RICERCA ITALIANA     

Adipokines, inflammatory cytokines and regulatory T cells role in accelerated atherosclerosis and metabolic syndrome pathogenesis, over the course of systemic lupus erythematosus

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune, multisystemic disorder, characterized by a broad spectrum of clinical and immunological manifestations. Many studies have been shown the SLE patients to have a 5 to 10-fold increased risk for cardio-vascular diseases (CVD) compared with age-matched general population, and strikingly this tendency towards CVD is higher for young fertile women. It is now well established that atherosclerosis is characterized by a persistent low-grade inflammatory state. Indeed, immune cell activation is involved in all stages of atherogenesis.
Recently, there is an increasing interest to the role of metabolic syndrome in SLE. Metabolic syndrome is characterized by insulin-resistance (IR), obesity, dyslipidaemia and hypertension and represents an independent cardiovascular and death risk factor from all cause. As in atherogenesis, inflammation plays a crucial role in development and maintenance of insulin resistance, thereby leading to metabolic syndrome.
In the immunoflogistic setting of SLE, the pathogenesis of atherosclerosis and metabolic syndrome may be based on a common inflammatory cellular and molecular milieu, involving proinflammatory cytokines, adipokines and regulatory T cells.
Adipokines leptin, resistin, visfatin and adiponectin, recently discovered peptides secreted by adipose tissue and immune cells, are involved in atherogenesis, metabolic syndrome pathogenesis and inflammation. Many reports suggest an adipokines involvement in autoimmunity.
Proinflammatory cytokines as TNF-alfa, IL-1, IL-6, INF-gamma, and IL-10 seem play a pivotal role in SLE phatogenesis. Moreover, these molecules exert a proatherogenitic action and lead to insuline-resistence.
Tregs are subset of T lymphocytes, which play a central role in inducing and maintaining immunologic tolerance. Deficiency or dysfunction of these cells lead to autoimmunity. It has been recently evaluated the role of Tregs in SLE, showing a reduced Tregs circulating levels in SLE patients. Furthermore, Tregs seem to be reduced in atherosclerosis. Leptin has shown to be able to inhibit Tregs function and proliferation.
AIM: To evaluate in SLE patients the cellular and molecular network involved in atherosclerosis and metabolic syndrome pathogenesis. Moreover, to estimate the metabolic syndrome prevalence among SLE patients.
UNITS PROGRAM: UNIT I: Enrolment of 70 SLE patients and 30 consecutive controls. Laboratory evaluation. UNIT II: Ultrasonogarphic evaluation for flow-mediated vasorelaxion, arterial stiffness and intima-media thickening. Regulatory T cells analysis. Statistical analysis. UNIT III: Serum dosage of adipokines and cytokines.
According to the Helsinki convention, the project will be submitted to University "Campus Bio-Medico" of Rome Ethical Committee evaluation for consent.
ENROLMENT AND CLINICAL EVALUATION: 70 SLE patients and 30 consecutive controls will be enrolled, between 2008 and 2009 in Unit I. Blood specimens, morning and 24 hour urine specimens will be collected after an overnight fast. Anamnesis and physical examination will be collected through a case sheet, concerning: fertility or menopausal status, cardio-vascular risk, and metabolic syndrome. SLE patients will be evaluated for disease activity. All subject will be female. SLE patients will fulfil ACR criteria and controls have no history of autoimmune disease. Exclusion criteria will be: pregnancy, estroprogestinic assumption, nephropaty, liver diseases and cancer.
LABORATORY EVALUATION: Exams will be performed in order to define metabolic syndrome, cardiovascular risk, SLE disease activity. Lab evaluation will involve determination of autoantibodies, lupus anti-coagulant, and thrombophilic screening. Regulatory T cells will be quantify from peripheral bood, using citofluorimeter. Serum adipokines and cytokines will be dosed using available ELISA kits.
ULTRASONOGRAPHIC EVALUATION: Will be evaluated common carotid intima-media thickening and arterial stiffness, and omeral artery flow-mediated vasorelation degree. <<<

Principal Investigator

Antonella Maria Vittoria Afeltra Università "Campus Bio-Medico" ROMA

Research Objectives

GENERAL OBJECTIVE.
The aim of the project is to evaluate the cellular and molecular network that underlines the condition of subclinical atherosclerosis and metabolic syndrome, over the course of systemic lupus erythematosus (SLE).

SPECIFIC OBJECTIVES.
Particular attention will be lent to evaluate in SLE patients:
- the adipokines and inflammatory cytokines serum levels, compared to healthy subjects, and their correlation with disease activity scores, metabolic syndrome presence and the endothelial dysfunction and/or damage condition;
- the peripheral blood regulatory T cell Foxp3+ amount and their potential correlation with disease activity scores and the endothelial dysfunction and/or damage grade;
- the possible correlation between adipokines and inflammatory cytokines serum levels and the peripheral blood regulatory T cell Foxp3+ amount, assessing their role in the pathogenesis of SLE;
- the metabolic syndrome prevalence and the potential correlation between any parameter of the syndrome and the disease activity scores and/or inflammation serological markers;
- the presence of endothelial dysfunction and/or damage grade and its possible correlation with the disease activity scores. <<<

First Results

Several epidemiological studies have shown, during the last years, an increased prevalence and a premature onset of cardiovascular disease (CVD), due to atherosclerotic lesions, in patients affected by systemic lupus erythematosus (SLE). Nowadays, CVD represents the main cause of morbidity and mortality among SLE patients. Indeed, many researchers suggested that, in addition to traditional cardiovascular risk factors, over the course of SLE others conditions could be implied in the atherosclerosis (ATS) process: autoantibodies, immunocomplexes, proinflammatory cytokines, oxidative stress, endothelial apoptosis, and steroidal and immunosuppressive treatment.
The ultrasonographic (US) study for intima-media thickness (IMT) determination has been demonstrated to be an effective diagnostic tool for estimation of future cerebral-cardiovascular events onset.
Moreover, echographic parameters of vascular stiffness and the flow-mediated vasodilation (VMD), allow to investigate the presence and severity of endothelial dysfunction, considered the early stage of ATS process. In the light of these evidences, we believe that our project will show an higher endothelial dysfunction and damage degree in SLE patients compared to healthy subjects. This datum may confirm the practical utility of US evaluation also in SLE patients. Thus, the matched US indices of damage and endothelial dysfunction assessment may be suggested as ATS screening tool in lupic population, allowing an early identification of prone subjects in order to improve preventive and therapeutic strategies.
Metabolic syndrome (MS) is currently considered a low degree inflammatory state and it could represent an indipendent risk factor for CVD, osteoporosis and malignancies. In the immunoflogistic setting of SLE, the hyperexpression of proinflammatory molecules could lead to a reduced insulin sensitivity and subsequently to MS onset. Therefore, we believe to obtain an increased insulin resistance status and a higher MS prevalence in SLE patients compared to control group. Considering the above mentioned assumptions, it is possible to hypothesized a positive correlation between MS parameters and inflammatory markers and disease activity scores. Moreover, being MS an independent risk factor for CVD, we expect to see a higher damage and endothelial dysfunction state in SLE patients affected by MS.
Recent reports suggest a common cellular and molecular network in ATS and MS onset, over the course of SLE, and adipokines, inflammatory cytokines and regulatory T cells seem to be involved.
Several in vitro and in vivo experimental models have shown a pro-atherogenic, insulin resistance inducing and immune system activating role of adipokines. Moreover, it has been suggested that the adipokines hyperexpression may interfere with immunotolerance processes. Indeed, we expect elevated serum levels of adipokines in SLE patients and their correlation with worst disease condition. Mainly, adipokines serum levels may be related to high disease activity scores, and elevated inflammatory haematological indices and inflammatory serum cytokines levels.
Considering the regulatory T cells immunomodulating function, and the in vitro leptin inhibitory ability on these cells, we can assume a lower Tregs peripheral blood amount in those patients with leptin hyperexpression. A preliminary (pilot) study, performed by Unit I (not yet published data), has shown that there is a difference in adipokines levels between SLE patients and healthy subjects, in particular during fertile age. Our purpose is to confirm this finding in a wider case study, increasing the statistical power, and improving the knowledge about molecular and cellular network that underlines ATS and MS in SLE.
If our previous hypothesis will be confirmed, the gain understanding of adipokines, cytokines and regulatory T cells links would represent a relevant goal in the SLE chronic inflammation processes knowledge.
Therefore our project could allow:
- to evaluate, by the colourcodedDoppler, an easy and non invasive tool, early phases of ATS
- to optimize, assessing the MS coexistence, the cardiovascular risk management, in order to improve SLE patients prognosis, particularly among fertile women
- to understand those mechanisms that maintain the chronic inflammatory state in SLE, potentially adding new disease activity indices
Eventually, the development of therapies aimed to prevent ATS and MS, and to modulate the low grade inflammatory systemic status in SLE patients, will result in clinical benefits. These will include a reduction in morbidity and mortality and a better quality of life for these patients. <<<

Timescale

24 months

National and international background

Systemic lupus erythematosus (SLE) is an autoimmune, multisystemic disorder, characterized by a broad spectrum of clinical and immunological manifestations. Many studies have been shown the SLE patients to have a 5 to 10-fold increased risk for cardio-vascular diseases (CVD) compared with age-matched general population, and strikingly this propensity towards CVD is higher for young fertile women. Traditional risk factors for atherosclerosis (sex, age, smoke, hypertension, diabetes, dyslipidaemia, obesity, thrombophilic status), alone, doesn’t explain this phenomenon. Pathogenetic events occuring in SLE, such as auto-antibodies production, chronic inflammation, oxidative stress, endothelial apoptosis, renal diseases and treatment-related factors, should be investigated in order to establish their role in atherosclerosis. It is now well established that atherosclerosis is characterized by a persistent low-grade inflammatory state, in which immune cell activation is involved in all stages of atherogenesis. Endothelial dysfunction is defined as the impaired vasorelaxation response to endothelium-dependent vasodilatators such as nitric oxide (NO) or acetylcholine and is also associated with the loss of antithrombotic and anti-inflammatory properties of healthy endothelium. This condition is involved in the early stages of atherogenesis and plays a pivotal role in the initiation and propagation of the atherosclerosis process. Many markers are available to assess the endothelial function. Ultrasonography gives information about endothelial function evaluated by arterial stiffness parameters (expressing the low vascular elasticity) and by the flow-mediated vasodilation (FMD). FMD can be assessed by measuring the dilation of brachial artery in response to stimuli for endothelial nitric oxide production such as a charge of shear stress by temporal inflation of brachial cuff. This index is able to predict acute cardiovascular events. The presence of subclinical atherosclerosis ca be detected by measuring the intima-media thickening (IMT), an ultrasonographic index of cardiovascular risk. Several studies showed that SLE patients have impaired endothelial function compared to healthy controls when assessed as both a reduction of FMD or an increased vascular stiffness. Thus, SLE patients have higher IMT values than controls.
Recently, there is an increasing interest to the role of metabolic syndrome in autoimmune disease. Metabolic syndrome is characterized by insulin-resistance (IR), obesity, dyslipidaemia and hypertension. At present, the most widely accepted metabolic syndrome definition are four: WHO, EGIR, ATPIII/NCEP, IFD. It has been shown that non diabetic people with the metabolic syndrome have an increased risk of death from all causes. Moreover, metabolic syndrome is an independent risk factor for CVD. IR plays a key role in pathogenesis of metabolic syndrome and is linked to disturbances in glucose and lipid homeostasis (impaired fasting glucose, impaired glucose tolerance and diabetes type 2, dyslipidaemia), visceral fat expansion and cardiovascular dysfunction (hypertension). IR is defined as high plasma insulin (in the upper quartile of non diabetic population) or as HOMA-IR (a IR index from fasting plasma glucose and insulin). As in atherogenesis, inflammation plays a crucial role in developing and maintenance of insulin resistance, thereby leading to metabolic syndrome. Metabolic syndrome has been shown to be a thrombophilic status, associated to hyperexpression of prothrombotic factors as plasminoger activator inhibitor (PA-I). Recent reports suggest that patients with autoimmune disease may have an increased risk to develop metabolic syndrome. Indeed, in these disease, chronic inflammation, poor physical activity and steroids can lead to IR. Two studies reported increased insulin levels and a high prevalence of IR among SLE-patients. Recently, Chang et al., using WHO criteria, demonstrated in SLE an higher prevalence of metabolic syndrome compared with controls.
In the immunoflogistic setting of SLE, the pathogenesis of atherosclerosis and metabolic syndrome may be based on a common inflammatory cellular and molecular milieu, involving proinflammatory cytokines, adipokines and regulatory T cells.
Adipokinesn leptin, resistin, visfatin and adiponectin are recently discovered peptide, secreted by adipose tissue and immune cells. Leptin, in physiological conditions, acts as anorexigenic stimulus on hypothalamic interneuron and its serum levels are closely correlated with overall adipose mass. Leptin exerts a pro-inflammatory action and its excess semms to be involved in autoimmunity. They are well established the pro-atherogenic proprieties of leptin. In humans, leptin correlates with common carotid intima-media thickness (IMT), a marker of subclinical atherosclerosis. Thus, the WOSCOPS prospective trial demonstrated that hyperleptinemia is an independent risk factor for cardiovascular events. Two studies shown higher serum leptin in SLE patients than in controls. Adiponectin has a central role in regulating insulin-sensitivity and appears to act as an anti-inflammatory and anti-atherogenic molecule. Low plasma adiponectin is now recognized to be an indipdendent risk factor of cardiovascular morbidity. Thus, plasma adiponectin inversely correlates with IMT and directly with VMD, an ultrasonographic index of endothelial function. In SLE patients, adiponectin levels were shown to be increased. Resitin, in humans, is secreted by immune cells and was initially considered to be a potent link between obesity and insuline-resistance. Later studies did not support this original hypotesis. Resistin belongs to the family of resistin-like molecules (RELMs) or molecules “found in inflammatory zone” (FIZZ). The experimental studies suggest that resistin may act as pro-inflammatory and pro-atherogenic cytokine. In fact, resistin levels are increased in non-SLE subjects with CVD and correlate with IMT. Visfatin, a recently discovered adipokine secreted primary by visceral white adipose tissue (WAT), exert an isulin-mimetic action, binding to insulin receptor. The adipokine was initially discovered as pre-B-cell colony enhancing factor (PBEF) and shows an immuno-stimulating action.
In mice models and in SLE patients many abnormalities of cytokine network have been descried. Interleukin 10 (IL-10) is secreted by T helper 2 lymphocytes. This cytokine is able to facilitate B cells proliferation and differentiation and is involved in SLE pathogenesis. In SLE patients, increased levels of IL-10 have been shown and correlate with disease activity index and title of anti ds-DNA autoantibodies. Interferon gamma (INF-gamma) is primarily secreted by T lymphocytes and NK cells. Mice prone to SLE knocked-out for INF-gamma are protected to autoantibodies and immunmocomplex mediated damage. Many reports described an hyperexpression of INF-gamma, but not a correlation with disease activity index. Interleukin 6 is a pleiotrophic molecule. Its pharmacologic blockage in mice prone to SLE ameliorate disease activity. In vitro, IL-6 facilitates autoantibodies production by B cells from SLE patients. In SLE have been shown increased levels of IL-6 correlated with C-reactive protein. Many reports suggest the implication of TNF-alpha in SLE pathogenesis. In SLE patients, this molecule is hyperexpressed during glomerulonephritis, and may be involved in determining local damage. Interleukin 1 (IL-1) may be able to perpetuate the SLE related chronic inflammatory status. Indeed, in vitro, anti-dsDNA autoantibodies stimulate the IL-1 release. Many reports suggest that proinflammatory cytokines are involved in atherogenesis and in the pathogenesis of metabolic syndrome.
Work in the field of regulatory T cells (Tregs) immunology was greatly enhanced by the discovery of Treg-specific markers, as Foxp3. Tregs are subset of T lymphocytes, which play a central role in inducing and maintaining immunologic tolerance and the termination of immune response. Deficiency or dysfunction of these cells lead to autoimmunity or aggravated pathogen induced inflammation. Tregs exist in form of naturally occurring Tregs (nTregs) or inducible Tregs (iTregs). Among nTregs, the functionally most important cells are CD4+CD25high/Foxp3+ (Tregs Foxp3+), which express, as surface phenotype, the CD4, high levels of CD25 and Foxp3, a transcription factor specifically expressed by these kind of nTregs. It has been recently evaluated the role of nTregs in SLE. In mice models, Tregs depletion exacerbates lupus-like systemic syndromes. SLE patients has lower levels peripheral bllod Tregs then controls. It has been demonstrated a decrease in the suppressive function of Tregs Foxp3+ from peripheral blood of SLE patient as compared with normal donors.
Recently a T regs downregulation it has been shown to be involved in atherogenesis. In mice models, atherosclerosis is prevented by adoptive transfer of ex-vivo expanded Tregs. Low numbers of TregsFoxp3+are present in all developmental stages of human atherosclerotic lesions. High frequency of nTregs has been described in patients with ST-segment elevation myocardial infarction. Interestly, it has been demonstrated a relation between leptin and Tregs. In multiple sclerosis, cephalorachidial fluid leptin increase correlates with reduced number of nTregs and a in recent study leptin has been shown to modulate the proliferation end function of nTregs. <<<