Objective The Fli-1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE) in both human patients and animal models. Dysregulation of interleukin 6 (IL-6) is also associated with SLE. We investigated whether Fli-1 directly regulates the expression of IL-6. Methods Sera were collected from wild-type and Fli-1 heterozygous (Fli-1+/−) MRL/lpr mice and the concentration of IL-6 was measured by ELISA. Expression of IL-6 in the kidney was measured by real-time PCR. T cells were isolated from wild-type and Fli-1+/− MRL/lpr mice and stimulated with CD3/CD28 beads, and the concentration of IL-6 in the supernatants was measured by ELISA. MS1 endothelial cells were transfected with Fli-1 and control siRNA, and the production of IL-6 was compared after lipopolysaccharides (LPS) stimulation. A chromatin immunoprecipitation (ChIP) assay was performed to determine whether Fli-1binds to the IL-6 promoter region. Transient transfections with the NIH 3T3 cell line were performed to study if Fli-1 regulates the expression of IL-6. Results Fli-1+/− MRL/lpr mice had significantly decreased IL-6 in sera and reduced expression of IL-6 in kidneys compared to wild-type littermates. The T cells isolated from Fli-1+/− MRL/lpr mice produced less IL-6. Inhibiting the expression of Fli-1 in endothelial cells resulted in reduced production of IL-6. The ChIP assay revealed direct binding of Fli-1 to three regions within the IL-6 promoter. Fli-1 activated transcription from the IL-6 promoter in a dose-dependent manner. Conclusion Fli-1 directly regulates IL-6 expression as one of possible mechanisms for the protective effect in lupus of decreased Fli-1 expression.

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.

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.

Friend leukemia integration-1, a member of the Ets family of transcription factors, is highly expressed in splenocytes. Dysregulation of Fli-1 occurs in Systemic Lupus Erythematosus patients and lupus prone mice. Systemic Lupus Erythematosus is an autoimmune disease utilizing the innate and adaptive immune response. Reduction of Fli-1 expression in NZM 2410 and MRL/lpr lupus prone murine models resulted in decreased severity of disease. Studies of Toll-like receptor 7 and 8 reveal various implications in autoimmunity. Inflammatory cytokine IL-6 plays a pivotal role in the pathogenesis of SLE. The role of Fli-1 in Toll-like receptor signaling pathways in splenocytes is unknown. In this study, we generated Fli-1 heterozygous knockout (Fli-1+/-) B6 mice that expressed decreased levels of Fli-1 and investigated the impact of Fli-1 on splenocyte development and cytokine production. Spleen cells were isolated from Fli-1+/- B6 mice and wild-type controls. Examination of cell populations in the spleen revealed unaffected populations of B, T, Natural Killers and Dendritic cells between Fli1+/- mice and wild-type littermates. Spleen cells isolated from Fli-1+/- mice produced increased levels of IL-6 compared to wild-type mice upon stimulation with Toll-like receptor 7 ligand R848. Our results suggest that Fli-1 is involved in IL-6 cytokine production in spleen cells.

The Fli-1 transcription factor plays a significant role in the pathogenesis of systemic lupus erythematosus (SLE). Fli-1 expression is significantly increased in patients with lupus nephritis and lupus-prone mice with reduced Fli-1 expression (Fli-1+/-) live longer and have significantly less nephritis compared to wild type controls. Recently, 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. In clinical settings, G-CSF increases the inflammatory response and worsens tissue damage. 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 endothelial cells transfected with Fli-1 specific siRNA. The murine G-CSF promoter contains numerous putative Fli-1 binding sites and five regions within the proximal promoter were found to be 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. In conclusion, we have discovered that Fli-1 plays a major role in the regulation of inflammatory cytokines and chemokines and inflammatory disease pathogenesis.