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Is cellular senescence involved in cystic fibrosis?

cystic fibrosis sasp cellular senescence

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#1 Engadin

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Posted 30 April 2019 - 06:04 PM


ABSTRACT

Pulmonary disease is the main cause of the morbidity and mortality of patients affected by cystic fibrosis (CF). The lung pathology is dominated by excessive recruitment of neutrophils followed by an exaggerated inflammatory process that has also been reported to occur in the absence of apparent pathogenic infections. Airway surface dehydration and mucus accumulation are the driving forces of this process. The continuous release of reactive oxygen species and proteases by neutrophils contributes to tissue damage, which eventually leads to respiratory insufficiency. CF has been considered a paediatric problem for several decades. Nevertheless, during the last 40 years, therapeutic options for CF have been greatly improved, turning CF into a chronic disease and extending the life expectancy of patients. Unfortunately, chronic inflammatory processes, which are characterized by a substantial release of cytokines and chemokines, along with ROS and proteases, can accelerate cellular senescence, leading to further complications in adulthood. The alterations and mechanisms downstream of CFTR functional defects that can stimulate cellular senescence remain unclear. However, while there are correlative data suggesting that cellular senescence may be implicated in CF, a causal or consequential relationship between cellular senescence and CF is still far from being established. Senescence can be both beneficial and detrimental. Senescence may suppress bacterial infections and cooperate with tissue repair. Additionally, it may act as an effective anticancer mechanism. However, it may also promote a pro-inflammatory environment, thereby damaging tissues and leading to chronic age-related diseases. In this review, we present the most current knowledge on cellular senescence and contextualize its possible involvement in CF.

 

 

BACKGROUND

Cellular senescence was first described in human fibroblasts as a process that limits cell proliferation [1]. It has been well established that the senescence response observed in this pioneering study was a consequence of the progressive shortening of telomeres [2]. However, these findings are only the tip of the iceberg in terms of a complex biological response that (in addition to telomere shortening) can be triggered by a multitude of cell-intrinsic and cell-extrinsic stresses, including DNA damage, epigenetic changes, oxidative stress, chronic mitogenic signalling, oncogene activation and inactivation, and the loss of tumour suppressors [3]. Currently, cellular senescence is understood to be a state of irreversible cell cycle arrest in which cells undergo distinctive phenotypic alterations, including profound chromatin and secretome changes, and tumour suppressor pathway activation [4]. Cellular senescence cannot be characterized by a unique biomarker or phenotypic alteration because senescent cells display different alterations depending on the senescence type (e.g., replicative, DNA damage-induced or oncogene-induced), cellular origin (e.g., fibroblast or endothelial), organism (e.g., mouseor human) and temporal factors (early, full or late senescence) [45]. For this reason, the characterization of cellular senescence is commonly performed by means of a combination of multiple biomarker measurements [6], including the activity of senescence-associated β-galactosidase (SAβGAL), the increased expression of cell cycle inhibitors (p16, p53, and p21), the presence of DNA damage foci containing activated γH2AX (a marker of the DNA damage response), the decondensation of peri-centromeric satellite DNA (a phenomenon termed senescence-associated distension of satellite, SADS), a lack of markers of proliferation (Ki67 expression or BrdU incorporation), the loss and relocalization of the nuclear protein high mobility group box 1 (HMGB1), staining for lipofuscins, and morphological changes (enlarged and irregular cells). Cellular senescence is thus a state of the cell that is clearly different from quiescence, which is defined as a “reversible non-proliferating state” that is due to cell cycle arrest [37]. However, cellular quiescence and senescence share several common signalling pathways. For instance, SAβGAL reactivity is observed in both senescence and quiescence even though reactivity is clearly higher in senescence [8]. Additionally, the tumour suppressor p53 plays a critical role in both reversible quiescence and irreversible senescence [9]. Maximal activation of p53 leads to quiescence, whereas its partial activation leads to senescence under certain conditions [10]. The mammalian target of rapamycin (mTOR) serine/threonine protein kinase can instead force the cell fate towards senescence over quiescence [81112]. Moreover, these two processes may present distinctive molecular mechanisms that underlie cell cycle arrest. In fact, during senescence, the cell cycle is permanently blocked by p53-dependent p21 activation or by p16 [13], whereas during quiescence, cell cycle arrest is mostly mediated by the CDK inhibitor p27 [914]. Another distinctive feature of most senescent cells is the development of a secretory phenotype, termed the senescence-associated secretory phenotype (SASP), which includes several proteins involved in inflammation processes, proteases, and haemostatic and growth factors [15].

 

Senescence can be both beneficial and detrimental. On the one hand, as mentioned above, senescence acts as an effective anticancer mechanism by preventing malignant transformation and by limiting tumour progression. Moreover, acute senescence can be beneficial during embryonic development, wound healing and tissue repair [16]. However, excessive accumulation of senescent cells has been demonstrated in a multitude of aged tissues. Increased resistance to apoptosis, decreased senescence immunosurveillance, an increased rate of senescence-inducing damage and “bystander effects” (a phenomenon in which senescent cells propagate the senescence of neighbouring cells through the SASP or through other uncharacterized mechanisms) have been proposed as mechanisms to explain why there is a consistent accumulation of senescent cells in ageing and in age-related diseases [17]. Excessive accumulation of senescent cells may thus promote a pro-inflammatory environment mediated by the SASP, which contributes to reduced tissue regeneration and organ dysfunction [418]. In fact, senescent cells that persist for a prolonged time within tissues lead to deterioration processes and chronic age-related diseases [16]. This phenomenon is related to the inflammatory status promoted by the senescent cells combined with the exhaustion of the stem cell pools and the resulting impairment in regenerative capacity. Indeed, the accumulation of senescent cells was recently observed in a transgenic mouse model (Sox2−TK mice) in which adult SOX2+ stem cells were depleted by the administration of ganciclovir [19]. Nevertheless, cellular senescence may also contribute to stem cell exhaustion through the SASP and the bystander effect [20], thus suggesting the existence of a vicious circle that contributes to tissue degeneration.

 

Importantly, lung aging in mice has been reported to be associated with higher levels of IL-6 and transforming growth factor beta (TGF-β) expression and the increased expression of the senescence-inducing cyclin-dependent kinase inhibitors (CDKi) p16 and p21 both in airway epithelia [21] and in vascular smooth muscle cells [22]. Senescent cells have been observed in several pathologies, including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) [16]. The lungs of COPD patients resemble those of subjects exhibiting normal lung aging, namely, increased expression of senescence-associated soluble markers and increased expression of p16 and p21, as reported in airway epithelial cells and in vascular endothelial cells [21]. Similarly, several biomarkers of cell senescence, such as increased p21 expression and SAβGAL activity, have been found in IPF alveolar, bronchial and mesenchymal cells both in humans and in mice [23]. Of note, a senescence-like phenotype has also been proposed in cystic fibrosis (CF). In fact, primary bronchial epithelial cells obtained from 9 patients with CF showed increased levels of p16 and DNA damage response markers, such as phospho-histone 2AX and phospho-checkpoint kinase 2 [23]. In the same study, the authors showed that neutrophil elastase (NE) was able to induce p16 accumulation in primary normal bronchial epithelial cells in a dose-dependent manner [23]. Moreover, neutrophils obtained from the bronchoalveolar lavage fluid (BALF) of patients with CF expressed p21 [24], which has been reported to modulate the delayed neutrophil apoptosis observed in CF [25]. Airborne particulate matter increased p21 expression in CF bronchial epithelial cells through mitochondrial stress activation [26]. Although the presence of senescent cells in CF airways is far from firmly established and although data regarding this point are only correlative, these results suggest that the excessive NE release observed in CF lungs might accelerate the senescence process in CF.

 

CF is an inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a chloride channel that is widely expressed in human epithelia [27]. Mutations affecting CFTR expression or function lead to defective chloride efflux followed by sodium absorption by the amiloride-sensitive epithelial Na + channels (ENaC). This process underlies dehydration, particularly within the bronchial lumina of CF patients. Dehydration of airway surface liquid impairs mucociliary clearance, favouring inflammation processes that are dominated by neutrophil infiltrate [28]. CF patients present chronic lung inflammation, which has been observed in young subjects and animal models in the absence of apparent bacterial infections [29]. After bacterial infections, mainly sustained by Pseudomonas aeruginosa, inflammation is amplified, leading to the exaggerated recruitment of neutrophils within the bronchial lumen, which unfortunately produces an ineffective antibacterial response [30]. The inflammatory process is mainly driven by the activation of the nuclear factor (NF)-κB [31], which in turn leads to the overexpression of the neutrophil chemoattractant interleukin (IL)-8 (CXCL-8) [32].

 

In normal subjects, lung maturity peaks between 20 and 25 years of age. After this period, lung functions gradually decline in terms of the forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC), ultimately leading to respiratory insufficiency over time [16]. Similar to CF conditions, ageing has been reported to modify lung structure by decreasing mucociliary clearance, reducing the inner surface area for exchanging gas and causing a constitutive pro-inflammatory status. This process is mainly mediated by reactive oxygen species (ROS) and proteases, in particular NE, that are released by innate immune cells. The accumulation of ROS leads to mitochondrial oxidative damage, decreasing the mitochondrial copy number in animal models [33]. Increased levels of ROS are associated with the augmented activity of the tumour suppressor protein p53 and subsequent upregulation of p21 and p16 protein expression both in vitro and in vivo [3435]. By means of BALF obtained from healthy individuals from discontinuous age groups, neutrophil recruitment was demonstrated to increase with age [36]. This process promotes the continuous release of NE, which has been shown to degrade elastin, impairing pulmonary elastic recoil. The basal pro-inflammatory status due to continuous oxidative stress signalling fosters the release of cytokines and chemokines, particularly IL-6 and IL-8 [36]. Similarly, CF is characterized by the recruitment of a large number of neutrophils, which leads to the sequential, ROS- and protease-driven degradation of lung parenchyma [37]. The appearance is that of CF somehow accelerating the physiological ageing process, leading to a prematurely aged microenvironment that further exacerbates the decline in lung function.

 

Ageing and senescence in chronic inflammatory diseases are becoming increasingly recognized by the scientific community. Because the life expectancy of CF patients is considerably increasing due to novel therapeutic options, further study is necessary to investigate whether the senescence process plays a role in the chronic inflammation of CF.

 

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CONCLUSIONS

Since the first report of CF that was published in 1938, in which Dorothy Andersen investigated 49 children showing a pathology characterized by exocrine pancreatic insufficiency associated with lung disease [115], the survival of patients with CF has risen from a few months to over 40 years [116]. The increased life expectancy is now promoted further by a new class of drugs termed CFTR modulators.

Over the last decade, CFTR modulator science has greatly advanced. Currently, the number of CF patients who are candidates for CFTR modulator therapy is higher than 50% and may reach up to 90% within a few years [117]. Ivacaftor (VX-770) was the first effective CFTR modulator to draw attention to this new class of molecules [118119]. Ivacaftor corrects gating-defective mutations of CFTR, such as the G551D variant, which is found in approximately 4–5% of CF patients. The major evidence for ivacaftor was reported in terms of clinical benefit: the drug significantly improved the percent predicted FEV1 by more than 10% and reduced the risk of pulmonary exacerbations by more than 50% [120]. Lumacaftor (VX-809) is a corrector molecule that is able to facilitate the trafficking and plasma membrane localization of the F508del CFTR [68121]. The F508del is the most common CFTR mutation. Among Caucasian CF patients worldwide, the frequency of F508del mutation varies from a minimum of 20% in Turkey to a maximum of 100% in the Faroe Islands of Denmark [108]. In US, its frequency is almost 72% in Caucasian population, 31–44% in African Americans, and 18% in Iranians [122123124]. Although both in vitro and in vivo studies have proven that lumacaftor can improve the maturation and function of the F508del CFTR protein [68121], lumacaftor monotherapy has shown insufficient clinical benefits in CF adults who are homozygous for the F508del CFTR, despite a moderate but significant reduction in sweat chloride [67]. A lumacaftor derivative, termed tezacaftor, in combination with ivacaftor, led to a modest improvement in the FEV1, a reduction in the pulmonary exacerbation risk and an improvement in weight in a cohort of CF subjects who were homozygous for the F508del CFTR [125126]. Furthermore, there are currently several new CFTR corrector and potentiator molecules in clinical trials, and these studies are expected to provide further insights into increasing CFTR expression and function in patients [127].

Unfortunately, these drugs will not be able to regenerate exhausted lung tissues, and whether the release of SASP and senescence signalling reported in CF lung epithelia can be affected by CFTR modulators is unclear. However, the extended life expectancy of CF subjects can promote senescence, which is early activated in patients with CF. Thus, in addition to the correction of CFTR dysfunction, the new challenge in treating older patients might be to control the accelerated aging processes, which are associated with inflammation, tissue damage and cancer development. While there are consistent data to support the hypothesis that cellular senescence may be implicated in CF (Table 2), the alterations and mechanisms downstream of the CFTR functional defects that can stimulate senescence in the lung and in other organs affected by the disease remain unknown. Most importantly, the role of cellular senescence in CF has not been investigated thus far. It is expected that the inflammation driven by an excess of SASP-producing cells may play a deleterious role in the progression of the disease (Fig. 1). However, we cannot exclude the possibility that senescent cells may play a beneficial role, suppressing bacterial infections and cooperating with tissue repair. This gap in knowledge is clearly a limit for testing bioactive compounds that have already been proven to have senolytic activity in clinical settings [3128129130]. The recent discovery that azithromycin exhibits senolytic activity in human fibroblasts [131] further supports our hypothesis to target senescent cells in CF. Indeed, azithromycin is an antibiotic used to treat patients with CF and is also known to have an anti-inflammatory effects [132133]. The evidence of a senolytic activity by azithromycin suggests that the anti-inflammatory effects may be the consequence of the selective removal of senescent lung fibroblasts and the subsequent decline in the production of SASP. To fill the gap and unravel the mechanisms involved, it would be useful to study the effects of senescent cell removal in mouse models of CF engineered with a transgene that allows the selective removal and visualization of senescent cells (e.g., by breeding with p16-3MR mice). This strategy has been successfully used to demonstrate the deleterious role of cellular senescence in atherosclerotic [134] and osteoarthritis [135] mouse models, and it is likely that this will be the key to understanding the role of cellular senescence in CF.

 

Rest at source: https://respiratory-...2931-019-0993-2


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Also tagged with one or more of these keywords: cystic fibrosis, sasp, cellular senescence

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