Therapeutic and Adverse Effects of a Non-Steroidal Glucocorticoid Receptor Ligand in a Mouse Model of Multiple Sclerosis

-methyl-ethylammonium chloride (CpdA), a dissociating non-steroidal GR ligand, in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).. Administration of high-dose CpdA to mice was lethal while treatment of EAE with low to intermediate amounts of CpdA dissolved in water significantly ameliorated the disease. The beneficial effect of CpdA required expression of the GR in T cells and was achieved by down regulating LFA-1 and CD44 on peripheral Th cells and by repressing IL-17 production.. Hence, non-steroidal GR ligands require careful analysis prior to their translation into new therapeutic concepts

Glucocorticoid Receptor-Deficient Foxp3+ Regulatory T Cells Fail to Control Experimental Inflammatory Bowel Disease

Activation of the immune system increases systemic adrenal-derived glucocorticoid (GC) levels which downregulate the immune response as part of a negative feedback loop. While CD4+ T cells are essential target cells affected by GC, it is not known whether these hormones exert their major effects on CD4+ helper T cells, CD4+Foxp3+ regulatory T cells (Treg cells), or both. Here, we generated mice with a specific deletion of the glucocorticoid receptor (GR) in Foxp3+ Treg cells. Remarkably, while basal Treg cell characteristics and in vitro suppression capacity were unchanged, Treg cells lacking the GR did not prevent the induction of inflammatory bowel disease in an in vivo mouse model. Under inflammatory conditions, GR-deficient Treg cells acquired Th1-like characteristics and expressed IFN-gamma, but not IL-17, and failed to inhibit pro-inflammatory CD4+ T cell expansion in situ. These findings reveal that the GR is critical for Foxp3+ Treg cell function and suggest that endogenous GC prevent Treg cell plasticity toward a Th1-like Treg cell phenotype in experimental colitis. When equally active in humans, a rationale is provided to develop GC-mimicking therapeutic strategies which specifically target Foxp3+ Treg cells for the treatment of inflammatory bowel disease

Investigation of therapy strategies of multiple sclerosis by using Experimental Autoimmune Encephalomyelitis

No abstract availabl

Acid Sphingomyelinase Is Required for Protection of Effector Memory T Cells against Glucocorticoid-Induced Cell Death

The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs. The Journal of Immunology, 2011, 187: 4509-4516

Single-cell profiling reveals heterogeneity and functional patterning of GPCR expression in the vascular system

G-protein-coupled receptor (GPCR) expression is extensively studied in bulk cDNA, but heterogeneity and functional patterning of GPCR expression in individual vascular cells is poorly understood. Here, we perform a microfluidic-based single-cell GPCR expression analysis in primary smooth muscle cells (SMC) and endothelial cells (EC). GPCR expression is highly heterogeneous in all cell types, which is confirmed in reporter mice, on the protein level and in human cells. Inflammatory activation in murine models of sepsis or atherosclerosis results in characteristic changes in the GPCR repertoire, and we identify functionally relevant subgroups of cells that are characterized by specific GPCR patterns. We further show that dedifferentiating SMC upregulate GPCRs such as Gpr39, Gprc5b, Gprc5c or Gpr124, and that selective targeting of Gprc5b modulates their differentiation state. Taken together, single-cell profiling identifies receptors expressed on pathologically relevant subpopulations and provides a basis for the development of new therapeutic strategies in vascular diseases

A CD28 superagonistic antibody elicits 2 functionally distinct waves of T cell activation in rats

Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function–associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein–coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1–phosphate–directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4+CD25+FoxP3+ regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation

Treatment of MOG_35–55 induced EAE by CpdA.

<p>(A) EAE was induced in C57Bl/6 mice followed by treatment with 15 mg/kg CpdA (dissolved in 20% ethanol), 100 mg/kg Dex or PBS as a control (con) on 3 consecutive days starting at an average clinical score of 2 (marked by arrows); n = 11; statistical analysis: days 11–22. The cross indicates that all animals of this group either died or had to be sacrificed for ethical reasons. The experiment was repeated twice with similar results. (B) CpdA was dissolved in water and therapeutically applied at doses of 5 mg/kg or 1.5 mg/kg for 3 consecutive days after the mice had developed an average clinical score of 3 (marked by arrows); treatment with the solvent alone (con) served as a control; n = 18/6/19; statistical analysis: days 13–23. The experiment was repeated five times with similar results. (C) EAE was induced in C57Bl/6 and GR^lckCre mice followed by treatment with CpdA dissolved in water at a dose of 5 mg/kg or the solvent alone (con). Therapy was started at an average score of 2 (defined as day 0); n = 6; statistical analysis: days 1–10. The experiment was repeated twice with similar results. (D) C57Bl/6 wildtype or GR^lckCre mice were treated with 5 mg/kg or 15 mg/kg CpdA dissolved in water or PBS as a control on 3 consecutive days. On the following morning surviving mice were sacrificed and the cellularity of the spleen was determined by microscopic counting. n = 3/12. **: p<0.01, ***: p<0.001, n.s.: p>0.05.</p

CpdA-induced apoptosis is independent of the GR.

<p>(A,B) Thymocytes were isolated either from GRN^+/+ or GRN^−/− E18.5 embryos and cultured in the absence (con) or presence of different concentrations of Dex (A) or CpdA (B) for 24 hrs. Apoptosis was assessed by flow cytometry based on AnnexinV/7-AAD staining. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3. (C,D) WEHI 7.1 cells stably transduced with a retrovirus encoding a GR-specific shRNA (GR-siRNA), Bcl-2 or the empty retroviral vector LMP, were cultured in the absence (con) or presence of different Dex concentrations (C) or 10⁻⁵ M CpdA (D) for 24 hrs. Apoptosis was assessed by flow cytometry based on AnnexinV/7-AAD staining. Survival of untreated cells was set as 100% for each time point to correct for spontaneous apoptosis. n = 3−5. *: p<0.05, **: p<0.01, n.s.: p>0.05.</p

Mechanism of CpdA action in the treatment of EAE.

<p>(A) EAE was induced in C57Bl/6 mice followed by treatment with 1.5 mg/kg or 5 mg/kg CpdA dissolved in water for 3 consecutive days after the mice had developed an average clinical score of 3. On the following morning the mice were sacrificed and the leukocytes isolated from the spinal cord. Cellularity was determined by microscopic counting; staining for AnnexinV binding and surface expression of LFA-1 on CD3⁺CD4⁺ Th cells was performed by flow cytometry. n = 3−7. (B) The spleen was isolated from the same animals as in panel A and the leukocytes analyzed for AnnexinV binding as well as LFA-1 and CD44 surface expression on CD3⁺CD4⁺ Th cells by flow cytometry. n = 3−8. (C, D) Splenocytes from the same animals as in panels A and B were cultured in the presence or absence of ConA or MOG_35–55 peptide. Proliferation was measured by ³[H]-thymidine incorporation assay and expressed as a proliferation index relative to the values obtained in the absence of any stimulus (C); IL-17 and IFNγ levels in the supernatant were determined by ELISA (D); n = 7−10. *: p<0.05, ***: p<0.001, n.s.: p>0.05.</p