Glycogen Dynamics Drives Lipid Droplet Biogenesis during Brown Adipocyte Differentiation.

Browning induction or transplantation of brown adipose tissue (BAT) or brown/beige adipocytes derived from progenitor or induced pluripotent stem cells (iPSCs) can represent a powerful strategy to treat metabolic diseases. However, our poor understanding of the mechanisms that govern the differentiation and activation of brown adipocytes limits the development of such therapy. Various genetic factors controlling the differentiation of brown adipocytes have been identified, although most studies have been performed using in vitro cultured pre-adipocytes. We investigate here the differentiation of brown adipocytes from adipose progenitors in the mouse embryo. We demonstrate that the formation of multiple lipid droplets (LDs) is initiated within clusters of glycogen, which is degraded through glycophagy to provide the metabolic substrates essential for de novo lipogenesis and LD formation. Therefore, this study uncovers the role of glycogen in the generation of LDs.

Nuclear Receptor Subfamily 1 Group D Member 1 Regulates Circadian Activity of NLRP3 Inflammasome to Reduce the Severity of Fulminant Hepatitis in Mice.

BACKGROUND & AIMS: The innate immune system responds not only to bacterial signals, but also to non-infectious danger-associated molecular patterns that activate the NLRP3 inflammasome complex after tissue injury. Immune functions vary over the course of the day, but it is not clear whether these changes affect the activity of the NLRP3 inflammasome. We investigated whether the core clock component nuclear receptor subfamily 1 group D member 1 (NR1D1, also called Rev-erbα) regulates expression, activity of the NLRP3 inflammasome, and its signaling pathway. METHODS: We collected naïve peritoneal macrophages and plasma, at multiple times of day, from Nr1d1-/- mice and their Nr1d1+/+ littermates (controls) and analyzed expression NLRP3, interleukin 1ß (IL1B, in plasma), and IL18 (in plasma). We also collected bone marrow-derived primary macrophages from these mice. Levels of NR1D1 were knocked down with small hairpin RNAs in human primary macrophages. Bone marrow-derived primary macrophages from mice and human primary macrophages were incubated with lipopolysaccharide (LPS) to induce expression of NLRP3, IL1B, and IL18; cells were incubated with LPS and adenosine triphosphate to activate the NLRP3 complex. We analyzed caspase 1 activity and cytokine secretion. NR1D1 was activated in primary mouse and human macrophages by incubation with SR9009; some of the cells were also incubated with an NLRP3 inhibitor or inhibitors of caspase 1. Nr1d1-/- mice and control mice were given intraperitoneal injections of LPS to induce peritoneal inflammation; plasma samples were isolated and levels of cytokines were measured. Nr1d1-/- mice, control mice, and control mice given injections of SR9009 were given LPS and D-galactosamine to induce fulminant hepatitis and MCC950 to specifically inhibit NLRP3; plasma was collected to measure cytokines and a marker of liver failure (alanine aminotransferase); liver tissues were collected and analyzed by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. RESULTS: In peritoneal macrophages, expression of NLRP3 and activation of its complex varied with time of day (circadian rhythm)-this regulation required NR1D1. Primary macrophages from Nr1d1-/- mice and human macrophages with knockdown of NR1D1 had altered expression patterns of NLRP3, compared to macrophages that expressed NR1D1, and altered patterns of IL1B and 1L18 production. Mice with disruption of Nr1d1 developed more-severe acute peritoneal inflammation and fulminant hepatitis than control mice. Incubation of macrophage with the NR1D1 activator SR9009 reduced expression of NLRP3 and secretion of cytokines. Mice given SR9009 developed less-severe liver failure and had longer survival times than mice given saline (control). CONCLUSIONS: In studies of Nr1d1-/- mice and human macrophages with pharmacologic activation of NR1D1, we found NR1D1 to regulate the timing of NLRP3 expression and production of inflammatory cytokines by macrophages. Activation of NR1D1 reduced the severity of peritoneal inflammation and fulminant hepatitis in mice.

Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways.

RATIONALE: A crucial step in atherogenesis is the infiltration of the subendothelial space of large arteries by monocytes where they differentiate into macrophages and transform into lipid-loaded foam cells. Macrophages are heterogeneous cells that adapt their response to environmental cytokines. Th1 cytokines promote monocyte differentiation into M1 macrophages, whereas Th2 cytokines trigger an "alternative" M2 phenotype. OBJECTIVE: We previously reported the presence of CD68(+) mannose receptor (MR)(+) M2 macrophages in human atherosclerotic plaques. However, the function of these plaque CD68(+)MR(+) macrophages is still unknown. METHODS AND RESULTS: Histological analysis revealed that CD68(+)MR(+) macrophages locate far from the lipid core of the plaque and contain smaller lipid droplets compared to CD68(+)MR(-) macrophages. Interleukin (IL)-4-polarized CD68(+)MR(+) macrophages display a reduced capacity to handle and efflux cellular cholesterol because of low expression levels of the nuclear receptor liver x receptor (LXR)α and its target genes, ABCA1 and apolipoprotein E, attributable to the high 15-lipoxygenase activity in CD68(+)MR(+) macrophages. By contrast, CD68(+)MR(+) macrophages highly express opsonins and receptors involved in phagocytosis, resulting in high phagocytic activity. In M2 macrophages, peroxisome proliferator-activated receptor (PPAR)γ activation enhances the phagocytic but not the cholesterol trafficking pathways. CONCLUSIONS: These data identify a distinct macrophage subpopulation with a low susceptibility to become foam cells but high phagocytic activity resulting from different regulatory activities of the PPARγ-LXRα pathways.

Clinical relevance of soluble HLA class I molecules in Waldenstrom Macroglobulinemia.

OBJECTIVES: Waldenstrom Macroglobulinemia (WM) is a B-cell neoplasm characterised by secretion of IgM by lymphoplasmacytic bone marrow cells and by cytopenias and hypogammaglobulinemia in a subset of patients. Beta-2 microglobulin (b2m) is a major prognostic factor in WM and the heavy chain of HLA class I molecules, which are known to have immunosuppressive properties and have been implicated in the pathogeny of several malignancies. METHODS: We assessed the serum levels of the total soluble HLA-I molecules and the HLA-Gs molecules in 105 patients with IgM-related disorders [WM (n = 42) and IgM MGUS (n = 63)], and compared the results to 41 healthy subjects. RESULTS: We found higher levels of HLA-Is in WM, compared to IgM MGUS and healthy donors. HLA-Gs levels were similar in WM and in IgM MGUS, but higher than in healthy donors. The association between HLA-Is at the cut-off of 1.8 microg/mL and known markers of poor prognosis was then evaluated among WM patients using univariate and multivariate methods. Based on this, high HLA-Is level was strongly associated with high serum beta2M level >3 mg/L [OR = 2, (CI 95% 1.1-5.7); P = 0.04], age > 65 yrs [OR = 1.5, (CI 95% 0.5-4.1), P = 0.06] and haemoglobin < or =11.5 g/dL [OR = 3.3, (CI 95% 1.2-9.7); P = 0.03]. High levels of serum HLA-Is were also found in patients with cryoglobulinemia, however irrespectively of WM or IgM-MGUS status. CONCLUSION: Together our results suggest a possible role for soluble MHC class I molecules in WM disease. Further investigations are necessary to further demonstrate the prognostic impact of soluble MHC class I molecules in Waldenstrom Macroglobulinemia.

Soluble HLA-G molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic cells.

HLA-G molecules are known to exert immunosuppressive action on DC maturation and on NK cells, and can in consequence inhibit respectively T cell responses and NK cytolysis. In this study, we show that monocyte-derived DC, differentiated in the presence of GM-CSF and IL-4, are sensitive to soluble (s) HLA-G molecules during LPS/IFN-gamma maturation as demonstrated by the decrease of CD80 and HLA-DR expressions and IL-12 secretion. Moreover, DC pretreated with sHLA-G were found to activate NK/DC crosstalk less than non-treated DC. Early activation of NK cells co-cultured with autologous DC was diminished as assessed by CD69 expression. The IFN-gamma production was impaired whereas a slight inhibition of the NK cell cytotoxicity against Daudi cell line was observed. Since sHLA-G is expressed in grafts or sites of tumour proliferation, its indirect action on NK cells via DC could constitute a pathway of early inhibition for both innate and specific immune responses.

Expression of functional soluble human leucocyte antigen-G molecules in lymphoproliferative disorders.

Membrane-bound and soluble human leucocyte antigen-G (sHLA-G) molecules display immunotolerant properties favouring tumour cell escape from immune surveillance. sHLA-G molecules have been detected in several tumour pathologies; this study aimed to evaluate sHLA-G expression in lymphoproliferative disorders. sHLA-G plasma level was significantly increased in 110 of 178 newly diagnosed lymphoid proliferations cases i.e. 59% of chronic lymphocytic leukaemia, 65% of B non-Hodgkin lymphomas (NHL) and 58% of T-NHL. To assess the mechanisms involved in this secretion, the differential effect of cytokines was tested in in vitro cultures of NHL cells. A significant induction of sHLA-G level was shown in T-NHL in contrast with B-NHL and normal equivalent cells, after cytokine stimulation with (i) interferongamma (IFNgamma), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor, (ii) IL-10 and (iii) transforming growth factor beta. An impact of microenvironment on sHLA-G expression was found in B-NHL as shown by the in vitro effect of addition of normal monocytes. Finally, a functional effect of sHLA-G molecules purified from pathologic plasma was demonstrated by their strong capacity to inhibit T-cell proliferation at concentrations currently observed during these disorders. These results suggest that functional sHLA-G molecules are upregulated in lymphoproliferative disorders which can support their potential immunomodulatory role during this pathology.

Soluble HLA-G molecules increase during acute leukemia, especially in subtypes affecting monocytic and lymphoid lineages.

Human leukocyte antigen G (HLA-G) molecules exhibit immunomodulatory properties corresponding to nonclassic class I genes of the major histocompatibility complex. They are either membrane-bound or solubly expressed during certain tumoral malignancies. Soluble human leukocyte antigen G (sHLA-G) molecules seem more frequently expressed than membrane-bound isoforms during hematologic malignancies, such as lymphoproliferative disorders. Assay of these molecules by enzyme-linked immunosorbent assay in patients suffering from another hematologic disorder (acute leukemia) highlights increased sHLA-G secretion. This increased secretion seems more marked in acute leukemia subtypes affecting monocytic and lymphoid lineages such as FABM4 and FABM5, as well as both B and T acute lymphoblastic leukemia (ALL). Moreover, this study uses in vitro cytokine stimulations and reveals the respective potential roles of granulocyte-macrophage colony-stimulating factor and interferon-gamma in increasing this secretion in FABM4 and ALL. Correlations between sHLA-G plasma level and clinical biologic features suggest a link between elevated sHLA-G level and 1) the absence of anterior myelodysplasia and 2) high-level leukocytosis. All these findings suggest that sHLA-G molecules could be a factor in tumoral escape from immune survey during acute leukemia.

Capacity of myeloid and plasmacytoid dendritic cells especially at mature stage to express and secrete HLA-G molecules.

Human leukocyte antigen (HLA-G), a class Ib major histocompatibility complex molecule, is potentially relevant in the immune response through its various immune cell functions. Its expression noticed in some malignancies has also been shown on macrophages and dendritic cells (DC) in tumoral and inflammatory diseases. As DC constitute a key component in the immune response, this work aimed at assessing the expression of HLA-G at transcriptional and proteic levels during differentiation and maturation of the different DC subsets. We show that HLA-G transcription was induced during CD34+-derived DC differentiation and is associated with a cell-surface expression in half of cases and with a substantial secretion of soluble HLA-G in all cases. Results were very similar for monocyte-derived DC, but there was still a weak HLA-G cell-surface expression and a lower level of secretion. On the contrary, HLA-G transcription was weak in plasmacytoid DC without any HLA-G cell-surface expression and with a basal level of secretion. The mechanisms involved in HLA-G expression appear transcriptional and post-transcriptional. However, the amount of HLA-G transcripts and the expression of the protein are not related. HLA-G expression or secretion by DC may have negative consequences on the function of effective immune cells and also on DC themselves via the interaction with inhibitory receptors expressed by these cells. The capacity of DC to express or secrete HLA-G should be studied in the context of cellular therapy using DC in addition to its suppressive action in immune response.

Soluble HLA-G molecules are increased in lymphoproliferative disorders.

The immunomodulatory properties of soluble human leukocyte antigen G (sHLA-G) explain its potential interest in malignancies. HLA-G frequently transcribed in lymphoproliferative disorders is rarely expressed at cell surface. In this article, we will demonstrate that the plasmatic level of soluble HLA-G was significantly increased in 70% of B chronic lymphocytic leukemia, 53% of non-Hodgkin B lymphoma (B-NHL), and 45% of T-NHL. To explain this variable secretion, the HLA-G secreting cell was searched and was identified as tumoral T4 lymphocytes only in one patient with Sezary syndrome. To approach the mechanisms involved in sHLA-G secretion, the potential role of cytokines has been studied in vitro on T lymphomas. A significant increase of sHLA-G level is observed after activation by cytokines associated with a small increase in the quantity of transcripts using real-time polymerase chain reaction, suggesting an involvement of both transcriptional and post-transcriptional mechanisms. Western Blot analysis reveals no evident variation of the protein expression whatever the conditions, suggesting a continuous secretion and a low intracellular storage. The frequency of the sHLA-G secretion associated to its inhibiting role on T cells and natural killer cells during tumoral lymphoid malignancies suggests a potential role of these molecules as escape mechanism from antitumoral response.

Soluble HLA-G inhibits human dendritic cell-triggered allogeneic T-cell proliferation without altering dendritic differentiation and maturation processes.

The role of the nonclassical human leukocyte antigen (HLA) class Ib molecule HLA-G in immune tolerance was first reported at maternofetal interface. This immunomodulating role could be exerted more generally in tumoral or post-transplantation situations in inhibiting natural killer (NK) and T-lymphocyte mediated lysis. Among the different transcripts resulting from alternative splicing, the mainly secreted isoform, HLA-G5, corresponds to complete molecule and has been demonstrated to be elevated in melanomas and in serum from heart-transplanted patients. As dendritic cells expressed ILT4, an inhibitory receptor capable of interacting with HLA-G, we have studied the effect of soluble HLA-G (HLA-G5) on differentiation, maturation, apoptosis and function of monocyte or CD34+-derived dendritic cells (DC). Soluble HLA-G did not alter differentiation, maturation or apoptosis of DC whatever their origin. On the other hand, an inhibitory effect of HLA-G5 on T lymphocytes proliferation was found in 53% of mixed leukocyte reactions (MLR) and was variable in intensity. These data demonstrate an indirect way of HLA-G5 action on DC occurring via T lymphocytes that reinforces the immune inhibitory role of soluble HLA-G capable to be secreted during tumoral malignancies or following heart transplantation.

HLA-G and lymphoproliferative disorders.

The immunomodulatory properties of the HLA-G molecule explain its relevance in malignancies. Our investigations in lymphoproliferative disorders show (i) a frequent and variable distribution of alternatively spliced HLA-G mRNA isoforms, (ii) a rare cell surface expression in diffuse large cell lymphomas with HLA class I loss in half of cases, and (iii) an increased serum level of sHLA-G in half of cases. The potential role of the microenvironment and/or tumoral process in HLA-G expression is discussed in the light of these data. HLA-G rather through its soluble isoform might provide a new way of immune evasion for lymphoid proliferations.