Protein translocation and retro-translocation across the endoplasmic reticulum are crucial to inflammatory effector CD4(+) T cell function

Effector CD4(+) T cells can be classified by the cytokines they secrete, with T helper 1 (Th1) cells generating interferon (IFN)gamma and Th17 cells secreting interleukin (IL)-17. Both Th1 and Th17 cells are strongly implicated in the initiation and chronicity of autoimmune diseases such as multiple sclerosis. The endoplasmic reticulum (ER) has been implicated as a potentially crucial site in regulating CD4(+) T cell function. Secretory and transmembrane proteins are shuttled into the ER via the Sec61 translocon, where they undergo appropriate folding; misfolded proteins are retro-translocated from the ER in a p97-dependent manner. Here, we provide evidence that both processes are crucial to the secretion of inflammatory cytokines from effector CD4(+) T cells. The pan-ER inhibitor eeeyarestatin-1 (ESI), which interferes with both Sec61 translocation and p97 retro-translocation, inhibited secretion of interferon (IFN)gamma, interleukin (IL)-2 and tumor necrosis factor (TNF)alpha from Th1 cells in a dose-dependent manner. Selective inhibition of Sec61 by Apratoxin A (ApraA) revealed that ER translocation is crucial for Th1 cytokine secretion, while inhibition of p97 by NMS-873 also inhibited Th1 function, albeit to a lesser degree. By contrast, none of ESI, ApraA or NMS-873 could significantly reduce IL-17 secretion from Th17 cells. ApraA, but not NMS-873, reduced phosphorylation ApraA had modest effects on activation of the Th17 transcription factor Stat3, while NMS-873 had no effect. Interestingly, NMS-873 was able to reduce disease severity in CD4(+) T cell-driven experimental autoimmune encephalomyelitis (EAE). Together, our data indicate that CD4(+) T cell function, and Th1 cell function in particular, is dependent on protein translocation and dislocation across the ER.Peer reviewe

Dimethyl Sulfoxide Induces Both Direct and Indirect Tau Hyperphosphorylation

Dimethyl sulfoxide (DMSO) is widely used as a solvent or vehicle for biological studies, and for treatment of specific disorders, including traumatic brain injury and several forms of amyloidosis. As Alzheimer’s disease (AD) brains are characterized by deposits of β-amyloid peptides, it has been suggested that DMSO could be used as a treatment for this devastating disease. AD brains are also characterized by aggregates of hyperphosphorylated tau protein, but the effect of DMSO on tau phosphorylation is unknown. We thus investigated the impact of DMSO on tau phosphorylation in vitro and in vivo. One hour following intraperitoneal administration of 1 or 2 ml/kg DMSO in mice, no change was observed in tau phosphorylation. However, at 4 ml/kg, tau was hyperphosphorylated at AT8 (Ser202/Thr205), PHF-1 (Ser396/Ser404) and AT180 (Thr231) epitopes. At this dose, we also noticed that the animals were hypothermic. When the mice were maintained normothermic, the effect of 4 ml/kg DMSO on tau hyperphosphorylation was prevented. On the other hand, in SH-SY5Y cells, 0.1% DMSO induced tau hyperphosphorylation at AT8 and AT180 phosphoepitopes in normothermic conditions. Globally, these findings demonstrate that DMSO can induce tau hyperphosphorylation indirectly via hypothermia in vivo, and directly in vitro. These data should caution researchers working with DMSO as it can induce artifactual results both in vivo and in vitro

Perturbations de la transmission dopaminergique chez les souris présentant une réduction de nurr1

Le facteur de transcription Nurrl est un récepteur nucléaire orphelin hautement impliqué dans le développement du système dopaminergique. Son expression persistante à l'âge adulte soulève toutefois de nombreuses questions quant à son rôle exact dans le cerveau mature. L'objectif de cette étude était d'évaluer les effets d'une diminution partielle du facteur de transcription Nurrl sur la transmission dopaminergique chez des souris adultes. Nous avons d'abord observé qu'une réduction partielle de Nurrl n'influence pas le comportement locomoteur des souris en conditions basales. Par contre, l'administration aiguë d'amphétamine chez les souris Nurrl (+/-) induit une brève augmentation de l'activité locomotrice précédant l'apparition marquée de mouvements verticaux et stéréotypés. La modulation de différents marqueurs tels que Nurrl, Nur77, Nor-1 et l'ENK fut également étudiée. De façon générale, nos résultats démontrent des modifications plus ou moins importantes de l'expression de ce neuropeptide et de ces récepteurs nucléaires en présence ou non du psychostimulant. Enfin, l'ensemble de ces résultats suggèrent qu'une réduction partielle de Nurrl induit des changements importants dans la transmission dopaminergiqu

Interferon-β suppresses murine Th1 cell function in the absence of antigen-presenting cells.

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals

IFN-β suppresses Th1 cell proliferation.

<p>A. WT CD4⁺CD62L^hi T cells were labeled with CFSE and stimulated for 5 days with either soluble anti-CD3 plus irradiated splenocytes (APCs), or with plate-bound anti-CD3+anti-CD28, under Th1 or Th17 conditions, with the indicated concentrations of IFN-β. CFSE dilution was assessed by flow cytometry. Data representative of three experiments. B. WT or <i>IFNAR1-/-</i> CD4⁺CD62L^hi T cells were labeled with CFSE and stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions with the indicated concentrations of IFN-β for 5 days. CFSE dilution was assessed by flow cytometry. Data representative of two experiments. Gates represent the percentage of cells that underwent at least one division.</p

IFN-β suppresses the encephalitogenic potential of Th1 cells.

<p>CD4⁺CD62L^hi T cells were isolated from female 2D2 mouse spleens and lymph nodes, and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, in the presence or absence of 100 U mL^-1 of IFN-β, for 5 days. They were then transferred to female, 6-week old, <i>Rag1-/-</i> mice (5x10⁶ cells/mouse). Recipient mice were monitored for clinical signs of EAE. n = 5 Th1, n = 4 Th1+IFN-β. Right graph, linear regression curves of the disease courses. The slopes are significantly different between the disease courses (p<0.0006). B. Brains and spinal cords were isolated from mice in (A) at d32 and single mononuclear cell suspensions were obtained. The frequency of CNS-infiltrating CD4⁺ cells was assessed by flow cytometry. * p<0.05, two-tailed Student’s <i>t</i> test.</p

IFN-β regulates Stat1 and Stat4 expression on Th cells in the absence of APCs.

<p>CD4⁺CD62L^hiCD25^- T cells were sorted from the spleens and lymph nodes of C57BL6/J mice. They were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 or Th17 conditions, with the indicated concentrations of IFN-β, for 48 hours, and cell lysates were generated. A. Expression of Stat1 and pStat1(Y701) were assessed by Western blot. Representative of two experiments. B. Expression of Stat4 and pStat4(Y693) were assessed by Western blot. Representative of three experiments. GAPDH, loading control.</p

IFN-β suppresses cytokine secretion from Th1 cells.

<p>A. CD4⁺CD62L^hi T cells were sorted from the spleens and lymph nodes of C57BL6/J mice and were stimulated with plate-bound anti-CD3+anti-CD28 under Th1 conditions, with the indicated concentrations of IFN-β, for 5 days. Cells were restimulated for 48 hours under Th1 conditions with the same concentration of IFN-β as at the initial stimulation. A. Generation of IFN-γ and IL-2 was assessed by intracellular cytokine staining and flow cytometry. Representative of three experiments. B. Expression of Tim-3 and PD-1 were assessed by flow cytometry. Representative of three experiments. C. Expression of T-bet was assessed by flow cytometry. Representative data from one of three mice assessed individually. Solid line with open histogram, Th1; dashed line with open histogram, Th1 + 10 U mL^-1 IFN-β; dotted line with open histogram, Th1 + 100 U mL^-1 IFN-β; shaded histogram, FMO control. Data in all panels gated on CD4⁺ cells.</p

Th1 cells express the type I interferon receptor.

<p>A. Naïve (CD62L^hi) and effector (CD62L^lo) CD4⁺ and CD8⁺ C57BL/6J T cells were assessed for surface expression of Ifnar1 by flow cytometry. Open histogram, Ifnar1; shaded histogram, fluorescence minus one (FMO) control. B. Th1 and Th17 cells were cultured and assessed for surface expression of Ifnar1 after 5 days. Solid line with open histogram, Th1; Dotted line with open histogram, Th17; shaded histogram, FMO control. Data representative of one of three experiments.</p

Effect of DMSO on tau phosphorylation in 3R human tau transfected SH-SY5Y cells following 1 h exposure.

<p>Cell lysate protein extracts were separated by SDS-PAGE and levels of tau phosphorylation were determined using antibodies directed at the (A) AT8, (B) PHF-1, or (C) AT180 phosphoepitopes, and (D) total tau. Relative immunoreactive band intensities are expressed as a percent of control from a total Tau ratio and are displayed for each phosphoepitope and total tau. For each condition, 2 representative data are displayed with control (n  = 9) and 0.1% DMSO (n  = 9). Data are expressed as mean ± SD. *, ** denote <i>p</i><0.05 and <i>p</i><0.01, versus control, respectively; Student’s t test.</p