Monocyte subset functional alterations with increased cardiovascular risk factors

Abstract

Atherogenesis is dependent upon monocyte influx into the vessel wall. In humans, three monocyte subsets exist, classical, intermediate and non-classical. Of these monocyte subsets, clinically, the intermediate monocytes are considered to be a potential treatment target in cardiovascular disease (CVD), due their significant elevation and inflammatory nature. However, whether their elevation in CVD would translate to a functional contribution to atherogenesis is currently unknown. In addition, intermediates are also known to adhere more to endothelium than the classical subset as described in murine models. However, adhesion does not necessarily equate to migration as classical subset migrates at a greater rate to the plaque (in response to chemokines such as CCL2/MCP-1) than other subsets. Further research in understanding the contribution of each monocyte subset in CVD is needed. Here we aimed to determine the phenotype of monocyte subsets in individuals (who were generally healthy) relative to their lipid levels, as monocytes displaying an atherogenic phenotype in those with perturbed lipid levels would indicate whether the monocyte functional changes are occurring in the circulation, prior to entry into the vessel wall. First, we determined the inflammatory nature of monocyte subsets by assessing the monocyte subset cytokine production and expression of inflammatory/anti-inflammatory markers (Chapter 4). The findings from cytokine production (upon LPS stimulation) and surface marker expression combined suggest that intermediates and non-classicals are more inflammatory than the classical subset. This is consistent with the literature but we extend the understanding to show that this remains the case even when including individuals with an altered lipid profile. However, for the intermediates and non-classicals, being more inflammatory than classicals does not mean that they will have the greatest contribution to atherosclerotic plaque development as their level of recruitment into the plaque is also a factor. Recruitment depends on two key steps, adhesion and migration – that are regulated by adhesion molecules and chemokine receptors. Therefore, we next assessed monocyte subset adhesive and migratory nature by the expression of various adhesion molecules and chemokine receptors. From assessment of expression of adhesion molecules (Chapter 5) and chemokine receptors (Chapter 6) on monocyte subsets, we found that they differentially express various adhesion molecules and chemokine receptors which ultimately, would result in their distinct trafficking patterns and thus differential functional consequences. This suggests that all monocyte subsets possess a potential to adhere or migrate into the plaque and thus, raises a question whether targeting intermediates alone would sufficiently reduce CVD risk. Further, we assessed whether the changes in cytokine production and surface marker (inflammatory, adhesion molecule and chemokine receptor) expression occurs in a gradual or distinct manner at the subset level by flow cytometry. Our findings indicate that the changes in cytokine production and surface marker expression levels varied incrementally from one subset to another. We also assessed monocytes at the cellular level by flow cytometry, where we suggest that the monocytes in some individuals are likely to be entering the circulation in a pro-atherogenic state as differences were evident in the classical monocytes of one individual compared to another. This finding adds to the growing body of evidence that challenges the paradigm that monocytes are just precursors to macrophages and dendritic cells. Importantly, we found that monocyte expression of pro-atherogenic markers varies between the participants with the degree of spread (coefficient of variance) for the monocytes being comparable. This suggests that no one particular monocyte subset displays an increased proatherogenic phenotype but instead, all do, and this acquisition of a pro-atherogenic phenotype by monocytes occurs even before they differentiate into the intermediate subset. We further explored the relationship between the pro-atherogenic marker expression by the monocyte subsets and we found that participants’ pro-atherogenic marker expression by one subset correlated with that of the next. This indicates that the pro-atherogenic state acquired throughout differentiation (from classical to non-classical) is dictated by the pro-atherogenic profile of the emerging classical subset. This finding suggests that no particular subset is proatherogenic but rather all, as they recapitulate the pro- therogenic phenotype of a precursor, the classical subset or perhaps, bone marrow cells. The monocyte subset pro-atherogenic state (increased monocyte inflammation, adhesion or migration) was related to the participants’ lipid levels. The key result was that not only the cytokines, but also the expression of most of the pro-atherogenic markers (M1 markers, adhesion molecules, chemokine receptors) on monocyte subsets, were inversely associated with participants’ Apo A1 (or HDL-C) levels and, importantly, this was evident for all monocyte subsets. This finding suggests that altered lipid levels may be a key factor promoting monocyte pro-atherogenic changes. Lastly, we assessed whether the relationship between the monocyte pro-atherogenic state (chemokine receptor expression) and participants’ lipid levels could be seen functionally by in vitro static migration assay in response to chemokines, as a preliminary study (Chapter 6). Interestingly, we observed an inverse relationship (trend only) with monocyte subset migration and participants’ cholesterol and LDL-C levels suggesting that the higher levels of cholesterol and LDL-C negatively impact on the degree of monocyte migration in these participants. Thus, overall it is noteworthy that the changes in expression of chemokine receptors on monocyte subsets may not necessarily translate to direct changes in theirmigration in vitro. Taken together, the findings from the present study provide a fundamental change in the understanding of the importance of monocytes in atherosclerosis (particularly, their role and functional contribution i.e. adoption of pro-atherogenic phenotype) as we have shifted the paradigm from the need to target the intermediate monocyte subset, to targeting specific functions of monocytes overall. Importantly, with the changes evident in individuals that have perturbed lipids, but no diagnosed CVD, this study has drawn attention to proatherogenic changes that are occurring quite early sub-clinically in association with an altered lipid profile. This could be contributing to plaque development as monocytes would enter the vessel wall primed to become pro-atherogenic macrophages.