Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Impact of reactive oxygen species (ROS) on the control of parasite loads and inflammation in Leishmania amazonensis infection

Background: Reactive oxygen species (ROS) protect the host against a large number of pathogenic microorganisms. ROS have different effects on parasites of the genus Leishmania: some parasites are susceptible to their action, while others seem to be resistant. The role of ROS in L. amazonensis infection in vivo has not been addressed to date. Methods: In this study, C57BL/6 wild-type mice (WT) and mice genetically deficient in ROS production by phagocytes (gp91phox−/− ) were infected with metacyclic promastigotes of L. amazonensis to address the effect of ROS in parasite control. Inflammatory cytokines, parasite loads and myeloperoxidase (MPO) activity were evaluated. In parallel, in vitro infection of peritoneal macrophages was assessed to determine parasite killing, cytokine, NO and ROS production. Results: In vitro results show induction of ROS production by infected peritoneal macrophages, but no effect in parasite killing. Also, ROS do not seem to be important to parasite killing in vivo, but they control lesion sizes at early stages of infection. IFN-γ, TNF-α and IL-10 production did not differ among mouse strains. Myeloperoxidase assay showed augmented neutrophils influx 6 h and 72 h post - infection in gp91phox−/− mice, indicating a larger inflammatory response in gp91phox−/− even at early time points. At later time points, neutrophil numbers in lesions correlated with lesion size: larger lesions in gp91phox−/− at earlier times of infection corresponded to larger neutrophil infiltrates, while larger lesions in WT mice at the later points of infection also displayed larger numbers of neutrophils. Conclusion: ROS do not seem to be important in L. amazonensis killing, but they regulate the inflammatory response probably by controlling neutrophils numbers in lesions.

Role of endogenous Fas (CD95/Apo-1) ligand in balloon-induced apoptosis, inflammation, and neointima formation

BACKGROUND: Fas (CD95/Apo-1) ligand (FasL)-induced apoptosis in Fas-bearing cells is critically involved in modulating immune reactions and tissue repair. Apoptosis has also been described after mechanical vascular injury such as percutaneous coronary intervention. However, the relevance of cell death in this context of vascular repair remains unknown. METHODS AND RESULTS: To determine whether FasL-induced apoptosis is causally related to neointimal lesion formation, we subjected FasL-deficient (generalized lymphoproliferative disorder [gld], C57BL/6J) and corresponding wild-type (WT) mice to carotid balloon distension injury, which induces marked endothelial denudation and medial cell death. FasL expression in WT mice was induced in injured vessels compared with untreated arteries (P<0.05; n=5). Conversely, absence of functional FasL in gld mice decreased medial and intimal apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling [TUNEL] index) at 1 hour and 7 days after balloon injury (P<0.05; n=6). In addition, peritoneal macrophages isolated from gld mice showed no apoptosis and enhanced migration (P<0.05; n=4). In parallel, we observed increased balloon-induced macrophage infiltrations (anti-CD68) in injured arteries of FasL-deficient animals (P<0.05; n=6). Together with enhanced proliferation (bromodeoxyuridine index; P<0.05), these events resulted in a further increase in medial and neointimal cells (P<0.01; n=8) with thickened neointima in gld mice (intima/media ratio, x3.8 of WT; P<0.01). CONCLUSIONS: Our data identify proapoptotic and antiinflammatory effects of endogenous FasL as important factors in the process of neointimal lesion formation after balloon injury. Moreover, they suggest that activation of FasL may decrease neointimal thickening after percutaneous coronary intervention.

Progression of atherosclerosis in ApoE-deficient mice that express distinct molecular forms of TNF-alpha

TNFalpha (TNF) critically regulates inflammation-driven atherosclerosis. Because the transmembrane (tmTNF) and soluble (sTNF) forms of TNF possess distinct immuno-modulatory properties, we hypothesized that they might differentially regulate atherosclerosis progression. Three groups of male ApoE(-/-) mice were studied: one expressing wild-type TNF (WT-TNF); one expressing exclusively a mutated non-cleavable form of TNF (KI-TNF); and one deficient in TNF (KO-TNF). Mice aged 5 weeks were fed the high-fat diet for 5 (T5) and 15 weeks (T15) or a standard chow diet for 15 weeks. At T5, in mice fed the high-fat diet, no significant differences in lesion area were observed among the three groups, either in valves or in aortas. At T15, lesion areas in valves were significantly lower in KO-TNF mice compared with those in WT-TNF mice, whereas in KI-TNF mice, they were intermediate between KO- and WT-TNF mice but not significantly different from these two groups. In aortas, lesions in KI-TNF were comparable to those of KO-TNF, both being significantly lower than those in WT-TNF. Theses differences were not linked to circulating lipids, or to macrophage, actin, and collagen contents of lesions. At T15, in mice fed the chow diet, lesion areas in valves and the aortic arch were not significantly different between the three groups. Levels of IL-6, IFNgamma, IL-10, and Foxp3 mRNAs in spleens and production of IL-6, IL-10, MCP-1, RANTES, and TNFR-2 by peritoneal macrophages at T15 of the high-fat diet showed a decrease in pro-inflammatory status, more marked in KO-TNF than in KI-TNF mice. Apoptosis was reduced only in KO-TNF mice. In conclusion, these data show that TNF effects on atherosclerosis development are detectable at stages succeeding fatty streaks and that wild-type TNF is superior to tmTNF alone in promoting atherosclerosis. TNF-dependent progression of atherosclerosis is probably linked to the differential production of pro-inflammatory mediators whether tmTNF is preponderant or essentially cleaved. Copyright (c) 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley ; Sons, Ltd.

Annexin-1 regulates macrophage IL-6 and TNF via glucocorticoid-induced leucine zipper

Annexin-1 (ANXA1) is a mediator of the anti-inflammatory actions of endogenous and exogenous glucocorticoids (GC). The mechanism of ANXA1 effects on cytokine production in macrophages is unknown and is here investigated in vivo and in vitro. In response to LPS administration, ANXA1(-/-) mice exhibited significantly increased serum IL-6 and TNF compared with wild-type (WT) controls. Similarly, LPS-induced IL-6 and TNF were significantly greater in ANXA1(-/-) than in WT peritoneal macrophages in vitro. In addition, deficiency of ANXA1 was associated with impairment of the inhibitory effects of dexamethasone (DEX) on LPS-induced IL-6 and TNF in macrophages. Increased LPS-induced cytokine expression in the absence of ANXA1 was accompanied by significantly increased LPS-induced activation of ERK and JNK MAPK and was abrogated by inhibition of either of these pathways. No differences in GC effects on MAPK or MAPK phosphatase 1 were observed in ANXA1(-/-) cells. In contrast, GC-induced expression of the regulatory protein GILZ was significantly reduced in ANXA1(-/-) cells by silencing of ANXA1 in WT cells and in macrophages of ANXA1(-/-) mice in vivo. GC-induced GILZ expression and GC inhibition of NF-kappaB activation were restored by expression of ANXA1 in ANXA1(-/-) cells, and GILZ overexpression in ANXA1(-/-) macrophages reduced ERK MAPK phosphorylation and restored sensitivity of cytokine expression and NF-kappaB activation to GC. These data confirm ANXA1 as a key inhibitor of macrophage cytokine expression and identify GILZ as a previously unrecognized mechanism of the anti-inflammatory effects of ANXA1.

Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons.

BACKGROUND Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown. OBJECTIVE We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients. METHODS We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes. RESULTS We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation-related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients. CONCLUSION We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.

Disruption of SIRPα signaling in macrophages eliminates human acute myeloid leukemia stem cells in xenografts

Although tumor surveillance by T and B lymphocytes is well studied, the role of innate immune cells, in particular macrophages, is less clear. Moreover, the existence of subclonal genetic and functional diversity in some human cancers such as leukemia underscores the importance of defining tumor surveillance mechanisms that effectively target the disease-sustaining cancer stem cells in addition to bulk cells. In this study, we report that leukemia stem cell function in xenotransplant models of acute myeloid leukemia (AML) depends on SIRPα-mediated inhibition of macrophages through engagement with its ligand CD47. We generated mice expressing SIRPα variants with differential ability to bind human CD47 and demonstrated that macrophage-mediated phagocytosis and clearance of AML stem cells depend on absent SIRPα signaling. We obtained independent confirmation of the genetic restriction observed in our mouse models by using SIRPα-Fc fusion protein to disrupt SIRPα-CD47 engagement. Treatment with SIRPα-Fc enhanced phagocytosis of AML cells by both mouse and human macrophages and impaired leukemic engraftment in mice. Importantly, SIRPα-Fc treatment did not significantly enhance phagocytosis of normal hematopoietic targets. These findings support the development of therapeutics that antagonize SIRPα signaling to enhance macrophage-mediated elimination of AML.

Molecular survival strategies of Echinococcus multilocularis in the murine host

Larval infection with Echinococcus multilocularis starts with the intrahepatic postoncospheral development of a metacestode that-at its mature stage-consists of an inner germinal and an outer laminated layer (GL ; LL). In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the LL. For instance, the LL has been proposed to protect the GL from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exsudate cells contributes to periparasitic immunosuppression, explaining why iNOS deficienct mice exhibit a significantly lower susceptibility towards experimental infection. The intense periparasitic granulomatous infiltration indicates a strong host-parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell deficient mouse strains. Carbohydrate components of the LL, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host. I.e., the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta+CD4+T cells, and in the absence of interactions between CD40 and CD40 ligand. Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 and other (not yet published) purified parasite metabolites suppress ConA and antigen-stimulated splenocyte proliferation. Infected mouse macrophages (AE-MØ) as antigen presenting cells (APC) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ. As AE-MØ fully maintain their capacity to appropriately process antigens, a failure in T cell receptor occupancy by antigen-Ia complex or/and altered co-stimulatory signals can be excluded. Studying the status of accessory molecules implicated in T cell stimulation by MØ, it could be shown that B7-1 (CD80) and B7-2 (CD86) remained unchanged, whereas CD40 was down-regulated and CD54 (=ICAM-1) slightly up-regulated. FACS analysis of peritoneal cells revealed a decrease in the percentage of CD4+ and CD8+T cells in AE-infected mice. Taken together the obstructed presenting-activity of AE-MØ appeared to trigger an unresponsiveness of T cells leading to the suppression of their clonal expansion during the chronic phase of AE infection. Interesting information on the parasite survival strategy and potential can be obtained upon in vitro and in vivo treatment. Hence, we provided very innovative results by showing that nitazoxanide, and now also, respectively, new modified compounds may represent a useful alternative to albendazole. In the context of chemotherapeutical repression of parasite growth, we searched also for parasite molecules, whose expression levels correlate with the viability and growth activity of E. multilocularis metacestode. Expression levels of 14-3-3 and II/3-10, relatively quantified by realtime reverse transcription-PCR using a housekeeping gene beta-actin, were studied in permissive nu/nu and in low-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro-treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide for a period of 8 days resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels,

TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases

Triggering receptor expressed on myeloid cells-1 (TREM-1) potently amplifies acute inflammatory responses by enhancing degranulation and secretion of proinflammatory mediators. Here we demonstrate that TREM-1 is also crucially involved in chronic inflammatory bowel diseases (IBD). Myeloid cells of the normal intestine generally lack TREM-1 expression. In experimental mouse models of colitis and in patients with IBD, however, TREM-1 expression in the intestine was upregulated and correlated with disease activity. TREM-1 significantly enhanced the secretion of relevant proinflammatory mediators in intestinal macrophages from IBD patients. Blocking TREM-1 by the administration of an antagonistic peptide substantially attenuated clinical course and histopathological alterations in experimental mouse models of colitis. This effect was also seen when the antagonistic peptide was administered only after the first appearance of clinical signs of colitis. Hence, TREM-1-mediated amplification of inflammation contributes not only to the exacerbation of acute inflammatory disorders but also to the perpetuation of chronic inflammatory disorders. Furthermore, interfering with TREM-1 engagement leads to the simultaneous reduction of production and secretion of a variety of pro-inflammatory mediators such as TNF, IL-6, IL-8 (CXCL8), MCP-1 (CCL2), and IL-1beta. Therefore, TREM-1 may also represent an attractive target for the treatment of chronic inflammatory disorders.

The immunomodulatory sphingosine 1-phosphate analog FTY720 reduces lesion size and improves neurological outcome in a mouse model of cerebral ischemia

Cerebral ischemia is accompanied by fulminant cellular and humoral inflammatory changes in the brain which contribute to lesion development after stroke. A tight interplay between the brain and the peripheral immune system leads to a biphasic immune response to stroke consisting of an early activation of peripheral immune cells with massive production of proinflammatory cytokines followed by a systemic immunosuppression within days of cerebral ischemia that is characterized by massive immune cell loss in spleen and thymus. Recent work has documented the importance of T lymphocytes in the early exacerbation of ischemic injury. The lipid signaling mediator sphingosine 1-phosphate-derived stable analog FTY720 (fingolimod) acts as an immunosuppressant and induces lymphopenia by preventing the egress of lymphocytes, especially T cells, from lymph nodes. We found that treatment with FTY720 (1mg/kg) reduced lesion size and improved neurological function after experimental stroke in mice, decreased the numbers of infiltrating neutrophils, activated microglia/macrophages in the ischemic lesion and reduced immunohistochemical features of apoptotic cell death in the lesion.

Generation and functional characterization of ovine bone marrow-derived macrophages.

A method for the culturing and propagation of ovine bone marrow-derived macrophages (BMM) in vitro is described. Bone marrow cells from sterna of freshly slaughtered sheep were cultured in hydrophobic (teflon foil) bags in the presence of high serum concentrations (20% autologous serum and 20% fetal calf serum). During an 18 day culture period in the absence of added conditioned medium, and without medium change, a strong enrichment of mononuclear phagocytes was achieved. Whereas the number of macrophages increased four to fivefold during this time, granulocytes, lymphoid cells, stem cells and undifferentiated progenitor cells were reduced to less than 3% of their numbers at Day 0. This resulted in BMM populations of 94 +/- 3% purity. These cells had morphological and histochemical characteristics of differentiated macrophages, and they performed functions similar to those of non-activated, unprimed human monocyte-derived macrophages. Thus, they avidly ingested erythrocytes coated with IgG of heterologous or homologous origin. They expressed a modest level of procoagulant activity, but upon triggering with lipopolysaccharide (LPS), a marked increase in cell-associated procoagulant activity was observed. LPS triggering promoted the secretion of interleukin-1, as evidenced by measurement of murine thymocyte costimulatory activity, and transforming growth factor-beta. Using the mouse L929 cell cytotoxicity assay as an indication of tumor necrosis factor (TNF) activity, no TNF activity was detected in the same supernatants, a result possibly due to species restriction. BMM generated low levels of O2- upon triggering with phorbol 12-myristate 13-acetate (PMA). On the other hand, no O2- production was observed upon stimulation with zymosan opsonized with ovine or human serum. Using luminol-enhanced chemiluminescence (CL) as a more sensitive indicator of an oxidative burst, both PMA or zymosan were able to trigger CL, but the response was subject to partial inhibition by sodium azide, an inhibitor of myeloperoxidase. This points to non-macrophage cells contributing also to the CL response, and is consistent with the view that unprimed BMM elicit a low oxidative burst upon triggering with strong inducers of a burst. Our functional characterization now allows us to apply priming and activation protocols and to relate their effect to functional alterations.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages.

Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1) and a human alveolar epithelial type II cell line (A549). In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis) and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis). Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint(-/-)) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1(-/-) mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1(-/-) mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1(-/-) mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1(-/-) mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.