Anquan Liu

Induction of Dendritic Cell–Mediated T-Cell Activation by Modified but Not Native Low-Density Lipoprotein in Humans and Inhibition by Annexin A5 Involvement of Heat Shock Proteins

Objective—Atherosclerosis is an inflammatory disease, where activated immunocompetent cells, including dendritic cells (DCs) and T cells are abundant in plaques. Low-density lipoprotein modified either by oxidation (oxLDL) or by human group X-secreted phospholipase A2 (LDLx) and heat shock proteins (HSP), especially HSP60 and 90, have been implicated in atherosclerosis. We previously reported that Annexin A5 inhibits inflammatory effects of phospholipids, decreases vascular inflammation and improves vascular function in apolipoprotein E−/− mice. Here, we focus on the LDLx effects on human DCs and T cells. Approach and Results—Human DCs were differentiated from peripheral blood monocytes, stimulated by oxLDL or LDLx. Naive autologous T cells were cocultured with pretreated DCs. oxLDL and LDLx, in contrast to LDL, induced DC-activation and T-cell proliferation. T cells exposed to LDLx-treated DCs produced interferon-&ggr;, interleukin (IL)-17 but not IL-4 and IL-10. Annexin A5 abrogated LDLx effects on DCs and T cells and increased production of transforming growth factor-&bgr; and IL-10. Furthermore, IL-10 producing T cells suppressed primary T-cell activation via soluble IL-10, transforming growth factor-&bgr;, and cell–cell contact. Lentiviral-mediated shRNA knock-down HSP60 and 90 in DCs attenuated the effect of LDLx on DCs and subsequent T-cell proliferation. Experiments on DC and T cells derived from carotid atherosclerotic plaques gave similar results. Conclusions—Our data show that modified forms of LDL such as LDLx but not native LDL activate human T cells through DCs. HSP60 and 90 contribute to such T-cell activation. Annexin A5 promotes induction of regulatory T cells and is potentially interesting as a therapeutic agent.

Naturally Occurring Human Phosphorylcholine Antibodies Are Predominantly Products of Affinity-Matured B Cells in the Adult

Phosphorylcholine (PC) is a classic T-independent Ag that is exposed on apoptotic cells, oxidized phospholipids, and bacterial polysaccharides. Experimental as well as epidemiological studies have over the past decade implicated Abs against PC (anti-PC) as anti-inflammatory and a strong protective factor in cardiovascular disease. Although clinically important, little is known about the development of anti-PC in humans. This study was conceived to dissect the human anti-PC repertoire and generate human mAbs. We designed a PC-specific probe to identify, isolate, and characterize PC-reactive B cells from 10 healthy individuals. The donors had all mounted somatically mutated Abs toward PC using a broad variety of Ig genes. PC-reactive B cells were primarily found in the IgM+ memory subset, although significant numbers also were detected among naive, IgG+, and CD27+CD43+ B cells. Abs from these subsets were clonally related, suggesting a common origin. mAbs derived from the same donors exhibited equivalent or higher affinity for PC than the well-characterized murine T-15 clone. These results provide novel insights into the cellular and molecular ontogeny of atheroprotective PC Abs, thereby offering new opportunities for Ab-based therapeutic interventions.

IgM antibodies against malondialdehyde and phosphorylcholine are together strong protection markers for atherosclerosis in systemic lupus erythematosus: Regulation and underlying mechanisms

OBJECTIVES Phosphorylcholine (PC) and malondialdehyde (MDA) are generated during lipid peroxidation and form adducts with proteins as albumin as studied herein. Atherosclerosis and cardiovascular disease (CVD) are increased in systemic lupus erythematosus (SLE). We here investigate the role and regulation of IgM antibodies against PC (anti-PC) and MDA (anti-MDA). METHODS IgM anti-PC and anti-MDA in SLE patients (n=114) were compared with age- and sex-matched population-based controls (n=108). Common carotid intima-media thickness (IMT) and plaque occurrence were determined by B-mode ultrasound. Plaques were graded according to echogenicity (potentially vulnerability). Production of IgM anti-PC and anti-MDA by B cells was determined by ELISA and ELISPOT. The effect of anti-PC and anti-MDA on macrophage uptake of apoptotic cells and oxidative stress was studied by flow cytometry. RESULTS Above 66rd percentile together, IgM anti-PC and anti-MDA were striking protection markers for plaque prevalence and echolucency in SLE (OR: 0.08, CI: 0.01-0.46 and OR: 0.10, CI: 0.01-0.82), respectively, and risk markers for plaque prevalence when below 33rd percentile: OR: 3.79, CI: (1.10-13.00). In vitro, IgM anti-PC and anti-MDA were much higher when B cells were co-cultured with CD3 T cells. Anti-HLA-, anti-CD40 antibody or CD40 silencing abolished these effects. Uptake of apoptotic cells was increased by IgM anti-PC and anti-MDA. MDA induced increased oxidative stress, which was inhibited by IgM anti-MDA. CONCLUSIONS Unexpectedly, both IgM anti-MDA and IgM anti-PC are T-cell dependent and especially together, are strong protection markers for atherosclerosis in SLE. Underlying mechanisms include increased phagocytosis of apoptotic cells and decrease of oxidative stress.

Oxidized Low‐Density Lipoprotein (OxLDL)–Treated Dendritic Cells Promote Activation of T Cells in Human Atherosclerotic Plaque and Blood, Which Is Repressed by Statins: microRNA let‐7c Is Integral to the Effect

Background Activated T cells and dendritic cells (DCs) are colocalized in atherosclerotic plaques in association with plaque rupture. Oxidized low‐density lipoprotein (oxLDL) promotes immune activation and inflammation. We studied the effects of statins (atorvastatin and simvastatin) on human DC maturation and T‐cell activation. Methods and Results Human peripheral blood monocytes were differentiated to DCs and stimulated with oxLDL. T cells were isolated from carotid endarterectomy specimens from patients undergoing carotid endarterectomy or from healthy individuals. Naïve T cells were cocultured with pretreated DCs. The effects of statin were studied. OxLDL induced DC maturation and T‐cell activation. OxLDL induced atherogenic heat shock proteins (HSP) 60 and 90 and decreased potentially atheroprotective heat shock protein 27, effects restored by atorvastatin. T cells exposed to oxLDL‐treated DCs produced interferon‐γ and interleukin (IL)‐17. Atorvastatin and simvastatin suppressed the DC maturation showing lower expression of CD80, CD83, and CD86, and limited their production of tumor necrosis factor‐α, IL‐1β and IL‐6, and increased transforming growth factor‐β and IL‐10 secretion. Statin‐treated DCs inhibited Th1 and/or Th17 polarization by downregulation of transcriptional factors T‐bet and RORγt expression, and induced T regulatory cells with IL‐10 production. OxLDL‐induced miRNA let7c and phosphorylation of Akt and ERK were repressed by statins. Let‐7c had a pivotal role in mediating effect of oxLDL. Experiments on T cells derived from carotid atherosclerotic plaques or healthy individuals showed similar results. Conclusions Statins repress human DC maturation induced by oxLDL, limit T‐cell activation, and repress an atherogenic heat shock protein profile and promote induction of T regulatory cells. MicroRNA let‐7c is integral to the effects.

Induction of Dendritic Cell–Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60

Background Atherosclerosis is characterized by the presence of activated immune‐competent cells including dendritic cells (DCs) and T cells, dead cells, and oxidized low‐density lipoprotein. HSP60 (Heat shock protein 60) has been implicated in atherosclerosis. A plasma protein, Annexin A5, has atheroprotective properties. Methods and Results Human DCs differentiated from peripheral blood monocytes were treated with human HSP60 or HSP90 and autologous T cells were cocultured with these pretreated DCs (mDCs). HSP60 induced mDCs and T‐cell activation as determined by FACScan (Fluorescence associated cell scan), gene‐activation, and cytokine production. HSP60‐induced T‐cell activation was partly major histocompatibility complex class II–dependent. T cells exposed to HSP60‐treated mDCs produced interferon‐γ, interleukin‐17, but not transforming growth factor‐β. HSP60 did not promote expression of Toll‐like receptors 2 or 4. HSP90 promoted mDCs maturation but had no effect on T‐cell activation. Annexin A5 inhibited HSP60‐proinflammatory Th1/Th17 effects on mDCs and T cells, and partly bound HSP60. Further, Annexin A5 inhibited HSP‐induced activation of mDCs and also oxidized low‐density lipoprotein–induced HSP‐production from mDCs. Experiments on mDCs and T cells derived from carotid atherosclerotic plaques from patients with symptomatic carotid disease gave similar results as from blood donors. Conclusions HSP60 induces mDCs activation and partly major histocompatibility complex class II–dependent activation of blood‐ and plaque‐derived T cells, which is mostly of Th1/Th17 type. HSP60 could thus be an important T‐cell antigen in plaques, and also mediate oxidized low‐density lipoproteins immunogenic effects on DC‐T‐cell activation, promoting plaque rupture and clinical manifestations of cardiovascular disease. Annexin A5 inhibits both oxidized low‐density lipoprotein–induced HSP60, and HSP60‐mediated immune activation, which suggests a potential therapeutic role.

Human IgM Antibodies to Malondialdehyde Conjugated With Albumin Are Negatively Associated With Cardiovascular Disease Among 60‐Year‐Olds

Background Malondialdehyde (MDA) is generated during lipid peroxidation as in oxidized low‐density lipoprotein, but antibodies against oxidized low‐density lipoprotein show variable results in clinical studies. We therefore studied the risk of cardiovascular disease (CVD) associated with IgM antibodies against MDA conjugated with human albumin (anti‐MDA). Methods and Results In a 5‐ to 7‐year follow‐up of 60‐year‐old men and women from Stockholm County previously screened for cardiovascular risk factors (2039 men, 2193 women), 209 incident CVD cases (defined as new events of coronary heart disease, fatal and nonfatal myocardial infarction, ischemic stroke, and hospitalization for angina pectoris) and 620 age‐ and sex‐matched controls were tested for IgM anti‐MDA by ELISA. Antibody peptide/protein characterization was done using a proteomics de novo sequencing approach. After adjustment for smoking, body‐mass index, type 2 diabetes mellitus, hyperlipidemia, and hypertension, an increased CVD risk was observed in the low IgM anti‐MDA percentiles (below 10th and 25th) (odds ratio and 95% CI: 2.0; 1.19–3.36 and 1.67; 1.16–2.41, respectively). Anti‐MDA above the 66th percentile was associated with a decreased CVD risk (odds ratio 0.68; CI: 0.48–0.98). After stratification by sex, associations were only present among men. IgM anti‐MDA levels were lower among cases (median [interquartile range]: 141.0 [112.7–164.3] versus 147.4 [123.5–169.6]; P=0.0177), even more so among men (130.6 [107.7–155.3] versus 143.0 [120.1–165.2]; P=0.001). The IgM anti‐MDA variable region profiles are distinctly different and also more homologous in their content (correlates strongly with fewer peptides) than control antibodies (not binding MDA). Conclusions IgM anti‐MDA is a protection marker for CVD. This finding could have diagnostic and therapeutic implications.

THU0226 Differential susceptibility of TH17 and T regulatory cells to apoptosis in systemic lupus erythematosus patients – the modulatory effects of statin

Background Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder. Patients with SLE have accelerated cardiovascular disease. Recent studies show there are more Th17 while less T regulatory (Treg) cells in the SLE patients. Th17/Treg imbalance may contribute to the pathogenesis of SLE. Objectives To investigate the underlying mechanisms of Th17/Treg imbalance, we test the proportion and susceptibility of Th17 and Treg to apoptosis, and the modulatory effects of statin in the SLE patients. Methods Totally 17 SLE patients and 20 gender- and age-matched control subjects were enrolled for this study. Peripheral blood mononuclear cells were isolated, either analyzed ex vivo, or cultured in the conditions to induce Th17 and/or Treg polarization. The proportion of Th17/Treg cells and frequency responding to apoptosis were analyzed by multiple color flow cytometry. Cytokines in cell culture supernatants and plasma were tested by ELISA. T cell polarization-related transcription factors were detected by quantitative real time PCR. Results The proportion of Th17 (CD4+IL17+) cells were higher in SLE patients, both in ex vivo and in the Th17-polarizing cultures. While the frequencies of Treg (CD4+CD25+CD127dim/-) cells were lower in the corresponding populations. Higher levels of IL17 and IL6 were detected in plasma of SLE patients. Responding to CD95-induced apoptosis the frequency of CD4+IL17+ cells from SLE patients was substantially lower, but that of CD4+CD25+CD127dim/- cells from Treg-polarizing cultures was considerably higher. With treatment of atorvastatin, CD4+IL17+ cell population in cultures derived from SLE patients showed an increased susceptibility to CD95-induced apoptosis. However, the CD4+CD25+CD127dim/- cell population had reduced response to apoptosis. Accordingly, the ratio of transcription factor RORC/FoxP3 decreased in T cell cultures of SLE patients. Conclusions Th17 cells were more resistant than Treg cells to CD95-induced apoptosis in SLE as compared to control subjects. Statins counteracted the dysregulated susceptibility of SLE T cells to apoptosis. Our findings reveal a novel mechanism underlying the imbalance of Th17/Treg and show a potential interest to the treatment of the patients with SLE. Disclosure of Interest None declared

AB0142 Igm antibodies against phosphorylcholine promote polarization of t regulatory cells from patients with atherosclerotic plaques, systemic lupus erythematosus and healthy donors: a novel immunological concept

Background IgM antibodies against Phosphorylcholine (anti-PC) are negatively associated with atherosclerosis, cardiovascular disease (CVD) and systemic lupus erythematosus (SLE) where the risk of CVD and atherosclerosis is very high. We here study effects of IgM anti-PC on Th17 and T regulatory cells (Tregs). Objectives Immunomodulation in atherosclerosis and SLE could have a huge impact on disease prevention and treatment. Methods Mononuclear leukocytes were isolated from peripheral blood (PBMC) obtained from healthy blood donors, from six SLE patients with age- and sex-matched controls and from symptom-giving human atherosclerotic plaques. The proportion of Th17 (CD4+CCR6+) and Treg (CD4+CD25+CD127dim/-) cells were determined by flow cytometry in CD4+T cells after 6 days culture with Th17 or Treg-polarizing cytokines, with PMA and Ionomycin stimulation. IgM anti-PC were extracted from total IgM, with flow-through IgM as controls. Dendritic cells (DC) were differentiated from PBMC. Antibody peptide/protein characterization was done by a proteomics de novo sequencing approach. Results IgM anti-PC increased significantly the proportion of Tregs from healthy donors, SLE patients and from atherosclerotic plaque cells while control antibodies did not. T cells from SLE patients had a significantly lower proportion of Tregs and higher proportion of Th17 cells as compared to matched controls. IgM anti-PC but not control antibodies significantly reduced production of IL-17 and TNF-alpha in cell culture from SLE patients and from atherosclerotic plaque cells. IgM anti-PC interacted with CD40 and kept DCs in an immature stage potentially being tolerogenic. We identify differences on the IgM peptide expression level in anti-PC compared to control antibodies. Conclusions IgM anti-PC increase Tregs and having low levels could contribute to both SLE and atherosclerosis (and CVD) and could thus represent a novel underlying mechanism in these conditions. This finding could also have therapeutic implications. Disclosure of Interest None declared