A benzenediamine derivate FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting myeloid dendritic cell-secreted BAFF.

Myeloid dendritic cells (DCs) can produce B-cell-activating factor (BAFF) that modulates survival and differentiation of B cells and plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). Toll-like receptor 4 (TLR4) signaling has important functions in the process of BAFF production. Our previous study showed that a benzenediamine derivate FC-99 possesses anti-inflammation activity and directly interacts with interleukin-1 receptor-associated kinase 4 (IRAK4), which was a pivotal molecule in TLR4 signaling. In this study, we demonstrated that FC-99 attenuated lupus nephritis in the MRL/lpr mice. FC-99 also decreased the levels of total immunoglobulin G (IgG), total IgG2a and IgM in sera, as well as the activation of B cells in the spleens of MRL/lpr mice. Moreover, FC-99 inhibited abnormal activation of myeloid DCs in spleens and reduced the levels of BAFF in sera, spleens, and kidneys of MRL/lpr mice. Furthermore, upon TLR4 stimulation with lipopolysaccharide in vitro, FC-99 inhibited IRAK4 phosphorylation, as well as the activation and BAFF production in murine bone marrow-derived DCs. These data indicate that FC-99 attenuates lupus nephritis in MRL/lpr mice via inhibiting DC-secreted BAFF, suggesting that FC-99 may be a potential therapeutic candidate for the treatment of SLE.

A novel benzenediamine derivate rescued mice from experimental sepsis by attenuating proinflammatory mediators via IRAK4.

We designed and synthesized a novel benzenediamine derivate, FC-99, that was tested for its ability to protect mice from experimental sepsis. Moreover, we sought to determine whether FC-99 could control a bacterial infection and to clarify the mechanism by which FC-99 inhibited LPS-activated macrophages. The effects of FC-99 on inflammation were evaluated in two experimental sepsis models and in cultured macrophages. Microarrays and docking and molecular dynamics simulations were used to determine the target of FC-99. Surface plasmon resonance and molecular detection were performed to confirm the direct interaction of FC-99 with its target. FC-99 protected mice from experimental sepsis. The mice that received FC-99 exhibited a diminished inflammatory response, had a lower local bacterial burden, and experienced a significantly improved survival rate. Genome-wide transcriptional profiling of FC-99-treated macrophages identified IRAK4 as a drug-regulated gene involved in LPS/TLR4 signaling. A computer search and calculations indicated that IRAK4 directly interacted with FC-99. Surface plasmon resonance, IRAK4-regulated signaling pathway analysis, and gene expression profiling of proinflammatory mediators confirmed the direct interaction between FC-99 and IRAK4. FC-99 is a potential therapeutic molecule for sepsis that alleviated experimental sepsis by directly inhibiting IRAK4 activation, which represents a novel target for sepsis therapy.

Novel benzenediamine derivative FC99 ameliorates zymosan-induced arthritis by inhibiting RORγt expression and Th17 cell differentiation.

Increased IL-17-producing helper T (Th17) cells have been observed in patients with rheumatoid arthritis (RA). The retinoic-acid-related orphan nuclear receptor (RORγt) is the master regulator of Th17 cells. Our previous research showed that FC99 possesses anti-inflammation activity. However, to date the effects of FC99 on RORγt expression in Th17 cell differentiation have not been investigated yet. In the present study, we found that FC99 significantly attenuated arthritis-like symptoms, i.e., suppressing the development of paw edema in zymosan-induced arthritis (ZIA) mice. H&E staining showed that the infiltration of inflammatory cells in ankle synovial tissues was significantly suppressed. FC99 also reduced the mRNA levels of pro-inflammatory cytokines in ankle synovial tissues as shown by Q-PCR analysis. The protein levels of the pro-inflammatory cytokines in sera were also suppressed after FC99 treatment. Moreover, FC99 decreased the RORγt mRNA level in spleen tissues. Th17 cell percentage was significantly decreased in spleens and draining lymph nodes (dLNs). The mRNA and protein levels of IL-17A and IL-23 were reduced after FC99 treatment in ZIA mice. Furthermore, in vitro experiments showed that FC99 inhibited the expression of IL-6 in LPS-induced RAW264.7 cells and BMDCs. Moreover, FC99 significantly inhibited the RORγt expression in PMA-induced CD4(+) T cells and LPS-induced RAW264.7 cells. These data indicate that FC99 improves arthritis-like pathological symptoms in vivo and in vitro, which might be related to the inhibition of RORγt expression in Th17 cells. Our findings suggest that FC99 may be a potential therapeutic candidate for the treatment of RA and other inflammatory disorders.

FC-99 reduces macrophage tenascin-C expression by upregulating miRNA-494 in arthritis.

The excessive production of inflammatory mediators by inflammatory cells contributes to the pathogenesis of rheumatoid arthritis. Tenascin-C (TN-C) is expressed in rheumatoid joint, and is associated with levels of inflammatory mediators. FC-99 (N1-[(4-methoxy)methyl]-4-methyl-1,2-Benzenediamine), a novel 1,2-benzenediamine derivative, was previously reported to block the prolonged expression of key rheumatoid arthritis inflammatory cytokines and relieve zymosan-induced joint inflammation. However, the specific mechanism is unknown. This study aimed to examine the effects of FC-99 on TN-C expression and inflammation and investigate its possible molecular mechanism. The results showed that FC-99 treatment reduced the high expression of TN-C in ankle joints of arthritis mice. Besides, FC-99 reduced the increased number of macrophages in arthritis mice, while did not change the number of synovioblasts. Concomitantly, expression of TN-C in synovial fibroblasts exhibited no difference between control and ZIA groups, and was not apparently altered following FC-99 treatment, while FC-99 decreased TN-C expression in macrophages both in vivo and in vitro. Meanwhile, TargetScan and luciferase assays indicated that TN-C was negatively regulated by miR-494. Transfection assay further demonstrated that FC-99 inhibited TN-C by targeting miR-494. Furthermore, the reduction of miR-494 mimic on expression of TN-C was associated with NF-κB pathway. Similarly, the down-regulation of FC-99 on TN-C was considerably decreased when NF-κB pathway was inhibited. These results indicated that FC-99 relieved macrophages inflammation via the miR-494/TN-C/NF-κB pathway, finally leading to the relief of inflammation in arthritis. The findings suggested that FC-99 might be a potential therapeutic candidate for the treatment of rheumatoid arthritis.

FC-99 ameliorates sepsis-induced liver dysfunction by modulating monocyte/macrophage differentiation via Let-7a related monocytes apoptosis.

BACKGROUND: The liver is a vital target for sepsis-related injury, leading to inflammatory pathogenesis, multiple organ dysfunction and high mortality rates. Monocyte-derived macrophage transformations are key events in hepatic inflammation. N1-[(4-methoxy)methyl]-4-methyl-1,2-benzenediamine (FC-99) previously displayed therapeutic potential on experimental sepsis. However, the underlying mechanism of this protective effect is still not clear. RESULTS: FC-99 treatment attenuated the liver dysfunction in septic mice that was accompanied with reduced numbers of pro-inflammatory Ly6Chi monocytes in the peripheral blood and CD11b+F4/80lo monocyte-derived macrophages in the liver. These effects were attributed to the FC-99-induced apoptosis of CD11b+ cells. In PMA-differentiated THP-1 cells, FC-99 repressed the expression of CD11b, CD14 and caspase3 and resulted in a high proportion of Annexin V+ cells. Moreover, let-7a-5p expression was abrogated upon CLP stimulation in vivo, whereas it was restored by FC-99 treatment. TargetScan analysis and luciferase assays indicated that the anti-apoptotic protein BCL-XL was targeted by let-7a-5p. BCL-XL was inhibited by FC-99 in order to induce monocyte apoptosis, leading to the impaired monocyte-to-macrophage differentiation. MATERIALS AND METHODS: Murine acute liver failure was generated by caecal ligation puncture surgery after FC-99 administration; Blood samples and liver tissues were collected to determine the monocyte/macrophage subsets and the induction of apoptosis. Human acute monocytic leukemia cell line (THP-1) cells were pretreated with FC-99 followed by phorbol-12-myristate-13-acetate (PMA) stimulation, in order to induce monocyte-to-macrophage differentiation. The target of FC-99 and the mechanistic analyses were conducted by microarrays, qRT-PCR validation, TargetScan algorithms and a luciferase report assay. CONCLUSIONS: FC-99 exhibits potential therapeutic effects on CLP-induced liver dysfunction by restoring let-7a-5p levels.

A new benzenediamine derivative modulates Toll-like receptors-induced myeloid dendritic cells activation and ameliorates lupus-like syndrome in MRLlpr/lpr mice.

Modulators of the over-activation of myeloid dendritic cells (mDCs) by Toll-like receptors (TLRs) have an advantage in the treatment of systemic lupus erythematosus (SLE). This study was designed to evaluate the effects of FC-99, a novel benzenediamine derivative, on TLR-induced activation of mDCs, and to assess the efficacy of FC-99 in a murine model of SLE. In vitro, FC-99 inhibited the phenotypic (CD40 and MHC-II) and functional activation (IL-12 and CXCL10) of mDCs induced by TLR ligands. In vivo, MRLlpr/lpr mice displayed renal diseases associated with increased levels of proteinuria and immunoglobulin, which were ameliorated by FC-99. Enhanced accumulation and activation of mDCs in lymphoid organs was also impaired by FC-99. Additionally, FC-99 inhibited the activation of IκB-α and upregulated the expression of TNFα-induced protein 3 (TNFAIP3) in vitro and in vivo. These results indicate that FC-99 modulates TLR-induced activation of mDCs and ameliorates lupus-like syndrome in MRLlpr/lpr mice. This effect is closely associated with the inhibition of IκB-α and upregulation of TNFAIP3.

A novel small molecule compound possesses immunomodulatory properties on bone marrow-derived dendritic cells via TLR7 signaling pathway and alleviates the development of SLE.

Dendritic cells (DCs) play an important role in the development and maintenance of immune tolerance. Activation of TLR7, which is expressed in DCs, is thought to contribute to the complex pathophysiology of systemic lupus erythematosus (SLE). In this study, we analyzed the in vitro and in vivo function of a novel small-molecule compound, FC-99, which was previously reported to have immunomodulatory functions. We found that FC-99 inhibited the expression of CD40 and inflammatory mediators (IL-6, IL-12, and CXCL-10), as well as R848-induced phosphorylation of IκB-α. We also present evidence that FC-99 is remarkably efficacious in the treatment of murine lupus. Interestingly, FC-99 affected the maturation and percentage of DCs in lupus-prone mice. Therefore, FC-99 may serve as a potential drug candidate for treatment of SLE.

Benzenediamine analog FC-99 inhibits TLR2 and TLR4 signaling in peritoneal macrophage in vitro.

AIM: Inflammatory bowel disease (IBD) is an inflammatory disorder, characterized by abnormally increased expression of Toll-like receptors TLR2 and TLR4 in the colon and increased pro-inflammatory cytokine production by macrophages. MAIN METHODS: In the present study, we explored the effect of FC-99, a novel benzenediamine analog, on dextran sulfate sodium (DSS)-induced mouse colitis and investigated its potential mechanism. KEY FINDINGS: The results revealed that FC-99 improved the colon morphology and the clinical parameters in DSS-induced mouse colitis. FC-99 inhibited the increase of DSS-induced T helper cells (Th) 1 and Th17 and enhanced the number of regulatory T cells (Treg) in mesenteric lymph nodes (MLN), but had no effect on Th2 cells. FC-99 also suppressed the DSS-induced secretion of interleukin (IL)-1ß, IL-6, and the tumor necrosis factor (TNF)-α in the colon and hindered the infiltration of macrophages into colon lamina propria. Flow cytometric analysis also confirmed that FC-99 reduced CD11b(+)F4/80(+) colon macrophages, and down-regulated TNF-α level in situ. Moreover, FC-99 inhibited concentration-dependently the expression of TNF-α and IL-6 in vitro from mouse peritoneal macrophages, which were induced by TLR ligands: PamCSK4 and peptidoglycan (PGN, TLR2 ligand) as well as LPS (TLR4 ligand). Of note, FC-99 also suppressed the activation of TLR2 and TLR4 signaling pathways and the downstream nuclear factor-κB (NF-κB) in the DSS-induced mouse colitis. SIGNIFICANCE: FC-99 improved the condition of DSS-induced mouse colitis by inhibiting the activation of TLR2 and TLR4 signaling pathways in macrophage. These results suggest that FC-99 may be developed as a new therapeutic drug for IBD.

A benzenediamine derivative fc-99 attenuates lupus-like syndrome in MRL/lpr mice related to suppression of pDC activation.

Systemic lupus erythematosus (SLE) is an autoimmune disease with prominent chronic inflammatory aspects. Plasmacytoid dendritic cells (pDCs), which are the principal interferon-α (IFN-α)-producing cells, have known to be critically involved in SLE pathogenesis. Our previous research demonstrated that a benzenediamine derivative FC-99 possessed anti-inflammatory activities. However, the effects of FC-99 on SLE have not been investigated to date. In this study, we found that FC-99 attenuated lupus-like pathological symptoms and lupus nephritis as well as the expression of pro-inflammatory cytokines in kidneys of MRL/lpr mice. FC-99 also decreased both the total IgM, total IgG and anti-dsDNA IgG levels in sera and the activation of B cells in the PBMCs and spleens of MRL/lpr mice. Moreover, FC-99 inhibited the abnormal activation and number of pDCs from PBMCs and spleens and levels of IFN-α in MRL/lpr mice. Notably, FC-99 significantly suppressed the expression of IFN-inducible genes in peripheral blood mononuclear cells (PBMCs) and spleens from MRL/lpr mice. As expected, in vitro experiments demonstrated that FC-99 decreased both the activation and IFN-α production of pDCs and inhibited IRAK4 phosphorylation in pDCs upon TLR7 and TLR9 stimulation. We further confirm that the inhibition of FC-99 on B cell activation depended on level of pDCs-secreting IFN-α. These data indicate that FC-99 attenuated lupus-like syndrome in MRL/lpr mice related to suppression of pDC activation, especially pDCs-secreting IFN-α. This study suggests that FC-99 may be a potential therapeutic candidate for the treatment of SLE.