Adi Mor

Hypoxia controls CD4+CD25+ regulatory T-cell homeostasis via hypoxia-inducible factor-1α†

Recent data suggest that hypoxia and its principal molecular signature HIF‐1 (hypoxia‐inducing factor‐1) may tune down inflammation by dictating anti‐inflammatory programs. We tested the effects of hypoxia and HIF‐1α on the homeostasis of naturally occurring regulatory T cells (Treg) and their transcriptional activator Foxp3. Hypoxia induced a time‐dependent increase in HIF‐1α in mouse and human T cells. Hypoxia upregulated the expression of Foxp3 in Jurkat T cells, human and murine mononuclear cells. The effects of hypoxia on Foxp3 expression were HIF‐1α‐dependent as they were abolished upon transfection with short‐interfering RNAs for HIF‐1α and promoted by HIF‐1α overexpression. Hypoxia increased the potency of Treg, as hypoxic CD4+CD25+ lymphocytes were more effective than normoxic cells in suppressing the proliferation of CD4+CD25− effectors. In vivo expression of HIF‐1α achieved by hydrodynamic injection of the respective naked DNA similarly induced an increase in Foxp3 expression and an increase in the number of functionally active Foxp3+CD4+CD25+ Treg. Thus, hypoxia dictates an anti‐inflammatory program by driving expression of HIF‐1α that acts to increase the number and suppressive properties of naturally occurring CD4+CD25+ Treg.

N-Ras or K-Ras inhibition increases the number and enhances the function of Foxp3 regulatory T cells

Naturally occurring regulatory T cells (Treg) driven by their transcriptional controller Foxp3 are compromised in immune‐mediated disorders and confer protection when adoptively transferred. We examined the Ras‐inhibitory effect on functional determinants of Treg in vivo and in vitro. Ras was inhibited in Jurkat T cells by transfection with a dominant‐negative form of Ras, or by shRNA for N‐Ras, K‐Ras, and H‐Ras, or by farnesylthiosalycylic acid, a small‐molecule inhibitor. Except for H‐Ras transduction with shRNA, each inhibitory mode increased expression of Foxp3 and nuclear factor of activated T cell proteins, and surface expression of CD25. Ras inhibition in PBMC and spleen‐derived lymphocytes reproduced these findings. The heightened Foxp3 expression reflected both increased basal cellular protein and peripheral conversion of non‐Treg to Treg. Ras inhibition enhanced Treg‐induced suppression; thus, when adoptively transferred to mice, Ras‐inhibited Treg reduced the incidence of diabetes. Inhibition of Foxp3 by respective siRNA reversed the enhancement. Thus, inhibition of the N‐ or K‐Ras isoform triggers an anti‐inflammatory effect by up‐regulating, via Foxp3 elevation, the numbers and functional suppressive properties of Treg.

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 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.

Ras inhibition increases the frequency and function of regulatory T cells and attenuates type-1 diabetes in non-obese diabetic mice.

Regulatory T cells (Treg) reportedly suppress diabetes in non-obese diabetic (NOD) mice, and the frequency and functional suppressive properties of Treg were found to be upregulated by inhibition of Ras. We thus postulated that treatment with the Ras inhibitor 5-fluoro-farnesylthiosalicylic acid (F-FTS), a derivative of S-farnesylthiosalicylic acid (FTS), would attenuate diabetes development in NOD mice. To test this hypothesis we assayed Foxp3, total Ras, and GTP-Ras in NOD splenocytes following their exposure to FTS and F-FTS in vitro. In splenocytes exposed to FTS, and even more so to F-FTS, Foxp3 expression was increased and GTP-Ras expression reduced. We also injected NOD mice intraperitoneally with F-FTS and assessed both their Treg pools and the occurrence of diabetes. The treated mice showed a significant increase in the frequency of spleen-cell-derived Foxp3+ Treg, and their Treg were more effective than Treg from untreated NOD controls in suppressing the proliferation of effector T cells. Moreover, the F-FTS treatment also attenuated the development of diabetes in the NOD mice. The mice were then killed and their insulin and cytokine levels assayed. The treated mice showed an increase in circulating insulin but no change in cytokine concentrations. One of the mechanisms underlying our novel finding that treatment with a Ras inhibitor ameliorates the development of experimental type-1 diabetes could thus conceivably be an augmentation in the frequency and functional suppressive properties of Treg. Ras inhibition might therefore be worth developing as a new treatment modality in patients with type 1 diabetes.

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.

Angiostatic and angiogenic chemokines in systemic sclerosis: an overview

In systemic sclerosis (SSc), the dysregulation of several molecular pathways seem to have a role in the disease pathogenesis. Either angiogenesis and vasculogenesis are disturbed and impaired, and an imbalance between angiogenic and angiostatic factors may be involved in the genesis and maintenance of vasculopathy. Aberrant immune system activation and function involves both B and T cells, as well as many different chemokines and cytokines. Particularly, chemokines are central to the initiation and maintenance of inflammatory responses as well as angiogenesis and fibrosis. Increased expression of several chemokines as CXCL4 (platelet factor 4), CXCL8 (IL8), CXCL5 (ENA-78), CCL5 (RANTS), CXCL9 (MIG), CCL24, CXCL10 IP-10), CXCL12, CXCL16 (SRPSDX), CCL2 (MCP-1), CCL19 (MIP-3β/ELC), CCL24 (Eotaxin 2), suggests a complex mechanism by which many immune cell types, including T cells, macrophages and neutrophils are recruited to the skin in SSc patients. Many of these chemokines have redundant roles, possibly to ensure recruitment of specific cell types. Several studies have shown a synergistic effect of combinations of these chemokines in cell recruitment, emphasizing the importance of understanding global chemokine expressions. urthermore, chemokines can be detected in peripheral blood compared with cytokines or growth factors. The utility of cytokines as biomarkers has been investigated but longitudinal studies are necessary to clarify their clinical utility for the evaluation of disease activity, therapeutic effects on skin sclerosis or interstitial lung disease and risk stratification of SSc patients. An effective therapeutic agent, able to interfere with complex chemokine networks, is warranted to attenuate perivascular inflammation, dysregulated angiogenesis and the evolution of skin and internal organ fibrosis, is the most ambitious goal for the scientific research of the future.

Elevated Levels of Eotaxin-2 in Serum of Fibromyalgia Patients

FMS patients demonstrate an altered profile of chemokines relative to healthy controls (HC). Eotaxin-2 is a potent chemoattractant distributed in a variety of tissues. The aim of the study was to compare serum levels of eotaxin-2 between FMS patients and HC and to examine a potential correlation between eotaxin-2 levels and clinical parameters of FMS. Methods. 50 patients with FMS and 15 HC were recruited. Data on the severity of FMS symptoms and depression were collected. Serum levels of eotaxin-2 (ELISA) were determined in all participants. High-sensitive CRP (hs-CRP) was measured in the FMS group. Results. The FMS cohort included predominantly females (84%), mean age of 49, and mean disease duration of 6 years. FMS patients exhibited significantly higher eotaxin-2 levels (pg/ml) versus HC: 833 (±384) versus 622 (±149), p=0.04. Mean hs-CRP level among FMS patients was 4.8 ± 6 mg/l, a value not indicative of acute inflammation. No correlation was found between eotaxin-2 and hs-CRP levels. No correlation was found between eotaxin-2 and severity measures of FMS or depression. Conclusion. Eotaxin-2 does not appear to be a candidate for a disease activity biomarker in FMS. Further research is warranted into the role of this chemokine in the pathophysiology of the FMS.