Adhered macrophages as an additional marker of cardiomyocyte injury in biopsies of patients with dilated cardiomyopathy

doi:10.21203/rs.3.rs-2237638/v1

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Adhered macrophages as an additional marker of cardiomyocyte injury in biopsies of patients with dilated cardiomyopathy

https://doi.org/10.21203/rs.3.rs-2237638/v1

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Background. As shown recently, macrophages have proved most suitable for the chronic stage of myocarditis (MCI).

The study aim. Accordingly, the aim of this study was to determine the number of CD68(+) macrophages adhered to injured cardiac myocytes in biopsy of patients with dilated cardiomyopathy (DCM) and clinical suspicion of MCI.

Patients and Methods. The study group consisted of 181 patients with DCM (159M, 22F, mean duration of symptoms was 1.8 years) and decreased left ventricular ejection fraction (< 40%). Immunohistochemistry on cryostat sections was performed using a Leica Bond Max autostainer and murine monoclonal antihuman antibodies anti-CD68(+) macrophages (cloneEBM11) from DAKO, Denmark. The total number of CD68(+) macrophages and the number of CD68 macrophages attached to injured cardiomyocytes were counted in at least 10 high power fields under 400x magnification and converted into the number of cells/mm².

Results. Two expression patterns of CD68(+) macrophages were observed: those localized freely in the interstitial space only, and cells attached to injured cardiomyocytes. The mean number of total CD68(+) macrophages and the number of those adhered to injured cardiomyocytes were 8.92 ± 4.29 and 0.89 ± 1.57, respectively. As far as CD68(+) macrophages adhered to injured cardiac myocytes are concerned, 72 out of 181 (39.8%) samples presented these cells. Importantly, in 7 positive cases for the presence of adhered macrophages, the average of total CD68(+) cells was < 14cells/mm² (7 out of 81, 8.64%). Spearman’s rank coefficient showed a significant correlation between the count of the total number of CD68(+) macrophages and CD68(+) cells directly adhered to injured cardiac myocytes (r = 0.53, P < 0.001). Both the total number of CD68(+) macrophages and the number of CD68(+) cells directly adhered correlated negatively with cTnT in the serum of DCM patients (Spearman’s rho, r = -0.45, P < 0.001 and r = - 0.31, P = 0.009, respectively).

Conclusion. The use of the count of CD68(+) macrophages by immunohistochemistry which are attached to injured cardiomyocytes may be useful for the diagnosis of chronic inflammation in endomyocardial biopsy in patients with DCM, especially in those who did not meet the criteria for MCI.

Heart failure

Dilated cardiomyopathy

Myocarditis

Endomyocardial biopsy

CD68(+) Macrophages

Dilated cardiomyopathy (DCM) is related to a broad range of progressive disorders characterized by pathological myocardial remodeling in a diffuse or multifocal fashion manifested clinically by impaired cardiac contractility [1]. The end stage of the disease leads to symptomatic heart failure (HF) in most patients and is related to high morbidity and mortality rates [2, 3].

It has generally been assumed that chronic immune response may play an important role in the development and disease progression [2, 4]. Accordingly, we recently reported that a higher number of CD68-positive macrophages in biopsy specimens significantly predicted clinical progression of HF due to DCM in patients after one-year follow-up [5]. These observations confirmed the previously reported impact of macrophages on the prognosis in DCM [6].

Cardiac tissue contains large numbers of resident and infiltrative macrophages which play a major role in immune response and post-inflammatory tissue remodeling [7, 8]. Macrophage family cells have marked phenotypic heterogeneity, which is related to their inflammatory responses [9]. In addition, macrophages are not “permanently differentiated”. They are characterized by plasticity and are able to shift their transcriptional profile depending on the local environment [10, 11, 12].

So far all data on macrophages in the myocardium of DCM patients have been based on counting the total number of these cells. However, considering the fact that histological detection of myocarditis (MCI) requires the presence of cell infiltration with myocyte cell necrosis, the detection of CD68(+) macrophages that are in direct contact with injured cardiac myocytes may be another important feature suggesting ongoing inflammation. However, the definition of universal histological criteria for ongoing MCI is still open to debate.

In the context of the above-mentioned cell necrosis, cardiac troponin T (cTnT) is a specific serum marker of myocardial injury suggesting ongoing myocardial injury [13, 14]. In addition, it may serve as a marker for HF progression [15]. The prevalence of elevated troponin T is associated with adverse outcomes in chronic HF [16, 17].

Considering the above, the aim of this study was to determine the number of CD68(+) macrophages (total number vs. those that were in direct contact with injured cardiac myocytes) in biopsy sections in the relation to plasma concentrations of cardiac troponin T (cTnT) of patients with DCM with a clinical suspicion of MCI.

Patients

We studied right ventricular biopsy specimens of 181 consecutive patients with DCM and a clinical suspicion of MCI (159M, 22F). The mean duration of symptoms was 1.8 years. All biopsied patients presented with functional class II and III HF symptoms (New York Heart Association; NYHA) and decreased left ventricular ejection fraction (< 40%) on echo study. Each patient underwent standard clinical assessment, including a detailed history and physical examination, routine blood studies, thyroid function testing, echocardiography, coronary angiography and endomyocardial biopsy. Cardiac cTnT was measured using the EDTA-plasma samples by electrochemiluminescence (Elecsys 2010 analyzer, Roche Diagnostics, Germany). The lowest detection limit for cTnT was < 0.01ng/mL. All patients were on typical therapeutic regimens for HF prior to biopsy. All patients gave their informed consent. The protocol was approved by the institutional Ethics Committee.

Endomyocardial Biopsy Procedure

Endomyocardial biopsy was performed using Cordis bioptome (Cordis Corp., Miami, US). The minimum number of five specimens was obtained from the right side of the ventricular septum. The specimens were promptly immersed in sterile saline and transported on ice to the laboratory. All specimens were fixed for 20 min in cold acetone, immersed in embedding medium (OCT compound, Miles Inc, Elkhart, IN), and stored in liquid nitrogen until tested. Cryostat specimens were cut serially into sections of 5 µm thickness, air-dried at room temperature and assayed.

Immunohistochemistry

Immunohistochemistry on cryostat sections was performed using a Leica Bond Max autostainer (Leica Biosystems). The protocol was based on the antibody detection and the samples were stained using the hematoxylin kit (BOND Polymer Refine Red Detection; DS9390; Leica Biosystems). The frozen sections were incubated with murine monoclonal antihuman antibodies anti-CD68(+) macrophages (cloneEBM11) from DAKO, Glostrup, Denmark. In addition, immunohistochemistry for HLA-class II (DR antigens), HLA-class I (ABC antigens), and CD3(+) T lymphocytes was also performed. As a negative control, clone M0PC-21 from Leica Biosystems was used. As a positive control, liver biopsy specimens from patients with chronic active hepatitis were used. The total number of CD68(+) macrophages and the number of CD68 macrophages attached to injured cardiomyocytes were counted in at least 10 high power fields (HPF) under 400x magnification and finally presented as a mean with standard deviation. In addition, the number of cells counted was converted into the number of cells/mm². All counts were made using the Eclipse Ni-U microscope and NIS Elements software from Nikon.

Statistical analysis

All parametric data were expressed as mean ± SD. Non-normally distributed data were analyzed by non-parametric tests and presented as median (1st -3rd quartile). To assess the relationship between quantitative data, the Spearman’s rank coefficient was used. Differences were considered statistically significant when p < 0.05. Statistical analyses were performed using PS IMAGO 4.0 (IBM SPSS Statistics 24 software package).

Baseline patient characteristics are given in Table 1. None of the patients presented with hypertension, valvular disease except for relative mitral and/or tricuspid regurgitation, ischemic or systemic heart disease. In this immunohistochemistry study on cryostat sections we observed two expression patterns of CD68(+) macrophages, i.e. localized freely in the interstitial space (Fig. 1A and 1B) only, and the cells attached to inured cardiomyocytes (Fig. 2A and 2B). The mean number of total CD68(+) macrophages and the number of those adhered to injured cardiomyocytes were 8.92 ± 4.29 and 0.89 ± 1.57, respectively. In addition, we revealed an average of total CD68(+) cell number > 4.5 cells per HPF (> 14cells/mm²) in 100 out of 181 (55.2%) patients. As far as the CD68(+) macrophages adhered to injured cardiac myocytes are concerned, 72 out of 181 (39.8%) samples presented these cells on cryostat sections. Importantly, in 7 positive cases for the presence of adhered macrophages (7 out of 81, 8.64%), the average of total CD68(+) cells was < 14cells/mm².

Table 1

Baseline Patient Characteristics
Age, y	41.3 ± 9.9
Sex, male / female	159 / 22
Atrial fibrillation, n (%)	26 (14.3)
BMI, kg/m²	25.6 ± 3.9
NYHA, n, II / III	161 / 20
EF, %	30.1 ± 9.5
LVDD, mm	65.8 ± 8.8
LVSD, mm	49.1 ± 8.8
MR, score, median (1st -3rd quartile)	1.2 (1.0–2.2)
C-reactive protein (mg/L), median (1st -3rd quartile)	2.2 (1.2–4.2)
cTnT, ng/mL, median (1st -3rd quartile)	0.005 (0.005–0.10)
NT-proBNP (pmol/L), median (1st -3rd quartile)	497.7 (142.7–1156.5)
Values are expressed as mean ± SD or as indicated

Based on the immunohistological criteria for MCI published previously [18], only 49 out of 181 (27%) cases presented with MCI in this cohort of patients. Spearman’s rank coefficient showed a significant correlation between the count of the total number of CD68 macrophages and CD68 cells directly adhered to injured cardiac myocytes (r = 0.53, P < 0.001). Importantly, both the total number of CD68 (+) macrophages and the number of CD68 cells directly adhered correlated negatively with cTnT in the serum of DCM patients (Spearman’s rho, r= -0.45, P < 0.001 and r = − 0.31, P = 0.009, respectively).

The results of the present study contribute to the available data regarding the use of macrophage infiltration in endomyocardial biopsy as a marker of MCI, especially in the context of chronic (ongoing) form of the disease. Accordingly, this study demonstrated that CD68(+) macrophages could adopt two forms, i.e. the first was characterized by their presence in the interstitial space, without direct contact with the surrounding cardiomyocytes and the second showed macrophages attached to injured cardiomyocytes using immunohistochemistry. Both of these forms were inversely proportional to cTnT serum levels, which suggested insufficient clearance of phagocytic activity of these cells in DCM patients [5].

Currently, there is no doubt that inflammation plays a pivotal role in the pathogenesis of DCM [4, 18]. However, despite the evolving understanding and diagnostic criteria of myocardial inflammation, universal histological criteria for a chronic form of MCI using endomyocardial biopsy have not been established yet [4, 19, 20]. Partially, it stems from the fact that histological detection of inflammation requires the presence of a high index of inflammatory cell infiltrations combined with the signs of myocyte injury which can be hampered by the focal nature of the disease, especially in its chronic form [21]. On the other hand, myocyte injury per se is a pathophysiological heterogeneous mechanism, including local ischemia as a major cause [22, 23, 24].

Based on the immunohistological criteria for MCI published previously [18, 25], only 27% of patients with cut-off values for CD68(+) > 14cells/mm2 can be considered positive for having macrophage infiltrations, which indicated MCI in our cohort of patients. These findings are consistent with the results of previous studies by Baccouche H et al., where active MCI in endomyocardial biopsy was found in 26% of cases [26].

Other authors and our previous paper recently reported that a higher number of CD68-positive macrophages in biopsy specimens significantly predicted clinical progression of HF due to DCM in patients after one-year follow-up [5, 6]. Moreover, accumulated clinical and experimental data suggest that chronic inflammation may be responsible for the development and disease progression in at least some of them [4].

In conclusion, the use of count of CD68(+) macrophages attached to injured cardiomyocytes using immunohistochemistry may be useful for the diagnosis of chronic active MCI in endomyocardial biopsy in patients with DCM, especially in those who did not meet the criteria for MCI.

Ethics approval and consent to participate : NN-6501-7/07
Consent for publication not applicable
Availability of data and materials.The datasets used and/or analysed 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. This study was supported by a grants from the Medical University of Silesia No: KNW-1-156/N/0/O and KNW-1-003/K/1/O
Authors' contributions
ERW -the conception and design of study, AB –acquisition of date, DD –analysis and interpretation of date, RW –final approval of the version to be sunmitted
Acknowledgements not applicable
Authors' information (optional)

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No competing interests reported.

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Adhered macrophages as an additional marker of cardiomyocyte injury in biopsies of patients with dilated cardiomyopathy

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Abstract

Figures

Background

Methods

Patients

Endomyocardial Biopsy Procedure

Immunohistochemistry

Statistical analysis

Results

Discussion

Declarations

References

Additional Declarations

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