Quercetin attenuates adipogenesis and fibrosis in human skeletal muscle.

Age-associated increase in ectopic fat degeneration and fibrosis in the skeletal muscle contribute to muscle degradation and weakness. Quercetin is a bioactive flavonoid with anti-inflammatory and anti-obesity effects. Thus, we aimed to investigate the effects of quercetin on adipogenesis and fibrosis in the human skeletal muscle, which have not yet been elucidated. Human muscle-derived PDGFRα+/CD201+ cells (mesenchymal progenitors) were incubated with various concentrations of quercetin (0, 0.3, 1, and 3 µM) under adipogenic or fibrogenic conditions. Lipid accumulation was visualized via Oil Red O staining. The expression of genes implicated in adipocyte or fibroblast differentiation and activation of signaling pathways was analyzed. The quercetin-treated PDGFRα+/CD201+ cells showed attenuated lipid accumulation and adipogenic gene expression (CEBPA and ADIPOQ) via the inhibition of CREB phosphorylation under adipocyte differentiation conditions. Additionally, quercetin treatment significantly attenuated the expression of fibrogenic genes (TIMP1, ACTA2, COL1A1 and COL3A1) by inhibiting Smad2 phosphorylation. Quercetin suppressed the differentiation of muscle-derived PDGFRα+/CD201+ cells to adipocytes and fibroblasts at concentrations achievable by dietary and dietary supplement intake, which indicated its preventive or therapeutic effect against the loss of muscle quality.

Necrostatin-7 suppresses RANK-NFATc1 signaling and attenuates macrophage to osteoclast differentiation.

Osteoclasts play a crucial role in osteolytic bone diseases, such as osteoporosis, rheumatoid arthritis, periodontitis, Paget's disease of bone and bone metastatic tumors. Therefore, controlling osteoclast differentiation and function has been considered a promising therapeutic strategy. Here, we show that necrostatin (Nec)-7, an inhibitor of programmed necrosis, strongly suppressed receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclastogenesis and bone resorption, without compromising macrophage colony-stimulating factor (M-CSF)-supported survival and growth of osteoclast precursor cells. Accordingly, Nec-7 significantly decreased the levels of RANKL-induced osteoclastogenic marker genes, such as cathepsin K. Mechanistically, Nec-7 neither affected MAPK nor NF-κB activation; however, it strongly inhibited the RANKL receptor (RANK) to nuclear factor of activated T cells c1 (NFATc1) signaling. Lentiviral expression of RANK in bone marrow-derived macrophages significantly restored osteoclastogenesis and NFATc1 amplification in Nec-7-treated cells. In this study, we revealed that Nec-7-sensitive pathways are crucially involved in osteoclast formation and function. Investigation of the molecular mechanism(s) through which Nec-7 inhibits RANK-NFATc1 signaling axis may lead to the development of new therapeutic strategies for bone disease.

Inhibition of MMP-2-Mediated Mast Cell Invasion by NF-κB Inhibitor DHMEQ in Mast Cells.

BACKGROUND: Stimulation with antigen and IgE is known to activate NF-κB in mast cells. In the present research, we studied the role of NF-κB on cellular migration in mast cell-like RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) using the NF-κB inhibitor (-)-DHMEQ. METHODS: A Matrigel invasion chamber was used to evaluate cell migration. A PCR array was used to screen the expression of 84 key genes involved in cell migration. RESULTS: (-)-DHMEQ inhibited antigen/IgE-induced NF-κB activation and expressions of its target genes such as IL-6 and TNF-α. (-)-DHMEQ was found to inhibit in vitro invasion toward the antigen without any toxicity. We then looked for NF-κB-dependent genes that would be important for mast cell invasion using the PCR array. (-)-DHMEQ was found to lower the expression of matrix metalloproteinase (MMP)-2. The MMP inhibitor GM6001 also inhibited cellular invasion toward the antigen. These effects of (-)-DHMEQ were obtained in both RBL-2H3 cells and BMMCs. CONCLUSIONS: These findings indicate that (-)-DHMEQ suppressed mast cell migration via the inhibition of NF-κB-regulated MMP-2 expression.

Intestinal epithelial cell-derived semaphorin 7A negatively regulates development of colitis via αvß1 integrin.

The intestinal immune system is constantly challenged by commensal bacteria; therefore, it must maintain quiescence via several regulatory mechanisms. Although intestinal macrophages (Ms) have been implicated in repression of excessive inflammation, it remains unclear how their functions are regulated during inflammation. In this study, we report that semaphorin 7A (Sema7A), a GPI-anchored semaphorin expressed in intestinal epithelial cells (IECs), induces IL-10 production by intestinal Mϕs to regulate intestinal inflammation. Sema7A-deficient mice showed severe signs of dextran sodium sulfate-induced colitis due to reduced intestinal IL-10 levels. We further identified CX3CR1(+)MHC class II(int)F4/80(hi)CD11b(hi) Mϕs as the main producers of IL-10 via αvß1 integrin in response to Sema7A. Notably, Sema7A was predominantly expressed on the basolateral side of IECs, and its expression pattern was responsible for protective effects against dextran sodium sulfate-induced colitis and IL-10 production by Mϕs during interactions between IECs and Mϕs. Furthermore, we determined that the administration of recombinant Sema7A proteins ameliorated the severity of colitis, and these effects were diminished by IL-10-blocking Abs. Therefore, our findings not only indicate that Sema7A plays crucial roles in suppressing intestinal inflammation through αvß1 integrin, but also provide a novel mode of IL-10 induction via interactions between IECs and Mϕs.

The novel multi-cytokine inhibitor TO-207 specifically inhibits pro-inflammatory cytokine secretion in monocytes without affecting the killing ability of CAR T cells.

Cancer immunotherapy using chimeric antigen receptor-armed T (CAR T) cells have been shown to improve outcomes significantly in patients with hematological malignancies. However, cytokine release syndrome (CRS) remains a risk. CRS is characterized by the excessive activation of CAR T cells and macrophages. Signs and symptoms of CRS are usually resolved after steroid administration, but steroids abrogate the expansion and persistence of CAR T cell populations. Tocilizumab is a humanized monoclonal antibody (mAb) that attenuates CRS without significant loss of CAR T cell activity. However, interleukin-6 (IL-6)/IL-6 receptor (IL-6R) blockade alone cannot relieve CRS symptoms fully, and novel treatments are needed to prevent or cure CRS. TO-207 is an N-benzoyl-L-phenylalanine derivative that significantly inhibits inflammatory cytokine production in human monocyte and macrophage-specific manner. We investigated whether TO-207 could inhibit cytokine production without impairing CAR T cell function in a CRS-simulating co-culture system.

Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K.

Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome-plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases.

Necrostatin-1 protects against reactive oxygen species (ROS)-induced hepatotoxicity in acetaminophen-induced acute liver failure.

Excessive acetaminophen (APAP) use is one of the most common causes of acute liver failure. Various types of cell death in the damaged liver are linked to APAP-induced hepatotoxicity, and, of these, necrotic cell death of hepatocytes has been shown to be involved in disease pathogenesis. Until recently, necrosis was commonly considered to be a random and unregulated form of cell death; however, recent studies have identified a previously unknown form of programmed necrosis called receptor-interacting protein kinase (RIPK)-dependent necrosis (or necroptosis), which is controlled by the kinases RIPK1 and RIPK3. Although RIPK-dependent necrosis has been implicated in a variety of disease states, including atherosclerosis, myocardial organ damage, stroke, ischemia-reperfusion injury, pancreatitis, and inflammatory bowel disease. However its involvement in APAP-induced hepatocyte necrosis remains elusive. Here, we showed that RIPK1 phosphorylation, which is a hallmark of RIPK-dependent necrosis, was induced by APAP, and the expression pattern of RIPK1 and RIPK3 in the liver overlapped with that of CYP2E1, whose activity around the central vein area has been demonstrated to be critical for the development of APAP-induced hepatic injury. Moreover, a RIPK1 inhibitor ameliorated APAP-induced hepatotoxicity in an animal model, which was underscored by significant suppression of the release of hepatic enzymes and cytokine expression levels. RIPK1 inhibition decreased reactive oxygen species levels produced in APAP-injured hepatocytes, whereas CYP2E1 expression and the depletion rate of total glutathione were unaffected. Of note, RIPK1 inhibition also conferred resistance to oxidative stress in hepatocytes. These data collectively demonstrated a RIPK-dependent necrotic mechanism operates in the APAP-injured liver and inhibition of this pathway may be beneficial for APAP-induced fulminant hepatic failure.