Oxy210, a Semi-Synthetic Oxysterol, Exerts Anti-Inflammatory Effects in Macrophages via Inhibition of Toll-like Receptor (TLR) 4 and TLR2 Signaling and Modulation of Macrophage Polarization

Abstract

Inflammatory responses by the innate and adaptive immune systems protect against infections and are essential to health and survival. Many diseases including atherosclerosis, osteoarthritis, rheumatoid arthritis, psoriasis, and obesity involve persistent chronic inflammation. Currently available anti-inflammatory agents, including non-steroidal anti-inflammatory drugs, steroids, and biologics, are often unsafe for chronic use due to adverse effects. The development of effective non-toxic anti-inflammatory agents for chronic use remains an important research arena. We previously reported that oral administration of Oxy210, a semi-synthetic oxysterol, ameliorates non-alcoholic steatohepatitis (NASH) induced by a high-fat diet in APOE*3-Leiden.CETP humanized mouse model of NASH and inhibits expression of hepatic and circulating levels of inflammatory cytokines. Here, we show that Oxy210 also inhibits diet-induced white adipose tissue inflammation in APOE*3-Leiden.CETP mice, evidenced by the inhibition of adipose tissue expression of IL-6, MCP-1, and CD68 macrophage marker. Oxy210 and related analogs exhibit anti-inflammatory effects in macrophages treated with lipopolysaccharide in vitro, mediated through inhibition of toll-like receptor 4 (TLR4), TLR2, and AP-1 signaling, independent of cyclooxygenase enzymes or steroid receptors. The anti-inflammatory effects of Oxy210 are correlated with the inhibition of macrophage polarization. We propose that Oxy210 and its structural analogs may be attractive candidates for future therapeutic development for targeting inflammatory diseases.

Oxy210, a Semi-Synthetic Oxysterol, Inhibits Profibrotic Signaling in Cellular Models of Lung and Kidney Fibrosis

“…In earlier reports, we described the beneficial effects of Oxy210, a dual inhibitor of Hh and TGF-β signaling, on profibrotic responses observed in HSCs in vitro, and on the progression of liver fibrosis in vivo, using a humanized mouse model of NASH [ 22 ]. We also reported considerable anti-inflammatory effects of Oxy210 in vitro in macrophages and in vivo, in the liver, adipose tissue [ 23 ], and plasma of mice [ 22 ]. In the present follow-up report, we investigate the effects of Oxy210 in cellular models of lung and kidney fibrosis.…”

“…In the NASH mouse model, oral administration of Oxy210 formulated in mouse food was well-tolerated over 16 weeks of continuous dosing and ameliorated several hallmarks of NASH, including hepatic inflammation, fibrosis, apoptosis, and lipid deposition in a dose-dependent manner, resulting in improved hepatic function ([ 22 ] and unpublished results). Given the potential for pro-inflammatory side effects associated with the systemic inhibition of TGF-β signaling, we were initially surprised to find that Oxy210 exerted anti-inflammatory effects in the liver, adipose tissue [ 23 ], and plasma of the mice, evidenced by reduced inflammatory cytokine expression and lowered cytokine levels in circulation [ 22 ]. In subsequent studies, we examined the effects of Oxy210 on macrophages in vitro and determined that Oxy210 can directly exert significant anti-inflammatory effects by inhibiting toll-like receptor (TLR) signaling in macrophages [ 23 ].…”

“…Given the potential for pro-inflammatory side effects associated with the systemic inhibition of TGF-β signaling, we were initially surprised to find that Oxy210 exerted anti-inflammatory effects in the liver, adipose tissue [ 23 ], and plasma of the mice, evidenced by reduced inflammatory cytokine expression and lowered cytokine levels in circulation [ 22 ]. In subsequent studies, we examined the effects of Oxy210 on macrophages in vitro and determined that Oxy210 can directly exert significant anti-inflammatory effects by inhibiting toll-like receptor (TLR) signaling in macrophages [ 23 ]. Notably, TGF-β signaling remains unaffected by Oxy210 in macrophages, confirmed by the absence of inhibitory effects on Smad2/3 phosphorylation, which preserves the anti-inflammatory effects of TGF-β ligands in these cells [ 23 ].…”

“…In subsequent studies, we examined the effects of Oxy210 on macrophages in vitro and determined that Oxy210 can directly exert significant anti-inflammatory effects by inhibiting toll-like receptor (TLR) signaling in macrophages [ 23 ]. Notably, TGF-β signaling remains unaffected by Oxy210 in macrophages, confirmed by the absence of inhibitory effects on Smad2/3 phosphorylation, which preserves the anti-inflammatory effects of TGF-β ligands in these cells [ 23 ]. This cell-selective inhibition of TGF-β signaling by Oxy210 is new and different from other known modalities of TGF-β inhibition, such as small molecule TGF-β receptor 1 (TGFβRI/ALK5) inhibitors or TGF-β neutralizing antibodies, which tend to suppress TGF-β signaling across all cell types [ 21 ] with the potential for adverse events mentioned earlier.…”

“…This cell-selective inhibition of TGF-β signaling by Oxy210 is new and different from other known modalities of TGF-β inhibition, such as small molecule TGF-β receptor 1 (TGFβRI/ALK5) inhibitors or TGF-β neutralizing antibodies, which tend to suppress TGF-β signaling across all cell types [ 21 ] with the potential for adverse events mentioned earlier. Encouraged by Hh and TGF-β inhibitory activity in HSCs and the combined antifibrotic and anti-inflammatory profile observed in APOE*3-Leiden.CETP mice [ 22 , 23 ], in the current study, we aim to demonstrate that Oxy210 can produce similar effects in cellular models of lung and kidney fibrosis driven by Hh and TGF-β signaling. Given additional favorable attributes of Oxy210 (oral bioavailability, drug-like pharmacokinetic and safety profiles, facile and scalable preparation, etc.…”