Therapeutic strategies in management of atherosclerosis by manipulation of anti-atherogenic B1a lymphocytes
thesisposted on 27.02.2017 by Hosseini, Hamid
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Abstract Atherosclerosis is a chronic inflammatory disease of large elastic and muscular arteries in which lesions are characterized by the deposition of cholesterol, leukocyte influx, smooth muscle cell proliferation, cell debris and collagen accumulation in the intima. Inflammatory cells of the innate and adaptive immune systems contribute to lesion development and progression. Cardiovascular diseases such as heart attacks and strokes are the leading causes of death globally with underlying of atherosclerosis. Their current treatment is restricted to lipid-lowering statins which are not sufficient in preventing and remitting atherosclerosis. Therefore new additional anti-inflammatory therapeutic strategy is required in the management of atherosclerosis. Earlier studies showed a protective role for total B cells; however further studies by our group and others showed a pro-atherogenic role for B2 cells and atheroprotective role for B1a cells. B cells have been shown to accumulate in atherosclerotic lesions in very low numbers. Understanding the role of B cells in atherosclerosis is rather complex as there are two main subsets of B cells, B1 and B2 B cells, each with differing origin, development, differentiation, localization and effector functions. B1a cells mainly produce Natural IgM antibodies while B2 cells mainly produce IgG antibodies. Splenectomy of Apolipoprotein E knockout mice (ApoE-/-) resulted in a preferential depletion in peritoneal B1a cell population and enhanced atherosclerosis. Our group showed that reconstitution of B1a cells by adoptive transfer rescued the aggravated atherosclerosis in splenectomised ApoE-/- mice. In another study we showed that B2 cells are atherogenic as adoptive transfer of B2 cells into lymphocyte-deficient ApoE-/-Rag2-/-c-/- mice and in B cell-deficient ApoE-/-MT mice exacerbated atherosclerosis. These studies established opposing roles of B cell subsets in atherosclerosis development in ApoE-/- atherogenic mouse model. B2 cells were identified as atherogenic whereas B1 cells were atheroprotective. Atherogenicity of B2 cells was confirmed by further studies from our laboratory on BAFF-R deficient ApoE-/- mice that are selectively deficient in B2 but not B1 cells. BAFF-R knockouts were selected for study because BAFF is a growth and maturation factor for B2 cells but not B1 cells. Additional confirmation was provided when we used monoclonal antibody to BAFF-R to selectively deplete B2 cells and spare B1a cell. This antibody not only attenuated the development of atherosclerosis but also suppressed the established atherosclerosis. To further extend these observations the studies presented in this thesis explores therapeutic strategies to expand B1a cells to suppress atherosclerosis development. These findings may have potential for clinical translation. Firstly I investigated the role of toll-like receptors (TLRs) and CD40 in activating atheroprotective B1a cells. Peritoneal B1a cells were depleted by splenectomy from ApoE-/- mice, then mice adoptively received B1a cells from C57Bl/6 wild type (WT), TLR2-/-, TLR4-/-, TLR9-/-, MyD88-/- and CD40-/- mice and fed a high fat (HFD) diet for 8 weeks. B1a cells from WT, TLR2-/-, TLR9-/- and CD40-/- mice suppressed atherosclerosis whilst B1a cells from TLR4-/- or MyD88-/- were without affect. Adoptively transferred B1a cells from WT but not TLR4-/- or MyD88-/- mice elevated IgM in plasma and atherosclerotic lesions. This was associated with reductions in lesion oxLDL, macrophages, T cells and proinflammatory cytokines. Also lesion apoptotic cell numbers were reduced by B1a cells from WT but not TLR4-/- or MyD88-/- mice and lesion anti-inflammatory cytokines IL-10 and TGF-β1 were increased. Polyclonal natural IgM produced by TLR-stimulated B1a cells during development of atherosclerosis not only neutralised oxLDL and facilitated removal of lesion apoptotic cells but also bound to leukocytes, CD3 and CD4 T cells and reduced local inflammation in atherosclerotic lesions. Next, I examined whether activating B1a cells with apoptotic cells (AC) as well as phosphatidylserine liposomes (PSL) could enhance their protective actions during atherosclerosis. Intraperitoneally administration of both ACs and PSL attenuated atherosclerosis in ApoE-/- mice and the effect was dependent on B1a cells as splenectomy abrogated these effects. Atheroprotection was associated with reductions in lesion macrophages and CD4+ and CD8+ T cell accumulation was well as reductions in proinflammatory cytokines and increases in anti-inflammatory cytokines. ACs and PSLs also increased B1a cell numbers including TIM-1+ B1a cells without affecting other lymphocyte populations. Total plasma IgM, anti-leukocyte, anti-CD3, anti-CD4 and anti-oxLDL IgM were also significantly elevated. The elevated IgM in developing atherosclerotic lesions were associated with reductions in lesion MDA-LDL (oxLDL), apoptotic cell numbers and lesion necrotic core size. Thirdly I explored the role of TIM-1 receptor, a receptor for phosphatidylserine (PS) expressed on regulatory B cells. I examined the capacity of anti-TIM-1 RMT1-10 low affinity agonist monoclonal antibody (mAb) to expand B1a cells to inhibit progression and development of established atherosclerosis. Six-week old male ApoE-/- mice were treated with anti-TIM-1 RMT1-10 mAb and fed a HFD for 8 weeks. B1a TIM-1+IgM+ B cells and B1a TIM-1+IgM+IL-10+ B cells were selectively expanded. These effects reduced lesion size and markedly increased plasma and lesion IgM and decreased lesion oxidatively modified LDL (oxLDL). This was associated with reduction of CD4+ and CD8+ T cells, macrophages and monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), proinflammatory cytokines expression, apoptotic cell numbers and necrotic cores in atherosclerotic lesions. Splenectomy indicated that these effects were B1a cell-dependent. B1a cell stimulation in-vitro with the anti-TIM-1 mAb promoted dose-response B1a cell proliferation and IgM production. In an intervention study to determine whether anti-TIM-1 mAb treatment could attenuate developed atherosclerosis progression, 6 week-old ApoE-/- mice were fed a HFD for 6 weeks, and treated with anti-TIM-1 mAb for another 8 weeks while continuing the HFD. Treatment also increased B1a TIM-1+IgM+ B cells, B1a TIM-1+IgM+IL-10+ B cells and IgM levels and greatly attenuated atherosclerosis progression. Taken together, my findings provide persuasive data for the expansion of atheroprotective B1a B cells in the prevention of atherosclerosis development and the suppression of already established atherosclerosis. The latter finding suggests potential for clinical translation.