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

## TL;DR

Repeated low-level inflammation in mouse brain endothelial cells activates the TNF-CXCL10 pathway, leading to changes in cell function that could contribute to neurological disorders.

## Contribution

This study identifies the TNF-CXCL10 signaling pathway as a novel target in cerebrovascular disease due to chronic low-level inflammation.

## Key findings

- Repeated low-level TNF exposure upregulates ICAM1 and CXCL10 at both mRNA and protein levels.
- Repeated inflammation increases endothelial cell proliferation and apoptosis, with CXCL10 knockdown reducing caspase activity.
- Modulation of the TNF-CXCL10 pathway may serve as a therapeutic target for neurovascular disease.

## 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.

Intermittent, mild pro‐inflammatory systemic disease contributes to cerebrovascular pathology, however the mechanism of low‐level exposure causing injury remains unclear. Our RNA‐Seq analysis reveals continuous activation of tumour necrosis factor (TNF)‐Nuclear Factor kappa B (NF‐κB) signalling and multiple downstream inflammatory mediators in mouse cerebral endothelial cells across various injury models. In vitro studies utilising a model of repeated low‐level inflammation in a mouse brain endothelial cell line show upregulation of the TNF‐ CXCL10 pathway, accompanied by altered endothelial function. Modulating this pathway may provide a broad‐spectrum therapeutic target for cerebrovascular disease.