) Analysis of the surface expression of CD24, CD27, and CD44 of γδ thymocytes of B6 (grey bar) or Cfl1<sup>nf/nf</sup> mice (black bar) (<i>n</i> = 4 independent experiments with ≥4 mice per group). (C) In vitro activation of splenic γδ T cells of Cfl1<sup>nf/nf</sup> (black bar) and control mice (grey bar) by plate-bound CD3 and CD28 antibodies for 24 h. Determination of the T-cell activation markers CD25 (left bar chart) and CD69 (right bar chart) by flow cytometry. (D) Age- and sex-matched Cfl1<sup>nf/nf</sup> (red line) and Cfl1<sup>+/+</sup> (black line) mice at 7 weeks of age were used for an IMQ-induced psoriasis-like model. Over 6 days, prior to topical application of IMQ, scores of individual parameters such as scaling, back skin thickness, and erythema formation were measured and the accumulated PASI was calculated. (E) Flow cytometric analysis of IL-17A and RORγt expression in γδ T cells of skin-draining LNs. Cytokine production was assessed after 6 days of topical application of IMQ containing Aldara crème (Sham) or control crème (Aldara) (experiment with ≥4 mice per group). Data are represented as mean ± SEM. **** <i>p</i> < 0.0001; *** <i>p</i> < 0.001; ** <i>p</i> < 0.01; * <i>p</i> < 0.05; Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005380#pbio.2005380.s006" target="_blank">S1 Data</a>. Cfl1, cofilin-1; IMQ, imiquimod; ns, not significant; PASI, psoriasis area severity index.</p