Defective apoptotic cell contractility provokes sterile inflammation, leading to liver damage and tumour suppression

Abstract

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating Aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour suppressive mechanism.

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

“…Treatment of 14 day old male mice with 400 µg DEN by IP injection results in the development of HCC with high penetrance [1, 17], with evidence of spontaneous apoptosis in tumours [30]. ROCK1nc mice were previously shown to develop fewer HCC tumours than ROCK1wt mice, suggesting that tumour development was affected by the alterations in apoptotic cell morphologies due to absence of caspase-mediated ROCK1 activation [1]. Similar to there being more steatosis in ROCK1nc livers following acute DEN-treatment [1], there was a significantly (p < 0.001, Chi-square test) higher proportion of high grade (≥ 3) steatotic HCC tumours in ROCK1nc livers relative to ROCK1wt livers (Figure 3A).…”

“…A genetically modified mouse model in which a single amino acid change in the DETD caspase cleavage site that rendered ROCK1 insensitive to caspase cleavage revealed the obligatory role of ROCK1 cleavage and activation for MLC phosphorylation, the generation of cellular contractile force and the morphological aspects of apoptotic cell death [1]. Treatment of mice expressing the non-cleavable ROCK1 (ROCK1nc) with the liver selective hepatotoxin diethylnitrosamine (DEN) [17] that undergoes CYP2E1-mediated oxidation to become an active genotoxin [18] resulted in greater acute liver damage and neutrophil recruitment relative to mice expressing wild-type ROCK1 (ROCK1wt) [1]. Apoptotic ROCK1nc cells released more of the DAMP protein high mobility group B1 (HMGB1) than ROCK1wt cells, and DEN-induced acute liver damage and neutrophil recruitment could be significantly reduced by inhibiting HMGB1 or its cognate pattern recognition receptor (PRR) toll-like receptor 4 (TLR4) [1].…”

“…Treatment of mice expressing the non-cleavable ROCK1 (ROCK1nc) with the liver selective hepatotoxin diethylnitrosamine (DEN) [17] that undergoes CYP2E1-mediated oxidation to become an active genotoxin [18] resulted in greater acute liver damage and neutrophil recruitment relative to mice expressing wild-type ROCK1 (ROCK1wt) [1]. Apoptotic ROCK1nc cells released more of the DAMP protein high mobility group B1 (HMGB1) than ROCK1wt cells, and DEN-induced acute liver damage and neutrophil recruitment could be significantly reduced by inhibiting HMGB1 or its cognate pattern recognition receptor (PRR) toll-like receptor 4 (TLR4) [1]. Taken together, these findings indicated that the inability of cells to undergo typical apoptotic morphological changes due to the absence of caspase-mediated ROCK1 activation induced a form of ICD.…”

“…We previously showed in Julian et al . [1] 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).…”

Orchestration of Force Generation and Nuclear Collapse in Apoptotic Cells

“…Activated ROCK then mediates phosphorylation of myosin II Regulatory Light Chain (MRLC), causing a hyperactivation of non-muscle myosin II, from which originates the dynamic blebbing of the dying cell membrane, and eventually the formation of the apoptotic bodies [ 12 , 13 , 14 , 15 ]. Interestingly, the inhibition of apoptotic cell contractility through the expression of a non-cleavable form of ROCK has recently been shown to delay the elimination of cell debris, which leads to sterile inflammation and has been associated with tumour suppression [ 16 ].…”