Alarmin-activated B cells accelerate murine atherosclerosis after myocardial infarction via plasma cell-immunoglobulin-dependent mechanisms.

AIMS: Myocardial infarction (MI) accelerates atherosclerosis and greatly increases the risk of recurrent cardiovascular events for many years, in particular, strokes and MIs. Because B cell-derived autoantibodies produced in response to MI also persist for years, we investigated the role of B cells in adaptive immune responses to MI. METHODS AND RESULTS: We used an apolipoprotein-E-deficient (ApoE-/-) mouse model of MI-accelerated atherosclerosis to assess the importance of B cells. One week after inducing MI in atherosclerotic mice, we depleted B cells using an anti-CD20 antibody. This treatment prevented subsequent immunoglobulin G accumulation in plaques and MI-induced accelerated atherosclerosis. In gain of function experiments, we purified spleen B cells from mice 1 week after inducing MI and transferred these cells into atherosclerotic ApoE-/- mice, which greatly increased immunoglobulin G (IgG) accumulation in plaque and accelerated atherosclerosis. These B cells expressed many cytokines that promote humoural immunity and in addition, they formed germinal centres within the spleen where they differentiated into antibody-producing plasma cells. Specifically deleting Blimp-1 in B cells, the transcriptional regulator that drives their terminal differentiation into antibody-producing plasma cells prevented MI-accelerated atherosclerosis. Alarmins released from infarcted hearts were responsible for activating B cells via toll-like receptors and deleting MyD88, the canonical adaptor protein for inflammatory signalling downstream of toll-like receptors, prevented B-cell activation and MI-accelerated atherosclerosis. CONCLUSION: Our data implicate early B-cell activation and autoantibodies as a central cause for accelerated atherosclerosis post-MI and identifies novel therapeutic strategies towards preventing recurrent cardiovascular events such as MI and stroke.

Follicular B Cells Promote Atherosclerosis via T Cell-Mediated Differentiation Into Plasma Cells and Secreting Pathogenic Immunoglobulin G.

OBJECTIVE: B cells promote or protect development of atherosclerosis. In this study, we examined the role of MHCII (major histocompatibility II), CD40 (cluster of differentiation 40), and Blimp-1 (B-lymphocyte-induced maturation protein) expression by follicular B (FO B) cells in development of atherosclerosis together with the effects of IgG purified from atherosclerotic mice. APPROACH AND RESULTS: Using mixed chimeric Ldlr-/- mice whose B cells are deficient in MHCII or CD40, we demonstrate that these molecules are critical for the proatherogenic actions of FO B cells. During development of atherosclerosis, these deficiencies affected T-B cell interactions, germinal center B cells, plasma cells, and IgG. As FO B cells differentiating into plasma cells require Blimp-1, we also assessed its role in the development of atherosclerosis. Blimp-1-deficient B cells greatly attenuated atherosclerosis and immunoglobulin-including IgG production, preventing IgG accumulation in atherosclerotic lesions; Blimp-1 deletion also attenuated lesion proinflammatory cytokines, apoptotic cell numbers, and necrotic core. To determine the importance of IgG for atherosclerosis, we purified IgG from atherosclerotic mice. Their transfer but not IgG from nonatherosclerotic mice into Ldlr-/- mice whose B cells are Blimp-1-deficient increased atherosclerosis; transfer was associated with IgG accumulating in atherosclerotic lesions, increased lesion inflammatory cytokines, apoptotic cell numbers, and necrotic core size. CONCLUSIONS: The mechanism by which FO B cells promote atherosclerosis is highly dependent on their expression of MHCII, CD40, and Blimp-1. FO B cell differentiation into IgG-producing plasma cells also is critical for their proatherogenic actions. Targeting B-T cell interactions and pathogenic IgG may provide novel therapeutic strategies to prevent atherosclerosis and its adverse cardiovascular complications.

CD4+ natural killer T cells potently augment aortic root atherosclerosis by perforin- and granzyme B-dependent cytotoxicity.

RATIONALE: CD4(+) natural killer T (NKT) cells augment atherosclerosis in apolipoprotein E-deficient (ApoE)(-/-) mice but their mechanisms of action are unknown. OBJECTIVES: We investigated the roles of bystander T, B, and NK cells; NKT cell-derived interferon-γ, interleukin (IL)-4, and IL-21 cytokines; and NKT cell-derived perforin and granzyme B cytotoxins in promoting CD4(+) NKT cell atherogenicity. METHODS AND RESULTS: Transfer of CD4(+) NKT cells into T- and B-cell-deficient ApoE(-/-)Rag2(-/-) mice augmented aortic root atherosclerosis by ≈75% that was ≈30% of lesions in ApoE(-/-) mice; macrophage accumulation similarly increased. Transferred NKT cells were identified in the liver and atherosclerotic lesions of recipient mice. Transfer of CD4(+) NKT cells into T-, B-cell-deficient, and NK cell-deficient ApoE(-/-)Rag2(-/-)γC(-/-) mice also augmented atherosclerosis. These data indicate that CD4(+) NKT cells can exert proatherogenic effects independent of other lymphocytes. To investigate the role of NKT cell-derived interferon-γ, IL-4, and IL-21 cytokines and perforin and granzyme B cytotoxins, CD4(+) NKT cells from mice deficient in these molecules were transferred into NKT cell-deficient ApoE(-/-)Jα18(-/-) mice. CD4(+) NKT cells deficient in IL-4, interferon-γ, or IL-21 augmented atherosclerosis in ApoE(-/-)Jα18(-/-) mice by ≈95%, ≈80%, and ≈70%, respectively. Transfer of CD4(+) NKT cells deficient in perforin or granzyme B failed to augment atherosclerosis. Apoptotic cells, necrotic cores, and proinflammatory VCAM-1 (vascular cell adhesion molecule) and MCP-1 (monocyte chemotactic protein) were reduced in mice receiving perforin-deficient NKT cells. CD4(+) NKT cells are twice as potent as CD4(+) T cells in promoting atherosclerosis. CONCLUSIONS: CD4(+) NKT cells potently promote atherosclerosis by perforin and granzyme B-dependent apoptosis that increases postapoptotic necrosis and inflammation.

Gene therapy delivery of myelin oligodendrocyte glycoprotein (MOG) via hematopoietic stem cell transfer induces MOG-specific B cell deletion.

The various mechanisms that have been described for immune tolerance govern our ability to control self-reactivity and minimize autoimmunity. However, the capacity to genetically manipulate the immune system provides a powerful avenue to supplement this natural tolerance in an Ag-specific manner. We have previously shown in the mouse model of experimental autoimmune encephalomyelitis that transfer of bone marrow (BM) transduced with retrovirus encoding myelin oligodendrocyte glycoprotein (MOG) promotes disease resistance and CD4(+) T cell deletion within the thymus. However, the consequence of this strategy on B cell tolerance is not known. Using BM from IgH(MOG) mice that develop MOG-specific B cell receptors, we generated mixed chimeras together with BM-encoding MOG. In these animals, the development of MOG-specific B cells was abrogated, resulting in a lack of MOG-specific B cells in all B cell compartments examined. This finding adds a further dimension to our understanding of the mechanisms of tolerance that are associated with this gene therapy approach to treating autoimmunity and may have important implications for Ab-mediated autoimmune disorders.

Cytotoxic and proinflammatory CD8+ T lymphocytes promote development of vulnerable atherosclerotic plaques in apoE-deficient mice.

BACKGROUND: Heart attacks and strokes, leading causes of deaths globally, arise from thrombotic occlusion of ruptured vulnerable atherosclerotic plaques characterized by abundant apoptosis, large necrotic cores derived from inefficient apoptotic cell clearance, thin fibrous caps, and focal inflammation. The genesis of apoptosis and necrotic cores in these vulnerable atherosclerotic plaques remains unknown. Cytotoxic CD8(+) T lymphocytes represent up to 50% of leukocytes in advanced human plaques and dominate early immune responses in mouse lesions, yet their role in atherosclerosis also remains unresolved. METHODS AND RESULTS: CD8(+) T-lymphocyte depletion by CD8α or CD8ß monoclonal antibody in apolipoprotein E-deficient mice fed a high-fat diet ameliorated atherosclerosis by reducing lipid and macrophage accumulation, apoptosis, necrotic cores, and monocyte chemoattractant protein 1, interleukin 1ß, interferon γ, and vascular cell adhesion molecule 1. Transfer of CD8(+) T cells into lymphocyte-deficient, apolipoprotein E-deficient mice partially reconstituted CD8(+) T cells in lymphoid compartments and was associated with CD8(+) T-cell infiltration in lesions, increased lipid and macrophage accumulation, apoptotic cells, necrotic cores, and interleukin 1ß in atherosclerotic lesions. Transfer of CD8(+) T cells deficient in perforin, granzyme B, or tumor necrosis factor α but not interferon γ failed to increase atherosclerotic lesions despite partial reconstitution in the lymphoid system and the presence in atherosclerotic lesions. Macrophages, smooth muscle cells, and endothelial cells were identified as apoptotic targets. CONCLUSIONS: We conclude that CD8(+) T lymphocytes promote the development of vulnerable atherosclerotic plaques by perforin- and granzyme B-mediated apoptosis of macrophages, smooth muscle cells, and endothelial cells that, in turn, leads to necrotic core formation and further augments inflammation by tumor necrosis factor α secretion.

Human amniotic epithelial cells suppress relapse of corticosteroid-remitted experimental autoimmune disease.

BACKGROUND AIMS: Multiple sclerosis (MS) is considered to be a T-cell-mediated disease. Although MS remits with corticosteroid treatment, the disease relapses on discontinuation of therapy. Human amniotic epithelial cells (hAEC) from the placenta are readily accessible in large quantities and have anti-inflammatory properties. Previously we reported that hAEC given near disease onset ameliorated clinical signs and decreased myelin oligodendrocyte glycoprotein (MOG)-specific immune responses in MOG-induced experimental autoimmune encephalomyelitis (EAE), an experimental MS model. METHODS: To examine the therapeutic effect of hAEC in a clinically relevant setting, we first treated MOG peptide-induced EAE mice with a corticosteroid, prednisolone, in drinking water to induce remission. hAEC were then infused intravenously into the remitted mice. Anti-MOG antibodies in serum were detected by enzyme-linked immunoassay. Splenocyte proliferation was assessed by (3)H-thymidine incorporation. Immune cell subpopulations in spleens and lymph nodes and secreted cytokines in splenocyte culture were quantified by flow cytometry. Central nervous system histology was examined with the use of hematoxylin and eosin, Luxol fast blue and immunostaining. RESULTS: With cessation of prednisolone treatment, hAEC delayed EAE relapse for 7 days, and, after another 7 days, largely remitted disease in six of eight responder mice. Splenocyte proliferation was suppressed, anti-MOG35-55 antibodies in serum were decreased and interleukin-2 and interleukin-5 production by splenocytes were elevated after hAEC treatment. In the central nervous system, hAEC-treated mice had decreased demyelination and fewer macrophages in the inflammatory infiltrates. hAEC treatment also increased CD4(+)CD25(+)FoxP3(+) regulatory T cells in inguinal lymph nodes. CONCLUSIONS: These data demonstrate that the therapeutic effects of hAEC after corticosteroid treatment in an MS model probably are the consequence of peripheral immunoregulation. We suggest that hAEC may have potential as a cell therapy for remitted MS.

Potently immunosuppressive 5-fluorouracil-resistant mesenchymal stromal cells completely remit an experimental autoimmune disease.

We treated mice with 5-fluorouracil (5-FU) to isolate a quiescent and undifferentiated mesenchymal stromal cell (MSC) population from the bone marrow. We examined these 5-FU-resistant MSCs (5-FU-MSCs) free from hematopoietic components for CFU fibroblasts (CFU-Fs) and assessed their immunosuppressive potential in vitro and in vivo. We differentiated fibroblastic CFU-Fs (Fibro-CFU-Fs) from mixed CFU-Fs, based on the absence of in situ expression of CD11b and CD45 hematopoietic markers, as well as on their differentiation capacity. Fibro-CFU-Fs were associated with increased numbers of large-sized Fibro-CFU-Fs (≥9 mm(2)) that displayed enhanced capacity for differentiation into adipogenic and osteogenic mesenchymal lineages. Administration of these 5-FU-resistant CD11b(-)CD45(-) MSCs 6 d after myelin oligodendrocyte glycoprotein (MOG) immunization completely remitted MOG-induced experimental autoimmune encephalomyelitis after initial development of mild disease. The remission was accompanied by reduced CNS cellular infiltration and demyelination, as well as a significant reduction in anti-MOG Ab and splenocyte proliferation to MOG. MOG-stimulated splenocytes from these mice showed elevated levels of Th2 cytokines (IL-4, IL-5, and IL-6) and decreased IL-17. Compared with untreated MSCs, 5-FU-MSCs demonstrated potent immunosuppression of Con A-stimulated splenocytes in vitro, even at a 1:320 MSC/splenocyte ratio. Immunosuppression was accompanied by elevated IL-1ra, IL-10, and PGE(2). Blocking IL-1ra, IL-10, and PGE(2), but not IL-6, heme oxygenase-1, and NO, attenuated 5-FU-MSC-induced immunosuppression. Together, our findings suggested that immunosuppression by 5-FU-MSC is mediated by a combination of elevated IL-1ra, IL-10, and PGE(2), anti-inflammatory Th2 cytokines, and decreased IL-17. Our findings suggested that 5-FU treatment identifies a population of potently immunosuppressive 5-FU-MSCs that have the potential to be exploited to remit autoimmune diseases.

Cytokine therapy with interleukin-2/anti-interleukin-2 monoclonal antibody complexes expands CD4+CD25+Foxp3+ regulatory T cells and attenuates development and progression of atherosclerosis.

BACKGROUND: CD4+CD25+Foxp3+ regulatory T cells (Tregs) attenuate atherosclerosis, but their therapeutic application by adoptive transfer is limited by the need for their expansion in vitro and limited purity. Recently, an interleukin (IL)-2/anti-IL-2 neutralizing monoclonal antibody (IL-2/anti-IL-2 mAb) complex has been shown to expand these Tregs. We examined the capacity of a modified IL-2/anti-IL-2 mAb treatment to expand Tregs and inhibit both the progression and development of developed atherosclerosis. METHODS AND RESULTS: Six-week old apolipoprotein E-deficient mice fed a high-fat diet for 8 weeks were administered IL-2/anti-IL-2 mAb commencing 2 weeks after starting the diet. Tregs in the spleen, lymph node, and liver were selectively expanded without affecting CD4+, CD8+, or natural killer cells. Tregs were increased in lesions and lesion size reduced. CD4+ T-cells, macrophages, mature dendritic cells, proliferating cell nuclear antigen+ cells, and monocyte chemoattractant protein-1 and vascular cell adhesion molecule-1 were reduced. In anti-CD3-stimulated splenocytes, proliferation and secretion of Th1, Th2, and Th17 (IL-17) cytokines and IL-1ß were reduced. To determine whether treatment attenuated progression of developed atherosclerosis, 6-week-old apolipoprotein E-deficient mice were fed a high-fat diet for 6 weeks, followed by IL-2/anti-IL-2 mAb treatment for 6 weeks while continuing the high-fat diet. Treatment also increased Tregs without affecting CD4+, CD8+, or natural killer cells, suppressed inflammation, and greatly attenuated progression of atherosclerosis. CONCLUSIONS: IL-2/anti-IL-2 mAb treatment in vivo attenuates atherosclerosis via selective Tregs expansion. The findings suggest that cytokine-based IL-2/anti-IL-2 mAb complex therapy could represent an attractive approach for treating atherosclerosis, because it markedly attenuates progression as well as development, by modulating its immunoinflammatory component.

A novel serogenetic approach determines the community prevalence of celiac disease and informs improved diagnostic pathways.

BACKGROUND: Changing perspectives on the natural history of celiac disease (CD), new serology and genetic tests, and amended histological criteria for diagnosis cast doubt on past prevalence estimates for CD. We set out to establish a more accurate prevalence estimate for CD using a novel serogenetic approach. METHODS: The human leukocyte antigen (HLA)-DQ genotype was determined in 356 patients with 'biopsy-confirmed' CD, and in two age-stratified, randomly selected community cohorts of 1,390 women and 1,158 men. Sera were screened for CD-specific serology. RESULTS: Only five 'biopsy-confirmed' patients with CD did not possess the susceptibility alleles HLA-DQ2.5, DQ8, or DQ2.2, and four of these were misdiagnoses. HLA-DQ2.5, DQ8, or DQ2.2 was present in 56% of all women and men in the community cohorts. Transglutaminase (TG)-2 IgA and composite TG2/deamidated gliadin peptide (DGP) IgA/IgG were abnormal in 4.6% and 5.6%, respectively, of the community women and 6.9% and 6.9%, respectively, of the community men, but in the screen-positive group, only 71% and 75%, respectively, of women and 65% and 63%, respectively, of men possessed HLA-DQ2.5, DQ8, or DQ2.2. Medical review was possible for 41% of seropositive women and 50% of seropositive men, and led to biopsy-confirmed CD in 10 women (0.7%) and 6 men (0.5%), but based on relative risk for HLA-DQ2.5, DQ8, or DQ2.2 in all TG2 IgA or TG2/DGP IgA/IgG screen-positive subjects, CD affected 1.3% or 1.9%, respectively, of females and 1.3% or 1.2%, respectively, of men. Serogenetic data from these community cohorts indicated that testing screen positives for HLA-DQ, or carrying out HLA-DQ and further serology, could have reduced unnecessary gastroscopies due to false-positive serology by at least 40% and by over 70%, respectively. CONCLUSIONS: Screening with TG2 IgA serology and requiring biopsy confirmation caused the community prevalence of CD to be substantially underestimated. Testing for HLA-DQ genes and confirmatory serology could reduce the numbers of unnecessary gastroscopies.

B1a B lymphocytes are atheroprotective by secreting natural IgM that increases IgM deposits and reduces necrotic cores in atherosclerotic lesions.

RATIONALE: Aggravated atherosclerosis in B lymphocyte-deficient chimeric mice and reduced atherosclerosis after transfer of unfractionated spleen B lymphocytes into splenectomized mice have led to the widely held notion that B lymphocytes are atheroprotective. However, B lymphocytes can be pathogenic, because their depletion by anti-CD20 antibody ameliorated atherosclerosis, and transfer of B2 lymphocytes aggravated atherosclerosis. These observations raise the question of the identity of the atheroprotective B-lymphocyte population. OBJECTIVE: The purpose of the study was to identify an atheroprotective B-lymphocyte subset and mechanisms by which they confer atheroprotection. METHODS AND RESULTS: Splenectomy of apolipoprotein E-deficient mice selectively reduced peritoneal B1a lymphocytes, plasma IgM, and oxidized low-density lipoprotein IgM levels and lesion IgM deposits. These reductions were accompanied by increased oil red O-stained atherosclerotic lesions and increased necrotic cores, oxidized low-density lipoproteins, and apoptotic cells in lesions. Plasma lipids, body weight, collagen, and smooth muscle content were unaffected. Transfer of B1a lymphocytes into splenectomized mice increased peritoneal B1a lymphocytes; restored plasma IgM, oxidized low-density lipoprotein IgM levels, and lesion IgM deposits; and potently attenuated atherosclerotic lesions, with reduced lesion necrotic cores, oxidized low-density lipoprotein, and apoptotic cells. In contrast, transfer of B1a lymphocytes that cannot secrete IgM failed to protect against atherosclerosis development in splenectomized mice despite reconstitution in the peritoneum. CONCLUSIONS: B1a lymphocytes are an atheroprotective B-lymphocyte population. Our data suggest that natural IgM secreted by these lymphocytes offers protection by depositing IgM in atherosclerotic lesions, which reduces the necrotic cores of lesions.

Thymic gene transfer of myelin oligodendrocyte glycoprotein ameliorates the onset but not the progression of autoimmune demyelination.

Tolerance induction, and thus prevention of autoimmunity, is linked with the amount of self-antigen presented on thymic stroma. We describe that intrathymic (i.t.) delivery of the autoantigen, myelin oligodendrocyte glycoprotein (MOG), via a lentiviral vector (LV), led to tolerance induction and prevented mice from developing fulminant experimental autoimmune encephalomyelitis (EAE). This protective effect was associated with the long-term expression of antigen in transduced stromal cells, which resulted in the negative selection of MOG-specific T cells and the generation of regulatory T cells (Tregs). These selection events were effective at decreasing T-cell proliferative responses and reduced Th1 and Th17 cytokines. In vivo, this translated to a reduction in inflammation and demyelination with minimal, or no axonal loss in the spinal cords of treated animals. Significantly intrathymic delivery of MOG to mice during the priming phase of the disease failed to suppress clinical symptoms despite mice being previously treated with a clearing anti-CD4 antibody. These results indicate that targeting autoantigens to the thymic stroma might offer an alternative means to induce the de novo production of tolerant, antigen-specific T cells; however, methods that control the number and or the activation of residual autoreactive cells in the periphery are required to successfully treat autoimmune neuroinflammation.

Conventional B2 B cell depletion ameliorates whereas its adoptive transfer aggravates atherosclerosis.

Atherosclerosis is a chronic inflammatory arterial disease characterized by focal accumulation of lipid and inflammatory cells. It is the number one cause of deaths in the Western world because of its complications of heart attacks and strokes. Statins are effective in only approximately one third of patients, underscoring the urgent need for additional therapies. B cells that accumulate in atherosclerotic lesions and the aortic adventitia of humans and mice are considered to protect against atherosclerosis development. Unexpectedly, we found that selective B cell depletion in apolipoprotein E-deficient (ApoE(-/-)) mice using a well-characterized mAb to mouse CD20 reduced atherosclerosis development and progression without affecting the hyperlipidemia imposed by a high-fat diet. Adoptive transfer of 5 × 10(6) or 5 × 10(7) conventional B2 B cells but not 5 × 10(6) B1 B cells to a lymphocyte-deficient ApoE(-/-) Rag-2(-/-) common cytokine receptor γ-chain-deficient mouse that was fed a high-fat diet augmented atherosclerosis by 72%. Transfer of 5 × 10(6) B2 B cells to an ApoE(-/-) mouse deficient only in B cells aggravated atherosclerosis by >300%. Our findings provide compelling evidence for the hitherto unrecognized proatherogenic role of conventional B2 cells. The data indicate that B2 cells can potently promote atherosclerosis development entirely on their own in the total absence of all other lymphocyte populations. Additionally, these B2 cells can also significantly augment atherosclerosis development in the presence of T cells and all other lymphocyte populations. Our findings raise the prospect of B cell depletion as a therapeutic approach to inhibit atherosclerosis development and progression in humans.

Current understanding of the role of B cell subsets and intimal and adventitial B cells in atherosclerosis.

PURPOSE OF REVIEW: Inflammation, in addition to high cholesterol is a major factor contributing to atherosclerosis-associated adverse cardiovascular events. Thus, there is a pressing need for additional therapeutic strategies to reduce inflammation, by targeting immune cells and cytokines. Here we review B cell subsets and adventitial and intimal B cells in atherosclerosis development and discuss potential B cell-targeted anti-inflammatory therapies for atherosclerosis. RECENT FINDINGS: B cell subsets can have opposing proatherogenic and atheroprotective roles in atherosclerosis. CD-20-targeted B cell depletion has been shown to decrease murine atherosclerotic lesions. The accumulation of intimal and adventitial B cells associated with atherosclerotic lesions is consistent with their participation in local inflammatory responses. As B2 B cells are proatherogenic, blocking its survival factor B cell activating factor may selectively delete this proatherogenic subset. SUMMARY: Both intimal and adventitial B cells appear important in atherosclerosis. B2 B cells are proatherogenic and other subsets such as regulatory B cells are antiatherogenic. Future B cell-targeted therapy for atherosclerosis should be customized to selectively deplete damaging B2 B cells while sparing or expanding protective B cell subsets.

Crosstalk between cytotoxic CD8+ T cells and stressed cardiomyocytes triggers development of interstitial cardiac fibrosis in hypertensive mouse hearts.

Aims: Cardiac fibrosis is central to heart failure (HF), especially HF with preserved ejection fraction (HFpEF), often caused by hypertension. Despite fibrosis causing diastolic dysfunction and impaired electrical conduction, responsible for arrhythmia-induced sudden cardiac death, the mechanisms are poorly defined and effective therapies are lacking. Here we show that crosstalk between cardiac cytotoxic memory CD8+ T cells and overly stressed cardiomyocytes is essential for development of non-ischemic hypertensive cardiac fibrosis. Methods and results: CD8 T cell depletion in hypertensive mice, strongly attenuated CF, reduced cardiac apoptosis and improved ventricular relaxation. Interaction between cytotoxic memory CD8+ T cells and overly stressed cardiomyocytes is highly dependent on the CD8+ T cells expressing the innate stress-sensing receptor NKG2D and stressed cardiomyocytes expressing the NKG2D activating ligand RAE-1. The interaction between NKG2D and RAE-1 results in CD8+ T cell activation, release of perforin, cardiomyocyte apoptosis, increased numbers of TGF-ß1 expressing macrophages and fibrosis. Deleting NKG2D or perforin from CD8+ T cells greatly attenuates these effects. Activation of the cytoplasmic DNA-STING-TBK1-IRF3 signaling pathway in overly stressed cardiomyocytes is responsible for elevating RAE-1 and MCP-1, a macrophage attracting chemokine. Inhibiting STING activation greatly attenuates cardiomyocyte RAE-1 expression, the cardiomyocyte apoptosis, TGF-ß1 and fibrosis. Conclusion: Our data highlight a novel pathway by which CD8 T cells contribute to an early triggering mechanism in CF development; preventing CD8+ T cell activation by inhibiting the cardiomyocyte RAE-1-CD8+ T cell-NKG2D axis holds promise for novel therapeutic strategies to limit hypertensive cardiac fibrosis.

Cell division autoantigen 1 enhances signaling and the profibrotic effects of transforming growth factor-ß in diabetic nephropathy.

Cell division autoantigen 1 (CDA1) modulates cell proliferation and transforming growth factor-ß (TGF-ß) signaling in a number of cellular systems; here we found that its levels were elevated in the kidneys of two animal models of diabetic renal disease. The localization of CDA1 to tubular cells and podocytes in human kidney sections was similar to that seen in the rodent models. CDA1 small interfering RNA knockdown markedly attenuated, whereas its overexpression increased TGF-ß signaling, modulating the expression of TGF-ß, TGF-ß receptors, connective tissue growth factor, collagen types I, III, IV, and fibronectin genes in HK-2 cells. CDA1 and TGF-ß together were synergistic in stimulating TGF-ß signaling and target gene expression. CDA1 knockdown effectively blocked TGF-ß-stimulated expression of collagen genes. This was due to its ability to modulate the TGF-ß type I, but not the type II, receptor, leading to increased phosphorylation of Smad3 and extracellular signal-regulated kinase mitogen-activated protein kinase. Furthermore, the Smad3 inhibitor, SIS3, markedly attenuated the activities of CDA1 in stimulating TGF-ß signaling as well as gene expression of collagens I, III, and IV. Thus, our in vitro and in vivo findings show that CDA1 has a critical role in TGF-ß signaling in the kidney.

Transplantation of autoimmune regulator-encoding bone marrow cells delays the onset of experimental autoimmune encephalomyelitis.

The autoimmune regulator (AIRE) promotes "promiscuous" expression of tissue-restricted antigens (TRA) in thymic medullary epithelial cells to facilitate thymic deletion of autoreactive T-cells. Here, we show that AIRE-deficient mice showed an earlier development of myelin oligonucleotide glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). To determine the outcome of ectopic Aire expression, we used a retroviral transduction system to over-express Aire in vitro, in cell lines and in bone marrow (BM). In the cell lines that included those of thymic medullary and dendritic cell origin, ectopically expressed Aire variably promoted expression of TRA including Mog and Ins2 (proII) autoantigens associated, respectively, with the autoimmune diseases multiple sclerosis and type 1 diabetes. BM chimeras generated from BM transduced with a retrovirus encoding Aire displayed elevated levels of Mog and Ins2 expression in thymus and spleen. Following induction of EAE with MOG(35-55), transplanted mice displayed significant delay in the onset of EAE compared with control mice. To our knowledge, this is the first example showing that in vivo ectopic expression of AIRE can modulate TRA expression and alter autoimmune disease development.

Hematopoietic stem cell gene therapy as a treatment for autoimmune diseases.

A key function of the immune system is to protect us from foreign pathogens such as viruses, bacteria, fungi and multicellular parasites. However, it is also important in many other aspects of human health such as cancer surveillance, tissue transplantation, allergy and autoimmune disease. Autoimmunity can be defined as a chronic immune response that targets self-antigens leading to tissue pathology and clinical disease. Autoimmune diseases, as a group of diseases that include type 1 diabetes, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, have no effective cures, and treatment is often based on long-term broad-spectrum immunosuppressive regimes. While a number of strategies aimed at providing disease specific treatments are being explored, one avenue of study involves the use of hematopoietic stem cells to promote tolerance. In this manuscript, we will review the literature in this area but in particular examine the relatively new experimental field of gene therapy and hematopoietic stem cell transplantation as a molecular therapeutic strategy to combat autoimmune disease.

Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin.

Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h (refs 2-6). During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. Here we show that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin.

Cholesterol involvement in the pathogenesis of neurodegenerative diseases.

Cholesterol, an essential component of cell membranes, plays an important role in the maintenance of cellular homeostasis and transmembrane communication within and between cellular compartments. In the brain that contains the highest levels of cholesterol in the body, cholesterol traffic occurs between nerve cells and between intracellular organelles in neurons to subserve normal brain function. Whereas glial cells produce the largest quantities of cholesterol, neurons also acquire cholesterol synthesized by astrocytes. The intracellular organelle endosomes and lysosomes receive and distribute cholesterol through the endocytic and retrograde transport pathways. However, deregulated cholesterol trafficking appears to be involved in the pathogenesis of Alzheimer's disease (AD), Parkinson's disease (PD) and Niemann-Pick disease type C (NPC) diseases. Under the pathological conditions of these neurodegenerative diseases, aberrant molecular interactions or particular depositions of cholesterol have been observed as critical causes to precipitate neuronal cell death. Here, we review the recent advances in terms of the role of cholesterol in healthy brain and molecular mechanisms of cholesterol involvement in AD, PD and NPC diseases. We discuss the different lines of evidence supporting different models of anomalous intracellular cholesterol trafficking with emphasis on cholesterol interactions with alpha-synuclein, NPC1 and NPC2 in AD, PD and NPC.