Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (H(II)), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn²⁺ cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a H(II)-preferring lipid, in the absence or presence of Mn²⁺, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged H(II)-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn²⁺, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.

Systemic lupus erythematosus is characterized by dysregulated activation of T and B cells and autoantibodies to nuclear antigens and, in some cases, lipid antigens. Liposomes with nonbilayer phospholipid arrangements induce a disease resembling human lupus in mice, including IgM and IgG antibodies against nonbilayer phospholipid arrangements. As the effect of these liposomes on the innate immune response is unknown and innate immune system activation is necessary for efficient antibody formation, we evaluated the effect of these liposomes on Toll-like receptor (TLR) signaling, cytokine production, proinflammatory gene expression, and T, NKT, dendritic, and B cells. Liposomes induce TLR-4- and, to a lesser extent, TLR-2/TLR-6-dependent signaling in TLR-expressing human embryonic kidney (HEK) cells and bone marrow-derived macrophages. Mice with the lupus-like disease had increased serum concentrations of proinflammatory cytokines, C3a and C5a; they also had more TLR-4-expressing splenocytes, a higher expression of genes associated with TRIF-dependent TLR-4-signaling and complement activation, and a lower expression of apoptosis-related genes, compared to healthy mice. The percentage of NKT and the percentage and activation of dendritic and B2 cells were also increased. Thus, TLR-4 and TLR-2/TLR-6 activation by nonbilayer phospholipid arrangements triggers an inflammatory response that could contribute to autoantibody production and the generation of a lupus-like disease in mice.

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Systemic lupus erythematosus (SLE) is a chronic multifactorial autoimmune disease; its pathology is mainly attributed to auto-antibodies that cause inflammation and tissue damage. Our group proposed that lipids associated in non-bilayer phospholipid arrangements (NPA) are involved in the development of SLE. BALB/c mice developed a disease very similar to human SLE when they received Mn2+ or drugs chlorpromazine, procainamide (which stabilize NPA in the plasma membrane), or liposomes bearing NPA induced by these drugs or by Mn2+. Recently, we found that the gene expression profile of mice with lupus was different to that found in healthy mice and was similar to patients with SLE: in mice with lupus genes involved in presentation of exogenous antigens, antibody production and TLR4 and NOD2 signaling and some genes encoded for complement proteins were over-expressed, while genes for NK cell recognition and apoptosis were under-expressed. In the present study, we used the mouse immunopathology miScriptTM miRNA PCR Array in order to compare mature microRNA profile between mice with lupus and healthy mice. The results indicated 9 microRNAs deregulated, 4 microRNAs were underexpressed (miR-155, miR-146b, miR-23, miR-let-7d) which can regulate positively transcription of RNAs associated with IL-6, IL-1 and 5 microRNAs were overexpressed (miR-205, miR-200a, miR-18a, miR-207 and miR-574) which can regulate negatively transcription of RNAs associated with apoptosis and T cell response. This results were validated used TaqMan microRNA Assays and were similar to those found in human LES. Then this study highlight the importance of microRNAs in regulate immune process that has implications in the pathogenesis of LES an inflammatory disease.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease where animal models are used to study its pathogenesis. We have developed a mouse model of an autoimmune disease that resembles human lupus by the injection of liposomes bearing non-bilayer phospholipid arrangements (NPA). We detected the presence of IgG antibodies against NPA in the serum of mice with lupus and patients with SLE. In this work, we determined by citofluorometry the presence of plasmacytoid dendritic cells (pDC) the main producer of type I interferons, and of NKT cells in the secondary lymphoid organs of mice 30 and 60 days after the injection of liposomes with or without NPA induced with 8 mM promazine. In both groups of mice injected with liposomes bearing NPA a significant increase of pDC cells (5-fold) was found which correlates with the high concentration of type I interferon previously detected in this mouse model of lupus and in patients with SLE. A significant increase of NKT (3-fold) was detected at 30 days, specifically in the NKT subpopulation CD4+ that is known to cooperate with B cells in response of lipid antigens; this increase suggests its probable involvement in adaptive immune responses, which lead to the production of anti-NPA IgG antibodies. The increase of pDC and NKT cells found by cytometry in secondary lymphoid organs in this work, suggests their involvement in the formation of anti-NPA IgG antibodies and the development of the disease resembling human lupus.

Non-bilayer phospholipid arrangements (NPA) are lipid associations different to the bilayer and are found on cell membranes; they participate in different cellular functions and are transient, but when they are stabilized by drugs such as chlorpromazine, they become immunogenic and induce the formation of anti-NPA antibodies. Mice that receive stabilized NPA share several characteristics with patients with systemic lupus erythematosus, such as the presence of anti-cardiolipin, anti-histone and anti-nuclear antibodies, lupus band, malar exanthema and glomerulonephritis. Mice with this lupus model have been shown to develop the disease as a consequence of the production of anti-NPA antibodies; in addition, most of these antibodies are of the IgG class and are produced mainly by the germinal center pathway. In the case of protein antigens, germinal centers are the sites where class-switch recombination, somatic mutation, memory generation and affinity maturation occur. To determine if affinity maturation, a hallmark of the antibodies produced via germinal center reactions, occurs in the anti-NPA IgG antibodies produced in germinal centers, we performed antigen-specific ELISAs, both in the absence and in the presence of urea. We observed that the percentage of urea-resistant (high affinity) anti-NPA IgG antibodies in relation to the total anti-NPA IgG antibodies increases over time, and it correlates with the number of NPA-specific germinal center B cells.

Phospholipids are the main structural constituents of all cell membranes, their form the smooth bilayer matrix that delimited cells in which membrane proteins are located. However, some anionic phospholipids can lead to the formation of nonbilayer phospholipid arrangements (NPA) within the bilayer. NPA are transient but when they are stabilized by the drugs chlorpromazine, procainamide or hydralazine, which produced lupus-like disease in humans, they induce a disease resembling human lupus in mice. Mice present IgM and IgG antibodies against NPA which appear 4 weeks before that autoantibodies against cardiolipin, histones, nuclear and lupus anticoagulant antibodies. Mice also present histopathological abnormalities in the skin and kidneys similar to those observed in systemic lupus erythematosus (SLE). We proposed that the recognition of NPA by anti-NPA antibodies and after by the classical complement pathway causes cell lysis exposing intracellular antigens with the consequent formation of autoantibodies. Antibodies against NPA have been described in patients with SLE, lepromatous leprosy and hypertensive disorders of pregnancy. The present investigation is a clinical study not randomized, prospective, in Mexican mestizos patients of any sex and age, with SLE or with secondary antiphospholipid syndrome in which we found that the elevated levels of anti-NPA antibodies could trigger autoimmunity in these diseases and we determined that high titers lead to increased activity and correlate with the expression of HLA-DR, HLA-DQ, HLA-A, HLA-B, IL-10 (-1082G/A, -819C/T, -592C/A) and PTPN22 (+1858C/T) alleles. All clinic assays were approved by the Committee of Bioethics of our Institutions.