Adaptive immune response in patients with atherosclerosis before and after coronary bypass surgery

Introduction

Today, atherosclerosis is one of the biggest challenges. Based on the Framingham Heart Study, the authors estimate that annual mortality due to clinical manifestations of atherosclerosis will reach 22 million by 2030 [1]. What is striking about this problem is that despite decades of research, the pathological mechanisms of atherosclerosis have not been clearly understood yet. Multifocal atherosclerotic disease affecting different arterial systems is associated with a wide variety of clinical symptoms. Coronary atherosclerosis accounting for about 60% of cases has been reported as a risk factor for ischemic heart disease (IHD) [2]. Atherosclerosis is an inflammatory disease, in which immunocompetent cells migrate into the vascular sub-endothelium, contributing to the build-up of atherosclerotic plaque. As with any inflammatory response, atherosclerosis is associated with activation of innate immunity (the first line of defense) and acquired immunity mediated by antigen-specific mechanisms [3].

Many literature publications focus on the involvement of adaptive immune response in patients with atherosclerosis; however, there are only a few studies that have described changes in the immune status after coronary artery bypass surgery.

The aim of the study was to evaluate changes in the immune status before and after coronary artery bypass surgery.

Materials and Methods

It was an open-label, cross-sectional study. It was conducted at the Rostov State Medical University of the Russian Ministry of Health between 2017 and 2019. Cardiac surgical patients with coronary atherosclerosis were enrolled. The Clinical Immunology and Allergology Research Institute of the Rostov State Medical University was responsible for clinical pathology. This study followed the principles of the WMA Declaration of Helsinki and was approved by the local ethics committee. The study included 70 patients with coronary atherosclerosis (group I) undergoing pre-operative direct coronary angiography and coronary artery bypass surgery supported by the clinical guidelines for the management of patients with stable IHD. The patients underwent a beating heart bypass. To compare the adaptive immune responses, 30 volunteers (group II) with no clinical and instrumental signs of IHD were studied. Both groups consisted of age-matched male patients (57.02±1.7 and 54.9±1.3 years, respectively, P = 0.33). IHD patients with evidence of an ongoing or prior infection were excluded from the study. Diabetes mellitus, rheumatic diseases, connective tissue disorders, benign and malignant neoplasms, and bad health habits were among the exclusion criteria. The immune status was assessed prior to surgery, on postoperative days 4–5, 9–10, and 28–30, i.e. in the early postoperative period. The lymphocyte phenotyping panels (CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD3⁺CD25⁺, CD3⁺CD45⁺, CD3⁺CD95⁺, CD4⁺CD25⁺, CD4⁺CD154⁺ subpopulations) were obtained by laser flow cytometry. Foxp3 and Granzyme B+ expression profiles were assessed in the peripheral mononuclear cells isolated by standard Ficoll-Verografin gradient centrifugation. For labeling, mononuclear cells were permeabilized with the IntraPrep Permeabilization Reagent (Immunotech) and stained with anti-Foxp3 (eBioscience) and anti-Granzyme B (Serotec) monoclonal antibodies. Samples were analyzed using the Cytomics FC 500 Flow Cytometer (Beckman Coulter).

B-lymphocytes (CD19⁺, CD19⁺CD40⁺) were identified by flow cytometric analysis (Beckman Coulter Cytomics FC 500) using specific monoclonal antibodies. IgA, IgG, and IgM were quantified by radial gel immunodiffusion using anti-heavy chain monospecific antibodies (Microgen, Russia). The values were expressed as g/L.

Serum circulating immune complexes (CICs) were detected by polyethylene glycol precipitation. Spectrophotometry was used for the quantitative evaluation of CICs at 450 nm, with the recorded values expressed in standard units. The data collected were statistically analyzed using the Statistica 12.0 package (StatSoft, USA). The descriptive statistics were summarized including mean (M) and its standard error (m). The mean difference between the groups was estimated by the Mann-Whitney test, and the Wilcoxon rank sum test was used to compare surgical outcomes over time. The distribution of study variables was analyzed using the Shapiro-Wilk test assessing whether the variables were normally distributed. The P-value <0.05 was considered statistically significant.

Results

The analysis of T-lymphocyte counts revealed a statistically significant increase in the relative numbers of the CD3⁺ subpopulation in the IHD group compared to the control group. The expression of early activation markers (CD3⁺CD25⁺) was increased in the IHD patients, whereas HLA DR expression on T-lymphocytes was found to be decreased. Moreover, the patients from the IHD group had significantly decreased apoptosis marker expression levels and annexin-positive cells. A significant increase in circulating CD45R0⁺ memory T-lymphocytes was shown in the IHD group vs. the control group. IHD was also associated with a decline in a naïve T lymphocyte (CD45RA⁺) pool (Table 1).

Analysis of the T-lymphocyte subpopulation was equivocal, with a relative increase in CD4⁺-lymphocytes in the IHD patients. No differences in CD3⁺CD8⁺ lymphocyte counts between the groups were reported, and no intergroup changes in immunoreactive insulin (IRI) levels may be considered predictable. The IHD group was remarkable for reliably higher concentrations of intracellular Granzyme B and CD4⁺ T-lymphocytes expressing early activation marker (CD25⁺), as well as for an increase in CD4⁺ cells known to express CD154⁺ (CD40L) on their surfaces, which is essential in activating B-lymphocytes. However, immunoregulatory CD4⁺CD25⁺Foxp3⁺ T cells significantly decreased (Table 2).

In the IHD group, the adaptive humoral immune response was associated with an increase in CD19⁺ cells, circulating B-lymphocytes with CD40-receptor mediating cell-to-cell communication, and higher production of the primary classes of immunoglobulins (IgA, IgM, IgG) (Table 3).

A comparison of the immune responses in both groups demonstrated the following differences. The dissociation of T-lymphocyte activation, maturation and apoptosis, suppression of immunoregulation and upregulation of cell cooperation could be observed in the IHD patients.

Coronary artery bypass grafting (CABG) remains the gold standard procedure for IHD, however, as any surgical intervention, it affects all body systems for regulating homeostasis, including the immune system. Due to the fundamental contribution the immune system makes to all stages of atherogenesis, it is considered appropriate to scrutinize the CABG-related mechanisms of immune response in IHD patients and compare between the groups based on recurrent adverse events.

Changes in Humoral and Cell-Mediated Immune Responses

Analysis of T-lymphocyte values within 1 month of CABG demonstrated slight variations in CD3⁺-lymphocytes, as well as in cells expressing early (CD3⁺CD25⁺) and late (CD3⁺HLA DR⁺) activation markers. However, these variations were not statistically significant. It is noteworthy to characterize the lymphocytes expressing the apoptosis marker CD95: relative and absolute counts of CD3⁺CD95⁺ cells significantly decreased by postoperative days 4, 5, and 12, and returned to baseline one month after the intervention. Similar changes were observed in annexin-stained cells. No significant trends were reported for CD45RO⁺ T-cells, while CD45RA⁺ naïve T-lymphocyte counts statistically significantly decreased within the same period and 12 days following the CABG, and returned to baseline one month after the intervention (Table 4).

T-cell subpopulation profiles contained no significant changes in CD3⁺CD4⁺-lymphocytes within a month post CABG. However, CD3⁺CD8⁺ cytotoxic lymphocytes showed a statistically significant decrease by postoperative days 4–5 and 10–12. These changes resulted in a significant increase in IRI levels on days 4, 5, and 12 of the intervention. Those time points were also remarkable for a decrease in the functional activity of CD8⁺-lymphocytes equivalent to Granzyme B (CD8⁺Gr⁺) concentrations. CD3⁺CD4⁺-cells expressing early activation marker (CD25⁺), CD4⁺CD25⁺Foxp3⁺ lymphocytes, CD154⁺ (CD40L) expressing cells remained unchanged within one month of follow-up (Table 5).

Starting from day 12 up to one month of the intervention, the adaptive humoral immune response was characterized by an increase in B-cells. From days 4–5 onwards, B-lymphocytes expressing CD40 receptor mediating cell-to-cell communication increased and remained significantly elevated after 1 month of the follow-up. It should be noted that the functional activity of B-cells producing immunoglobulins A, M, and G did not change significantly within one-month follow-up. Significant trends could be demonstrated in CIC profiles, with their levels increasing on days 4–5 postoperatively and gradually decreasing by the end of the follow-up period (Table 6).

Thus, the results of follow-up immune response monitoring in the patients subjected to CABG are characterized by varying patterns at different time points and demonstrate the differentiated involvement of adaptive defense mechanisms.

Discussion

In this study, the IHD patients were characterized by a decrease in peripheral naïve T-lymphocytes bound to antigen-presenting cells to differentiate into different T-helper subpopulations. It should be emphasized that in addition to a decrease of circulating naïve T-cells, the IHD patients had a higher proportion of CD4⁺CD45R0⁺ memory T-lymphocytes subpopulation compared to the control group. The obtained results are consistent with the authors who demonstrated the correlation between memory T-lymphocyte counts and the severity of coronary atherosclerosis, with the highest CD4⁺CD45R0⁺ levels in patients with acute IHD [4][5].

Characterization of T-cell activation performance was inconclusive and related to immunogenic dysregulation. There was an increase in T-lymphocytes expressing early activation marker CD25⁺ and a decrease in T-lymphocytes expressing late activation marker HLA DR⁺. The results presented in this paper are in line with the findings published by Golderova et al. (2011), who reported an increase in CD25-expressing T-lymphocytes in patients with multifocal coronary artery disease with no relevant changes in single-vessel coronary lesions [6].

An elevated expression of this marker indicates the potential for the T helper-mediated activation of effector mechanisms. This was confirmed by the evidence of the increased IL-2 (CD25) receptor expression on CD4+ T-cells. The obtained data are compatible with the results of the authors who have shown the increase in this marker specific for more severe forms of IHD (acute coronary syndrome and unstable angina pectoris) and its association with increased serum IL-6 and IFN-γ [7][8]. A decrease in T-lymphocytes expressing late activation marker HLA DR may be due to the fact that different stages of atherosclerosis are characterized by morphologies showing CD3+HLADR+lymphocytes in atheroma but not in peripheral blood flow [9]. There are publications reporting a decrease in HLA DR expression on peripheral lymphocytes in stable IHD and an increase related to myocardial infarction and unstable angina [8][10].

Although CD4⁺ T-lymphocytes expressing early activation marker CD 25⁺ increased, a decrease in CD4⁺CD25⁺Foxp3⁺ cells performing as an immunoregulatory subpopulation was observed. A decrease in the peripheral counts of these cells is one of the contributors to the development of autoimmune aggression [11]. It is associated with more severe coronary atherosclerosis [12]. Regulatory T cells (Treg cells) display a suppressor function by synthesizing anti-inflammatory cytokines IL-10, IL-35, and THFR-1β [5]. Moreover, oxidized low-density lipoproteins (LDLs) have been found to inhibit the expression of the Foxp3 regulator in CD4⁺CD25⁺-lymphocytes, resulting in that they lose their immunoregulatory function [13]. Moreover, the patients with atherosclerosis had lower immunosuppressive activity of CD4⁺CD25⁺Foxp3⁺ along with elevated serum TNF-α [14]. It should be highlighted that a similar pattern has been reported in this paper and is consistent with the authors who established a decrease in CD4⁺CD25⁺Foxp3⁺ cells in patients with acute coronary syndrome without ST-segment elevation [15] and angina pectoris [16]. T-lymphocyte responses to upregulation stimuli facilitate further IHD-specific immunopathogenesis pathways associated with proliferation or apoptosis. In atherosclerotic IHD, dissociation of these processes was disclosed as contrasted with the control group. A CD3⁺ subpopulation expressing an apoptosis marker (CD95⁺) and annexin-stained CD3⁺ cells, i.e. those involved in the programmed cell death process, decreased significantly [6]. This was also suggested by Pasqui et al. (2003), who showed this correlation in patients with myocardial infarction vs. patients with stable angina [8]. The apoptosis/proliferation imbalance with a gain of apoptosis inhibition and higher proliferative activity of T-lymphocytes maintains progressive inflammation in atherosclerosis and may be indicative of autoimmune response, including to endogenous antigens [17][18].

In this study, elevated CD154 expression on CD4⁺ lymphocytes, as well as CD40 expression on B-lymphocytes, reflects the activation of T-B interaction, with the latter to be upregulated. A coreceptor molecule on T-lymphocytes and a receptor on B-lymphocytes transform the B-cell population into plasma cells actively producing different classes of immunoglobulins, mostly IgG, whereas the absence of such interaction largely results in IgM production. In addition, this interaction contributes to the upregulation of cytotoxic T-lymphocytes and increases their effector potential [19]. This suggestion is consistent with the results of this study, which demonstrates an increase in both absolute counts of CD8+ lymphocytes and their cytolytic activity mediated by intracellular Granzyme B levels [20].

Analysis of CD3⁺ lymphocyte profiles showed that the IHD group experienced greater apoptosis inhibition post CABG, as well as a decrease in annexin-positive cells and naïve T-lymphocytes, which may be related to the use of glucocorticosteroids in the early postoperative period and surgical stress. The early postoperative period was remarkable for slight cyclic changes in Treg cells, which probably reflects a decrease in the proinflammatory immune response as evidenced by a decrease in cytotoxic T-lymphocyte counts and an increase in IRI levels. CD4⁺CD25⁺Foxp3⁺ lymphocytes are known to control the processes of proliferation/apoptosis, and a decrease in Treg cells is associated with their imbalance [11]. This suggestion could be demonstrated by a decrease of both CD3⁺CD95⁺ cells and T-lymphocytes involved in the programmed cell death vs. reference values.

There were insignificant changes in CD154 T-lymphocytes in the early postoperative period compared to baseline elevations. An increase in B-lymphocytes and CD40 B-cells was also reported. These changes were probably due to the persistent autoimmune response to oxidized LDLs.

Conclusion

Thus, the dissociation of T-lymphocyte activation, maturation and apoptosis, suppression of immunoregulation, and upregulation of cell cooperation could be observed in coronary artery disease. The results of follow-up immune response monitoring in the patients subjected to CABG are characterized by varying patterns at different time points and demonstrate the differentiated involvement of adaptive defense mechanisms.