Pathological changes in the lungs and lymphatic organs of 12 COVID-19 autopsy cases

Abstract Systematic autopsy and comprehensive pathological analyses of COVID-19 decedents should provide insights into the disease characteristics and facilitate the development of novel therapeutics. In this study, we report the autopsy findings from the lungs and lymphatic organs of 12 COVID-19 decedents—findings that evaluated histopathological changes, immune cell signature and inflammatory factor expression in the lungs, spleen and lymph nodes. Here we show that the major pulmonary alterations included diffuse alveolar damage, interstitial fibrosis and exudative inflammation featured with extensive serous and fibrin exudates, macrophage infiltration and abundant production of inflammatory factors (IL-6, IP-10, TNFα and IL-1β). The spleen and hilar lymph nodes contained lesions with tissue structure disruption and immune cell dysregulation, including lymphopenia and macrophage accumulation. These findings provide pathological evidence that links injuries of the lungs and lymphatic organs with the fatal systematic respiratory and immune malfunction in critically ill COVID-19 patients.

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INTRODUCTION
Coronaviruses are non-segmented positive-sense RNA viruses that are constantly transmitted from animals to humans [1]. Although most coronaviruses cause mild respiratory diseases in humans, the epidemics of severe acute respiratory syndromeassociated coronavirus (SARS-CoV) and the Middle East respiratory syndrome-associated coronavirus (MERS-CoV) totaled over 10 000 cumulative cases in the past two decades, with mortality rates of 10% for SARS-CoV and 37% forMERS-CoV [2,3]. Since the end of 2019, COVID-19, caused by a novel coronavirus SARS-CoV-2, has caused a global pandemic [2,3]. Although COVID-19 is generally an acute self-limited disease featured with pneumonia, approximately 15% of COVID-19 patients, especially those of an older age and with preexisting medical conditions, can rapidly develop fatal C The Author(s) 2020. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. systematic conditions, such as acute respiratory distress syndrome and severe cardiovascular or renal injuries, among which 5%-6% of cases may progress into critical status with high mortality [4,5].
Thus, the virological, epidemiological and clinical characteristics of COVID-19 have been extensively investigated and the clinical features profiled. However, COVID-19-associated pathological alterations and the underlying translational relevance are still to be elucidated. Recent investigations through full autopsy or minimally invasive autopsy of COVID-19 decedents by us and other laboratories [6][7][8][9][10][11][12][13][14][15][16], revealed that SARS-CoV-2 hijacked angiotensin converting (Continued from previous page) enzyme 2 (ACE2) for entry into target cells [17] to cause severe pathologic changes in the lungs and multiple extrapulmonary organs/tissues. While clinical observations supported that most of the critically ill COVID-19 patients manifested severe immune disorders [4,5], which might result in damages to the lungs and multiple extrapulmonary organs, the changes of immune cell signature in the lungs and lymphatic organs such as the spleen and lymph nodes have not been fully investigated. To profile pathological changes underlying the severe respiratory distress syndrome and immune dysfunction in critically ill COVID-19 patients, we performed systematic autopsy and comprehensive pathological analyses of 12 deceased patients with COVID-19. Our findings provide new evidence on pathological changes associated with COVID-19 and suggest clinical approaches against the disease.

Clinical characteristics of the COVID-19 autopsy cases
A total of 12 autopsy cases in this study included seven males (58.3%) and five females (41.7%). The age of the patients was between 51 and 88 years (mean: 70.6 years/median: 72.5 years). All 12 cases had been diagnosed with SARS-CoV-2 infection with pneumonia-like symptoms and abnormalities on chest images when admitted (

Pulmonary immune signature
Immunohistochemical staining showed the SARS-CoV-2 spike protein located in alveolar and bronchial epithelia from the lungs of COVID-19 cases, but not in control pulmonary tissues from a patient who died of heart attack ( Fig. 2A and  B). Serial sectioning further demonstrated that the SARS-CoV-2 spike protein and ACE2 were co-expressed in alveolar and bronchial epithelia of the COVID-19 cases ( Fig. 2A and B), confirming that ACE2-expressing epithelial cells could be the target of SARS-CoV-2 in the lungs. Investigation of pulmonary immune cell signature showed that in contrast with the minor infiltration of CD4-or CD8-positive T lymphocytes or CD20-positive B lymphocytes in alveolar space or the lung interstitial compartment (Fig. 2C), extensive infiltration of CD68-positive macrophages was found in the areas with diffuse alveolar damage in COVID-19  patients (Fig. 2C). Interleukin 6 (IL-6), interferon gamma-induced protein 10 (IP-10), tumor necrosis factor (TNF) α and interleukin 1β (IL-1β) implicated as inflammatory factors associated with COVID-19 progression were detected in the alveoli with diffuse immune cell infiltration (Fig. 2D).

Pathological changes in the spleen and hilar lymph nodes
Since a number of severe COVID-19 patients manifested symptoms of immune disorders and lymphopenia, we investigated the pathological changes in lymphatic organs including the spleen and hilar lymph nodes in autopsy cases. The postmortem spleens were generally contracted with shrinking capsules (Fig. 3A). Mixed thrombi, anemic infarction and hemorrhage were found in the contracted spleens ( Fig. 3B and C). Moreover, COVID-19 spleens showed atrophic white pulp and relatively enlarged red pulp (Fig. 3D). Lymphoid follicles in white pulp were decreased or absent as compared to control spleens from patients with abdominal trauma necessitating splenectomy (Fig. 3E-G). The proportion of CD20-positive B lymphocytes was reduced in COVID-19 spleens (100%) compared to the control trauma spleens (Fig. 3H-J). Some of the COVID-19 spleens showed decreased CD3-positive T lymphocytes and Ki67-positive cells relative to the control trauma spleens ( Fig. 3K-P). Focal lymphocyte apoptosis and macrophage enrichment were noted in the COVID-19 spleens, which might contribute to the lymphocyte reduction. The predominant changes in the hilar lymph nodes were mildly to moderately expanded subcapsular sinuses infiltrated with CD68-positive macrophages in COVID-19 cases in comparison with the control lung carcinoma cases without tumor metastasis into hilar lymph nodes (Fig. 4A-D). The superficial cortex and paracortex in COVID-19 cases were generally not identifiable with different proportions of necrotic and apoptotic lymphocytes. Interstitial vessels were congested in some COVID-19 cases. The proportions of CD3-positive T lymphocytes and CD20positive B lymphocytes in the lymph node cortex had no apparent changes (Fig. 4E-H).

DISCUSSION
COVID-19 patients have been reported to manifest distinct clinical manifestations, disease courses and heterogeneous pathological alterations. Most published papers include limited numbers of COVID-19 autopsy cases. Herein, we systematically evaluated the pathological changes in the lungs and lymphatic organs from 12 COVID-19 autopsy cases. We combined macroscopic and microscopic examinations of the lungs, spleen and lymph nodes, and determined immune cell signature and inflammatory factor production in the lungs. As major targets of SARS-CoV-2, the lungs of COVID-19 decedents showed lesions of diffuse alveolar damage, interstitial fibrosis and exudative inflammation with Immune cells infiltrated in alveoli extensive serous and fibrin exudates, macrophage infiltration and abundant production of inflammatory factors. SARS-CoV-2 and ACE2 were co-localized in the alveoli and bronchioles. The spleen of COVID-19 decedents manifested lymphocyte reduction but macrophage enrichment. Our work together with previous literature provides a comprehensive overview of histopathological changes in COVID-19 patients, implying that acute respiratory distress syndrome, multiple organ injuries, comorbidities like immune and coagulation disorders, as well as underlying diseases could result in the death of the criticial ill COVID-19 patients [8][9][10][11]13,14,16,18,19]. In addition, most of the cases in our cohort were deceased elder patients comparable to published COVID-19 biopsy/autopsy reports [10,16]. Comorbidities in our cohort were also consistent with clinical observational studies [20]. Thus, the corroboration of pathological findings with analyses of the   serological and clinical data enables a better evaluation of COVID-19 disease progression and outcome.

RESEARCH ARTICLE
A recent study confirmed that SARS-CoV-2 uses coronavirus spike glycoproteins to bind ACE2 for cell entry [21]. Our findings revealed a close association between SARS-CoV-2 infection and ACE2 expression in the lungs, supporting that ACE2-positive cells are targeting cells for SARS-CoV-2 in humans. In agreement with our findings, SARS-CoV-2 infection in ACE2-positive pneumocytes may cause diffuse alveolar damage, hyaline membranes and inflammatory cell infiltration, serving as the pathological basis of blood-air barrier disruption and the generation of lethal hypoxemia. Further investigations are warranted to evaluate the systematic distribution and organotropism of SARS-CoV-2, as well as its association with COVID-19 disease progression and patient outcome.
Lymphopenia has been known as a common manifestation in COVID-19 patients [4]. The decreased proportion of lymphocytes in the spleen shown in our study suggests that SARS-CoV-2 infection may impair the survival of lymphocytes and disrupt lymphocyte-mediated immune reaction. We propose that the following mechanisms may contribute to lymphopenia in COVID-19: (i) the engulfing of lymphocytes by macrophages is frequently observed in the spleen in the postmortem COVID-19 specimens, suggesting that macrophage-mediated phagocytosis may contribute to lymphopenia; (ii) the increase of proinflammatory factors, which has been implicated in disrupting the maintenance and proliferation of lymphocytes, is a common event during COVID-19 progression [22,23]. The use of glucocorticoid in COVID-19 patients may cause immune suppression and lymphocyte reduction. In contract with the reduced proportion of lymphocytes, the proportion of macrophages was significantly increased in alveolar space and the spleen, frequently observed in COVID-19 autopsy cases [18]. These results provide pathological evidence for the development of therapeutics aimed at promoting lymphopoiesis and neutralizing pro-inflammatory signals to prevent cytokine storm in severe COVID-19 patients.

Autopsy cases and control specimens
The autopsy cases in this study were the first consecutive 12 cases from Jinyintan Hospital, Wuhan, China. All decedents met the diagnostic criteria for COVID-19 and the presence of SARS-CoV-2 in all autopsy cases was confirmed by polymerase chain reaction (PCR) tests. Basic patient information and clinical data were obtained from electronic medical records from Jinyintan Hospital, Wuhan, China as summarized in Table 1. Pulmonary tissues from a patient who died from sudden heart death were used as control for comparison with the lungs from COVID-19 patients. Spleens from patients with blunt or penetrating abdominal trauma necessitating a splenectomy were used as control tissues for comparison with the spleens from COVID-19 patients. The control hilar lymph nodes were derived from lung carcinoma patients receiving hilar lymph node dissection.

Autopsy procedures
The study was approved by the ethics committee of Jinyintan Hospital, Wuhan, China, and was performed with written consent from patient family members in accordance with the regulations issued by the National Health Commission of China and the Helsinki Declaration. All autopsy procedures were performed in an isolated workspace with adequate negative pressure ventilation in accordance with the Provisional Guidelines on Autopsy Practice for Deaths Associated with COVID-19 in China [19,24]. All staff members had been adequately trained before autopsy examination and were equipped with appropriate personal protective equipment during the practice. None of the staff members involved in this study developed COVID-19. To minimize autolysis, decedents were promptly stored at 4 • C after death and the range of the postmortem interval (time of death to time of autopsy) was 4-24 hours. Autopsy materials were collected, fixed in 4% neutral formaldehyde for at least 24 hours and sampled as formalin-fixed, paraffin-embedded (FFPE) tissues for histopathological analyses and SARS-CoV-2 RNA detection.

Statistical analysis
All statistical analyses were performed using SPSS (Statistical Package for the Social Sciences) version 13.0 software (SPSS Inc.). Categorical variables were described as frequency rates and percentages. Continuous variables were described using the mean and median values. The means for continuous variables were compared using independent group t tests when the data was normally distributed. For unadjusted comparisons, a 2-sided α of less than .05 was considered statistically significant.