Repeated Low-Level Inflammatory Challenge Leads to Alterations in the TNF-CXCL10 Signalling Pathway in Mouse Cerebral Endothelial Cells In Vitro

Abstract

The mechanism by which chronic systemic inflammation contributes to cerebral endothelial dysfunction and neurological disorders is unclear, although endothelial inflammatory signalling is considered a cornerstone of this process. Here, we have performed transcriptomic analysis of published RNASeq datasets and identified consistent upregulation of the Tumour Necrosis Factor—C-X-C Motif Chemokine Ligand 10 (TNF-CXCL10) signalling pathway in mouse cerebral endothelial cells following a single inflammatory challenge. We subsequently investigated the effects of repeated low-level inflammation on the modulation of this pathway in a mouse cerebral endothelial cell line, analysing the effect on markers of endothelial cell activation and changes in cellular function, as a potential mechanism underlying the cerebrovascular response to low-level systemic inflammation. Mouse cerebral endothelial cells (bEnd.3) were exposed to hour-long treatments with phosphate buffered saline (PBS), a single low concentration of TNF (0.5 ng/mL), repeated low-concentration TNF (0.5 ng/mL, 1 h × 4 days) or a single cumulative concentration of TNF (2.0 ng/mL). RNA and protein were extracted 4 and 24 h after the final treatment for analysis of gene/protein expression using qRT-PCR and western blotting. Repeated inflammatory challenge significantly upregulated both Intercellular Adhesion Molecule 1 (ICAM1) and CXCL10 at the mRNA and protein levels. Signal transducer and activator of transcription 1 (STAT1) and phosphorylated-STAT1 (pSTAT1) protein levels were also increased at 4 and 24 h. Differentially, tumor necrosis factor receptor-associated factor 2 (TRAF2) and Interferon gamma (IFNγ) gene expression were decreased at 4 h, returning to control levels at 24 h. Functional analysis revealed significant increases in endothelial cell proliferation and apoptosis in the presence of repeated TNF exposure. CXCL10 knockdown with small interfering RNA (siRNA) reduced mean caspase 3/7 activity induced by the repeated inflammatory paradigm. These data suggest an upregulation of the TNF-CXCL10 pathway in response to low-level repetitive inflammation in mouse cerebral endothelial cells. Modulation of this pathway may represent a broad therapeutic target for neurovascular disease.