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Total antibody deficiency in B cell sufficient mice enhances atherosclerosis
Abstracts / Atherosclerosis 241 (2015) e1ee31
Methods: Combined pharmacological and immuno-histochemistry approaches were used to define the anti-apoA-1 IgG target on NRVC and the potential signaling pathways involved in the response. Results: Blocking CD14 with specific antibodies suppressed the anti-apoA1 IgG binding to the cells and was followed by the complete abrogation of the anti-apoA-1 IgG-induced chronotropic response on NRVC. Pharmacological inhibition of src fully abrogated the anti-apoA-1 IgG-induced chronotropic response. Furthermore, MR-dependent sensitization of NRVC to anti-apoA-1 IgG chronotropic effect was accompanied by CD14 relocation within lipid rafts and specific activation of Pi3Kinase pathway through phosphorylation of AKT. Conclusion: Those results point to CD14 as one of the key target of antiapoA-1 IgG, necessary to mediate their positive chronotropic response on NRVC through an aldosterone and src-dependent pathway. Our results also indicate that the MR-dependent sensitization of NRVC requires the CD14 localisation into lipids raft, unravelling a novel non-genomic pathway downstream MR activation, as well as a novel modulator involved in heart rate regulation in vitro.
EAS-0566. APOLIPOPROTEIN E IN HUMANS AND MICE IMPACTS THE DIFFERENTIATION OF NAIVE CD4 T CELLS TOWARD EFFECTOR SUBSETS F. Bonacina 1, F. Pellegatta 2, L. Grigore 2, K. Garlaschelli 2, A. Baragetti 2, A.L. Catapano 1, G.D. Norata 1. 1 Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy; 2 SISA Center for the study of Atherosclerosis, Bassini Hospital, Cinisello Balsamo, Italy Immunity plays a major role during atherosclerosis with a prevalence of effector (TEM) versus central (TCM) memory T cells in both mice and humans. Aim of this work was to investigate how dysplipidemia related to apolipoprotein E deficiency (ApoE) affects naïve(TN) to TEM CD4+T cell polarization. ApoE KO mice present increased levels of circulating TEM(CD4+CD44+CD62L-) compared with C57Bl/6J mice which are correlated with the extension of atherosclerotic plaques in the aortic sinus. ApoE deficiency also affects T cells functionality; in fact i) upon stimulation with anti-CD3 and anti CD-28, lymphocytes from ApoE KO mice proliferate more compared to C57Bl/6J (4,5±0,3x10^4 vs 3,2±0,2x10^4 mitotic events,p¼0,0005);ii) following skin graft allotransplantation a faster rejection (after 10 days 100%vs62,5% rejection,p¼0,002), associated with the presence of TEM cells with increased migratory activity toward inflammatory chemokines is observed in ApoE KO mice;iii) conjugates formation between Tcells from ApoE KO and C57Bl/6J mice with CBA dendritic cells (DCs) was similar pointing to a role for ApoE in DCs on T cells polarization. In humans, circulating CD4þTcells subsets (FACS analysis for CD3,CD4,CD45RO,CD45RA,CCR7,CCR5,CXCR3,HLA-DR) were investigated. Percentage of plasma TEM were significantly correlated to cholesterol levels independently of other metabolic and cardiovascular risk factors and resulted increased in carriers of apoE4 isoform(ε4/2,4/3,4/4) compared to apoE2(ε2/2,2/3) and apoE3(ε3/3) carriers (13,69±4,0% vs 10,28±4,9% and 11,05±4,4%,p<0,01 and p<0,05). Furthermore, following mixed lymphocyte reaction(MLR), ApoE4 presented increased TEM levels compared to ApoE2 and ApoE3 carriers. All together our data suggest that ApoE mimetics might represent a target to limit the activation of DCs-TEM axis.
EAS-0586. TOTAL ANTIBODY DEFICIENCY IN B CELL SUFFICIENT MICE ENHANCES ATHEROSCLEROSIS
e7
and anti-atherogenic actions reported. The aim of this study was to specifically target antibodies but not other B cell functions and determine the effect on mouse atherosclerosis. Methods: We created MB1-Cre x XBP1fl/fl mice that have B cell specific deficiency in the unfolded protein response transcription factor XBP1, previously reported to result in a specific antibody secretion defect. Specific antibody responses were assessed by ELISA and ELISpot after immunization with NP-chicken gamma globulin. Chimeric Ldlr/ mice with bone marrow from MB1-Cre x XBP1fl/fl or control mice were fed a western diet for 8 weeks. Results: B cell XBP1 deficiency led to significantly attenuated antibody secretion after NP-CGG immunization. Analysis of ldlr/ chimeric mice revealed enhanced atherosclerosis progression in MB1-Cre x XBP1fl/fl recipients. These mice displayed severely attenuated serum IgM, IgG and IgE levels, but intact spleen, lymph node and peritoneal B cells. There were no differences in weights or total cholesterol levels. Antibody deficiency was associated with enhanced pro-atherogenic Th1 CD4+ T cell immunity. Conclusions: Antibodies have an overall protective effect on atherosclerosis and B2 cell antibodies are not responsible for enhanced atherosclerosis in IgM-deficient mice. Other B cell functions may be pathogenic in atherosclerosis.
EAS-0690. OXLDL-INDUCED APOPTOTIC DENDRITIC CELLS AS A NOVEL THERAPY FOR ATHEROSCLEROSIS V. Frodermann, G.H.M. van Puijvelde, L. Wierts, H.M. Lagraauw, A. Foks, P.J. van Santbrink, I. Bot, J. Kuiper, S.C.A. de Jager. Biopharmaceutics, Leiden Academic Center for Drug Research/Leiden University, Leiden, Netherlands Aim: Induction of tolerance to disease-specific antigens forms a powerful treatment for atherosclerosis. Apoptotic cell clearance has been shown to induce tolerance. Although this occurs in atherosclerotic lesions and has been shown to contribute to reducing local inflammation, it is insufficient to generate tolerance. This could be due to a reduced emigration capacity of dendritic cells (DCs) from cholesterol-rich lesions. Here we hypothesized that direct intravenous administration of oxLDL-loaded apoptotic (apopox-)DCs could modulate atherosclerosis by inducing peripheral tolerance. Methods: Apoptosis in DCs was induced by 0.1 mM epoxomicin (apopctrlDCs) or 50 mg/mL oxLDL (apopox-DCs). These apoptotic DCs were adoptively transferred into male LDLrKO mice on a Western-type diet to assess effects on atherosclerotic lesion progression. All animal work was approved by the Ethics Committee for Animal Experiments of Leiden University and conforms to Dutch government guidelines. Results: Apoptotic-DCs were preferentially cleared by splenic marginal zone macrophages and DCs, however, only apopox-DCs resulted in increased tolerogenic CD103+ DCs, with a concomitant increase in regulatory T cells. Interestingly, both types of apoptotic-DCs induced an immediate 40% decrease in Ly-6Chi monocyte numbers and a 50% decrease in CCL2 levels, but only apopox-DC-treatment resulted in long-term effects. Only apopox-DC-treatment prevented lesion progression by 28% and these lesions displayed enhanced stability, determined by a robust 45% increase in collagen content. Conclusions: Our findings clearly show that apopox-DC-treatment significantly decreases lesion progression and increases lesion stability. These findings may translate into a safe treatment of patients with established cardiovascular diseases using patient-derived oxLDL-induced apoptotic DCs.
A. Sage, D. Murphy, L. Masters, L.L. Baker, A.J. Finigan, Z. Mallat. Division of Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom
EAS-0783. MULTIPLE NOVEL REGULATORY MECHANISMS OF IL-18 BY THE LIPIDACTIVATED NUCLEAR RECEPTOR LXR
Background and aim: Extensive studies have revealed an important protective function for IgM antibodies from B1a cells in atherosclerosis, whereas the role of B2 cells and IgG antibodies is uncertain, with both pro-
M. Gage 1, B. Pourcet 1, T. Leon 1, A. Valledor 2, I. Pineda-Torra 1. 1 Division of Medicine, University College London, London, United Kingdom; 2 School of Biology, University or Barcelona, Barcelona, Spain
Methods: Combined pharmacological and immuno-histochemistry approaches were used to define the anti-apoA-1 IgG target on NRVC and the potential signaling pathways involved in the response. Results: Blocking CD14 with specific antibodies suppressed the anti-apoA1 IgG binding to the cells and was followed by the complete abrogation of the anti-apoA-1 IgG-induced chronotropic response on NRVC. Pharmacological inhibition of src fully abrogated the anti-apoA-1 IgG-induced chronotropic response. Furthermore, MR-dependent sensitization of NRVC to anti-apoA-1 IgG chronotropic effect was accompanied by CD14 relocation within lipid rafts and specific activation of Pi3Kinase pathway through phosphorylation of AKT. Conclusion: Those results point to CD14 as one of the key target of antiapoA-1 IgG, necessary to mediate their positive chronotropic response on NRVC through an aldosterone and src-dependent pathway. Our results also indicate that the MR-dependent sensitization of NRVC requires the CD14 localisation into lipids raft, unravelling a novel non-genomic pathway downstream MR activation, as well as a novel modulator involved in heart rate regulation in vitro.
EAS-0566. APOLIPOPROTEIN E IN HUMANS AND MICE IMPACTS THE DIFFERENTIATION OF NAIVE CD4 T CELLS TOWARD EFFECTOR SUBSETS F. Bonacina 1, F. Pellegatta 2, L. Grigore 2, K. Garlaschelli 2, A. Baragetti 2, A.L. Catapano 1, G.D. Norata 1. 1 Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy; 2 SISA Center for the study of Atherosclerosis, Bassini Hospital, Cinisello Balsamo, Italy Immunity plays a major role during atherosclerosis with a prevalence of effector (TEM) versus central (TCM) memory T cells in both mice and humans. Aim of this work was to investigate how dysplipidemia related to apolipoprotein E deficiency (ApoE) affects naïve(TN) to TEM CD4+T cell polarization. ApoE KO mice present increased levels of circulating TEM(CD4+CD44+CD62L-) compared with C57Bl/6J mice which are correlated with the extension of atherosclerotic plaques in the aortic sinus. ApoE deficiency also affects T cells functionality; in fact i) upon stimulation with anti-CD3 and anti CD-28, lymphocytes from ApoE KO mice proliferate more compared to C57Bl/6J (4,5±0,3x10^4 vs 3,2±0,2x10^4 mitotic events,p¼0,0005);ii) following skin graft allotransplantation a faster rejection (after 10 days 100%vs62,5% rejection,p¼0,002), associated with the presence of TEM cells with increased migratory activity toward inflammatory chemokines is observed in ApoE KO mice;iii) conjugates formation between Tcells from ApoE KO and C57Bl/6J mice with CBA dendritic cells (DCs) was similar pointing to a role for ApoE in DCs on T cells polarization. In humans, circulating CD4þTcells subsets (FACS analysis for CD3,CD4,CD45RO,CD45RA,CCR7,CCR5,CXCR3,HLA-DR) were investigated. Percentage of plasma TEM were significantly correlated to cholesterol levels independently of other metabolic and cardiovascular risk factors and resulted increased in carriers of apoE4 isoform(ε4/2,4/3,4/4) compared to apoE2(ε2/2,2/3) and apoE3(ε3/3) carriers (13,69±4,0% vs 10,28±4,9% and 11,05±4,4%,p<0,01 and p<0,05). Furthermore, following mixed lymphocyte reaction(MLR), ApoE4 presented increased TEM levels compared to ApoE2 and ApoE3 carriers. All together our data suggest that ApoE mimetics might represent a target to limit the activation of DCs-TEM axis.
EAS-0586. TOTAL ANTIBODY DEFICIENCY IN B CELL SUFFICIENT MICE ENHANCES ATHEROSCLEROSIS
e7
and anti-atherogenic actions reported. The aim of this study was to specifically target antibodies but not other B cell functions and determine the effect on mouse atherosclerosis. Methods: We created MB1-Cre x XBP1fl/fl mice that have B cell specific deficiency in the unfolded protein response transcription factor XBP1, previously reported to result in a specific antibody secretion defect. Specific antibody responses were assessed by ELISA and ELISpot after immunization with NP-chicken gamma globulin. Chimeric Ldlr/ mice with bone marrow from MB1-Cre x XBP1fl/fl or control mice were fed a western diet for 8 weeks. Results: B cell XBP1 deficiency led to significantly attenuated antibody secretion after NP-CGG immunization. Analysis of ldlr/ chimeric mice revealed enhanced atherosclerosis progression in MB1-Cre x XBP1fl/fl recipients. These mice displayed severely attenuated serum IgM, IgG and IgE levels, but intact spleen, lymph node and peritoneal B cells. There were no differences in weights or total cholesterol levels. Antibody deficiency was associated with enhanced pro-atherogenic Th1 CD4+ T cell immunity. Conclusions: Antibodies have an overall protective effect on atherosclerosis and B2 cell antibodies are not responsible for enhanced atherosclerosis in IgM-deficient mice. Other B cell functions may be pathogenic in atherosclerosis.
EAS-0690. OXLDL-INDUCED APOPTOTIC DENDRITIC CELLS AS A NOVEL THERAPY FOR ATHEROSCLEROSIS V. Frodermann, G.H.M. van Puijvelde, L. Wierts, H.M. Lagraauw, A. Foks, P.J. van Santbrink, I. Bot, J. Kuiper, S.C.A. de Jager. Biopharmaceutics, Leiden Academic Center for Drug Research/Leiden University, Leiden, Netherlands Aim: Induction of tolerance to disease-specific antigens forms a powerful treatment for atherosclerosis. Apoptotic cell clearance has been shown to induce tolerance. Although this occurs in atherosclerotic lesions and has been shown to contribute to reducing local inflammation, it is insufficient to generate tolerance. This could be due to a reduced emigration capacity of dendritic cells (DCs) from cholesterol-rich lesions. Here we hypothesized that direct intravenous administration of oxLDL-loaded apoptotic (apopox-)DCs could modulate atherosclerosis by inducing peripheral tolerance. Methods: Apoptosis in DCs was induced by 0.1 mM epoxomicin (apopctrlDCs) or 50 mg/mL oxLDL (apopox-DCs). These apoptotic DCs were adoptively transferred into male LDLrKO mice on a Western-type diet to assess effects on atherosclerotic lesion progression. All animal work was approved by the Ethics Committee for Animal Experiments of Leiden University and conforms to Dutch government guidelines. Results: Apoptotic-DCs were preferentially cleared by splenic marginal zone macrophages and DCs, however, only apopox-DCs resulted in increased tolerogenic CD103+ DCs, with a concomitant increase in regulatory T cells. Interestingly, both types of apoptotic-DCs induced an immediate 40% decrease in Ly-6Chi monocyte numbers and a 50% decrease in CCL2 levels, but only apopox-DC-treatment resulted in long-term effects. Only apopox-DC-treatment prevented lesion progression by 28% and these lesions displayed enhanced stability, determined by a robust 45% increase in collagen content. Conclusions: Our findings clearly show that apopox-DC-treatment significantly decreases lesion progression and increases lesion stability. These findings may translate into a safe treatment of patients with established cardiovascular diseases using patient-derived oxLDL-induced apoptotic DCs.
A. Sage, D. Murphy, L. Masters, L.L. Baker, A.J. Finigan, Z. Mallat. Division of Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom
EAS-0783. MULTIPLE NOVEL REGULATORY MECHANISMS OF IL-18 BY THE LIPIDACTIVATED NUCLEAR RECEPTOR LXR
Background and aim: Extensive studies have revealed an important protective function for IgM antibodies from B1a cells in atherosclerosis, whereas the role of B2 cells and IgG antibodies is uncertain, with both pro-
M. Gage 1, B. Pourcet 1, T. Leon 1, A. Valledor 2, I. Pineda-Torra 1. 1 Division of Medicine, University College London, London, United Kingdom; 2 School of Biology, University or Barcelona, Barcelona, Spain