ILDR2 Is a Novel B7-like Protein That Negatively Regulates T Cell Responses.

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer.

Celecoxib enhances the anti-inflammatory effects of farnesylthiosalicylic acid on T cells independent of prostaglandin E(2) production.

Celecoxib (Celebrex(®)), a non-steroidal anti-inflammatory drug and selective cyclooxygenase-2 inhibitor, is widely used to treat arthritis and other inflammatory disorders. Awareness of its anti-proliferative properties has prompted another indication for its use, in preventing colon polyps in high-risk populations. Farnesylthiosalicylic acid (FTS; Salirasib(®)), designed to inhibit oncogenic Ras and currently under evaluation in phase I/II and II clinical trials, was recently shown by our group to exert anti-inflammatory effects on both lymphocytes and mast cells. Here we examined whether celecoxib combined with FTS would enhance this anti-inflammatory activity. While each drug separately inhibited Ras activation in these cells, their combination yielded more marked inhibition as well as further inhibition of ERK phosphorylation, lymphocyte adhesion, and interleukin-2 secretion. The inhibitory effects, moreover, were independent of prostaglandin E(2) secretion. These data point to the promising potential of combined treatment with celecoxib and FTS for inflammatory disorders involving lymphocytes.

Ras inhibition by FTS attenuates brain tumor growth in mice by direct antitumor activity and enhanced reactivity of cytotoxic lymphocytes.

A major concern in targeted drug therapy is that the inhibition of receptors and signaling molecules in tumor cells may also affect similar components in the tumor microenvironment or in the immune system, with undefined consequences for inhibition of tumor growth. One example is given by the Ras inhibitor salirasib (Farnesythiosalycilic acid, FTS), which in addition to its antitumor activity in mice and humans also exhibits anti-inflammatory activity. Here we show three major effects through which Ras inhibition by FTS provides a favorable antitumor environment in immune-competent mice with subcutaneous or intracranial tumors. First, FTS exhibited antitumor activity in intracranial immune-competent tumor-bearing mice and increased their survival relative to tumor-bearing immune-compromised mice. Second, FTS induced an increase in regulatory T cells in mouse splenocytes, in which Foxp3+ T cells did not interfere with the tumor growth inhibitory effects of FTS. Third, FTS induced an increase in antitumor cytotoxic T-cell reactivity in glioma cells by downregulating their own expression of Foxp3. This downregulation induced a TGF-ß-associated mechanism in glioma cells altering the tumor microenvironment and causing reduced resistance of the tumor to the immune system. These results are important as they might explain some of the major beneficial effects of Ras inhibitors. They may provide an experimental framework for examination of the impact of other anticancer drugs on cancer and the immune system.

Prevention of induced colitis in mice by the ras antagonist farnesylthiosalicylic acid.

BACKGROUND: Ras proteins are crucial for cell differentiation and proliferation. Targeting Ras with farnesylthiosalicylic acid (FTS), a Ras antagonist, has been suggested as a therapeutic strategy in proliferative and inflammatory diseases. AIMS: To examine the role of Ras and the therapeutic potential of FTS in experimental colitis. METHODS: Colitis was induced in 26 mice by adding 2.5% dextran sodium sulfate to their drinking water for 7 days during which 12 study mice were treated with FTS and 14 control mice were given normal saline. Two additional controls included 10 naïve mice treated with FTS and 7 naïve non-treated mice. The animals were followed clinically and sacrificed after 7 days. Their colons were isolated for histological assessment and for measurement of myeloperoxidase activity (MPO), tumor necrosis factor-α(TNF-α), and interleukin-1ß(Il-1ß) levels. Ras and activated Ras expression was determined by immunoblotting assays. T cell populations in the colon and spleen were analyzed by flow-cytometry. RESULTS: FTS induced a 2.1-fold reduction in activated Ras levels (P < 0.004). FTS-treated mice had lower disease activity scores (3.9 ± 1.7 vs. 7.5 ± 2.3, P < 0.001), and lower levels of MPO activity (1.65 ± 0.6 vs. 2.6 ± 0.8 units/g, P < 0.007), Il-1ß (2.4 ± 3.6 vs. 24.3 ± 17.5 pg/mg, P < 0.01) and TNF-α (0.63 ± 0.5 vs. 1.9 ± 1 pg/mg, P < 0.04). FTS increased regulatory T cell population in the spleen (1.9 ± 0.4-fold, P < 0.04), and decreased effector T cell populations in the colon and spleen by 24 ± 3% (P < 0.03) and 27 ± 1% (P < 0.02), respectively. FTS had no remarkable side effects. CONCLUSIONS: Ras is involved in the inflammatory processes of induced colitis in mice and its inhibition by FTS ameliorates the severity of the inflammation.

Ras inhibition induces insulin sensitivity and glucose uptake.

BACKGROUND: Reduced glucose uptake due to insulin resistance is a pivotal mechanism in the pathogenesis of type 2 diabetes. It is also associated with increased inflammation. Ras inhibition downregulates inflammation in various experimental models. The aim of this study was to examine the effect of Ras inhibition on insulin sensitivity and glucose uptake, as well as its influence on type 2 diabetes development. METHODS AND FINDINGS: The effect of Ras inhibition on glucose uptake was examined both in vitro and in vivo. Ras was inhibited in cells transfected with a dominant-negative form of Ras or by 5-fluoro-farnesylthiosalicylic acid (F-FTS), a small-molecule Ras inhibitor. The involvement of IκB and NF-κB in Ras-inhibited glucose uptake was investigated by immunoblotting. High fat (HF)-induced diabetic mice were treated with F-FTS to test the effect of Ras inhibition on induction of hyperglycemia. Each of the Ras-inhibitory modes resulted in increased glucose uptake, whether in insulin-resistant C2C12 myotubes in vitro or in HF-induced diabetic mice in vivo. Ras inhibition also caused increased IκB expression accompanied by decreased expression of NF-κB . In fat-induced diabetic mice treated daily with F-FTS, both the incidence of hyperglycemia and the levels of serum insulin were significantly decreased. CONCLUSIONS: Inhibition of Ras apparently induces a state of heightened insulin sensitization both in vitro and in vivo. Ras inhibition should therefore be considered as an approach worth testing for the treatment of type 2 diabetes.

The combined treatment of Copaxone and Salirasib attenuates experimental autoimmune encephalomyelitis (EAE) in mice.

EAE is a common animal model for multiple sclerosis (MS). Immunomodulatory treatments such as glatiramer acetate (GA, Copaxone) are beneficial in EAE but are not universally effective in the clinic. The Ras inhibitor farnesylthiosalycylic acid (FTS, Salirasib), efficiently ameliorate EAE as well. Here we demonstrate a synergistic beneficial effect of the combined GA and FTS treatment on EAE; 22.5% of the combined-treatment mice developed disease compared to 87.5%, 77.5% and 82.5% of mice treated with vehicle, GA and FTS, respectively, results supported by MRI, histological, immunological and biochemical data. Such a combined treatment may improve clinical outcome in MS patients.

Ras inhibition attenuates pancreatic cell death and experimental type 1 diabetes: possible role of regulatory T cells.

Regulatory T cells (Treg) play a crucial role in the maintenance of immune homeostasis and prevention of autoimmune diseases. Ras inhibition by 5-fluoro-farnesylthiosalicylic acid (F-FTS) was recently shown to increase the number and boost the suppressive function of Treg, thereby reducing the incidence of experimental diabetes in non-obese diabetic (NOD) mice. To investigate the effect of F-FTS on pancreatic beta cells and the possible involvement of Treg in such an effect, we evaluated the incidence of diabetes and assayed the pancreatic expression of Foxp3, cleaved caspase 3, and Ras-GTP expression in NOD mice treated with different doses of F-FTS. The treated mice showed attenuated progression of experimental diabetes, accompanied by an increase in serum insulin. Daily injections of F-FTS led to an increase in both the number and the migratory capacity of pancreatic Foxp3(+)CD4(+)CD25(+) Treg, while cleaved caspase 3 in the pancreas were significantly decreased, indicating reduced apoptosis. The Treg population induced by F-FTS helped to preserve pancreatic beta-cell viability in the presence of effector T cells. These findings suggest that inhibition of Ras by F-FTS in mice with experimental diabetes upregulates the Treg pool, which infiltrates the pancreas and attenuates the apoptotic cell death of beta cells. It thus appears that F-FTS induces Treg to play a protective role in the progression of experimental type-1 diabetes, suggesting that these cells represent a potential target for the treatment of this disorder.

Transplanted myogenic progenitor cells express neuronal markers in the CNS and ameliorate disease in experimental autoimmune encephalomyelitis.

This study explores the potential of non-neural progenitor cells for CNS cell therapy. Muscle progenitor cells (MPCs), transplanted either intraventricularly or intraperitonealy, incorporated into the CNS of EAE-induced but not of naïve mice. Some of the migrating MPCs expressed the neuronal marker beta-III-Tubulin and gained neuronal morphology. Co-treatment of transplanted mice with the immunomodulatory agent glatiramer acetate (GA, Copaxone) resulted in improved MPCs incorporation and differentiation towards the neuronal pathway. The therapeutic potential of myogenic progenitor cells was demonstrated by amelioration of clinical symptoms and reduced mortality in EAE mice, as well as by expression of IL-10, TGF-beta, and the neurotrophin-BDNF.