Activation of senescence in critically ill patients: mechanisms, consequences and therapeutic opportunities.

Whereas aging is a whole-organism process, senescence is a cell mechanism that can be triggered by several stimuli. There is increasing evidence that critical conditions activate cell senescence programs irrespective of patient's age. In this review, we briefly describe the basic senescence pathways and the consequences of their activation in critically ill patients. The available evidence suggests a paradigm in which activation of senescence can be beneficial in the short term by rendering cells resistant to apoptosis, but also detrimental in a late phase by inducing a pro-inflammatory and pro-fibrotic state. Senescence can be a therapeutic target. The use of drugs that eliminate senescent cells (senolytics) or the senescence-associated phenotype (senomorphics) will require monitoring of these cell responses and identification of therapeutic windows to improve the outcome of critically ill patients.

Age-dependent effect of the IFIH1/MDA5 gene variants on the risk of critical COVID-19.

MDA5, encoded by the IFIH1gene, is a cytoplasmic sensor of viral RNAs that triggers interferon (IFN) antiviral responses. Common and rare IFIH1 variants have been associated with the risk of type 1 diabetes and other immune-mediated disorders, and with the outcome of viral diseases. Variants associated with reduced IFN expression would increase the risk for severe viral disease. The MDA5/IFN pathway would play a critical role in the response to SARS-CoV-2 infection mediating the extent and severity of COVID-19. Here, we genotyped a cohort of 477 patients with critical ICU COVID-19 (109 death) for three IFIH1 functional variants: rs1990760 (p.Ala946Thr), rs35337543 (splicing variant, intron 8 + 1G > C), and rs35744605 (p.Glu627Stop). The main finding of our study was a significant increased frequency of rs1990760 C-carriers in early-onset patients (< 65 years) (p = 0.01; OR = 1.64, 95%CI = 1.18-2.43). This variant was also increased in critical vs. no-ICU patients and in critical vs. asymptomatic controls. The rs35744605 C variant was associated with increased blood IL6 levels at ICU admission. The rare rs35337543 splicing variant showed a trend toward protection from early-onset critical COVID-19. In conclusion, IFIH1 variants associated with reduced gene expression and lower IFN response might contribute to develop critical COVID-19 with an age-dependent effect.

Transcriptomic clustering of critically ill COVID-19 patients.

BACKGROUND: Infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may cause a severe disease, termed coronavirus disease 2019 (COVID-19), with significant mortality. Host responses to this infection, mainly in terms of systemic inflammation, have emerged as key pathogenetic mechanisms and their modulation has shown a mortality benefit. METHODS: In a cohort of 56 critically ill COVID-19 patients, peripheral blood transcriptomes were obtained at admission to an intensive care unit (ICU) and clustered using an unsupervised algorithm. Differences in gene expression, circulating microRNAs (c-miRNAs) and clinical data between clusters were assessed, and circulating cell populations estimated from sequencing data. A transcriptomic signature was defined and applied to an external cohort to validate the findings. RESULTS: We identified two transcriptomic clusters characterised by expression of either interferon-related or immune checkpoint genes, respectively. Steroids have cluster-specific effects, decreasing lymphocyte activation in the former but promoting B-cell activation in the latter. These profiles have different ICU outcomes, despite no major clinical differences at ICU admission. A transcriptomic signature was used to identify these clusters in two external validation cohorts (with 50 and 60 patients), yielding similar results. CONCLUSIONS: These results reveal different underlying pathogenetic mechanisms and illustrate the potential of transcriptomics to identify patient endotypes in severe COVID-19 with the aim to ultimately personalise their therapies.

Effects of IFIH1 rs1990760 variants on systemic inflammation and outcome in critically ill COVID-19 patients in an observational translational study.

Background: Variants in IFIH1, a gene coding the cytoplasmatic RNA sensor MDA5, regulate the response to viral infections. We hypothesized that IFIH1 rs199076 variants would modulate host response and outcome after severe COVID-19. Methods: Patients admitted to an intensive care unit (ICU) with confirmed COVID-19 were prospectively studied and rs1990760 variants determined. Peripheral blood gene expression, cell populations, and immune mediators were measured. Peripheral blood mononuclear cells from healthy volunteers were exposed to an MDA5 agonist and dexamethasone ex-vivo, and changes in gene expression assessed. ICU discharge and hospital death were modeled using rs1990760 variants and dexamethasone as factors in this cohort and in-silico clinical trials. Results: About 227 patients were studied. Patients with the IFIH1 rs1990760 TT variant showed a lower expression of inflammation-related pathways, an anti-inflammatory cell profile, and lower concentrations of pro-inflammatory mediators. Cells with TT variant exposed to an MDA5 agonist showed an increase in IL6 expression after dexamethasone treatment. All patients with the TT variant not treated with steroids survived their ICU stay (hazard ratio [HR]: 2.49, 95% confidence interval [CI]: 1.29-4.79). Patients with a TT variant treated with dexamethasone showed an increased hospital mortality (HR: 2.19, 95% CI: 1.01-4.87) and serum IL-6. In-silico clinical trials supported these findings. Conclusions: COVID-19 patients with the IFIH1 rs1990760 TT variant show an attenuated inflammatory response and better outcomes. Dexamethasone may reverse this anti-inflammatory phenotype. Funding: Centro de Investigación Biomédica en Red (CB17/06/00021), Instituto de Salud Carlos III (PI19/00184 and PI20/01360), and Fundació La Marató de TV3 (413/C/2021).

Patients with severe COVID-19 often need mechanical ventilation to help them breathe and other types of intensive care. The outcome for many of these patients depends on how their immune system reacts to the infection. If the inflammatory response triggered by the immune system is too strong, this can cause further harm to the patient. One gene that plays an important role in inflammation is IFIH1 which encodes a protein that helps the body to recognize viruses. There are multiple versions of this gene which each produce a slightly different protein. It is possible that this variation impacts how the immune system responds to the virus that causes COVID-19. To investigate, Amado-Rodríguez, Salgado del Riego et al. analyzed the IFIH1 gene in 227 patients admitted to an intensive care unit in Spain for severe COVID-19 between March and December 2020. They found that patients with a specific version of the gene called TT experienced less inflammation and were more likely to survive the infection. Physicians typically treat patients with moderate to severe COVID-19 with corticosteroid drugs that reduce the inflammatory response. However, Amado-Rodríguez, Salgado del Riego et al. found that patients with the TT version of the IFIH1 gene were at greater risk of dying if they received corticosteroids. The team then applied the distribution of IFIH1 variants among different ethnic ancestries to data from a previous clinical trial, and simulated the effects of corticosteroid treatment. This 'mock' clinical trial supported their findings from the patient-derived data, which were also validated by laboratory experiments on immune cells from individuals with the TT gene. The work by Amado-Rodríguez, Salgado del Riego et al. suggests that while corticosteroids benefit some patients, they may cause harm to others. However, a real-world clinical trial is needed to determine whether patients with the TT version of the IFIH1 gene would do better without steroids.

Mechanical ventilation promotes lung tumour spread by modulation of cholesterol cell content.

BACKGROUND: Mechanical stretch of cancer cells can alter their invasiveness. During mechanical ventilation, lungs may be exposed to an increased amount of stretch, but the consequences on lung tumours have not been explored. METHODS: To characterise the influence of mechanical ventilation on the behaviour of lung tumours, invasiveness assays and transcriptomic analyses were performed in cancer cell lines cultured in static conditions or under cyclic stretch. Mice harbouring lung melanoma implants were submitted to mechanical ventilation and metastatic spread was assessed. Additional in vivo experiments were performed to determine the mechanodependent specificity of the response. Incidence of metastases was studied in a cohort of lung cancer patients that received mechanical ventilation compared with a matched group of nonventilated patients. RESULTS: Stretch increases invasiveness in melanoma B16F10luc2 and lung adenocarcinoma A549 cells. We identified a mechanosensitive upregulation of pathways involved in cholesterol processing in vitro, leading to an increase in pro-protein convertase subtilisin/kexin type 9 (PCSK9) and LDLR expression, a decrease in intracellular cholesterol and preservation of cell stiffness. A course of mechanical ventilation in mice harbouring melanoma implants increased brain and kidney metastases 2 weeks later. Blockade of PCSK9 using a monoclonal antibody increased cell cholesterol and stiffness and decreased cell invasiveness in vitro and metastasis in vivo. In patients, mechanical ventilation increased PCSK9 abundance in lung tumours and the incidence of metastasis, thus decreasing survival. CONCLUSIONS: Our results suggest that mechanical stretch promote invasiveness of cancer cells, which may have clinically relevant consequences. Pharmacological manipulation of cholesterol endocytosis could be a novel therapeutic target in this setting.

Biotrauma during ultra-low tidal volume ventilation and venoarterial extracorporeal membrane oxygenation in cardiogenic shock: a randomized crossover clinical trial.

BACKGROUND: Cardiogenic pulmonary oedema (CPE) may contribute to ventilator-associated lung injury (VALI) in patients with cardiogenic shock. The appropriate ventilatory strategy remains unclear. We aimed to evaluate the impact of ultra-low tidal volume ventilation with tidal volume of 3 ml/kg predicted body weight (PBW) in patients with CPE and veno-arterial extracorporeal membrane oxygenation (V-A ECMO) on lung inflammation compared to conventional ventilation. METHODS: A single-centre randomized crossover trial was performed in the Cardiac Intensive Care Unit (ICU) at a tertiary university hospital. Seventeen adults requiring V-A ECMO and mechanical ventilation due to cardiogenic shock were included from February 2017 to December 2018. Patients were ventilated for two consecutive periods of 24 h with tidal volumes of 6 and 3 ml/kg of PBW, respectively, applied in random order. Primary outcome was the change in proinflammatory mediators in bronchoalveolar lavage fluid (BALF) between both ventilatory strategies. RESULTS: Ventilation with 3 ml/kg PBW yielded lower driving pressures and end-expiratory lung volumes. Overall, there were no differences in BALF cytokines. Post hoc analyses revealed that patients with high baseline levels of IL-6 showed statistically significant lower levels of IL-6 and IL-8 during ultra-low tidal volume ventilation. This reduction was significantly proportional to the decrease in driving pressure. In contrast, those with lower IL-6 baseline levels showed a significant increase in these biomarkers. CONCLUSIONS: Ultra-low tidal volume ventilation in patients with CPE and V-A ECMO may attenuate inflammation in selected cases. VALI may be driven by an interaction between the individual proinflammatory profile and the mechanical load overimposed by the ventilator. Trial registration The trial was registered in ClinicalTrials.gov (identifier NCT03041428, Registration date: 2nd February 2017).

Activation of p21 limits acute lung injury and induces early senescence after acid aspiration and mechanical ventilation.

The p53/p21 pathway is activated in response to cell stress. However, its role in acute lung injury has not been elucidated. Acute lung injury is associated with disruption of the alveolo-capillary barrier leading to acute respiratory distress syndrome (ARDS). Mechanical ventilation may be necessary to support gas exchange in patients with ARDS, however, high positive airway pressures can cause regional overdistension of alveolar units and aggravate lung injury. Here, we report that acute lung injury and alveolar overstretching activate the p53/p21 pathway to maintain homeostasis and avoid massive cell apoptosis. A systematic pooling of transcriptomic data from animal models of lung injury demonstrates the enrichment of specific p53- and p21-dependent gene signatures and a validated senescence profile. In a clinically relevant, murine model of acid aspiration and mechanical ventilation, we observed changes in the nuclear envelope and the underlying chromatin, DNA damage and activation of the Tp53/p21 pathway. Absence of Cdkn1a decreased the senescent response, but worsened lung injury due to increased cell apoptosis. Conversely, treatment with lopinavir and/or ritonavir led to Cdkn1a overexpression and ameliorated cell apoptosis and lung injury. The activation of these mechanisms was associated with early markers of senescence, including expression of senescence-related genes and increases in senescence-associated heterochromatin foci in alveolar cells. Autopsy samples from lungs of patients with ARDS revealed increased senescence-associated heterochromatin foci. Collectively, these results suggest that acute lung injury activates p53/p21 as an antiapoptotic mechanism to ameliorate damage, but with the side effect of induction of senescence.

Cellular and molecular features of senescence in acute lung injury.

A wide range of insults can trigger acute injury in the lungs, which eventually may lead to respiratory failure and death of patients. Current treatment relies mainly on supportive measures and mechanical ventilation. Even so, survivors frequently develop important sequels that compromise quality of life. In the search for new approaches to prevent and treat acute lung injury, many investigations have focused on molecular and cellular pathways which could exert a pathogenic role in this disease. Herein, we review recent findings in the literature suggesting that cellular senescence could be involved in lung injury and discuss the potential use of senotherapies to prevent disease progression.