There is a growing body of evidence regarding the negative impact of air pollution (especially its organic part) on the functioning of the cardiovascular system (18, 19). However, the role of inorganic constituents of the particulate matter on the development of different cardiovascular diseases (CVD) including atherosclerosis remains less clear. The main finding of our study is that the two oxides: SiO2 and Fe2O3 (essential components of the inorganic part of airborne particulate pollutants) influence the atherogenesis and lesions morphology in apoE-/- mice model in different fashion. Our data indicate, that the macrophages are the common target of NPs pro-atherosclerotic actions, resulting in altered phenotype and activation of these cells (Figs. 4 and 5).
Macrophages are the pivotal cells of innate immunity and key regulators of the inflammatory response, both in terms of the antibacterial action as well as the protection against tissue damage (resolution of inflammation) (20). In general, macrophages can be polarized into two different phenotypes: classically activated type M1, characterized by high-antigen-presenting capacity and production of pro-inflammatory cytokines, and type M2, which displays anti-inflammatory actions (16). Consequently, M1 macrophages promote adaptive Th1-oriented immune responses against various pathogens, while M2 cells stimulate Th2 branch of immunity and plays a role in resolution of inflammation and wound healing (21). It is well recognized that M1 macrophages maintain atherogenic low-grade inflammation in the vascular wall and their increased content in atherosclerotic lesions promotes plaque instability. On the contrary, M2 cells have been found to be associated with atherosclerosis regression (20).
It has been shown in vitro that nanoparticles formed by silica and iron compounds can stimulate macrophages to induce reactive oxygen species (ROS) production, activation of NLRLP3 inflammasome and cytokine release (22–24). What is more, several studies indicated a strong ability of silica NPs (SiNPs) to differentiate macrophages towards M1 phenotype (25–27). Our data are in line with these data and extend the observations of the influence of SiNPs on atherogenesis in apoE-/- mice model. We evidenced, to our best knowledge for the first time, that the progression of atherosclerosis in apoE-/- mice exposed to SiNPs was associated with the skewing macrophage polarization in atherosclerotic plaques and peritoneum towards M1 phenotype, as evidenced by immunohistochemical and molecular measurements (Figs. 4, 5 and 6).
By far only one study examined the influence of SiNPs on the progression of atherosclerosis in apoE-/- mice (14). In this study, the macrophage phenotype was not investigated, while the acceleratory effect of SiNPs on the progression of atherosclerosis has been attributed to lipid accumulation in macrophages caused by endoplasmic reticulum (ER) stress-mediated upregulation of CD36 expression. Indeed, ER stress has been recently linked to the regulation of macrophage differentiation towards M1 phenotype (28, 29). It is tempting to hypothesize that ER stress may represent a link between SiNPs action and modulation of macrophage polarization. However, ER stress has been also found to contribute to the alternative activation of these cells towards M2 phenotype, thus the role of ER in regulating macrophage phenotype is more intricate (30), while other mechanisms of the influence of SiNPs on the macrophage phenotype may also be involved in the process. More, it has been demonstrated, that SiO2 could polarize primary macrophages toward the M1 phenotype by up-regulation of CD14, a co-receptor of toll-like receptor 4 (TLR4), which involves NFκB pathway, similarly to bacterial lipopolysaccharide LPS (25).
An interesting mechanism of SiNP's influence on the macrophage phenotype may be the enhancement of mitochondrial dysfunction and deregulation of cell metabolism (31). Our study does not make it possible to verify this assumption. On the other hand, in vitro stimulation of endothelial cells (HMEC-1) by low concentrations of SiO2, but not Fe2O3 results in mitochondrial damage (our unpublished data). The answer to the question whether any of the abovementioned mechanisms are responsible for the SiNP-elicited changes in the macrophage polarization in apoE−/− mice requires further research.
Several in vitro studies have shown toxic effects of various iron compounds on cells involved in atherogenesis, such as endothelial cells, smooth muscles and cells of the immune system (i.a. macrophages and lymphocytes) (32–34). They were mainly attributed to the ability of iron compounds to increase the production of ROS, which is widely recognized as one of the most important mechanisms damaging the vessel wall and contributing the progression of atherosclerotic lesions (35). To our knowledge, this study is the first to test the effect of inhaled administration of iron nanoparticles on the development of atherosclerosis in vivo. We evidenced the differences in the SiO2 - and Fe2O3-mediated effects in terms of their influence on the development and morphology of atherosclerotic plaques (Figs. 2 and 3). The SiO2 augmented the atherogenesis in the aortas of apoE-/- mice (Fig. 2), but did not change the content of macrophages in plaques (Fig. 3). On the other hand, Fe2O3 did not influence the development of atherosclerotic plaques (Fig. 2), but significantly increased their overall CD68-positive cell content (Fig. 3). Surprisingly, this was associated with decreased proportions of both M1 and M2 cells, however, the latter did not reach the statistical significance (Fig. 4B). One can speculate, that FeNPs induce complex changes in lesion morphology, possibly including the de-differentiation of different cell types within the aortic wall. The literature on the in vivo effect of iron NPs on macrophage polarization is sparse and still inconclusive. In recent in vivo study NPs of ferrumxytol – a carbohydrate-coated, superparamagnetic iron oxide nanoparticle – inhibited tumor growth by inducing M1 macrophage polarization. (36). It therefore seems that the macrophage response to FeNP might largely depend on the experimental model.
In contrast to the effect on M1, our results regarding the effect of NPs on the abundance of M2 macrophages are less conclusive. The decreased level of arginase-1 mRNA in aortas of SiNPs-treated mice might reflect decreased content of M2 macrophages, however, this is neither confirmed by histochemical staining in plaques nor by flow cytometry of peritoneal macrophages isolated from SiNPs-treated mice. Clearly, the influence of NPs on the macrophage polarization requires verification in further studies.
T lymphocytes were shown to play important role in the early stages of atherosclerosis development. It has been demonstrated that the disruption of the balance between pro-atherogenic T-helper 1 and anti-atherogenic T-helper 2 cells, as well as between Th17 and regulatory T lymphocytes (Treg) may contribute to atherosclerosis (21). Previous reports have shown that the exposure to air pollution PMs affects T cells populations in mice by promoting Th1 and Th17 response (37–39). Moreover, several studies indicate that air pollutants could reduce the level of suppressor regulatory T cells in vivo (40, 41). In our setting both silica and iron NPs promoted polarization of T lymphocytes in the spleen towards Th17 cells, thus shifting the balance between Th17 and Treg in favour of activation of inflammatory and immune response (Fig. 7). Both compounds also increased Th1/Th2 ratios, however this effect depended more on decrease of Th2 cells and was less pronounced.
Another difference in the action of SiNPs and FeNPs was revealed by the measurements of the levels of selected cytokines in the plasma. Inhaled administration of SiNPs, but not FeNPs, was associated with a significant increase in plasma levels of the CCL11 (eotaxine). The rise of this eosinophil-specific chemokine may suggest the greater role of these cells in the pulmonary inflammatory response induced by SiO2, as compared to Fe2O3. Interestingly, it has been shown, that silica nanoparticles in particular may act as adjuvants to enhance allergic diseases in mice (42). Whether and to what extent eosinophils could be involved in atherogenesis enhanced by inhaled SiNPs remains to be tested.
Clearly, our study has a major limitation regarding the NPs delivery system. Although the whole body exposure system, allowing for unrestrained movement of animals during inhalation and reducing their stress during experimental procedures, is one of the best solutions enabling for the consistent environmental exposure to administered materials, it has several drawbacks. The compounds are not delivered directly to the respiratory tract thus nor the estimation of the exact quantity of air inhaled by the individual mice neither the precise determination of the exposure dose is impossible. Moreover, mice exposed to NPs in a chamber may cluster in a group, while part of the airborne material can settle on their hair or cage bottom and ultimately end up in the digestive tract. Collectively, these factors could lead to variation in the amount of aspirated compounds among animals (43, 44). To address these issues we expanded the size of the experimental groups, as indicated by the power analysis. Our study can also provide important, but only an indirect indication that inhalation with NPs, via influence on macrophage content and/or phenotype in atherosclerotic plaques may reduce plaque stability and increase the risk of plaque rupture. As the plaques in apoE−/− mice are not prone to the rupture, this model is not relevant for assessment of risk of rupture and association of such complication with inhalation of NPs should be estimated directly in other experimental models.