Itzhak Mendel

Modified phospholipids as anti-inflammatory compounds

Purpose of review Oxidized phospholipids (OxPLs) are abundantly found at sites of inflammation and are considered to play an active role in the modulation of the immune response. Whereas most studies attributed a proinflammatory role to OxPLs, recent studies demonstrate that some products of phospholipid oxidation may in fact exhibit anti-inflammatory properties. This study summarizes the proinflammatory and anti-inflammatory properties of OxPLs and sheds light on the therapeutic potential of OxPL derivatives or analogs for treatment of chronic inflammatory disorders. Recent findings OxPLs may inhibit activation of several Toll-like receptors and can epigenetically reduce the capacity of dendritic cells to function as mature, fully functional immunostimulatory cells. These data demonstrate that OxPLs can induce anti-inflammatory effects. Moreover, VB-201, an orally available synthetic phospholipid analog of the Lecinoxoid family, was found to attenuate inflammation in various preclinical animal models and is currently employed in a phase II clinical trial in psoriasis. Summary Chemical or biological modifications of phospholipids yield various products, some of which may exhibit anti-inflammatory properties. Identification of such species and generation of more stable/potent anti-inflammatory OxPL variants may represent a novel approach for the treatment of immune-mediated diseases such as psoriasis, atherosclerosis, multiple sclerosis and rheumatoid arthritis.

Activated T cells express the OX40 ligand: requirements for induction and costimulatory function

OX40/OX40 ligand (OX40L) interactions have been shown to exert potent costimulatory effects on T‐cell activation. OX40 expression is transiently up‐regulated on T cells following T‐cell receptor engagement, while OX40L is expressed on antigen‐presenting cells following activation. Although expression of the OX40L by T cells has been reported, the requirements for induction of OX40L on T cells have not been studied in detail. Here, we demonstrate that the OX40L can be induced on murine CD4+ and CD8+ T cells after 6 days of culture under T helper type 1 (Th1) conditions, but not under Th2 conditions. Induction of OX40L expression required a high concentration of interleukin‐12 (IL‐12), was not seen in the presence of interferon‐γ, and was dependent on signal transducer and activator of transcription type 4 (STAT4). Notably, induction of OX40L on T cells was only seen at very low concentrations of antigen or anti‐CD3. T‐cell‐expressed OX40L was fully capable of delivering a potent costimulatory signal that enhanced the proliferation of CD4+ T cells as well as promoted their differentiation to Th2 cells. OX40L expression could also be induced on CD4+ T cells in vivo following immunization with low‐dose antigen and an IL‐12 inducer. OX40/OX40L interactions between antigen‐specific T cells may occur in T‐cell zones in lymph node and spleen when OX40L expression has diminished on APC. Costimulation by T‐cell‐expressed OX40L may result in deviation of a Th1 response to a Th2 response under conditions where T cells are exposed to low concentrations of foreign or autoantigens in the presence of high concentrations of IL‐12.

The IL-10-producing competence of Th2 cells generated in vitro is IL-4 dependent.

Previous studies have suggested that activation of CD4+ T cells in the presence of IL‐10 results in the generation of a population of T cells, T regulatory 1 (Tr1) cells, that primarily produce IL‐10 and TGF‐β, but not IL‐4. The relationship between Tr1 cells and conventional Th2 cells remains unclear. We were not successful in our attempts to generate significant numbers ofantigen‐specific T cells that secreted IL‐10, but not IL‐4, by culture in the presence of IL‐10. The small numbers of cells that produced IL‐10 only were completely dependent on the presence of IL‐4 for their generation. In a polyclonal model, the development of IL‐10 only producers was completely dependent on the presence of signal transducer and activator of transcription 6. Studies with myelin basic protein‐specific T cells derived from an IL‐4‐deficient mouse confirmed the absolute requirement for IL‐4 for the generation of IL‐10 producers under all culture conditions. These IL‐10‐producing Th2 cells failed to inhibit EAE in an adoptive transfer model and were pathogenic when transferred to immunodeficient mice. Collectively, our results raise doubts about the existence of a unique population of CD4+ regulatory T cells that can be generated by activation in the presence of IL‐10.

A Lecinoxoid, an oxidized phospholipid small molecule, constrains CNS autoimmune disease

Oxidized phospholipids (Ox-PLs) are generated in abundance at sites of inflammation. Recent studies have indicated that Ox-PLs may also exhibit anti-inflammatory activities. In this study, we investigated the beneficial effect of VB-201, a pure synthetic Ox-PL analog that we synthesized, on the development of a central nervous system (CNS) autoimmune inflammatory disease, in vivo. Oral administration of VB-201 ameliorated the severity of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide MOG35-55, and restrained the encephalogenicity of MOG35-55-specific T-cells. Our data presents a novel prospect for the role of Ox-PL analogs in CNS inflammatory diseases.

Inhibition of monocyte chemotaxis by VB-201, a small molecule lecinoxoid, hinders atherosclerosis development in ApoE−/− mice

OBJECTIVE Monocytes are motile cells which sense inflammatory stimuli and subsequently migrate to sites of inflammation. Key players in host defense, monocytes have nevertheless been implicated as requisite mediators of several chronic inflammatory diseases. Inhibition of monocyte chemotaxis is therefore an attractive anti-inflammatory strategy. Oxidized phospholipids (OxPL) are native regulators of inflammation, yet their direct effect on monocyte chemotaxis is poorly defined. In this study, we investigated the direct effect of natural and synthetic phospholipids on monocyte chemotaxis. METHODS Exploring various phospholipids using in vitro chemotaxis assays, we found that the natural phospholipid 1-palmitoyl-2-glutaryl phosphatidylcholine (PGPC) can decrease monocyte chemotaxis by 50%, while other tested OxPL had no effect. We generated a library of synthetic OxPL designated lecinoxoids, which was screened for anti-inflammatory properties. RESULTS AND CONCLUSIONS VB-201, a small-molecule lecinoxoid, exhibited up to 90% inhibition of monocyte chemotaxis in vitro. Molecular analysis revealed that the effect of VB-201 was not restricted to a specific chemotactic ligand or receptor, and resulted from inhibition of signaling pathways required for monocyte chemotaxis. Interestingly, VB-201 did not inhibit monocyte adhesion or phagocytosis and had no effect on chemotaxis of CD4(+) T-cells or neutrophils. In vivo, oral treatment with VB-201 reduced monocyte migration in a peritonitis model and inhibited atheroma development in ApoE(-/-) mice, without affecting cholesterol or triglyceride levels. Our findings highlight a novel role played by native and synthetic phospholipids in regulation of monocyte chemotaxis. The data strengthen the involvement of phospholipids as key signaling molecules in inflammatory settings and demonstrate their potential therapeutic applicability.

A novel protective model against experimental allergic encephalomyelitis in mice expressing a transgenic TCR-specific for myelin oligodendrocyte glycoprotein

Myelin oligodendrocyte glycoprotein (MOG) is an important autoantigen in multiple sclerosis and in experimental autoimmune encephalomyelitis (EAE). We generated a T cell receptor (TCR) transgenic (Tg) mouse expressing a TCR derived from an encephalitogenic T cell clone specific for MOG(35-55). This mouse failed to develop EAE spontaneously and developed mild EAE at late onset when immunized with MOG(35-55). The Tg T cells produced large amounts of IL-4 when stimulated with MOG(35-55) and underwent FAS/FAS-L-mediated activation-induced cell death when stimulated with MOG(35-55) and IL-12. The unique phenotype of these autoantigen-specific T cells may represent an important mechanism of protection against autoimmune disease.

Differentiated Th1 autoreactive effector cells can induce experimental autoimmune encephalomyelitis in the absence of IL-12 and CD40/CD40L interactions.

IL-12 plays a critical role in the priming of Th1 responses to bacterial/parasitic antigens and autoantigens. Several studies have demonstrated a dependency on CD40/CD40L interactions and IL-12 for maintenance of both antibacterial/parasitic and autoreactive Th1 cells in vivo. However, it is still unclear if fully differentiated Th1 effectors require continued stimulation by IL-12. We demonstrate that the proliferative response and IFN-gamma production by a fully differentiated T cell line specific for myelin oligodendrocyte glycoprotein are completely independent of IL-12 and CD40/CD40L interactions. The capacity of this line to adoptively transfer experimental autoimmune encephalomyelitis is also independent of IL-12 and CD40/CD40L. These results have important implications regarding the therapeutic usefulness of blockade of IL-12 or the CD40/CD40L pathway for treatment of autoimmune disease.

Newly Characterized Motile Sperm Domain-Containing Protein 2 Promotes Human Breast Cancer Metastasis

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women worldwide. Breast cancer metastasis results in poor prognosis and increased mortality, but the mechanisms of breast cancer metastasis are yet to be fully resolved. Identifying distinctive proteins that regulate metastasis might be targeted to improve therapy in breast cancer. We previously described MOSPD2 as a surface membrane protein that regulates monocyte migration in vitro. In this study, we demonstrate for the first time that MOSPD2 has a major role in breast cancer cell migration and metastasis. MOSPD2 expression was highly elevated in invasive and metastatic breast cancer while it was absent or residual in normal tissue and in primary in situ tumors. In vitro experiments showed that silencing MOSPD2 in different breast cancer cell lines significantly inhibited cancer cell chemotaxis migration. Mechanistically, we found that silencing MOSPD2 profoundly abated phosphorylation events that are involved in breast tumor cell chemotaxis. In vivo, MOSPD2‐silenced breast cancer cells exhibited marked impaired metastasis to the lungs. These results indicate that MOSPD2 plays a key role in the migration and metastasis of breast cancer cells and may be used to prevent the spreading of breast cancer cells and to mediate their death.

Treatment with lecinoxoids attenuates focal and segmental glomerulosclerosis development in nephrectomized rats

Focal segmental glomerulosclerosis (FSGS) is a scarring process associated with chronic low‐grade inflammation ascribed to toll‐like receptor (TLR) activation and monocyte migration. We developed synthetic, small‐molecule lecinoxoids, VB‐201 and VB‐703, that differentially inhibit TLR‐2‐ and TLR‐4‐mediated activation and monocyte migration. The efficacy of anti‐inflammatory lecinoxoid treatment on FSGS development was explored using a 5/6 nephrectomy rat model. Five‐sixths of nephrectomized rats were treated with lecinoxoids VB‐201, VB‐703 or PBS, for 7 weeks. Upon sacrifice, albumin/creatinine ratio, glomerulosclerosis, fibrosis‐related gene expression and the number of glomerular and interstitial monocyte were evaluated. Treatment of nephrectomized rats with lecinoxoids ameliorated glomerulosclerosis. The percentage of damaged glomeruli, glomerular sclerosis and glomeruli fibrotic score was significantly reduced following VB‐201 and VB‐703 treatment. VB‐703 attenuated the expression of fibrosis hallmark genes collagen, fibronectin (FN) and transforming growth factor β (TGF‐β) in kidneys and improved albumin/creatinine ratio with higher efficacy than did VB‐201, but only VB‐201 significantly reduced the number of glomerular and interstitial monocytes. These results indicate that treatment with TLR‐2, and more prominently, TLR‐4 antagonizing lecinoxioids, is sufficient to significantly inhibit FSGS. Moreover, inhibiting monocyte migration can also contribute to treatment of FSGS. Our data demonstrate that targeting TLR‐2‐TLR‐4 and/or monocyte migration directly affects the priming phase of fibrosis and may consequently perturb disease parthogenesis.

Identification of Motile Sperm Domain–Containing Protein 2 as Regulator of Human Monocyte Migration

Binding of chemokines to their cognate receptors on monocytes instigates a cascade of events that directs these cells to migrate to sites of inflammation and cancerous tissues. Although targeting of selected chemokine receptors on monocytes exhibited preclinical efficacy, attempts to translate these studies to the clinic have failed thus far, possibly due to redundancy of the target receptor. We reveal that motile sperm domain–containing protein 2 (MOSPD2), a protein with a previously unknown function, regulates monocyte migration in vitro. This protein was found to be expressed on the cytoplasmic membrane of human monocytes. Silencing or neutralizing MOSPD2 in monocytes restricted their migration when induced by different chemokines. Mechanistically, silencing MOSPD2 inhibited signaling events following chemokine receptor ligation. When tested for expression in other immune cell subsets, MOSPD2 was apparent also, though less abundantly, in neutrophils, but not in lymphocytes. Thus, in the presence of neutralizing Abs, neutrophil migration was inhibited to some extent whereas lymphocyte migration remained intact. In view of these results, we suggest MOSPD2 as a potential target protein for treating diseases in which monocyte and neutrophil accumulation is correlated with pathogenesis.