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Hao Liu,
Peter S Yang,
Tina Zhu,
Justin Manuel, Jianhua Zhang,
Wei He,
Itay Shalev,
Li Zhang,
Myron I Cybulsky,
David R Grant,
M James Phillips,
Gary A Levy
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ABSTRACT: Fibrinogen-like protein 2 (FGL2), a novel effector molecule of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), mediates its suppressive activity through binding to low affinity Fcγ receptors expressed on antigen presenting cells (APCs). FGL2 has been implicated in the pathogenesis of viral hepatitis, xeno- and allotransplant rejection, and rheumatoid arthritis. Here we fully analyzed the structure-function relationships of recombinant murine FGL2 generated in COS-7 cells and identified the receptor binding domains. Native FGL2 exists as an oligomer with a molecular weight of approximately 260kDa, while under reducing conditions, FGL2 has a molecular weight of 65kDa suggesting that native FGL2 is composed of four monomers. By site-directed mutation, cysteines at positions 94, 97, 184 and 187, found in the coiled-coil domain were shown to be crucial for FGL2 oligomerization. Monomeric FGL2 had a lower affinity binding to APCs, but increased immunosuppressive activity compared to oligomeric FGL2. Deglycosylation demonstrated that sugar moieties are critical for maintaining solubility of FGL2. SWISS-MODEL analysis suggested that FGL2 has a similar tertiary structure with other members of the fibrinogen family such as fibrinogen and tachylectin. Mutational analysis of cysteine residues and Western blots suggested an asymmetric bouquet-shaped quaternary structure for oligomeric FGL2, resembling many pattern-recognition molecules in the lectin pathway of innate immunity. The functional motifs of FGL2 were mapped to the C terminal globular domain, using a peptide blockade assay. These results collectively define the biochemical and immunological determinants of FGL2, an important immunosuppressive molecule of Treg providing important insights for designing FGL2-related therapeutics.
The international journal of biochemistry & cell biology 11/2012; · 4.89 Impact Factor
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ABSTRACT: The outbreak of severe acute respiratory syndrome (SARS) in 2003 reinforces the potential of lethal pandemics of respiratory viral infections. The underlying mechanisms of SARS are still largely undefined. Long pentraxin PTX3, a humoral mediator of innate immunity, has been reported to have anti-viral effects. We examined the role of PTX3 in coronavirus murine hepatitis virus strain 1 (MHV-1)-induced acute lung injury, a previously reported animal model for SARS. PTX3-deficient mice (129/SvEv/C57BL6/J) and their wild-type (WT) littermates were intranasally infected MHV-1. These mice were also treated with recombinant PTX3. Effects of PTX3 on viral binding and infectivity were determined in vitro. Cytokine expression, severity of lung injury, leukocyte infiltration and inflammatory responses were examined in vivo. In PTX3 WT mice, MHV-1 induced PTX3 expression in the lung and serum in a time-dependent manner. MHV-1 infection led to acute lung injury with greater severity in PTX3-deficient mice than that in WT mice. PTX3 deficiency enhanced early infiltration of neutrophils and macrophages in the lung. PTX3 bound to MHV-1 and MHV-3 and reduced MHV-1 infectivity in vitro. Administration of recombinant PTX3 significantly accelerated viral clearance in the lung, attenuated MHV-1-induced lung injury, and reduced early neutrophil influx and elevation of inflammatory mediators in the lung. Results from this study indicate a protective role of PTX3 in coronaviral infection-induced acute lung injury.
Laboratory Investigation 06/2012; 92(9):1285-96. · 3.64 Impact Factor
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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.
Journal of Virology 12/2010; 84(23):12419-28. · 5.40 Impact Factor
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ABSTRACT: CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.
Nucleic Acids Research 10/2010; 38(19):6684-96. · 8.03 Impact Factor
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Julian L Leibowitz,
Rajiv Srinivasa,
Shawn T Williamson,
Ming Ming Chua,
Mingfeng Liu,
Samantha Wu,
Hyojeung Kang,
Xue-Zhong Ma, Jianhua Zhang,
Itay Shalev,
Robert Smith,
Melville J Phillips,
Gary A Levy,
Susan R Weiss
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ABSTRACT: We report here investigation into the genetic basis of mouse hepatitis virus strain 1 (MHV-1) pneumovirulence. Sequencing of the 3' one-third of the MHV-1 genome demonstrated that the genetic organization of MHV-1 was similar to that of other strains of MHV. The hemagglutinin esterase (HE) protein was truncated, and reverse transcription-PCR (RT-PCR) studies confirmed previous work that suggested that the MHV-1 HE is a pseudogene. Targeted recombination was used to select chimeric viruses containing either the MHV-1 S gene or genes encoding all of the MHV-1 structural proteins, on an MHV-A59 background. Challenge studies in mice demonstrated that expression of the MHV-1 S gene within the MHV-A59 background (rA59/S(MHV-1)) increased the pneumovirulence of MHV-A59, and mice infected with this recombinant virus developed pulmonary lesions that were similar to those observed with MHV-1, although rA59/S(MHV-1) was significantly less virulent. Chimeras containing all of the MHV-1 structural genes on an MHV-A59 background were able to reproduce the severe acute respiratory syndrome (SARS)-like pathology observed with MHV-1 and reproducibly increased pneumovirulence relative to rA59/S(MHV-1), but were still much less virulent than MHV-1. These data suggest that important determinants of pneumopathogenicity are contained within the 3' one-third of the MHV-1 genome, but additional important virulence factors must be encoded in the genome upstream of the S gene. The severity of the pulmonary lesions observed correlates better with elevated levels of inflammatory cytokines than with viral replication in the lungs, suggesting that pulmonary disease has an important immunological component.
Journal of Virology 09/2010; 84(18):9278-91. · 5.40 Impact Factor
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ABSTRACT: Although it is becoming increasingly common to accept livers from older donors for transplantation, old livers are more damaged by hepatic ischemia and reperfusion injury (HIRI) than young livers. We hypothesized that this age-related susceptibility to HIRI is due to increased hepatocellular apoptosis driven by tumor necrosis factor alpha (TNFalpha). Young (6-week-old) and old (60-week-old) mice underwent 60 minutes of hepatic ischemia and increasing periods of reperfusion. TNFalpha was determined by enzyme-linked immunosorbent assay. Liver injury (enzyme release), apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling staining, cytochrome C release, and caspase activation), and necrosis (hematoxylin and eosin staining) were assessed. We assessed the impact of apoptosis by blocking TNFalpha production or effect (pentoxifylline and TNFalpha receptor knockout), inhibiting apoptotic pathways (caspase inhibition), or imposing a hepatic protective strategy [glucose infusion with ischemic preconditioning (Glc/PC)]. In comparison with young livers, old livers subjected to HIRI had more pronounced liver aspartate aminotransferase release (6200 versus 3900 U/L, P = 0.02), necrosis (45% versus 25%, P = 0.03), and apoptosis with increased 30-minute TNFalpha release (19.02 versus 10.62 pg/mg, P = 0.03). Eliminating TNFalpha production reversed the effect of age, as did inhibition of apoptotic pathways with caspase inhibition. Glc/PC of old mice attenuated TNFalpha release (9.56 versus 19.02 pg/mg, P = 0.001) and age-related exaggerated HIRI and improved survival (60% versus 0%). In conclusion, the age-related susceptibility to HIRI is driven by an exaggerated induction of TNFalpha-dependent hepatocellular apoptosis. Targeting the apoptotic cascade has implications for the older donor liver population.
Liver Transplantation 11/2009; 15(11):1594-604. · 3.39 Impact Factor
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Itay Shalev,
Kit Man Wong,
Katharina Foerster,
Yi Zhu,
Cecilia Chan,
Asif Maknojia, Jianhua Zhang,
Xue-Zhong Ma,
Xiao Chun Yang,
Julia Fang Gao,
Hao Liu,
Nazia Selzner,
David A Clark,
Oyedele Adeyi,
M James Phillips,
Reginald R Gorczynski,
David Grant,
Ian McGilvray,
Gary Levy
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ABSTRACT: 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 11/2008; 49(2):387-97. · 11.66 Impact Factor
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Nadine De Albuquerque,
Ehtesham Baig,
Xuezhong Ma, Jianhua Zhang,
William He,
Andrea Rowe,
Marlena Habal,
Mingfeng Liu,
Itay Shalev,
Gregory P Downey,
Reginald Gorczynski,
Jagdish Butany,
Julian Leibowitz,
Susan R Weiss,
Ian D McGilvray,
M James Phillips,
Eleanor N Fish,
Gary A Levy
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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.
Journal of Virology 12/2006; 80(21):10382-94. · 5.40 Impact Factor
