Ramzi Khattar

Self-renewing resident arterial macrophages arise from embryonic CX3CR1+ precursors and circulating monocytes immediately after birth

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1+ precursors and postnatally from bone marrow–derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

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.

Proteasome Inhibition In Vivo Promotes Survival in a Lethal Murine Model of Severe Acute Respiratory Syndrome

ABSTRACT Ubiquitination is a critical regulator of the host immune response to viral infection, and many viruses, including coronaviruses, encode proteins that target the ubiquitination system. To explore the link between coronavirus infection and the ubiquitin system, we asked whether protein degradation by the 26S proteasome plays a role in severe coronavirus infections using a murine model of SARS-like pneumonitis induced by murine hepatitis virus strain 1 (MHV-1). In vitro, the pretreatment of peritoneal macrophages with inhibitors of the proteasome (pyrrolidine dithiocarbamate [PDTC], MG132, and PS-341) markedly inhibited MHV-1 replication at an early step in its replication cycle, as evidenced by inhibition of viral RNA production. Proteasome inhibition also blocked viral cytotoxicity in macrophages, as well as the induction of inflammatory mediators such as IP-10, gamma interferon (IFN-γ), and monocyte chemoattractant protein 1 (MCP-1). In vivo, intranasal inoculation of MHV-1 results in a lethal pneumonitis in A/J mice. Treatment of A/J mice with the proteasome inhibitor PDTC, MG132, or PS-341 led to 40% survival (P < 0.01), with a concomitant improvement of lung histology, reduced pulmonary viral replication, decreased pulmonary STAT phosphorylation, and reduced pulmonary inflammatory cytokine expression. These data demonstrate that inhibition of the cellular proteasome attenuates pneumonitis and cytokine gene expression in vivo by reducing MHV-1 replication and the resulting inflammatory response. The results further suggest that targeting the proteasome may be an effective new treatment for severe coronavirus infections.

The regulatory T cell effector molecule fibrinogen‐like protein 2 is necessary for the development of rapamycin‐induced tolerance to fully MHC‐mismatched murine cardiac allografts

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long‐term immunosuppression. To investigate mechanisms of rapamycin‐induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC‐mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4+ and CD8+ immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin‐induced, donor‐specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen‐like protein 2 (FGL2). Depletion of Treg cells with anti‐CD25 (PC61) and treatment with anti‐FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co‐expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual‐staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg‐associated genes (foxp3, lag3, tgf‐β and fgl2) had increased expression and pro‐inflammatory genes (ifn‐γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin‐induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.

Lipopolysaccharide and Tumor Necrosis Factor Alpha Inhibit Interferon Signaling in Hepatocytes by Increasing Ubiquitin-Like Protease 18 (USP18) Expression

ABSTRACT Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-α. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-α/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-α and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states. IMPORTANCE Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.

Role of Regulatory T Cells (Treg) and the Treg Effector Molecule Fibrinogen-like Protein 2 in Alloimmunity and Autoimmunity

CD4+CD25+Foxp3+ regulatory T cells (Treg) are critical to the maintenance of immune tolerance. Treg are known to utilize a number of molecular pathways to control immune responses and maintain immune homeostasis. Fibrinogen-like protein 2 (FGL2) has been identified by a number of investigators as an important immunosuppressive effector of Treg, which exerts its immunoregulatory activity by binding to inhibitory FcγRIIB receptors expressed on antigen-presenting cells including dendritic cells, endothelial cells, and B cells. More recently, it has been suggested that FGL2 accounts for the immunosuppressive activity of a highly suppressive subset of Treg that express T cell immunoreceptor with Ig and ITIM domains (TIGIT). Here we discuss the important role of Treg and FGL2 in preventing alloimmune and autoimmune disease. The FGL2–FcγRIIB pathway is also known to be utilized by viruses and tumor cells to evade immune surveillance. Moving forward, therapies based on modulation of the FGL2–FcγRIIB pathway hold promise for the treatment of a wide variety of conditions ranging from autoimmunity to cancer.

Overexpression of Fibrinogen-Like Protein 2 Promotes Tolerance in a Fully Mismatched Murine Model of Heart Transplantation.

Fibrinogen‐like protein 2 (FGL2) is an immunomodulatory protein that is expressed by regulatory T cells (Tregs). The objective of this study was to determine if recombinant FGL2 (rFGL2) treatment or constitutive FGL2 overexpression could promote transplant tolerance in mice. Although rFGL2 treatment prevented rejection of fully mismatched cardiac allografts, all grafts were rejected after stopping treatment. Next, we generated FGL2 transgenic mice (fgl2Tg) that ubiquitously overexpressed FGL2. These mice developed normally and had no evidence of the autoimmune glomerulonephritis seen in fgl2−/− mice. Immune characterization showed fgl2Tg T cells were hypoproliferative to stimulation with alloantigens or anti‐CD3 and anti‐CD28 stimulation, and fgl2Tg Tregs had increased immunosuppressive activity compared with fgl2+/+ Tregs. To determine if FGL2 overexpression can promote tolerance, we transplanted fully mismatched cardiac allografts into fgl2Tg recipients. Fifty percent of cardiac grafts were accepted indefinitely in fgl2Tg recipients without any immunosuppression. Tolerant fgl2Tg grafts had increased numbers and proportions of Tregs and tolerant fgl2Tg mice had reduced proliferation to donor but not third party antigens. These data show that tolerance in fgl2Tg recipients involves changes in Treg and T cell activity that contribute to a higher intragraft Treg–to–T cell ratio and acceptance of fully mismatched allografts.

Inhibition of the Fibrinogen-Like Protein 2:FcγRIIB/RIII immunosuppressive pathway enhances antiviral T-cell and B-cell responses leading to clearance of lymphocytic choriomeningitis virus clone 13

Persistent viruses evade immune detection by interfering with virus‐specific innate and adaptive antiviral immune responses. Fibrinogen‐like protein‐2 (FGL2) is a potent effector molecule of CD4+ CD25+ FoxP3+ regulatory T cells and exerts its immunosuppressive activity following ligation to its cognate receptor, FcγRIIB/RIII. The role of FGL2 in the pathogenesis of chronic viral infection caused by lymphocytic choriomeningitis virus clone‐13 (LCMV cl‐13) was assessed in this study. Chronically infected fgl2+/+ mice had increased plasma levels of FGL2, with reduced expression of the maturation markers, CD80, CD86 and MHC‐II on macrophages and dendritic cells and impaired production of neutralizing antibody. In contrast, fgl2−/− mice or fgl2+/+ mice that had been pre‐treated with antibodies to FGL2 and FcγRIIB/RIII and then infected with LCMV cl‐13 developed a robust CD4+ and CD8+ antiviral T‐cell response, produced high titred neutralizing antibody to LCMV and cleared LCMV. Treatment of mice with established chronic infection with antibodies to FGL2 and FcγRIIB/RIII was shown to rescue the number and functionality of virus‐specific CD4+ and CD8+ T cells with reduced total and virus‐specific T‐cell expression of programmed cell death protein 1 leading to viral clearance. These results demonstrate an important role for FGL2 in viral immune evasion and provide a rationale to target FGL2 to treat patients with chronic viral infection.

Hepatitis C virus

Hepatitis C virus (HCV) remains a leading cause of chronic liver disease worldwide, with an estimate…