Prognostic Value of Cellular Senescence, Lymphatic Proliferation, and Histological Findings in the Follow-up of Post-covid-19 Interstitial Lung Disease Sequelae.

Prognostic Value of Cellular Senescence, Lymphatic Proliferation, and Histological Findings in the Follow-up of Post-covid-19 Interstitial Lung Disease Sequelae. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Prognostic Value of Cellular Senescence, Lymphatic Proliferation, and Histological Findings in the Follow-up of Post-covid-19 Interstitial Lung Disease Sequelae. María Florencia Pilia, Irene Sansano, Diego Varona, Marina Sánchez-Calleja, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4654047/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract BACKGROUND In some patients post COVID-19 interstitial lung disease (ILD) sequelae persist beyond 6 months after hospital discharge. Little is known about the pathophysiology of this condition. This study aimed to determine the prognostic value of certain histopathological findings, cellular senescence, and lymphatic proliferation in patients with ILD sequelae. METHODS This prospective observational study of patients hospitalized at Vall d’Hebron University Hospital due to COVID-19 pneumonia and presenting respiratory symptoms, radiological alterations, and pulmonary function test impairment during the 3-month follow-up visit after discharge. Lung cryobiopsies were performed, and the histopathological findings and expression of senescence and lymphatic proliferation (P16 and D2-40) were analyzed. RESULTS Between March 2020 and February 2021, 4,332 patients were hospitalized at Vall d’Hebron University Hospital due to COVID-19 pneumonia, and 1,403 were visited in the Respiratory Clinic 3 months after discharge. The first 66 patients presenting with respiratory symptoms, radiological alterations, and decreased pulmonary function tests during the post hospitalization follow-up underwent cryobiopsy for diagnostic purposes. Multivariate regression showed that Masson bodies in the 3-month cryobiopsy were related to a higher forced vital capacity at 6 months whereas higher expression of senescence and lymphatic proliferation markers, such as P-16 and D2-40, in the histological samples were related to decreased carbon monoxide transfer test values at 6 months. CONCLUSION Senescence and lymphatic proliferation are related to worse pulmonary function outcomes in the mid-term follow-up of patients with post-COVID-19 ILD. Interstitial lung disease lung fibrosis SARS-CoV-2 cellular senescence COVID-19 sequalae lymphatic proliferation cryobiopsy Figures Figure 1 BACKGROUND On March 11, 2020, the World Medical Organization declared a worldwide pandemic due to SARS-CoV-2, a new strain of the coronavirus family. SARS-CoV-2 can cause a wide spectrum of respiratory illnesses ranging from the common cold to severe pneumonia and acute respiratory distress syndrome( 1 ). While the prevalence of post COVID-19 sequelae, such as interstitial lung disease (ILD), after hospital discharge varies according to the published series, most authors coincide that these findings are related to the severity of acute pneumonia( 2 , 3 ) In most patients high-resolution computed tomography (HRCT) images in the 6- and 12-month follow-up visits after hospitalization due to COVID-19 pneumonia show progressive improvement of the main findings, such as ground glass opacities (GGO), septal thickening, consolidations, and reticular patterns( 4 ). However, a substantial group of patients remain with respiratory symptoms and present decreased lung function values and radiological alterations in HRCT images long after hospital discharge. In this context, it seems mandatory to identify and explore the pathophysiology behind this torpid evolution in this subgroup of patients. While the most consistent histological finding in severe COVID-19 patients is diffuse alveolar damage, some patients develop postinfectious organizing pneumonia (OP) that persists for months( 5 ). OP is a non-specific pulmonary reaction to a pulmonary insult that consist of fibroblastic plugs that fill the airspace (Masson bodies) often along with mild lymphoplasmacytic infiltration of the interstitium. Other inflammatory cells, such as mast cells, may play a role in the disease ( 6 ). However, beyond these findings, other key underlying factors, such as cellular senescence or lymphatic proliferation, may deserve further attention. Cellular senescence refers to a state of irreversible growth arrest in which cells lose their ability to divide and function properly( 7 – 10 ). Cellular senescence is a complex phenomenon that encompasses maladaptive tissue repair, decreased regeneration and chronic inflammation showing similarities with ILD and lung fibrosis ( 7 , 11 ). P-16, also known as cyclin-dependent kinase inhibitor 2A, is a protein that plays a critical role in cellular senescence. Pulmonary lymphatic vessels are classically found along the peribronchial vascular sheaths, interlobular septa, and pleural connective tissue. Small lymphatic channels are observed within the lobules of normal human lung in the interstitial space around small blood vessels and occasionally emerging from the interalveolar interstitium( 12 ). Some authors have reported proliferation of these vessels in distal parenchyma in animal models of diffuse alveolar damage( 13 ). Others have observed lung lymphatic vessels in proximity to alveolar spaces in patients with idiopathic pulmonary fibrosis( 14 ). The objective of the present study was to determine the prognostic value of the histologic characteristics, cellular senescence and lymphatic proliferation in tissue samples obtained during the follow-up of patients with ILD sequelae after COVID-19 pneumonia. METHODS A prospective observational study was carried out on all the patients hospitalized due to COVID-19 pneumonia and attending the post-COVID-19 Respiratory Clinic at Vall d’Hebron University Hospital in Barcelona, Spain. Three months after hospital discharge all the patients that had required hospitalization within the context of SARS-CoV-2 pneumonia were offered pulmonary function testing, including spirometry and the carbon monoxide transfer test (DLCO), a 6-minute walking test, HRCT and a follow-up visit in the Respiratory Clinic. Adult patients ≥ 18 years old that had been hospitalized due to COVID-19 pneumonia and presenting respiratory symptoms, radiological alterations and pulmonary function test impairment at the 3-month follow-up visit after discharge were included. The clinical characteristics of the patients were analyzed during hospitalization followed by evaluation of lung function tests, and radiological and histological findings at 3 months, and clinical, lung function and radiological characteristics at 6 months after hospital discharge. Patient recruitment occurred during the first and second waves of the COVID-19 pandemic in Spain (March 2020 to February 2021), primarily involving the original strain of SARS-CoV-2. The Alpha and Beta variants began to emerge towards the end of this period but were not dominant. In the follow-up visit at 3 months, the first 66 patients attending the Respiratory Clinic and fulfilling the inclusion criteria underwent a bronchoscopy with transbronchial cryobiopsy. In patients with HRCT abnormalities, transbronchial cryobiopsy was performed if they had dyspnea (grade > 2 on the Modified Medical Research Council Dyspnea Scale) or lung function abnormalities (FVC or DLCO between 40%-70% of predicted value, or > 3% desaturation in the 6-minute walk test). Cryobiopsy was also considered in patients with normal FVC and FEV 1 but significant symptoms or radiological findings, based on a multidisciplinary evaluation. We opted to perform cryobiopsies at 3 months instead of 6 months due to the limited understanding of COVID-19 pulmonary sequelae during the early stages of the pandemic. This early intervention facilitated the detection of significant histopathological alterations, enabling timely therapeutic interventions and informed clinical management. While acknowledging the benefits of prolonged follow-up, our primary objective was to address immediate uncertainties and enhance patient outcomes during this critical period. The study protocol, including the criteria for performing cryobiopsies, was approved by the Ethics Committee of Vall d’Hebron Hospital (Reference: PR(AG)50/2019). All patients provided written informed consent before participating in the study. Pulmonary function tests Spirometry and DLCO were performed using a MasterLab device (MasterLab, Jaeger, Germany) following European Respiratory Society (ERS) and American Thoracic Society guidelines( 15 ). The reference values used were those proposed by the Global Lung Initiative. 6-minute walking test The 6-minute walking test was performed according to ERS guidelines( 15 ). Standardized instructions and encouragement were given during the test. HRCT HRCT was performed in all patients with 1 mm slices at 10 mm intervals in maximum inspiration. All the HRCT images were analyzed by a chest radiologist. The following radiological findings were analyzed in the HRCTs: GGO, septal and subpleural lines, honeycombing and pulmonary consolidations. The extent of the radiological findings was classified into 3 grades based on the number of affected segments: grade 1 (1 to 3 affected segments), 2 (4 to 9) and 3 (more than 9). Bronchoscopy and cryobiopsy All procedures were performed as described previously( 16 ). Single-use Ambu aScope 4 Broncho Large (Ambu Corp) and 2.4-mm single-use Erbe cryoprobes were employed, using ERBEKRYO 2 cryosurgical technology (ERBE Medizintechnik). According to the radiological findings, the most affected lung was chosen for the transbronchial cryobiopsy. A negative polymerase chain reaction assay for SARS-CoV-2 RNA (nasopharyngeal swab) was required before all procedures. Histopathological and immunohistochemistry findings The cryobiopsies were reviewed by two senior pathologists (IS and SRyC). The presence or absence of Masson bodies, interstitial inflammation and other histopathological findings were recorded along with a descriptive diagnosis for each patient. The interstitial inflammatory infiltrate was graded as mild, moderate, or severe, and the cells were characterized using immunohistochemical markers: CD20 (L26), CD3 (2GV6), CD138 (B-A38), CD4 (SP35), and CD8 (SP57). Mast cells were stained with tryptase (G3), counted per high power field (HPF), and a mean value was calculated per patient. The presence of senescent cells and lymphatic induction was confirmed using immunohistochemical markers P16 (CINtec histology) and podoplanin (D2-40), respectively. P16 expression was considered relevant in pneumocytes, fibroblasts, or other mesenchymal cells, and graded as absent (no positive cells), mild (1 positive cell/HPF), moderate (2–5 cells/HPF), and severe (> 5 cells/HPF). Only lymphatic vessels unrelated to the bronchial wall, septum, or pleura were considered. Their abundance was described using a semiquantitative scale: absent (0), mild (1/HPF), moderate (2–3/HPF), and severe (> 3/HPF). All immunohistochemistry was performed using a Benchmark Ultra stainer with the UltraView DAB kit, following datasheet staining protocols. Statistical analysis All the variables obtained at 3 and 6 months were described according to the type of variable. For continuous variables, the mean, standard deviation (SD), median, minimum (min) and maximum (max) were reported, and for categorical variables the number of cases (N) and the percentage (%) were reported by category. Non-parametric tests were used to assess whether there were statistically significant differences among the variables. The Wilcoxon test was used to compare the continuous variables with the histological variables with two categories. When these were compared with the variables with three categories, the Kruskal-Wallis test was used. For the comparison of the categorical variables with those of histology, the Chi-square test was used. Bivariate analysis of the different clinical outcomes was carried out and logistic models were performed. Multivariate models of the definitions of clinical outcomes were also analyzed. The software R version 4.1.1 was used. Ethical approval and consent to participate The study was approved by the Ethics Committee of the Vall d´Hebron Hospital (Reference number: PR (AG) 50/2019). All patients provided written informed consent prior to participating. RESULTS Between March 2020 and February 2021, 4,332 patients were hospitalized in a university hospital due to COVID-19 pneumonia. Of this group, 1,403 patients were visited in the Respiratory Clinic 3 months after discharge. The first 66 patients in correlative chronological order presenting respiratory symptoms, radiological alterations and decreased pulmonary function test values in the post hospitalization follow-up underwent a fibrobronchoscopy with a cryobiopsy for diagnostic purposes. Out of the 66 initial patients who underwent cryobiopsy, all 66 received HRCT follow-up at 3 months, and 48 also had HRCT at 6 months. The reasons for the lack of radiologic follow-up in the remaining 18 patients are as follows: 5 patients had HRCT performed outside the 6-month window (all showed improvement on HRCT); follow-up HRCT were not performed for 2 patients due to the normalization of pulmonary function and resolution of dyspnea; 1 patient died from systemic cryptococcosis; and 10 patients were lost to follow-up and did not have follow-up HRCT. Basal characteristics of the study population The basal characteristics of the study population are summarized in Table 1 . Thirty-two (48.48%) patients were female with a mean (SD) age of 58.22 (9.78) years. The mean body mass index was 27.89 (3.90) kg/m 2 , and 15 (22.73%), 13 (19.70%) and 38 (57.57%) were current smokers, ex-smokers and never smokers, respectively. Forty-two (63.64%) patients required admission in the intensive care unit with a mean stay of 18.70 (17.90) days. Thirty-five (53.03%) patients required mechanical ventilation. The mean hospital stay per patient was 33.06 (38.11) days. The data regarding the treatment received during and after hospitalization are summarized in Table 2. Eighty-one percent (54) of patients underwent cardiorespiratory rehabilitation based on the treating physician's discretion. Pulmonary function testing along the follow-up visits Table 3 summarizes the pulmonary function test results corresponding to the follow-up visits at 3 and 6 months after hospital discharge. The mean forced vital capacity (FVC) at 3 months was 3.03 liters (SD 1.03) and 3.20 liters (0.84) (p=0.003) at 6 months. The mean FVC in the first second was 2.48 liters (0.84) at 3 months and 2.64 liters (0.69) (p=0.010) at 6 months. The DLCO at 3 months was 56.77% (14.50) of the predicted value and 67.11% (16.46) (p= <0.001) at 6 months. HRCT images along the follow-up visits The radiological findings of the HRCTs corresponding to the 3- and 6-month follow-up visits after hospital discharge are shown in Table 4. Sixty-six (100%) patients presented GGO at 3 months compared to 44 patients (91.67) (p=0.134) at 6 months. In regard to the grade of extension of the GGO at 3 months, no patient presented grade 0, 9 (14.64%) presented grade 1, 29 (43.94%) grade 2 and 28 (42.42%) presented grade 3, while 4 (8.33%), 13 (27.08%), 29 (60.42%) and 2 (4.17%) patients presented grades 0, 1, 2 and 3 GGO at 6 months, respectively (p < 0.001). Sixty-four (96.97%) patients presented septal/subpleural lines at 3 months after hospital discharge compared to 42 (87.50%) (p=0.131) patients at 6 months. With respect to the extension of septal/subpleural lines 2 (3.03%), 27 (40.91%), 29 (43.94%) and 8 (12.12%) patients presented grades 0, 1, 2 and 3 at 3 months, respectively compared to 6 (12.5%), 25 (52.08%), 15 (31.25%) and 2 (4.17%) at 6 months (p=0.010). Histological features of the lung cryobiopsies The histological characteristics evaluated are summarized in Table 5. Twenty-two (33.33%) patients presented Masson bodies. Regarding interstitial inflammation, 15 (22.73%) patients showed none, while 33 (50%) patients presented mild inflammation, 15 (22.73%) moderate inflammation and 3 (4.55%) patients presented severe inflammation. Immunohistochemical characterization of the inflammatory infiltrate revealed that it was mostly composed of CD3 cells and that CD4/CD8 was >1. The mean number of mastocytes per HPF was 60.05. Thirteen patients (19.70%) presented bronchiolar metaplasia, 12 (18.18%) pigmented macrophages, and 5 (7.58%) presented interstitial giant cells. Regarding senescence, defined as the abundance of cells expressing P16, in 4 (6.06%) patients it was absent, in 39 (59.09%) mild, in 18 (27.27%) it was moderate and in 5 (27.27%) patients senescence was severe. Lymphatic vessels proliferation, labeled with D2-40, was mild, moderate, and severe in the samples of 25 (37.88%), 23 (34.85%) and 18 (27.27%) patients, respectively (Fig 1). Factors predicting mid-term outcomes after hospital discharge Multivariate analyses showed that patients with Masson bodies in the cryobiopsy at 3 months were less likely to have an FVC (%) < 80 at the 6-month visit (p=0.04). P16 and D2-40 in the histological samples at 3 months after hospitalization were related to DLCO values < 70% at 6 months (p=0.02, 0.04). The multivariate regression model confirmed that lymphatic proliferation and senescence values in the histological samples at 3 months after hospitalization were independently related to DLCO values < 70% at 6 months (p=0.04) (Table 6). DISCUSSION The present study showed that OP, senescence and lymphatic proliferation in pulmonary cryobiopsies in patients with respiratory sequelae after hospitalization due to COVID-19 pneumonia, can predict the evolution of lung function during the following months. The only pathological finding associated with good prognosis was OP defined as the presence of intralveolar Masson bodies. Increased expression of P16 and D2-40 in the cryobiopsy performed at 3 months after hospital discharge was independently related to lower DLCO values at 6 months. Senescence reflects the indelible mark of aging and is defined as a cellular program that induces stable growth arrest along with diverse phenotypic alterations, such as genomic instability, telomere attrition, chromatin remodeling, metabolic reprogramming, increased autophagy, decreased mitophagy and the up-regulation of a complex proinflammatory secretome(17). This growth arrest can be triggered by a wide spectrum of insults including telomere attrition, DNA damage, chromatin alterations and oncogene activation(11). While cellular senescence plays a key role in distinct physiologic protector processes, such a tumor progression, the secretome may also be involved in the appearance of lung fibrosis(7,9,10). This pathway has been described in depth in lung fibroblasts, alveolar type 2 cells and immune cells, which play a key role in lung fibrosis. In this context, in the present study, the measurement of P-16 values in lung tissue in patients presenting interstitial lung abnormalities at 3 months after COVID-19 pneumonia showed that increased levels of this cell cycle inhibitor were independently related to lower DLCO values at 6 months, confirming its utility as a predictor of poor outcomes in post-COVID-19 ILD sequelae. DLCO reflects the gas exchange between the lungs and the pulmonary vascular bed and is the parameter that probably best defines correct physiological functioning of the lungs. This is the first time that the prognostic value of a cycle cell inhibitor such as P16 has been assessed as a mid-term predictor of poor outcomes in ILD, confirming the biological plausibility proposed previously (18,19). In parallel, the presence and distribution of lymphatic vessels in normal lungs has been the subject of much controversy(20). Reports involving animal and human studies have described lymphatic vessels in the peribronchovascular interstitium but not in the parenchyma between alveoli (13,21,22). In contrast to the distribution of lymphatic vessels in normal lungs, de novo lymph angiogenesis has been described in diffuse alveolar damage(23–27). In this line, Chemaly et al .(28) described lymphatic vessels in the proximity of alveolar spaces with a greater perimeter and area with worsening stages of idiopathic pulmonary fibrosis(29). Lymphatic vessels have been related to the fibrotic process and the maintenance of lung injury(20,23). In our study moderate and intense expression of D2-40 antibodies were independently related to lower DLCO values at 6 months, also confirming, the utility of DLCO as a predictor of poor outcomes in ILD. Again, this is the first time that the potential predictive role of D2-40 is described in post-COVID-related ILD. OP secondary to SARS-CoV-2 is the most frequently described finding in HRCT and lung tissue samples in post-COVID-19 ILD sequelae(30,31). Different post-COVID-19 patient cohorts have demonstrated an overall good response of post-COVID-19 OP to systemic corticosteroids, although the dosage and duration of treatment are still controversial(32). Our study revealed that the presence of Masson bodies in lung cryobiopsy (which is the histological hallmark of OP) at 3 months was related to higher FVC values at the 6-month follow-up. This finding coincides with previous reports, describing a good response of OP to systemic steroids and progressive spontaneous improvement of post-COVID-19 ILD sequelae. Thus, our results confirm that Masson bodies are a marker of good prognosis in post-COVID-19 ILD sequelae. Therefore, cryobiopsy results significantly impacted therapeutic management, informing decisions like corticosteroid use based on detailed histopathological findings, thus improving medium-term outcomes for post-COVID-19 patients. The severity of inflammation in the lung tissue sample was not related to the lung function outcomes at follow-up. In parallel, immunohistochemical characterization of the inflammatory infiltrate revealed a predominant CD3 reactive pattern. Contrary to what has been found in COVID-19 autopsies, our patients showed a normal number of mastocytes per HPF(6). This could be due to the fact that the cryobiopsies were performed three months after the acute pneumonia. Approximately 15% of the patients in our study had some form of respiratory comorbidity, with asthma being the most prevalent, accounting for more than half of these cases. Current scientific evidence indicates that asthma does not significantly increase the risk of developing more severe SARS-CoV-2 infection(33). Consequently, the presence of asthma and other respiratory comorbidities in our cohort is unlikely to impact the overall results. This context is crucial for interpreting our findings and indicates that the observed effects are not influenced by an increased severity of COVID-19 due to respiratory comorbidities This study presents some limitations. First, the patient sample is limited, and therefore, a possible lack of power could prevent the observation of other prognostic properties related to the presence of the P16 or D2-40 antigen. Second, the lack of histologic follow-up samples at 6 months does not allow full understanding of the long-term meaning of these markers or the underlying physio pathological process that they represent. Third, our study lacks a control group, which could have contributed to the validation of the potential utility of the findings. However, obtaining healthy lung tissue in order to test experimental biomarkers seems ethically unapproachable. In summary, this is the first study aimed at analyzing the prognostic utility of senescence and lymphatic vessels and other histological characteristics such as OP within the context of post-COVID-19 ILD sequelae. While the P16 and D2-40 expression in lung tissue was related to decreased DLCO values at mid-term follow-up, OP was related to higher FVC values. These findings suggest the potential utility of P16 and D2-40 as markers of bad prognosis in a wide spectrum of patients presenting lung fibrosis. Further studies are necessary to confirm and deepen the understanding of the role of senescence and lymphatic proliferation in ILD. Declarations Ethics approval and consent to participate The study was approved by the Ethics Committee of the Vall d´Hebron Hospital (Reference number: PR (AG) 50/2019). All patients provided written informed consent prior to participating Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests IO declares to have received honoraria in the last three years for participating as a speaker in meetings sponsored by Astrazeneca, Boehringuer-Ingelheim, Chiesi, and Novartis and as a consultant for Astrazeneca, GlaxoSmithKlein, Puretech and Sanofi. He has received financial aid from Astrazeneca, Bial and Chiesi for congress attendance and has received grants from Sanofi for research projects. IS declares to have received honoraria in the last three years for participating as a speaker in meetings sponsored by Astrazeneca, Boehringuer-Ingelheim, Roche, Pfizer and Takeda and has received financial aid from Takeda for congress attendance. XM declares to have received honoraria in the last three years for participating as a speaker in meetings sponsored by Astrazeneca. GlaxoSmithKlein, Novartis and Sanofi as a consultant for Astrazeneca, GlaxoSmithKlein and Sanofi. He has received financial aid from Astrazeneca, Novartis, Menarini, GlaxoSmithKlein and FAES for congress attendance. MFP, DV, MSC, CRM, DE, MC, MJC, SRyC declare that they have no competing interests. Funding This project was supported by the Fundació Catalana de Pneumologia (FUCAP), Instituto de Salud Carlos III (PI21/01046) and Fondo Europeo de Desarrollo Regional (FEDER). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. MFP is a researcher supported by the Contratos Predoctorales de Formación en Investigación en Salud (PFis) programme from Instituto de Salud Carlos III (FI22/00262). Authors' contributions IO, IS, SRyC, MFP, XM, MJC, CRM, DV, MC and DE contributed substantially to the study design, data analysis and interpretation, and the writing of the manuscript. The authors had approved the submitted version and had agreed to be personally accountable for their own contributions, ensuring that any questions related to the accuracy or integrity of the work were appropriately investigated and resolved. MSC, IS and SRyC analyzed and interpreted the histological samples. 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El-Chemaly S, Malide D, Zudaire E, Ikeda Y, Weinberg BA, Pacheco-Rodriguez G, et al. Abnormal lymphangiogenesis in idiopathic pulmonary fibrosis with insights into cellular and molecular mechanisms. Proc Natl Acad Sci U S A [Internet]. 2009 Mar 10 [cited 2023 Mar 28];106(10):3958–63. Available from: https://pubmed.ncbi.nlm.nih.gov/19237567/ Sanchez-Ramirez DC, Normand K, Yang Z, Torres-Castro R. Long-Term Impact of COVID-19: A Systematic Review of the Literature and Meta-Analysis. Biomedicines [Internet]. 2021 Aug 1 [cited 2023 Mar 28];9(8). Available from: https://pubmed.ncbi.nlm.nih.gov/34440104/ Polak SB, Van Gool IC, Cohen D, von der Thüsen JH, van Paassen J. A systematic review of pathological findings in COVID-19: a pathophysiological timeline and possible mechanisms of disease progression. Mod Pathol [Internet]. 2020 Nov 1 [cited 2023 Mar 28];33(11):2128–38. Available from: https://pubmed.ncbi.nlm.nih.gov/32572155/ Dhooria S, Chaudhary S, Sehgal IS, Agarwal R, Arora S, Garg M, et al. High-dose versus low-dose prednisolone in symptomatic patients with post-COVID-19 diffuse parenchymal lung abnormalities: an open-label, randomised trial (the COLDSTER trial). Eur Respir J [Internet]. 2022 Feb 1 [cited 2023 Mar 28];59(2). Available from: https://pubmed.ncbi.nlm.nih.gov/34887325/ Muñoz X, Pilia F, Ojanguren I, Romero-Mesones C, Cruz MJ. Is asthma a risk factor for COVID-19? Are phenotypes important? ERJ Open Res [Internet]. 2020 Nov 12 [cited 2020 Dec 19];00216–2020. Available from: /pmc/articles/PMC7667724/?report=abstract Tables Tables 1 to 6 are available in the Supplementary Files section Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4654047","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330928869,"identity":"4a6df785-7a85-4af1-805b-7a607663e42f","order_by":0,"name":"María Florencia Pilia","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Florencia","lastName":"Pilia","suffix":""},{"id":330928871,"identity":"cf917d6f-dff9-4cc4-868e-1e4b86a9d7b6","order_by":1,"name":"Irene Sansano","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"","lastName":"Sansano","suffix":""},{"id":330928872,"identity":"492ef159-4d09-41e6-a6be-38802c01b85f","order_by":2,"name":"Diego Varona","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Varona","suffix":""},{"id":330928873,"identity":"b1bd2a49-b0d6-41a8-ad19-a72dddc0103f","order_by":3,"name":"Marina Sánchez-Calleja","email":"","orcid":"","institution":"Universitat Autònoma de Barcelona (UAB)","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Sánchez-Calleja","suffix":""},{"id":330928874,"identity":"94a0ea5e-23b0-4c7b-8786-8e6f187cc229","order_by":4,"name":"Christian Romero-Mesones","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"Christian","middleName":"","lastName":"Romero-Mesones","suffix":""},{"id":330928875,"identity":"d738a791-408b-4909-a465-541ddac97528","order_by":5,"name":"David Espejo","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Espejo","suffix":""},{"id":330928876,"identity":"159079f6-30ea-4313-8d55-2b14b52a7fc0","order_by":6,"name":"Mario Culebras","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"Mario","middleName":"","lastName":"Culebras","suffix":""},{"id":330928877,"identity":"748ae8ee-a4bf-4c1c-ba21-76707e592c88","order_by":7,"name":"María Jesús Cruz","email":"","orcid":"","institution":"Vall d’Hebron Institut de Recerca (VHIR)","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Jesús","lastName":"Cruz","suffix":""},{"id":330928878,"identity":"f1cca0b7-2d50-458f-80bf-823e7093e894","order_by":8,"name":"Xavier Muñoz","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"","lastName":"Muñoz","suffix":""},{"id":330928879,"identity":"eae14430-5f90-481d-8269-1344ca1028cd","order_by":9,"name":"Iñigo Ojanguren","email":"data:image/png;base64,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","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":true,"prefix":"","firstName":"Iñigo","middleName":"","lastName":"Ojanguren","suffix":""},{"id":330928880,"identity":"9b7ca5ec-a844-4fa6-95a4-4550ed0941ab","order_by":10,"name":"Santiago Ramón","email":"","orcid":"","institution":"Hospital Universitari Vall d´Hebron","correspondingAuthor":false,"prefix":"","firstName":"Santiago","middleName":"","lastName":"Ramón","suffix":""}],"badges":[],"createdAt":"2024-06-28 10:24:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4654047/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4654047/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61347979,"identity":"ca7467e7-cc36-48cd-a726-cc561b230231","added_by":"auto","created_at":"2024-07-29 18:19:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":987625,"visible":true,"origin":"","legend":"\u003cp\u003eHistological sections of lung cryobiopsies.\u003c/p\u003e\n\u003cp\u003eP-16: nuclear and cytoplasmic expression in alveolar cells (40x). D2-40 Cytoplasmic expression in alveolar cells (20x). Semiquantitative determination of P-16: A1: mild; A2: moderate, A3: severe. Semiquantitative determination of D2-40: B1: mild; B2: moderate; B3: severe.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4654047/v1/5c1a4b0f44ec08bc47eaa15f.png"},{"id":75118105,"identity":"30195c89-1dd2-4dd0-8cde-15b23c8ef120","added_by":"auto","created_at":"2025-01-30 16:38:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1543635,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4654047/v1/ebd5c62d-21f4-4642-b55b-7425e2df2e3f.pdf"},{"id":61347978,"identity":"a3d976d1-5729-4541-a7c3-707d1010aa30","added_by":"auto","created_at":"2024-07-29 18:19:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":141602,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4654047/v1/e07820ca59ee8f7e3115592c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePrognostic Value of Cellular Senescence, Lymphatic Proliferation, and Histological Findings in the Follow-up of Post-covid-19 Interstitial Lung Disease Sequelae.\u003c/p\u003e","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eOn March 11, 2020, the World Medical Organization declared a worldwide pandemic due to SARS-CoV-2, a new strain of the coronavirus family. SARS-CoV-2 can cause a wide spectrum of respiratory illnesses ranging from the common cold to severe pneumonia and acute respiratory distress syndrome(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). While the prevalence of post COVID-19 sequelae, such as interstitial lung disease (ILD), after hospital discharge varies according to the published series, most authors coincide that these findings are related to the severity of acute pneumonia(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn most patients high-resolution computed tomography (HRCT) images in the 6- and 12-month follow-up visits after hospitalization due to COVID-19 pneumonia show progressive improvement of the main findings, such as ground glass opacities (GGO), septal thickening, consolidations, and reticular patterns(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). However, a substantial group of patients remain with respiratory symptoms and present decreased lung function values and radiological alterations in HRCT images long after hospital discharge. In this context, it seems mandatory to identify and explore the pathophysiology behind this torpid evolution in this subgroup of patients.\u003c/p\u003e \u003cp\u003eWhile the most consistent histological finding in severe COVID-19 patients is diffuse alveolar damage, some patients develop postinfectious organizing pneumonia (OP) that persists for months(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). OP is a non-specific pulmonary reaction to a pulmonary insult that consist of fibroblastic plugs that fill the airspace (Masson bodies) often along with mild lymphoplasmacytic infiltration of the interstitium. Other inflammatory cells, such as mast cells, may play a role in the disease (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, beyond these findings, other key underlying factors, such as cellular senescence or lymphatic proliferation, may deserve further attention.\u003c/p\u003e \u003cp\u003eCellular senescence refers to a state of irreversible growth arrest in which cells lose their ability to divide and function properly(\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Cellular senescence is a complex phenomenon that encompasses maladaptive tissue repair, decreased regeneration and chronic inflammation showing similarities with ILD and lung fibrosis (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). P-16, also known as cyclin-dependent kinase inhibitor 2A, is a protein that plays a critical role in cellular senescence.\u003c/p\u003e \u003cp\u003ePulmonary lymphatic vessels are classically found along the peribronchial vascular sheaths, interlobular septa, and pleural connective tissue. Small lymphatic channels are observed within the lobules of normal human lung in the interstitial space around small blood vessels and occasionally emerging from the interalveolar interstitium(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Some authors have reported proliferation of these vessels in distal parenchyma in animal models of diffuse alveolar damage(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Others have observed lung lymphatic vessels in proximity to alveolar spaces in patients with idiopathic pulmonary fibrosis(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe objective of the present study was to determine the prognostic value of the histologic characteristics, cellular senescence and lymphatic proliferation in tissue samples obtained during the follow-up of patients with ILD sequelae after COVID-19 pneumonia.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eA prospective observational study was carried out on all the patients hospitalized due to COVID-19 pneumonia and attending the post-COVID-19 Respiratory Clinic at Vall d\u0026rsquo;Hebron University Hospital in Barcelona, Spain. Three months after hospital discharge all the patients that had required hospitalization within the context of SARS-CoV-2 pneumonia were offered pulmonary function testing, including spirometry and the carbon monoxide transfer test (DLCO), a 6-minute walking test, HRCT and a follow-up visit in the Respiratory Clinic.\u003c/p\u003e\n\u003cp\u003eAdult patients\u0026thinsp;\u0026ge;\u0026thinsp;18 years old that had been hospitalized due to COVID-19 pneumonia and presenting respiratory symptoms, radiological alterations and pulmonary function test impairment at the 3-month follow-up visit after discharge were included. The clinical characteristics of the patients were analyzed during hospitalization followed by evaluation of lung function tests, and radiological and histological findings at 3 months, and clinical, lung function and radiological characteristics at 6 months after hospital discharge. Patient recruitment occurred during the first and second waves of the COVID-19 pandemic in Spain (March 2020 to February 2021), primarily involving the original strain of SARS-CoV-2. The Alpha and Beta variants began to emerge towards the end of this period but were not dominant.\u003c/p\u003e\n\u003cp\u003eIn the follow-up visit at 3 months, the first 66 patients attending the Respiratory Clinic and fulfilling the inclusion criteria underwent a bronchoscopy with transbronchial cryobiopsy. In patients with HRCT abnormalities, transbronchial cryobiopsy was performed if they had dyspnea (grade\u0026thinsp;\u0026gt;\u0026thinsp;2 on the Modified Medical Research Council Dyspnea Scale) or lung function abnormalities (FVC or DLCO between 40%-70% of predicted value, or \u0026gt;\u0026thinsp;3% desaturation in the 6-minute walk test). Cryobiopsy was also considered in patients with normal FVC and FEV\u003csub\u003e1\u003c/sub\u003e but significant symptoms or radiological findings, based on a multidisciplinary evaluation. We opted to perform cryobiopsies at 3 months instead of 6 months due to the limited understanding of COVID-19 pulmonary sequelae during the early stages of the pandemic. This early intervention facilitated the detection of significant histopathological alterations, enabling timely therapeutic interventions and informed clinical management. While acknowledging the benefits of prolonged follow-up, our primary objective was to address immediate uncertainties and enhance patient outcomes during this critical period. The study protocol, including the criteria for performing cryobiopsies, was approved by the Ethics Committee of Vall d\u0026rsquo;Hebron Hospital (Reference: PR(AG)50/2019). All patients provided written informed consent before participating in the study.\u003c/p\u003e\n\u003ch3\u003ePulmonary function tests\u003c/h3\u003e\n\u003cp\u003eSpirometry and DLCO were performed using a MasterLab device (MasterLab, Jaeger, Germany) following European Respiratory Society (ERS) and American Thoracic Society guidelines(\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). The reference values used were those proposed by the Global Lung Initiative.\u003c/p\u003e\n\u003cp\u003e6-minute walking test\u003c/p\u003e\n\u003cp\u003eThe 6-minute walking test was performed according to ERS guidelines(\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e). Standardized instructions and encouragement were given during the test.\u003c/p\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eHRCT\u003c/h2\u003e\n \u003cp\u003eHRCT was performed in all patients with 1 mm slices at 10 mm intervals in maximum inspiration. All the HRCT images were analyzed by a chest radiologist.\u003c/p\u003e\n \u003cp\u003eThe following radiological findings were analyzed in the HRCTs: GGO, septal and subpleural lines, honeycombing and pulmonary consolidations. The extent of the radiological findings was classified into 3 grades based on the number of affected segments: grade 1 (1 to 3 affected segments), 2 (4 to 9) and 3 (more than 9).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eBronchoscopy and cryobiopsy\u003c/h2\u003e\n \u003cp\u003eAll procedures were performed as described previously(\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e). Single-use Ambu aScope 4 Broncho Large (Ambu Corp) and 2.4-mm single-use Erbe cryoprobes were employed, using ERBEKRYO 2 cryosurgical technology (ERBE Medizintechnik). According to the radiological findings, the most affected lung was chosen for the transbronchial cryobiopsy. A negative polymerase chain reaction assay for SARS-CoV-2 RNA (nasopharyngeal swab) was required before all procedures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eHistopathological and immunohistochemistry findings\u003c/h2\u003e\n \u003cp\u003eThe cryobiopsies were reviewed by two senior pathologists (IS and SRyC). The presence or absence of Masson bodies, interstitial inflammation and other histopathological findings were recorded along with a descriptive diagnosis for each patient.\u003c/p\u003e\n \u003cp\u003eThe interstitial inflammatory infiltrate was graded as mild, moderate, or severe, and the cells were characterized using immunohistochemical markers: CD20 (L26), CD3 (2GV6), CD138 (B-A38), CD4 (SP35), and CD8 (SP57). Mast cells were stained with tryptase (G3), counted per high power field (HPF), and a mean value was calculated per patient. The presence of senescent cells and lymphatic induction was confirmed using immunohistochemical markers P16 (CINtec histology) and podoplanin (D2-40), respectively. P16 expression was considered relevant in pneumocytes, fibroblasts, or other mesenchymal cells, and graded as absent (no positive cells), mild (1 positive cell/HPF), moderate (2\u0026ndash;5 cells/HPF), and severe (\u0026gt;\u0026thinsp;5 cells/HPF). Only lymphatic vessels unrelated to the bronchial wall, septum, or pleura were considered. Their abundance was described using a semiquantitative scale: absent (0), mild (1/HPF), moderate (2\u0026ndash;3/HPF), and severe (\u0026gt;\u0026thinsp;3/HPF). All immunohistochemistry was performed using a Benchmark Ultra stainer with the UltraView DAB kit, following datasheet staining protocols.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical analysis\u003c/h2\u003e\n \u003cp\u003eAll the variables obtained at 3 and 6 months were described according to the type of variable. For continuous variables, the mean, standard deviation (SD), median, minimum (min) and maximum (max) were reported, and for categorical variables the number of cases (N) and the percentage (%) were reported by category. Non-parametric tests were used to assess whether there were statistically significant differences among the variables. The Wilcoxon test was used to compare the continuous variables with the histological variables with two categories. When these were compared with the variables with three categories, the Kruskal-Wallis test was used. For the comparison of the categorical variables with those of histology, the Chi-square test was used. Bivariate analysis of the different clinical outcomes was carried out and logistic models were performed. Multivariate models of the definitions of clinical outcomes were also analyzed. The software R version 4.1.1 was used.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe study was approved by the Ethics Committee of the Vall d\u0026acute;Hebron Hospital (Reference number: PR (AG) 50/2019). All patients provided written informed consent prior to participating.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eBetween March 2020 and February 2021, 4,332 patients were hospitalized in a university hospital due to COVID-19 pneumonia. Of this group, 1,403 patients were visited in the Respiratory Clinic 3 months after discharge. The first 66 patients in correlative chronological order presenting respiratory symptoms, radiological alterations and decreased pulmonary function test values in the post hospitalization follow-up underwent a fibrobronchoscopy with a cryobiopsy for diagnostic purposes. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOut of the 66 initial patients who underwent cryobiopsy, all 66 received HRCT follow-up at 3 months, and 48 also had HRCT at 6 months.\u0026nbsp;The reasons for the lack of radiologic follow-up in the remaining 18 patients are as follows: 5 patients had HRCT performed outside the 6-month window (all showed improvement on HRCT); follow-up HRCT were not performed for 2 patients due to the normalization of pulmonary function and resolution of dyspnea; 1 patient died from systemic cryptococcosis; and 10 patients were lost to follow-up and did not have follow-up HRCT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBasal characteristics of the study population \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe basal characteristics of the study population are summarized in Table 1\u003cstrong\u003e.\u003c/strong\u003e Thirty-two (48.48%) patients were female with a mean (SD) age of 58.22 (9.78) years. The mean body mass index was 27.89 (3.90) kg/m\u003csup\u003e2\u003c/sup\u003e, and 15 (22.73%), 13 (19.70%) and 38 (57.57%) were current smokers, ex-smokers and never smokers, respectively. Forty-two (63.64%) patients required admission in the intensive care unit with a mean stay of 18.70 (17.90) days. Thirty-five (53.03%) patients required mechanical ventilation. The mean hospital stay per patient was 33.06 (38.11) days. \u0026nbsp;The data regarding the treatment received during and after hospitalization are summarized in Table 2.\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003eEighty-one percent (54) of patients underwent cardiorespiratory rehabilitation based on the treating physician\u0026apos;s discretion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePulmonary function testing along the follow-up visits \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 3 summarizes the pulmonary function test results corresponding to the follow-up visits at 3 and 6 months after hospital discharge. The mean forced vital capacity (FVC) at 3 months was 3.03 liters (SD 1.03) and 3.20 liters (0.84) (p=0.003) at 6 months. The mean FVC in the first second was 2.48 liters (0.84) at 3 months and 2.64 liters (0.69) (p=0.010) at 6 months. The DLCO at 3 months was 56.77% (14.50) of the predicted value and 67.11% (16.46) (p= \u0026lt;0.001) at 6 months. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHRCT images along the follow-up visits \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe radiological findings of the HRCTs corresponding to the 3- and 6-month follow-up visits after hospital discharge are shown in Table 4. Sixty-six (100%) patients presented GGO at 3 months compared to 44 patients (91.67) (p=0.134) at 6 months. In regard to the grade of extension of the GGO at 3 months, no patient presented grade 0, 9 (14.64%) presented grade 1, 29 (43.94%) grade 2 and 28 (42.42%) presented grade 3, while 4 (8.33%), 13 (27.08%), 29 (60.42%) and 2 (4.17%) patients presented grades 0, 1, 2 and 3 GGO at 6 months, respectively (p \u0026lt; 0.001). Sixty-four (96.97%) patients presented septal/subpleural lines at 3 months after hospital discharge compared to 42 (87.50%) (p=0.131) patients at 6 months. With respect to the extension of septal/subpleural lines 2 (3.03%), 27 (40.91%), 29 (43.94%) and 8 (12.12%) patients presented grades 0, 1, 2 and 3 at 3 months, respectively compared to 6 (12.5%), 25 (52.08%), 15 (31.25%) and 2 (4.17%) at 6 months (p=0.010). \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological features of the lung cryobiopsies \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe histological characteristics evaluated are summarized in Table 5. Twenty-two (33.33%) patients presented Masson bodies. Regarding interstitial inflammation, 15 (22.73%) patients showed none, while 33 (50%) patients presented mild inflammation, 15 (22.73%) moderate inflammation and 3 (4.55%) patients presented severe inflammation. Immunohistochemical characterization of the inflammatory infiltrate revealed that it was mostly composed of CD3 cells and that CD4/CD8 was \u0026gt;1. The mean number of mastocytes per HPF was 60.05. Thirteen patients (19.70%) presented bronchiolar metaplasia, 12 (18.18%) pigmented macrophages, and 5 (7.58%) presented interstitial giant cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding senescence, defined as the abundance of cells expressing P16, in 4 (6.06%) patients it was absent, in 39 (59.09%) mild, in 18 (27.27%) it was moderate and in 5 (27.27%) patients senescence was severe. Lymphatic vessels proliferation, labeled with D2-40, was mild, moderate, and severe in the samples of 25 (37.88%), 23 (34.85%) and 18 (27.27%) patients, respectively (Fig 1). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFactors predicting mid-term outcomes after hospital discharge\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultivariate analyses showed that patients with Masson bodies in the cryobiopsy at 3 months were less likely to have an FVC (%) \u0026lt; 80 at the 6-month visit (p=0.04).\u003c/p\u003e\n\u003cp\u003eP16 and D2-40 in the histological samples at 3 months after hospitalization were related to DLCO values \u0026lt; 70% at 6 months (p=0.02, 0.04). The multivariate regression model confirmed that lymphatic proliferation and senescence values in the histological samples at 3 months after hospitalization were independently related to DLCO values \u0026lt; 70% at 6 months (p=0.04) (Table 6).\u0026nbsp;\u003c/p\u003e"},{"header":" DISCUSSION ","content":"\u003cp\u003eThe present study showed that OP, senescence and lymphatic proliferation in pulmonary cryobiopsies in patients with respiratory sequelae after hospitalization due to COVID-19 pneumonia, can predict the evolution of lung function during the following months. The only pathological finding associated with good prognosis was OP defined as the presence of intralveolar Masson bodies. Increased expression of P16 and D2-40 in the cryobiopsy performed at 3 months after hospital discharge was independently related to lower DLCO values at 6 months. Senescence reflects the indelible mark of aging and is defined as a cellular program that induces stable growth arrest along with diverse phenotypic alterations, such as genomic instability, telomere attrition, chromatin remodeling, metabolic reprogramming, increased autophagy, decreased mitophagy and the up-regulation of a complex proinflammatory secretome(17). This growth arrest can be triggered by a wide spectrum of insults including telomere attrition, DNA damage, chromatin alterations and oncogene activation(11). While cellular senescence plays a key role in distinct physiologic protector processes, such a tumor progression, the secretome may also be involved in the appearance of lung fibrosis(7,9,10). This pathway has been described in depth in lung fibroblasts, alveolar type 2 cells and immune cells, which play a key role in lung fibrosis. In this context, in the present study, the measurement of P-16 values in lung tissue in patients presenting interstitial lung abnormalities at 3 months after COVID-19 pneumonia showed that increased levels of this cell cycle inhibitor were independently related to lower DLCO values at 6 months, confirming its utility as a predictor of poor outcomes in post-COVID-19 ILD sequelae. DLCO reflects the gas exchange between the lungs and the pulmonary vascular bed and is the parameter that probably best defines correct physiological functioning of the lungs. This is the first time that the prognostic value of a cycle cell inhibitor such as P16 has been assessed as a mid-term predictor of poor outcomes in ILD, confirming the biological plausibility proposed previously\u0026nbsp;(18,19).\u003c/p\u003e\n\u003cp\u003eIn parallel, the presence and distribution of lymphatic vessels in normal lungs has been the subject of much controversy(20). Reports involving animal and human studies have described lymphatic vessels in the peribronchovascular interstitium but not in the parenchyma between alveoli\u0026nbsp;(13,21,22). In contrast to the distribution of lymphatic vessels in normal lungs, \u003cem\u003ede novo\u003c/em\u003e lymph angiogenesis has been described in diffuse alveolar damage(23\u0026ndash;27). In this line, Chemaly \u003cem\u003eet al\u003c/em\u003e.(28)\u0026nbsp;described lymphatic vessels in the proximity of alveolar spaces with a greater perimeter and area with worsening stages of idiopathic pulmonary fibrosis(29). Lymphatic vessels have been related to the fibrotic process and the maintenance of lung injury(20,23). In our study moderate and intense expression of D2-40 antibodies were independently related to lower DLCO values at 6 months, also confirming, the utility of DLCO as a predictor of poor outcomes in ILD. Again, this is the first time that the potential predictive role of D2-40 is described in post-COVID-related ILD.\u003c/p\u003e\n\u003cp\u003eOP secondary to SARS-CoV-2 is the most frequently described finding in HRCT and lung tissue samples in post-COVID-19 ILD sequelae(30,31). Different post-COVID-19 patient cohorts have demonstrated an overall good response of post-COVID-19 OP to systemic corticosteroids, although the dosage and duration of treatment are still controversial(32). Our study revealed that the presence of Masson bodies in lung cryobiopsy (which is the histological hallmark of OP) at 3 months was related to higher FVC values at the 6-month follow-up. This finding coincides with previous reports, describing a good response of OP to systemic steroids and progressive spontaneous improvement of post-COVID-19 ILD sequelae. Thus, our results confirm that Masson bodies are a marker of good prognosis in post-COVID-19 ILD sequelae. Therefore, cryobiopsy results significantly impacted therapeutic management, informing decisions like corticosteroid use based on detailed histopathological findings, thus improving medium-term outcomes for post-COVID-19 patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe severity of inflammation in the lung tissue sample was not related to the lung function outcomes at follow-up. \u0026nbsp;In parallel, immunohistochemical characterization of the inflammatory infiltrate revealed a predominant CD3 reactive pattern. Contrary to what has been found in COVID-19 autopsies, our patients showed a normal number of mastocytes per HPF(6). This could be due to the fact that the cryobiopsies were performed three months after the acute pneumonia.\u003c/p\u003e\n\u003cp\u003eApproximately 15% of the patients in our study had some form of respiratory comorbidity, with asthma being the most prevalent, accounting for more than half of these cases. Current scientific evidence indicates that asthma does not significantly increase the risk of developing more severe SARS-CoV-2 infection(33). Consequently, the presence of asthma and other respiratory comorbidities in our cohort is unlikely to impact the overall results. This context is crucial for interpreting our findings and indicates that the observed effects are not influenced by an increased severity of COVID-19 due to respiratory comorbidities\u003c/p\u003e\n\u003cp\u003eThis study presents some limitations. First, the patient sample is limited, and therefore, a possible lack of power could prevent the observation of other prognostic properties related to the presence of the P16 or D2-40 antigen. Second, the lack of histologic follow-up samples at 6 months does not allow full understanding of the long-term meaning of these markers or the underlying physio pathological process that they represent. Third, our study lacks a control group, which could have contributed to the validation of the potential utility of the findings. However, obtaining healthy lung tissue in order to test experimental biomarkers seems ethically unapproachable.\u003c/p\u003e\n\u003cp\u003eIn summary, this is the first study aimed at analyzing the prognostic utility of senescence and lymphatic vessels and other histological characteristics such as OP within the context of post-COVID-19 ILD sequelae. While the P16 and D2-40 expression in lung tissue was related to decreased DLCO values at mid-term follow-up, OP was related to higher FVC values. These findings suggest the potential utility of P16 and D2-40 as markers of bad prognosis in a wide spectrum of patients presenting lung fibrosis. Further studies are necessary to confirm and deepen the understanding of the role of senescence and lymphatic proliferation in ILD. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003e\u003cu\u003eEthics approval and consent to participate\u0026nbsp;\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of the Vall d´Hebron Hospital (Reference number: PR (AG) 50/2019). All patients provided written informed consent prior to participating\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eConsent for publication\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eAvailability of data and materials\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eCompeting interests\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eIO declares to have received honoraria in the last three years for participating as a speaker in meetings sponsored by Astrazeneca, Boehringuer-Ingelheim, Chiesi, and Novartis and as a consultant for Astrazeneca, GlaxoSmithKlein, Puretech and Sanofi. He has received financial aid from Astrazeneca, Bial and Chiesi for congress attendance and has received grants from Sanofi for research projects. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIS declares to have received honoraria in the last three years for participating as a speaker in meetings sponsored by Astrazeneca, Boehringuer-Ingelheim, Roche, Pfizer and Takeda and has received financial aid from Takeda for congress attendance. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eXM declares to have received honoraria in the last three years for participating as a speaker in meetings sponsored by Astrazeneca. GlaxoSmithKlein, Novartis and Sanofi as a consultant for Astrazeneca, GlaxoSmithKlein and Sanofi. He has received financial aid from Astrazeneca, Novartis, Menarini, GlaxoSmithKlein and FAES for congress attendance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMFP, DV, MSC, CRM, DE, MC, MJC, SRyC declare that they have no competing interests.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis project was supported by the Fundació Catalana de Pneumologia (FUCAP), Instituto de Salud Carlos III (PI21/01046) and Fondo Europeo de Desarrollo Regional (FEDER).\u0026nbsp;The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMFP is a researcher supported by the Contratos Predoctorales de Formación en Investigación en Salud (PFis) programme from Instituto de Salud Carlos III (FI22/00262).\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eAuthors' contributions\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eIO, IS, SRyC, MFP, XM, MJC, CRM, DV, MC and DE contributed substantially to the study design, data analysis and interpretation, and the writing of the manuscript. \u0026nbsp;The authors had approved the submitted version and had agreed to be personally accountable for their own contributions, ensuring that any questions related to the accuracy or integrity of the work were appropriately investigated and resolved. MSC, IS and SRyC analyzed and interpreted the histological samples. DV reviewed and compared the radiological tests. MC performed the cryobiopsy.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eTo T, Viegi G, Cruz A, Taborda-Barata L, Asher I, Behera D, et al. A global respiratory perspective on the COVID-19 pandemic: Commentary and action proposals. European Respiratory Journal. 2020 Jul 1;56(1).\u003c/li\u003e\n \u003cli\u003eHuang C, Huang L, Wang Y, Li X, Ren L, Gu X, et al. 6-month consequences of COVID-19 in patients discharged from hospital: a cohort study. The Lancet. 2023 Jun 17;401(10393):e21\u0026ndash;33.\u003c/li\u003e\n \u003cli\u003eVijayakumar B, Tonkin J, Devaraj A, Philip KEJ, Orton CM, Desai SR, et al. CT Lung Abnormalities after COVID-19 at 3 Months and 1 Year after Hospital Discharge. Radiology. 2022 May 1;303(2):444\u0026ndash;54.\u003c/li\u003e\n \u003cli\u003eVijayakumar B, Tonkin J, Devaraj A, Philip KEJ, Orton CM, Desai SR, et al. CT Lung Abnormalities after COVID-19 at 3 Months and 1 Year after Hospital Discharge. Radiology. 2022 May 1;303(2):444\u0026ndash;54.\u003c/li\u003e\n \u003cli\u003eKing CS, Mannem H, Kukreja J, Aryal S, Tang D, Singer JP, et al. Lung Transplantation for Patients With COVID-19. Chest [Internet]. 2022 Jan 1 [cited 2023 Aug 9];161(1):169\u0026ndash;78. Available from: https://pubmed.ncbi.nlm.nih.gov/34418410/\u003c/li\u003e\n \u003cli\u003eSchaller T, M\u0026auml;rkl B, Claus R, Sholl L, Hornick JL, Giannetti MP, et al. Mast cells in lung damage of COVID-19 autopsies: A descriptive study. Vol. 77, Allergy: European Journal of Allergy and Clinical Immunology. John Wiley and Sons Inc; 2022. p. 2237\u0026ndash;9.\u003c/li\u003e\n \u003cli\u003eXie C, Ya Likun MM, Luo Q li, Dong J cheng. Role of cellular senescence in inflammatory lung diseases. Cytokine Growth Factor Rev [Internet]. 2023 Apr 1 [cited 2023 Aug 9];70:26\u0026ndash;40. Available from: https://pubmed.ncbi.nlm.nih.gov/36797117/\u003c/li\u003e\n \u003cli\u003eCampisi J. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell [Internet]. 2005 Feb 25 [cited 2023 Aug 9];120(4):513\u0026ndash;22. Available from: https://pubmed.ncbi.nlm.nih.gov/15734683/\u003c/li\u003e\n \u003cli\u003eAghali A, Ngassie MLK, Pabelick CM, Prakash YS. Cellular Senescence in Aging Lungs and Diseases. Cells [Internet]. 2022 Jun 1 [cited 2023 Aug 9];11(11). Available from: https://pubmed.ncbi.nlm.nih.gov/35681476/\u003c/li\u003e\n \u003cli\u003eTorrance BL, Haynes L. Cellular senescence is a key mediator of lung aging and susceptibility to infection. 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Abnormal lymphangiogenesis in idiopathic pulmonary fibrosis with insights into cellular and molecular mechanisms. Proc Natl Acad Sci U S A [Internet]. 2009 Mar 10 [cited 2024 Mar 25];106(10):3958\u0026ndash;63. Available from: https://www.pnas.org/doi/abs/10.1073/pnas.0813368106\u003c/li\u003e\n \u003cli\u003eCoates AL, Wanger J, Cockcroft DW, Culver BH, Carlsen KH, Diamant Z, et al. ERS technical standard on bronchial challenge testing: General considerations and performance of methacholine challenge tests. European Respiratory Journal. 2017 May 1;49(5).\u003c/li\u003e\n \u003cli\u003eLoor K, Culebras M, Sansano I, \u0026Aacute;lvarez A, Berastegui C, de Gracia J. Optimization of Transbronchial Cryobiopsy in Lung Transplant Recipients. Ann Thorac Surg. 2019 Oct;108(4):1052\u0026ndash;8.\u003c/li\u003e\n \u003cli\u003eKuilman T, Michaloglou C, Mooi WJ, Peeper DS. The essence of senescence. Genes Dev [Internet]. 2010 Nov 15 [cited 2023 Mar 28];24(22):2463\u0026ndash;79. Available from: https://pubmed.ncbi.nlm.nih.gov/21078816/\u003c/li\u003e\n \u003cli\u003eMart\u0026iacute;nez-Zamudio RI, Robinson L, Roux PF, Bischof O. SnapShot: Cellular Senescence Pathways. Cell [Internet]. 2017 Aug 10 [cited 2023 Mar 28];170(4):816-816.e1. Available from: https://pubmed.ncbi.nlm.nih.gov/28802049/\u003c/li\u003e\n \u003cli\u003eMu\u0026ntilde;oz-Esp\u0026iacute;n D, Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol [Internet]. 2014 [cited 2023 Mar 28];15(7):482\u0026ndash;96. Available from: https://pubmed.ncbi.nlm.nih.gov/24954210/\u003c/li\u003e\n \u003cli\u003eTrivedi A, Reed HO. The lymphatic vasculature in lung function and respiratory disease. Front Med (Lausanne) [Internet]. 2023 [cited 2023 Aug 9];10. Available from: https://pubmed.ncbi.nlm.nih.gov/36999077/\u003c/li\u003e\n \u003cli\u003eMarchetti C, Poggi P, Clement MG, Aguggini G, Piacentini C, Icaro‐Cornaglia A. Lymphatic capillaries of the pig lung: TEM and SEM observations. Anat Rec [Internet]. 1994 [cited 2023 Mar 28];238(3):368\u0026ndash;73. Available from: https://pubmed.ncbi.nlm.nih.gov/8179218/\u003c/li\u003e\n \u003cli\u003ePusztaszeri MP, Seelentag W, Bosman FT. Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues. J Histochem Cytochem [Internet]. 2006 Apr [cited 2023 Mar 28];54(4):385\u0026ndash;95. Available from: https://pubmed.ncbi.nlm.nih.gov/16234507/\u003c/li\u003e\n \u003cli\u003eYamashita M, Iwama N, Date F, Chiba R, Ebina M, Miki H, et al. Characterization of lymphangiogenesis in various stages of idiopathic diffuse alveolar damage. Hum Pathol. 2009 Apr;40(4):542\u0026ndash;51.\u003c/li\u003e\n \u003cli\u003eKambouchner M, Bernaudin JF. Intralobular pulmonary lymphatic distribution in normal human lung using D2-40 antipodoplanin immunostaining. J Histochem Cytochem [Internet]. 2009 Jul [cited 2023 Mar 28];57(7):643\u0026ndash;8. Available from: https://pubmed.ncbi.nlm.nih.gov/19289553/\u003c/li\u003e\n \u003cli\u003eMatsui K, Nagy-Bojarsky K, Laakkonen P, Krieger S, Mechtler K, Uchida S, et al. Lymphatic microvessels in the rat remnant kidney model of renal fibrosis: aminopeptidase p and podoplanin are discriminatory markers for endothelial cells of blood and lymphatic vessels. J Am Soc Nephrol [Internet]. 2003 Aug 1 [cited 2023 Mar 28];14(8):1981\u0026ndash;9. Available from: https://pubmed.ncbi.nlm.nih.gov/12874451/\u003c/li\u003e\n \u003cli\u003eMandal R V., Mark EJ, Kradin RL. Organizing pneumonia and pulmonary lymphatic architecture in diffuse alveolar damage. Hum Pathol [Internet]. 2008 Aug [cited 2023 Mar 28];39(8):1234\u0026ndash;8. Available from: https://pubmed.ncbi.nlm.nih.gov/18602671/\u003c/li\u003e\n \u003cli\u003eEl-Chemaly S, Levine SJ, Moss J. Lymphatics in lung disease. Ann N Y Acad Sci [Internet]. 2008 [cited 2023 Mar 28];1131:195\u0026ndash;202. Available from: https://pubmed.ncbi.nlm.nih.gov/18519971/\u003c/li\u003e\n \u003cli\u003eEl-Chemaly S, Pacheco-Rodriguez G, Ikeda Y, Malide D, Moss J. Lymphatics in idiopathic pulmonary fibrosis: new insights into an old disease. Lymphat Res Biol. 2009;7(4):197\u0026ndash;203.\u003c/li\u003e\n \u003cli\u003eEl-Chemaly S, Malide D, Zudaire E, Ikeda Y, Weinberg BA, Pacheco-Rodriguez G, et al. Abnormal lymphangiogenesis in idiopathic pulmonary fibrosis with insights into cellular and molecular mechanisms. Proc Natl Acad Sci U S A [Internet]. 2009 Mar 10 [cited 2023 Mar 28];106(10):3958\u0026ndash;63. Available from: https://pubmed.ncbi.nlm.nih.gov/19237567/\u003c/li\u003e\n \u003cli\u003eSanchez-Ramirez DC, Normand K, Yang Z, Torres-Castro R. Long-Term Impact of COVID-19: A Systematic Review of the Literature and Meta-Analysis. Biomedicines [Internet]. 2021 Aug 1 [cited 2023 Mar 28];9(8). Available from: https://pubmed.ncbi.nlm.nih.gov/34440104/\u003c/li\u003e\n \u003cli\u003ePolak SB, Van Gool IC, Cohen D, von der Th\u0026uuml;sen JH, van Paassen J. A systematic review of pathological findings in COVID-19: a pathophysiological timeline and possible mechanisms of disease progression. Mod Pathol [Internet]. 2020 Nov 1 [cited 2023 Mar 28];33(11):2128\u0026ndash;38. Available from: https://pubmed.ncbi.nlm.nih.gov/32572155/\u003c/li\u003e\n \u003cli\u003eDhooria S, Chaudhary S, Sehgal IS, Agarwal R, Arora S, Garg M, et al. High-dose versus low-dose prednisolone in symptomatic patients with post-COVID-19 diffuse parenchymal lung abnormalities: an open-label, randomised trial (the COLDSTER trial). Eur Respir J [Internet]. 2022 Feb 1 [cited 2023 Mar 28];59(2). Available from: https://pubmed.ncbi.nlm.nih.gov/34887325/\u003c/li\u003e\n \u003cli\u003eMu\u0026ntilde;oz X, Pilia F, Ojanguren I, Romero-Mesones C, Cruz MJ. Is asthma a risk factor for COVID-19? Are phenotypes important? ERJ Open Res [Internet]. 2020 Nov 12 [cited 2020 Dec 19];00216\u0026ndash;2020. Available from: /pmc/articles/PMC7667724/?report=abstract\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 6 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Interstitial lung disease, lung fibrosis, SARS-CoV-2, cellular senescence, COVID-19 sequalae, lymphatic proliferation, cryobiopsy","lastPublishedDoi":"10.21203/rs.3.rs-4654047/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4654047/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn some patients post COVID-19 interstitial lung disease (ILD) sequelae persist beyond 6 months after hospital discharge. Little is known about the pathophysiology of this condition. This study aimed to determine the prognostic value of certain histopathological findings, cellular senescence, and lymphatic proliferation in patients with ILD sequelae.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis prospective observational study of patients hospitalized at Vall d’Hebron University Hospital due to COVID-19 pneumonia and presenting respiratory symptoms, radiological alterations, and pulmonary function test impairment during the 3-month follow-up visit after discharge. Lung cryobiopsies were performed, and the histopathological findings and expression of senescence and lymphatic proliferation (P16 and D2-40) were analyzed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetween March 2020 and February 2021, 4,332 patients were hospitalized at Vall d’Hebron University Hospital due to COVID-19 pneumonia, and 1,403 were visited in the Respiratory Clinic 3 months after discharge. The first 66 patients presenting with respiratory symptoms, radiological alterations, and decreased pulmonary function tests during the post hospitalization follow-up underwent cryobiopsy for diagnostic purposes. Multivariate regression showed that Masson bodies in the 3-month cryobiopsy were related to a higher forced vital capacity at 6 months whereas higher expression of senescence and lymphatic proliferation markers, such as P-16 and D2-40, in the histological samples were related to decreased carbon monoxide transfer test values at 6 months.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSenescence and lymphatic proliferation are related to worse pulmonary function outcomes in the mid-term follow-up of patients with post-COVID-19 ILD.\u003c/p\u003e","manuscriptTitle":"Prognostic Value of Cellular Senescence, Lymphatic Proliferation, and Histological Findings in the Follow-up of Post-covid-19 Interstitial Lung Disease Sequelae.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-29 18:19:19","doi":"10.21203/rs.3.rs-4654047/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c3bf8932-2243-47eb-b1e7-75d19e9a3b97","owner":[],"postedDate":"July 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-30T16:38:17+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-29 18:19:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4654047","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4654047","identity":"rs-4654047","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}