The role of neutrophils in telomere induced senescence via bystander effects

PhD ThesisSenescence is classically defined as a state of permanent cell-cycle arrest. Senescence can occur in response to various stresses, which have been shown to act mainly through the activation of a DNA damage response (DDR). Senescence is characterised not solely by a cell-cycle arrest but also by increased production of Reactive Oxygen Species (ROS) and the development of a Senescence-Associated Secretory Phenotype (SASP). SASP components include growth factors, cytokines, chemokines and immune modulators and have been shown to contribute to senescence in an autocrine manner but also impact on the tissue microenvironment trough paracrine effects. Several studies have linked the SASP with immune surveillance suggesting that Natural Killer cells, monocytes and T lymphocytes CD4+ can effectively eliminate senescent cells. However, the interaction between neutrophils (the first innate immune responders to infection or injury) and cellular senescence has not yet been investigated. In this thesis, I have shown that neutrophils induce premature senescence in human fibroblasts in a telomere-dependent manner. My data indicates that hydrogen peroxide released by neutrophils damages telomeric DNA, thereby accelerating the rate of telomere shortening and contributing to the early onset of senescence. Consistently, pre-treatment with the antioxidant enzyme catalase, prevents neutrophil-induced telomere shortening and premature senescence. In addition, overexpression of the catalytic subunit of telomerase (hTERT), which maintains telomere length in cultured fibroblasts, is able to bypass neutrophil-induced premature senescence. In accordance with my in vitro results, I have shown that following acute liver injury (using CCl4) which is characterised by neutrophil infiltration, mouse hepatocytes show increased markers of telomere dysfunction, which can be prevented by neutralisation of neutrophils. Importantly, I have found that during the ageing process or after injection with lipopolysaccharide (LPS), mouse livers experience increased neutrophil infiltrations which positively correlate with markers of telomere-dysfunction. Finally, I have shown that senescent cells secrete factors which act as a neutrophil chemoattractant and that neutrophils preferentially induce cell-death in senescent cells but not young cells. These data suggest for the first time that neutrophils play an important role in the immune clearance of senescent cells. viii Altogether, my data propose that neutrophils act as a double-edged sword: on one hand, they can induce senescence by accelerating telomere shortening; on the other hand, they can be recruited to sites where senescent cells are present and accelerate their specific clearance

How to model mutually exclusive events based on independent causal pathways in Bayesian network models

This is supported by ERC project ERC-2013-AdG339182-BAYES_KNOWLEDGE

Cytoplasmic innate immune sensing by the caspase-4 non-canonical inflammasome promotes cellular senescence

Cytoplasmic recognition of microbial lipopolysaccharides (LPS) in human cells is elicited by the caspase-4 and caspase-5 noncanonical inflammasomes, which induce a form of inflammatory cell death termed pyroptosis. Here we show that LPS-mediated activation of caspase-4 also induces a stress response promoting cellular senescence, which is dependent on the caspase-4 substrate gasdermin-D and the tumor suppressor p53. Furthermore, we found that the caspase-4 noncanonical inflammasome is induced and assembled in response to oncogenic RAS signaling during oncogene-induced senescence (OIS). Moreover, targeting caspase-4 expression in OIS showed its critical role in the senescence-associated secretory phenotype and the cell cycle arrest induced in cellular senescence. Finally, we observed that caspase-4 induction occurs in vivo in mouse models of tumor suppression and ageing. Altogether, we are showing that cellular senescence is induced by cytoplasmic LPS recognition by the noncanonical inflammasome and that this pathway is conserved in the cellular response to oncogenic stress.This work was funded by Cancer Research UK (CRUK) (C47559/A16243 Training & Career Development Board - Career Development Fellowship), the University of Edinburgh Chancellor’s Fellowship R42576 MRC, and the Ministry of Science and Innovation of the Government of Spain (Proyecto PID2020-117860GB-I00 financiado por MCIN/ AEI /10.13039/501100011033). J.C.A. was supported by CRUK, the University of Edinburgh and is supported by the Spanish National Research Council (CSIC). P.H., I.F.D and N.T. were funded by the University of Edinburgh. A.Q. was funded by CRUK. J.F.P and A.B.L. are funded by NIH grants: 1R01AG068048-01; P01 AG062413; 1UG3 CA268103-01. J.B. was funded by BBSRC (BB/K017314/1). V.S-B is supported by funding from the University of Edinburgh and Medical Research Council (MC_UU_00009/2). F.R.M is funded by a Wellcome Trust Clinical Research Fellowship through the Edinburgh Clinical Academic Track (ECAT) (203913/Z/16/Z). M.M. was supported by CRUK Edinburgh Centre Award (C157/A25140). V.G.B. is funded by CRUK (C157/A24837) and the University of Edinburgh

Neutrophils induce paracrine telomere dysfunction and senescence in ROS‐dependent manner

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro-inflammatory phenotype, thought to contribute to aging and age-related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age-related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non-immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS-dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil-induced senescence may be beneficial during aging and age-related disease

Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk

Abstract: Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially

Mitochondria-to-nucleus retrograde signaling drives formation of cytoplasmic chromatin and inflammation in senescence

Cellular senescence is a potent tumor suppressor mechanism but also contributes to aging and aging-related diseases. Senescence is characterized by a stable cell cycle arrest and a complex proinflammatory secretome, termed the senescence-associated secretory phenotype (SASP). We recently discovered that cytoplasmic chromatin fragments (CCFs), extruded from the nucleus of senescent cells, trigger the SASP through activation of the innate immunity cytosolic DNA sensing cGAS-STING pathway. However, the upstream signaling events that instigate CCF formation remain unknown. Here, we show that dysfunctional mitochondria, linked to down-regulation of nuclear-encoded mitochondrial oxidative phosphorylation genes, trigger a ROS-JNK retrograde signaling pathway that drives CCF formation and hence the SASP. JNK links to 53BP1, a nuclear protein that negatively regulates DNA double-strand break (DSB) end resection and CCF formation. Importantly, we show that low-dose HDAC inhibitors restore expression of most nuclear-encoded mitochondrial oxidative phosphorylation genes, improve mitochondrial function, and suppress CCFs and the SASP in senescent cells. In mouse models, HDAC inhibitors also suppress oxidative stress, CCF, inflammation, and tissue damage caused by senescence-inducing irradiation and/or acetaminophen-induced mitochondria dysfunction. Overall, our findings outline an extended mitochondria-to-nucleus retrograde signaling pathway that initiates formation of CCF during senescence and is a potential target for drug-based interventions to inhibit the proaging SASP