EVOLUTION: Of Mice . . .

Cellular programs coupled to cycles of epithelial–mesenchymal transitions (EMTs) play critical roles during embryogenesis, as well as during tissue development, remodeling, and repair. Research over the last decade has established the importance of an ever-expanding list of master EMT transcription factors, whose activity is regulated by STAT3 and function to stimulate the rapid transition of cells between epithelial and mesenchymal phenotypes. Importantly, inappropriate reactivation of embryonic EMT programs in carcinoma cells underlies their metastasis to distant organ sites, as well as their acquisition of stem cell-like and chemoresistant phenotypes operant in eliciting disease recurrence. Thus, targeted inactivation of master EMT transcription factors may offer new inroads to alleviate metastatic disease. Here we review the molecular, cellular, and microenvironmental factors that contribute to the pathophysiological activities of STAT3 during its regulation of EMT programs in human carcinomas.

STAT3 and epithelial-mesenchymal transitions in carcinomas.

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.

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Murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in A/J mice

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.

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

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. In this study, our objective was to identify differentially regulated proteins in HCC through a quantitative proteomic approach using iTRAQ. More than 600 proteins were quantitated of which 59 proteins were overexpressed and 92 proteins were underexpressed in HCC as compared to adjacent normal tissue. Several differentially expressed proteins were not implicated previously in HCC. A subset of these proteins (six each from upregulated and downregulated groups) was further validated using immunoblotting and immunohistochemical labeling. Some of the overexpressed proteins with no previous description in the context of HCC include fibroleukin, interferon induced 56 kDa protein, milk fat globule-EGF factor 8, and myeloidassociated differentiation marker. Interestingly, all the enzymes of urea metabolic pathway were dramatically downregulated. Immunohistochemical labeling confirmed differential expression of fibroleukin, myeloid associated differentiation marker and ornithine carbamoyl transferase in majority of HCC samples analyzed. Our results demonstrate quantitative proteomics as a robust discovery tool for the identification of differentially regulated proteins in cancers.

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A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma.

Immune coagulation is a major contributor to the pathogenesis of xenograft rejection, viral-induced hepatocellular injury and cytokine-induced fetal loss syndrome. In this study, we investigated the contribution of the novel gene product, fibrinogen-like protein 2 (fgl2) prothrombinase, in mediating immune injury in experimental and human acute allograft rejection. Using a mouse heterotopic cardiac transplant model, mouse fgl2 (mfgl2)/fibroleukin mRNA transcripts and protein were highly expressed in macrophages, CD4- and CD8-positive T lymphocytes, and endothelial cells in rejecting cardiac allografts in association with deposits of fibrin. Although mfgl2-deficient mice rejected allografts at similar rates to littermate controls, survival of grafts from mfgl2-deficient mice were prolonged and deposition of intravascular fibrin was diminished. Treatment of wild-type mice with a neutralizing anti-fgl2 Ab ameliorated histological evidence for allorejection and intravascular fibrin deposition, and resulted in an increase in graft survival. To address further the relevance of fgl2 in acute allograft rejection, we examined kidney biopsies from patients who had undergone renal transplantation. Human fgl2 mRNA transcripts and protein were markedly expressed mainly in renal tubule cells, infiltrating lymphoid cells including macrophages, CD8 + T cells, mature B cells (plasma cells), and endothelial cells. Dual staining showed fibrin deposition was localized mainly to blood vessels, in the glomerulus and interstitium and the lumen of tubules, and occurred in association with human fgl2 expression. These data collectively suggest that fgl2 accounts for the fibrin deposition seen in both experimental and human allograft rejection and provide a rationale for targeting fgl2 as adjunctive therapy to treat allograft rejection.

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Role of fibrinogen-like protein 2 prothrombinase/fibroleukin in experimental and human allograft rejection.

Signal transducer and activator of transcription 1alpha (STAT1 alpha) is reported to be essential for IFN-gamma and IFN-alpha regulated gene expression, while STAT1 beta, an alternate splice-form, mediates only IFN-alpha-dependent gene expression. STAT3 alpha and STAT3 beta splice forms are also differentially activated in response to cytokines including IL-6 and IL-10. The aim of this study was to investigate whether the STAT activation will predict the host immune response to viral infection and possibly a therapeutic target for the treatment of viral infection. Mouse hepatitis virus type 3 (MHV-3) resistant strain (A/J) and sensitive mouse strains (BalB/cJ) were infected intraperitoneally (i.p.) with 100 plaque form units (pfu) of MHV-3. The mice were sacrificed at the indicated times, and livers and spleens were immediately frozen in liquid nitrogen. Nuclear extracts proteins were detected by immunoblotting. STAT1 and STAT3 activation in spleen increased 24 to 72 hr following MHV-3 infections in both sensitive and resistant mouse strains. However, over this time period, the ratio of activated alpha to beta splice-form for STAT1 and STAT3 increased above 1.0 in resistant A/J mice, while the ratio fell to <0.3 in MHV-3 sensitive Balb/cJ and C3H/HeJ strains. Activated STAT1 alpha/beta and STAT3 alpha/beta ratio in liver were similar in resistant and sensitive mouse strains. Treatment of sensitive Balb/cJ mice with neutralizing anti-TGF-beta antibody could increase the STAT1 alpha/beta ratio to <1.0 in spleens, predicting enhanced rates of survival. These results suggested that ratio of activated STAT1 alpha/beta and STAT3 alpha/beta in mixed leukocytes from spleen predict the outcome to MHV-3 infection, and may be an important marker and therapeutic target for modification of host immune response to virus infection.

Feili Gong

Papers

Role of fibrinogen-like protein 2 prothrombinase/fibroleukin in experimental and human allograft rejection.

STAT1 and STAT3 α/β splice form activation predicts host responses in mouse hepatitis virus type 3 infection