Machine Learning Identifies Key Proteins in Primary Sclerosing Cholangitis Progression and Links High CCL24 to Cirrhosis.

Primary sclerosing cholangitis (PSC) is a rare, progressive disease, characterized by inflammation and fibrosis of the bile ducts, lacking reliable prognostic biomarkers for disease activity. Machine learning applied to broad proteomic profiling of sera allowed for the discovery of markers of disease presence, severity, and cirrhosis and the exploration of the involvement of CCL24, a chemokine with fibro-inflammatory activity. Sera from 30 healthy controls and 45 PSC patients were profiled with proximity extension assay, quantifying the expression of 2870 proteins, and used to train an elastic net model. Proteins that contributed most to the model were tested for correlation to enhanced liver fibrosis (ELF) score and used to perform pathway analysis. Statistical modeling for the presence of cirrhosis was performed with principal component analysis (PCA), and receiver operating characteristics (ROC) curves were used to assess the useability of potential biomarkers. The model successfully predicted the presence of PSC, where the top-ranked proteins were associated with cell adhesion, immune response, and inflammation, and each had an area under receiver operator characteristic (AUROC) curve greater than 0.9 for disease presence and greater than 0.8 for ELF score. Pathway analysis showed enrichment for functions associated with PSC, overlapping with pathways enriched in patients with high levels of CCL24. Patients with cirrhosis showed higher levels of CCL24. This data-driven approach to characterize PSC and its severity highlights potential serum protein biomarkers and the importance of CCL24 in the disease, implying its therapeutic potential in PSC.

Targeting CCL24 in Inflammatory and Fibrotic Diseases: Rationale and Results from Three CM-101 Phase 1 Studies.

BACKGROUND: Overexpression of C-C motif chemokine ligand 24 (CCL24) is associated with inflammatory and fibrotic diseases, including primary sclerosing cholangitis (PSC), systemic sclerosis, metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH). CM-101 is a humanized monoclonal antibody that neutralizes CCL24 to attenuate inflammation and fibrosis in preclinical models. Here we report the results from two Phase 1a studies investigating the safety and tolerability of intravenous (IV) and subcutaneous (SC) CM-101 in healthy participants, and in one Phase 1b study of IV and SC CM-101 in patients with MASLD without evidence of MASH. METHODS: In each dose group (0.75 mg/kg, 2.5 mg/kg, 5.0 mg/kg, and 10.0 mg/kg) of the single-center, double-blind, placebo-controlled Phase 1a IV study, healthy volunteers were randomized 3:1 to receive a single IV infusion of CM-101 or placebo. In another Phase 1a, single-center, double-blind placebo-controlled study, healthy volunteers were randomized 3:1 to receive a single SC injection of CM-101 5.0 mg/kg or placebo. In the multicenter, double-blind, placebo-controlled Phase 1b MASLD study, patients with MASLD without evidence of MASH were randomized 3:1 to receive the following: cohort 1, IV CM-101 2.5 mg/kg or placebo, and cohort 2, SC CM-101 5.0 mg/kg or placebo every three weeks for 12 weeks. The primary endpoints (for all these studies) were safety, tolerability, and serum pharmacokinetic parameters of CM-101. RESULTS: In each study, adverse events were rare and mild to moderate. The CM-101 pharmacokinetics profile was typical of a monoclonal antibody, with a terminal half-life of approximately 19 days when given IV and approximately 17 days when given as SC injection. In patients with MASLD without evidence of MASH, CM-101 was associated with decreased serum levels of inflammatory, fibrotic, and collagen turnover biomarkers. CONCLUSIONS: In healthy volunteers and patients with MASLD without evidence of MASH, IV and SC CM-101 was well tolerated at doses ranging from 0.75 mg/kg to10.0 mg/kg and engaged its target (i.e., CCL24), indicating therapeutic potential in treating inflammatory and fibrotic diseases. CLINICAL TRIAL RETROSPECTIVELY REGISTRATION: NCT06025851, NCT06037577, and NCT06044467. Date of registration: September 2023.

Serum CCL24 as a Biomarker of Fibrotic and Vascular Disease Severity in Systemic Sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a heterogeneous disease, characterized by variable tissue and vascular fibrosis in the context of autoimmune activation. CCL24 (or Eotaxin2) has been shown to promote microangiopathic, proinflammatory, and profibrotic processes in preclinical models of SSc. Here, we study serum CCL24 levels in a real-life cohort of patients with SSc, to determine its distribution across disease features and its value in predicting disease progression and related mortality. METHODS: Serum CCL24 was assessed in an observational cohort of consecutively enrolled patients with SSc. A high CCL24 cutoff was defined based on its distribution in a matched cohort of healthy controls. Disease progression and mortality were analyzed from the date of serum assessment. RESULTS: Two-hundred thirteen consecutively enrolled patients with SSc were included in this analysis. Median disease duration was six years (interquartile range 3-14), 28.6% of patients presented with interstitial lung disease (ILD), 46.9% had digital ulcers, and 25.3% showed high CCL24 serum concentration. High-CCL24 patients were more frequently male and positive for anti-scl-70, with a diagnosis of ILD and synovitis (P < 0.05 for all). Notably, high-CCL24 patients had lower diffusion of carbon monoxide and higher prevalence of digital ulcers, telangiectasias, and calcinosis (P < 0.05 for all). In a longitudinal setting, high CCL24 was associated with greater lung function decline and with higher disease-related mortality. CONCLUSION: Serum CCL24 is a biomarker of disease severity across fibrotic and vascular disease manifestations. These data support the development of therapies targeting CCL24 as a novel comprehensive therapeutic target in SSc.

The Role of CCL24 in Primary Sclerosing Cholangitis: Bridging Patient Serum Proteomics to Preclinical Data.

Primary sclerosing cholangitis (PSC) is an inflammatory and fibrotic biliary disease lacking approved treatment. We studied CCL24, a chemokine shown to be overexpressed in damaged bile ducts, and its involvement in key disease-related mechanisms. Serum proteomics of PSC patients and healthy controls (HC) were analyzed using the Olink® proximity extension assay and compared based on disease presence, fibrosis severity, and CCL24 levels. Disease-related canonical pathways, upstream regulators, and toxicity functions were elevated in PSC patients compared to HC and further elevated in patients with high CCL24 levels. In vitro, a protein signature in CCL24-treated hepatic stellate cells (HSCs) differentiated patients by disease severity. In mice, CCL24 intraperitoneal injection selectively recruited neutrophils and monocytes. Treatment with CM-101, a CCL24-neutralizing antibody, in an α-naphthylisothiocyanate (ANIT)-induced cholestasis mouse model effectively inhibited accumulation of peribiliary neutrophils and macrophages while reducing biliary hyperplasia and fibrosis. Furthermore, in PSC patients, CCL24 levels were correlated with upregulation of monocyte and neutrophil chemotaxis pathways. Collectively, these findings highlight the distinct role of CCL24 in PSC, influencing disease-related mechanisms, affecting immune cells trafficking and HSC activation. Its blockade with CM-101 reduces inflammation and fibrosis and positions CCL24 as a promising therapeutic target in PSC.

CCL24 regulates biliary inflammation and fibrosis in primary sclerosing cholangitis.

ˆCCL24 is a pro-fibrotic, pro-inflammatory chemokine expressed in several chronic fibrotic diseases. In the liver, CCL24 plays a role in fibrosis and inflammation, and blocking CCL24 led to reduced liver injury in experimental models. We studied the role of CCL24 in primary sclerosing cholangitis (PSC) and evaluated the potential therapeutic effect of blocking CCL24 in this disease. Multidrug resistance gene 2-knockout (Mdr2-/-) mice demonstrated CCL24 expression in liver macrophages and were used as a relevant experimental PSC model. CCL24-neutralizing monoclonal antibody, CM-101, significantly improved inflammation, fibrosis, and cholestasis-related markers in the biliary area. Moreover, using spatial transcriptomics, we observed reduced proliferation and senescence of cholangiocytes following CCL24 neutralization. Next, we demonstrated that CCL24 expression was elevated under pro-fibrotic conditions in primary human cholangiocytes and macrophages, and it induced proliferation of primary human hepatic stellate cells and cholangiocytes, which was attenuated following CCL24 inhibition. Correspondingly, CCL24 was found to be highly expressed in liver biopsies of patients with PSC. CCL24 serum levels correlated with Enhanced Liver Fibrosis score, most notably in patients with high alkaline phosphatase levels. These results suggest that blocking CCL24 may have a therapeutic effect in patients with PSC by reducing liver inflammation, fibrosis, and cholestasis.

A blocking monoclonal antibody to CCL24 alleviates liver fibrosis and inflammation in experimental models of liver damage.

BACKGROUND & AIMS: C-C motif chemokine ligand 24 (CCL24) is a chemokine that regulates inflammatory and fibrotic activities through its receptor, C-C motif chemokine receptor (CCR3). The aim of the study was to evaluate the involvement of the CCL24-CCR3 axis in liver fibrosis and inflammation and to assess the potential of its blockade, by a monoclonal anti-CCL24 antibody, as a therapeutic strategy for non-alcoholic steatohepatitis (NASH) and liver fibrosis. METHODS: Expression of CCL24 and CCR3 was evaluated in liver biopsies and blood samples. CCL24 involvement in NAFLD/NASH pathogenesis was assessed in Ccl24 knockout mouse using the methionine-choline deficient (MCD) diet experimental model. Antifibrotic and anti-inflammatory effects of CM-101 were tested in the MCD and STAM mouse models and in the thioacetamide (TAA) model in rats. Liver enzymes, liver morphology, histology and collagen deposition, as well as fibrosis- and inflammation-related protein expression were assessed. Activation of hepatic stellate cells (HSCs) was evaluated in the human LX2 cell line. RESULTS: Patients with NASH and advanced NAFLD exhibited significant expression of both CCL24 and CCR3 in liver and blood samples. In the experimental MCD-diet model, Ccl24 knockout mice showed an attenuated liver damage response compared to wild-type mice, exhibiting reduced histological NAFLD activity scores and fibrosis, as well as lower levels of liver enzymes. Blocking CCL24 using CM-101 robustly reduced liver damage in 3 experimental animal models (MCD, STAM and TAA), as demonstrated by attenuation of liver fibrosis and NAFLD activity score. Furthermore, blocking CCL24 by CM-101 significantly inhibited CCL24-induced HSC motility, α-SMA expression and pro-collagen I secretion. CONCLUSION: Our results reveal that blocking CCL24 significantly attenuates liver fibrosis and inflammation and may have a potential therapeutic effect in patients with NASH and/or liver fibrosis. LAY SUMMARY: CCL24 is a chemokine that regulates inflammation and fibrosis. It was found to be significantly expressed in patients with non-alcoholic steatohepatitis, in whom it regulates profibrotic processes in the liver. Herein, we show that blockade of CCL24 using a monoclonal antibody robustly attenuated liver fibrosis and inflammation in animal models, thus suggesting a potential therapeutic role for an anti-CCL24 agent.

Blockade of CCL24 with a monoclonal antibody ameliorates experimental dermal and pulmonary fibrosis.

OBJECTIVES: We aimed to assess the expression of the CCL24 chemokine in systemic sclerosis (SSc) and to evaluate the possible pathogenic implications of the CCL24/CCR3 axis using both in vitro and in vivo models. We further investigated the efficacy of an anti-CCL24 monoclonal antibody (mAb), CM-101, in inhibiting cell activation as well as dermal and pulmonary inflammation and fibrosis in experimental animal models. METHODS: We used ELISA and fluorescence immunohistochemistry to determine CCL24 levels in serum and CCL24/CCR3 expression in skin biopsies of SSc patients. Skin fibroblasts and endothelial cells treated with CCL24 or SSc serum with or without CM-101 were used to follow cell activation and differentiation. Prevention and treatment in vivo bleomycin (BLM)-induced models were used to evaluate experimental dermal and pulmonary fibrosis progression following treatment with the CM-101 mAb. RESULTS: CCL24 circulating levels were significantly elevated in SSc patients. CCL24/CCR3 expression was strongly increased in SSc skin. Blockade of CCL24 with CM-101 significantly reduced the activation of dermal fibroblasts and their transition to myofibroblasts induced by SSc serum. CM-101 was also able to significantly inhibit endothelial cell activation induced by CCL24. In BLM-induced experimental animal models, CM-101 profoundly inhibited both dermal and pulmonary fibrosis and inflammation. CONCLUSIONS: CCL24 plays an important role in pathological processes of skin and lung inflammation and fibrosis. Inhibition of CCL24 by CM-101 mAb can be potentially beneficial for therapeutic use in SSc patients.

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.

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.

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.

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.

Clopidogrel attenuates atheroma formation and induces a stable plaque phenotype in apolipoprotein E knockout mice.

AIM: Clopidogrel is a widely used anti-thrombotic for the prevention of stent thrombosis and cardiovascular events in patients with coronary atherosclerosis. Clopidogrel has been shown to exhibit anti-inflammatory effects that are related to the attenuated activation of platelets. Atherosclerosis is a complex process in which the immune system and the endothelium appear to play a prominent role. Herein, we tested the hypothesis that clopidogrel will influence plaque size and composition in the atherosclerosis prone apolipoprotein E knockout (apoE KO) mouse model. METHODS AND RESULTS: Eight week old mice were fed daily with either PBS, 1 mg or 2 mg of clopidogrel for 10 weeks. Plaque size was evaluated in the aortic sinus and cellular and humoral responses were studied as well as splenic and bone marrow endothelial progenitors by FACS. Treatment with either 1 mg and 2 mg of clopidogrel significantly reduced plaque size and augmented its stability by increasing atheromatous fibrous area. Whereas antigen specific oxLDL immune response was not influenced by clopidogrel feeding, the number of atheroprotective regulatory CD4+CD25+ T cells was significantly increased. Moreover, clopidogrel treatment resulted in a prominent rise in splenic but not bone marrow derived Sca-1+/flk-1+ endothelial progenitors. CONCLUSION: Clopidogrel significantly reduces atheroma burden and stabilizes aortic sinus plaques in apoE KO mice. These effects may partially be mediated by upregulation of the regulatory T cell pool and splenic endothelial progenitor cells. These findings may expand the potential applications of clopidogrel in human subjects.

Hypoxia controls CD4+CD25+ regulatory T-cell homeostasis via hypoxia-inducible factor-1alpha.

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-1alpha on the homeostasis of naturally occurring regulatory T cells (Treg) and their transcriptional activator Foxp3. Hypoxia induced a time-dependent increase in HIF-1alpha 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-1alpha-dependent as they were abolished upon transfection with short-interfering RNAs for HIF-1alpha and promoted by HIF-1alpha 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-1alpha 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-1alpha 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 effect of HMG-CoA reductase inhibitors on naturally occurring CD4+CD25+ T cells.

Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) are in widespread use due to their LDL reducing properties and concomitant improvement of clinical outcome in patients with and without preexisting atherosclerosis. Considerable evidence suggests that immune mediated mechanisms play a dominant role in the beneficial effects of statins. Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) have a key role in the prevention of various inflammatory and autoimmune disorders by suppressing immune responses. We tested the hypothesis that statins influence the circulating number and the functional properties of Tregs. We studied the effects of in vivo and in vitro statin treatment of human and murine mononuclear cells on the number of Tregs and the expression level of their master transcription regulator, Foxp3. Atorvastatin, but not mevastatin nor pravastatin, treatment of human peripheral blood mononuclear cells (PBMCs) increased the number of CD4(+)CD25(high) cells, and CD4(+)CD25(+)Foxp3(+) cells. These Tregs, induced by atorvastatin, expressed high levels of Foxp3, which correlated with an increased regulatory potential. Furthermore, co-culture studies revealed that atorvastatin induced CD4(+)CD25(+)Foxp3(+) Tregs were derived from peripheral CD4(+)CD25(-)Foxp3(-) cells. Simvastatin and pravastatin treatment in hyperlipidemic subjects increased the number of Tregs. In C57BL/6 mice however, no effect of statins on Tregs was evident. In conclusion, statins appear to significantly influence the peripheral pool of Tregs in humans. This finding may shed light on the mechanisms governing the plaque stabilizing properties of statins.

Role of naturally occurring CD4+ CD25+ regulatory T cells in experimental atherosclerosis.

OBJECTIVE: Naturally occurring CD4+ CD25+ regulatory T cells (Tregs) exert suppressive effects on effector CD4 cells and downregulate experimental autoimmune disorders. We investigated the importance and potential role of Tregs in murine atherogenesis. METHODS AND RESULTS: Tregs were investigated comparatively between aged and young apolipoprotein E-knockout (ApoE-KO) mice and age-matched C57BL/6 littermates. The effect of oxidized LDL (oxLDL) was tested on the functional suppressive properties of Tregs from ApoE-KO and C57BL/6 mice. Tregs, CD4+ CD25- cells, and saline were infused into ApoE-KO mice to study their effects on atherogenesis. Treg numbers were reduced in atherosclerotic compared with nonatherosclerotic ApoE-KO mice. The functional suppressive properties of Tregs from ApoE-KO mice were compromised in comparison with those from their C57BL/6 littermates. Thus, oxLDL attenuated the suppressive properties of Tregs from C57BL/6 mice and more so in ApoE-KO mice. Transfer of Tregs from age-matched ApoE-KO mice resulted in significant attenuation of atherosclerosis compared with that after delivery of CD4+ CD25+/- T cells or phosphate-buffered saline. CONCLUSIONS: CD4+ CD25+ Tregs may play a protective role in the progression of atherosclerosis and could be considered a therapeutic tool if results from human studies can solidify observations in murine models.

Altered status of CD4(+)CD25(+) regulatory T cells in patients with acute coronary syndromes.

AIMS: Considerable evidence supports the role of innate and adaptive immunity in the progression and destabilization of the atheromatous plaque. Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) are a subpopulation of lymphocytes that are capable of suppressing the progression of experimental autoimmune disorders. We have hypothesized that peripheral numbers and function of Tregs would be deranged in patients with acute coronary syndromes (ACS). METHODS AND RESULTS: Peripheral numbers of Tregs were evaluated by FACS employing labelled antibodies to CD4 and CD25. Functional suppressive properties of Tregs were assayed by establishing a triple-cell culture in which purified Tregs were incubated with irradiated antigen-presenting cells and anti-CD3-activated responder T cells. Proliferation in the presence or absence of oxidized LDL (oxLDL) was evaluated by thymidine incorporation. mRNA and protein content of foxp3, a master transcriptional regulator of Tregs, were determined for all subjects. Patients with ACS exhibited significantly reduced numbers of peripheral Tregs as compared with patients with stable angina and normal coronary artery subjects. Moreover, oxLDL induced a more profound reduction in Treg numbers in patients with ACS. Tregs in ACS patients were significantly compromised as their ability to suppress responder CD4(+)CD25(-) T-cell proliferation was attenuated. mRNA and protein content of foxp3 were significantly reduced in purified Tregs obtained from patients with ACS. CONCLUSION: In patients with ACS, naturally occurring CD4(+)CD25(+) Treg numbers are reduced and their functional properties compromised. These findings may aid in understanding the mechanisms leading to culprit plaque associated T-cell activation in patients with ACS.

Expression, crystallization and phasing of vacuolar H(+)-ATPase subunit C (Vma5p) of Saccharomyces cerevisiae.

The expression, crystallization and phasing of subunit C (Vma5p) of the yeast (Saccharomyces cerevisiae) vacuolar proton-translocating ATPase (V-ATPase) is described. The expressed protein consists of 412 residues: 392 from the reading frame of Vma5p and 20 N-terminal residues originating from the plasmid. Diffraction-quality crystals were obtained using the hanging-drop and sitting-drop vapour-diffusion methods assisted by streak-seeding, with PEG 3350 as precipitant. The crystals formed in hanging drops diffracted to 1.80 A and belong to space group P4(3)2(1)2(1), with unit-cell parameters a = b = 62.54, c = 327.37 A, alpha = beta = gamma = 90 degrees. The structure was solved using SIRAS with a Lu(O2C2H3)2 heavy-atom derivative.