key: cord-0962890-74c8egr5
authors: Middleton, Elizabeth A.; Zimmerman, Guy A.
title: COVID-19-Associated Acute Respiratory Distress Syndrome: Lessons from Tissues and Cells
date: 2021-05-26
journal: Crit Care Clin
DOI: 10.1016/j.ccc.2021.05.004
sha: 28050191e1db9337313d0642714f5975777bd925
doc_id: 962890
cord_uid: 74c8egr5

Reports examining lung histopathology in COVID-19 infection provide an essential body of information for clinicians and investigators. SARS-CoV-2-induced lung injury is complex, involving the airways, alveoli, and pulmonary vessels. Although no anatomic marker is specific the signature histologic lesion is diffuse alveolar damage (DAD). The biologic and molecular mechanisms that drive this pattern of injury are unknown, and the relationship of SARS-CoV-2-induced DAD to physiologic alterations and clinical outcomes in COVID-19-associated acute respiratory distress syndrome is undefined. Additional histologic patterns that may be variant phenotypes have been reported. These are key issues for future clinical and experimental research.

Patients with coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) frequently develop acute, precipitous respiratory failure with features of the Acute Respiratory Distress Syndrome (ARDS) (1) . Understanding the biology, mechanisms of acute lung injury, and pathophysiology of COVID-19-associated ARDS is essential for its rational management (1, 2) , but these elements are largely uncharacterized.

ARDS is a common, complex, and lethal syndrome that is caused by a spectrum of infectious J o u r n a l P r e -p r o o f and non-infectious insults (3, 4) . COVID-19-associated ARDS may be, in large part, similar to ARDS of other etiologies (5, 6) . Alternatively, it may have novel features and it is possible, and perhaps likely, that COVID-19-associated ARDS represents a unique phenotype (7, 8) . It is also possible that there is biologic and physiologic heterogeneity and that there are sub-phenotypes of COVID-19-associated ARDS, as there are in classical ARDS (a term that will be used to indicate ARDS unrelated to COVID-19) (2, 4, 7) .

Histopathology and clinical cell biology are fundamental to understanding human diseases and for elucidation of their mechanisms and physiologic consequences, and may be critical for precise characterization of new or emerging syndromes. For the clinician, description of the anatomic pathology provides correlates for interpretation of imaging and other diagnostic measures, understanding pathophysiology, and formulating therapeutic strategies; for the translational investigator, human histopathology is a basis for devising "reduced" experimental models and for evaluating outcomes in surrogate in vivo experiments; for medical scientists new to a field, histopathology can provide an unbiased sense of complexity of a disease and insights regarding the cellular and molecular issues that underpin it. Linking pathologic patterns to clinical variables can be particularly informative (9) . In this article we profile available information on the pathology of SARS-CoV-2 pneumonia, focusing on histology and cellular characterization and lessons and questions that these studies provide regarding COVID-19associated ARDS.

Overview of the Pathology of SARS-CoV-2 Pneumonia J o u r n a l P r e -p r o o f Current synthesis of the pathology of SARS-CoV-2 pneumonia is based on reports of autopsies, more limited postmortem sampling, surgically-excised lung tissue, and cytologic analysis.

Multiple studies of documented SARS-CoV-2 infection from Asia, Europe, and the U.S. have appeared. Early cases and case series are summarized in reviews (10) (11) (12) (13) . We also discuss findings reported in selected early and more recent primary reports.

Macroscopic features of the lung in COVID-19 are non-specific and include edema, hemorrhage, and thrombosis (12) . Lung weights are substantially increased. In an international report of 68 autopsies, the combined lung weight was > 1,300g (normal average 840g) in 92% of cases (14) .

In a smaller series the mean weight of lungs from subjects with COVID-19 (1,681 ± 49g) was greater than that of uninfected control lungs (1,045 ± 91g) but lower than that of patients dying of influenza-associated ARDS (2,404 ± 560g) (15) . Macroscopic involvement is frequently patchy but there can be extensive consolidation, corresponding to a spectrum of patterns on diagnostic imaging (11, 12, 16) .

In early case reports and series the most commonly reported histologic finding, by far, was diffuse alveolar damage (DAD) (10) (11) (12) (13) , including cellular features of the acute, exudative, and proliferative, or organizing, phases (Box 1; Figure 1 ). More recent autopsy series, some multicenter involving relatively large numbers of patients, extend early findings and frequently also emphasize pulmonary vascular involvement and apparent temporal evolution of acute lung injury (14, 15, (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) . Tracheobronchial injury and inflammation, independent of intubation and mechanical ventilation, has been documented in addition to alveolar involvement (14, 30) . The conclusion is that SARS-CoV-2 pneumonia is a complex respiratory disorder involving the tracheobronchial, alveolar, and vascular compartments (11, 12, 14) .

Evidence for viral infection of tracheobronchial and alveolar epithelial cells by ultrastructure, immunohistochemistry, or in situ hybridization has been a consistent finding in COVID-19; detection of viral particles or markers in endothelial and immune cells has been reported in some, but not all, studies (11-15, 22, 23, 28, 30, 32, 33-35) . Angiotensin-converting enzyme 2 (ACE2), a requisite component of the molecular system by which SARS-CoV-2 enters host cells, was detected on alveolar epithelial and endothelial cells (15) . Persistent viral infection may drive ongoing focal alveolar injury and clinical manifestations (14, 23) but is absent in the organizing phase of DAD in some subjects (32) .

Histologic patterns that vary from the dominant DAD phenotype have been reported (10, 11, 14, 22, 25, (33) (34) (35) including acute fibrinous and organizing pneumonia (AFOP) (10, 36) . They may be sentinel subsets that indicate biologic heterogeneity, potentially underlying clinical heterogeneity (2) .

In addition to the respiratory system, multiple studies have examined other organs and tissues.

While we do not review extra-pulmonary features of SARS-CoV-2 infection here they indicate, along with clinical manifestations and circulating biomarkers, that SARS-CoV-2 infection is frequently a systemic syndrome (1, (10) (11) (12) (13) 16) .

There are a number of caveats regarding current reports of the pathology of COVID-19. The lung tissue examined to date has largely been from elderly subjects, frequently with a variety of comorbidities, consistent with the well-known susceptibility to severe SARS-CoV-2 infection and increased mortality in this population. Common features of the lung pathology could therefore be due in part to responses of the aged, multiply -compromised lung. Observations in non-J o u r n a l P r e -p r o o f human primates indicate that DAD occurs across the age spectrum and in otherwise healthy lungs in response to SARS-CoV-2 infection, however (see below). A second issue is that most of the lung tissue from patients with COVID-19 examined so far is from patients that expired well after onset of the clinical illness, often after days or weeks of medical intervention and ICU support. Thus, the early patterns of lung involvement in SARS-CoV-2 infection in humans are not known. Tissue from patients undergoing lung resection while in the undiagnosed, apparently early, phase of COVID-19 infection provides useful but limited information (37) (38) (39) .

Experimental animal models (40) will likely be essential in addressing this unknown feature.

Experimental animal studies will also likely be the only avenue immediately available for determining patterns of histologic and cellular evolution in SARS-CoV-2-induced acute lung injury, although molecular imaging or histology-specific biomarkers (21, 31) may be informative in humans in the future. Finally, virtually no patients in reports of COVID-19 lung histopathology have been ascertained to have ARDS according to the Berlin definition (3, 4, 9) or other consensus criteria, although relevant clinical and physiologic data are available for some. Therefore, it is currently necessary to extrapolate existing histopathologic findings to COVID-19associated ARDS.

DAD: The signature but nonspecific histologic pattern of classical ARDS and COVID-19 lung injury.

DAD is a specific constellation of histologic features that defines a characteristic but nonspecific pattern of response to acute or sub-acute lung injury (17) (Box 1; Figure 1 ). It is the signature pathologic lesion and is central in current concepts of ARDS, although not all patients with J o u r n a l P r e -p r o o f classical ARDS have DAD on autopsy or diagnostic lung biopsy examination and the physiologic syndrome of ARDS can be associated with other histologic patterns (3, 4, 9, 21) . The presence of hyaline membranes is a requisite criterion for DAD diagnosis, and other histologic features are used to determine evolutionary stages of the response (21) .

As noted previously, DAD is a consistent, almost ubiquitous, finding when lungs from decedents with COVID-19 are examined (10-15, 22-32, 35) . DAD is also a key component of the acute lung injury induced by coronaviruses in Middle East respiratory syndrome (MERS-CoV) and severe acute respiratory syndrome (SARS-CoV) infection (11, 12, 24) . DAD in lungs of SARS-CoV-2-infected patients could not be differentiated from DAD in acute lung injury induced by other insults when early case reports and case series were reviewed (12) .

A focused study concluded that DAD in COVID-19 infection is morphologically indistinguishable from DAD of other causes (29) . This determination was based on consensus blinded analysis of histopathology of lungs from hospitalized patients with SARS-CoV-2 infection, SARS-CoV-2infected patients who died in the community, and historic hospitalized and outpatient controls previously determined to have DAD induced by sepsis, lung infection, or other conditions, including hospitalized controls assessed to have clinical ARDS. The findings were interpreted as indicating that DAD -without distinctive histologic features that differentiate it from DAD of other etiologies -is the primary manifestation of COVID-19-associated lung injury in subjects who die in the hospital or in the community. A corollary was that mechanical ventilation and high inspired oxygen concentration are not primary drivers of the histologic changes (29) . Both supportive interventions induce the DAD phenotype (3, 17) .

Correlation of the histologic pattern of DAD with physiologic data and clinical variables in classical ARDS has been informative (9, 21, 41, 42) . Evolution of histologic features of DAD in patients with classical ARDS suggests a time-dependent set of biologic events that alter physiologic variables and might be modified by time-sensitive interventions (41) . Cellular features indicating evolution of acute lung injury appear to also correlate with duration of disease in COVID-19 (14, 22, 28, 32) . A recent histologic and transcriptomic analysis extending findings in one of the early major COVID-19 autopsy series suggests distinct phases of immune pathology -varying in interferon -stimulated genes, cytokines, viral load, and cellular injuryand natural progression culminating in DAD (31) . If verified, this may provide a basis for timing of administration of targeted therapeutics in clinical trials and, ultimately, in practice.

Evaluation of the impact of DAD on physiologic variables and response to supportive measures in COVID-19-associated ARDS (5, 6) requires future study.

Examination of the complex relationship between DAD and classical ARDS indicates that the presence of DAD influences clinical outcomes (21) . ARDS with DAD detected by open lung biopsy is associated with higher mortality than is ARDS in the absence of DAD (43) . This may also be true of COVID-19-associated ARDS, but it is unclear if lung biopsy will be widely utilized in COVID-19 because of risk of viral dissemination and other concerns, underscoring the potential benefits of biomarkers and molecular imaging techniques that reflect this histologic pattern (21, 31) .

Although DAD in COVID-19 and DAD in acute lung injury of other etiologies are indistinguishable by light microscopy (12, 29) , they may be quite different at the molecular J o u r n a l P r e -p r o o f level. Future analysis of clinical lung tissue and BAL samples, and samples from in vivo and in vitro experimental models, may reveal unique biochemical and cellular features that influence evolution of the alveolar injury or its resolution and repair (4).

Leukocytes are a common feature of DAD (Box 1), classically as a prominent interstitial infiltrate (17) . Inflammatory cells also concentrate in microvessels in numbers greater than those seen in the normal lung, suggesting intravascular activation and sequestration, and accumulate in the alveolar spaces in some cases (4, 18, 19) .

In COVID-19-associated DAD, perivascular and interstitial mononuclear cell infiltrates of variable intensity composed primarily of CD4+ and CD8+ lymphocytes were commonly reported in early studies and were assessed to be consistent with viral infection (10) (11) (12) (13) . Macrophages may have particular activities that drive acute lung injury early in the evolution of SARS-CoV-2 infection (31, 37-39, 44, 45) . Limited early reports of bronchoalveolar lavage (BAL) sampling also indicated predominant lymphocytic and mononuclear cell inflammation, including plasma cells and macrophages (46) (47) (48) . Although characteristic of viral infection, mononuclear leukocyte infiltrates are also found in acute lung injury of other etiologies including oxygen toxicity (17) and malaria-associated ARDS (49) .

"Angiocentric" accumulation of lymphocytes in the alveolar perivascular interstitium and microvessels was observed in lung tissue from patients with COVID-19 (15) and SARS-CoV-2infected primates (45) . CD68 + macrophages and an activated T cell signature correlated with J o u r n a l P r e -p r o o f DAD in a detailed study of lungs from COVID-19 decedents involving histology, immunophenotyping, and transcriptomics (31) . The activities and mechanisms of accumulation of alveolar lymphocytes in COVID-19 are unknown; they are suggested to contribute to both viral clearance and acute lung injury based on histologic and blood analysis (31, 50) . In influenza infection signaling interplay between lymphocyte subsets, injured alveolar cells, and fibroblasts influence immune cell accumulation and lung dysfunction, suggesting that similar events may occur in COVID-19 (51) . Circulating platelet-lymphocyte and platelet-monocyte aggregates were increased in the blood of COVID-19 patients compared to samples from control subjects (52, 53) , indicating one potential mechanism of lung accumulation of mononuclear leukocytes and demonstrating interaction of key immune effector cells (54) .

Myeloid leukocyte subsets may also have critical effector activities in COVID-19 pneumonia (44, 47, 55) . Neutrophils (polymorphonuclear leukocytes, PMNs) are hallmarks of classical ARDS induced by a variety of common infectious and non-infectious triggers (20) , but their involvement in SARS-CoV-2 lung infection is unclear. Neutrophils were infrequently identified in early COVID-19 autopsies, surgical cases, and BAL samples (12, 38, 39, 46, 48) . They were much less frequently detected by quantitative scoring in alveoli of COVID-19 patients than in the lungs of patients dying of influenza (15) . Nevertheless, neutrophils were present in lung tissue from some subjects with COVID-19 (10, 12, 14, 22, (33) (34) (35) (36) (37) 56) . One possibility is that neutrophils in these samples were indicators of superimposed bacterial pneumonia, and it was suggested that they are not effector cells in the primary inflammatory response to SARS-CoV-2 since they are not typically found in the lung in uncomplicated viral infection (12) . Autopsy evidence (4, (17) (18) (19) 21) . Endothelial dysfunction, with endothelial cells injured but largely intact by ultrastructural examination, contributes to increased alveolar capillary permeability to protein and interstitial and alveolar edema in the exudative phase of DAD; alterations in the endothelial network can be dramatic in the later proliferative phase (18, 19) . Thrombosis of pulmonary vessels is a common feature of both the exudative and proliferative phases. In systematic studies of lung vessels by postmortem angiography and histology correlated with antemortem balloon arteriography, vascular occlusion was detected in as many as 95% of cases. By microscopic examination platelet-fibrin thrombi were commonly observed in alveolar capillaries and arterioles in acute ARDS, and platelet-fibrin thrombi in microvessels and laminated fibrin clots in pre-acinar and intracinar arteries were detected across the time line spectrum. The pulmonary vascular bed was found to be extensively remodeled in later proliferative and fibrotic phases of ARDS in lungs from some patients (19) .

Vasculopathy is also a component of acute lung injury in COVID-19, and it is suggested that SARS-CoV-2 induces a distinct vascular endotype of ARDS (8) . Pulmonary vascular injury was identified in 16/23 early reports of acute lung injury in COVID-19 (11) . In a quantitative histologic study that also employed micro-CT, ultrastructural analysis, and molecular assays, J o u r n a l P r e -p r o o f lungs from decedents with COVID-19 respiratory failure or ARDS secondary to H1N1 influenza were equally likely to have thrombi in precapillary pulmonary arteries, but alveolar-capillary microthrombi were 9 times more prevalent in patients with COVID-19. Structurally-deformed capillaries with evidence for intususseptive angiogenesis were also significantly more frequent in the lungs of subjects with COVID-19 compared to those with influenza, although the number of patients in each group was relatively small (15) . In a different analysis platelet and/or fibrin microthrombi were identified in lungs from 84% of 68 patients with COVID-19, a number of whom had large vessel pulmonary thrombi and extra-pulmonary thrombotic involvement (14) . "Endotheliitis" (also termed "endothelialitis") has been identified in lungs from patients with COVID-19 (15, 33) and SARS-CoV-2-infected rhesus macaques (45) . The term implies endothelial injury and dysfunction with a component of perivascular inflammation, but there is not yet a consensus definition or rigorous determination if it is commonly present in COVID-19 pneumonia or represents a unique histologic sub-phenotype. Endothelial damage, swelling, and vacuolization demonstrated by light and electron microscopy have been attributed to endothelial cell infection by SARS-CoV-2 via ACE2, but in some studies viral particles or antigen were detected in alveolar epithelial cells but not in capillary endothelium (11, 15, 22, 24, 32, 33, 36, 56) . Perivascular leukocyte involvement has been reported as lymphocytic angiocentric inflammation (15, 45) , lymphocytic and myeloid cell accumulation in different patients (33) , intravascular fibrin deposition with septal accumulation of neutrophils (34), and neutrophilic "capillaritis" (22, 60) . In one report focal microthrombi were found, but no histologic evidence J o u r n a l P r e -p r o o f of endotheliitis (56) . Perivascular inflammation did not reliably separate COVID-19 from other causes of DAD in a comparative analysis (29) . Thus, there are many questions related to pulmonary endotheliitis in COVID-19 to be resolved. In addition, there are multiple fundamental issues to be explored. For example, it may be critical to know if SARS-CoV-2induced acute lung injury differentially affects recently-described alveolar endothelial cell subtypes (64) . Direct contributions of injured endothelial cells to other facets of alveolar damage and inflammation (Figure 1) , and to viral containment versus spread, are also yet to be examined.

Thrombocytopenia, which is common and often profound in critically-ill patients with classical ARDS (49) , is generally mild in COVID-19 although it can be progressive in non-survivors (65) .

Platelets have dual roles as hemostatic and immune effector cells in the lung and other tissues (54) , and have thromboinflammatory activities in ARDS (49) . Aggregates of platelets and platelet-fibrin thrombi have been commonly observed in microvessels in SARS-CoV-2-infected lungs (10, 14, 24-26, 62, 63) , and molecular signatures associated with platelet activation, aggregation, and adhesion were detected in infected macaques (45) . Altered platelet reactivity was detected in mild and severe COVID-19 infection (52, 53, 62) . Platelet-neutrophil aggregates, a marker of platelet activation (54) , were observed in COVID-19 lungs and blood (35, 53, 62) . Platelets trigger NETosis and are effectors of pathologic clotting (49, 60, 65) and may contribute to NET formation and immunothrombosis in COVID-19 (61, 62, 66) (see above).

Megakaryocytes, the precursors of platelets, are present in normal and injured lungs and generate platelets in the pulmonary compartment (reviewed in 49). Megakaryocytes, including J o u r n a l P r e -p r o o f cells actively producing platelets, were detected in lung microvessels of decedents of COVID-19 (10, 26) . Two autopsy series that included patients dying of COVID-19 or of classical ARDS found increased numbers of megakaryocytes in the pulmonary vasculature of subjects with COVID-19

infection, although only one comparison reached statistical significance (63, 67) .

Megakaryocyte transcript signatures were increased in blood from patients (68) and macaques (45) infected with SARS-CoV-2. It is unknown if megakaryocytes in the lungs of patients with COVID-19 have anti-SARS-CoV-2 activities that may influence COVID-19 infection, as with other viruses (69) . Conversely, novel functions of megakaryocytes may contribute to acute lung injury or repair in COVID-19 (49) . Circulating megakaryocytes with a strong interferon-associated molecular signature were one of three cell types reported to be hallmarks of severe COVID-19

and were linked to inflammatory markers in plasma (68) .

Dysregulated systemic hemostasis may contribute to pulmonary vasculopathy in COVID-19 (65) .

As markers, elevation of plasma D-dimer concentration has been widely-reported, and increased thrombin generation and relative impairment in fibrinolysis have been demonstrated in severe COVID-19 (6, 65, 70, 71) . Increased circulating platelet-monocyte aggregates were found in blood samples from patients with severe COVID-19 pneumonia, particularly those requiring mechanical ventilation. Platelets from these patients induced tissue factor expression by monocytes in vitro (52) , suggesting a mechanism for pulmonary and systemic thromboinflammation (54) .

The casual links between deranged coagulation and macro-and microthrombosis in COVID-19

have not been elucidated (65) , establishing key priorities for basic and clinical investigation. As Pulmonary coinfection in COVID-19 pneumonia: A common phenotype Microbial co-infection is common in COVID-19-induced acute lung injury, based on autopsy series in which the frequency of suspected or documented bacterial or fungal coinfection, usually termed superimposed bronchopneumonia or superinfection, was 13 -79% (11, 14, 23-25, 27, 30, 32, 35, 63) . The histologic pattern has been described as dense accumulations of neutrophils in alveoli and airways, alveolar hemorrhage, and vascular congestion on a background of DAD with lymphocytic interstitial infiltrates (27) . Special stains, microbial cultures and molecular techniques have been used to confirm histologic findings and identify a variety of non-COVID-19 pathogens (14, 27, 30) although in some reports the diagnosis was made exclusively on the pattern of neutrophilic bronchopneumonia with DAD and, in others, coinfection was not detected (26) . Frequent coinfection was also reported in a recent study of blood and airway myeloid leukocyte subsets in patients with severe COVID-19 lung involvement (55) . For comparison, coinfection was present in 26% of 100 fatal cases of pandemic influenza A; DAD was a ubiquitous underlying histologic pattern (75) . In contrast, coinfection was not frequently reported in the SARS and MERS pandemics (30) .

In classical ARDS, DAD, acute neutrophilic pneumonia due to bacterial or fungal pathogens, and DAD together with acute neutrophilic pneumonia are each distinct histologic phenotypes that carry physiologic significance and have different clinical outcomes (21) . This may also be true for COVID-19-associated ARDS, an issue that should be addressed in future clinical and experimental investigations. Application of metagenomic techniques will also be revealing.

Animal models: experimental correlates and consistent histopathology Specific findings in primate models of SARS-CoV-2 lung infection are mentioned above.

Although overt clinical signs do not develop short-term, histopathology in cynomolgus macaques is consistent with that in human COVID-19 pneumonia (Figure 1) . Foci of pulmonary consolidation were found in 2 of 4 animals (young and aged) four days after infection with SARS-CoV-2 isolated from a human subject (57) . The major histologic changes included areas of acute lung injury with features of exudative and proliferative DAD, indicating that this pattern of response is a facet of the biology of SARS-CoV-2 lung infection that can develop rapidly and supporting the conclusion that DAD is not exclusively secondary to respiratory therapy measures (29) . DAD histopathology was also observed in animals infected in parallel with MERS-CoV for comparison. SARS-CoV-2 antigen was detected in AEC I and AEC II in areas of DAD, a proximity that suggested that SARS-CoV-2 infection drives evolution of the DAD histologic pattern (57) . Similarly, SARS-CoV-2 antigen and viral RNA were detected in focal areas of DAD in human COVID-19 pneumonia (14, 32) .

Histologic changes of DAD also occur in rhesus macaques infected with SARS-CoV-2. Hyaline membranes, alveolar edema, fibrin deposition, interstitial infiltrates and AEC II hyperplasia J o u r n a l P r e -p r o o f were observed in multifocal areas of involvement 3 days after infection (59) . In a second study, hyaline membranes were present together with interstitial and alveolar inflammatory infiltrates and edema two days after infection (58) . In a study to examine age as a variable, histologic findings were generally similar but the specific components of interstitial inflammation and edema were more severe in aged rhesus macaques than in younger animals 7 days after SARS-CoV-2 infection (76) . In cynomolgus macaques, age did not affect the histologic pattern at an early time point but aged animals shed SARS-CoV-2 longer after infection (57) .

Additional animal models of COVID-19 have been developed, including experimental infection of rodents (40) . Severe disease has been reported in some, depending on the animal species, genetic background, and conditions of viral challenge (40, (77) (78) (79) . To date, studies of mice have largely described alveolar interstitial infiltrates and edema, recapitulating some but not all of the histologic determinants of DAD (Figure 1) . Acute lung injury with hyaline membranes and other DAD features were reported in two recently-described murine models (77, 79) .

While the rapidly expanding repertoire of primate and small animal models will provide correlates of human COVID-19 and experimental infrastructure for mechanistic studies and for evaluation of therapeutics and vaccines, none yet developed is a faithful surrogate for human COVID-19 -associated ARDS (40) . As existing models are further refined and others developed for this purpose lung histology will be a critical variable, as it has been in experimental acute lung injury of other etiologies (80) . A recently-reported model in standard laboratory mouse strains infected with a murine-adapted SARS-CoV-2 was interpreted to have features of ARDS based on assessment of acute lung injury and DAD by histologic scoring (77) , although J o u r n a l P r e -p r o o f oxygenation and other key determinants of ARDS (3, 4, 80) were not examined. The severity of injury was greater in BALB/c than in C57/BL6 mice.

In January of 2020 nothing was known of the anatomic basis for respiratory failure caused by the novel SARS-CoV-2 virus. Since that time examination of lung tissue from patients with COVID-19 has yielded a body of information with clinical and basic relevance, demonstrating the unique value of analysis of pathologic anatomy in emerging and re-emerging infectious diseases (11) . The findings provide an initial foundation for understanding pathophysiologic features of COVID-19-associated ARDS (1, 2, 5, 6, 16) and priorities for future investigations.

It is clear that SARS-CoV-2 infection can cause injury from the tracheobronchial epithelium to the pleura (10) (11) (12) (13) . Injury to the alveoli characterized by DAD is central to COVID-19 respiratory failure, consistent with the histopathology of severe infections caused by other respiratory viruses including SARS-CoV, MERS-CoV, and influenza (11, 12, 17, 75) . Features of DAD were also prominent in lungs of patients reported in the original description of ARDS, several of whom were thought to have fatal viral pneumonia (81) . Pulmonary vascular and microvascular involvement is common in COVID-19 pneumonia and may have unique features. DAD and its microvascular component are histologic counterparts for altered compliance, dead space, and oxygenation and for perfusion and other imaging abnormalities in subjects with COVID-19associated ARDS (6) . Further study may provide additional useful insights, as it has in classical ARDS (19, 21, 41, 42) . While the common histopathologic and cellular features of established COVID-19 acute lung injury are becoming clear, the early events in alveolar damage, the J o u r n a l P r e -p r o o f temporal progression to DAD, and the cellular and molecular mechanisms involved are obscure.

Further definition of these features may have therapeutic significance. Current anatomic findings with clinical correlates suggest that pathways inherent to both the pathogen and the host contribute to COVID-19-associated ARDS (12, 15, 31, 82, 83) .

Findings from examination of COVID-19 lung tissue raise many additional questions and priorities for investigation, some of which we've identified previously, and potential controversies. The frequency, mechanisms, and physiologic significance of histologic patterns that vary from established DAD (10, 11, 14, 22, 25, 27, (33) (34) (35) (36) are open questions that need to be resolved. The specific issue of "endotheliitis", which is currently based on a relatively small number of diverse observations, needs further definition. The extent to which alveolar microvascular involvement is greater, or not different, compared to that of classical ARDS (15, 29) , potential SARS-CoV-2-specific mechanisms of alveolar vasculopathy (15, 45, 79) , and whether COVID-19-associated ARDS is a distinct vascular endotype of ARDS (8) each merit additional examination. Similarly, the specific contributions of neutrophils, NETs, platelets, and megakaryocytes to the pathogenesis of SARS-CoV-2 pneumonia and COVID-19-associated ARDS, in the broader context of activities of key immune effector cells, are unresolved issues with possible therapeutic relevance. Histopathologic analysis amplified by the tools of modern biology and molecular immunology (15, 31) , in parallel with rigorous animal models (40) and reduced experimental approaches including isolated cell-based assays and organoid preparations, has the potential to provide needed answers and contribute to vetting of emerging hypotheses (84) . Such insights may then refine our understanding of the place of COVID-19 acute lung injury in the broader spectrum of ARDS (2-4, 7-9). DAD was originally characterized as a nonspecific response to acute or sub-acute alveolar injury incited by a variety of insults, alone or in combination. Specific histologic features identifying early "exudative" and later "proliferative" phases, often progressing to extensive interstitial fibrosis, were described (17) (see Figure 1 ). Ultrastructural analysis confirmed and extended observations by light microscopy (18, 19) . Hyaline membranes, alveolar epithelial cell Type I (AEC I; Type I pneumocyte) injury and loss, interstitial and alveolar edema, and interstitial and alveolar inflammatory infiltrates are key histologic features of the acute exudative phase. The inflammatory infiltrate was originally described to be mononuclear (17) , but neutrophils are prominent in classical DAD of many causes (4, 20) . Fibrin thrombi are common in alveolar capillaries and pulmonary arterioles. Platelets are frequently detected in microvascular thrombi, especially when specific markers are utilized (17) (18) (19) 21) . Intraalveolar fibrin, hemorrhage, and cellular debris are variably seen. Ultrastructural studies reveal denuded alveolar epithelial basement membrane and cellular details of epithelial and endothelial injury (18, 19) . The organizing, proliferative phase is indicated by alveolar epithelial cell type II (AEC II; Type II pneumocyte) hyperplasia and by interstitial fibrosis that can be severe (17) (18) (19) .

Currently, hyaline membranes are considered the required feature for pathologic diagnosis of DAD in classical ARDS, and the other elements of the histologic pattern are used to establish its evolutionary stage (21) . 

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Early histologic findings of pulmonary SARS-CoV-2 infection detected in a surgical specimen

Pulmonary pathology of early-phase 2019 novel Coronavirus (COVID-19) pneumonia in two patients with lung cancer

Pulmonary pathology of early-phase COVID-19 pneumonia in a patient with a benign lung lesion

Animal models for COVID-19

Chronology of histological lesions in acute respiratory distress syndrome with diffuse alveolar damage: a prospective cohort study of clinical autopsies

Comparison of the Berlin definition for acute respiratory distress syndrome with autopsy

The presence of diffuse alveolar damage on open lung biopsy is associated with mortality in patients with acute respiratory distress syndrome: a systematic review and meta-analysis

Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages

Vascular disease and thrombosis in SARS-CoV-2-infected rhesus macaques

Exuberant plasmocytosis in bronchoalveolar lavage specimen of the first patient requiring extracorporeal membrane oxygenation for SARS-CoV-2 in Europe

Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19

Bronchoalveolar lavage findings in severe COVID-19 pneumonia

Amicus or adversary revisited: platelets in acute lung injury and acute respiratory distress syndrome

Immunological and inflammatory profiles in mild and severe cases of COVID-19

Exuberant fibroblast activity compromises lung function via ADAMTS4

Platelet activation and plateletmonocyte aggregate formation trigger tissue factor expression in patients with severe COVID-19

Platelet gene expression and function in patients with COVID-19

Platelets in pulmonary immune responses and inflammatory lung diseases

COVID-19 severity associates with pulmonary redistribution of CD 1C + DCs and inflammatory transitional and nonclassical monocytes

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model

SARS-CoV-2 infection protects against rechallenge in rhesus macaques

Respiratory disease in rhesus macaques inoculated with SARS-CoV-2

Targeting potential drivers of COVID-19: neutrophil extracellular traps

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Immunothrombotic dysregulation in COVID-19 pneumonia is associated with respiratory failure and coagulopathy

Megakaryocytes and platelet-fibrin thrombi characterize multi-organ thrombosis at autopsy in COVID-19: a case series

Capillary cell-type specialization in the alveolus

Coagulation abnormalities and thrombosis in patients infected with SARS-CoV-2 and other pandemic viruses

Complement and tissue factorenriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis

Increased number of pulmonary megakaryocytes in COVID-19 patients with diffuse alveolar damage: an autopsy study with clinical correlation and review of the literature

Longitudinal multi-omics analyses identify responses of megakaryocytes, erythroid cell, and plasmablasts as hallmarks of severe COVID-19

Human megakaryocytes possess intrinsic antiviral immunity through regulated induction of IFITM3

COVID-19 and sepsis are associated with different abnormalities in plasma procoagulant and fibrinolytic activity

COVID-19-associated coagulopathy: biomarkers of thrombin generation and fibrinolysis leading the outcome

Complement activation in patients with COVID-19: A novel therapeutic target

Complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition

pandemic influenza A (H1N1): pathology and pathogenesis of 100 fatal cases in the United States

Age-related rhesus macaque models of COVID-19

A mouse-adapted SARS-CoV-2 induces acute lung injury and mortality in standard laboratory mice

Ad26 vaccine protects against SARS-CoV-2 severe clinical disease in hamsters

COVID-19 treatments and pathogenesis including anosmia in K18-hACE2 mice

An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals

Acute respiratory distress in adults

Uncontrolled innate and impaired adaptive immune responses in patients with COVID-19 acute respiratory distress syndrome

Biological Mechanisms of COVID-19 acute respiratory distress syndrome

Circuits between infected macrophages and T cells in SARS-CoV-2 pneumonia