Stefano Bruscoli

A New Dexamethasone-Induced Gene of the Leucine Zipper Family Protects T Lymphocytes from TCR/CD3-Activated Cell Death

By comparing mRNA species expressed in dexamethasone (DEX)-treated and untreated murine thymocytes, we have identified a gene, glucocorticoid-induced leucine zipper (GILZ), encoding a new member of the leucine zipper family. GILZ was found expressed in normal lymphocytes from thymus, spleen, and lymph nodes, whereas low or no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. In thymocytes and peripheral T cells, GILZ gene expression is induced by DEX. Furthermore, GILZ expression selectively protects T cells from apoptosis induced by treatment with anti-CD3 monoclonal antibody but not by treatment with other apoptotic stimuli. This antiapoptotic effect correlates with inhibition of Fas and Fas ligand expression. Thus, GILZ is a candidate transcription factor involved in the regulation of apoptosis of T cells.

GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations

GITR (glucocorticoid‐induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8+ and CD4+ peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti‐CD3 stimulus. Furthermore, expression of the main activation‐induced receptor (IL‐2Rα) and production of IL‐2 and IFN‐γ were increased more with a GITR costimulus than with anti‐CD3 alone. GITR stimulation also enhanced anti‐CD3‐induced ERK phosphorylation, suggesting that GITR is involved in MAPK‐pathway activation. Interestingly, CD4+CD25+ regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR–/– CD4+CD25+ and GITR+/+ CD4+CD25+ cells were equally able to exert suppressor activity on CD4+CD25– responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.

Glucocorticoid-induced leucine zipper (GILZ)/NF-κB interaction: role of GILZ homo-dimerization and C-terminal domain

Glucocorticoid-induced leucine zipper (GILZ) is a 137 amino acid protein, rapidly induced by treatment with glucocorticoids (GC), characterized by a leucine zipper (LZ) domain (76–97 amino acids), an N-terminal domain (1–75 amino acids) and a C-terminal PER domain (98–137 amino acids) rich in proline and glutamic acid residues. We have previously shown that GILZ binds to and inhibits NF-κB activity. In the present study we used a number of mutants with the aim of defining the GILZ molecular domains responsible for GILZ/p65NF-κB interaction. Results, obtained by in vitro and in vivo co-immunoprecipitation (Co-IP) and by transcriptional activity experiments, indicate that GILZ homo-dimerization, through the LZ domain, as well as the C-terminal PER domain, particularly the 121–123 amino acids, are both necessary for GILZ interaction with NF-κB, inhibition of transcriptional activity and of IL-2 synthesis.

Cloning, chromosomal assignment and tissue distribution of human GILZ, a glucocorticoid hormone-induced gene.

Cloning, chromosomal assignment and tissue distribution of human GILZ, a glucocorticoid hormone-induced gene

Role of glucocorticoid-induced TNF receptor family gene (GITR) in collagen-induced arthritis

In rheumatoid arthritis (RA), a widespread autoimmune/inflammatory joint disease, early activation of effector CD4+ T lymphocytes, and cytokine production is followed by recruitment of other inflammatory cells, production of a range of inflammation mediators, tissue damage, and disease. GITR (glucocorticoid‐induced TNFR family‐related gene), a costimulatory molecule for T lymphocytes, increases CD4+CD25− effector T cell activation while inhibiting suppressor activity of CD4+CD25+ T regulatory (Treg) cells. We analyzed the role of GITR in type II collagen (CII) ‐induced arthritis (CIA) using GITR− /− and GITR+ / + mice. Results indicate significantly less CIA induction in GITR−/− mice than in GITR+/+ mice, with marked differences in erythema, edema, neutrophil infiltration, joint injury, and bone erosion. Production of IFNγ, IL‐6, TNFα, MIP‐1α, and MIP‐2, inducible NOS (iNOS), COX‐2, and nitrotyrosine poly‐ADP‐ribose (PAR) were also less in CII‐treated GITR−/− mice. Although CD4+CD25+ Treg cells from GITR+/+ and GITR−/− CII‐challenged mice exerted similar suppressor activity in vitro, GITR triggering abrogated GITR+/+ Treg suppressor activity and costimulated CD4+CD25− GITR+/+ effector cells. Furthermore, Treg cells from GITR−/− protected more than Treg cells from GITR+/+ mice against CIA when cotransferred with Treg‐depleted splenocytes from arthritic GITR+/+ animals into severe combined immunodeficient (SCID) mice. In conclusion, GITR plays a critical role in the immunological response against CII and in the development of CIA. Cuzzocrea, S., Ayroldi, E., Di Paola, R., Agostini, M., Mazzon, E., Bruscoli, S., Genovese, T., Ronchetti, S., Caputi, A. P., Riccardi, C. Role of glucocorticoid‐induced TNF receptor family gene (GITR) in collagen‐induced arthritis. FASEB J. 19, 1253–1265 (2005)

GITR modulates innate and adaptive mucosal immunity during the development of experimental colitis in mice

Background: Uncontrolled T cell activation and abnormal function of the innate immune system against normal enteric bacterial flora play a critical part in the pathogenesis of inflammatory bowel disease (IBD). Therefore, pharmacological strategies directed to restore the normal responsiveness of the immune system could be efficacious in the treatment of these pathological conditions. Glucocorticoid-induced tumour necrosis factor receptor (GITR)-related gene is a member of the tumour necrosis factor receptor superfamily that is constitutively expressed at high levels on regulatory T cells and at low levels on unstimulated T cells, B cells and macrophages. GITR triggering leads to activation of T effectors and reversal of suppressive function of regulatory T cells. Aim: To investigate the role of GITR in the development of experimental colitis in mice. Results: Using GITR−/− mice, GITR deletion protected against 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis by reducing innate immune responses and effector T cell activity. Effector T cells isolated from GITR−/− mice were less effective than T cells isolated from GITR+/+ mice to transfer colitis in immunodeficient mice. Blocking the GITR/ligand for GITR (GITRL) signal by giving soluble GITR prevented TNBS-induced colitis in normal GITR+/+ and also in lymphocyte-deficient SCID mice. Conclusions: Collectively, these data suggest that GITR plays a critical part in regulating both acquired and innate mucosal immune responses during the development of experimental colitis in mice. Therefore, targeting the GITR/GITRL system signalling may represent a potential pharmacological tool for the treatment of IBD.

Peroxisome Proliferator-Activated Receptor-α Contributes to the Anti-Inflammatory Activity of Glucocorticoids

Glucocorticoids (GCs) are effective anti-inflammatory agents widely used in the therapeutic approach to treatment of acute and chronic inflammatory diseases. Previous results suggest that peroxisome proliferator-activated receptor-α (PPAR-α), an intracellular transcription factor activated by fatty acids, plays a role in the control of inflammation. With the aim of characterizing the role of PPAR-α in GC-mediated anti-inflammatory activity, we tested the efficacy of dexamethasone (DEX), a synthetic GC specific for glucocorticoid receptor, in an experimental model of lung inflammation, carrageenan-induced pleurisy, comparing mice lacking PPAR-α (PPAR-αKO) with wild-type (WT) mice. We also tested the possible synergism of combined treatment with DEX and clofibrate, a PPAR-α agonist. Results indicate that DEX-mediated anti-inflammatory activity is weakened in PPAR-αKO mice compared with WT controls, and that is increased in WT mice when combined with PPAR-α agonist treatment. In particular, DEX was less effective in PPAR-αKO, compared with WT mice, as evaluated by inhibition of NF-κB, of TNF-α production, of cell migration, of cycloxygenase-2 (COX-2) and inducible nitric-oxide synthase activation. Interestingly enough, macrophages from PPAR-αKO were less susceptible to DEX-induced COX-2 inhibition in vitro compared with WT mice. However, PPAR-α transfection in PPAR-αKO macrophages, with consequent receptor expression, resulted in reconstitution of susceptibility to DEX-induced COX-2 inhibition to levels comparable with that obtained in WT macrophages. It is noteworthy that the DEX effect on macrophages in vitro was significantly increased in WT cells when combined with PPAR-α agonist treatment. These results indicate that PPAR-α can contribute to the anti-inflammatory activity of GCs.

GILZ promotes production of peripherally induced Treg cells and mediates the crosstalk between glucocorticoids and TGF-β signaling.

Regulatory T (Treg) cells expressing the transcription factor forkhead box P3 (FoxP3) control immune responses and prevent autoimmunity. Treatment with glucocorticoids (GCs) has been shown to increase Treg cell frequency, but the mechanisms of their action on Treg cell induction are largely unknown. Here, we report that glucocorticoid-induced leucine zipper (GILZ), a protein induced by GCs, promotes Treg cell production. In mice, GILZ overexpression causes an increase in Treg cell number, whereas GILZ deficiency results in impaired generation of peripheral Treg cells (pTreg), associated with increased spontaneous and experimental intestinal inflammation. Mechanistically, we found that GILZ is required for GCs to cooperate with TGF-β in FoxP3 induction, while it enhances TGF-β signaling by binding to and promoting Smad2 phosphorylation and activation of FoxP3 expression. Thus, our results establish an essential GILZ-mediated link between the anti-inflammatory action of GCs and the regulation of TGF-β-dependent pTreg production.

Silymarin suppress CD4+ T cell activation and proliferation: effects on NF-κB activity and IL-2 production.

Silymarin, a mixture of bioactive flavonolignans isolated from Silybum marianum, exhibits anti-carcinogenic, anti-inflammatory and cytoprotective effects. In this study, the in vitro immunomodulatory activity of silymarin was investigated using CD4+ splenocytes from C57/Bl6 mice. Proliferation assay revealed that silymarin, at 50 microM concentration, significantly inhibited CD4+ cells proliferation. ELISA analyses indicated that silymarin significantly inhibited IL-2 and IFN-gamma production. Immunofluorescence staining performed on the mouse hybridoma T cell line (3DO) revealed a block of nuclear translocation of transcription factor kappaB (NF-kappaB), which is known to be responsible for IL-2 transcriptional activation. Moreover, silymarin inhibited p65/NF-kappaB phosphorylation in CD4+ T cell. These results suggest that silymarin is able to inhibit T cell activation and proliferation, notably acting on pathways of NF-kappaB activation/translocation.

Long Glucocorticoid-induced Leucine Zipper (L-GILZ) Protein Interacts with Ras Protein Pathway and Contributes to Spermatogenesis Control

Background: Understanding how spermatogenesis occurs in mammals is not yet fully understood. Results: L-GILZ deficiency in germ cells leads to complete loss of germ cell lineage resulting in male sterility. Conclusion: Our study identifies L-GILZ as an important factor for spermatogenesis. Significance: Identification of genes critical for maintenance of spermatogenesis is pivotal for diagnosis and treatment of male infertility. Correct function of spermatogonia is critical for the maintenance of spermatogenesis throughout life, but the cellular pathways regulating undifferentiated spermatogonia proliferation, differentiation, and survival are only partially known. We show here that long glucocorticoid-induced leucine zipper (L-GILZ) is highly expressed in spermatogonia and primary spermatocytes and controls spermatogenesis. Gilz deficiency in knock-out (gilz KO) mice leads to a complete loss of germ cell lineage within first cycles of spermatogenesis, resulting in male sterility. Spermatogenesis failure is intrinsic to germ cells and is associated with increased proliferation and aberrant differentiation of undifferentiated spermatogonia and with hyperactivity of Ras signaling pathway as indicated by an increase of ERK and Akt phosphorylation. Spermatogonia differentiation does not proceed beyond the prophase of the first meiotic division due to massive apoptosis associated with accumulation of unrepaired chromosomal damage. These results identify L-GILZ as a novel important factor for undifferentiated spermatogonia function and spermatogenesis.