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Abstract Background S100A9 has been shown to be important for the function of so called Myeloid Derived Suppressor Cells (MDSC). Cells with a similar phenotype are also involved in pro-inflammatory processes, and we therefore wanted to investigate the gene expression and function of these cells in animals that were either subjected to chronic inflammation, or inoculated with tumors. Methods CD11b+Ly6C++ and Ly6G+ cells were isolated from spleen, tumor tissue or inflammatory granulomas. S100A9, Arginase 1 and iNOS gene expression in the various CD11b+ cell populations was analyzed using Q-PCR. The suppressive activity of the CD11b+ cell populations from different donors was studied in co-culture experiments. Results S100A9 was shown to be expressed mainly in splenic CD11b+Ly6C+G+ cells both at the RNA and protein level. Arginase I and iNOS expression could be detected in both CD11b+Ly6C+Ly6G+ and CD11b+Ly6C+G-/C++G- derived from tumors or a site of chronic inflammation, but was very low in the same cell populations isolated from the spleen. CD11b+ cells isolated from mice with peritoneal chronic inflammation were able to stimulate T lymphocytes, while CD11b+ cells from mice with peritoneal tumors suppressed T cell growth. Conclusion An identical CD11b+Ly6C++G- cell population appears to have the ability to adopt immune stimulatory or immune suppressive functions dependent on the presence of a local inflammatory or tumor microenvironment. Thus, there is a functional plasticity in the CD11b+Ly6C++G- cell population that cannot be distinguished with the current molecular markers.

Background Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune dysfunction. A hallmark of SSc is the excessive accumulation of extracellular matrix in the skin and in internal organs. There is a high and unmet medical need for novel therapies in this disease. The pathogenesis of SSc is complex and still poorly understood, but the innate immune system has emerged as an important factor in the disease. SSc patients show increased numbers of macrophages/monocytes in the blood and in the skin compared to healthy individuals and these cells are important sources of profibrotic cytokines and chemokines. Paquinimod belongs to a class of orally active quinoline-3-carboxamide (quinoline) derivatives with immunomodulatory properties and has shown effects in several models of autoimmune/inflammatory disorders. Paquinimod is currently in clinical development for treatment of SSc. The immunomodulatory effects of paquinimod is by targeting the myeloid cell compartment via the S100A9 protein. Objective In this study we investigate whether targeting of myeloid cells by paquinimod can effect disease development in an experimental model of SSc, the tight skin 1 (Tsk-1) mouse model. Methods Seven weeks old female B6.Cg- Fbn1 Tsk /J (Tsk-1) mice were treated with vehicle or paquinimod at the dose of 5 or 25 mg/kg/day in the drinking water for 8 weeks. The effect of paquinimod on the level of skin fibrosis and on different subpopulations within the myeloid cell compartment in skin biopsies were evaluated by using histology, immunohistochemisty, a hydroxyproline assay and real-time PCR. Furthermore, the level of IgG in serum from treated animals was also analysed. The statistical analyses were performed using Mann-Whitney nonparametric two tailed rank test. Results The results show that treatment with paquinimod reduces skin fibrosis measured as reduction of skin thickness and decreased number of myofibroblasts and total hydroxyproline content. The effect on fibrosis was associated with a polarization of macrophages in the skin from a pro-fibrotic M2 to a M1 phenotype. Paquinimod treatment also resulted in a reduced TGFβ-response in the skin and an abrogation of the increased auto-antibody production in this SSc model. Conclusions Paquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system. [ABSTRACT FROM AUTHOR]

Abstract: Quinoline-3-carboxamides are currently in clinical development for treatment of both autoimmune disease and cancer. Carboxamides such as ABR-215757 (5757) have shown efficacy in several in vivo mouse models of human inflammatory autoimmune disease. Some microbial infections in mice cause GM-CSF dependent accumulation of dendritic cells expressing TNFα and inducible nitric oxide synthase (iNOS; Tip-DCs) in lymphoid organs. Functionally similar DCs develop in GM-CSF stimulated bone marrow (BM) cell cultures and offered an in vitro model that allowed us to study the impact of 5757 on cellular development of relevance for in vivo inflammatory conditions. We show in here that addition of 5757 to such cultures, in a dose-dependent way increased the frequency of DCs, while it reduced the frequency of Gr-1+ cells by inhibiting their proliferation. This effect was specific as the compound neither influenced DC development from myeloid progenitors, nor the development of granulocytes in G-CSF stimulated BM cell cultures. Importantly, we also show that 5757 treatment reduced the accumulation of Gr-1+ cells during inflammation in vivo. We therefore propose that this compound may ameliorate autoimmune disease by blocking proliferation of Gr-1+ cells during inflammation-induced mobilization of myeloid cells. [Copyright &y& Elsevier]

Despite more than 25 years of research, the molecular targets of quinoline-3-carboxamides have been elusive although these compounds are currently in Phase II and III development for treatment of autoimmune/inflammatory diseases in humans. Using photoaffinity cross-linking of a radioactively labelled quinoline-3-carboxamide compound, we could determine a direct association between human S100A9 and quinoline-3-carboxamides. This interaction was strictly dependent on both Zn++ and Ca++. We also show that S100A9 in the presence of Zn++ and Ca++ is an efficient ligand of receptor for advanced glycation end products (RAGE) and also an endogenous Toll ligand in that it shows a highly specific interaction with TLR4/MD2. Both these interactions are inhibited by quinoline-3-carboxamides. A clear structure-activity relationship (SAR) emerged with regard to the binding of quinoline-3-carboxamides to S100A9, as well as these compounds potency to inhibit interactions with RAGE or TLR4/MD2. The same SAR was observed when the compound's ability to inhibit acute experimental autoimmune encephalomyelitis in mice in vivo was analysed. Quinoline-3-carboxamides would also inhibit TNFα release in a S100A9-dependent model in vivo, as would antibodies raised against the quinoline-3-carboxamide-binding domain of S100A9. Thus, S100A9 appears to be a focal molecule in the control of autoimmune disease via its interactions with proinflammatory mediators. The specific binding of quinoline-3-carboxamides to S100A9 explains the immunomodulatory activity of this class of compounds and defines S100A9 as a novel target for treatment of human autoimmune diseases. [ABSTRACT FROM AUTHOR]

Natural killer T (NKT) lymphocytes rapidly produce several cytokines, including IL-4 and IFN-γ, upon activation, and act as regulatory cells at an early interphase of innate and adaptive immune responses. They have been implicated as important elements in diverse immune responses including the regulation of autoimmune disease, the immune response to infections, and the prevention of tumor metastasis. The broad spectrum of their activities suggested that functionally different subsets of NKT cells may exist. We demonstrate two functionally distinct splenic NKT populations identified by the expression of CD49b and CD69, respectively. Each NKT subset was represented by the amplified transgenic NKT cell population in a distinct transgenic mouse line expressing a CD1d-restricted TCR. CD49b CD69 NKT cells, termed NKT1 cells by us, were high producers of IFN-γ after stimulation, but essentially devoid of IL-4-synthesizing cells. Most NKT1 cells used diverse (non-Vα14-canonical) TCR. The CD69 CD49 NKT cell population, which we term NKT2, produced large quantities of IL-4 and substantial amounts of IFN-γ upon activation and were dominated by cells using the canonical Vα14-Jα18 T cell receptor. Knowledge of the unique roles of the different NKT cell subsets in specific situations will be essential for our understanding of NKT cell biology. [ABSTRACT FROM AUTHOR]

Natural killer (NK) T lymphocytes are thought to act as regulatory cells directing early events during immune responses. Murine NKT cells express inhibitory receptors of the Ly49 family. These receptors have a well-established and crucial role in modulating NK cell activities, but their physiological role in regulating NKT cells is not well understood, nor is the influence of major histocompatibility (MHC) ligands on endogenous Ly49 expression. We have further investigated how the expression of inhibitory NK receptors is regulated on NKT cells, and demonstrate a non-random expression of ligated Ly49 molecules on CD1d-restricted NKT cells. The nature of the T-cell receptor on the NKT cell crucially determines the profile of expressed Ly49 isoforms. Further, we show that MHC class I ligands efficiently modulate the expression levels of the inhibitory receptors, and the frequencies of cells positive for the Ly49 members. In addition, we find a several-fold increase in Ly49C/I-expressing NKT cells in adult thymus, apparently independent of MHC class I molecules. Abundant expression of Ly49 receptors on NKT cells, and the striking differences found in Ly49 isoform patterns on NKT-cell subsets differing in T-cell receptor expression, suggest that the pattern of Ly49 expression is tuned to fit the T-cell receptor and to emphasize further a role for these receptors in NKT immunity. [ABSTRACT FROM AUTHOR]

Introduction: Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9., Methods: Flow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively., Results: Cell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9., Conclusions: Patients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE.

A role for regulatory lymphocytes has been demonstrated in the pathogenesis of type 1 diabetes in the NOD mouse but the nature of these cells is debated. CD1d-restricted NKT lymphocytes have been implicated in this process. Previous reports of reduced diabetes incidence in NOD mice in which the numbers of NKT cells are artificially increased have been attributed to the enhanced production of IL-4 by these cells and a role for classical NKT cells, using the Valpha14-Jalpha18 rearrangement. We now show that overexpression in NOD mice of CD1d-restricted TCR Valpha3.2(+)Vbeta9(+) NKT cells producing high levels of IFN-gamma but low amounts of IL-4 leads to prevention of type 1 diabetes, demonstrating a role for nonclassical CD1d-restricted NKT cells in the regulation of autoimmune diabetes.