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.

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.

SARS-CoV-2 Spike Protein in Intestinal Cells of a Patient with Coronavirus Disease 2019 Multisystem Inflammatory Syndrome.

A previously healthy 12-year-old boy had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related multisystem inflammatory syndrome (MIS-C) that was rapidly fatal. Autopsy revealed the presence of a large intracardiac thrombus. SARS-CoV-2 spike protein was detected in intestinal cells, supporting the hypothesis that viral presence in the gut may be related to the immunologic response of MIS-C.

The Interferon-induced transmembrane protein 3 gene (IFITM3) rs12252 C variant is associated with COVID-19.

BACKGROUND AND AIMS: The interferon-induced transmembrane proteins play an important antiviral role by preventing viruses from traversing the cellular lipid bilayer. IFITM3 gene variants have been associated with the clinical response to influenza and other viruses. Our aim was to determine whether the IFITM3 rs12252 polymorphism was associated with the risk of developing severe symptoms of COVID-19 in our population. METHODS: A total of 288 COVID-19 patients who required hospitalization (81 in the intensive care unit) and 440 age matched controls were genotyped with a Taqman assay. Linear regression models were used to compare allele and genotype frequencies between the groups, correcting for age and sex. RESULTS: Carriers of the minor allele frequency (rs12252 C) were significantly more frequent in the patients compared to controls after correcting by age and sex (p = 0.01, OR = 2.02, 95%CI = 1.19-3.42). This genotype was non-significantly more common among patients who required ICU. CONCLUSIONS: The IFITM3 rs12252 C allele was a risk factor for COVID-19 hospitalization in our Caucasian population. The extent of the association was lower than the reported among Chinese, a population with a much higher frequency of the risk allele.

Lung Purinoceptor Activation Triggers Ventilator-Induced Brain Injury.

OBJECTIVES: Mechanical ventilation can cause ventilator-induced brain injury via afferent vagal signaling and hippocampal neurotransmitter imbalances. The triggering mechanisms for vagal signaling during mechanical ventilation are unknown. The objective of this study was to assess whether pulmonary transient receptor potential vanilloid type-4 (TRPV4) mechanoreceptors and vagal afferent purinergic receptors (P2X) act as triggers of ventilator-induced brain injury. DESIGN: Controlled, human in vitro and ex vivo studies, as well as murine in vivo laboratory studies. SETTING: Research laboratory. SUBJECTS: Wild-type, TRPV4-deficient C57BL/6J mice, 8-10 weeks old. Human postmortem lung tissue and human lung epithelial cell line BEAS-2B. INTERVENTION: Mice subjected to mechanical ventilation were studied using functional MRI to assess hippocampal activity. The effects of lidocaine (a nonselective ion-channel inhibitor), P2X-purinoceptor antagonist (iso-PPADS), or genetic TRPV4 deficiency on hippocampal dopamine-dependent pro-apoptotic signaling were studied in mechanically ventilated mice. Human lung epithelial cells (BEAS-2B) were used to study the effects of mechanical stretch on TRPV4 and P2X expression and activation. TRPV4 levels were measured in postmortem lung tissue from ventilated and nonventilated patients. MEASUREMENTS AND MAIN RESULTS: Hippocampus functional MRI analysis revealed considerable changes in response to the increase in tidal volume during mechanical ventilation. Intratracheal lidocaine, iso-PPADS, and TRPV4 genetic deficiency protected mice against ventilationinduced hippocampal pro-apoptotic signaling. Mechanical stretch in both, BEAS-2B cells and ventilated wild-type mice, resulted in TRPV4 activation and reduced Trpv4 and P2x expression. Intratracheal replenishment of adenosine triphosphate in Trpv4 mice abrogated the protective effect of TRPV4 deficiency. Autopsy lung tissue from ventilated patients showed decreased lung TRPV4 levels compared with nonventilated CONCLUSIONS:: TRPV4 mechanosensors and purinergic receptors are involved in the mechanisms of ventilator-induced brain injury. Inhibition of this neural signaling, either using nonspecific or specific inhibitors targeting the TRPV4/adenosine triphosphate/P2X signaling axis, may represent a novel strategy to prevent or treat ventilator-induced brain injury.

The Emerging Role of Neutrophils in Repair after Acute Lung Injury.

Neutrophils are key players in acute lung injury. Once recruited from the circulation, these cells release cytotoxic molecules that lead to tissue disruption, so their blockade has been advocated to prevent lung damage. However, lung injury also occurs during neutropenia and usually involves a very poor outcome. There is emerging evidence that neutrophils not only contribute to that early damage but also orchestrate later repair. Neutrophils promote epithelial proliferation and are a source of proteases, which are required for the processing of the collagen scar and facilitation of cell migration. This article reviews the effects of neutrophils in repair after acute lung injury, focusing on their role as biovectors for proteases and other molecules involved in tissue remodeling.

Impaired lung repair during neutropenia can be reverted by matrix metalloproteinase-9.

BACKGROUND: Neutrophils may cause tissue disruption during migration and by releasing cytotoxic molecules. However, the benefits of neutrophil depletion observed in experimental models of lung injury do not correspond with the poor outcome of neutropenic patients. METHODS: To clarify the role of neutrophils during repair, mice with ventilator induced lung injury (VILI) were rendered neutropenic after damage, and followed for 48 hours of spontaneous breathing. Lungs were harvested and inflammatory mediators and matrix metalloproteinases measured. Bronchoalveolar lavage fluid (BALF) from ventilated patients with acute respiratory distress syndrome, with or without neutropenia, was collected, the same mediators measured and their effects in an ex vivo model of alveolar repair studied. Finally, neutropenic mice were treated after VILI with exogenous matrix metalloproteinase-9 (MMP-9). RESULTS: Lungs from neutropenic animals showed delayed repair and displayed higher levels of tumour necrosis factor α, interferon γ and macrophage inflammatory protein 2, and absence of MMP-9. BALF from ventilated neutropenic patients with acute respiratory distress syndrome showed similar results. BALFs from neutropenic patients yielded a delayed closure rate of epithelial wounds ex vivo, which was improved by removal of collagen or addition of exogenous MMP-9. Lastly, treatment of neutropenic mice with exogenous MMP-9 after VILI reduced tissue damage without modifying cytokine concentrations. CONCLUSION: Release of MMP-9 from neutrophils is required for adequate matrix processing and lung repair.

Impact of Initial Ventilatory Strategy in Hematological Patients With Acute Respiratory Failure: A Systematic Review and Meta-Analysis.

OBJECTIVE: Acute respiratory failure in hematological patients is related to a high mortality. Noninvasive mechanical ventilation may benefit a subset of these patients, but the overall effect on mortality and the risks derived from its failure are unclear. Our objective was to review the impact of initial ventilatory strategy on mortality and the risks related to noninvasive mechanical ventilation failure in this group of patients. DATA SOURCES: Data sources, including PubMed and conference proceedings, were searched from the year 2000 to January 2015. STUDY SELECTION: We selected studies reporting mortality and the need for mechanical ventilation in hematological patients with acute respiratory failure. DATA EXTRACTION: Two trained reviewers independently conducted study selection, abstracted data, and assessed the risk of bias. Discrepancies between reviewers were resolved through discussion and consensus. The outcomes explored were all-cause mortality after mechanical ventilation and incidence of noninvasive mechanical ventilation failure. DATA SYNTHESIS: A random-effects model was used in all the analysis. Thirteen studies, involving 2,380 patients, were included. Use of noninvasive mechanical ventilation was related to a better outcome than initial intubation (risk ratio, 0.74; 95% CI, 0.65-0.84). Failure of noninvasive mechanical ventilation did not increase the overall risk of death (risk ratio, 1.02; 95% CI, 0.93-1.13). There were signs of publication bias and substantial heterogeneity among the studies. Compensation of this bias by using the trim-and-fill method showed a significant risk of death after noninvasive mechanical ventilation failure (risk ratio, 1.07; 95% CI, 1.00-1.14). Meta-regression analysis showed that the predicted risk of death for the noninvasive mechanical ventilation group acted as a significant moderator, with a higher risk of death after noninvasive mechanical ventilation failure in those studies reporting lower predicted mortality. CONCLUSIONS: Noninvasive mechanical ventilation is associated with a lower risk of death in hematological patients with respiratory failure. Noninvasive mechanical ventilation failure may worsen the prognosis, mainly in less severe patients.

Impact of Recruitment on Static and Dynamic Lung Strain in Acute Respiratory Distress Syndrome.

BACKGROUND: Lung strain, defined as the ratio between end-inspiratory volume and functional residual capacity, is a marker of the mechanical load during ventilation. However, changes in lung volumes in response to pressures may occur in injured lungs and modify strain values. The objective of this study was to clarify the role of recruitment in strain measurements. METHODS: Six oleic acid-injured pigs were ventilated at positive end-expiratory pressure (PEEP) 0 and 10 cm H2O before and after a recruitment maneuver (PEEP = 20 cm H2O). Lung volumes were measured by helium dilution and inductance plethysmography. In addition, six patients with moderate-to-severe acute respiratory distress syndrome were ventilated with three strategies (peak inspiratory pressure/PEEP: 20/8, 32/8, and 32/20 cm H2O). Lung volumes were measured in computed tomography slices acquired at end-expiration and end-inspiration. From both series, recruited volume and lung strain (total, dynamic, and static) were computed. RESULTS: In the animal model, recruitment caused a significant decrease in dynamic strain (from [mean ± SD] 0.4 ± 0.12 to 0.25 ± 0.07, P < 0.01), while increasing the static component. In patients, total strain remained constant for the three ventilatory settings (0.35 ± 0.1, 0.37 ± 0.11, and 0.32 ± 0.1, respectively). Increases in tidal volume had no significant effects. Increasing PEEP constantly decreased dynamic strain (0.35 ± 0.1, 0.32 ± 0.1, and 0.04+0.03, P < 0.05) and increased static strain (0, 0.06 ± 0.06, and 0.28 ± 0.11, P < 0.05). The changes in dynamic and total strain among patients were correlated to the amount of recruited volume. An analysis restricted to the changes in normally aerated lung yielded similar results. CONCLUSION: Recruitment causes a shift from dynamic to static strain in early acute respiratory distress syndrome.

Exposure to mechanical ventilation promotes tolerance to ventilator-induced lung injury by Ccl3 downregulation.

Inflammation plays a key role in the development of ventilator-induced lung injury (VILI). Preconditioning with a previous exposure can damp the subsequent inflammatory response. Our objectives were to demonstrate that tolerance to VILI can be induced by previous low-pressure ventilation, and to identify the molecular mechanisms responsible for this phenomenon. Intact 8- to 12-wk-old male CD1 mice were preconditioned with 90 min of noninjurious ventilation [peak pressure 17 cmH2O, positive end-expiratory pressure (PEEP) 2 cmH2O] and extubated. Seven days later, preconditioned mice and intact controls were submitted to injurious ventilation (peak pressure 20 cmH2O, PEEP 0 cmH2O) for 2 h to induce VILI. Preconditioned mice showed lower histological lung injury scores, bronchoalveolar lavage albumin content, and lung neutrophilic infiltration after injurious ventilation, with no differences in Il6 or Il10 expression. Microarray analyses revealed a downregulation of Calcb, Hspa1b, and Ccl3, three genes related to tolerance phenomena, in preconditioned animals. Among the previously identified genes, only Ccl3, which encodes the macrophage inflammatory protein 1 alpha (MIP-1α), showed significant differences between intact and preconditioned mice after high-pressure ventilation. In separate, nonconditioned animals, treatment with BX471, a specific blocker of CCR1 (the main receptor for MIP-1α), decreased lung damage and neutrophilic infiltration caused by high-pressure ventilation. We conclude that previous exposure to noninjurious ventilation induces a state of tolerance to VILI. Downregulation of the chemokine gene Ccl3 could be the mechanism responsible for this effect.

Mechanical ventilation triggers hippocampal apoptosis by vagal and dopaminergic pathways.

RATIONALE: Critically ill patients frequently develop neuropsychological disturbances including acute delirium or memory impairment. The need for mechanical ventilation is a risk factor for these adverse events, but a mechanism that links lung stretch and brain injury has not been identified. OBJECTIVES: To identify the mechanisms that lead to brain dysfunction during mechanical ventilation. METHODS: Brains from mechanically ventilated mice were harvested, and signals of apoptosis and alterations in the Akt survival pathway were studied. These measurements were repeated in vagotomized or haloperidol-treated mice, and in animals intracerebroventricularly injected with selective dopamine-receptor blockers. Hippocampal slices were cultured and treated with micromolar concentrations of dopamine, with or without dopamine receptor blockers. Last, levels of dysbindin, a regulator of the membrane availability of dopamine receptors, were assessed in the experimental model and in brain samples from ventilated patients. MEASUREMENTS AND MAIN RESULTS: Mechanical ventilation triggers hippocampal apoptosis as a result of type 2 dopamine receptor activation in response to vagal signaling. Activation of these receptors blocks the Akt/GSK3ß prosurvival pathway and activates the apoptotic cascade, as demonstrated in vivo and in vitro. Vagotomy, systemic haloperidol, or intracerebroventricular raclopride (a type 2 dopamine receptor blocker) ameliorated this effect. Moreover, ventilation induced a concomitant change in the expression of dysbindin-1C. These results were confirmed in brain samples from ventilated patients. CONCLUSIONS: These results prove the existence of a pathogenic mechanism of lung stretch-induced hippocampal apoptosis that could explain the neurological changes in ventilated patients and may help to identify novel therapeutic approaches.

Impairment of autophagy decreases ventilator-induced lung injury by blockade of the NF-κB pathway.

Excessive lung stretch triggers lung inflammation by activation of the NF-κB pathway. This route can be modulated by autophagy, an intracellular proteolytic system. Our objective was to study the impact of the absence of autophagy in a model of ventilator-induced lung injury. Mice lacking Autophagin-1/ATG4B (Atg4b-/-), a critical protease in the autophagic pathway, and their wild-type counterparts were studied in baseline conditions and after mechanical ventilation. Lung injury, markers of autophagy, and activation of the inflammatory response were evaluated after ventilation. Mechanical ventilation increased autophagy and induced lung injury in wild-type mice. Atg4b-/- animals showed a decreased lung injury after ventilation, with less neutrophilic infiltration than their wild-type counterparts. As expected, autophagy was absent in mutant animals, resulting in the accumulation of p62 and ubiquitinated proteins. Activation of the canonical NF-κB pathway was present in ventilated wild-type, but not Atg4b-deficient, animals. Moreover, these mutant mice showed an accumulation of ubiquitinated IκB. High-pressure ventilation partially restored the autophagic response in Atg4b-/- mice and abolished the differences between genotypes. In conclusion, impairment of autophagy results in an ameliorated inflammatory response to mechanical ventilation and decreases lung injury. The accumulation of ubiquitinated IκB may be responsible for this effect.

Anti-inflammatory effects of clarithromycin in ventilator-induced lung injury.

BACKGROUND: Mechanical ventilation can promote lung injury by triggering a pro-inflammatory response. Macrolides may exert some immunomodulatory effects and have shown significant benefits over other antibiotics in ventilated patients. We hypothesized that macrolides could decrease ventilator-induced lung injury. METHODS: Adult mice were treated with vehicle, clarithromycin or levofloxacin, and randomized to receive mechanical ventilation with low (12 cmH2O, PEEP 2 cmH2O) or high (20 cmH2O, ZEEP) inspiratory pressures for 150 minutes. Histological lung injury, neutrophil infiltration, inflammatory mediators (NFκB activation, Cxcl2, IL-10) and levels of adhesion molecules (E-selectin, ICAM) and proteases (MMP-9 and MMP-2) were analyzed. RESULTS: There were no differences among groups after low-pressure ventilation. Clarithromycin significantly decreased lung injury score and neutrophil count, compared to vehicle or levofloxacin, after high-pressure ventilation. Cxcl2 expression and MMP-2 and MMP-9 levels increased and IL-10 decreased after injurious ventilation, with no significant differences among treatment groups. Both clarithromycin and levofloxacin dampened the increase in NFκB activation observed in non-treated animals submitted to injurious ventilation. E-selectin levels increased after high pressure ventilation in vehicle- and levofloxacin-treated mice, but not in those receiving clarithromycin. CONCLUSIONS: Clarithromycin ameliorates ventilator-induced lung injury and decreases neutrophil recruitment into the alveolar spaces. This could explain the advantages of macrolides in patients with acute lung injury and mechanical ventilation.

Iron accumulation drives fibrosis, senescence and the senescence-associated secretory phenotype.

Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.

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.

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.

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).

Variant-genetic and transcript-expression analysis showed a role for the chemokine-receptor CCR5 in COVID-19 severity.

The chemokine receptor CCR5 has been implicated in COVID-19. CCR5 and its ligands are overexpressed in patients. The pharmacological targeting of CCR5 would improve the COVID-19 severity. We sought to investigate the role of the CCR5-Δ32 variant (rs333) in COVID-19. The CCR5-Δ32 was genotyped in 801 patients (353 in the intensive care unit, ICU) and 660 healthy controls, and the deletion was significantly less frequent in hospitalysed COVID-19 than in healthy controls (p = 0.01, OR = 0.66, 95%CI = 0.49-0.88). Of note, we did not find homozygotes among the patients, compared to 1% of the controls. The CCR5 transcript was measured in leukocytes from 85 patients and 40 controls. We found a significantly higher expression of the CCR5 transcript among the patients, with significant difference when comparing the non-deletion carriers (controls = 35; patients = 81; p = 0.01). ICU-patients showed non-significantly higher expression than no-ICU cases. Our study points to CCR5 as a genetic marker for COVID-19. The pharmacological targeting of CCR5 should be a promising treatment for COVID-19.

Angiotensin-converting enzymes (ACE, ACE2) gene variants and COVID-19 outcome.

The Angiotensin system is implicated in the pathogenesis of COVID-19. First, ACE2 is the cellular receptor for SARS-CoV-2, and expression of the ACE2 gene could regulate the individuals susceptibility to infection. In addition, the balance between ACE1 and ACE2 activity has been implicated in the pathogenesis of respiratory diseases and could play a role in the severity of COVID-19. Functional ACE1/ACE2 gene polymorphisms have been associated with the risk of cardiovascular and pulmonary diseases, and could thus also contribute to the outcome of COVID-19. We studied 204 COVID-19 patients (137 non-severe and 67 severe-ICU cases) and 536 age-matched controls. The ACE1 insertion/deletion and ACE2 rs2285666 polymorphism were determined. Variables frequencies were compared between the groups by logistic regression. We also sequenced the ACE2 coding nucleotides in a group of patients. Severe COVID-19 was associated with hypertension male gender (p < 0.001), hypertension (p = 0.006), hypercholesterolaemia (p = 0.046), and the ACE1-DD genotype (p = 0.049). In the multiple logistic regression hypertension (p = 0.02, OR = 2.26, 95%CI = 1.12-4.63) and male gender (p = 0.002; OR = 3.15, 95%CI = 1.56-6.66) remained as independent significant predictors of severity. The ACE2 polymorphism was not associated with the disease outcome. The ACE2 sequencing showed no coding sequence variants that could explain an increased risk of developing COVID-19. In conclusion, an adverse outcome of COVID-19 was associated with male gender, hypertension, hypercholesterolemia and the ACE1 genotype. Our work suggested that the ACE1-I/D might influence COVID-19 severity, but the effect was dependent on the hypertensive status. This result requires further validation in other large cohorts.