Neutrophil Inltration Induces Myocardial Injury In COVID-19 Post-Mortem Cases

Objectives: The pathological features of severe cardiac injury induced by COVID-19 and relevant clinical features is unknown. Methods: This autopsy cohort study, including hearts from 26 deceased patients hospitalized in intensive care unit due to COVID-19, was conducted at four sites in Wuhan, China. Cases were divided into neutrophil-inltration group and no-neutrophil group according to histopathological identication of neutrophilic inltrates or not. Results: Among 26 cases, four cases had active myocarditis with histopathological examination. All cases with myocarditis accompanied with extensive neutrophil inltration, while cases without myocarditis did not. Detection rates of interleukin-6 (100% vs 4.6%) and tumor necrosis factor-a (100% vs 31.8%) in neutrophil-inltration group were signicantly higher compared to no-neutrophil group (p<0.05 for both). At admission, patients with neutrophil inltration in myocardium had signicantly higher baseline values of aspartate aminotransferase, D dimer and high-sensitivity C reactive protein compared to other 22 patients (p<0.05 for all). During hospitalization, patients with neutrophil inltration had a signicantly higher maximum of creatine kinase (CK)-MB (median 280.0 vs 38.7IU/L, p=0.04), and a quantitatively higher top Troponin I (median 1.112 vs 0.220ng/ml, p=0.56) than patients without neutrophil inltration. Conclusions: In hearts from deceased patients with severe COVID-19, active myocarditis was commonly inltrated with neutrophils. Cases with neutrophil-inltrated myocarditis had a series of severe abnormal laboratory tests at admission, and a high maximum of CK-MB during hospitalization. Role of neutrophil on severe heart injury and even systemic condition in COVID-19 should be emphasized.


Introduction
Coronavirus disease 2019 (COVID-19) outbreaks caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still repeatedly and intermittently occur around the whole world. Although COVID-19 is mainly characterized by the infection of lung and respiratory failure, cardiac injury with troponin elevation was demonstrated to be associated with the increased mortality (1,2). Several postmortem autopsy studies showed that the injury of heart tissue and cardiomyocytes including myocardial necrosis were common and non-speci c, but the rate of pathology-con rmed myocarditis was low (3,4). In addition, despite found in heart tissue, the presence of SARS-CoV-2 via reverse transcriptionpolymerase chain reaction (RT-PCR) was rarely detected in cardiomyocytes, which failed to qualify virus direct invasion as a primary cause of cardiac injury (5,6). To date, the de nite mechanism of pathological changes in heart induced by COVID-19 is still unclear.
A proportion of patients with COVID-19 was progressed to critically ill cases, and had a signi cantly higher mortality (7,8). Especially with the current rapid spread of Delta and Omicron variants, an increasing trend of severe cases with worse prognosis emerges (9). Critically ill patients experienced a long stay in intensive care unit (ICU), and were more prone to develop multiple organ dysfunction syndrome including heart (10,11), which may produce signi cant histological and immunological changes. Therefore, we intended to conduct a post-mortem pathological study among critically ill COVID-19 patients, to describe pathological features of hearts and explore the relationship between these changes and clinical characteristics.

Study population and specimen disposal
This autopsy cohort study included 26 deceased patients from Huoshenshan Hospital (n=8), Taikang Tongji Hospital (n=5), Zhongfaxincheng Hospital (n=5), and Wuhan Jinyintan Hospital (n=8), China during February 18th to April 4th, 2020. Patient hospitalization information has been described in our previous study (12). Brie y, all 26 patients were con rmed with COVID-19 by nasopharyngeal or pharyngeal PCR analyses of SARS-CoV-2 RNA and were hospitalized in ICU. Full autopsy was performed after patient death with the approval of the ethics committees and written consent of patient relatives in accordance with regulations issued by the National Health Commission of China and the Helsinki Declaration.
To minimize autolysis, decedents were promptly stored at 4℃ after death and the range of the postmortem interval (time of death to time of autopsy) was 4-24 hours. Autopsy materials were collected, xed in 4% neutral formaldehyde for at least 24 hours and sampled as formalin-xed, para n-embedded tissues for histopathological analyses.

Pathological Analysis
Autopsies of hearts were performed by two experienced pathologists and tissues at ventricles, atriums and epicardial coronary arteries were respectively collected for further analyses. A median of 25 fullthickness blocks of myocardium were examined histologically (range 11-40 blocks). The pathological changes of hearts were evaluated using hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) staining. H&E staining was performed according to the standard procedure. IHC staining was performed on routine automated diagnostic immunohistochemical staining devices (Roche, BenchMark-ultra). Myocarditis was de ned as microscopic ndings of multiple foci of increased leukocyte in ltration associated with myocyte injury, which was not due to some other cause(3). Number of myocardium-in ltrating mononuclear cells per mm 2 in high-power eld was counted in each sample with the most in ammation using IHC staining for CD4 (Zhongshan Jinqiao, #ZM-0418), CD8 (Zhongshan Jinqiao, #ZA-0508), CD20 (Zhongshan Jinqiao, #ZM-0039) and CD68 (Zhongshan Jinqiao, #ZM-0060). Primary antibodies used for IHC staining includes interleukin-6 (IL-6, Abcam, ab6672, 1:600) and tumor necrosis factor alpha (TNF-α, Cell Signal Technology, #8184, 1:20). Images were captured using a digital camera (DP73, Olympus) under a light microscope (BX43, Olympus). The diluent without primary antibodies was used as negative control for IHC staining.

Statistical Analysis
Clinical characteristics, laboratory tests, echocardiography results, complications during hospitalization, medications and invasive procedures of each patient were abstracted from hospitalization records and other forms of information. Time from syndrome onset to hospitalization were also collected. For laboratory tests including cardiac markers and in ammatory indicators, the baseline values at admission and maximum values during hospitalization were recorded.
Continuous variables were presented as mean ± standard deviation (SD) or median with range if nonparametric. Categorical data were presented as count with percentages (%). To identify the correlation between pathological ndings and clinical characteristics, the Kendall's tau-b index for bivariate correlation analysis was conducted. A 2-sided p value < 0.05 was considered to be statistically signi cant. All the statistical analyses were completed via IBM SPSS version 25 and R packages version 3.6.1.

Role of the funding source
Supported by Emergency Key Program of Guangzhou Laboratory, Grant No. EKPG21-32.

Results
A total of 26 patients admitted in ICU due to COVID-19 were included in this pathological study. General patient characteristics and main death causes were published elsewhere (12). In brief, the median age of study cohort was 68 years (range 53-88), and 50.0% (13 patients) were male. Median duration in ICU until deaths was 20 days (range 3-61). 20 patients had at least one comorbidity, including 10 with chronic cardiovascular diseases (including three coronary artery disease, one dilated cardiomyopathy, two valvular heart disease, one arrhythmia and three cardiac dysfunction), nine with hypertension, four with diabetes, and six with chronic pulmonary diseases. Most of 26 patients died of pulmonary injuries related to COVID-19.
Heart failure (HF) occurred in 10 (38.5%) patients. Atrial brillation (AF) was documented among six (23.1%) patients, which was the main type of new-onset arrhythmias during hospitalization. Due to the serious illness, various complications emerged during hospitalization in ICU, including respiratory failure, pleural effusion, pneumothorax, anemia, renal dysfunction and disseminated intravascular coagulation (DIC). 18 patients received anticoagulation treatment, and two patients received antiplatelet therapy. Also, multiple invasive procedures including non-end-stage endotracheal intubation, ventilator, deep vein puncture, bronchoscopy, dialysis and extracorporeal membrane oxygenation were intermittently or continuously used in critical situations. Treatment information was listed in Supplemental Materials (Table S1).

Pathological Findings
Active myocarditis with neutrophil in ltration As described in our previous work, a series of common pathological changes of hearts were found in all 26 patients, including myocardial cell degeneration and scattered necrosis, mild interstitial oedema and in ltration of monocytes, and lymphocytes and/or neutrophils (13). There were also cardiomyocyte hypertrophy, atrophy, and interstitial brosis of varying degrees based on underlying diseases. After morphological analysis of more heart tissue blocks, active myocarditis was found in only four (15.4%) cases. Surprisingly, neutrophilic in ltrates were detected in all four cases of myocarditis: diffuse neutrophilic in ltrates associated with adjacent cardiomyocyte degeneration or necrosis, involving bilateral ventricles and atriums, was found in rst two cases of myocarditis, the second case was accompanied with obvious myocardial interstitial oedema ( Figure 1A and 1B); the third and fourth cases of active myocarditis showed multiple small discrete foci of mixed in ammatory cells with visible neutrophils and mild lymphocytes associated with single-cell necrosis of cardiomyocytes, involving the left ventricle and atrium ( Figure 1C and 1D). Whereas the rest 22 cases without active myocarditis showed a small in ltration of scattered mononuclear cells rather than neutrophils in the myocardial interstitium. To further explore the severity and property of in ammation in myocardium, IHC staining was performed to detect the expression of TNF-α and IL-6 proteins in all cases. Positive expression of TNF-α and IL-6 in in ltrated in ammatory cells and myocardial interstitial cells was found in all four cases with neutrophil in ltration ( Figure 1E-L), while cases without neutrophil in ltration showed negative or mild expression of these in ammation related factors ( Figure 1M-P). Cases with neutrophil in ltration showed higher ratios of TNF-α (+) and IL-6 (+) than those in cases without neutrophil in ltration (TNF-α (+): 100% vs 31.8% (seven in 22 cases), P = 0.022; IL-6 (+): 100% vs 4.6% (one in 22 cases), P < 0.001, Table 1).
We further analyzed the immunologic characteristics of myocardium-in ltrating mononuclear cells using IHC staining for markers CD4 (marked helper T lymphocytes), CD8 (marked cytotoxic T lymphocytes), CD20 (marked B lymphocytes) and CD68 (marked monocytes and macrophages). All four cases with active myocarditis were dispersedly in ltrated of very mild CD4+, CD8+, CD20+ lymphocytes and single or small clusters of CD68+ macrophages. And this pattern was also seen in all other cases without active myocarditis ( Figure 2). The number of each subtype cells per mm 2 in high-power eld was counted and presented in Table 1. No signi cant difference of CD4+, CD8+, CD20+ or CD68+ cell density was found between cases with active myocarditis and cases without active myocarditis (P > 0.05 for all, Table 1).
Kendall's tau-b index showed that no signi cant correlation was found between number of each subtype of cell density and positive detection of TNF-α or IL-6 (P > 0.05 for all). Other various types of pathologic ndings were shown in Supplemental Materials ( Figure S1). Two of the four cases with neutrophil in ltration were found to have neutrophil-predominant endocarditis. Dilated cardiomyopathy with tricuspid valve infective endocarditis occurred in one case without neutrophil in ltration. Epicarditis with focal in ltration of mixed in ammatory cells occurred in three cases with neutrophil in ltration and in seven cases without neutrophil in ltration. Mixed thrombi were found in four cases without neutrophil in ltration, including one in left atrium, one in right atrium and two in right ventricle. Epicardial coronary arteriosclerosis was also found in nine of 26 cases, including the one case with neutrophil in ltration. There was no thrombotic occlusion or endarteritis of epicardial coronary in all 26 cases. Intravascular microthrombi in myocardial interstitial were observed under microscope in 12 (46.2%) cases, including all four cases with neutrophil in ltration and other eight cases without neutrophil in ltration. The detection rate of cardiac microthrombi in cases with neutrophil in ltration was signi cantly higher than that in cases without neutrophil in ltration (100% vs 36.4%, P = 0.02, Table 1).

Clinical characteristics of neutrophil in ltration group versus no-neutrophil in ltration group
In order to observe the dynamic changes of clinical characteristics in 26 deceased patients, baseline characteristics at admission, and the maximum values of a series of laboratory tests which re ected the severe medical conditions of patients, were both collected. Baseline characteristics including parameters of cardiac injury, in ammation, coagulation and liver function at admission were presented in Table 2. The median time from symptom onset to hospital admission in neutrophil in ltration cases was 20.5 days (range 13-26), which was signi cantly longer than that (10 days, range 1-24) cases without neutrophil in ltration (P = 0.02). In comparison with cases without neutrophil in ltration, cases with neutrophil in ltration in heart tissues had a signi cantly higher baseline value of aspartate aminotransferase (AST), D dimer or high-sensitivity C reactive protein (hsCRP) (P = 0.05 for all, Table 2). In terms of baseline cardiac markers, creatine kinase (CK) in cases with neutrophil in ltration was signi cantly higher than that in cases without neutrophil in ltration (median 277.5 (range 91.0-486.0) IU/L vs 36.0 (11.5-547.0) IU/L, P = 0.03), while CK-MB and brain natriuretic peptide (BNP) were similar between two groups (P > 0.05 for both, Table 2). However, baseline hypersensitive Troponin I (hsTnI) was available on only 16 (61.5%) of 26 patients. 10 (38.5%) patients were not examined for hsTnI until transferred into ICU (six patients) or presenting with symptoms indicating HF or AF (four patients).  Maximum values of laboratory tests during hospitalization were also compared between two groups ( Table 2) Cases with neutrophil in ltration had a quantitatively higher peak value of hsTnI during hospitalization compared to cases without neutrophil in ltration, although without statistical signi cance (median 1.112 (range 0.008-7.775) ng/ml vs 0.220 (0.008-8.749) ng/ml, P = 0.56). Other main laboratory parameters were comparable between two groups ( Table 2). HF occurred in one of the four cases with neutrophil in ltration, and AF occurred only in six cases without neutrophil in ltration.

Discussion
Since the pandemic of COVID-19 around the world, a considerable proportion of patients with COVID-19 developed into critically ill cases, and often experienced multiple organ failure (MOF) including not only lung but heart and other organs (14,15). To explore the speci c pathological changes of heart, we conducted this autopsy study of hearts from 26 critically ill patients who died of COVID-19 in Wuhan from February to April 2020 (whom could be deemed as the earliest series of severe COVID-19 cases all over the world). This study mainly reported that: 1) Although with a low rate, active myocarditis was commonly and speci cally accompanied with neutrophil in ltration; 2) The positively IHC detection rates of cytokines including TNF-α and IL-6 were signi cantly higher in cases with neutrophil in ltration compared to cases without neutrophil in ltration, but was not correlated with the extent of lymphocyte and macrophage in ltration; 3) Clinical data showed that cases with neutrophil in ltration had a signi cantly prolonged time from syndrome onset to hospitalization, and higher baseline levels of a series of laboratory tests including CK, AST, hsCRP and D dimer compared to cases without neutrophil in ltration at hospital admission. While almost 50% of 26 patients did not examine cardiac biomarkers until the exacerbation of illness; 4) In terms of the peak values of laboratory tests during hospitalization, cases with neutrophil in ltration had a signi cantly higher level of CK-MB, and a quantitatively higher level of hsTnI than those in cases without neutrophil in ltration.
Role of neutrophil: a new notion of COVID-19 related myocarditis?
In comparison with the dramatic pathological changes in lung (16,17), microscopic ndings from heart in COVID-19 were not intensive and unspeci c: previous post-mortem studies found various pathological manifestations, most of those focused on the necrosis, myocarditis, in ammatory in ltration and brin microthrombi (3,4,18). Compared with other autopsy studies, the scattered necrosis cardiomyocytes in our study were more common, probably because patients in our autopsy were all severe cases and had a signi cantly longer stay in ICU, which greatly increased risks of cardiac injury. Similar to already existed ndings (19), a small proportion of cases was found to have active myocarditis which indicated more severe injury of hearts. Surprisingly, neutrophil in ltration was found in all the four cases with myocarditis in our study, but was rarely found in cases without myocarditis. A series of trials have demonstrated that neutrophil in ltration into pulmonary tissues prominently caused the deterioration of COVID-19 (20)(21)(22)(23).
In ltrated neutrophils could release neutrophil extracellular traps (NETs), which are extracellular networks of chromatin and microbicidal proteins in response to SARS-CoV-2 infection, while excessive activation of NETs simultaneously results in lung cell deaths in critically ill patients (24)(25)(26)(27). NETs derived from neutrophils are responsible for multiple causes pathophysiological changes including microthrombi, angiotensin-converting enzyme 2 (ACE2) activity and oxidative stress (28,29). Moreover, NETs are identi ed to be correlated with cytokine storms: various cytokines could medicate the migration of neutrophil to injury sites (30); reversely, the generation of NETs subsequently stimulates the aggravation of cytokine storms such as Il-6 via IL-1β (20,31,32). In the present post-mortem study, the positive detection rates of IL-6 and TNF-α were signi cantly higher in four cases with neutrophil in ltration than those in the other 22 cases without neutrophil in ltration by IHC staining. Although the identi cation of NETs was not conducted, results from our study indirectly con rmed precious ndings, and to some extent identi ed the strong association of neutrophil and severe cardiac injury.
In all 26 cases, the presence of SARS-CoV-2 nucleic acids were found in only 5 heart tissues by real-time RT-PCR, which was reported previously (12). Interestingly, none of these 5 cases had pathologically diagnosed myocarditis. A pathological autopsy study from 39 deceased COVID-19 patients also reported the localization of virus infection was in interstitial cells or in ltrated macrophage in hearts, rather than cardiomyocytes (33). Also, other pathological studies from endomyocardial biopsy (EMB) or autopsy rarely reported direct invasion of SARS-CoV-2 into cardiomyocytes (34). Current evidences still fail to determine the key role of SARS-CoV-2 infection on cardiac injury, while in ammatory in ltration was now regarded as a preliminary cause of heart damage in severe COVID-19. The Dallas Criteria(35) only according to histological evidences has already been not fully suitable for diagnosis of myocarditis, and IHC analysis for in ammatory in ltration was particularly advocated (36). A comparison of CD3+ T cells and CD68+ macrophages between COVID-19 cases and noninfectious control cases were conducted in a state-of-art review (34). No signi cant difference of the total number of CD3+ and CD68+ cells was observed between two groups, while the number count of CD68+ cells was signi cantly higher in COVID-19 group than that in control group (34). Our study also found that the CD68+ macrophages were single or clustered in myocardium, but no difference of the number count of CD68 cell was found between cases with and without active myocarditis. Moreover, each case with myocarditis was in ltrated with neutrophils accompanied with distinct cytokines, which was not prevalent in cases without myocarditis. Therefore, based on previous ndings on association of neutrophil in ltration and critically ill COVID-19, we reasonably reckon that neutrophil and relevant in ammatory in ltration may be an essential cause of devastating heart damage into myocarditis. Some researchers believed glucocorticoid could act an immunomodulatory role on inhibiting cytokine storm and excessive immune response for purpose of improving therapeutic effect on critically ill patients (37,38). Besides, other speci c cytokine inhibitors, including IL-6 receptor inhibitor tocilizumab is being explored (39,40). Role of neutrophil in ltration on severe cardiac injury deserves more special attention in COVID-19.

Cardiac Biomarkers: A Warning Sign Of Severe Covid-19?
Several studies evaluating risk factors of poor prognosis in COVID-19 identi ed a series of laboratory factors may be predictors of in-hospital mortality, including AST, D dimer and hsCRP (41,42). Signi cant elevation of the baseline levels of these three parameters were also found in patients with neutrophil in ltration in hearts from our study. According to our ndings, a link may exist between a relatively severe situation (involving liver function, coagulation and in ammation) and pathological changes of heart tissues, indicating that COVID-19 could simultaneously cause damage of multiple organs although initiated in respiratory system. From our data, however, nearly 50% patients were not tested for cardiac biomarkers at admission until they were transferred to ICU or presented with relevant symptoms. This result may re ect that, under an emergent situation, inspections on heart may be easily neglected by physicians who mainly focused on treatment strategy on respiratory system. Furthermore, we found that cases with neutrophil in ltration had a relatively worse situation both on admission and during ICU stay. The peak level of CK-MB among the whole duration of hospitalization was signi cantly higher in cases with neutrophil in ltration than that in cases without neutrophil in ltration. Peak value of hsTnI was also found to be quantitatively higher in cases with neutrophil in ltration, although without statistical difference due to limited sample size.  Figure 1 Representative histological and IHC ndings from hearts Figure 1 shows the histological and IHC ndings from heart tissues. Figure   showed there was a scattered in ltration of very mild CD4+, CD8+, CD20+ lymphocytes and single or small clusters of CD68+ macrophages. This pattern was also seen in other two cases without myocarditis. The arrows denote the lymphocytes or macrophages. Scale bars represent 50 μm. IHC: immunohistochemical.

Supplementary Files
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