Mara Lennard Richard

Differential transcriptomic responses of Biomphalaria glabrata (Gastropoda, Mollusca) to bacteria and metazoan parasites, Schistosoma mansoni and Echinostoma paraensei (Digenea, Platyhelminthes)

A 70-mer-oligonucleotide-based microarray (1152 features) that emphasizes stress and immune responses factors was constructed to study transcriptomic responses of the snail Biomphalaria glabrata to different immune challenges. In addition to sequences with relevant putative ID and Gene Ontology (GO) annotation, the array features non-immune factors and unknown B. glabrata ESTs for functional gene discovery. The transcription profiles of B. glabrata (3 biological replicates, each a pool of 5 snails) were recorded at 12h post-wounding, exposure to Gram negative or Gram positive bacteria (Escherichia coli and Micrococcus luteus, respectively), or infection with compatible trematode parasites (Schistosoma mansoni or Echinostoma paraensei, 20 miracidia/snail), relative to controls, using universal reference RNA. The data were subjected to Significance Analysis for Microarrays (SAM), with a false positive rate (FPR) <or=10%. Wounding yielded a modest differential expression profile (27 up/21 down) with affected features mostly dissimilar from other treatments. Partially overlapping, yet distinct expression profiles were recorded from snails challenged with E. coli (83 up/20 down) or M. luteus (120 up/42 down), mostly showing up-regulation of defense and stress-related features. Significantly altered expression of selected immune features indicates that B. glabrata detects and responds differently to compatible trematodes. Echinostoma paraensei infection was associated mostly with down-regulation of many (immune-) transcripts (42 up/68 down), whereas S. mansoni exposure yielded a preponderance of up-regulated features (140 up/23 down), with only few known immune genes affected. These observations may reflect the divergent strategies developed by trematodes during their evolution as specialized pathogens of snails to negate host defense responses. Clearly, the immune defenses of B. glabrata distinguish and respond differently to various immune challenges.

The Fli-1 Transcription Factor Regulates the Expression of CCL5/RANTES

The friend leukemia insertion site 1 (Fli-1) transcription factor, an Ets family member, is implicated in the pathogenesis of systemic lupus erythematosus in human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival, and decreased infiltrating inflammatory cells into the kidney. Inflammatory chemokines, including CCL5, are critical for attracting inflammatory cells. In this study, decreased CCL5 mRNA expression was observed in kidneys of lupus-prone NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly decreased in endothelial cells transfected with Fli-1–specific small interfering RNA compared with controls. Fli-1 binds to endogenous Ets binding sites in the distal region of the CCL5 promoter. Transient transfection assays demonstrate that Fli-1 drives transcription from the CCL5 promoter in a dose-dependent manner. Both Ets1, another Ets family member, and Fli-1 drive transcription from the CCL5 promoter, although Fli-1 transactivation was significantly stronger. Ets1 acts as a dominant-negative transcription factor for Fli-1, indicating that they may have at least one DNA binding site in common. Systematic deletion of DNA binding sites demonstrates the importance of the sites located within a 225-bp region of the promoter. Mutation of the Fli-1 DNA binding domain significantly reduces transactivation of the CCL5 promoter by Fli-1. We identified a novel regulator of transcription for CCL5. These results suggest that Fli-1 is a novel and critical regulator of proinflammatory chemokines and affects the pathogenesis of disease through the regulation of factors that recruit inflammatory cells to sites of inflammation.

Fli-1 controls transcription from the MCP-1 gene promoter, which may provide a novel mechanism for chemokine and cytokine activation

Regulation of proinflammatory cytokines and chemokines is a primary role of the innate immune response. MCP-1 is a chemokine that recruits immune cells to sites of inflammation. Expression of MCP-1 is reduced in primary kidney endothelial cells from mice with a heterozygous knockout of the Fli-1 transcription factor. Fli-1 is a member of the Ets family of transcription factors, which are evolutionarily conserved across several organisms including Drosophilla, Xenopus, mouse and human. Ets family members bind DNA through a consensus sequence GGAA/T, or Ets binding site (EBS). Fli-1 binds to EBSs within the endogenous MCP-1 promoter by ChIP assay. In this study, transient transfection assays indicate that the Fli-1 gene actively promotes transcription from the MCP-1 gene promoter in a dose-dependent manner. Mutation of the DNA binding domain of Fli-1 demonstrated that Fli-1 activates transcription of MCP-1 both directly, by binding to the promoter, and indirectly, likely through interactions with other transcription factors. Another Ets transcription factor, Ets-1, was also tested, but failed to promote transcription. While Ets-1 failed to drive transcription independently, a weak synergistic activation of the MCP-1 promoter was observed between Ets-1 and Fli-1. In addition, Fli-1 and the NFκB family member p65 were found to interact synergistically to activate transcription from the MCP-1 promoter, while Sp1 and p50 inhibit this interaction. Deletion studies identified that EBSs in the distal and proximal MCP-1 promoter are critical for Fli-1 activation from the MCP-1 promoter. Together, these results demonstrate that Fli-1 is a novel regulator of the proinflammatory chemokine MCP-1, that interacts with other transcription factors to form a complex transcriptional mechanism for the activation of MCP-1 and mediation of the inflammatory response.

The Fli-1 transcription factor is a critical regulator for controlling the expression of chemokine C-X-C motif ligand 2 (CXCL2).

HighlightsInhibiting Fli‐1 in endothelial cells and monocytes results in decreased production of CXCL2.Fli‐1 binds to the endogenous promoter of CXCL2 in endothelial cells.Fli‐1 activates transcription from the CXCL2 promoter in a dose‐dependent manner.Fli‐1 activates transcription of CXCL2 through direct binding.An additive effect on CXCL2 activation is observed between NF‐&kgr;B and Fli‐1 transcription factors. ABSTRACT Mammalian cells produce inflammatory cytokines and chemokines in response to innate immune signals and their expression is tightly regulated. Chemokine (C‐X‐C motif) ligand 2 (CXCL2), also known as macrophage inflammatory protein 2‐alpha (MIP2‐alpha), is an inflammatory chemokine belonging to the CXC chemokine family. CXCL2 is chemotactic for neutrophils and elevated expression of CXCL2 is associated with many inflammatory and autoimmune diseases. The Fli‐1 gene belongs to the large Ets transcription factor family, whose members regulate a wide variety of cellular functions including the immune response. In this study, we demonstrate that endothelial cells transfected with Fli‐1 specific siRNA produce significantly less CXCL2 compared to cells transfected with control siRNA after stimulation by the Toll‐like receptor (TLR) 4 ligands, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF‐&agr;). The production of CXCL2 in endothelial cells stimulated with LPS stimulation is dose‐dependent. We found that Fli‐1 binds to the CXCL2 promoter as established by Chromatin immunoprecipitation (ChIP) assay. Transient transfection assays show that Fli‐1 drives transcription from the CXCL2 promoter in a dose‐dependent manner and Fli‐1 regulates the expression of CXCL2 largely by directly binding to the promoter. Targeted knockdown and transient transfection experiments suggest that both Fli‐1 and the p65 subunit of NF‐&kgr;B affect the activation of CXCL2 in an additive manner. These results indicate that Fli‐1 is a novel, critical transcription factor that regulates the expression of the inflammatory chemokine CXCL2.

Acetylation impacts Fli-1-driven regulation of granulocyte colony stimulating factor.

Fli‐1 has emerged as a critical regulator of inflammatory mediators, including MCP‐1, CCL5, and IL‐6. The cytokine, granulocyte colony stimulating factor (G‐CSF) regulates neutrophil precursor maturation and survival, and activates mature neutrophils. Previously, a significant decrease in neutrophil infiltration into the kidneys of Fli‐1+/‐ lupus‐prone mice was observed. In this study, a significant decrease in G‐CSF protein expression was detected in stimulated murine and human endothelial cells when expression of Fli‐1 was inhibited. The murine G‐CSF promoter contains numerous putative Fli‐1 binding sites and several regions within the proximal promoter are significantly enriched for Fli‐1 binding. Transient transfection assays indicate that Fli‐1 drives transcription from the G‐CSF promoter and mutation of the Fli‐1 DNA binding domain resulted in a 94% loss of transcriptional activation. Mutation of a known acetylation site, led to a significant increase in G‐CSF promoter activation. The histone acetyltransferases p300/CBP and p300/CBP associated factor (PCAF) significantly decrease Fli‐1 specific activation of the G‐CSF promoter. Thus, acetylation appears to be an important mechanism behind Fli‐1 driven activation of the G‐CSF promoter. These results further support the theory that Fli‐1 plays a major role in the regulation of several inflammatory mediators, ultimately affecting inflammatory disease pathogenesis.

Mouse models of lupus: what they tell us and what they don’t

Lupus is a complex heterogeneous disease characterised by autoantibody production and immune complex deposition followed by damage to target tissues. Animal models of human diseases are an invaluable tool for defining pathogenic mechanisms and testing of novel therapeutic agents. There are perhaps more applicable murine models of lupus than any other human disease. There are spontaneous models of lupus, inducible models of lupus, transgenic-induced lupus, gene knockout induced lupus and humanised mouse models of lupus. These mouse models of lupus have contributed significantly to our knowledge of the pathogenesis of lupus and served as valuable preclinical models for proof of concept for new therapies. Despite their utility, mouse models of lupus have their distinct limitations. Although similar, mouse and human immune systems are different and thus one cannot assume a mechanism for disease in one is translatable to the other. Efficacy and toxicity of compounds can vary significantly between humans and mice, also limiting direct translation. Finally, the heterogeneous aspects of human lupus, both in clinical presentation, underlying pathogenesis and genetics, are not completely represented in current mouse models. Thus, proving a therapy or mechanism of disease in one mouse model is similar to proving a mechanism/therapy in a limited subset of human lupus. These limitations, however, do not marginalise the importance of animal models nor the significant contributions they have made to our understanding of lupus.

A critical role for the transcription factor Fli-1 in lupus development by regulating expression of Interleukin 6

The Fli‐1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE), both in humans and in animal models. Dysregulation of interleukin‐6 (IL‐6) is also associated with SLE. The purpose of this study was to investigate whether Fli‐1 directly regulates the expression of IL‐6.

A Critical Role of the Transcription Factor Fli-1 in Murine Lupus Development by Regulation of Interleukin-6 Expression: Fli-1 Regulates IL-6 Expression in Murine Lupus

The Fli‐1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE), both in humans and in animal models. Dysregulation of interleukin‐6 (IL‐6) is also associated with SLE. The purpose of this study was to investigate whether Fli‐1 directly regulates the expression of IL‐6.