Research February 2018 | Volume 126 | Issue 2
Concentrated Ambient PM2.5-Induced Inflammation and Endothelial Dysfunction in a Murine Model of Neural IKK2 Deficiency
1Department of Medicine Cardiology Division, University of Maryland School of Medicine, Baltimore, Maryland, USA
2Department of Environmental Health, School of Public Health, Fudan University, Shanghai, China
3Department of Electrocardiography, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, China
4Department of Occupational and Environmental Health, School of Public Health, Nantong University, Nantong, China
5Department of Pathology, Hubei University of Science and Technology, Xianning, Hubei, China
6Department of Endocrinology, The People’s Hospital of Zhengzhou University (Henan Provincial People’s Hospital), Zhengzhou, Henan, China
7Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai, China
8Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, Maryland, USA
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- Exposure to ambient fine particulate matter (PM2.5) is associated with cardiovascular mortality, but underlying pathophysiologic mechanisms are not fully understood. Hypothalamic inflammation, characterized by the activation of Inhibitor kappaB kinase 2/Nuclear factor kappaB (IKK2/NF–κB) signaling pathway, may play an important role in the pathogenesis of cardiovascular diseases. We recently demonstrated that hypothalamic inflammation is increased in mice exposed to concentrated ambient PM2.5 (CAP).
- In the present study, we used a neuron-specific IKK2 knockout mouse model to examine the role of neural IKK2 expression and hypothalamic inflammation in the pathophysiologic effects of PM2.5.
- We assessed inflammatory and vascular responses in Nestin–creIKK2flox/flox (IKK2Neu–KO) and littermate Nestin–creIKK2flox/+ (control) mice after 4 mo of exposure to filtered air (FA) or CAP.
- CAP exposure was associated with significantly higher tumor necrosis factor-α (TNFα) and interleukin (IL)-6 mRNA in the hypothalamus of control mice, but not IKK2Neu–KO mice. In addition, CAP exposure–induced increases in bronchoalveolar lavage fluid (BALF) leukocytes, pulmonary macrophage infiltration and IL-6 expression, plasma TNFα and IL–1β levels, adipose macrophage infiltration and IL–1β expression, and endothelial dysfunction were reduced or absent in IKK2Neu–KO mice compared with controls.
- Our findings support a role of neural IKK2 in CAP exposure–induced local and systemic pro-inflammatory cytokine expression, pulmonary and adipose inflammation, and endothelial dysfunction, thus providing insight into pathophysiologic mechanisms that may mediate effects of PM2.5 exposure. https://doi.org/10.1289/EHP2311
Received: 8 June 2017
Revised: 11 December 2017
Accepted: 12 December 2017
Published: 5 February 2018
Address correspondence to Z. Ying, HSFII S022, 20 Penn St., Baltimore, MD 21201, USA. Telephone: 410-328-2063. Fax: 410-328-1048. Email: firstname.lastname@example.org
The authors declare they have no actual or potential competing financial interests.
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Exposure to ambient fine particulate matter (PM2.5) is associated with increased risk of cardiovascular morbidity and mortality (Brook et al. 2010). However, while the majority of inhaled PM2.5 are known to deposit in the airway and not enter the systemic circulation (U.S. EPA 2009), mechanisms that link PM2.5 with cardiovascular effects remain elusive. The putative mechanisms for this include a) egress from the lung of PM2.5 components, b) autonomic nervous system (ANS) dysfunction, and c) inflammation (Brook et al. 2010). Notably, despite that inhaled nanoparticles have been identified in remote organs, the pathophysiological role of these nanoparticles remain controversial, as their concentrations are low and inconsistent (Kreyling et al. 2009; Oberdörster et al. 2002; Oberdörster and Utell 2002; Semmler-Behnke et al. 2008). PM2.5 exposure is associated with decreased heart rate variability (Brook 2005; Chen and Hwang 2005; Godleski et al. 2000; Gong et al. 2008; Magari et al. 2001; Sivagangabalan et al. 2011; Tankersley et al. 2004), indicating sympathetic nervous system (SNS) activation and/or parasympathetic nervous system withdrawal (Brook 2008; Karemaker 1999). However, because the nervous system can adapt to chronic stimulation, ANS dysfunction has been thought to play a trivial role in the mediation of the long-term health effects of PM2.5 exposure (Brook et al. 2010). Evidence that cardiovascular effects of PM2.5 are mediated by inflammation is stronger, given the established role of PM2.5 in the of various cardiovascular diseases and evidence of inflammatory responses to PM2.5 exposure in humans (Peters et al. 2001; Pope 2000; Utell et al. 2002) and various animal models (Kennedy et al. 1998; Quay et al. 1998; van Eeden et al. 2001; Vogel et al. 2005).
Putative effect mechanisms that involve ANS dysfunction or inflammation are not mutually exclusive, but may affect the same or different stages of pathogenesis, and may have synergistic effects (Brook et al. 2010). For example, ANS dysfunction may impact various inflammatory responses (Lambert et al. 2015; Pavlov and Tracey 2017), and increases in circulating pro-inflammatory cytokines/mediators resulting from PM2.5–related pulmonary or systemic inflammation may cause ANS dysfunction (Dunn 2000; Turnbull and Rivier 1999; Zhang et al. 2003). More importantly, there is emerging evidence that ANS dysfunction may also play a critical role in the long-term regulation of cardiovascular function (DiBona 2013; Froeschl et al. 2013; Simonds and Cowley 2013), which appears to be dependent on an inflammatory response in the hypothalamus. For example, rats and mice fed a high-fat diet (HFD) showed rapid increases in hypothalamic inflammation, which may contribute to HFD feeding-induced long-term effects, including obesity and insulin resistance (Thaler et al. 2012). Inhibition or deletion of IKK2 in some hypothalamic neurons was sufficient to abolish HFD feeding-induced ANS dysfunction, and subsequently, normalized blood pressure in a mouse model (Purkayastha et al. 2011). Collectively, these data have highlighted a crucial role of an IKK2-dependent hypothalamic inflammatory response in the pathophysiology of various cardiovascular diseases.
The IKK2/NF–κB signaling pathway is pivotal in both acute and chronic inflammatory responses (Pahl 1999). Previous mechanistic studies by our laboratory (Kampfrath et al. 2011; Wang et al. 2017) and others (Feng et al. 2017; Jeong et al. 2017; Song et al. 2017; Zhang et al. 2017) have demonstrated that exposure to ambient PM2.5 activates the IKK2/NF–κB pathway, which is believed to play a key role the pathophysiologic effects of PM2.5 (Brook et al. 2010) in various tissues. Notably, we recently reported that chronic exposure of C57Bl/6J mice to ambient PM2.5 resulted in activation of IKK2/NF–κB pathway in the hypothalamus, abnormal SNS activation, and hypertension (Ying et al. 2014), while in the KKay mouse model of type 2 diabetes, intracerebroventricular injection of an IKK2 inhibitor reduced PM2.5 exposure–induced abnormalities in glucose homeostasis (Liu et al. 2014). These findings suggest that an IKK2-dependent hypothalamic inflammatory response may be crucial in the development of adverse health effects in response to ambient PM2.5 exposure. In the present study, to verify the role of hypothalamic inflammation in the pathophysiologic effects of ambient PM2.5, we exposed Nestin–creIKK2flox/flox (IKK2Neu–KO) mice and littermate controls (Nestin–creIKK2flox/+) to filtered air (FA) or concentrated ambient PM2.5 (CAP), and assessed their inflammatory and vascular responses.
University of Maryland, Baltimore (UMB) is an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited institution. All procedures of this study were approved by the Institutional Animal Care and Use Committee at UMB, and all the animals were treated humanely and with regard for alleviation of suffering. Nestin-cre transgenic mice in the C57Bl/6J background were obtained from Jackson Laboratories (Stock No.: 003,771). IKK2flox/flox mice were generated as previously described (Li et al. 2003) and back-crossed with C57Bl/6J for three generations. Nestin–creIKK2flox/flox (IKK2Neu–KO) and littermate Nestin–creIKK2flox/+ (control) mice were generated through in-house crossing of Nestin–creKK2flox/+ and IKK2flox/flox. The pups were genotyped at the age of 1 wk by polymerase chain reaction (PCR), and the genotyping results were verified before exposure to FA/CAP.
Concentrated Ambient PM2.5 Exposure
IKK2Neu–KO and control mice (male, 8–12 wk old) were grouped into four age-matched groups and randomly designated to be subjected to exposure to FA (six IKK2Neu–KO and seven controls) or CAP (seven IKK2Neu–KO and seven controls) from May 2016 to September 2016 for a total duration of 4 mo in a mobile trailer with a 12-h light/12-h dark cycle, temperatures of 18–25°C, and relative humidity of 40–60%. Because we failed to observe CAP exposure–induced impairment of glucose tolerance in female mice, male mice only were used in the present study. The mobile trailer was located on the campus of the UMB. Animal exposure and the monitoring of exposure atmosphere and ambient aerosol were performed as previously described using a versatile aerosol concentration enrichment system that was modified for long-term exposures (Geller et al. 2005; Maciejczyk et al. 2005). The exposure protocol comprised exposures for 6 h/d, 5 d/wk (no exposure took place during weekends). Ambient aerosol and exposure atmosphere were monitored as previously described (Maciejczyk et al. 2005). The elemental composition of CAP was determined by inductively coupled plasma mass spectroscopy (for trace element analysis as previously described (Mirowsky et al. 2013; Wang et al. 2017).
Bronchoalveolar Lavage and Lung Histopathology
After euthanasia by overdose of isoflurane, the mouse trachea was cannulated, and the right primary bronchus was closed off with a ligature. Through the tracheal cannula, 0.5 mL sterile phosphate-buffered saline with 0.1 mM ethylenediaminetetraacetic acid (EDTA) was instilled and withdrawn to recover bronchoalveolar lavage fluid (BALF). This was repeated twice. The total number of cells in the collected BALF (around 1.5 mL) was estimated using a hemocytometer. Cytospin slides were prepared using Shandon Cytospin 3™ (Shandon) and stained with Diff-Quik solution (EMS). Differential cell counts for neutrophils, eosinophils, macrophages/monocytes, and lymphocytes were assessed by a pathologist who was blinded to the grouping.
Following BALF collection, the right lung was harvested. About one-third were cut and fixed with 4% paraformaldehyde, and the rest were snap frozen in liquid nitrogen and then kept at −80°C. To assess the inflammation in the lung, tissue blocks were embedded in paraffin, 5–μm-thick sections were cut, and the sections were subjected to hematoxylin and eosin staining. Three consecutive sections per sample were used for histopathology. Images covering all the tissue area were taken by a laboratory technician who was blinded to the grouping, and all images were then sent to and quantitated by the pathologist (blinded to the grouping, too).
Plasma Cytokine Analysis
To rapidly harvest blood samples for cytokine analysis, mouse retroorbital blood samples were collected in EDTA-treated tubes and subjected to centrifugation for 15 min at 2,000×g using a refrigerated centrifuge. The supernatants (plasma) were then transferred to new tubes and stored at −80°C until assessments of cytokine levels. Plasma cytokine levels were assessed using BD™ Cytometric Bead Array Kit (BD Biosciences) per manufacturer’s instructions. Briefly, 25 μL/mouse plasma were incubated with the beads, and signaling was quantified by flow cytometry.
Real-Time Polymerase Chain Reactions
Mouse heads were removed before bronchoalveolar lavage. Hypothalamus, hippocampus, cortex, and olfactory bulb were immediately isolated. Epididymal adipose tissues were harvested after bronchoalveolar lavage. All these samples were snap frozen in liquid nitrogen and kept at −80°C. Total RNA was extracted and purified using the Trizol reagent (Invitrogen) from epididymal adipose tissue, lung, hypothalamus, hippocampus, cortex, and olfactory bulb. The quality of RNA was assessed by determination of the ratio of absorbance at 260 nm to absorbance at 280 nm by nanodrop. Two micrograms of total DNase-treated RNA were reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kits (Applied Biosystems™) per manufacturer’s instruction. Real-time PCR was performed using LightCycler® 480 SYBR Green I Master in the LightCycler® (Roche). Reactions were performed in a total volume of 10 μL containing 1 μL cDNA, 0.2 μM of each primer, and 5 μL of the SYBR® Green reaction mix. The ampliﬁcation protocol was as follows: 95°C/5 min (95°C/10 s, 60°C/20 s, and 72°C/30 s)×45. Following ampliﬁcation, a dissociation curve analysis was performed to ensure purity of PCR product. The speciﬁc sense and antisense primers for tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL–1β, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (the reference housekeeping gene) were previously described (Chen et al. 2017). In each sample, 2ΔΔCt was calculated and used to represent its gene expression level.
Standard techniques were performed with primary antibodies: mouse Anti-IKK2 (Millipore), mouse Anti–β–Actin (Sigma-Aldrich), and mouse Anti-GAPDH (Cell Signaling Technology). Signals were detected by chemiluminescence and analyzed by densitometry.
Vascular Function Assay
After euthanasia, mouse thoracic aorta was quickly removed and cleaned in physiological salt solution (PSS) containing (mM): NaCl, 130; NaHCO3, 14.9; KCl, 4.7; KH2PO4, 1.18; MgSO4–7H2O, 1.18; CaCl2–2H2O, 1.56; EDTA, 0.026; and glucose, 5.5. The aorta was cut into 2-mm rings. The aortic rings were then mounted in a muscle bath containing PSS at 37°C and bubbled with 95% O2–5% CO2. Isometric force generation was recorded with a Multi Myograph System (Danish Myo Technology). A resting tension of 4 mN was imposed on each ring, and the rings were allowed to equilibrate for 1 h. To test vascular function, the contractile responses of aortic rings to phenylephrine (PE) in the absence or presence of an NOS inhibitor ω–nitro–L–arginine methyl ester (L-NAME; 100 μM) were assessed in an accumulative manner. To analyze the endothelial function, aortic rings were precontracted with PE (1 μM), and acetylcholine (ACh) or sodium nitroprusside (SNP) was added in an accumulative manner.
All data are expressed as means±standard errors of the mean (SEMs) unless noted otherwise. Statistical tests were performed using one-way or two-way analysis of variance with Bonferroni post tests or unpaired t-tests using GraphPad Prism (version 4.1.2; GraphPad Software). The significance level was set at p<0.05.
The average PM2.5 concentrations in the FA and CAP chambers were 3.6±1.9 and 71.6±33.2 μg/m3, respectively. Table 1 shows the elemental composition of CAP and ambient PM2.5. Although identifying PM2.5 emission sources is generally beyond the scope of this paper, the relatively high ratio of Na/Al reflects more contribution by the marine source than the crustal source, which is consistent with the geographic proximity of the study site to the ocean (Huang et al. 1999), whereas the relatively high sulfur suggests that the study site was most strongly affected by secondary aerosols, which are likely to include emissions from coal-fired utility boilers located regionally (Huang et al. 1999).
|Mean (ng/m3)||SD||Mean (%)||SD|
Note: CAP, concentrated ambient particulate matter (PM)2.5; SD, standard deviation.
Characterization of IKK2Neu-KO and Evaluation of Pro-Inflammatory Cytokines after Exposure to Filtered Air or Concentrated Ambient PM2.5
In order to examine the role of neural inflammation in the PM2.5 exposure–related pathophysiology, we generated Nestin–creIKK2flox/flox (IKK2Neu–KO) and littermate Nestin–creIKK2flox/+ (control). Except for lower body weight at 12 wk of age (mean 22.3±1.1 vs. 20.3±0.6 g for controls and IKK2Neu–KO, respectively, n=8/group, student’s t-test p<0.05), adult IKK2Neu–KO mice were similar in appearance to adult control mice. Figure 1A shows the deletion of IKK2 in the brain of IKK2Neu–KO. To confirm the tissue-specific deletion of IKK2 in IKK2Neu–KO, we assessed IKK2 protein levels in different tissues. Figures 1B and C reveal that compared to control, IKK2Neu–KO had significantly decreased expression of IKK2 in the brain but not lung, heart, and epididymal adipose tissue, corroborating the neuron-specific deletion of IKK2 in these mice. Consistent with our previous study (Ying et al. 2014), Figures 1D and E show that CAP exposure significantly increased the hypothalamic expression of TNFα and IL-6 mRNAs. Neural IKK2 knockout did not significantly alter the hypothalamic expression levels of TNFα and IL-6 mRNAs in FA-exposed mice, but significantly reduced them in CAP-exposed mice. Given that recent studies have suggested that some components of ambient PM2.5 may enter the brain through the olfactory system (Maher et al. 2016), we isolated olfactory bulb, cortex, and hippocampus, and assessed their expression of TNFα and IL-6 mRNAs. Except for a marked trend of increase in the expression of IL-6 mRNA in the hippocampus of CAP-exposed control mice vs. that of FA-exposed control mice, no any other remarkable difference was observed (Figures 1F–K).
Evaluation of Pulmonary Inflammation after Exposure to Filtered Air or Concentrated Ambient PM2.5
Central inflammation has been implicated in the regulation of peripheral inflammatory responses (Liu et al. 2014), and pulmonary inflammation is widely believed to be essential in the pathophysiology due to exposure to ambient PM2.5 (Brook et al. 2010). To determine whether central inflammation plays a role in the inflammatory response of the lung to PM2.5 inhalation, we performed BALF cell differentiation. Figures 2A–D reveal that exposure to CAP significantly increased BALF total cells and macrophages in control mice. Consistent with many previous studies (Filep et al. 2016; Yoshizaki et al. 2017), although neutrophil infiltration is the hallmark of inflammation, we did not observe significant increase in BALF neutrophils in CAP-exposed mice (Figure 2D). This may be a reflection of the crucial role of neutrophils in acute but not chronic inflammatory response. BALF cell numbers were not significantly different between CAP- vs. FA-exposed neural IKK2–deficient mice, which suggests that CAP exposure–induced pulmonary inflammation may be regulated by a central mechanism. To confirm this, we performed lung histological analysis. The percentage of alveolar macrophages was significantly higher in CAP-exposed vs. FA-exposed IKK2Neu–KO mice and control mice, but was significantly lower in CAP-exposed IKK2Neu–KO mice vs. CAP-exposed controls (Figure 2E–F). Pulmonary TNFα and IL-6 expression was higher in CAP-exposed vs. FA mice, though the difference was significant only for controls (Figure 2G–I). In addition, pulmonary IL-6 mRNA expression was significantly lower in CAP-exposed IKK2Neu–KO mice than in CAP-exposed controls (Figure 2H).
Evaluation of Systemic and Adipose Inflammation after Exposure to Filtered Air or Concentrated Ambient PM2.5
Systemic inflammation is one of the putative mechanisms that mediate the development of extrapulmonary abnormalities due to exposure to ambient PM2.5 (Brook et al. 2010). In control mice, CAP vs. FA exposure was associated with higher plasma TNFα, IL–1β, and IL-6 levels (significant for IL–1β and IL-6 only) (Figure 3A–C). In CAP-exposed IKK2Neu–KO mice, plasma TNFα, IL–1β, and IL-6 were lower than in CAP-exposed controls (significant for TNFα and IL–1β only). In addition, IL-6 was significantly higher in CAP- vs. FA-exposed IKK2Neu–KO mice. (Figure 3C). In addition, exposure to ambient PM2.5 has been shown to induce marked inflammation in adipose tissues, and the latter is believed to be subsequent to systemic inflammation and play a role in the PM2.5 exposure–related abnormalities in energy and glucose metabolism (Brook et al. 2010). CAP vs. FA exposure was associated with significantly higher IL–1β mRNA expression in epididymal adipose tissue from both IKK2Neu–KO and control mice, but was not associated with significant differences in TNFα or IL-6 expression (Figure 4A–C). IL–1β mRNA expression in adipose tissue was significantly lower in FA-exposed IKK2Neu–KO mice vs. FA controls and in CAP-exposed IKK2Neu–KO mice vs. CAP controls (Figure 4C). Macrophage infiltration in epididymal adipose tissue was significantly higher in CAP-exposed IKK2Neu–KO and control mice relative to FA-exposed IKK2Neu–KO and control mice, respectively, but was significantly lower in CAP-exposed IKK2Neu–KO mice than in CAP-exposed controls (Figure 4D–E).
Evaluation of Endothelial Dysfunction after Exposure to Filtered Air or Concentrated Ambient PM2.5
Endothelial dysfunction may contribute to PM2.5 exposure–related cardiometabolic abnormalities, such as insulin resistance and hypertension (Brook et al. 2010). CAP vs. FA exposure was associated with a significantly higher aortic contractile response to PE in both IKK2Neu–KO and control mice, though the maximal PE response was significantly lower in CAP-exposed IKK2Neu–KO mice than in CAP-exposed controls (Figure 5A and Table 2). As increased contractile response may result from increased contractility of vascular smooth muscle and/or decreased endothelium-dependent relaxation, we next assessed the aortic response to an endothelium-dependent vasodilator, Ach. CAP vs. FA exposure was associated with a significantly reduced Ach-induced aortic relaxation in both IKK2Neu–KO and control mice, though the maximal effect was significantly lower in CAP-exposed IKK2Neu–KO mice than in CAP-exposed controls (Figure 5B and Table 2). In contrast, we did not observe any significant effect of CAP exposure and neural IKK2 knockout on aortic contractile response to PE in the presence of a NOS inhibitor L-NAME (Figure 5C) and aortic relaxation by endothelium-independent vasodilator SNP (Figure 5D). Together, these findings support a role of central inflammation in the mediation of ambient PM2.5 exposure–induced endothelial dysfunction.
Note: Complete data are shown in Figure 5. Ach, acetylcholine; CAP, concentrated ambient particulate matter (PM)2.5; FA, filtered air; L-NAME, L–NG–Nitroarginine methyl ester; PE, phenylephrine; SNP, sodium nitroprusside.
*p<0.05 vs. FA.
#p<0.05 vs. control, analysis of variance (ANOVA).
Findings from experimental studies of mice suggest that exposure to ambient PM2.5 may induce central inflammation characterized by activation of the IKK2/NF–κB signaling pathway in the hypothalamus (Liu et al. 2014; Ying et al. 2014). However, to our knowledge, the role of central inflammation in pathophysiologic responses to ambient PM2.5 exposure has not yet been systemically investigated. In the present study, we exploited a mouse model with reduced neuronal IKK2 expression to examine the role of central inflammation in PM2.5 exposure–induced peripheral inflammation and endothelial dysfunction. The main findings from the present study are that neural IKK2 reduction appears to inhibit CAP exposure–induced increases in a) hypothalamic TNFα and IL-6 mRNA expression; b) pulmonary, systemic, and adipose tissue inflammatory responses; and c) endothelial dysfunction. These findings provide further support for a role of central inflammation in the development of adverse cardiometabolic responses to ambient PM2.5 exposure.
Central inflammation, particularly in the hypothalamus, has been shown to play an important role in the pathogenesis of various cardiometabolic diseases such as obesity, hypertension, and diabetes (Han et al. 2016). We previously reported that exposure to ambient PM2.5 was associated with increased expression of pro-inflammatory cytokines and activation of IKK2 in the mouse hypothalamus (Ying et al. 2014). As in our previous study, we demonstrated that CAP exposure increased expression of pro-inflammatory cytokines in the hypothalamus. In addition, we demonstrated that these responses to CAP exposure were significantly inhibited or abolished in mice with reduced neural IKK2 expression. We found that, on average, IKK2 protein expression was only 31±12% lower in the brains of IKK2Neu–KO mice compared with Nestin–creIKK2flox/+ controls, consistent with neuron-specific knockout by Nestin-cre (Tronche et al. 1999) and the presence of cells other than neurons in brain, including astrocytes and microglia that may play a role in local inflammatory responses (Douglass et al. 2017; Jassam et al. 2017). Nonetheless, the partial reduction in brain IKK2 expression appeared to be sufficient to reduce or eliminate CAP exposure–induced expression of TNFα and IL-6 mRNA in the hypothalamus. A recent study reported hypothalamic inflammation induced by a single-day exposure to an HFD was inhibited in vagotomized mice, suggesting that the vagal afferent nerve may be responsible for transmitting gut-derived inflammatory signals to the hypothalamus (Waise et al. 2015). Further studies are needed to determine whether a similar mechanism might be involved in CAP-induced hypothalamic inflammation.
In addition to systemic circulation, foreign materials may also gain access to the brain via the olfactory mucosa, known as olfactory bulb transmission (Tjälve et al. 1996). Given the previous demonstration of ambient particles in the brain (Maher et al. 2016) and increasing evidence that exposure to ambient PM2.5 correlates to various brain insults (Underwood 2017), olfactory bulb transmission has been suspected to be an important mechanism for the access of ambient PM2.5 to the brain (Underwood 2017). In the present study, CAP vs. FA exposure was associated with significantly higher expression of the pro-inflammatory cytokines TNFα and IL-6 in the hypothalamus, whereas in the olfactory bulb, TNFα expression was higher in CAP- vs. FA-exposed control mice, but not significantly, and there was a nonsignificant decrease in IL-6 expression. Additional research is needed to clarify the potential for olfactory bulb transmission of PM2.5 to the brain, and its influence, if any, on central inflammation.
Another finding from the present study was that increases in plasma TNFα, IL–1β, and IL-6 in CAP- vs. FA-exposed control mice were reduced or eliminated in IKK2Neu–KO mice. This suggests a potential role of the central nervous system in inflammatory responses to CAP, consistent with our previous finding that intracerebrovascular administration of an IKK2 inhibitor was associated with reduced systemic inflammatory responses to CAP exposure (Liu et al. 2014). Moreover, as hypothalamic inflammation has been shown to correlate to ANS dysfunction (Han et al. 2016), these present data are also consistent with increasing evidence that ANS regulates systemic inflammatory responses (Lambert et al. 2015; Pavlov and Tracey 2017). Our finding that CAP exposure–induced pulmonary inflammation was reduced in IKK2Neu–KO mice compared with controls (Figure 2), consistent with involvement of a central neural mechanism in pathophysiologic responses to PM2.5 exposure, may seem inconsistent with compelling evidence that pulmonary inflammation in response to ambient PM2.5 is induced through toll-like receptors (TLRs)- and/or pattern recognition receptors (PRRs)-dependent mechanisms (Bekki et al. 2016; Fuertes et al. 2013; Shoenfelt et al. 2009; Zhao et al. 2012). However, previous studies have also reported evidence of close cross-talk between the brain and lung (Davison et al. 2012; Engel et al. 2015; Winklewski et al. 2014).
Exposure to ambient PM2.5 has been associated with endothelial dysfunction in humans (O’Neill et al. 2005; Wauters et al. 2013) and various animal models (Davel et al. 2012; Lei et al. 2005; Ying et al. 2013), and effects of PM2.5 on endothelial function have been implicated in the pathogenesis of cardiometabolic diseases associated with PM2.5 exposure, such as atherosclerosis, hypertension, and type 2 diabetes mellitus (Corban et al. 2017). As such, endothelial dysfunction is widely believed to be central in the pathophysiology due to exposure to PM2.5 (Brook et al. 2010). In the present study, although CAP exposure was associated with evidence of endothelial dysfunction in both control and IKK2Neu–KO mice, the response was significantly less pronounced in the mice with reduced neural IKK2 expression compared with controls, consistent with a role of central inflammation in PM2.5 exposure–related effects. Many studies have demonstrated that systemic inflammation may lead to endothelial dysfunction (Piccardi et al. 2017).
Although the present study supports a role of neural IKK2 in pathophysiologic responses to ambient PM2.5 exposure, it has a number of important limitations. These include the fact that we did not evaluate whether specific types of neurons, or neurons in specific locations, are responsible for differences in responses to CAPs between control and IKK2Neu–KO mice. This will require additional experiments targeting different neurons, particularly pro-opiomelanocortin and agouti-related peptide neurons in the hypothalamus and those in the nucleus ambiguus and dorsal nucleus of the brainstem, and concomitantly measuring effects of exposure to CAP. In addition, we did not investigate specific mechanisms through which neural IKK2 might mediate systemic inflammation or other effects of PM2.5 exposure, or directly assess neural mechanisms linking ambient PM2.5 with central inflammation.
In summary, our findings support a role of neural IKK2 in CAP exposure–induced local and systemic pro-inflammatory cytokine expression, pulmonary and adipose inflammation, and endothelial dysfunction, and thus advance knowledge our understanding of the contribution of central inflammation to the pathophysiologic effects of ambient PM2.5 exposure.
This work was supported by the National Institutes of Health (R01ES024516 to Z. Ying), the American Heart Association (13SDG17070131 to Z. Ying), and the National Natural Science Foundation of China (81770805 to Z. Ying, 81500216 to M. Chen, and 81302452 to L. Qiu).
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