key: cord-0947804-rfcqkgfa
authors: Mueller, Karin Anne Lydia; Langnau, Carolin; Günter, Manina; Pöschel, Simone; Gekeler, Sarah; Petersen-Uribe, Álvaro; Kreisselmeier, Klaus-Peter; Klingel, Karin; Bösmüller, Hans; Li, Bo; Jaeger, Philippa; Castor, Tatsiana; Rath, Dominik; Gawaz, Meinrad Paul; Autenrieth, Stella E
title: Numbers and phenotype of non-classical CD14(dim)CD16(+) monocytes are predictors of adverse clinical outcome in patients with coronary artery disease and severe SARS-CoV-2 infection
date: 2020-11-03
journal: Cardiovasc Res
DOI: 10.1093/cvr/cvaa328
sha: ce8e786caf2954752409c7b8ace030c00511eda7
doc_id: 947804
cord_uid: rfcqkgfa

AIMS: To elucidate the prognostic role of monocytes in the immune response of patients with coronary artery disease (CAD) at risk for life-threatening heart and lung injury as major complications of SARS-CoV-2 infection. METHODS AND RESULTS: From February to April 2020, we prospectively studied a cohort of 96 participants comprising 47 consecutive patients with CAD and acute SARS-CoV-2 infection (CAD+SARS-CoV-2), 19 CAD patients without infections, and 30 healthy controls. Clinical assessment included blood sampling, echocardiography, and electrocardiography within 12 hours of admission. Respiratory failure was stratified by the Horovitz Index (HI) as moderately/severely impaired when HI ≤ 200 mmHg. The clinical endpoint (EP) was defined as HI ≤ 200 mmHg with subsequent mechanical ventilation within a follow-up of 30 days. The numbers of CD14(dim)CD16(+) non-classical monocytes in peripheral blood were remarkably low in CAD+SARS-CoV-2 compared to CAD patients without infection and healthy controls (p < 0.0001). Moreover, these CD14(dim)CD16 monocytes showed decreased expression of established markers of adhesion, migration, and T cell activation (CD54, CD62L, CX3CR1, CD80, HLA-DR). Decreased numbers of CD14(dim)CD16(+) monocytes were associated with the occurrence of EP. Kaplan-Meier curves illustrate that CAD+SARS-CoV-2 patients with numbers below the median of CD14(dim)CD16(+) monocytes (median 1443 cells/mL) reached EP significantly more often compared to patients with numbers above the median (log-rank 5.03, p = 0.025). CONCLUSION: Decreased numbers of CD14(dim)CD16(+) monocytes are associated with rapidly progressive respiratory failure in CAD+SARS-CoV-2 patients. Intensified risk assessments comprising monocyte sub- and phenotypes may help to identify patients at risk for respiratory failure. TRANSLATIONAL PERSPECTIVE: Patients with coronary artery disease (CAD) are at risk of life-threatening heart and lung injury accelerated by the pro-inflammatory and pro-thrombotic immune response during SARS-CoV-2 infection. We found substantially low numbers of CD14(dim)CD16(+) non-classical monocytes with an altered phenotype suggesting impaired migration behaviour and T cell activation capacity in peripheral blood of SARS-CoV-2 patients with CAD, compared to CAD patients without infection or healthy controls. Decreased numbers of CD14(dim)CD16(+) monocytes predicted rapidly progressive respiratory failure (Horovitz index ≤ 200 mmHg) with subsequent mechanical ventilation. Therefore, early sub- and phenotyping of CD14(dim)CD16(+) monocytes using simple flow cytometry might predict worsening of respiratory failure at an early stage of SARS-CoV-2 infection in high-risk CAD patients, who require an extensive heart failure and anti-thrombotic therapy to improve their clinical outcome.

Patients with coronary artery disease (CAD) are at risk to develop severe courses of SARS-CoV-2 infection comprising life-threatening heart and lung injury which are some of the most frequent complications according to the current assessment of the European Society of Cardiology. 1, 2 SARS-CoV-2 infection leads to a pro-inflammatory and pro-thrombotic immune response which, in the presence of CAD, can often lead to acute coronary syndrome with subsequent impairment of left or right ventricular function. 3, 4 An accompanying myocarditis caused by SARS-CoV-2 infection has also been described in the literature. 5, 6 Viral myocarditis comprises a broad spectrum of symptoms ranging from asymptomatic to most severe clinical courses resulting in congestive heart failure and inflammatory cardiomyopathy with potentially life-threatening functional impairment of the ventricle and poor prognosis. 3, 5, 7 Patients with CAD are also particularly at risk for acute right heart failure due to pre-existing right heart and diastolic dysfunction as well as elevated pulmonary artery pressure. 1, 2 Right heart dysfunction is triggered by SARS-CoV-2 infection as pneumonic infiltrates and lung involvement, often associated with progressive respiratory failure and acute respiratory distress syndrome (ARDS), lead to an additional increase of pulmonary pressure and tricuspid regurgitation resulting in right heart overload and finally failure. 3 Furthermore, patients are at risk to develop pulmonary embolisms due to infect-associated coagulopathy resulting in acute right heart failure and disseminated intravascular coagulation. 4 These clinical scenarios explain the increased rate of organ failure, admissions to the intensive care unit (ICU) with rapidly progressive respiratory failure, and mortality in cardiovascular patients when SARS-CoV-2 infection occurs. [1] [2] [3] 7 Chronic alterations of inflammatory mediators, like C-reactive protein (CRP), pro-inflammatory cytokines, or adhesion molecules trigger atherogenesis and -progression in CAD. 8 Up-regulation of the involved cytokines and chemokines recruit inflammatory cells like monocytes to the vessel wall causing atherosclerotic lesions. 8 Inflammatory cells like monocytes and macrophages have been implicated in many inflammatory heart diseases, e.g. CAD, myocardial infarction, myocarditis, and heart failure. [8] [9] [10] In humans, three distinct monocyte subpopulations have been classified based on their surface receptor expression into classical (CD14 + CD16 -), intermediate (CD14 + CD16 + ), and nonclassical (CD14 dim CD16 + ) monocytes. 11, 12 Classical monocytes secrete soluble mediators and differentiate into monocyte-derived dendritic cells (DCs) and therefore show a rather pro-inflammatory phenotype. Intermediate monocytes are specialized in antigen presentation, whereas non-classical monocytes are important for the anti-viral immune responses. [11] [12] [13] Non-classical monocytes predominantly remain in the vascular system and migrate along the endothelium. This process is termed patrolling and is mediated by their expression of the adhesion-related receptor CX3CR1 among others. 14, 15 An athero-protective role and also antiinflammatory and pro-homeostatic effects of these patrolling non-classical monocytes have recently been suggested. 14 During infections viral RNA and DNA is recognized by non-classical (CD14 dim CD16 + ) monocytes via TLR7 expression leading to the production of TNF-α, IL-1β, and CCL3. 14 In addition, in HIV-infected patients, a central role of TNF overproduction by non-classical monocytes has been proposed, indicating that they could be considered as key players in the immune hyperactivation of the disease. 12 Moreover, in viraemic HIV-infected patients a pivotal role by TNF overproduction was shown for non-classical monocytes, indicating that they might be considered as a major actor in the immune hyperactivation of the disease. 16 Furthermore, patients with HIV and subclinical atherosclerosis show altered expression of activation markers on non-classical monocytes. 17 Coronavirus disease 2019 (COVID-19) has spread rapidly worldwide and is associated with significant mortality, especially in risk groups with poor prognostic features, such as CAD. 18 In hospitalized patients infected with SARS-CoV-2, the causative agent of COVID-19, pneumonia, sepsis, respiratory failure and ARDS are common complications. [18] [19] [20] The pathophysiology of SARS-CoV-2 is characterized by an early production of proinflammatory cytokines (tumour necrosis factor (TNF), IL-6, and IL-1β) described as a cytokine storm, resulting in an increased risk of vascular hyperpermeability and, if long-lasting, multiorgan failure, and eventually death. 20 This is mediated after entry of the coronavirus and the release of RNA as genomic material into the cell by activation of TLRs. The frequency of monocytes is reduced in patients with severe SARS-CoV-2 infection 21 , however, the different monocyte subsets were not analysed in detail yet. On the other hand, increased frequencies of non-classical monocytes have been associated with the occurrence of acute coronary syndromes in the general population. 22 Thus, we analysed numbers of monocyte subsets and their surface marker expression in CAD patients with and without SARS-CoV-2 infection in order to determine prognostic markers for treatment options.

From February to April 2020 we prospectively studied a consecutive cohort of 96 participants. Out of these, 47 consecutive patients with pre-existing CAD and acute SARS-CoV-2 infection (CAD+SARS- Figure 1 shows the gating strategy of human monocyte subsets. Data analysis was done with FlowJo software V.10.6.2 (Tree Star).

Specimens were gained from heart and lung tissue of healthy controls, CAD, and CAD+SARS-CoV-2 patients and fixed in 4 % buffered formaldehyde for immunohistology. Paraffin-embedded EMB were stained with hematoxylin/eosin (H&E) and analyzed by light microscopy. 26 

We determined clinical and laboratory baseline characteristics in relation to measured monocyte 

We prospectively studied a consecutive cohort of 96 participants from February to April 2020. 47 out of 96 patients with pre-existing CAD and acute SARS-CoV-2 infection (CAD + SARS-CoV-2) were admitted with progressive respiratory symptoms. 19 patients with pre-existing stable CAD without any infections were matched to CAD + SARS-CoV-2 patients, while 30 healthy participants served as controls. Baseline characteristics and demographics of the overall cohort are given in (Table 3) . This was the pre-defined clinical endpoint (EP) during a follow-up of 30 days.

We analysed subtypes of monocytes in an intensified risk assessment of patients with CAD and SARS-CoV-2 infection to elucidate their prognostic impact during the cause of the disease. Therefore, we stratified monocytes in peripheral blood by their expression of CD14 and CD16 into classical As current literature suggested gender-specific immune response in patients with SARS-CoV-2 infection, we stratified our data by gender to detect differences between men and women in our cohort. [28] [29] [30] Interestingly, there were no statistically relevant, gender-specific differences regarding monocyte subtypes in the overall cohort and in the comparison of controls vs CAD vs CAD + SARS-CoV-2 patients (supplementary Fig. 2A -C). (Fig. 2E) . These data suggest impaired migration behaviour and T cell activation capacity of the remaining CD14 dim CD16 + monocytes.

In subgroup analysis of ICU patients, assessing the markers described above, we detected an even . 2B-D) .

In a subgroup of patients we performed histological and immunohistochemical analysis of heart and lung tissue (Figure 3 ). Representative heart tissue sections from healthy controls, CAD, and CAD+SARS-CoV-2 patients (n=4 in each group) were stained with anti-CD68, anti-CD14, and anti-CD16. We identified distinct expression patterns of these CD68 + , CD14 + , and CD16 + inflammatory cells infiltrating the myocardium to maintain local inflammation in CAD+SARS-CoV-2 compared to healthy controls and CAD patients (Figure 3 A) . Interestingly, we could detect a dramatically increased number of CD68 + , CD14 + , and CD16 + cells in CAD+SARS-CoV-2 patients compared to healthy controls and CAD patients (Figure 3 A) . These findings suggest that non-classical monocytes and macrophages migrate to affected myocardial tissue and maintain local inflammation in SARS-Cov-2 related myocarditis (Fig. 3 A) .

Analysis of affected lung tissue showed similar results. Here, we found substantially higher numbers of CD68 + , CD14 + , and CD16 + inflammatory cells in CAD+SARS-CoV-2 patients compared to healthy controls (Fig. 3 B) . more often compared to patients with cell counts above the median (log-rank 5.03, p=0.025) (Fig. 4B) .

Remarkably, considering the median number of CD14 dim CD16 + monocytes from ICU patients in relation to all CAD + SARS-CoV-2 patients, Kaplan-Meier analysis revealed that patients with a CD14 dim CD16 + monocyte cell count below the median of 388 cell/mL develop most frequently respiratory failure (log-rank 14.18, p<0.0001) (Fig. 4C) . Additionally, we performed gender-specific

Kaplan-Meier analysis ( Supplementary Fig. 4A 

In contrast to other studies on COVID-19 18, 19 we focussed on patients with pre-existing coronary artery disease, which are prone to progressive heart and lung failure when suffering from SARS-CoV- showed an increased frequency of this subsets in peripheral blood and even higher frequencies in severe pulmonary syndrome patients from ICU. 32 However, we cannot compare our data showing reduced numbers of CD14 + CD16 + monocytes in CAD + SARS-CoV-2 patients compared to CAD patients with this study of Zhou et al. 32 as CAD patients are characterized by doubling of intermediate monocytes which is even associated with cardiovascular outcomes. 33 Zhou et al. do not give information about comorbidities of the cohort, especially not of presence or absence of CAD. 32 We showed that in infected CAD patients with progressive respiratory failure not only the numbers of monocytes were substantially reduced, but also their function was impaired. More precise, these patients showed less expression of adhesion and activation markers on non-classical monocytes when 

None of the authors has any conflict of interest to declare. Our study complies with the Declaration of Helsinki, our locally appointed ethics committee has approved the research protocol, and informed consent has been obtained from all participants.

For original data, please contact Karin Anne Lydia Mueller, k.mueller@med.uni-tuebingen.de.

Alvaro I, Alfranca A, Sanchez-Madrid F, Munoz-Calleja C, Soriano JB, Ancochea J, Martin-Gayo E. COVID-19 severity associates with pulmonary redistribution of CD1c+ DC and inflammatory transitional and nonclassical monocytes. J Clin Invest 2020. 36.

Zheng YY, Ma YT, Zhang JY, Xie X. COVID-19 and the cardiovascular system. Nat Rev Cardiol 2020;17:259-260. Table 1 . Baseline characteristics of the overall cohort (n = 96) Numbers and phenotype of non-classical CD14 dim CD16 + monocytes are predictors of adverse clinical outcome in patients with coronary artery disease and severe SARS-CoV-2 infection coronary artery blood monocytes progressive respiratory failure

ESC Guidance for the Diagnosis and Management of CV Disease during the COVID-19

COVID-19): Epidemiology, Clinical Spectrum and Implications for the Cardiovascular Clinician

Coronaviruses and the cardiovascular system: acute and long-term implications

Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy

SARS-CoV-2: a potential novel etiology of fulminant myocarditis

Deisolating COVID-19 Suspect Cases: A Continuing Challenge

Association of Cardiac Injury With Mortality in Hospitalized Patients With COVID-19 in Wuhan, China

Immunity and Inflammation in Atherosclerosis

Cardioimmunology: the immune system in cardiac homeostasis and disease

Monocytes in atherosclerosis: subsets and functions

Nonclassical Monocytes in Health and Disease

Human Monocyte Subsets and Phenotypes in Major Chronic Inflammatory Diseases

Patrolling Mechanics of Non-Classical Monocytes in Vascular Inflammation

Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors

CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation

Pivotal role of M-DC8(+) monocytes from viremic HIV-infected patients in TNFalpha overproduction in response to microbial products

Loss of CXCR4 on non-classical monocytes in participants of the Women's Interagency HIV Study (WIHS) with subclinical atherosclerosis

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

COVID-19 cytokine storm: the interplay between inflammation and coagulation

Dysregulation of immune response in patients with COVID-19 in Wuhan, China

Association of monocyte subsets with vulnerability characteristics of coronary plaques as assessed by 64-slice multidetector computed tomography in patients with stable angina pectoris

Oxygenation Saturation Index Predicts Clinical Outcomes in ARDS

Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging

Assessment of pulmonary artery pressure by echocardiography-A comprehensive review

Molecular pathology of inflammatory cardiomyopathy

Myocarditis: the Dallas criteria

Gender Differences in Patients With COVID-19: Focus on Severity and Mortality

Coronavirus COV-19/SARS-CoV-2 affects women less than men: clinical response to viral infection

Decrease of Non-Classical and Intermediate Monocyte Subsets in Severe Acute SARS-CoV-2 Infection

Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe COVID-19 patients

Shift of monocyte subsets along their continuum predicts cardiovascular outcomes

Immune cell subset differentiation and tissue inflammation

PTT (s) 24 (22

ACE -Angiotensin Converting Enzyme, Afib -atrial fibrillation, ALT -alanine amino-tranferase, ARB -Angiotensin II Receptor Blockers, ASA -Acetylsalicylic acid , AST -aspartate-aminotransferase, BMI -body mass index, CAD -coronary artery disease, CK -creatinine kinase, CRP -C-reactive protein, GFR-MDRD -glomerular filtration rate, Hb -hemoglobin, hs TNI -High sensitive Troponin I, INRinternational normalized ratio, LDH -lactate dehydrogenase, NT-pro-BNP -N-terminal pro-brain natriuretic peptide

Continuous, not normally distributed variables are expressed as median and IQR and were compared using Mann-Whitney U test for two group comparison and Kruskal-Wallis test for three group comparison, where applicable. Continuous parameters were dichotomized at established cut-off values if necessary. Categorical data are presented as total numbers and proportions and were analysed by chi-squared test. Comparisons were considered statistically significant if two-sided p-value was <0.05. ACE -Angiotensin Converting Enzyme, Afib -atrial fibrillation, ALT -alanine aminotranferase, ARB -Angiotensin II Receptor Blockers, ASA -Acetylsalicylic acid, AST -aspartate-aminotransferase, BMI -body mass index, CAD -coronary artery disease, CK -creatinine kinase, CRP -C-reactive protein, GFR-MDRD -glomerular filtration rate, Hb -hemoglobin, hs TNI -High sensitive Troponin I, INR -international normalized ratio, LDH -lactate dehydrogenase, NT-pro-BNP -N-terminal pro-brain natriuretic peptide

/dL) 13 (11.2-13.8)

Platelets (1000/µL)

Continuous, not normally distributed variables are expressed as median and IQR and were compared using Mann-Whitney U test for two group comparison and Kruskal-Wallis test for three group comparison, where applicable. Continuous parameters were dichotomized at established cut-off values if necessary. Categorical data are presented as total numbers and proportions and were analysed by chi-squared test. Comparisons were considered statistically significant if two-sided p-value was <0.05. ACE -Angiotensin Converting Enzyme, Afib -atrial fibrillation, ALT -alanine aminotranferase, ARB -Angiotensin II Receptor Blockers, ASA -Acetylsalicylic acid , AST -aspartate-aminotransferase, BMI -body mass index, CAD -coronary artery disease, CK -creatinine kinase, CRP -C-reactive protein, GFR-MDRD -glomerular filtration rate, Hb -hemoglobin, Hs TNI -High sensitive Troponin I, INR -international normalized ratio, LDH -lactate dehydrogenase, NT-pro-BNP -N-Terminal pro-brain natriuretic peptide