Inhibiting the oligomerisation and glycosaminoglycan binding of the chemokine CCL5 reduces experimental liver injury in vivo

Abstract

Aims: We recently identified the chemokine CCL5 as a mediator of the interaction between immune cells and hepatic stellate cells. Notably, a competitive antagonist of CCL5 receptors ameliorated liver injury in vivo (Berres et al. JCI 2010). We here investigate whether inhibition of CCL5 oligomerisation and glycosaminoglycan binding by a mutated CCL5 protein (44AANA47-CCL5) does also ameliorate liver cell injury and fibrosis. Methods: Liver injury was induced in C57BL/6 mice by intraperitoneal injection of CCl4 (chronic: 0.6mg/kg, twice a week, 6 weeks; acute: 0.8mg/kg, single dose). Simultaneously, mice received either ANAA-CCL5 (chronic: 10µg i.p./day; acute: 10µg i.p./12h) or vehicle. Fibrosis was analyzed by Sirius red staining and hydroxyproline measurement. The intrahepatic mRNA expression of fibrosis and inflammation related genes were determined by quantitative RT-PCR and infiltration of immune cells was assessed by FACS analysis and immunocytochemistry. In vitro, HSC were stimulated with conditioned media of T-cell enriched splenocytes. Results: AANA-CCL5 treated mice displayed a significantly reduced degree of acute liver injury (liver cells necrosis, transaminases) and fibrosis (Sirus red positive area and hydroxyproline, both P<0.01). Ameliorated fibrosis by AANA-CCL5 was associated with a decreased expression of fibrosis related genes, decreased SMA and a reduction of infiltrating immune cells. In acute model AANA-CCL5 mice displayed a reduced immune cell infiltration and mRNA levels of TNF, IL–1 and CCL3 (P<0.05). In vitro, conditioned medium of T-cell enriched splenocytes of AANA-CCL5 treated mice reduced the chemotaxis of HSC (P<0.001) and their proliferation (P<0.05). Conclusions: The data provide evidence that inhibition of oligomerisation of chemokines could be a new therapeutic strategy for the treatment of acute and chronic liver injuries which is probably associated with fewer side effects than chemokine receptor antagonists.