Sex differences in coronavirus disease 2019 myocarditis

Abstract

Myocarditis is frequently caused by viral infections, but animal models that closely resemble human disease suggest that virus-triggered autoimmune disease is the most likely cause of myocarditis. Myocarditis is a rare condition that occurs primarily in men under age 50. The incidence of myocarditis rose at least 15x during the COVID-19 pandemic from 1–10 to 150–400 cases/100,000 individuals, with most cases occurring in men under age 50. COVID-19 vaccination was also associated with rare cases of myocarditis primarily in young men under 50 years of age with an incidence as high as 50 cases/100,000 individuals reported for some mRNA vaccines. Sex differences in the immune response to COVID-19 are virtually identical to the mechanisms known to drive sex differences in myocarditis pre-COVID based on clinical studies and animal models. The many similarities between COVID-19 vaccine-associated myocarditis to COVID-19 myocarditis and non-COVID myocarditis suggest common immune mechanisms drive disease.

1. Introduction

Viral infections such as coxsackievirus, influenza, cytomegalovirus, hepatitis C, HIV and parvovirus are the leading cause of viral myocarditis worldwide.¹ Although commonly triggered by infections, the cardiac inflammation that defines myocarditis is largely driven by an autoimmune response to damaged heart tissue rather than being due directly to viral infection.^2–5 It is perhaps not surprising that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is linked to myocarditis because viral strains with a similar homology, SARS-CoV-1 and Middle Eastern respiratory syndrome coronavirus (MERS-CoV), were also associated with cases of myocarditis.^6,7 Unexpectedly, vaccines developed to protect against SARS-CoV-2-mediated coronavirus disease 2019 (COVID-19) were associated with cases of myocarditis, pericarditis, and/or perimyocarditis as a rare side effect.^5,8 Rare cases of vaccine-associated myocarditis and perimyocarditis had been previously reported for smallpox vaccines in particular.⁹ SARS-CoV-2 is estimated to have increased the incidence of viral myocarditis at least 15x to around 150–400 cases/100,000 individuals compared to pre-COVID-19 pandemic levels of around 1–10/100,000 individuals.^8,10–12 We recently reviewed COVID-19 myocarditis and pericarditis.⁵ The aim of this review is to briefly summarize what is known about sex differences in COVID-19 myocarditis.

2. Sex differences in COVID-19

Sex differences have been reported for COVID-19 with more cis-men (hereafter referred to as men) (60%) hospitalized than cis-women (hereafter referred to as women) (40%).^13,14 Importantly, male sex was identified as a factor that increased the risk of death in patients hospitalized for COVID-19,^14–16 with men dying at twice the rate of women.¹⁷ The spike protein of SARS-CoV-2 binds angiotensin converting enzyme II (ACE2) and is cleaved by human type II transmembrane serine protease 2 (TMPRSS2) facilitating viral entry into the cytosol.¹⁸ It has been reported that ACE2 expression is higher on cells from males than females and that testosterone can increase ACE2 expression on human airway smooth muscle cells in culture,^19,20 which could be a possible explanation for the observed sex differences- although many studies reported no sex difference.²¹ Additionally, no sex differences were identified in SARS-CoV-2 levels based on PCR tests in patients with COVID-19 for most large clinical studies.^14–16 These findings suggest that if sex differences in receptor expression exist they are not conferring a clear advantage in viral replication that can be detected by this method (i.e., PCR). Perhaps part of the reason for divergent findings related to ACE2 cell expression levels relate to the fact that ACE2 and angiotensin (Ang)II receptor genes are located on the X chromosome and may be more highly expressed in females if they escape X inactivation, particularly healthy females, and have been found to be downregulated by the male Y chromosome gene Sry.²² Thus, if SARS-CoV-2 infection and testosterone increase ACE2 expression in males while females already have higher expression there may appear to be no overall sex difference. However, most studies do not clearly delineate sex differences in the context of age and race/ethnicity, which are factors that strongly influence sex differences in disease.,²³ adding to the difficulty of interpreting sex differences. For example, myocarditis occurs primarily in White men under age 50,²⁴ and so data need to be analyzed according to sex, age and race/ethnicity to draw better conclusions about sex differences. ^24,25

Despite these considerations, the immune response to SARS-CoV-2 has been found to display sex differences. Sex differences in the innate and adaptive immune response in healthy individuals and in response to infectious agents are well described (summarized in ^21,24). In general, estrogen promotes B cell and plasma cell differentiation and proliferation resulting in higher antibody (and autoantibody) levels following infection.²⁶ Females also have elevated T cell responses (CD3, CD4, CD8) as well as regulatory T (Treg) cells.²⁷ In contrast, men and male animals often respond to infection with higher innate immune cell activation.^25,28–31 This is likely due to the fact that estrogen response elements downregulate many proinflammatory innate immune gene pathways like the TLR-4/inflammasome pathways.²⁷ Consistent with these known sex differences in the immune response to infection, men with COVID-19 were found to have elevated C-reactive protein (CRP), Toll-like receptor (TLR)4/inflammasome/NOD-like receptor family pyrin domain containing 3 (NLRP3)-derived interleukin (IL)-1β and IL-18, IL-6, and IL-8 levels as well as a greater ratio of neutrophils to T cells.^14,32–36 Another potential source of elevated innate immune factors in COVID-19 involves the downregulation of ACE2 following SARS-CoV-2 binding which leads to the release of reactive oxygen species (ROS) from mitochondria that activate TLRs leading to release of tumor necrosis factor (TNF)α and IL-6.^37,38 Patients hospitalized for COVID-19 often have bacterial infections that increase the risk of death. One study of 1,553 patients found that the majority of patients with bacterial coinfections were men (70%), indicating sex differences in the immune response to co-infection following SARS-CoV-2 infection.³⁹ Additionally, sociocultural gender differences are likely to influence the outcome of SARS-CoV-2 infection, but gender influences have not been well studied in the context of COVID-19 (see discussion in reviews^21,40,41).

Another important factor affecting susceptibility to COVID-19 is age. Early in the pandemic it was recognized that the greatest risk factor for death from COVID-19 was age, especially from age 70 and older.^42,43 Because children, especially very young children, have underdeveloped immune systems they are typically more susceptible to infections/ viral infections than adults. However, with COVID-19 it has been found that children have a significantly lower susceptibility to infection.⁴⁴ Several studies found elevated anti-viral immune responses in children (e.g., interferon-γ/IFNγ) compared to adults after SARS-CoV-2 infection, but studies conflict.⁴⁵ An animal model of COVID-19 in mice found that IFNs were higher in younger than older mice following infection with a mouse-adapted strain of SARS-CoV-2, providing support for the clinical observations.³⁰ When children develop COVID-19 they have symptoms including fever, vomiting and diarrhea that may progress to severe respiratory problems, multi-system inflammatory syndrome in children (MIS-C) and potentially acute hepatitis.^44,46,47 Most studies have not found a sex/gender difference in children with COVID-19.^48,49

3. Pathogenic mechanisms in myocarditis

We, and others, recently reviewed key pathways in the pathogenesis of myocarditis.^{5,11,24,50–52} Investigators for many studies during the pandemic required the presence of necrosis in biopsies and elevated cardiac troponin in addition to cardiac inflammation for a diagnosis of myocarditis. However, animal models that are highly translational and closely resemble clinical myocarditis (e.g., autoimmune myocarditis induced with complete Freund’s adjuvant or with mild viral infections such as coxsackievirus B3/CVB3)^53,54 do not display overt/severe histologic necrosis that lead to elevated serum cardiac troponin or high levels of viral replication that would cause cardiac damage. Evidence from these and other myocarditis animal models demonstrate that the key factor driving myocarditis is the immune response to the virus rather than viral replication itself. This is likely why diverse viruses, pathogens, toxins and adjuvants cause myocarditis using the same immune mechanisms. This is important, because the immune response to the virus is what leads to sex differences in myocarditis. There are no sex differences in viral replication in the heart.^28,55 In further support of this idea, many common viruses like SARS-CoV-2 cause myocarditis yet the disease is rare indicating it is not due to virus alone but other factors like an autoimmune response that arises from viral infection. Additionally, studies of experimental autoimmune myocarditis (EAM), that does not use virus to induce disease, have found the same sex differences in disease and that the same mechanisms drive disease (summarized in ^24,56) Importantly, the Dallas criteria, which was developed for the diagnosis of myocarditis based on histology from endomyocardial biopsies, does not require the presence of necrosis in biopsy sections but only that there are changes indicating cardiomyocyte damage such as vacuolization.⁵⁷ Severe myocardial damage (i.e., necrosis) is typically assessed clinically by examining serum cardiac troponin levels.^42,58 However, myocarditis often occurs without overt necrosis so that serum troponin can be a poor indicator of the presence of myocarditis, even severe myocarditis.^59,60

Overall, innate immune activation of complement pathways by virus or adjuvants leads to elevated complement C3 which binds to and upregulates CD11b (also named complement receptor 3/CR3) on neutrophils, mast cells and macrophages (Figure 1), as previously reviewed ^24,56. Cells that express CD11b also express TLR4 leading to the cardiac production of IL-1β and IL-18 (Figure 1, Figure 2). Susceptibility to dilated cardiomyopathy (DCM) following myocarditis depends at least in part on mast cell activation leading to release of enzymes like Serpin A3n (also called α1-anti-chymotrypsin), which activates the cytokines and matrix metalloproteinases that lead to remodeling and fibrosis (Figure 2). Activation of these TLR4+ CD11b+ cells during the innate immune response and during acute myocarditis promotes proinflammatory and profibrotic pathways including alternatively activated M2b macrophages that express TLR4 and release IL-1β and IL-18 driving a mixed T helper (Th)1/Th2 adaptive response (Figure 1, Figure 2). IL-1β increases IL-6 which is critical in the development of the profibrotic Th17 adaptive immune response that further promotes remodeling leading to DCM. Thus, all these pathways increase inflammation and remodeling that lead to more severe myocarditis and progression to DCM in males.

Figure 1. Innate immune pathways important in the pathogenesis of myocarditis.

Left: Activation of the three complement pathways by virus leads to elevated levels of C3 in the serum which breaks down to activate mast cells via the anaphylatoxin receptors and CD11b (also known as complement receptor 3) and macrophages. Right: Active virus or viral particles as well as damaged self-tissue upregulate Toll-like receptor 4 (TLR4) on mast cells and macrophages in the heart leading to a mixed T helper (Th)1, Th2 and Th17 (not shown) proinflammatory and profibrotic immune response that increases myocarditis in males and promotes progression to dilated cardiomyopathy (DCM) in males. Mast cells additionally release enzymes like Serpin A3n (α1-antichymotrypsin) that activate cytokines and matrix metalloproteinases (MMPs) that promote remodeling and fibrosis which are necessary for the development of DCM. These pathways are increased in males with myocarditis.

Figure 2. Adaptive immune pathways important in the pathogenesis of myocarditis.

Mast cell (left) and macrophage (right) activation during the innate immune response to virus/viral antigens and self-tissue are critical drivers of a mixed Th1/Th2 and Th17 (not shown) immune response that increases acute inflammation during myocarditis and promotes a M2b/Th2-type immune response that leads to remodeling, fibrosis and dilated cardiomyopathy. These pathways are increased in males with myocarditis.

4. Sex differences in COVID-19 myocarditis

According to the latest statistics for the United States, men have an increased incidence of most cardiovascular diseases including atherosclerosis, myocardial infarction, myocarditis, DCM, and heart failure.^24,61–63 In contrast, females have increased hypertension after age 65 but only bypass males for most other cardiovascular diseases including stroke after age 85^24,61–63 We recently updated our 2013 review article on sex differences in myocarditis^5,24 but did not focus on sex differences in COVID-19 myocarditis which is the topic of this review. Myocarditis occurs more often in men under the age of 50 with a sex ratio of 2–4:1 males to females, while more women develop myocarditis after menopause.^64–66 Pericarditis also primarily occurs in males under 50 years of age with a sex ratio of 2:1 males to females.⁶⁷ Based on animal models, pericarditis almost always appears with myocarditis where it is termed myopericarditis or perimyocarditis. Most studies of COVID-19 report a male dominance of around 60% males to 40% females, and a very similar sex ratio has been observed for COVID-19 myocarditis of around 60–70% males to 30–40% females.^{13,14,68–70}

ACE2 has been reported to be expressed on cardiomyocytes, pericytes (cells present along the walls of capillaries), macrophages and mast cells with lower expression on fibroblasts and endothelial cells (Figure 3).^52,71–73 Cardiomyocytes, pericytes, macrophages and mast cells also express TMPRSS2 as well as other accessory proteins (i.e., neuropilin-1 receptor/NRP1, CD147, integrin α5β1, and cathepsin B/L) needed for viral infection by SARS-CoV-2 (reviewed in ^74–76) (Figure 3). Activation of resident macrophages and mast cells by ACE2 and other accessory receptors by SARS-CoV-2 could lead to the production of innate cytokines such as IL-1β, IL-6 and IL-18 that are associated with the development of myocarditis in males (Figure 3).

Figure 3. COVID-19 myocarditis.

SARS-CoV-2 binds to cells in the heart including cardiomyocytes and pericytes which express ACE2 and other accessory receptors needed for viral entry. Mast cells and macrophages also express these receptors and can be activated to release cytokines including interleukin (IL)-1β, IL-18 and IL-6 that have been found to be increased in males with myocarditis pre-COVID and during COVID-19.

Based on autopsy tissue from patients with COVID-19 myocarditis and pericarditis, CD68+ macrophages make up the primary infiltrate with fewer T and B cells.^24,77, This is also the characteristic immune composition found in non-COVID clinical myocarditis (lymphocytic myocarditis) and what is observed in animal models of viral and autoimmune myocarditis.^8,56 Also similar to the known pathogenesis of myocarditis/pericarditis prior to COVID-19 and animal models of myocarditis, patients with COVID-19 have strongly activated complement and TLR4/NLRP3 inflammasome-pathways with corresponding elevated IL-1β and IL-18 levels.^78–81 TLR4/IL-1β is more highly expressed on macrophages and mast cells in males with myocarditis than females.²⁸ TLR4/IL-1β levels were shown to be increased by testosterone during myocarditis by the removal of testes (primary producers of testosterone) via gonadectomy which decreased circulating testosterone levels, myocardial inflammation and cardiac TLR4/IL-1β levels. This result was reversed by performing gonadectomy and treating mice with a slow-release testosterone pellet showing that testosterone increase cardiac TLR4 and IL-1β levels during myocarditis(summarized in ^24,29,50). Importantly, in mouse models cardiac IL-1β levels directly correlate to cardiac inflammation (but not viral levels), echocardiography-derived global longitudinal strain (GLS), and serum soluble ST2 (sST2) also known as IL-1 receptor-like 1/IL-1RL1 which is a biomarker of heart failure, but only in males.⁶⁴ In patients with biopsy confirmed myocarditis, elevated serum sST2 levels correlate to New York Heart Association classification, but only in men under age 50.⁶⁴ IL-1β itself has low expression in serum but strongly upregulates IL-6 which drives Th17 responses and is a serum biomarker of heart failure. IL-6 and Th17 responses are increased in men and animal models of myocarditis.⁸² Importantly, elevated serum IL-6 was found in nearly 80% of COVID-19 cases in one study that had cardiac dysfunction based on echocardiography-derived GLS.⁸³ In a BALB/c mouse model of COVID-19 the virus was passaged through the lung six times to increase viral tropism for the lung and SARS-CoV-2 was detected in the heart at day 3 and 5 along with elevated IL-1β and IL-6.⁸⁴ In a Syrian hamster model of COVID-19, male hamsters developed diffuse cardiac inflammation but not focal inflammatory foci typical of myocarditis. However, enumeration of cells using immunohistochemistry revealed higher numbers of CD15+ cells (a marker of myeloid cells such as granulocytes, neutrophils, eosinophils, mast cells and macrophages), CD68+ macrophages and fewer CD4 and CD8 T cells in the heart of male SARS-CoV-2 infected hamsters compared to females.^85,86 Thus, sex differences in the cardiac immune response have been found in the Syrian hamster model of SARS-CoV-2-induced myocarditis providing further evidence of clinical observations.

5. Sex differences in COVID-19 vaccine-associated myocarditis

Not long after COVID-19 vaccination began in the general population, case reports appeared identifying myocarditis and pericarditis or perimyocarditis as a side effect of vaccination. After the establishment of vaccine programs, many large, population-based epidemiology studies were conducted both by countries and internationally, reporting myocarditis/pericarditis as a rare side effect of COVID-19 vaccination, especially the new mRNA platform vaccines (reviewed in ⁸). The incidence of myocarditis/pericarditis varied widely depending on the vaccine type and how many doses were administered. The highest rates of myocarditis/pericarditis was reported for the Moderna mRNA vaccine, with an overall incidence (men and women of all ages) of nearly 10/100,000 or around 50/100,000 in males under 40 years of age.^56,87 The studies were in agreement that the greatest risk of developing myocarditis occurs after the second vaccine dose in young males aged 12–39 years with the mRNA vaccine platforms (e.g., Moderna, Pfizer). There were far fewer reports of myocarditis in individuals past age 50, mirroring the same sex and age groups known for myocarditis prior to COVID-19. Although few studies analyzed data by race, when they did, vaccine-associated myocarditis occurred primarily in White individuals which is the same demographic as pre-COVID myocarditis.^64,88

The diagnosis and treatment of COVID-vaccine associated myocarditis/pericarditis is the same as conventional myocarditis, although the severity is typically mild (although most cases of myocarditis are also mild).^8,11 Analysis of the biopsies from patients with COVID-19 vaccine-associated myocarditis reveal a predominant macrophage infiltrate with fewer T cells- the same composition as other forms of myocarditis.^8,56 Additionally, antibodies directed against the IL-1 receptor have been found in patients with COVID-19 vaccine-associated myocarditis,¹³ suggesting that the TLR4/IL-1β pathway is also important in the pathogenesis of this type of myocarditis.

The many similarities between COVID-19 vaccine-associated myocarditis to COVID-19 myocarditis and non-COVID myocarditis suggest common mechanisms may drive disease. All forms of myocarditis are rare even though many of the viruses that cause myocarditis are common such as coxsackieviruses, influenza, cytomegalovirus and coronaviruses. Many viruses that cause myocarditis such as CVB3 target mitochondria to obtain a replicative advantage and are released in extracellular vesicles that contain live virus or virus particles that can activate the innate immune response (i.e., resident mast cells and macrophages).⁸⁹ These extracellular vesicles that contain virus or virus particles may resemble the new mRNA vaccine platforms that house the mRNA spike protein within a lipoprotein vesicle and may activate an immune response that leads to myocarditis. An important question is why the inflammation would target the heart. Animal models that most closely resemble clinical myocarditis/DCM are autoimmune models induced using either inactivated bacteria (i.e., Mycobacterium tuberculosis) or a mild viral infection (i.e., MCMV, CVB3) as an adjuvant and cardiac myosin or damaged heart protein as the self-tissue.^2–4,56 This suggests that something about the lipid vesicle component of the vaccine may be interpreted as ‘self’ by resident innate immune cells at the site of vaccination which respond to the viral/self threat in a sex-specific, autoinflammatory manner.

6. Conclusions

Myocarditis and pericarditis associated with COVID-19 in the US increased around 15x compared to pre-COVID levels. In adults, myocarditis/pericarditis occurs predominantly in males under the age of 50 regardless of the cause. This demographic is also reported for myocarditis/pericarditis associated with COVID-19 or COVID-19 vaccination, providing a clue to how live viruses or virus antigens/particles may cause myocarditis. Animal models of viral and autoimmune myocarditis provide valuable information about the pathogenesis of clinical disease and suggest that autoimmunity is important and may provide a common explanation for the development of myocarditis following SARS-CoV-2 and COVID-19 vaccination.

Table 1.

Similarities between COVID-19 and COVID-19-vaccine myocarditis

	COVID-19 myocarditis	COVID-19 vaccines
Incidence	Relatively rare 150–400/1000,000	Rare 10/100,000 but up to 50/100,000 in males under 50 years of age
Sex	Male dominant	Male dominant
Age	Under 50 years, 12–39	Under 50 years, 12–39
Race	White predominantly	White predominantly
Myocardial Biopsy	Macrophages dominate with fewer T and B cells	Macrophages dominate with fewer T and B cells
Evidence of TLR4/IL- 1 receptor activation	TLR4, IL-1β, IL-18, IL-6	Antibodies against IL-1 receptor
ACE and other receptors for SARS-CoV-2 entry	Expressed on resident mast cells and macrophages at site of infection and in the heart	Expressed on resident mast cells and macrophages at vaccination site and heart

Highlights.

• Common viruses like SARS-CoV-2 induce autoimmune viral myocarditis which is rare

• SARS-CoV-2 increased myocarditis cases at least 15-fold during the pandemic

• The immune response to SARS-CoV-2 differs by sex increasing disease in males

• Pre-COVID vs. COVID-19 myocarditis almost identical in immune response

• Same sex and age for vaccine and COVID myocarditis indicate common mechanisms

Abbreviations

AngII

angiotensin II

ACE2

angiotensin converting enzyme II

CR3

complement receptor 3

COVID-19

SARS-CoV-2-mediated coronavirus disease 2019

CVB

coxsackievirus B

CRP

C-reactive protein

DCM

dilated cardiomyopathy

EAM

experimental autoimmune myocarditis

GLS

global longitudinal strain

IL-1RL1

IL-1 receptor-like 1

MERS-CoV

Middle Eastern respiratory syndrome coronavirus

MIS-C

multi-system inflammatory syndrome in children

NLRP3

NOD-, LRR- and pyrin domain-containing protein 3

ROS

reactive oxygen species

SARS-CoV-2

severe acute respiratory syndrome coronavirus 2

sST2

soluble ST2 also known as IL-1RL1

TMPRSS2

type II transmembrane serine protease 2

Th

T helper cells

TLR4

Toll-like receptor 4

TNFα

tumor necrosis factor-alpha

Treg

regulatory T cells