Justin Manuel

Targeted Deletion of fgl2 Leads to Impaired Regulatory T Cell Activity and Development of Autoimmune Glomerulonephritis

Mice with targeted deletion of fibrinogen-like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild-type recipients reconstituted with fgl2−/− bone marrow. These data implicate FGL2 as an important immunoregulatory molecule and led us to identify the underlying mechanisms. Deficiency of FGL2, produced by CD4+CD25+ regulatory T cells (Treg), resulted in increased T cell proliferation to lectins and alloantigens, Th 1 polarization, and increased numbers of Ab-producing B cells following immunization with T-independent Ags. Dendritic cells were more abundant in fgl2−/− mice and had increased expression of CD80 and MHCII following LPS stimulation. Treg cells were also more abundant in fgl2−/− mice, but their suppressive activity was significantly impaired. Ab to FGL2 completely inhibited Treg cell activity in vitro. FGL2 inhibited dendritic cell maturation and induced apoptosis of B cells through binding to the low-affinity FcγRIIB receptor. Collectively, these data suggest that FGL2 contributes to Treg cell activity and inhibits the development of autoimmune disease.

Ecology of danger-dependent cytokine-boosted spontaneous abortion in the CBA x DBA/2 mouse model. I. Synergistic effect of LPS and (TNF-alpha + IFN-gamma) on pregnancy loss.

Problem:  Previous data have shown ‘danger’ signals, such as bacterial lipopolysaccharide (LPS) acting via toll‐like (tlr) receptors are required for early pregnancy failure in several murine abortion models. Indeed, the abortion rate increased in the CBA × DBA/2 model after a gestation day (gd) 7.5 injection of tumour necrosis factor (TNF)‐α + interferon (IFN)‐γ only if the LPS‐tlr signalling pathway was intact. High rates of cytokine‐boosted abortion >80% loss can be achieved in certain animal colonies, that have a high endogenous (spontaneous) rate of resorption (30–50%). A specific role for LPS has been postulated to determine both the endogenous and cytokine‐boosted losses.

LPS-induced murine abortions require C5 but not C3, and are prevented by upregulating expression of the CD200 tolerance signaling molecule.

Problem  Lipopolysaccharide (LPS) acts via tlr4 to promote Th1 cytokine secretion and abortions. LPS is an essential co‐factor in spontaneous abortion in the CBA × DBA/2 model and in stress‐triggered abortions. In the CBA × DBA/2 model, C3a, C5a, and fgl2 prothrombinase participate in triggering inflammation that terminates embryo viability. As fgl2 prothrombinase (via thrombin) can generate C5a, it was predicted that LPS‐driven abortions (which require fgl2) would be independent of C3. CD200Fc can prevent abortions in the CBA × DBA/2 model, but an action through Fc could not be excluded.

ORIGINAL ARTICLE: LPS-Induced Murine Abortions Require C5 but not C3, and are Prevented by Upregulating Expression of the CD200 Tolerance Signaling Molecule

Problem  Lipopolysaccharide (LPS) acts via tlr4 to promote Th1 cytokine secretion and abortions. LPS is an essential co‐factor in spontaneous abortion in the CBA × DBA/2 model and in stress‐triggered abortions. In the CBA × DBA/2 model, C3a, C5a, and fgl2 prothrombinase participate in triggering inflammation that terminates embryo viability. As fgl2 prothrombinase (via thrombin) can generate C5a, it was predicted that LPS‐driven abortions (which require fgl2) would be independent of C3. CD200Fc can prevent abortions in the CBA × DBA/2 model, but an action through Fc could not be excluded.

FGL2/Fibroleukin mediates hepatic reperfusion injury by induction of sinusoidal endothelial cell and hepatocyte apoptosis in mice

BACKGROUND & AIMS Sinusoidal endothelial cell (SEC) and hepatocyte death are early, TNF-α mediated events in ischemia and reperfusion of the liver (I/Rp). We previously reported that TNF-α induced liver injury is dependent on Fibrinogen like protein 2 (FGL2/Fibroleukin) and showed that FGL2 binding to its receptor, FcγRIIB, results in lymphocyte apoptosis. In this study we examine whether I/Rp is induced by specific binding of FGL2 to FcγRIIB expressed on SEC. METHODS Hepatic ischemia and reperfusion was induced in wild type (WT) mice and in mice with deletion or inhibition of FGL2 and FcRIIB. Liver injury was determined by AST release, necrosis and animal death. Apoptosis was evaluated with caspase 3 and TUNEL staining. RESULTS FGL2 deletion or inhibition resulted in decreased liver injury as determined by a marked reduction in both levels of AST and ALT and hepatocyte necrosis. Caspase 3 staining of SEC (12% vs. 75%) and hepatocytes (12% vs. 45%) as well as TUNEL staining of SEC (13% vs. 60%, p=0.02) and hepatocytes (18% vs. 70%, p=0.03), markers of apoptosis, were lower in Fgl2(-/-) compared to WT mice. In vitro incubation of SEC with FGL2 induced apoptosis of SEC from WT mice, but not FcγRIIB(-/-) mice. Deletion of FcγRIIB fully protected mice against SEC and hepatocyte death in vivo. Survival of mice deficient in either Fgl2(-/-) (80%) or FcγRIIB(-/-) (100%) was markedly increased compared to WT mice (10%) which were subjected to 75min of total hepatic ischemia (p=0.001). CONCLUSIONS FGL2 binding to the FcγRIIB receptor expressed on SEC is a critical event in the initiation of the hepatic reperfusion injury cascade through induction of SEC and hepatocyte death.

LPS-induced occult loss in mice requires FGL2.

Problem  FGL2 prothrombinase is required for spontaneous abortion (resorptions) in the CBA × DBA/2 model, and for abortions in C57Bl/6 (B6) mice triggered by Salmonella enteritidis lipopolysaccharide (LPS). Unlike abortions, occult losses in B6 mice, which begin before gestation day 9.5 in mice, do not require the tumor necrosis factor‐α receptor type 1, and may be triggered by either Salmonella enteritidis or Escherichia coli LPS. Heterozygous matings of fgl2+/− × fgl2+/− B6 mice also have a high spontaneous occult loss of fgl2−/− and to a lesser extent, fgl2+/− embryos caused by hemorrhage between trophoblast and Reichert’s membrane. However, the frequency of such losses appears to vary among breeding periods and between laboratories.

ORIGINAL ARTICLE: LPS-Induced Murine Abortions Require C5 but not C3, and are Prevented by Upregulating Expression of the CD200 Tolerance Signaling Molecule: COMPLEMENT AND CD200 IN ABORTION

Problem  Lipopolysaccharide (LPS) acts via tlr4 to promote Th1 cytokine secretion and abortions. LPS is an essential co‐factor in spontaneous abortion in the CBA × DBA/2 model and in stress‐triggered abortions. In the CBA × DBA/2 model, C3a, C5a, and fgl2 prothrombinase participate in triggering inflammation that terminates embryo viability. As fgl2 prothrombinase (via thrombin) can generate C5a, it was predicted that LPS‐driven abortions (which require fgl2) would be independent of C3. CD200Fc can prevent abortions in the CBA × DBA/2 model, but an action through Fc could not be excluded.

Replication and Oncolytic Activity of an Avian Orthoreovirus in Human Hepatocellular Carcinoma Cells

Oncolytic viruses are cancer therapeutics with promising outcomes in pre-clinical and clinical settings. Animal viruses have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. We isolated an avian orthoreovirus (ARV-PB1), and tested it against a panel of hepatocellular carcinoma cells. We found that ARV-PB1 replicated well and induced strong cytopathic effects. It was determined that one mechanism of cell death was through syncytia formation, resulting in apoptosis and induction of interferon stimulated genes (ISGs). As hepatitis C virus (HCV) is a major cause of hepatocellular carcinoma worldwide, we investigated the effect of ARV-PB1 against cells already infected with this virus. Both HCV replicon-containing and infected cells supported ARV-PB1 replication and underwent cytolysis. Finally, we generated in silico models to compare the structures of human reovirus- and ARV-PB1-derived S1 proteins, which are the primary targets of neutralizing antibodies. Tertiary alignments confirmed that ARV-PB1 differs from its human homolog, suggesting that immunity to human reoviruses would not be a barrier to its use. Therefore, ARV-PB1 can potentially expand the repertoire of oncolytic viruses for treatment of human hepatocellular carcinoma and other malignancies.

Diet-induced adipose tissue expansion is mitigated in mice with a targeted inactivation of mesoderm specific transcript (Mest)

Interindividual variation of white adipose tissue (WAT) expression of mesoderm specific transcript (Mest), a paternally-expressed imprinted gene belonging to the α/β-hydrolase fold protein family, becomes apparent among genetically inbred mice fed high fat diet (HFD) and is positively associated with adipose tissue expansion (ATE). To elucidate a role for MEST in ATE, mice were developed with global and adipose tissue inactivation of Mest. Mice with homozygous (MestgKO) and paternal allelic (MestpKO) inactivation of Mest were born at expected Mendelian frequencies, showed no behavioral or physical abnormalities, and did not perturb expression of the Mest locus-derived microRNA miR-335. MestpKO mice fed HFD showed reduced ATE and adipocyte hypertrophy, improved glucose tolerance, and reduced WAT expression of genes associated with hypoxia and inflammation compared to littermate controls. Remarkably, caloric intake and energy expenditure were unchanged between genotypes. Mice with adipose tissue inactivation of Mest were phenotypically similar to MestpKO, supporting a role for WAT MEST in ATE. Global profiling of WAT gene expression of HFD-fed control and MestpKO mice detected few differences between genotypes; nevertheless, genes with reduced expression in MestpKO mice were associated with immune processes and consistent with improved glucose homeostasis. Ear-derived mesenchymal stem cells (EMSC) from MestgKO mice showed no differences in adipogenic differentiation compared to control cells unless challenged by shRNA knockdown of Gpat4, an enzyme that mediates lipid accumulation in adipocytes. Reduced adipogenic capacity of EMSC from MestgKO after Gpat4 knockdown suggests that MEST facilitates lipid accumulation in adipocytes. Our data suggests that reduced diet-induced ATE in MEST-deficient mice diminishes hypoxia and inflammation in WAT leading to improved glucose tolerance and insulin sensitivity. Since inactivation of Mest in mice has minimal additional effects aside from reduction of ATE, an intervention that mitigates MEST function in adipocytes is a plausible strategy to obviate obesity and type-2-diabetes.

Novel Transforming Growth Factor Betas (TGFβ2) in Pregnancy and Cancer

A number of cytokines may be present at the fetomaternal interface (1), and these may determine whether the implanted mammalian conceptus will succeed or fail. Certain cytokines have the potential to compromise the pregnancy and cause abortion:tumor necrosis factor α (TNFα), interleukin-1 (IL-1), and interferon γ (IFNγ). This may involve activation of natural killer (NK) lineage cells into lymphokine-activated killer (LAK) cells that have the capacity to damage fetal trophoblast cells (2–7) that lie at the fetomaternal interface and are crucial for placentation and embryo survival (8). Other cytokines have been postulated to be favorable to survival of the pregnancy. Three of these—interleukin-10 (IL-10), granulocyte-macrophage colony stimulating factor (GM-CSF), and transforming growth factor β (TGFβ)—appear to counter those cytokine-dependent processes that are antagonistic to pregnancy. IL-10 does not block generation of LAK cells in response to interleukin-2 (IL-2) (9–13), but can inhibit release of IL-2, TNFα, and related cytokines that are abortogenic. IL-10-deficient mice give birth to babies that are smaller than normal, but apparently do not have a higher resorption (abortion) rate (14). This may be due to the well-known phenomenon of redundancy, whereby several different cytokines produce the same effects, and this preserves a degree of protection when one cytokine is missing. GM-CSF, which is produced by fetal trophoblast (15, 16), has been shown to inhibit the generation of antitrophoblast LAK-like cells in vivo (17 and Clark, Chaouat, Mogil, Wegmann, manuscript submitted). Further, a potent immunosuppressive molecule closely related to TGFβ2 that blocks LAK generation and macrophage activation and cytokine release is also present at the fetomaternal interface (7, 18–20). These TGFβ2-like molecules appear to be released by bone marrow-derived natural suppressor cells and to have unusual molecular properties (20, 21).