Type I interferon mediates ET-1 and IP10, but not KC release induced by LPS in vivo.

PM George, NW Bartlett, NS Kirkby, JA Mitchell. Imperial College, London, UK

We have shown that the potent vasoconstrictor peptide endothelin(ET)-1 is induced in vascular smooth muscle cells by type II(Woods et al., 1999; Wort et al., 2009) and type I(Badiger et al., 2012; George et al., 2012b) interferons (IFN). We have interpreted these observations as meaning that IFN–induced ET-1 is clinically relevant, particularly in pulmonary arterial hypertension (PAH) (George et al., 2012a). However, our evidence has focused on in vitro cell based experiments with, so far, no in vivo demonstration that IFN pathways are associated with ET-1 release. Systemic levels of ET-1 are increased experimentally in two animal models; (i) hypoxia driven PAH and (ii) LPS induced endotoxemia. LPS, via the pattern recognition receptor Toll like receptor (TLR)4 activates two adapter proteins which give rise to separate groups of genes. TLR4 activates TRIF, which is associated with IFN related genes, such as the prototypic IFN-inducible gene, IP10 (CXCL10). TLR4 also activates Myd88, which activates NFκB associated genes including KC (the mouse homologue of CXCL8). The purpose of this study was to investigate the role of type I IFN signalling in ET-1 induced by LPS in mice. Type I IFN signals through the highly selective IFNAR1 receptor. Here we injected C57/BL6 wild type and IFNAR1-/- gene deleted mice (on a C57/BL6 background), (male mice 8–10 wks old) with 1.25mg/ml LPS or 0.9% saline vehicle (at a dose of 8µl per gram body weight) for 4 hours. Mice were then humanely sacrificed and plasma was collected for the measurement of ET-1, IP10 and KC by ELISA. In wild type mice, LPS induced systemic release of ET-1, IP10 and KC. However, while in IFNAR1-/- mice, LPS-induced release of ET-1 (A) or IP10 (B) was greatly reduced, no effect was seen on KC (C) levels (Figure 1).

Figure 1; plasma levels of ET-1 (A), IP10 (B) or KC (C) in wild type (C57/BL6) or IFNAR1-/- mice after intraperitoneal LPS or vehicle administration. Data is mean ± SEM for n=6 mice. Data was analysed by One Way ANOVA followed by Bonferroni’s post-test; *p<0.05 vs. wild type: #p<0.05 vs. vehicle.

The data are the first to confirm the relative importance of ET-1 as an IFN inducible hormone and illustrate the critical important of type I IFN in its release in vivo. These observations substantiate our hypothesis that there exists an IFN/ET-1 axis and that it is of clinical relevance (George et al., 2012a).

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