Journal of Autoimmunity xxx (2015) 1e9
Contents lists available at ScienceDirect
Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm
CD4þCXCR3þ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus Marc Clement a, Nicolas Charles b, c, Brigitte Escoubet d, Kevin Guedj a, Marie-Paule Chauveheid e, Giuseppina Caligiuri a, c, Antonino Nicoletti a, c, Thomas Papo b, c, e, Karim Sacre b, c, e, * INSERM U1148, Universit e Paris Diderot, PRES Sorbonne Paris Cit e, Paris, France INSERM U1149, Universit e Paris Diderot, Laboratoire d'excellence INFLAMEX, PRES Sorbonne Paris Cit e, Paris, France c Departement Hospitalo-Universitaire FIRE (Fibrosis, Inflammation and Remodelling in Renal and Respiratory Diseases), Universit e Paris Diderot, PRES Sorbonne Paris Cit e, Paris, France d ^pital Bichat, Universit ^pitaux de Paris, Paris, France Departement de Physiologie, Ho e Paris Diderot, PRES Sorbonne Paris Cit e, Assistance Publique Ho e ^pital Bichat, Universit ^pitaux de Paris, Paris, France Departement de M edecine Interne, Ho e Paris Diderot, PRES Sorbonne Paris Cit e, Assistance Publique Ho a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2015 Received in revised form 1 July 2015 Accepted 1 July 2015 Available online xxx
Cardiovascular disease due to accelerated atherosclerosis is the leading cause of death in patients with systemic lupus erythematosus (SLE). Noteworthy, accelerated atherosclerosis in SLE patients appears to be independant of classical Framingham risk factors. This suggests that aggravated atherosclerosis in SLE patients may be a result of increased inflammation and altered immune responses. However, the mechanisms that mediate the acceleration of atherosclerosis in SLE remain elusive. Based on experimental data which includes both humans (SLE patients and control subjects) and rodents (ApoE/ mice), we herein propose a multi-step model in which the immune dysfunction associated with SLE (i.e. high level of IFN-a production by TLR 9-stimulated pDCs) is associated with, first, an increased frequency of circulating pro inflammatory CD4þCXCR3þ T cells; second, an increased production of CXCR3 ligands by endothelial cells; third, an increased recruitment of pro-inflammatory CD4þCXCR3þ T cells into the arterial wall, and fourth, the development of atherosclerosis. In showing how SLE may promote accelerated atherosclerosis, our model also points to hypotheses for potential interventions, such as pDCstargeted therapy, that might be studied in the future. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Lupus Accelerated atherosclerosis CXCR3þCD4þ T cells Plasmacytoid dendritic cells Chemokines
1. Introduction In patients with systemic lupus erythematosus (SLE), cardiovascular disease (CVD) caused by atherosclerosis occurs more frequently and with earlier onset as compared to general population [1e5]. Traditional cardiovascular risk factors do not fully account for such CVD increased propensity [2]. Interestingly, accelerated atherosclerosis is associated with longer disease duration, supporting the hypothesis that chronic SLE immune dysregulation promotes atherosclerosis [6]. Accordingly, SLE-specific
* Corresponding author. Department of Internal Medicine, Assistance Publique Hopitaux de Paris, University Paris Diderot, 46 rue Henri Huchard, 75018, Paris, France. E-mail address: [email protected] (K. Sacre).
inflammatory pathways may drive cardiovascular damage. However the specific mechanisms by which SLE pathogenesis contribute to accelerate atherosclerosis have not been directly addressed. Aberrant recognition of self-nucleic acids by plasmacytoid dendritic cells (pDCs) through innate immunoreceptors, such as endosomal Toll like receptors TLR7 and TLR9, is a key factor of SLE pathogenesis. The main consequence of innate recognition of nucleic acids by pDCS is the secretion of type I interferon (IFN) such as IFN-a which strongly contributes to SLE development [7e9]. In addition to SLE pathogenesis, evidence indicates that pDCs play an important role in atherosclerosis development. Indeed, IFNa-producing pDCs are increased in atherosclerotic plaques and pDC depletion is found protective in murine atherosclerosis models [10e12]. Moreover SLE patients with raised type I IFNs signature
http://dx.doi.org/10.1016/j.jaut.2015.07.001 0896-8411/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: M. Clement, et al., CD4þCXCR3þ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.07.001
2
M. Clement et al. / Journal of Autoimmunity xxx (2015) 1e9
have increased carotid intima media thickness and coronary calcification score [13]. Chronic inflammation and T cell activation are thought to play a central role in the development of atherosclerosis. Activated T cells are prominent in early atherosclerotic lesions, suggesting that atherosclerosis is initiated by vessel-infiltrating T cells [14]. Among the chemokine receptors involved in tissue migration of T cells, CXCR3 e and its ligands: CXCL9, CXCL10 and CXCL11 e have been associated with atherosclerosis [15,16]. Interestingly, abrogation of type I IFN pathways in atherosclerosis-prone mice decreases plaque severity and T cell infiltration [12]. Of note CXCR3 is up-regulated on memory CD4þ T cells in SLE patients [17]. Given the above observations, we hypothesized that T cells expressing CXCR3 might play a key role in the pathogenesis of atherosclerosis associated with SLE and that this role might be proximally driven by IFN-a-producing pDCs. The following studies provide compelling data in support of this hypothesis and give a comprehensive picture of how lupus immunopathogenesis may promote accelerated atherosclerosis. 2. Materials and methods 2.1. Studied populations From February 2012 to March 2013, 51 consecutive patients with SLE patients and 18 healthy controls were recruited at the Department of Internal Medicine of Bichat Hospital (Paris Diderot University, France). All SLE patients fulfilled at least four of the American College of Rheumatology criteria for SLE [18]. Exclusion criteria consisted of known coronary disease or symptoms of cardiovascular diseases (angina, arrhythmia, congestive heart failure, stroke, or peripheral arterial disease). Controls were volunteer health-workers who had prospectively undergone vascular ultrasound imaging and were considered with respect to age, sex and Framingham score. None had coronary disease or symptoms suggestive of CVD. Extensive screening for conventional cardiovascular risk factors (age, sex, smoking status, family history of coronary artery disease, body-mass index, waist circumference, hypertension, diabetes mellitus, dyslipidemia, Framingham score for 10-year risk of heart attack) was undertaken in all cases. SLE-related data were also analyzed: nephritis, thrombosis, central nervous system involvement, Raynaud's phenomenon, valvular heart disease, pulmonary hypertension and medication including hormonal contraception, statins, antiplatelet, anticoagulant, hydroxychloroquine, glucocorticoid, immunosuppressive treatments. A family history of coronary artery disease was defined when a first-degree relative had suffered a myocardial infarction or stroke before the age of 55 years in males or before the age of 65 years in females. Height and weight were measured, and the body-mass index was computed as the weight in kilograms divided by the square of the height in meters. Subjects were considered to have hypertension if they repeatedly had a systolic blood pressure (SBP) of at least 140 mm Hg or a diastolic blood pressure of at least 90 mm Hg. All patients with hypertension were receiving antihypertensive medications. Diabetes mellitus was defined by a persistent finding of fasting hyperglycemia or the need for antidiabetic drug therapy. Dyslipidemia was defined as a low density lipoprotein cholesterol (LDL-C) >1.3 g/L. The risk for cardiovascular events was calculated as the absolute risk within the next 10 years using the Framingham risk equation, which includes age, sex, total cholesterol level, high-density lipoprotein cholesterol level, smoking history, and SBP. Systemic lupus erythematosus disease activity was assessed
using the SELENA-SLEDAI score [19]. The diagnosis of antiphospholipid syndrome (APS) was based on a history of venous and/or arterial thromboses or recurrent miscarriages in the presence of antiphospholipid antibodies in accordance with published criteria [20]. Blood tests included complete blood count, creatinine, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glycated hemoglobin, homocysteine, 25(OH)-D3 vitamin, lupus serology (anti-DNA antibodies, complement) and antiphospholipid antibodies (anticardiolipin and anti-b2GP1 antibodies, lupus anticoagulant). Urine sample was assayed for creatinine, red blood cells and proteins. The local ethics committee approved the study (Institutional Review Board e IRB 00006477 e of HUPNVS, Paris 7 University, APHP). All patients provided written informed consent. 2.2. Vascular assessment Vascular ultrasound study was performed in the context of care, in a temperature-controlled room after a 15 min rest (Vivid 7, General Electric, Horten, Norway). A single investigator (BE) conducted vascular measurements in SLE patients and controls on the same day as the blood collection for immunological study. All data were analyzed offline (EchoPACTM, General Electric Ultrasound). Investigator was unaware of the immunological outcome assessments. Internal carotid wall thickness (ICWT) was measured at the carotid bulb level at end diastole, as gated on ECG. Right and left values were averaged for each patient. Carotid plaques were defined as thickness greater than 2 mm. 2.3. PBMCs collection Human blood samples were collected into EDTA and PBMCs were prepared using a ficoll hypaque gradient. Cells were washed in phosphate buffered saline (PBS) and cryopreserved in FCS containing 10% DMSO. 2.4. Flow cytometry The panels of antibodies used for phenotypic detection and intracellular cytokine detection are described in Supplementary Table 1. T cell cytokine assays were performed in vitro on 5 105 cells after stimulation with or without anti-CD3 (1 mg/ml) e anti-CD28 (2.5 mg/ml) antibodies (BD Pharmingen, San Jose, CA) for 6 h at 37 C in 5% CO2 in the presence of brefeldin A (GolgiPlug, BD Pharmingen, San Jose, CA) to block cytokine secretion. pDC cytokine assays were performed on 106 cells after stimulation with or without TLR-9 agonist, CpG-A ODN 2216 (5 mM; InvivoGen, San Diego, California, USA) for 5 h at 37 C in 5% CO2. Brefeldin A (GolgiPlug, BD Pharmingen, San Diego, CA, USA) was added during the final 3 h of stimulation. Stat1 phosphorylation assays were performed on 106 cells after stimulation or not with IFN-a (104 U/ ml; Tebu-Bio, Le Perray-en-Yvelines, France) for 15 min at 37 C. IFN-a T cell stimulation assays were performed on 106 cells after stimulation or not with IFN-a (104 U/ml; Tebu-Bio) at 37 C in 5% CO2 for 30 h. Phenotyping, cytokine and stat1 phosphorylation detections were performed by cell surface staining and subsequent intracellular staining following the manufacturer's instruction. FACS analysis was performed on one four-laser BD LSR- Fortessa (BD Pharmingen, San Jose, CA). Flow cytometer data were analyzed with FlowJo software v8-6 (Treestar, Ashland, OR, USA) and transferred into analysis and graphic software GraphPad Prism 5.0a software (La Jolla, CA, USA). The strategy used to gate the different subsets of PBMCs is shown in Supplementary Figs. 1e3.
Please cite this article in press as: M. Clement, et al., CD4þCXCR3þ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.07.001
M. Clement et al. / Journal of Autoimmunity xxx (2015) 1e9
3
2.5. Enzyme-linked immunosorbent assays (ELISAs)
2.10. Statistical analysis
Monokine induced by IFN-g (MIG)/CXCL9, IFN-g-induced protein 10 (IP-10)/CXCL10, interferon-inducible T-cell alpha chemoattractant (ITAC)/CXCL11 and TNF-a were detected in human and mice sera and culture supernatants with a commercially available enzyme-linked immunosorbent assay (ELISA) detection kit (R&D Systems Europe, Lille, France). dsDNA coated plates (Calbiotech) were incubated with 1/50 dilution of mouse serum in PBS containing 5% FCS (Invitrogen). Horseradish peroxidase-conjugated antibody to mouse IgG (goat anti mouse IgG-Fc fragment, Bethyl laboratories) was used for detection. Colorimetric detection was with tetramethylbenzidine substrate (TMB, Invitrogen) and the optical density (OD) was measured at 450 nm.
Exact nonparametric two-tailed tests were used. The KruskaleWallis One Way Analysis of Variance on Ranks (ANOVA) or the ManneWhitney tests were used to compare continuous variables. The Dunn's multiple comparison test was used for statistical correction of multiple comparisons. The Fisher's exact test was used to compare dichotomous variables. The Pearson rank correlation test was used to determine correlations between variables, with r being the Pearson correlation coefficient. Statistical analysis was performed with GraphPad Prism 5.01 software. P values 2, n ¼ 29) and inactive (SLEDAI ¼ 0, n ¼ 22) disease and controls (n ¼ 18). SLEDAI ranged from 2 to 13 (median of 3) in SLE patients with active disease. (B) Progression overtime of serum level of CXCL9 (circle), CXCL10 (square) and CXCL11 (triangle) in three SLE-patients (SLE P1, P2, P3) who experienced a severe SLE flare (M0) and achieved complete remission during follow-up. SLEDAI score in SLE P1 was 27, 4 and 0 at M0, M3 and M6, respectively. SLEDAI score in SLE P2 was 25, 4, 2 and 2 at M0, M3, M6, and M12, respectively. SLEDAI score in SLE P3 was 2, 22, 4, 2 and 2 at M-7, M0, M3, M6, and M12, respectively. pg/ml referred to picogramm per milliliter. *, referred to p < 0.05; **, referred to p ¼
Contents lists available at ScienceDirect
Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm
CD4þCXCR3þ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus Marc Clement a, Nicolas Charles b, c, Brigitte Escoubet d, Kevin Guedj a, Marie-Paule Chauveheid e, Giuseppina Caligiuri a, c, Antonino Nicoletti a, c, Thomas Papo b, c, e, Karim Sacre b, c, e, * INSERM U1148, Universit e Paris Diderot, PRES Sorbonne Paris Cit e, Paris, France INSERM U1149, Universit e Paris Diderot, Laboratoire d'excellence INFLAMEX, PRES Sorbonne Paris Cit e, Paris, France c Departement Hospitalo-Universitaire FIRE (Fibrosis, Inflammation and Remodelling in Renal and Respiratory Diseases), Universit e Paris Diderot, PRES Sorbonne Paris Cit e, Paris, France d ^pital Bichat, Universit ^pitaux de Paris, Paris, France Departement de Physiologie, Ho e Paris Diderot, PRES Sorbonne Paris Cit e, Assistance Publique Ho e ^pital Bichat, Universit ^pitaux de Paris, Paris, France Departement de M edecine Interne, Ho e Paris Diderot, PRES Sorbonne Paris Cit e, Assistance Publique Ho a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 June 2015 Received in revised form 1 July 2015 Accepted 1 July 2015 Available online xxx
Cardiovascular disease due to accelerated atherosclerosis is the leading cause of death in patients with systemic lupus erythematosus (SLE). Noteworthy, accelerated atherosclerosis in SLE patients appears to be independant of classical Framingham risk factors. This suggests that aggravated atherosclerosis in SLE patients may be a result of increased inflammation and altered immune responses. However, the mechanisms that mediate the acceleration of atherosclerosis in SLE remain elusive. Based on experimental data which includes both humans (SLE patients and control subjects) and rodents (ApoE/ mice), we herein propose a multi-step model in which the immune dysfunction associated with SLE (i.e. high level of IFN-a production by TLR 9-stimulated pDCs) is associated with, first, an increased frequency of circulating pro inflammatory CD4þCXCR3þ T cells; second, an increased production of CXCR3 ligands by endothelial cells; third, an increased recruitment of pro-inflammatory CD4þCXCR3þ T cells into the arterial wall, and fourth, the development of atherosclerosis. In showing how SLE may promote accelerated atherosclerosis, our model also points to hypotheses for potential interventions, such as pDCstargeted therapy, that might be studied in the future. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Lupus Accelerated atherosclerosis CXCR3þCD4þ T cells Plasmacytoid dendritic cells Chemokines
1. Introduction In patients with systemic lupus erythematosus (SLE), cardiovascular disease (CVD) caused by atherosclerosis occurs more frequently and with earlier onset as compared to general population [1e5]. Traditional cardiovascular risk factors do not fully account for such CVD increased propensity [2]. Interestingly, accelerated atherosclerosis is associated with longer disease duration, supporting the hypothesis that chronic SLE immune dysregulation promotes atherosclerosis [6]. Accordingly, SLE-specific
* Corresponding author. Department of Internal Medicine, Assistance Publique Hopitaux de Paris, University Paris Diderot, 46 rue Henri Huchard, 75018, Paris, France. E-mail address: [email protected] (K. Sacre).
inflammatory pathways may drive cardiovascular damage. However the specific mechanisms by which SLE pathogenesis contribute to accelerate atherosclerosis have not been directly addressed. Aberrant recognition of self-nucleic acids by plasmacytoid dendritic cells (pDCs) through innate immunoreceptors, such as endosomal Toll like receptors TLR7 and TLR9, is a key factor of SLE pathogenesis. The main consequence of innate recognition of nucleic acids by pDCS is the secretion of type I interferon (IFN) such as IFN-a which strongly contributes to SLE development [7e9]. In addition to SLE pathogenesis, evidence indicates that pDCs play an important role in atherosclerosis development. Indeed, IFNa-producing pDCs are increased in atherosclerotic plaques and pDC depletion is found protective in murine atherosclerosis models [10e12]. Moreover SLE patients with raised type I IFNs signature
http://dx.doi.org/10.1016/j.jaut.2015.07.001 0896-8411/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: M. Clement, et al., CD4þCXCR3þ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.07.001
2
M. Clement et al. / Journal of Autoimmunity xxx (2015) 1e9
have increased carotid intima media thickness and coronary calcification score [13]. Chronic inflammation and T cell activation are thought to play a central role in the development of atherosclerosis. Activated T cells are prominent in early atherosclerotic lesions, suggesting that atherosclerosis is initiated by vessel-infiltrating T cells [14]. Among the chemokine receptors involved in tissue migration of T cells, CXCR3 e and its ligands: CXCL9, CXCL10 and CXCL11 e have been associated with atherosclerosis [15,16]. Interestingly, abrogation of type I IFN pathways in atherosclerosis-prone mice decreases plaque severity and T cell infiltration [12]. Of note CXCR3 is up-regulated on memory CD4þ T cells in SLE patients [17]. Given the above observations, we hypothesized that T cells expressing CXCR3 might play a key role in the pathogenesis of atherosclerosis associated with SLE and that this role might be proximally driven by IFN-a-producing pDCs. The following studies provide compelling data in support of this hypothesis and give a comprehensive picture of how lupus immunopathogenesis may promote accelerated atherosclerosis. 2. Materials and methods 2.1. Studied populations From February 2012 to March 2013, 51 consecutive patients with SLE patients and 18 healthy controls were recruited at the Department of Internal Medicine of Bichat Hospital (Paris Diderot University, France). All SLE patients fulfilled at least four of the American College of Rheumatology criteria for SLE [18]. Exclusion criteria consisted of known coronary disease or symptoms of cardiovascular diseases (angina, arrhythmia, congestive heart failure, stroke, or peripheral arterial disease). Controls were volunteer health-workers who had prospectively undergone vascular ultrasound imaging and were considered with respect to age, sex and Framingham score. None had coronary disease or symptoms suggestive of CVD. Extensive screening for conventional cardiovascular risk factors (age, sex, smoking status, family history of coronary artery disease, body-mass index, waist circumference, hypertension, diabetes mellitus, dyslipidemia, Framingham score for 10-year risk of heart attack) was undertaken in all cases. SLE-related data were also analyzed: nephritis, thrombosis, central nervous system involvement, Raynaud's phenomenon, valvular heart disease, pulmonary hypertension and medication including hormonal contraception, statins, antiplatelet, anticoagulant, hydroxychloroquine, glucocorticoid, immunosuppressive treatments. A family history of coronary artery disease was defined when a first-degree relative had suffered a myocardial infarction or stroke before the age of 55 years in males or before the age of 65 years in females. Height and weight were measured, and the body-mass index was computed as the weight in kilograms divided by the square of the height in meters. Subjects were considered to have hypertension if they repeatedly had a systolic blood pressure (SBP) of at least 140 mm Hg or a diastolic blood pressure of at least 90 mm Hg. All patients with hypertension were receiving antihypertensive medications. Diabetes mellitus was defined by a persistent finding of fasting hyperglycemia or the need for antidiabetic drug therapy. Dyslipidemia was defined as a low density lipoprotein cholesterol (LDL-C) >1.3 g/L. The risk for cardiovascular events was calculated as the absolute risk within the next 10 years using the Framingham risk equation, which includes age, sex, total cholesterol level, high-density lipoprotein cholesterol level, smoking history, and SBP. Systemic lupus erythematosus disease activity was assessed
using the SELENA-SLEDAI score [19]. The diagnosis of antiphospholipid syndrome (APS) was based on a history of venous and/or arterial thromboses or recurrent miscarriages in the presence of antiphospholipid antibodies in accordance with published criteria [20]. Blood tests included complete blood count, creatinine, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, glycated hemoglobin, homocysteine, 25(OH)-D3 vitamin, lupus serology (anti-DNA antibodies, complement) and antiphospholipid antibodies (anticardiolipin and anti-b2GP1 antibodies, lupus anticoagulant). Urine sample was assayed for creatinine, red blood cells and proteins. The local ethics committee approved the study (Institutional Review Board e IRB 00006477 e of HUPNVS, Paris 7 University, APHP). All patients provided written informed consent. 2.2. Vascular assessment Vascular ultrasound study was performed in the context of care, in a temperature-controlled room after a 15 min rest (Vivid 7, General Electric, Horten, Norway). A single investigator (BE) conducted vascular measurements in SLE patients and controls on the same day as the blood collection for immunological study. All data were analyzed offline (EchoPACTM, General Electric Ultrasound). Investigator was unaware of the immunological outcome assessments. Internal carotid wall thickness (ICWT) was measured at the carotid bulb level at end diastole, as gated on ECG. Right and left values were averaged for each patient. Carotid plaques were defined as thickness greater than 2 mm. 2.3. PBMCs collection Human blood samples were collected into EDTA and PBMCs were prepared using a ficoll hypaque gradient. Cells were washed in phosphate buffered saline (PBS) and cryopreserved in FCS containing 10% DMSO. 2.4. Flow cytometry The panels of antibodies used for phenotypic detection and intracellular cytokine detection are described in Supplementary Table 1. T cell cytokine assays were performed in vitro on 5 105 cells after stimulation with or without anti-CD3 (1 mg/ml) e anti-CD28 (2.5 mg/ml) antibodies (BD Pharmingen, San Jose, CA) for 6 h at 37 C in 5% CO2 in the presence of brefeldin A (GolgiPlug, BD Pharmingen, San Jose, CA) to block cytokine secretion. pDC cytokine assays were performed on 106 cells after stimulation with or without TLR-9 agonist, CpG-A ODN 2216 (5 mM; InvivoGen, San Diego, California, USA) for 5 h at 37 C in 5% CO2. Brefeldin A (GolgiPlug, BD Pharmingen, San Diego, CA, USA) was added during the final 3 h of stimulation. Stat1 phosphorylation assays were performed on 106 cells after stimulation or not with IFN-a (104 U/ ml; Tebu-Bio, Le Perray-en-Yvelines, France) for 15 min at 37 C. IFN-a T cell stimulation assays were performed on 106 cells after stimulation or not with IFN-a (104 U/ml; Tebu-Bio) at 37 C in 5% CO2 for 30 h. Phenotyping, cytokine and stat1 phosphorylation detections were performed by cell surface staining and subsequent intracellular staining following the manufacturer's instruction. FACS analysis was performed on one four-laser BD LSR- Fortessa (BD Pharmingen, San Jose, CA). Flow cytometer data were analyzed with FlowJo software v8-6 (Treestar, Ashland, OR, USA) and transferred into analysis and graphic software GraphPad Prism 5.0a software (La Jolla, CA, USA). The strategy used to gate the different subsets of PBMCs is shown in Supplementary Figs. 1e3.
Please cite this article in press as: M. Clement, et al., CD4þCXCR3þ T cells and plasmacytoid dendritic cells drive accelerated atherosclerosis associated with systemic lupus erythematosus, Journal of Autoimmunity (2015), http://dx.doi.org/10.1016/j.jaut.2015.07.001
M. Clement et al. / Journal of Autoimmunity xxx (2015) 1e9
3
2.5. Enzyme-linked immunosorbent assays (ELISAs)
2.10. Statistical analysis
Monokine induced by IFN-g (MIG)/CXCL9, IFN-g-induced protein 10 (IP-10)/CXCL10, interferon-inducible T-cell alpha chemoattractant (ITAC)/CXCL11 and TNF-a were detected in human and mice sera and culture supernatants with a commercially available enzyme-linked immunosorbent assay (ELISA) detection kit (R&D Systems Europe, Lille, France). dsDNA coated plates (Calbiotech) were incubated with 1/50 dilution of mouse serum in PBS containing 5% FCS (Invitrogen). Horseradish peroxidase-conjugated antibody to mouse IgG (goat anti mouse IgG-Fc fragment, Bethyl laboratories) was used for detection. Colorimetric detection was with tetramethylbenzidine substrate (TMB, Invitrogen) and the optical density (OD) was measured at 450 nm.
Exact nonparametric two-tailed tests were used. The KruskaleWallis One Way Analysis of Variance on Ranks (ANOVA) or the ManneWhitney tests were used to compare continuous variables. The Dunn's multiple comparison test was used for statistical correction of multiple comparisons. The Fisher's exact test was used to compare dichotomous variables. The Pearson rank correlation test was used to determine correlations between variables, with r being the Pearson correlation coefficient. Statistical analysis was performed with GraphPad Prism 5.01 software. P values 2, n ¼ 29) and inactive (SLEDAI ¼ 0, n ¼ 22) disease and controls (n ¼ 18). SLEDAI ranged from 2 to 13 (median of 3) in SLE patients with active disease. (B) Progression overtime of serum level of CXCL9 (circle), CXCL10 (square) and CXCL11 (triangle) in three SLE-patients (SLE P1, P2, P3) who experienced a severe SLE flare (M0) and achieved complete remission during follow-up. SLEDAI score in SLE P1 was 27, 4 and 0 at M0, M3 and M6, respectively. SLEDAI score in SLE P2 was 25, 4, 2 and 2 at M0, M3, M6, and M12, respectively. SLEDAI score in SLE P3 was 2, 22, 4, 2 and 2 at M-7, M0, M3, M6, and M12, respectively. pg/ml referred to picogramm per milliliter. *, referred to p < 0.05; **, referred to p ¼
