Sterile Inflammation in Mouse Lung Driven by Lipid Mediator Pathways Following MWCNT Exposure.

Recent studies from us and others have shown that nanoparticle (NP) exposure could modulate endoplasmic reticulum (ER) stress. Since ER stress is closely related to lipid droplet biogenesis, in this study, we investigated if multi-walled carbon nanotubes (MWCNTs) might influence lipid accumulation in THP-1 macrophages through the modulation of ER stress. Exposure to up to 64 μg/mL pristine MWCNTs (p-MWCNTs), hydroxylated MWCNTs (h-MWCNTs), and carboxylated MWCNTs (c-MWCNTs) led to NP internalization, which was associated with a modest increase of cytotoxicity. Oxidative stress was also induced, showing as increased intracellular reactive oxygen species (ROS) and decreased glutathione. Only h-MWCNTs and c-MWCNTs significantly promoted the release of interleukin-8 (IL-8), which was significantly higher compared with that after p-MWCNT exposure. All types of MWCNTs significantly induced lipid accumulation in THP-1 macrophages and more modestly in the co-culture model consisting of 16HBE human bronchial epithelial cells and THP-1 macrophages. MWCNT-induced lipid accumulation in THP-1 macrophages was decreased modestly by antioxidant N-acetyl-l-cysteine and more effectively by ER stress inhibitor 4-phenylbutyric acid. Moreover, MWCNT exposure promoted the expression of ER stress gene DDIT3 as well as ER stress protein p-chop, whereas the transcription factor XBP-1s was decreased. In addition, the expression of scavenger receptors, namely CD36 and MSR1, was also elevated after MWCNT exposure. In conclusion, this study suggested that MWCNT exposure could promote lipid accumulation in THP-1 macrophages, which could be related to the modulation of ER stress leading to upregulation of scavenger receptors.

Overwhelming evidence links inflammation to the pathogenesis of smoking-related pulmonary diseases, especially chronic obstructive pulmonary disease (COPD). Despite an increased understanding of the disease pathogenesis, mechanisms initiating smoking-induced inflammatory processes remain incompletely understood. To investigate the mechanisms that initiate and propagate smoke-induced inflammation, we used a well-characterised mouse model of cigarette smoke exposure, mice deficient for interleukin (IL)-1α, IL-1β and Toll-like receptor 4, and antibodies blocking granulocyte-macrophage colony-stimulating factor (GM-CSF). Studies were also pursued using intranasal delivery of human oxidised low-density lipoprotein (hOxLDL), a source of oxidised lipids, to investigate the inflammatory processes associated with impaired lipid homeostasis. We found that cigarette smoke exposure rapidly led to lipid accumulation in pulmonary macrophages, a defining feature of foam cells, which in turn released high levels of IL-1α. In smoke-exposed IL-1α-deficient mice, phospholipids accumulated in the bronchoalveolar lavage, a phenomenon also observed when blocking GM-CSF. Intranasal administration of hOxLDL led to lipid accumulation in macrophages and initiated an inflammatory process that mirrored the characteristics of cigarette smoke-induced inflammation. These findings identify a link between lipid accumulation in macrophages, inflammation and damaged surfactant, suggesting that the response to damaged pulmonary surfactant is a central mechanism that drives cigarette smoke-induced inflammation. Further investigations are required to explore the role of distorted lipid homeostasis in the pathogenesis of COPD.

Objective Environmental exposures exacerbate age-related pathologies, such as cardiovascular and neurodegenerative diseases. Nanoparticulates, and specifically carbon nanomaterials, are a fast-growing contributor to the category of inhalable pollutants, whose risks to health are only now being unraveled. The current study assessed the exacerbating effect of age on multiwalled-carbon nanotube (MWCNT) exposure in young and old C57BL/6 and ApoE−/− mice. Materials and methods Female C57BL/6 and apolipoprotein E-deficient (ApoE−/−) mice, aged 8 weeks and 15 months, were exposed to 0 or 40 µg MWCNT via oropharyngeal aspiration. Pulmonary inflammation, inflammatory bioactivity of serum, and neurometabolic changes were assessed at 24 h post-exposure. Results Pulmonary neutrophil infiltration was induced by MWCNT in bronchoalveolar lavage fluid in both C57BL/6 and ApoE−/−. Macrophage counts decreased with MWCNT exposure in ApoE−/− mice but were unaffected by exposure in C57BL/6 mice. Older mice appeared to have greater MWCNT-induced total protein in lavage fluid. BALF cytokines and chemokines were elevated with MWCNT exposure, but CCL2, CXCL1, and CXCL10 showed reduced responses to MWCNT in older mice. However, no significant serum inflammatory bioactivity was detected. Cerebellar metabolic changes in response to MWCNT were modest, but age and strain significantly influenced metabolite profiles assessed. ApoE−/− mice and older mice exhibited less robust metabolite changes in response to exposure, suggesting a reduced health reserve. Conclusions Age influences the pulmonary and neurological responses to short-term MWCNT exposure. However, with only the model of moderate aging (15 months) in this study, the responses appeared modest compared to inhaled toxicant impacts in more advanced aging models.

The majority of lung diseases occur with a sex bias in terms of prevalence and/or severity. Previous studies demonstrated that, compared with males, female mice develop greater eosinophilic inflammation in the airways after multiwalled carbon nanotube (MWCNT) exposure. However, the mechanism by which this sex bias occurs is unknown. Two immune cells that could account for the sex bias are type II innate lymphoid cells (ILC2s) and alveolar macrophages (AMs). In order to determine which immune cell type was responsible for MWCNT-induced airway eosinophil recruitment and subsequent sex differences in inflammation and disease, male and female C57BL/6 mice were exposed to MWCNTs (2 mg/kg) via oropharyngeal aspiration, and the respiratory immune response was assessed 7 d later. Greater eosinophilia and eotaxin 2 levels were observed in MWCNT-treated females and corresponded with greater changes in airway hyperresponsiveness than those in MWCNT-treated males. In MWCNT-treated females, there was a significant increase in the frequency of ILC2s within the lungs compared with control animals. However, depletion of ILC2s via α-CD90.2 administration did not decrease eosinophil recruitment 24 h and 7 d after MWCNT exposure. AMs isolated from control and MWCNT-treated animals demonstrated that M2a macrophage phenotype gene expression, ex vivo cytokine production, and activation of (p)STAT6 were upregulated to a significantly greater degree in MWCNT-treated females than in males. Our findings suggest that sex differences in AM phenotype development, not ILC2 signaling, are responsible for the observed female bias in eosinophilic inflammation after MWCNT inhalation.

Lipid homeostasis is reprogrammed in the presence of inflammation, which results in excessive lipid accumulation in macrophages, and leads to the formation of lipid-laden foam cells. We aimed to link an inflammation-responsive transcription factor CCAAT/enhancer-binding protein delta (CEBPD) with polarized macrophages and dissect its contribution to lipid accumulation. We found that CEBPD protein colocalized with macrophages in human and mouse (C57BL/6, Apoe-/-) atherosclerotic plaques and that Cebpd deficiency in bone marrow cells suppressed atherosclerotic lesions in hyperlipidemic Apoe-/- mice. CEBPD was responsive to modified low-density lipoprotein (LDL) via the p38MAPK/CREB pathway, and it promoted lipid accumulation in M1 macrophages but not in M2 macrophages. CEBPD up-regulated pentraxin 3 (PTX3), which promoted the macropinocytosis of LDL, and down-regulated ATP-binding cassette subfamily A member 1 (ABCA1), which impaired the intracellular cholesterol efflux in M1 macrophages. We further found that simvastatin (a HMG-CoA reductase inhibitor) could target CEBPD to block lipid accumulation in a manner not directly related to its cholesterol-lowering effect in M1 macrophages. This study underscores how CEBPD functions at the junction of inflammation and lipid accumulation in M1 macrophages. Therefore, CEBPD-mediated lipid accumulation in M1 macrophages could represent a new therapeutic target for the treatment of cardiovascular diseases.

Pulmonary exposure to multiwalled carbon nanotubes (MWCNTs) causes indirect systemic inflammation through unknown pathways. MWCNTs translocate only minimally from the lungs into the systemic circulation, suggesting that extrapulmonary toxicity may be caused indirectly by lung-derived factors entering the circulation. To assess a role for MWCNT-induced circulating factors in driving neuroinflammatory outcomes, mice were acutely exposed to MWCNTs (10 or 40 µg/mouse) via oropharyngeal aspiration. At 4 h after MWCNT exposure, broad disruption of the blood-brain barrier (BBB) was observed across the capillary bed with the small molecule fluorescein, concomitant with reactive astrocytosis. However, pronounced BBB permeation was noted, with frank albumin leakage around larger vessels (>10 µm), overlain by a dose-dependent astroglial scar-like formation and recruitment of phagocytic microglia. As affirmed by elevated inflammatory marker transcription, MWCNT-induced BBB disruption and neuroinflammation were abrogated by pretreatment with the rho kinase inhibitor fasudil. Serum from MWCNT-exposed mice induced expression of adhesion molecules in primary murine cerebrovascular endothelial cells and, in a wound-healing in vitro assay, impaired cell motility and cytokinesis. Serum thrombospondin-1 level was significantly increased after MWCNT exposure, and mice lacking the endogenous receptor CD36 were protected from the neuroinflammatory and BBB permeability effects of MWCNTs. In conclusion, acute pulmonary exposure to MWCNTs causes neuroinflammatory responses that are dependent on the disruption of BBB integrity.

Endothelin-1 (ET-1), a potent proatherogenic vasoconstrictive peptide, is known to promote macrophage foam cell formation via mechanisms that are not fully understood. Excessive lipid accumulation in macrophages is a major hallmark during the early stages of atherosclerotic lesions. Cholesterol homeostasis is tightly regulated by scavenger receptors (SRs) and ATP-binding cassette (ABC) transporters during the transformation of macrophage foam cells. The aim of this study was to investigate the possible mechanisms by which ET-1 affects lipid accumulation in macrophages. Our results demonstrate that oxidized low-density lipoprotein (oxLDL) treatment increases lipid accumulation in rat bone marrow-derived macrophages. Combined treatment with ET-1 and oxLDL significantly exacerbated lipid accumulation in macrophages as compared to treatment with oxLDL alone. The results of Western blotting show that ET-1 markedly decreased the ABCG1 levels via ET type A and B receptors and activation of the phosphatidylinositol 3-kinase pathway; however, ET-1 had no effect on the protein expression of CD36, SR-BI, SR-A, or ABCA1. In addition, real-time PCR analysis showed that ET-1 treatment did not affect ABCG1 mRNA expression. We also found that ET-1 decreases ABCG1 possibly due to the enhancement of the proteosome/calpain pathway-dependent degradation of ABCG1. Moreover, ET-1 significantly reduced the efficiency of the cholesterol efflux in macrophages. Taken together, these findings suggest that ET-1 may impair cholesterol efflux and further exacerbate lipid accumulation during the transformation of macrophage foam cells.

Foam cell formation is an important step for atherosclerosis (AS) progression. We investigated the mechanism by which the long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) regulates foam cell formation during AS progression.Methods and Results: An in vivo AS model was created by feeding ApoE-/-mice a high-fat diet. Oxidized low-density lipoprotein (ox-LDL)-stimulated macrophages were used as a cellular AS model. Interactions between NEAT1, miR-17-5p, itchy E3 ubiquitin protein ligase (ITCH) and liver kinase B1 (LKB1) were analyzed. NEAT1 and ITCH were highly expressed in clinical samples collected from 10 AS patients and in ox-LDL-treated macrophages, whereas expression of both miR-17-5p and LKB1 was low. ITCH knockdown inhibited ox-LDL-induced lipid accumulation and LDL uptake in macrophages. Mechanistically speakingly, ITCH promoted LDL uptake and lipid accumulation in macrophages by mediating LKB1 ubiquitination degradation. NEAT1 knockdown reduced LDL uptake and lipid accumulation in macrophages and AS progression in vivo. NEAT1 promoted ITCH expression in macrophages by acting as a sponge for miR-17-5p. Inhibition of miR-17-5p facilitated ox-LDL-induced increase in LDL uptake and lipid accumulation in macrophages, which was reversed by NEAT1/ITCH knockdown. NEAT1 accelerated foam cell formation during AS progression through the miR-17-5p/ITCH/LKB1 axis.

Macrophage metabolism contributes to the progression of metabolic diseases, and peroxisome proliferator-activated receptors (PPARs) play vital roles in macrophage metabolism and the treatment of metabolic diseases. However, the role of PPARs in metabolic reprogramming related to lipid accumulation in macrophages, a key pathological event in metabolic diseases, remains unclear. We aimed to identify PPAR-mediated metabolic reprogramming and potential therapeutic targets associated with lipid accumulation in macrophages. Following treatment with oleate, oleate + WY-14643 and oleate + pioglitazone to induce alterations in PPAR signaling, lipids and relevant metabolism, macrophage samples were analyzed employing an untargeted metabolomics based on gas chromatography-mass spectrometry. The metabolomics approach revealed that multiple metabolic pathways were altered during lipid accumulation in oleate-treated macrophages and responsive to WY-14643 and pioglitazone treatment. Notably, levels of most metabolites involved in amino acid metabolism and nucleotide metabolism were accumulated in oleate-treated macrophages, and these effects were alleviated or abolished by PPARA/G activation. Additionally, during oleate-induced lipid accumulation and lipid lowering with WY-14643 and pioglitazone in macrophages, levels of most amino acids were positively associated with neutral lipid, total cholesterol, cholesterol ester, total free fatty acid and triglyceride levels but negatively associated with expression of genes related to PPARA/G signaling. Furthermore, glycine was found to be a potential biomarker for assessing lipid accumulation and the lipid-lowering effects of PPARA/G in oleate-treated macrophages. The results of this study revealed a high correlation of amino acid metabolism with lipid accumulation and the lipid-lowering effects of PPARA/G in macrophages.

Epidemiological studies and in vivo animal models have shown that exposure to PFAS can lead to cardiovascular toxicity and promote atherosclerosis. In this study, we explored the effects of PFOA and PFOS exposure on lipid accumulation in macrophages and analyzed critical markers of foam cell formation, which are early precursors of atherosclerotic lesions. Our results demonstrate that PFOS and PFOA enhance lipid and cholesterol accumulation in human U937-derived macrophages, which is characteristic of foam cells. PFOS and PFOA induced the activity of the peroxisomeproliferator-activatedreceptor gamma (PPARγ) and treatment with a PPARγ antagonist partly reversed the accumulation of lipids after PFAS exposure. Furthermore, the results show that PFOS and PFOA activate (NF)-erythroid-derived 2 (E2)-related factor 2 (Nrf2) and induce markers of oxidative stress. Gene expression analysis revealed that mRNA levels of interleukin-1β (IL-1β) and plasminogen activator inhibitor-2 (PAI-2) were upregulated in a time- and concentration-dependent manner in PFOS- and PFOA-treated macrophages. The expression of other key atherosclerosis-related enzymes, including cytochrome P450 8B1 (CYP8B1) and lanosterol synthase (LSS),was downregulated, whereas the expression of cyclooxygenase 2 (COX-2) and aldo-keto reductase family 1 member C3 (AKR1C3) was induced by PFOS and PFOA. Additionally, elevated levels of matrix metalloproteinases (MMP)-1 and MMP-12 were found in PFOS- and PFOA-treated cells, which were associated with increased cell migration. Furthermore, PFOS and PFOA enhanced the expression of IL-1β when macrophages were activated; however, elevated levels of IL-6 and COX-2 in activated macrophages were repressed by PFOS and PFOA. Together, the findings indicate that PFAS exposure modifies immune responses and promotes lipid accumulation in macrophages, potentially contributing to foam cell and plaque formation in atherosclerosis.

Multi-walled carbon nanotubes (MWCNTs) are increasingly used in industry and in nanomedicine raising safety concerns, especially during unique life-stages such as pregnancy. We hypothesized that MWCNT exposure during pregnancy will increase vascular tissue contractile responses by increasing Rho kinase signaling. Pregnant (17-19 gestational days) and non-pregnant Sprague Dawley rats were exposed to 100 μg/kg of MWCNTs by intratracheal instillation or intravenous administration. Vasoactive responses of uterine, mesenteric, aortic and umbilical vessels were studied 24 hours post-exposure by wire myography. The contractile responses of the vessel segments were different between the pregnant and non-pregnant rats, following MWCNT exposure. Maximum stress generation in the uterine artery segments from the pregnant rats following pulmonary MWCNT exposure was increased in response to angiotensin II by 4.9 mN/mm2 (+118%), as compared to the naïve response and by 2.6 mN/mm2 (+40.7%) as compared to the vehicle exposed group. Following MWCNT exposure, serotonin induced approximately 4 mN/mm2 increase in stress generation of the mesenteric artery from both pregnant and non-pregnant rats as compared to the vehicle response. A significant contribution of the dispersion medium was identified as inducing changes in the contractile properties following both pulmonary and intravenous exposure to MWCNTs. Wire myographic studies in the presence of a Rho kinase inhibitor and RhoA and Rho kinase mRNA/protein expression of rat aortic endothelial cells were unaltered following exposure to MWCNTs, suggesting absent/minimal contribution of Rho kinase to the enhanced contractile responses following MWCNT exposure. The reactivity of the umbilical vein was not changed; however, mean fetal weight gain was reduced with dispersion media and MWCNT exposure by both routes. These results suggest a susceptibility of the vasculature during gestation to MWCNT and their dispersion media-induced vasoconstriction, predisposing reduced fetal growth during pregnancy.

In the lung, carcinogenesis is a multi-stage process that includes initiation by a genotoxic agent, promotion that expands the population of cells with damaged DNA to form a tumor, and progression from benign to malignant neoplasms. We have previously shown that Mitsui-7, a long and rigid multi-walled carbon nanotube (MWCNT), promotes pulmonary carcinogenesis in a mouse model. To investigate the potential exposure threshold and dose-response for tumor promotion by this MWCNT, 3-methylcholanthrene (MC) initiated (10 μg/g, i.p., once) or vehicle (corn oil) treated B6C3F1 mice were exposed by inhalation to filtered air or MWCNT (5 mg/m3) for 5 h/day for 0, 2, 5, or 10 days and were followed for 17 months post-exposure for evidence of lung tumors. Pulmonary neoplasia incidence in MC-initiated mice significantly increased with each MWCNT exposure duration. Exposure to either MC or MWCNT alone did not affect pulmonary neoplasia incidence compared with vehicle controls. Lung tumor multiplicity in MC-initiated mice also significantly increased with each MWCNT exposure duration. Thus, a significantly higher lung tumor multiplicity was observed after a 10-day MWCNT exposure than following a 2-day exposure. Both bronchioloalveolar adenoma and bronchioloalveolar adenocarcinoma multiplicity in MC-initiated mice were significantly increased following 5- and 10-day MWCNT exposure, while a 2-day MWCNT exposure in MC-initiated mice significantly increased the multiplicity of adenomas but not adenocarcinomas. In this study, even the lowest MWCNT exposure promoted lung tumors in MC-initiated mice. Our findings indicate that exposure to this MWCNT strongly promotes pulmonary carcinogenesis.

BackgroundMulti-walled carbon nanotubes (MWCNTs) are widely used in many disciplines due to their unique physical and chemical properties. Therefore, some concerns about the possible human health and environmental impacts of manufactured MWCNTs are rising. We hypothesized that instillation of MWCNTs impairs pulmonary function in C57BL/6 mice due to development of lung inflammation and fibrosis.MethodsMWCNTs were administered to C57BL/6 mice by oropharyngeal aspiration (1, 2, and 4 mg/kg) and we assessed lung inflammation and fibrosis by inflammatory cell infiltration, collagen content, and histological assessment. Pulmonary function was assessed using a FlexiVent system and levels of Ccl3, Ccl11, Mmp13 and IL-33 were measured by RT-PCR and ELISA.ResultsMice administered MWCNTs exhibited increased inflammatory cell infiltration, collagen deposition and granuloma formation in lung tissue, which correlated with impaired pulmonary function as assessed by increased resistance, tissue damping, and decreased lung compliance. Pulmonary exposure to MWCNTs induced an inflammatory signature marked by cytokine (IL-33), chemokine (Ccl3 and Ccl11), and protease production (Mmp13) that promoted the inflammatory and fibrotic changes observed within the lung.ConclusionsThese results further highlight the potential adverse health effects that may occur following MWCNT exposure and therefore we suggest these materials may pose a significant risk leading to impaired lung function following environmental and occupational exposures.

Immunomodulation of susceptibility to pneumococcal pneumonia infection in mouse lungs exposed to carbon nanoparticles via dysregulation of innate and adaptive immune responses

MicroRNA–21 attenuates BDE-209-induced lipid accumulation in THP-1 macrophages by downregulating Toll-like receptor 4 expression