Itay Shalev

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.

Making sense of regulatory T cell suppressive function.

Several types of regulatory T cells maintain self-tolerance and control excessive immune responses to foreign antigens. The major regulatory T subsets described over the past decade and novel function in transplantation will be covered in this review with a focus on CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells. Multiple mechanisms have been proposed to explain how Treg cells inhibit effector cells but none can completely explain the observed effects in toto. Proposed mechanisms to explain suppressive activity of Treg cells include the generation of inhibitory cytokines, induced death of effector cells by cytokine deprivation or cytolysis, local metabolic perturbation of target cells mediated by changes in extracellular nucleotide/nucleoside fluxes with alterations in intracellular signaling molecules such as cyclic AMP, and finally inhibition of dendritic cell functions. A better understanding of how Treg cells operate at the molecular level could result in novel and safer therapeutic approaches in transplantation and immune-mediated diseases.

Targeted Deletion of Fgl-2/Fibroleukin in the Donor Modulates Immunologic Response and Acute Vascular Rejection in Cardiac Xenografts

Background—Xenografts ultimately fail as a result of acute vascular rejection (AVR), a process characterized by intravascular thrombosis, fibrin deposition, and endothelial cell activation. Methods and Results—We studied whether targeted deletion of Fgl-2, an inducible endothelial cell procoagulant, (Fgl-2−/−) in the donor prevents AVR in a mouse-to-rat cardiac xenotransplantation model. By 3 days after transplant, Fgl-2+/+ grafts developed typical features of AVR associated with increased levels of donor Fgl-2 mRNA. Grafts from Fgl-2−/− mice had reduced fibrin deposition but developed cellular rejection. Treatment with a short course of cobra venom factor and maintenance cyclosporine resulted in long-term acceptance of both Fgl-2+/+ and Fgl-2−/− grafts. On withdrawal of cyclosporine, Fgl-2+/+ grafts developed features of AVR; in contrast, Fgl-2−/− grafts again developed acute cellular rejection. Rejecting Fgl-2+/+ hearts stained positively for IgG, IgM, C3, and C5b-9, whereas rejecting Fgl-2−/− hearts had minimal Ig and complement deposition despite xenoantibodies in the serum. Furthermore, serum containing xenoantibodies failed to stain Fgl-2−/− long-term treated hearts but did stain wild-type heart tissues. Treatment of Fgl-2−/− xenografts with mycophenolate mofetil and tacrolimus, a clinically relevant immune suppression protocol, led to long-term graft acceptance. Conclusions—Deletion of Fgl-2 ameliorates AVR by downregulation of xenoantigens and may facilitate successful clinical heart xenotransplantation.

Murine Hepatitis Virus Strain 1 Produces a Clinically Relevant Model of Severe Acute Respiratory Syndrome in A/J Mice

ABSTRACT Severe acute respiratory syndrome (SARS) is a life-threatening infectious disease which has been difficult to study and treat because of the lack of a readily available animal model. Intranasal infection of A/J mice with the coronavirus murine hepatitis virus strain 1 (MHV-1) produced pulmonary pathological features of SARS. All MHV-1-infected A/J mice developed progressive interstitial pneumonitis, including dense macrophage infiltrates, giant cells, and hyaline membranes, resulting in death of all animals. In contrast, other mouse strains developed only mild transitory disease. Infected A/J mice had significantly higher cytokine levels, particularly macrophage chemoattractant protein 1 (MCP-1/CCL-2), gamma interferon, and tumor necrosis factor alpha. Furthermore, FGL2/fibroleukin mRNA transcripts and protein and fibrin deposits were markedly increased in the lungs of infected A/J mice. These animals developed a less robust type I interferon response to MHV-1 infection than resistant C57BL/6J mice, and treatment with recombinant beta interferon improved survival. This study describes a potentially useful small animal model of human SARS, defines its pathogenesis, and suggests treatment strategies.

The novel CD4+CD25+ regulatory T cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis.

Fulminant viral hepatitis (FH) remains an important clinical problem in which the underlying pathogenesis is not well understood. Here, we present insight into the immunological mechanisms involved in FH caused by murine hepatitis virus strain 3 (MHV‐3), indicating a critical role for CD4+CD25+ regulatory T cells (Tregs) and production of the novel Treg effector molecule FGL2. Before infection with MHV‐3, susceptible BALB/cJ mice had increased numbers of Tregs and expression of fgl2 messenger RNA (mRNA) and FGL2 protein compared with resistant A/J mice. After MHV‐3 infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased, correlating with increased percentage of Tregs. Treatment with anti‐FGL2 antibody completely inhibited Treg activity and protected susceptible BALB/cJ mice against MHV‐3‐liver injury and mortality. Adoptive transfer of wild‐type Tregs into resistant fgl2−/− mice increased their mortality caused by MHV‐3 infection, whereas transfer of peritoneal exudate macrophages had no adverse effect. Conclusion: This study demonstrates that FGL2 is an important effector cytokine of Tregs that contributes to susceptibility to MHV‐3–induced FH. The results further suggest that targeting FGL2 may lead to the development of novel treatment approaches for acute viral hepatitis infection. (HEPATOLOGY 2009.)

The FGL2-FcγRIIB pathway: A novel mechanism leading to immunosuppression

Fibrinogen‐like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM‐derived DC and T‐cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2‐specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FcγR)IIB and FcγRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FcγRIIB and FcγRIII, or deficiency of these receptors, abrogated FGL2 binding. FGL2 inhibited the maturation of BMDC from FcγRIIB+/+ mice but not from FcγRIIB−/− mice and induced apoptosis in the FcγRIIB+ mouse B‐cell line (A20) but not the A20IIA1.6 cell line that does not express FcγRIIB. Recombinant FGL2 infused into FcγRIIB+/+ (C57BL/6J, H‐2b) mice but not FcγRIIB−/− mice inhibited rejection of fully mismatched BALB/cJ (H‐2d) skin allografts. The identification of specific receptor binding has important implications for the pathogenesis of immune‐mediated disease and suggests a potential for targeted FGL2 therapy.

Cytokine-induced hepatic apoptosis is dependent on FGL2/fibroleukin: the role of Sp1/Sp3 and STAT1/PU.1 composite cis elements.

Previous studies from our laboratory have shown that fulminant hepatitis caused by the mouse hepatitis virus, MHV-3, is dependent on production of the novel immune coagulant fgl2/fibroleukin. In this study, we investigate the role of IFN-γ and TNF-α in the induction of fgl2 expression and fgl2-dependent hepatic apoptosis. Infusion of IFN-γ in combination with TNF-α through the portal vein of fgl2+/+ mice led to widespread hepatic apoptosis and fibrin deposition. Livers from fgl2−/− mice were normal, although strong expression of the fgl2 knockout reporter gene Lac Z was seen in both resident hepatic macrophages and endothelial cells. In vitro, IFN-γ and TNF-α induced fgl2 expression in a macrophage and endothelial cell-specific manner. In macrophages (peritoneal and RAW 264.7 cells), IFN-γ, but not IFN-α, LPS, TNF-α, or IL-1 induced fgl2 mRNA transcription and protein expression, while in endothelial cells TNF-α, but not IFN-γ, induced fgl2 transcription. In addition, while TNF-α enhanced IFN-γ-induced macrophage fgl2 transcription, IFN-γ also enhanced TNF-α-induced endothelial cell fgl2 transcription. The induction of fgl2 by IFN-γ in macrophages involved a STAT1-dependent pathway, involving the composite cis elements Sp1/Sp3 and GAS/PU.1. The latter interacted with IFN-γ-dependent Sp1/Sp3, STAT1, and the ETS family of transcription factors member PU.1. The interaction of PU.1 with the IFN-γ-activated sequence/ETS family of transcription factors site determined the macrophage-specific induction of fgl2 by IFN-γ. Overall, this study demonstrates that IFN-γ and TNF-α induce hepatocyte apoptosis in vivo, which is dependent on induction of fgl2, and defines the molecular basis of transcription of fgl2 in vitro.

The novel immunoregulatory molecule FGL2: A potential biomarker for severity of chronic hepatitis C virus infection

BACKGROUND & AIMS This report describes the use of a novel sensitive and specific ELISA for the measurement of human fibrinogen-like protein 2 (FGL2/fibroleukin), a novel effector of natural regulatory T (Treg) cells, to predict the course of chronic hepatitis C viral infection (HCV). METHODS Plasma levels of FGL2 were measured in HCV patients and compared to healthy controls and to patients with alcoholic liver disease. RESULTS FGL2 levels were significantly higher in HCV patients (84.3+/-89.1 ng/ml, n=80) compared to healthy controls (36.4+/-21.9 ng/ml, n=30, p<0.001), to a subset of patients who cleared HCV following anti-viral treatment (16.6+/-19.7 ng/ml, n=32, p<0.001), and to patients with inactive alcoholic liver disease (18.8+/-17.4 ng/ml, n=24, p<0.001). Among HCV patients, plasma levels of FGL2 correlated significantly with the stage of fibrosis (p=0.001) and were significantly higher in patients with cirrhosis (164.1+121.8 ng/ml, n=60) compared to non-cirrhotics (57.7+/-52.8 ng/ml, n=20, p=0.001). Genotype 1 patients had significantly higher levels of FGL2 (98.1+/-100.3 ng/ml, n=60) compared to patients with genotype 2/3 (41.5+/-38.6 ng/ml, n=20, p=0.0008). Patients with genotype 2/3 had FGL2 levels similar to healthy controls (41.5+/-38.6 vs. 36.41+/-21.9 ng/ml, p=ns). Infiltrating lymphocytes in liver biopsies of HCV patients were positive for either FGL2 or FoxP3 (a marker of Treg cells) or expressed both markers. CONCLUSIONS This report documents the development of a sensitive ELISA for measurement of plasma levels of FGL2 an effector Treg cells, which correlates with the severity of HCV infection.

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.

Role of regulatory T cells in the promotion of transplant tolerance

Liver transplantation is now recognized as the most effective therapy for patients with end‐stage acute and chronic liver failure. Despite outstanding short‐term graft and patient survival, liver transplantation continues to face several major challenges, including poor long‐term graft survival due to chronic rejection and major side effects of long‐term immunosuppressive therapy (which is required for the prevention of rejection). The ability to produce a state of tolerance after transplantation would potentially obviate long‐term immunosuppression. Self‐tolerance and immune homeostasis involve both central and peripheral immunoregulatory mechanisms. To date, studies have shown that many subsets of regulatory T cells (Tregs) control immune responses to foreign and alloantigens. The identification of Tregs that are positive for CD4, CD25, and the transcription factor forkhead box (Foxp3) has resulted in major advances in our understanding of the immunology of rejection and the development of transplant tolerance. In this article, we focus on the importance of Tregs in tolerance induction in experimental models of liver transplantation. Furthermore, we discuss the therapeutic potential of Tregs for the promotion of tolerance in transplant patients and highlight recent clinical trials of Treg‐based therapies. Liver Transpl, 2012.