Icaritin inhibited cigarette smoke extract-induced CD8+ T cell chemotaxis enhancement by targeting the CXCL10/CXCR3 axis and TGF-β/Smad2 signaling

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is a disabling/fatal disease characterized by progressive pulmonary function decline, and there are currently few drugs that can effectively reverse the decline in lung function; therefore, it is necessary to find novel drug targets. CD8⁺ T cells might be a new therapeutic target for alleviating lung tissue destruction and improving pulmonary function in COPD. The CXCL10/CXCR3 axis is a pivotal chemotactic axis involved in the abnormal infiltration of CD8⁺ T cells into the lung tissue of COPD; thus, inhibition of this axis might be a potential method to suppress CD8⁺ T cell-mediated lung tissue destruction in COPD. However, few drugs have been reported to target CD8⁺ T cells and the CXCL10/CXCR3 axis. Icaritin (ICT), one of the major components of Epimedii Folium, has been reported to have antioxidative effects in a COPD model in vitro. Whether ICT also has effects on CD8⁺ T cells and the CXCL10/CXCR3 axis in COPD has never been investigated.

Purpose: This study aimed to investigate the effects of ICT on CD8⁺ T cell chemotaxis and the CXCL10/CXCR3 axis in interferon (IFN)-γ + cigarette smoke extract (CSE)-stimulated THP-1-derived macrophages, which simulated the pulmonary microenvironment of COPD, and then to determine the mechanisms.

Methods: The effects of ICT on the expression and secretion of CXCL9, CXCL10, and CXCL11 in THP-1-derived macrophages were measured by qRT-PCR and ELISA, and the effects of the supernatant of THP-1-derived macrophages treated with or without ICT on CD8⁺ T cell chemotaxis were also evaluated. Subsequently, the effects of ICT on the apoptosis and proliferation of CD8⁺ T cells were also assessed by EdU-488 assays and Annexin V/PI staining, respectively. Moreover, the mechanisms by which ICT inhibits the CXCL10/CXCR3 axis were investigated by RNA sequencing (RNA-seq) and KEGG pathway enrichment analysis.

Results: The present study showed that ICT (5 μM) significantly suppressed the expression and secretion of CXCL9, CXCL10, and CXCL11 in THP-1-derived macrophages after stimulation with IFN-γ + CSE and indirectly inhibited CD8⁺ T cell chemotaxis by reducing the secretion of the above chemokines. In addition, this study found that ICT had no significant effect on the proliferation of CD8⁺ T cells, and neither led to apoptosis. The results of the RNA-seq analysis illustrated that the transforming growth factor (TGF)-β signaling pathway was significantly downregulated after ICT intervention, and subsequent qRT-PCR and western blotting showed that ICT could significantly downregulate the TGF-β-Smad2 signaling pathway.

Conclusions: ICT reduced CD8⁺ T cell chemotaxis by inhibiting the CXCL10/CXCR3 axis, and these effects might be achieved by suppressing the TGF-β-Smad2 signaling pathway.

Keywords: CD8(+)T cells; CXCL10/CXCR3; Cigarette smoke extract; Icaritin; chemotaxis.