Immunogenic cell death in mice expressing caspase-resistant ROCK1 is not replicated by ROCK inhibitors

Abstract

The characteristic changes in morphology during apoptosis help facilitate "immunologically-silent" cell death by limiting the release of cellular contents and enabling efficient clearance of apoptotic cell bodies. Caspase-mediated cleavage of the ROCK1 protein kinase results in its constitutive activation, which drives the rapid and forceful contraction of apoptotic cells. We previously showed in Julian et al. that when ROCK1 was mutated to render it caspase-resistant, there was greater liver damage and neutrophil recruitment after acute treatment with the hepatotoxin diethylnitrosamine (DEN). We now show that acute DEN-induced liver damage resulted in greater induction of pro-inflammatory cytokines/chemokines, indicative of immunogenic cell death (ICD), in mice expressing non-cleavable ROCK1 (ROCK1nc). Hepatocellular carcinoma (HCC) tumours in ROCK1nc mice had higher grade steatosis, and more neutrophils and CD8+ T cells relative to mice expressing wild-type ROCK1 (ROCK1wt), indicating that spontaneous tumour cell death also was more immunogenic. Since the induction of ICD has been proposed to be tumour-suppressive, the effects of two structurally distinct pharmacological ROCK inhibitors on HCC tumours was examined. Both fasudil and AT13148 significantly decreased tumour numbers and area, but neither treatment resulted in greater numbers of neutrophils or CD8+ T cells to be recruited. In the context of acute DEN-induced liver damage, AT13148 reduced circulating platelet and lymphocyte numbers, and inhibited the recruitment of dendritic, natural killer and CD8+ T cells to livers. These observations indicate that there is an important role for ROCK1 cleavage to limit immunogenic cell death which was not replicated by systemic administration of pharmacological ROCK inhibitors. As a result, adjuvant administration of ROCK inhibitors with cancer therapeutics would be unlikely to result in therapeutic benefit by inducing ICD to increase anti-tumour immune responses.