Liposomes Bearing Non-Bilayer Phospholipid Arrangements Induce Specific IgG Anti-Lipid Antibodies by Activating NK1.1+, CD4+ T Cells in Mice.

Liposomes are artificial models of cellular membranes that are used as delivery systems for genes, drugs and protein antigens. We have previously used them to study the antigenic properties of their phospholipids. Here, we used them to induce the production of IgG anti-non-bilayer phospholipid arrangements (NPAs) antibodies in mice; these antibodies cause cell lysis and trigger a lupus-like disease in mice. We studied the mechanisms that lead to the production of these antibodies, and provide evidence that NK1.1+, CD4+ T cells respond to NPA-bearing liposomes and deliver the help required for specific B cell activation and antibody class-switching to IgG. We found increased numbers of IL-4-producing NK1.1+, CD4+ T cells in the secondary lymphoid organs of mice administered with NPAs, and these cells also expressed CD40L, which is required for B cell activation. Additionally, we isolated and purified NK1.1+, CD4+ T cells from spleens and determined that they over-expressed 40 genes, which are key players in inflammatory processes and B cell stimulation and have TRAF6 and UNC39B1 as key nodes in their network. These results show that liposomes are membrane models that can be used to analyze the immunogenicity of lipids.

Effect of B-NIPOx in Experimental Trypanosoma cruzi Infection in Mice.

Chagas disease is caused by Trypanosoma cruzi and represents a major public health problem, which is endemic in Latin America and emerging in the rest of the world. The two drugs that are currently available for its treatment, Benznidazole and Nifurtimox, are partially effective in the chronic phase of the disease. In this study, we designed and synthesized the benzyl ester of N-isopropyl oxamic acid (B-NIPOx), which is a non-polar molecule that crosses cell membranes. B-NIPOx is cleaved inside the parasite by carboxylesterases, releasing benzyl alcohol (a molecule with antimicrobial activity), and NIPOx, which is an inhibitor of α-hydroxy acid dehydrogenase isozyme II (HADH-II), a key enzyme in T. cruzi metabolism. We evaluated B-NIPOx cytotoxicity, its toxicity in mice, and its inhibitory activity on purified HADH-II and on T. cruzi homogenates. We then evaluated the trypanocidal activity of B-NIPOx in vitro and in vivo and its effect in the intestine of T. cruzi-infected mice. We found that B-NIPOx had higher trypanocidal activity on epimastigotes and trypomastigotes than Benznidazole and Nifurtimox, that it was more effective to reduce blood parasitemia and amastigote nests in infected mice, and that, in contrast to the reference drugs, it prevented the development of Chagasic enteropathy.

Interleukin 4 deficiency limits the development of a lupus-like disease in mice triggered by phospholipids in a non-bilayer arrangement.

Non-bilayer phospholipids arrangements (NPAs) are transient molecular associations different from lipid bilayers. When they become stable, they can trigger a disease in mice resembling human lupus, which is mainly characterized by the production of anti-NPA IgG antibodies. NPAs are stabilized on liposomes or cell bilayers by the drugs procainamide or chlorpromazine, which produce drug-induced lupus in humans. Here, we evaluated the participation of the TH 2 response, through its hallmark cytokine IL-4, on the development of the lupus-like disease in mice. Wild-type or IL-4 knockout BALB/c mice received liposomes bearing drug-induced NPAs, the drugs alone, or an anti-NPA monoclonal antibody (H308) to induce the lupus-like disease (the last two procedures stabilize NPAs on mice cells). IL-4 KO mice showed minor disease manifestations, compared to wild-type mice, with decreased production of anti-NPA IgG antibodies, no anti-cardiolipin, anti-histones and anticoagulant antibodies, and no kidney or skin lesions. In these mice, H308 was the only inducer of anti-NPA IgG antibodies. These findings indicate that IL-4 has a central role in the development of the murine lupus-like disease induced by NPA stabilization.

Lupresan, a new drug that prevents or reverts the formation of nonbilayer phospholipid arrangements that trigger a murine lupus resembling human lupus.

Non-bilayer phospholipid arrangements (NPA) are lipid associations different from the bilayer, formed by the interactions of conic anionic lipids and divalent cations that produce an inverted micelle which is inserted between the lipid layers, so the polar heads of the outer lipids spread and expose new antigens. Since these structures are transient, they are not immunogenic, but if they are stabilized by drugs, such as chlorpromazine, they become immunogenic and induce anti-NPA antibodies that trigger a lupus-like disease in mice. Chloroquine is a drug used for the treatment of lupus; chloroquine has a quinoline ring and two positive charges that interact with conic anionic lipids and prevent or revert the formation of NPA. However, the polyamine spermidine is more effective, since it has three positive charges and interacts with more lipids, but polyamines cannot be used as drugs, because they are highly toxic. Here we report the design and synthesis of Lupresan, an analogous of chloroquine with its quinoline ring but with three positive charges. Lupresan is more effective in preventing or reverting the formation of NPA than chloroquine or spermidine, and as a consequence, it decreased auto-antibody titers and healed the malar rash in mice with lupus to a greater extent than chloroquine. A drug as Lupresan could be used for the treatment of human lupus.

Corrigendum: Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus.

[This corrects the article on p. 396 in vol. 7, PMID: 27746783.].

Dysregulation of miR-155-5p and miR-200-3p and the Anti-Non-Bilayer Phospholipid Arrangement Antibodies Favor the Development of Lupus in Three Novel Murine Lupus Models.

Systemic lupus erythematosus (SLE) is characterized by deregulated activation of T and B cells, autoantibody production, and consequent formation of immune complexes. Liposomes with nonbilayer phospholipid arrangements (NPA), induced by chlorpromazine, procainamide, or manganese, provoke a disease resembling human lupus when administered to mice. These mice produce anti-NPA IgM and IgG antibodies and exhibit an increased number of TLR-expressing spleen cells and a modified gene expression associated with TICAM1-dependent TLR-4 signaling (including IFNA1 and IFNA2) and complement activation. Additionally, they showed a diminished gene expression related to apoptosis and NK cell activation. We hypothesized that such gene expression may be affected by miRNAs and so miRNA expression was studied. Twelve deregulated miRNAs were found. Six of them were common to the three lupus-like models. Their validation by qRT-PCR and TaqMan probes, including miR-342-3p, revealed that miR-155-5p and miR-200a-3p expression was statistically significant. Currently described functions for these miRNAs in autoimmune diseases such as SLE reveal their participation in inflammation, interferon production, germinal center responses, and antibody maturation. Taking into account these findings, we propose miR-155-5p and miR-200a-3p, together with the anti-NPA antibodies, as key players in the murine lupus-like models and possible biomarkers of the human SLE.

Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus.

Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1-, CD19-, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD-, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.