Xian K. Zhang

Objective: Bromodomain 4 (BRD4) could be a therapeutic target in various diseases. We sought to investigate its role in lupus nephritis (LN) progression and explore whether targeting BRD4 could serve as a therapeutic approach for LN. Methods: BRD4 expression in renal cells was evaluated by immunofluorescence. Inhibition of endothelial BRD4 in mice was conducted by using BRD4 short hairpin RNA adeno‐associated virus (shRNA‐AAV) targeting endothelial cells (ECs). Mouse lupus models were MRL/Lpr mice. Human glomerular ECs (GECs) coupled with RNA sequencing, chromatin immunoprecipitation polymerase chain reaction, and protein‐protein interaction assays were performed to define underlying mechanisms. Results: BRD4 expression was increased in renal ECs in patients with LN and in mouse lupus models. Bromodomain and extraterminal inhibitor NHWD870 treatment greatly improved the features of LN in MRL/Lpr mice as evidenced by reduced lesions, IgG and C3 deposition, immune infiltration, and improved ultrastructural morphology in renal tissues. BRD4 shRNA‐AAV treatment robustly ameliorated the hallmark features of LN in MRL/Lpr mice including proteinuria, histologic and ultrastructural morphology of kidney, and IgG and C3 deposition and immune infiltration in kidney in MRL/Lpr mice. Elevated BRD4 led to changes in many pathways linked to LN and promoted permeability and immune responses in GECs, which were associated with their interaction with FLI‐1. BRD4 shRNA‐AAV treatment attenuated FLI‐1 expression in MRL/Lpr mice. BRD4 and FLI‐1 were colocalized in renal ECs, and FLI‐1 expression was elevated in patients with LN. Conclusion: Elevated BRD4 in ECs is involved in LN pathogenesis, which is partly associated with its interaction with FLI‐1. Targeting endothelial BRD4 could be a therapeutic strategy for LN. image

Friend leukemia virus integration 1 (Fli-1), a member of the ETS family of transcription factors, plays an essential role in diverse biological processes. Recent studies have underscored the significance of Fli-1 in modulating inflammation and autoimmune diseases via the regulation of inflammatory responses. Specifically, Fli-1 exerts control over inflammatory processes, influencing key effectors and signaling pathways associated with conditions such as systemic lupus erythematosus, scleroderma, cancer, and sepsis. This review aims to summarize the emerging roles of Fli-1 in inflammation and autoimmune diseases, with a focus on elucidating the underlying molecular mechanisms and exploring the potential therapeutic implications.

The transcription factor Fli-1, a member of the ETS family of transcription factors, is implicated in the pathogenesis of lupus disease. Reduced Fli-1 expression in lupus mice leads to decreased renal Cxcl10 mRNA levels and renal infiltrating CXCR3+ T cells that parallels reduced renal inflammatory cell infiltration and renal damage. Inflammatory chemokine CXCL10 is critical for attracting inflammatory cells expressing the chemokine receptor CXCR3. The CXCL10/CXCR3 axis plays a role in the pathogenesis of various inflammatory diseases including lupus. Our data here demonstrate that renal CXCL10 protein levels are significantly lower in Fli-1 heterozygous MRL/lpr mice compared to wild-type MRL/lpr mice. Knockdown of Fli-1 significantly reduced CXCL10 secretion in mouse and human endothelial cells, and human mesangial cells, upon LPS or TNFα stimulation. The Fli-1 inhibitor, Camptothecin, significantly reduced CXCL10 production in human monocyte cells upon interferon stimulation. Four putative Ets binding sites in the Cxcl10 promoter showed significant enrichment for FLI-1; however, FLI-1 did not directly drive transcription from the human or mouse promoters, suggesting FLI-1 may regulate CXCL10 expression indirectly. Our results also suggest that the DNA binding domain of FLI-1 is necessary for regulation of human hCXCR3 promotor activity in human T cells and interactions with co-activators. Together, these results support a role for FLI-1 in modulating the CXCL10-CXCR3 axis by directly or indirectly regulating the expression of both genes to impact lupus disease development. Signaling pathways or drugs that reduce FLI-1 expression may offer novel approaches to lupus treatment.

Transcription factors E26 transformation-specific-1 (Ets-1) and Friend leukemia insertion site-1 (Fli-1) and type I interferon (IFN) have been implicated in systemic lupus erythematosus (SLE). We examined the expression of these genes in peripheral blood mononuclear cells (PBMCs) from Japanese patients with SLE and analyzed their association with SLE. We enrolled 53 Japanese patients with SLE, 42 patients with rheumatoid arthritis (RA), and 30 healthy donors (HDs) (as controls) in this study. PBMCs were collected from all participants, and the expressions of Ets-1, Fli-1, and three interferon-inducible genes (IFIGs) (interferon-inducible protein with tetratricopeptide 1 [IFIT1], interferon-inducible protein 44 [IFI44], and eukaryotic translation initiation factor 2 alpha kinase 2 [EIF2AK2]) were measured using real-time polymerase chain reaction (PCR). The relationships of each molecule with clinical symptoms, laboratory data, and treatments were analyzed. The expression of Ets-1 and Fli-1 was significantly lower in the PBMCs from patients with SLE than that in the PBMCs from patients with RA and HDs. The expression of the three IFIGs was significantly higher in the PBMCs from patients with SLE than that in the PBMCs from patients with RA and HDs. For patients with SLE, significantly positive correlations were found between Ets-1 and three IFIGs; a similar trend was observed between Fli-1 and IFIGs. IFIG expression in the PBMCs was significantly higher in patients with SLE than that in other participants, and the expression of Ets-1 and Fli-1 was positively associated with IFN expression. Therefore, it was suggested that Ets-1 and Fli-1 were associated with the pathophysiology of SLE by regulating the type I IFN pathway.

Graft-versus-host disease (GVHD), manifesting as either acute (aGVHD) or chronic (cGVHD), presents significant life-threatening complications following allogeneic hematopoietic cell transplantation. Here, we investigated Friend virus leukemia integration 1 (Fli-1) in GVHD pathogenesis and validated Fli-1 as a therapeutic target. Using genetic approaches, we found that Fli-1 dynamically regulated different T cell subsets in allogeneic responses and pathogenicity in the development of aGVHD and cGVHD. Compared with homozygous Fli1-deficient or WT T cells, heterozygous Fli1-deficient T cells induced the mildest GVHD, as evidenced by the lowest Th1 and Th17 cell differentiation. Single-cell RNA-Seq analysis revealed that Fli-1 differentially regulated CD4⁺ and CD8⁺ T cell responses. Fli-1 promoted the transcription of Th1/Th17 pathways and T cell receptor–inducible (TCR-inducible) transcription factors in CD4⁺ T cells, while suppressing activation- and function-related gene pathways in CD8⁺ T cells. Importantly, a low dose of camptothecin, topotecan, or etoposide acted as a potent Fli-1 inhibitor and significantly attenuated GVHD severity, while preserving the graft-versus-leukemia (GVL) effect. This observation was extended to a xenograft model, in which GVHD was induced by human T cells. In conclusion, we provide evidence that Fli-1 plays a crucial role in alloreactive CD4⁺ T cell activation and differentiation and that targeting Fli-1 may be an attractive strategy for treating GVHD without compromising the GVL effect.

Objective: To examine the therapeutic effects of camptothecin (CPT) and topotecan (TPT), inhibitors of transcription factor Fli‐1 and topoisomerase, on lupus nephritis in (NZB × NZW)F1 (NZBWF1) mice, and to examine the effects of CPT and TPT on inflammatory mediators in human renal cells.Methods: Female NZBWF1 mice were treated with vehicle, cyclophosphamide (CYC), CPT (1 mg/kg or 2 mg/kg), or TPT (0.03 mg/kg, 0.1 mg/kg, or 0. 3 mg/kg) by intraperitoneal injection twice a week, beginning at the age of 25 weeks (n = 8–10 mice per group). Blood and urine were collected for monitoring autoantibodies and proteinuria. Mice were euthanized at 40 weeks, and renal pathology scores were assessed. Human renal endothelial and mesangial cells were treated with CPT or TPT, and cytokine expression was measured.Results: None of the NZBWF1 mice treated with 1 mg/kg or 2 mg/kg of CPT or 0.3 mg/kg of TPT had proteinuria >100 mg/dl at the age of 40 weeks. One of 8 mice treated with 0.1 mg/kg of TPT and 1 of 10 mice treated with CYC had proteinuria >300 mg/dl, whereas 90% of the mice treated with vehicle had proteinuria >300 mg/dl. Compared to vehicle control, mice treated with 1 mg/kg or 2 mg/kg of CPT, 0.1 mg/kg or 0.3 mg/kg of TPT, or CYC had significantly prolonged survival, attenuated renal injury, diminished splenomegaly, reduced anti–double‐stranded DNA autoantibody levels, and reduced IgG and C3 deposits in the glomeruli (all P < 0.05). Human renal cells treated with CPT or TPT had reduced expression of Fli‐1 and decreased monocyte chemotactic protein 1 production following stimulation with interferon‐α (IFNα) or IFNγ.Conclusion: Our findings indicate that low‐dose CPT and TPT could be repurposed to treat lupus nephritis.

Friend leukemia virus integration 1 (Fli-1) is an ETS transcription factor and a critical regulator of inflammatory mediators, including MCP-1, CCL5, IL-6, G-CSF, CXCL2, and caspase-1. GM-CSF is a regulator of granulocyte and macrophage lineage differentiation and a key player in the pathogenesis of inflammatory/autoimmune diseases. In this study, we demonstrated that Fli-1 regulates the expression of GM-CSF in both T cells and endothelial cells. The expression of GM-CSF was significantly reduced in T cells and endothelial cells when Fli-1 was reduced. We found that Fli-1 binds directly to the GM-CSF promoter using chromatin immunoprecipitation assay. Transient transfection assays indicated that Fli-1 drives transcription from the GM-CSF promoter in a dose-dependent manner, and mutation of the Fli-1 DNA binding domain resulted in a significant loss of transcriptional activation. Mutation of a known phosphorylation site within the Fli-1 protein led to a significant increase in GM-CSF promoter activation. Thus, direct binding to the promoter and phosphorylation are two important mechanisms behind Fli-1–driven activation of the GM-CSF promoter. In addition, Fli-1 regulates GM-CSF expression in an additive manner with another transcription factor Sp1. Finally, we demonstrated that a low dose of a chemotherapeutic drug, camptothecin, inhibited expression of Fli-1 and reduced GM-CSF production in human T cells. These results demonstrate novel mechanisms for regulating the expression of GM-CSF and suggest that Fli-1 is a critical druggable regulator of inflammation and immunity.

The transcription factor Friend leukemia integration 1 (Fli-1) regulates the expression of numerous cytokines and chemokines and alters the progression of lupus nephritis in humans and in the MRL/MpJ-Fas^lpr (MRL/lpr) mouse model. Th17-mediated immune responses are notably important as they promote ongoing inflammation. The purpose of this study is to determine the impact of Fli-1 on expression of interleukin-17A (IL-17A) and the infiltration of immune cells into the kidney. IL-17A concentrations were measured by ELISA in sera collected from MRL/lpr Fli-1-heterozygotes (Fli-1^+/−) and MRL/lpr Fli-1^+/+ control littermates. Expression of IL-17A and related proinflammatory mediators was measured by real-time polymerase chain reaction (RT-PCR). Immunofluorescence staining was performed on renal tissue from MRL/lpr Fli-1^+/− and control littermates using anti-CD3, anti-CD4, and anti-IL-17A antibodies to detect Th17 cells and anti-CCL20 and anti-CD11b antibodies to identify CCL20⁺ monocytes. The expression of IL-17A in renal tissue was significantly reduced; this was accompanied by decreases in expression of IL-6, signal transducer and activator of transcription 3 (STAT3), and IL-1β. Likewise, we detected fewer CD3⁺IL-17⁺ and CD4⁺IL-17⁺ cells in renal tissue of MLR/lpr Fli-1^+/− mice and significantly fewer CCL20⁺CD11b⁺ monocytes. In conclusion, partial deletion of Fli-1 has a profound impact on IL-17A expression and on renal histopathology in the MRL/lpr mouse.

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.