In the present study, we investigated the effects of exposure to air pollution on cardiac morphology of lupus-prone NZBWF1 female mice. The exposure to PM2.5 was performed during a period of 4 months to evaluate the long-term effects of chronic exposure to air pollution. We started the exposure when animals were 3 months-old, the expected age for beginning of clinical manifestations of SLE in this model [18]. Our results corroborate preexisting data showing that exposure to air pollution may aggravate or be a risk factor for lupus disease and that cardiac alterations may be accelerated due to this exposure. In our study, the daily dose of PM2.5 was settled in 600ug/m³, referred to the estimated daily dose that citizens of big cities are exposed to [19, 20].
SLE is an autoimmune disease that affects various systems and has a higher prevalence among young adult women. Besides genetic factors, different environmental factors are being investigated as triggers for the development of SLE in predisposed individuals. Exposure to UV light and air pollution are being considered as important factors for SLE, and recently our group showed that lupus-prone mice present an aggravation of clinical manifestations of SLE [17].
The importance of environmental factors in SLE has been increasing because genomic studies showed that genetic mutations do not fully explain the development of lupus. Other environmental factors that may be linked with SLE include exposure to silica and infections, and life habits such as tabagism and alcohol consumption may also be associated with SLE [21]. Considering the large diversity of factors that may interfere with the triggering or the progression of lupus, animal studies can contribute significantly for the discovery of main risk factors and for the elucidation of physiopathological mechanisms of SLE [22].
SLE is a complex and multifactorial disease with diverse clinical manifestations including joint inflammation and neuronal, renal, cutaneous, gastrointestinal and hematological alterations. Also, SLE patients may present increased risk for cardiovascular diseases. A systematic review composed of 28 studies showed that the risk for the development of cardiovascular diseases in lupus patients is two-fold higher in comparison to healthy subjects and it is one of the main causes of death among SLE patients [23]. However, there is a lack of studies that focus on cardiovascular outcomes, especially those regarding the exposure to air pollution in individuals predisposed to SLE.
Hypertension has a high prevalence among SLE patients [24, 25, 26, 27, 28, 29, 30]. However, the mechanisms are not elucidated. Factors such as age, sex, ethnics and obesity combined with immune alterations, inflammation, impairments of renin-angiotensin system and side effects of drugs, and environmental factors, such as air pollution may contribute to this association [31, 32, 33, 34].
Renal function is important for the long-term control of arterial pressure. However, studies that investigated the association between renal alterations and cardiovascular diseases are controversial. For example, Ward and Studenski [35] and Petrin et al [36] verified that hypertension occurs independently from nephritis and more recently Shaharir and colleagues [30] observed that in a cohort of SLE patients, 53% were hypertensive, but did not present nephritis. Nevertheless, impairments in the glomerular filtration rate (GFR) and in the renal plasma flow are present in some SLE patients [37, 38].
In our previous study, in which lupus-prone female NZBWF1 mice were exposed to air pollution, we could observe decreased survival, increased circulating neutrophils, accelerated onset of proteinuria and increased kidney weight with expansion of the cortex [17]. Here, our aim was to evaluate morphological outcomes over cardiovascular system using the same model of lupus-prone female mice exposed to air pollution. In our study, we evaluated only lupus-prone female mice because in humans SLE is more prevalent in young adult women than men (ratio of 3:1), and this gender difference proportion is even higher in children (ratio of 9:1) [39, 40]. And although we could not perform an evaluation of cardiac function, morphological alterations in the heart were observed that have potential to promote dysfunctions.
Although there are studies demonstrating that air pollution contributes to increase in body weight and predisposition to metabolic syndrome [41, 42, 43], and showing a positive correlation of exposure to air pollution with increased body weight [44, 43], our results indicate absence of significant difference between the group exposed to air pollution and the control group. Some studies showed a protective effect of estrogen in females, resulting in resistance to weight gain [45], that may explain why we did not find increased body mass in the exposed group. Regarding the heart weight, evaluation of cardiac trophism was performed by dividing heart weight by body weight (normalization). However, we observed no differences between groups.
A recent study showed that NZBWF1 mice exhibit a spontaneous increase in arterial pressure at 34-weeks-old and this alteration are preceded by alterations in circulating levels of autoantibodies, in renal hemodynamic function and in glomerular injury. However, in this study, morphological alteration in the cardiovascular system were not accessed [46].
In the present study, the animals were evaluated with 28-weeks-old. Thus, in comparison with the study of Dent and colleagues [46], our animals were younger and, therefore, the alterations observed can be in an initial stage, probably been accelerated by the exposure to air pollution. Besides the cardiomyocytes hypertrophy, various alterations, such as apoptosis, fibrosis and alterations in coronal circulation could be observed and may explain the increased risk of cardiovascular outcomes [47]. Also, the quantification of the area composed by collagen fibers demonstrated an increase in the percentage of cardiac fibrosis in the animals exposed to air pollution for 4 months, corroborating previous data that demonstrate the influence of air pollution in the deposition of collagen and suggestive alterations in cardiac function [19, 11].
In hypertensive rats the chronic exposure to PM2.5 induced cardiac dysfunction end cardiomyocyte hypertrophy, and these alterations were subsequent to increased blood pressure and inflammation [48], indicating that the alterations observed in the present study can be associated with alterations in blood pressure and cardiac function.
The investigation of alterations in the myocardium was performed by the analysis of relative area of cardiomyocytes. Our results did not show increased heart weight, however in the evaluation of cardiac morphology, we observed that animals exposed to air pollution showed an increased surface area of cardiomyocytes, indicating that air pollution is able to promote cardiac hypertrophy. This result is similar to other studies published elsewhere that observed these alterations in rodents [19, 49].
Immunohistochemistry was used to evaluate inflammatory markers, cell death and growth factors. To evaluate the dynamics of endothelial growth we used the marker VEGF (vascular endothelial growth factor), which has an important role in the development of new vessels and in the increased vascular permeability [50]. In addition, serum levels of VEGF are used as an independent predictor of SLE activity. However, there are no studies showing alterations in cardiac muscle in SLE patients [51, 52]. Nevertheless, our results did not demonstrate any significant difference in VEGF marker when comparing the groups, diverging from studies that showed increased levels of VEGF associated with acute exposure to PM2.5 [53, 54]. However, the animal model, the dose and the time of exposure were different from our study.
One of the inflammatory markers analyzed was interleukin-17A (IL-17A), an important proinflammatory molecule. Their functions include the recruitment of neutrophils, responses against extracellular pathogens and induction of inflammation [55], and it is also associated with autoimmune diseases [56]. Studies evidenced that IL-17 blocking can decrease SLE manifestations [57], suggesting that IL-17 is an important molecule to be investigated in the present study. Our results demonstrate a tendency of increased expression of IL-17A in cardiac tissue. However, no statistical differences were observed between groups.
We also analyzed the interleukin IL-1β, a proinflammatory cytokine produced mainly by macrophages, monocytes and dendritic cells [58, 59]. In our results, we observed increased marker for IL-1β in mice exposed to PM2.5 when compared to control group. These data corroborate with previous studies where exposure to air pollution was directly associated with increased serum and molecular levels of IL-1β [60, 61].
We also evaluated the apoptosis in the cardiac tissue of animals exposed or not to PM2.5. Apoptosis is a mechanism of programmed cell death, where in physiological conditions is a system of maintenance of cells and replacement of tissues [62]. Pathological apoptosis, in the other hand, can be triggered by harming factors that cause cell injury. For example, PM2.5 was evidenced as an inductor of apoptosis in epithelial bronchial cells [63].
Caspase-3 is a largely utilized marker in immunohistochemistry for analysis of apoptosis [64]. Serum levels of caspase-3 and caspase-9 are increased in SLE patients when compared to healthy subjects [65]. As mentioned, in the present study we analyzed the expression of caspase-3 in cardiac tissue and our results demonstrate a tendency of increase. Caspase-3 was evaluated because intrinsic cell death is necessary for the development of cardiomyocyte hypertrophy [66]. However, this outcome corroborates results described previously [67, 68, 69] where was observed increased expression of caspase-3 in animals exposed to air pollution.
C3 complement is an inflammatory mediator that composes the complement system and is utilized as an important marker of SLE physiopathology. When activated, it can cause cytolysis, leukocyte recruitment and inflammation in tissue where it is deposited [70]. We performed the analysis of C3 expression and could observe increased deposition of C3 in the myocardium of animals that were exposed to air pollution. This outcome corroborates data found in literature that show increased deposition of immune complexes in different tissues, including the cardiac tissue, when the disease is active [71, 72].
The evaluation of cardiac impairment in animal models of spontaneous SLE (BXSB, MRL/lpr and NZBWF1) shows that all lineages develop lesions in coronary vessels, myocardial strokes characterized by cardiomyocyte necrosis and focal hemorrhage, infiltration of leucocytes and macrophages, and formation of cicatricial tissue. In our study we could not observe these alterations. Deposits of IgG and C3 complement are observed in atrium and ventricle vessels of all lupus-prone lineages and, therefore, immune complexes deposition may have an important role in the subjacent pathogenesis [73, 74].
Another important characteristic marker of SLE is the production of anti-DNA antibodies that is directly associated to disease activity, being used by many studies as a parameter for diagnosis [75, 76]. In this way, we performed an assay to detect these antibodies in our animals to verify the plausible interference of the exposure to PM2.5 in autoantibody production. We were expecting to obtain increased levels of anti-DNA antibodies. However, we did not observe statistical differences between exposed and control groups. Both groups produced anti-DNA antibodies confirming this SLE phenotype in NZBW animals.
In summary, in this present study, we could observe a strong influence of exposure to PM2.5 in the modulation of production of inflammatory markers in the myocardium of lupus-prone animals, and consequent morphological alterations in this tissue. However, the mechanisms involved are not elucidated. More studies with genetic and molecular tools are necessary for a better understanding of interaction between PM2.5 and cardiac manifestations in SLE.