Bronchial Aspirate-Based Profiling Identifies MicroRNA Signatures Associated With COVID-19 and Fatal Disease in Critically Ill Patients

Background: The pathophysiology of COVID-19-related critical illness is not completely understood. Here, we analyzed the microRNA (miRNA) profile of bronchial aspirate (BAS) samples from COVID-19 and non-COVID-19 patients admitted to the ICU to identify prognostic biomarkers of fatal outcomes and to define molecular pathways involved in the disease and adverse events. Methods: Two patient populations were included (n = 89): (i) a study population composed of critically ill COVID-19 and non-COVID-19 patients; (ii) a prospective study cohort composed of COVID-19 survivors and non-survivors among patients assisted by invasive mechanical ventilation (IMV). BAS samples were obtained by bronchoaspiration during the ICU stay. The miRNA profile was analyzed using RT-qPCR. Detailed biomarker and bioinformatics analyses were performed. Results: The deregulation in five miRNA ratios (miR-122-5p/miR-199a-5p, miR-125a-5p/miR-133a-3p, miR-155-5p/miR-486-5p, miR-214-3p/miR-222-3p, and miR-221-3p/miR-27a-3p) was observed when COVID-19 and non-COVID-19 patients were compared. In addition, five miRNA ratios segregated between ICU survivors and nonsurvivors (miR-1-3p/miR-124-3p, miR-125b-5p/miR-34a-5p, miR-126-3p/miR-16-5p, miR-199a-5p/miR-9-5p, and miR-221-3p/miR-491-5p). Through multivariable analysis, we constructed a miRNA ratio-based prediction model for ICU mortality that optimized the best combination of miRNA ratios (miR-125b-5p/miR-34a-5p, miR-199a-5p/miR-9-5p, and miR-221-3p/miR-491-5p). The model (AUC 0.85) and the miR-199a-5p/miR-9-5p ratio (AUC 0.80) showed an optimal discrimination value and outperformed the best clinical predictor for ICU mortality (days from first symptoms to IMV initiation, AUC 0.73). The survival analysis confirmed the usefulness of the miRNA ratio model and the individual ratio to identify patients at high risk of fatal outcomes following IMV initiation. Functional enrichment analyses identified pathological mechanisms implicated in fibrosis, coagulation, viral infections, immune responses and inflammation. Conclusions: COVID-19 induces a specific miRNA signature in BAS from critically ill patients. In addition, specific miRNA ratios in BAS samples hold individual and collective potential to improve risk-based patient stratification following IMV initiation in COVID-19-related critical illness. The biological role of the host miRNA profiles may allow a better understanding of the different pathological axes of the disease.We want particularly to acknowledge the patients, Biobank IdISBa and CIBERES Pulmonary Biobank Consortium (PT17/0015/0001), a member of the Spanish National Biobanks Network financed by the Carlos III Health Institute, with the participation of the Units of Intensive Care, Clinical Analysis and Pulmonology of Hospital Universitario Son Espases and Hospital Son Llatzer for their collaboration. This work was also supported by IRBLleida Biobank (B.0000682) and Plataforma Biobancos PT17/0015/0027/.Peer Reviewed"Article signat per 25 autors/es: Marta Molinero, Iván D. Benítez, Jessica González, Clara Gort-Paniello, Anna Moncusí-Moix, Fátima Rodríguez-Jara, María C. García-Hidalgo, Gerard Torres, J. J. Vengoechea, Silvia Gómez, Ramón Cabo, Jesús Caballero, Jesús F. Bermejo-Martin, Adrián Ceccato, Laia Fernández-Barat, Ricard Ferrer, Dario Garcia-Gasulla, Rosario Menéndez, Ana Motos, Oscar Peñuelas, Jordi Riera, Antoni Torres, Ferran Barbé and David de Gonzalo-Calvo* on behalf of the CIBERESUCICOVID Project (COV20/00110 ISCIII)"Postprint (published version

Prognostic implications of comorbidity patterns in critically ill COVID-19 patients: A multicenter, observational study

Background: The clinical heterogeneity of COVID-19 suggests the existence of different phenotypes with prognostic implications. We aimed to analyze comorbidity patterns in critically ill COVID-19 patients and assess their impact on in-hospital outcomes, response to treatment and sequelae. Methods: Multicenter prospective/retrospective observational study in intensive care units of 55 Spanish hospitals. 5866 PCR-confirmed COVID-19 patients had comorbidities recorded at hospital admission; clinical and biological parameters, in-hospital procedures and complications throughout the stay; and, clinical complications, persistent symptoms and sequelae at 3 and 6 months. Findings: Latent class analysis identified 3 phenotypes using training and test subcohorts: low-morbidity (n=3385; 58%), younger and with few comorbidities; high-morbidity (n=2074; 35%), with high comorbid burden; and renal-morbidity (n=407; 7%), with chronic kidney disease (CKD), high comorbidity burden and the worst oxygenation profile. Renal-morbidity and high-morbidity had more in-hospital complications and higher mortality risk than low-morbidity (adjusted HR (95% CI): 1.57 (1.34-1.84) and 1.16 (1.05-1.28), respectively). Corticosteroids, but not tocilizumab, were associated with lower mortality risk (HR (95% CI) 0.76 (0.63-0.93)), especially in renal-morbidity and high-morbidity. Renal-morbidity and high-morbidity showed the worst lung function throughout the follow-up, with renal-morbidity having the highest risk of infectious complications (6%), emergency visits (29%) or hospital readmissions (14%) at 6 months (p<0.01). Interpretation: Comorbidity-based phenotypes were identified and associated with different expression of in-hospital complications, mortality, treatment response, and sequelae, with CKD playing a major role. This could help clinicians in day-to-day decision making including the management of post-discharge COVID-19 sequelae.Financial support was provided by Instituto de Salud Carlos III (CIBERESUCICOVID, COV20/00110), co-funded by Fondo Europeo de Desarrollo Regional (FEDER), “Una manera de hacer Europa”, Centro de Investigación Biomédica en Red − Enfermedades Respiratorias (CIBERES) and Donation Program “estar preparados”, UNESPA, Madrid, Spain. JdB acknowledges receiving financial support from Instituto de Salud Carlos III (ISCIII; Miguel Servet 2019: CP19/00108), cofunded by the European Social Fund (ESF), “Investing in your future”. DdGC acknowledges receiving financial support from Instituto de Salud Carlos III (ISCIII; Miguel Servet 2019: CP20/00041), co-funded by the European Social Fund (ESF), “Investing in your future”. AC acknowledges receiving financial support from Instituto de Salud Carlos III (ISCIII; Sara Borrell 2021: CD21/00087).Peer ReviewedArticle signat per 71 autors/es: Iván D. Benítez (a,b,1), Jordi de Batlle (a,b,1), Gerard Torres (a,b), Jessica Gonzáalez (a,b), David de Gonzalo-Calvo (a,b), Adriano D.S. Targa (a,b), Clara Gort-Paniello (a,b), Anna Moncusí-Moix (a,b), Adrián Ceccato (b,c), Laia Fernández-Barat (b,d), Ricard Ferrer (b,e), Dario Garcia-Gasulla (f), Rosario Menéndez (b,g), Anna Motos (b,d), Oscar Peñuelas (b,h), Jordi Riera (b,e), Jesús F. Bermejo-Martin (b,i), Yhivian Peñasco (j), Pilar Ricart (k), María Cruz Martin Delgado(l), Luciano Aguilera(m), Alejandro Rodríguez(n), Maria Victoria Boado Varela (o), Fernando Suarez-Sipmann (p), Juan Carlos Pozo-Laderas (q), Jordi Solé-Violan (r), Maite Nieto (s), Mariana Andrea Novo (t), José Barberán (u), Rosario Amaya Villar (v), José Garnacho-Montero (w), Jose Luis García-Garmendia (x), José M. Gómez (y), José Ángel Lorente (b,h), Aaron Blandino Ortiz (z), Luis Tamayo Lomas (aa), Esther López-Ramos (ab), Alejandro Úbeda (ac), Mercedes Catalán-González (ad), Angel Sánchez-Miralles (ae), Ignacio Martínez Varela (af), Ruth Noemí Jorge García (ag), Nieves Franco (ah), Víctor D. Gumucio-Sanguino (ai), Arturo Huerta Garcia (aj), Elena Bustamante-Munguira (ak), Luis Jorge Valdivia (al), Jesús Caballero (am), Elena Gallego (an), Amalia Martínez de la Gándara (ao), Álvaro Castellanos-Ortega (ap), Josep Trenado (aq), Judith Marin-Corral (ar), Guillermo M Albaiceta (b,as), Maria del Carmen de la Torre (at), Ana Loza-Vázquez (au), Pablo Vidal (av), Juan Lopez Messa (aw), Jose M. Añon (b,ax), Cristina Carbajales Pérez (ay), Victor Sagredo (az), Neus Bofill (ba), Nieves Carbonell (bb), Lorenzo Socias(bc), Carme Barberá (bd), Angel Estella (be), Manuel Valledor Mendez (bf), Emili Diaz (bg), Ana López Lago (bh), Antoni Torres (b,d) and Ferran Barbé (a,b*), on behalf of the CIBERESUCICOVID Project (COV20/00110, ISCIII)2 // (a) Translational Research in Respiratory Medicine, University Hospital Arnau de Vilanova and Santa Maria, IRBLleida, Lleida, Spain; (b) CIBER of Respiratory Diseases (CIBERES), Institute of Health Carlos III, Madrid, Spain; (c) Critical Care Center, ParcTaulí Hospital Universitari, Institut d'Investigació i Innovació Parc Taulí I3PT, Sabadell, Spain; (d) Department of Pneumology, Hospital Clinic of Barcelona; August Pi i Sunyer Biomedical Research Institute−IDIBAPS, University of Barcelona, Barcelona, Spain; (e) Intensive Care Department, Vall d’Hebron Hospital Universitari. SODIR Research Group, Vall d’Hebron Institut de Recerca (VHIR), Barcelona, Spain; (f) Barcelona Supercomputing Center (BSC), Barcelona, Spain; (g) Pulmonology Service, University and Polytechnic Hospital La Fe, Valencia, Spain; (h) Hospital Universitario de Getafe, Madrid, Spain; Universidad Europea, Madrid, Spain; (i) Hospital Universitario Río Hortega de Valladolid, Valladolid, Spain; Group for Biomedical Research in Sepsis (BioSepsis), Instituto de Investigación Biomédica de Salamanca (IBSAL), Salamanca, Spain; (j) Servicio de Medicina Intensiva, Hospital Universitario Marqués de Valdecilla, Santander, Spain; (k) Servei de Medicina Intensiva, Hospital Universitari Germans Trias, Badalona, Spain; (l) Hospital Universitario Torrejón-Universidad Francisco de Vitoria, Madrid, Spain; (m) Servicio de Anestesiología y Reanimación, Hospital Universitario Basurto, Bilbao, Spain; (n) Critical Care Department, Hospital Joan XXIII, Tarragona, Spain; (o) Servicio de Medicina Intensiva, Hospital de Cruces, Baracaldo, Vizcaya, Spain; (p) Intensive Care Unit, Hospital Universitario La Princesa, Madrid, Spain; (q) UGC-Medicina Intensiva, Hospital Universitario Reina Sofia, Instituto Maimonides IMIBIC, Córdoba, Spain; (r) Critical Care Department, Hospital Dr. Negrín Gran Canaria, Las Palmas, Gran Canaria, Spain. Universidad Fernando Pessoa, Canarias, Spain; (s) Hospital Universitario de Segovia, Segovia, Spain; (t) Servei de Medicina Intensiva, Hospital Universitari Son Espases, Palma de Mallorca, Illes Balears, Spain; (u) Hospital Universitario HM Montepríncipe, Universidad San Pablo-CEU, Madrid, Spain; vIntensive Care Clinical Unit, Hospital Universitario Virgen de Rocío, Sevilla, Spain; (w) Intensive Care Clinical Unit, Hospital Universitario Virgen Macarena, Seville, Spain; (x) Intensive Care Unit, Hospital San Juan de Dios del Aljarafe, Bormujos, Sevilla, Spain; (y) Hospital General Universitario Gregorio Marañon, Madrid, Spain; (z) Servicio de Medicina Intensiva, Hospital Universitario Ramón y Cajal, Madrid, Spain; (aa) Critical Care Department, Hospital Universitario Río Hortega de Valladolid, Valladolid, Spain; (ab) Servicio de Medicina Intensiva, Hospital Universitario Príncipe de Asturias, Madrid, Spain; (ac) Servicio de Medicina Intensiva, Hospital Punta de Europa, Algeciras, Spain; (ad) Department of Intensive Care Medicine, Hospital Universitario 12 de Octubre, Madrid, Spain; (ae) Hospital de Sant Joan d’Alacant, Alacant, Spain; (af) Critical Care Department, Hospital Universitario Lucus Augusti, Lugo, Spain; (ag) Intensive Care Department, Hospital Nuestra Señora de Gracia, Zaragoza, Spain; (ah) Hospital Universitario de Móstoles, Madrid, Spain; (ai) Department of Intensive Care. Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, Barcelona, Spain. Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet de Llobregat, Barcelona, Spain; (aj) Pulmonary and Critical Care Division; Emergency Department, Clínica Sagrada Família, Barcelona, Spain; (ak) Department of Intensive Care Medicine, Hospital Clínico Universitario Valladolid, Valladolid, Spain; (al) Hospital Universitario de León, León, Spain; (am) Critical Care Department, Hospital Universitari Arnau de Vilanova; IRBLleida, Lleida, Spain; (an) Unidad de Cuidados Intensivos, Hospital Universitario San Pedro de Alcántara, Cáceres, Spain; (ao) Department of Intensive Medicine, Hospital Universitario Infanta Leonor, Madrid, Spain; (ap) Servicio de medicina intensiva. Hospital Universitario y Politécnico La Fe, Valencia, Spain; (aq) Servicio de Medicina Intensiva, Hospital Universitario Mútua de Terrassa, Terrassa, Barcelona, Spain; (ar) Critical Care Department, Hospital del Mar-IMIM, Barcelona, Spain; (as) Departamento de Biología Funcional. Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo; Instituto de Investigación Sanitaria del Principado de Asturias, Hospital Central de Asturias, Oviedo, Spain; (at) Hospital de Mataró de Barcelona, Spain; (au) Unidad de Medicina Intensiva, Hospital Universitario Virgen de Valme, Sevilla, Spain; (av) Complexo Hospitalario Universitario de Ourense, Ourense, Spain; (aw) Complejo Asistencial Universitario de Palencia, Palencia, Spain; (ax) Servicio de Medicina Intensiva. Hospital Universitario La Paz, IdiPAZ, Madrid, Spain; (ay) Intensive Care Unit, Hospital Álvaro Cunqueiro, Vigo, Spain; (az) Hospital Universitario de Salamanca, Salamanca, Spain; (ba) Department of Physical Medicine and Rehabilitation, Hospital Verge de la Cinta, Tortosa, Tarragona, Spain; (bb) Intensive Care Unit, Hospital Clínico y Universitario de Valencia, Valencia, Spain; (bc) Intensive Care Unit, Hospital Son Llàtzer, Palma de Mallorca, Illes Balears, Spain; (bd) Hospital Santa Maria; IRBLleida, Lleida, Spain; (be) Intensive Care Unit, University Hospital of Jerez. Medicine Department University of Cadiz. INiBICA, Spain; (bf) Hospital Universitario San Agustín, Asturias, Spain; (bg) Department of Medicine, Universitat Autónoma de Barcelona (UAB); Critical Care Department, Corporació Sanitària Parc Taulí, Sabadell, Barcelona, Spain; (bh) Department of Intensive care Medicine, Complejo Hospitalario Universitario de Santiago de Compostela, Santiago de Compostela, SpainPostprint (published version

M2 Cortex-Dorsolateral striatum stimulation reverses motor symptoms and synaptic deficits in Huntington's Disease

Huntington's disease (HD) is a neurological disorder characterized by motor disturbances. HD pathology is most prominent in the striatum, the central hub of the basal ganglia. The cerebral cortex is the main striatal afferent, and progressive cortico-striatal disconnection characterizes HD. We mapped striatal network dysfunction in HD mice to ultimately modulate the activity of a specific cortico-striatal circuit to ameliorate motor symptoms and recover synaptic plasticity. Multimodal MRI in vivo indicates cortico-striatal and thalamo-striatal functional network deficits and reduced glutamate/glutamine ratio in the striatum of HD mice. Moreover, optogenetically-induced glutamate release from M2 cortex terminals in the dorsolateral striatum (DLS) was undetectable in HD mice and striatal neurons show blunted electrophysiological responses. Remarkably, repeated M2-DLS optogenetic stimulation normalized motor behavior in HD mice and evoked a sustained increase of synaptic plasticity. Overall, these results reveal that selective stimulation of the M2-DLS pathway can become an effective therapeutic strategy in HD

Impact of time to intubation on mortality and pulmonary sequelae in critically ill patients with COVID-19: a prospective cohort study

We evaluated whether the time between first respiratory support and intubation of patients receiving invasive mechanical ventilation (IMV) due to COVID-19 was associated with mortality or pulmonary sequelae. Materials and methods: Prospective cohort of critical COVID-19 patients on IMV. Patients were classified as early intubation if they were intubated within the first 48 h from the first respiratory support or delayed intubation if they were intubated later. Surviving patients were evaluated after hospital discharge. Results: We included 205 patients (140 with early IMV and 65 with delayed IMV). The median [p25;p75] age was 63 [56.0; 70.0] years, and 74.1% were male. The survival analysis showed a significant increase in the risk of mortality in the delayed group with an adjusted hazard ratio (HR) of 2.45 (95% CI 1.29–4.65). The continuous predictor time to IMV showed a nonlinear association with the risk of in-hospital mortality. A multivariate mortality model showed that delay of IMV was a factor associated with mortality (HR of 2.40; 95% CI 1.42–4.1). During follow-up, patients in the delayed group showed a worse DLCO (mean difference of -¿10.77 (95% CI -¿18.40 to -¿3.15), with a greater number of affected lobes (+¿1.51 [95% CI 0.89–2.13]) and a greater TSS (+¿4.35 [95% CI 2.41–6.27]) in the chest CT scan. Conclusions: Among critically ill patients with COVID-19 who required IMV, the delay in intubation from the first respiratory support was associated with an increase in hospital mortality and worse pulmonary sequelae during follow-up.Postprint (published version

Pulmonary function and radiologic features in survivors of critical COVID-19: a 3-month prospective cohort

© 2021 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/BACKGROUND: More than 20% of hospitalized patients with COVID-19 demonstrate ARDS requiring ICU admission. The long-term respiratory sequelae in such patients remain unclear. RESEARCH QUESTION: What are the major long-term pulmonary sequelae in critical patients who survive COVID-19? STUDY DESIGN AND METHODS: Consecutive patients with COVID-19 requiring ICU admission were recruited and evaluated 3 months after hospitalization discharge. The follow-up comprised symptom and quality of life, anxiety and depression questionnaires, pulmonary function tests, exercise test (6-min walking test [6MWT]), and chest CT imaging. RESULTS: One hundred twenty-five patients admitted to the ICU with ARDS secondary to COVID- 19 were recruited between March and June 2020. At the 3-month follow-up, 62 patients were available for pulmonary evaluation. The most frequent symptoms were dyspnea (46.7%) and cough (34.4%). Eighty-two percent of patients showed a lung diffusing capacity of less than 80%. The median distance in the 6MWT was 400 m (interquartile range, 362-440 m). CT scans showed abnormal results in 70.2% of patients, demonstrating reticular lesions in 49.1% and fibrotic patterns in 21.1%. Patients with more severe alterations on chest CT scan showed worse pulmonary function and presented more degrees of desaturation in the 6MWT. Factors associated with the severity of lung damage on chest CT scan were age and length of invasive mechanical ventilation during the ICU stay. INTERPRETATION: Three months after hospital discharge, pulmonary structural abnormalities and functional impairment are highly prevalent in patients with ARDS secondary to COVID- 19 who required an ICU stay. Pulmonary evaluation should be considered for all critical COVID-19 survivors 3 months after discharge.This study was supported in part by the Instituto de Salud Carlos III [Grant CIBERESUCICOVID, COV20/00110] and was cofunded by European Regional Development Funds, “Una manera de hacer Europa.” D. d. G.-C. has received financial support from the Instituto de Salud Carlos III [Grant Miguel Servet 2020: CP20/00041], co-funded by the European Social Fund “Investing in Your Future.” L. P. acknowledges receiving financial support from the Ministry of Science, Innovation and Universities for the Training of University Lecturers (FPU19 / 03526).Peer ReviewedPostprint (author's final draft

One Year Overview and Follow-Up in a Post-COVID Consultation of Critically Ill Patients

The long-term clinical management and evolution of a cohort of critical COVID-19 survivors have not been described in detail. We report a prospective observational study of COVID-19 patients admitted to the ICU between March and August 2020. The follow-up in a post-COVID consultation comprised symptoms, pulmonary function tests, the 6-minute walking test (6MWT), and chest computed tomography (CT). Additionally, questionnaires to evaluate the prevalence of post-COVID-19 syndrome were administered at 1 year. A total of 181 patients were admitted to the ICU during the study period. They were middle-aged (median [IQR] of 61 [52;67]) and male (66.9%), with a median ICU stay of 9 (5-24.2) days. 20% died in the hospital, and 39 were not able to be included. A cohort of 105 patients initiated the follow-up. At 1 year, 32.2% persisted with respiratory alterations and needed to continue the follow-up. Ten percent still had moderate/severe lung diffusion (DLCO) involvement (<60%), and 53.7% had a fibrotic pattern on CT. Moreover, patients had a mean (SD) number of symptoms of 5.7 ± 4.6, and 61.3% met the criteria for post-COVID syndrome at 1 year. During the follow-up, 46 patients were discharged, and 16 were transferred to other consultations. Other conditions, such as emphysema (21.6%), COPD (8.2%), severe neurocognitive disorders (4.1%), and lung cancer (1%) were identified. A high use of health care resources is observed in the first year. In conclusion, one-third of critically ill COVID-19 patients need to continue follow-up beyond 1 year, due to abnormalities on DLCO, chest CT, or persistent symptoms.This study was supported in part by ISCIII (CIBERESUCICOVID, COV20/00110), co-funded by ERDF, “Una manera de hacer Europa,” donation program “Estar Preparados,” UNESPA, Madrid, Spain and Fundación Soria Melguizo (Madrid, Spain). DG-C had received financial support from Instituto de Salud Carlos III (Miguel Servet 2020: CP20/00041), co-funded by the European Social Fund (ESF)/“Investing in your future.” JB acknowledged receiving financial support from Instituto de Salud Carlos III (ISCIII; Miguel Servet 2019: CP19/00108), co-funded by the European Social Fund (ESF), “Investing in your future.

Identification of circulating microRNA profiles associated with pulmonary function and radiologic features in survivors of SARS-CoV-2-induced ARDS

There is a limited understanding of the pathophysiology of postacute pulmonary sequelae in severe COVID-19. The aim of current study was to define the circulating microRNA (miRNA) profiles associated with pulmonary function and radiologic features in survivors of SARS-CoV-2-induced ARDS. The study included patients who developed ARDS secondary to SARS-CoV-2 infection (n = 167) and a group of infected patients who did not develop ARDS (n = 33). Patients were evaluated 3 months after hospital discharge. The follow-up included a complete pulmonary evaluation and chest computed tomography. Plasma miRNA profiling was performed using RT-qPCR. Random forest was used to construct miRNA signatures associated with lung diffusing capacity for carbon monoxide (DLCO) and total severity score (TSS). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were conducted. DLCO < 80% predicted was observed in 81.8% of the patients. TSS showed a median [P25;P75] of 5 [2;8]. The miRNA model associated with DLCO comprised miR-17-5p, miR-27a-3p, miR-126-3p, miR-146a-5p and miR-495-3p. Concerning radiologic features, a miRNA signature composed by miR-9-5p, miR-21-5p, miR-24-3p and miR-221-3p correlated with TSS values. These associations were not observed in the non-ARDS group. KEGG pathway and GO enrichment analyses provided evidence of molecular mechanisms related not only to profibrotic or anti-inflammatory states but also to cell death, immune response, hypoxia, vascularization, coagulation and viral infection. In conclusion, diffusing capacity and radiological features in survivors from SARS-CoV-2-induced ARDS are associated with specific miRNA profiles. These findings provide novel insights into the possible molecular pathways underlying the pathogenesis of pulmonary sequelae.This work is supported by Instituto de Salud Carlos III (COV20/00110), co-funded by European Regional Development Fund (ERDF)/“A way to make Europe”. CIBERES is an initiative of the Instituto de Salud Carlos III. Suported by: Programa de donaciones “estar preparados” UNESPA (Madrid, Spain) and Fundación Francisco Soria Melguizo (Madrid, Spain). Supported by La Fundació La Marató de TV3, projecte amb codi 202108-30/-31. COVIDPONENT is funded by Institut Català de la Salut and Gestió de Serveis Sanitaris. MM is the recipient of a predoctoral fellowship (PFIS: FI21/00187) from Instituto de Salud Carlos III. MCGH is the recipient of a predoctoral fellowship from “University of Lleida”. DdGC has received financial support from Instituto de Salud Carlos III (Miguel Servet 2020: CP20/00041), co-funded by the European Social Fund (ESF)/“Investing in your future”. AC acknowledges receiving financial support from Instituto de Salud Carlos III (ISCIII; Sara Borrell 2021: CD21/00087). ENL and GL were funded by COVID1005 and ACT210085 from National Agency of Investigation & Development & Development (ANID), Chil

Genome-wide transcriptional profiling of pulmonary functional sequelae in ARDS- secondary to SARS-CoV-2 infection

Background: Up to 80% of patients surviving acute respiratory distress syndrome (ARDS) secondary to SARS-CoV- 2 infection present persistent anomalies in pulmonary function after hospital discharge. There is a limited un-derstanding of the mechanistic pathways linked to post-acute pulmonary sequelae. Aim: To identify the molecular underpinnings associated with severe lung diffusion involvement in survivors of SARS-CoV-2-induced ARDS. Methods: Survivors attended to a complete pulmonary evaluation 3 months after hospital discharge. RNA sequencing (RNA-seq) was performed using Illumina technology in whole-blood samples from 50 patients with moderate to severe diffusion impairment (DLCO<60%) and age- and sex-matched individuals with mild-normal lung function (DLCO≥60%). A transcriptomic signature for optimal classification was constructed using random forest. Transcriptomic data were analyzed for biological pathway enrichment, cellular deconvolution, cell/tissue-specific gene expression and candidate drugs. Results: RNA-seq identified 1357 differentially expressed transcripts. A model composed of 14 mRNAs allowed the optimal discrimination of survivors with severe diffusion impairment (AUC=0.979). Hallmarks of lung sequelae involved cell death signaling, cytoskeleton reorganization, cell growth and differentiation and the immune response. Resting natural killer (NK) cells were the most important immune cell subtype for the pre-diction of severe diffusion impairment. Components of the signature correlated with neutrophil, lymphocyte and monocyte counts. A variable expression profile of the transcripts was observed in lung cell subtypes and bodily tissues. One upregulated gene, TUBB4A, constitutes a target for FDA-approved drugs. Conclusions: This work defines the transcriptional programme associated with post-acute pulmonary sequelae and provides novel insights for targeted interventions and biomarker development.MCGH is the recipient of a predoctoral fellowship from the University of Lleida. MM is the recipient of a predoctoral fellowship (PFIS: FI21/00187) from Instituto de Salud Carlos III. AC is supported by Instituto de Salud Carlos III (Sara Borrell 2021: CD21/00087). DdGC has received financial support from Instituto de Salud Carlos III (Miguel Servet 2020: CP20/00041), co-funded by the European Social Fund (ESF) “Investing in your future”. IML is supported by a Miguel Servet contract (CPII20/00029) from the Instituto de Salud Carlos III, co-funded by the European Social Fund (ESF) “Investing in your future”. CIBERES is an initiative of the Instituto de Salud Carlos III. This work is supported by the Instituto de Salud Carlos III (COV20/00110), co-funded by the European Regional Development Fund (ERDF) “A way to make Europe”. Supported by: Programa de donaciones "estar preparados"; UNESPA (Madrid, Spain) and Fundación Francisco Soria Melguizo (Madrid, Spain). Funded by: La Fundació La Marató de TV3, project with code 202108–30/ 31. COVIDPONENT is funded by the Institut Català de la Salut and Gestió de Serveis Sanitaris. This research was funded in part by a grant (PI19/01805) from the Instituto de Salud Carlos III, co-funded by the European Regional Development Fund (ERDF) “A way to build Europe” and by the Fundación Rioja Salu

Multiplex protein profiling of bronchial aspirates reveals disease-, mortality- and respiratory sequelae-associated signatures in critically ill patients with ARDS secondary to SARS-CoV-2 infection

Introduction: Bronchial aspirates (BAS) obtained during invasive mechanical ventilation (IMV) constitutes a useful tool for molecular phenotyping and decision making. Aim: To identify the proteomic determinants associated with disease pathogenesis, all-cause mortality and respiratory sequelae in BAS samples from critically ill patients with SARS-CoV-2-induced ARDS. Methods: Multicenter study including 74 critically ill patients with COVID-19 and non-COVID-19 ARDS. BAS were obtained by bronchoaspiration after IMV initiation. Three hundred sixty-four proteins were quantified using proximity extension assay (PEA) technology. Random forest models were used to assess predictor importance. Results: After adjusting for confounding factors, CST5, NADK, SRPK2 and TGFa were differentially detected in COVID-19 and non-COVID-19 patients. In random forest models for COVID-19, CST5, DPP7, NADK, KYAT1 and TYMP showed the highest variable importance. In COVID-19 patients, reduced levels of ENTPD2 and PTN were observed in nonsurvivors of ICU stay, even after adjustment. AGR2, NQO2, IL-1a, OSM and TRAIL showed the strongest associations with in-ICU mortality and were used to construct a proteinbased prediction model. Kaplan-Meier curves revealed a clear separation in mortality risk between subgroups of PTN, ENTPD2 and the prediction model. Cox regression models supported these findings. In survivors, the levels of FCRL1, NTF4 and THOP1 in BAS samples obtained during the ICU stay correlated with lung function (i.e., DLCO levels) 3 months after hospital discharge. Similarly, Flt3L and THOP1 levels were correlated with radiological features (i.e., TSS). These proteins are expressed in immune and nonimmune lung cells. Poor host response to viral infectivity and an inappropriate reparative mechanism seem to be linked with the pathogenesis of the disease and fatal outcomes, respectively. Conclusion: BAS proteomics identified novel factors associated with the pathology of SARS-CoV-2-induced ARDS and its adverse outcomes. BASbased protein testing emerges as a novel tool for risk assessment in the ICU