Coronary heart disease (CHD) is the leading cause of death all over the world, and the prevalence of cardiovascular diseases is increasing year by year (Heidenreich et al., 2011; Chua et al., 2022). The major sensitive diagnostic methods for CHD currently include electrocardiography, ultrasound, angiography and cardiac damage markers (Infante et al., 2017). However, these tests are limited by their expensive price, or the time required for testing, etc. Searching for new sensitive indicators and early detection of CHD can effectively guide management to prevent CHD related death (Gatto and Prati, 2020; Chow et al., 2021).

Coronary atherosclerosis is the main cause of CHD. It has been recognized that atherosclerosis is a complex chronic inflammatory disorder mediated through both adaptive and innate immunity (Taleb, 2016; Ruparelia and Choudhury, 2020). Previous studies demonstrated that proinflammatory cytokines (such as IL-1, IL-6, IL-1β and TNF α) participated in the development of CHD by endothelial damage, induction of monocyte-endothelial adhesion and subendothelial migration (Lusis, 2000; Ridker, 2019), and some large clinical trials have focused on the effects of targeted proinflammatory cytokines inhibition therapy on CHD (Ridker et al., 2017; Ridker et al., 2019). Pro-inflammatory cytokines are usually produced by immune cells such as macrophages, monocytes and dendritic cells. White blood cell (WBC), as the body’s predominant immune cells, have traditionally been recognized as markers of acute or chronic inflammation. Some observational studies have reported WBC and its subpopulations (lymphocytes, neutrophils, monocytes, eosinophils, and basophils) are associated with CHD (Wheeler et al., 2004; Madjid and Fatemi, 2013). However, the evidence is largely based on small sample studies, which are prone to systematic bias and do not support a causal association.

Mendelian randomization (MR) study is one in which genetic variants are used to investigate the causal relationship of a biomarker on risk of diseases (Smith and Ebrahim, 2003; Ference et al., 2012; Dale et al., 2017; Luo et al., 2021). Compared to the traditional observational studies that are susceptible to confounding bias and selection bias due to confounding factors, differences in study subject selection, etc. MR approach can be thought of as an analogy to a randomized controlled clinical trial (RCT) without the main biases of classical epidemiological studies (Bennett and Holmes, 2017). Recently, there have been a few studies using this genetic approach with large genome-wide association studies (GWAS) datasets to find causal effects of novel risk factors for CHD such as diet-derived circulating antioxidants, low-density lipoprotein cholesterol level, adiposity and body fat distribution and so on. (Ference et al., 2012; Dale et al., 2017; Luo et al., 2021). Therefore, there is an urgent need for MR studies to identify the causal relationships between WBC cell counts and CHD to help find targeted prevention and therapy for CHD.

In the present study, we employed MR analysis using data generated from large GWAS analysis to examine whether WBC and its major subpopulations may be causally linked to CHD.

In order to develop CHD prevention strategies, it is important to identify and compare causal risk factors in populations. In this study, we evaluated the causality of blood cell counts on CHD by MR analyses, we found that WBC count and the neutrophil cell count were associated with an increased risk of CHD, Additionally, no causal relationship was found between blood counts among other WBC types and CHD.

Currently, researchers have recognized that inflammation is a crucial factor in the pathophysiology process and mechanisms of clinical complications of CVD (Libby et al., 2016). Due to the inexpensive and widely-available, WBC and its subpopulation counts test have been the most commonly used test for inflammation. A small number of studies claim no association between WBC counts and CHD. In NHEFS cohort study, the association between neutrophil count and CHD disappeared after adjusting for age, gender, smoking and other risk factors (Gillum et al., 2005). However, more studies have demonstrated that WBC counts were associated with CVD and non-CVD mortality (Wheeler et al., 2004; Madjid and Fatemi, 2013; Welsh et al., 2018; Chan et al., 2022). Among the elderly Japanese-American male population cohort with 8 years of follow-up, high WBC count and neutrophil count were confirmed to be related to increased risk of incident CHD (Karino et al., 2015). Furthermore, recent data from UK biobank has shown that elevated neutrophil counts are particularly associated with a higher risk of CVD among the general population (Welsh et al., 2018). Our study is the first MR to explore the relationship between WBC counts and CHD, which effectively reduces the bias of observational line studies and clarifies the causal relationship between the two. Consistent with most previous studies, our results also show a strong association between WBC and neutrophil count with CHD. Further studies are needed to determine cut-points of WBC and its subpopulation and anti-inflammatory treatment protocols for CHD patients in the future.

According to research on the mechanism, leukocytes are engaged in the atherosclerosis process. Leukocytes can interact with structurally intact but dysfunctional arterial endothelium, and the development of a proinflammatory and prothrombotic environment following endothelial dysfunction leads to increased recruitment of leukocytes, lipids, smooth muscle cells, fibroblasts, and platelets to the arterial wall. Repeated cycles of this process lead to proliferation of the intimal lining of the vessel wall and formation of atherosclerotic plaques (Brydon et al., 2006). In addition, adhesion molecules allow leukocytes to attach to the endothelium monolayer when inflammation occurs. In the arterial plaque, pro-inflammatory cytokines stimulate leukocyte migration into the intima (Ilhan and Kalkanli, 2015). Oxidative processes play a significant part in the etiology of atherosclerosis. It has been hypothesized that white blood cells counts are implicated in the oxidative destruction of LDL that promotes atherosclerosis. (Wang et al., 2013).

Circulating neutrophils also contributed to pathophysiology of vascular disorders, particularly in the development of atherosclerosis. In mice, the researchers discovered that the size of the growing lesion was proportional to the number of circulating neutrophils (Silvestre-Roig et al., 2020). Complex structures called neutrophil extracellular traps (NETs), are released from suicide neutrophils. These traps contain proteins of nuclear, granular, and cell membrane origin as well as chromatin. Both human and mouse atherosclerotic lesions and artery thrombi have been shown to contain these reticular structures (Döring et al., 2017). When neutrophils come into contact with cholesterol, they generate NETs, and these NETs trigger the release of cytokines from macrophages, activating T helper 17 cells and so boosting immune cell recruitment to atherosclerotic plaques (Warnatsch et al., 2015).

Our study has some limitations. First, there was some horizontal pleiotropy in our study, which may result in a biased estimation of causal inference. However, we performed MR-PRESSO and the results remained consistent after removing the outlier SNPs. Secondly, the samples in our study were European and East Asian, which limits our results from being generalizable to other ethnic groups. The sample size of the East Asian GWAS data used here (N = 78,744) is much smaller than the sample size of the European population GWAS data (N = 563,946). Future research is needed to explore racial differences further. Despite relative weaknesses, our study also has several strengths. Our study used a large sample of GWAS data to explore the association between blood cell counts and CHD, which avoid confounding measurement error and residuals in observational studies. And we performed a series of sensitivity analyses to assess heterogeneity and pleiotropy, and we also conducted MR-PRESSO to remove the outlier SNPs. In addition, the present study conducted MR analysis, which uses genetic IVs analysis to model the randomization process that underlies causal inference in RCTs, thus was less susceptible to reverse causality bias. (Smith and Ebrahim, 2003).

In conclusion, in this two-sample MR analysis, we demonstrated evidence for a potential causal link between WBC and neutrophil cells and CHD. In addition, causal relationships between blood cell counts among other WBC types and CHD were not found. In order to validate the results and investigate the mechanisms underlying these associations, future studies are still needed. The findings might help inform prevention strategies and suggest potential therapeutic targets for CHD.