Evidence of Autoinflammation as the Principal Mechanism of Myocardial Injury in SARS-CoV-2 PCR-Positive Autopsy Hearts

doi:10.21203/rs.3.rs-2703054/v1

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Evidence of Autoinflammation as the Principal Mechanism of Myocardial Injury in SARS-CoV-2 PCR-Positive Autopsy Hearts

https://doi.org/10.21203/rs.3.rs-2703054/v1

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Background

Disease from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains the third leading cause of death in the United States, after cancer and heart disease. Many patients infected with this virus develop cardiovascular complications including myocardial infarctions, stroke, arrhythmia, heart failure, and sudden cardiac death. Specifically, patients with SARS-CoV-2 have a high prevalence of severe myocardial injury (20–28%). The purpose of this study is to understand the primary mechanism of myocyte injury in patients infected with SARS-CoV-2.

Methods

We investigated a consecutive cohort of 84 medical examiner cases who died with PCR-positive SARS-CoV-2 (COVpos) infection prior to availability of therapy or vaccines. We compared them to a consecutive cohort of 42 age- and sex-matched controls who were PCR-negative for SARS-CoV-2 (COVneg). Formalin-fixed paraffin embedded sections of left and right ventricle were examined on each case using antibodies directed against CD42 (platelets), CD15 (myeloid cells), CD68 (monocytes), C4d, Fibrin, CD34 (stem cell antigen), CD56 (natural killer cells), and Myeloperoxidase (MPO) (neutrophils and NETs). Slides were scanned using an Aperio slide scanner and viewer and each digital slide was entirely examined at 5x,10x and 20x. Each slide was graded using a 0–3 scale where 3 indicates the marker was present in every field at 20x. We used a Welch 2-sample T-test to determine significance.

Results

We found a significant difference between COVpos and COVneg samples for all markers, all of which were significant at p < 0.001. The most prominent features were neutrophils (CD15, MPO) and MPO positive debris suggestive of NETS and were located in or around arterioles, venules, and capillaries. A similar distribution of platelets, monocytes, and C4d was seen in COVpos cases. Fibrin was found scattered in arterioles, venules, interstitial regions, and within ventricular cavities. CD34 highlighted vascular alterations of endothelial cells in some but not all cases.

Conclusion

Autoinflammation is the primary mechanism of myocyte injury observed in COVpos hearts. The significant increase in platelets, monocytes, and neutrophils and the presence of neutrophil NETs, C4d, and fibrin suggest that myocardial injury involves neutrophils, NETosis, coagulation, complement activation, and monocyte accumulation.

SARS-COV-2

COVID-19

innate immunity

heart

autoinflammation

neutrophils

inflammasome

heart

myocarditis

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) caused a global pandemic. As of December 29, 2022, 6,656,601 people died among 651,918,402 confirmed cases worldwide (1). Disease from SARS-CoV-2 remains the third leading cause of death in the United States, after cancer and heart disease (2). Many patients infected with this virus develop cardiovascular (CV) complications including myocardial infarctions, stroke, arrhythmia, heart failure, and sudden cardiac death. Specifically, patients with SARS-CoV-2 have a high prevalence of severe myocardial injury (20–28%) (2–4). The mechanism of cardiac injury in patients dying of SARS-CoV-2 after severe respiratory illness followed by cardiovascular complications is controversial. Direct viral infection as well as various indirect causes have been postulated (2–7). A recent study comparing 153,760 US SARS-C0V-2 infected veterans to a large cohort of normal controls showed the risk of cardiovascular outcomes hazard ratio for any prespecified cardiovascular outcomes regardless of acute presentation was 1.63 ± 0.09 (2). We hypothesized that innate immune activation (autoinflammation) and its consequences is the primary cause of cardiovascular disease in SARS-CoV-2 patients. We used immunohistochemistry and routine histology to assess our hypothesis.

Population

We investigated a consecutive sample of 100 PCR-positive SARS CoV-2 (COVpos) patients who underwent postmortem examination in 2020. This series of cases was collected prior to the availability of SARS-CoV-2 treatments or vaccines. Most of these patients were examined by the medical examiner because of death at home. The controls consisted of a contemporaneous consecutive population of 44 age- and sex-matched individuals who were PCR-negative for SARS CoV-2 (COVneg) and died during 2020.

Immunohistochemical Analysis

Formalin-fixed paraffin-embedded serial 4 micron thick sections of left and right ventricles were examined from each case using antibodies directed against CD42 (platelets), CD15 (myeloid cells), CD68 (monocytes and macrophages), CD 56 (natural killer cells), CD34 (stem cell antigen found on endothelial cells), C4d, fibrin, and myeloperoxidase (neutrophils and neutrophil extracellular traps (NETs). Hematoxylin and Eosin (H&E) stained sections were also examined. Samples were stained as a batch (with one stain and controls) using automated platforms and standardized reagents. A table of details is available on request. The stained slides were scanned using whole slide imaging (Aperio Techologies, Vista, CA). Each slide was accessed on an Aperio web viewer. Each slide was entirely viewed at 5x, 10x, and 20x. Slides were graded using a 0–3 scale where 3 indicated the marker was present in every field at 20x and 1 indicated the marker was present in any field at 10x. Grade 2 indicated that some fields at 20x showed staining, but many did not. The tissue distribution of the marker was also noted. All slides were examined by a single observer (MEHH). At review, it was noted that several cases were autolyzed and could not be included. Excluding these, we analyzed 84 COVpos and 42 COVneg cardiac samples. Areas of left and right ventricular tissue were similarly selected from each heart.

Statistical Analysis

We used a Welch 2-sample T-test to determine significance between COVpos and COVneg samples and adjusted for multiple comparisons using the False Discovery Rate method. (8, 9)

We found a significant difference between the COVpos and COVneg case for each of the markers examined. See Fig. 1.

Histologic Features

Careful review of H&E-stained slides of each case showed no cases of lymphocytic myocarditis according to established histologic criteria (10). No inflammation associated with individual cell myocyte necrosis was seen in any case. In some COVpos cases, small aggregates of mononuclear cells were seen, but few if any of these cells were lymphocytes. Scattered large ischemic myocardial areas were seen associated with typical inflammation in some cases. Fibrin aggregates were noted scattered in the myocardium of many COVpos cases (in and around capillaries, arterioles and venules). In many cases, fibrin clots were seen in arterioles and venules or within ventricular cavities. COVneg cases showed only rare fibrin aggregates and no significant intracavitary fibrin aggregates.

Immunohistochemical analysis (See Fig. 2, A-F)

We found a significant difference between COVpos and COVneg samples in the amount and pattern of staining for fibrin, CD42, CD68, CD15, MPO, and C4d. Figure 1 shows the differences in these markers between COVpos and COVneg cases. All differences were statistically significant (p < 0.001). There was no significant difference between the left and right ventricles for amount or pattern of staining in COVpos or COVneg cases (intracase variability was low). Among the COVpos cases, only 11/84 (13%) had no marker staining or H&E evidence of fibrin thrombi. The mean number of positive markers in the COVpos cases was 3.70 ± 1.74 markers/case. Among the COVneg cases, 20/42 (47%) had no marker staining or H&E evidence of fibrin thrombi. The mean number of positive markers in the COVneg cases was 0.95 ± 1.09 markers/case. Comparing the number of positive markers between COVpos and COVneg samples resulted in a statistically significant difference (p < 0.001)

CD15 (cells of myelogenous origin, monocytes): Fig. 2A

Abundant CD15 was found in arterioles, venules and capillaries in many COVpos cases, often associated with interstitial cells. In some cases, CD15 positive cells lined the endothelium resembling the CD42 (platelet) distribution. Areas of myocyte ischemia in some hearts had large deposits of CD15 positive cells associated with necrotic myocytes and debris. Some similar staining was found in COVneg hearts, in areas of previous myocyte damage. Mean grade for COVpos = 1.56 ± 0.91; Mean grade for COVneg = 0.98 ± 0.97, p < 0.001

MPO: Fig. 2B

MPO staining in COVpos cases was distributed in arterioles, venules, and scattered in the interstitium like CD15 staining. Interstitial staining suggests neutrophils, NETs (8). No similar staining was seen in COVneg cases. Mean grade for COVpos = 1.496 ± 1.052; Mean grade for COVneg = 0.550 ± 0.654, p < 0.001

CD 68 (macrophages, lysosomes): Fig. 2C

COVpos cases showed little morphologic evidence of macrophage activation since most stained cells were spindle shaped rather than plump. Small numbers of macrophages were found scattered through the interstitium like the CD15 staining pattern. Many cells were found in areas of myocyte damage and necrosis. Staining of COVneg cases was confined to areas of myocyte damage. Mean grade for COVpos = 1.73 ± 1.09; Mean grade for COVneg = 0.048 ± 0.215, p < 0.001

CD42 (platelets): Fig. 2D

Platelets were found mostly in venules and capillaries, but occasionally scattered within the interstitium. In some COVpos cases we observed a beading pattern of platelets along venules. Platelets were rarely found in COVneg cases. Mean grade for COVpos = 0.96 ± 0.84; Mean grade for COVneg = 0.000 ± 0.016, p < 0.001.

C4d (complement component): Fig. 2E

C4d was mainly found attached to arteriolar and venular endothelium in COVpos cases. Occasional cases had focal or general capillary staining, but this was uncommon. In addition, autostaining of necrotic myocytes was observed by H&E staining including subendocardial necrosis. Occasional arterioles had muscularis staining, highlighting necrosis of smooth muscle in these cases. Rare C4d staining was found in COVneg cases. Mean grade for COVpos = 0.662 ± 0.866; Mean grade for COVneg = 0.008 ± 0.052, p ≤ 0.001

Fibrin: Fig. 2F

Fibrin aggregates were found scattered in the myocardium of many COVpos cases. In addition, fibrin aggregates were found adherent to the endothelium of arterioles and venules, within arteriolar walls, and occasionally in subendocardial regions. Such staining rarely was found in COVneg cases. Mean grade for COVpos = 1.092 ± 0.895; Mean grade for COVneg = 0.108 ± 0.276, p < 0.001

CD56 (natural killer cells): not shown

CD56 was negative in all cases of both COVpos and COVneg samples. Controls run with the test cases showed appropriate positive and negative staining.

CD 34 (stem cell vascular antigen): not shown.

CD 34 staining patterns in many COVpos cases resembled the typical pattern of staining observed in heart biopsy sections with smooth outlines of most uninjured capillaries (11). This was especially true of COVneg cases. In a subset of COVpos cases, a feathering pattern of vascular staining was observed, suggestive of vascular proliferation. Some cases also showed a random, patchy lack of capillary staining in random areas, suggestive of capillary loss.

The overall pattern of immune factor staining in most of the COVpos cases supports the hypothesis that innate immune activation (autoinflammation) is a major mechanism for cardiac injury due to SARS-CoV-2 infection. (See Fig. 3)

In severe infection with SARS-CoV-2, the virus is likely disseminated to other organs via infected monocytes and circulating vesicles from infected cells (6). Because many ACE2 receptors are found in the heart, particularly on pericytes, the vasculature of the heart is a convenient target for viral proliferation and innate and adaptive immune system activation. (6, 12–15) Because of SARS-CoV-2’s molecular mimicry of humans’ proteins which have analogous peptide sequences, immune responses against SARS-CoV-2 can be directed towards human proteins. In particular, molecular mimicry was revealed between the human SARS-CoV-2 and heat shock proteins (Hsp), which has been linked to Guillain-Barré syndrome and other autoimmune illnesses. The autoimmune response of SARS-CoV-2 to Hsp further damages the vascular endothelial lining throughout target organs like the heart, leading to activation of vascular endothelium with subsequent damage, coagulation, complement activation, and leukocyte infiltration. (12–15)

Our study highlights the pivotal role of neutrophils in cardiac injury caused by SARS-CoV-2 infection. The increased presence of neutrophils and MPO in cells as well as in interstitial staining debris is evidence of NET formation. Other features include fibrin aggregates, platelets, monocytes, and complement (C4d), which were most prominent in or around venules and arterioles. This combination of factors was not seen in COVneg cases, suggesting this type of inflammation is unique to those infected with SARS-CoV-2.

Recent studies on the lung, serum, and hearts of patients hospitalized for SARS-CoV-2 disease have highlighted the pivotal role of neutrophils (16–18). Neutrophils, together with mononuclear cells, are the first cells attracted to the SARS-CoV-2-infected alveoli recruited by interferons, CCL2, IL-6, IL-1, and other cytokines. On site, they eradicate the virus-infected cells, produce proinflammatory mediators, and secrete various proteases (via NETosis or independently on NETs). One report provides evidence of massive degranulation of neutrophils in the serum. (17) Our study supports these findings as degranulation was found in the cardiac tissue, manifested by MPO positive debris in the interstitium (16). The majority of COVpos cases did not appear to have chronic myocardial injury upon histologic examination. Most cases had scattered areas of ischemic myocyte damage, confirmed by staining with C4d. Often this ischemic damage was localized to the subendocardial space. (16–18)

Many autopsy studies have been published since the SARS-CoV-2 pandemic started and illustrate a variety of pathologic features. A recent meta-analysis of 50 studies comprising 548 autopsy cases found that myocyte necrosis and edema were the most common cardiac findings (median prevalence 100% for necrosis; 55.5% for edema) in patients dying of SARS-CoV-2 infection (19). The analysis also showed evidence that SARS-CoV-2 can be found in the heart in cardiomyocytes and endothelial cells. The median prevalence of virus in these studies was 60.8%, although the amount of virus found in any study using immunohistochemistry or in situ hybridization was minimal. Direct viral infection of the heart may act to further trigger the autoinflammation that is prevalent in these cases. Further work is needed to understand the relationship between direct infection and autoinflammation.

Importantly, we saw no evidence of lymphocytic myocarditis in any COVpos or COVneg cases. The meta-analysis of cardiac findings in patients dying of SARS-CoV-2 infection emphasized the confusion associated with the diagnosis of myocarditis. In the meta analysis, no convincing evidence of lymphocytic myocarditis was found that could not be explained by the improved sampling that autopsy tissue permits relative to endomyocardial biopsy. The median prevalence of myocarditis was 0.0%. Our data support this conclusion. We saw no evidence of lymphocytic myocarditis in our study cases and all the inflammatory cells seen stained as monocytes, macrophages, or neutrophils.(19)

Some confusion about myocarditis in SARS-CoV-2 infection has been generated by the adoption of cardiovascular magnetic resonance (CMR) imaging criteria for myocarditis rather than histologic criteria.(20) CMR findings include T1 mapping abnormalities (suggesting diffuse myocardial changes such as diffuse fibrosis and/or edema); T2, short tau inversion recovery, or T2 mapping abnormalities (more specific for myocardial inflammation, as occurs in acute myocarditis); late gadolinium enhancement (LGE, suggestive of acute myocardial injury and/or myocardial fibrosis); or pericardial involvement—all of which can indicate cardiac pathologies associated with SARS-CoV-2 infection (4, 5, 6). None of these CMR findings are specific evidence of lymphocytic myocarditis according to the classic Dallas criteria (10). All could be related to the findings we report in which there is myocardial damage, ischemia, edema, fibrin thrombi, and cellular infiltrates of neutrophils, monocytes, and platelets. Our findings lend credence to the conclusions of several authors that CMR findings are more likely related to myocardial injury and edema than lymphocytic infiltration with associated myocyte damage. (4–6) We saw no evidence of lymphocytic myocarditis. By contrast, we found immunohistochemical evidence of coagulopathy, neutrophil infiltration, complement activation, and vascular injury. These features were seen only in COVpos samples and could be responsible for the imaging patterns detected.

Limitations

This is a retrospective, single institution study of a selected population without clinical information available. We selected markers based on our hypothesis that autoinflammation was the most important cause of cardiac damage. We did not investigate antibody-mediated responses or search directly for viral antigens. We plan on conducting future studies once we obtain permission to investigate these cases further by accessing their clinical records.

Our study provides morphologic evidence that systemic innate immune activation (autoinflammation) in the cardiovascular system is a major mechanism of cardiac injury in medical examiner cases dying with SARS CoV-2 infection compared to cases negative for this virus. The cases were collected prior to the availability of treatments or vaccines for SARS CoV-2 infection which could alter pathologic features of cardiac involvement.

SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus-2

H&E Hematoxylin and Eosin

CD cluster designation

NETS Neutrophil Extracellular Traps

MPO Myeloperoxidase

NK Natural Killer Cells

NLRP3 NLR family pyrin domain containing 3

NFkB nuclear factor kappa B

IL-6 Interleukin 6

hsCRP High sensitivity C reactive protein

IL-1 Interleukin 1

MIF Macrophage inhibitory factor

TNF Tumor necrosis factor

PAD4 Peptidylarginine deiminase 4

Hsp Heat Shock Protein

CCL-2 Chemokine ligand-2

CMR Cardiovascular Magnetic Resonance

Ethics approval and consent to participate: This study was approved by the Institutional Review Boards of Utah State Government, Department of Health; University of Utah School of Medicine, and Intermountain Healthcare. Letters of approval are available on request.

Consent for publication: not applicable

Availability of data and materials; The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests: The authors declare that they have no competing interests. Funding: The immunohistochemical analysis and digital imaging of the study was funded by the A. Lee Christensen Fund of the Urban Central Region Medical and Research Foundation of Intermountain Healthcare, Salt Lake City, Utah.

Authors' contributions: The contributions of the authors are as follows:

MEH participated in the study design and data analysis. She was the major author writing the manuscript and created the graphical illustration. EC and MB contributed to the planning of the study, defined which patient’s samples should be included, provided samples for analysis, and participated in the data analysis. GS participated in the study design and analysis of data. KS participated in the study design and analysis of data. MEHH participated in the study design, analysis of samples by histology and immunohistochemistry, analysis of data and writing of the manuscript. All authors read and approved the final manuscript.

Acknowledgements: The authors acknowledge the contributions of medical examiner office staff to the provision of samples for this study. They especially acknowledge the contributions of Janet Hansen and Amy Furness in the preparation of samples for immunohistochemical and digital imaging analysis.

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No competing interests reported.

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Version 1

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Evidence of Autoinflammation as the Principal Mechanism of Myocardial Injury in SARS-CoV-2 PCR-Positive Autopsy Hearts

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods

Population

Immunohistochemical Analysis

Statistical Analysis

Results

Immunohistochemical analysis (See Fig. 2, A-F)

CD15 (cells of myelogenous origin, monocytes): Fig. 2A

MPO: Fig. 2B

CD 68 (macrophages, lysosomes): Fig. 2C

CD42 (platelets): Fig. 2D

C4d (complement component): Fig. 2E

Fibrin: Fig. 2F

CD56 (natural killer cells): not shown

Discussion

Limitations

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1